Any given sperm sample shows a complex heterogeneity revealed at different levels. The most obvious intra-sample diversity can be directly seen under the bright field microscope as different motions are shown by each spermatozoon. A closest examination also reveals a mosaic of subpopulations regarding various morphological features. This heterogeneity of the sperm motility and morphology is objectively confirmed by automatized techniques of sperm tracking and analysis (i.e., CASA and CAMA) that classify the spermatozoa into different subpopulations according to specific kinetical parameters (Martínez-Pastor et al., 2011) and morphologies (Yániz et al., 2015; Soler et al., 2016). Moreover, the composition of sperm subpopulations regarding motility is dynamic throughout time. For instance, in vitro conditions for capacitation cause time-dependent changes in the spermatozoa at the cellular level that can be recorded as changes in motility, such as the acquisition of the hyperactive motility. This motility type involves vigorous movements under low-viscous conditions produced by asymmetrical and high-amplitude waves in the flagella and resulting in erratic swimming trajectories (Suarez, 2008). However, not all the spermatozoa acquire the hyperactive motility under capacitation conditions, reaching a maximum of around 20% in humans (Burkman, 1984). Furthermore, human spermatozoa also show a heterogeneous response to chemical inductors of hyperactivation. Ooi et al. (2014), following a single cell analysis by high-speed video recording of adhered human spermatozoa, showed that in response to the potent hyperactivation inducer 4-aminopyridine (4AP), multiple patterns of flagellar beating are produced in each spermatozoa, configuring complex and heterogeneous responses to this type of stimulus. Moreover, using the chemoattractant progesterone, another inductor of hyperactivation, Armon and Eisenbach (2011) showed a variety of responses of individual spermatozoa within few seconds immediately after the sperm–progesterone interaction. These observations suggest the existence of different sperm subpopulations, each with a specific sensitivity to respond by chemotaxis, a mechanism leading the spermatozoa to orient their swimming within a chemical gradient (Eisenbach, 1999). In accordance, only 2–12% of the human spermatozoa are able to align the direction of their swimming toward follicular fluid in a chemotactical response (Cohen-Dayag et al., 1994) and only ∼5% of human spermatozoa migrate toward the warmer temperature in response to thermotaxis (Bahat et al., 2012), a mechanism leading the spermatozoa to orient their swimming within a temperature gradient (Bahat et al., 2003).

Sperm heterogeneity has been reported at every level and it can be noticed almost in any study and analysis. Accordingly, a number of research studies hypothesize that only a small subpopulation of spermatozoa within the ejaculate retain the ability to achieve fertilization (Holt and Fazeli, 2015; Sakkas et al., 2015; Pérez-Cerezales et al., 2017). This determines that a high number of spermatozoa are needed to be placed directly onto the oocyte for achieving fertilization in vitro. The standard ratio for IVF in humans is 50,000 or more motile sperm per oocyte (Stephens et al., 2013); however, the lower limits of this ratio for achieving fertilization depend on the sperm quality. For example, in a study with subfertile male patients showing diverse sperm qualities, the use of 5000 sperm/oocyte resulted in 37% of zygotes from total oocytes, a percentage that was significantly increased to 60% when using 20,000 sperm/oocyte (Tournaye et al., 2002). The meta-analysis conducted on available publications reporting randomized controlled trials for the treatment of male subfertility evidences a large variability of fertilization ratios, ranging from 50,000 to 10 × 106 sperm/oocyte for IVF (Tournaye et al., 2002). In mice, this limit was reduced to 5 motile sperm/oocyte achieving 60% of fertilization by prolonging the capacitated status of the spermatozoa via creatine supplementation of the medium (Umehara et al., 2018). Therefore, these results also indicate a transient availability of capacitated and "fertile spermatozoa" within the semen. Overall, these data indicate that from the whole spermatozoa population, only a small fraction is susceptible of acquiring the capacity to achieve fertilization at a given time point under in vitro conditions.

This observable intra-sample heterogeneity is surely determined by underlying differences among spermatozoa at the cellular and molecular levels. Using discontinuous Percoll gradient, Buffone et al. (2004) found at the different fractions (45, 65, and 90% of Percoll) that human spermatozoa possessed different characteristics regarding quality (motility and morphology) and capacity to show hyperactive motility and protein tyrosine phosphorylation in response to incubation under capacitating conditions. A recent study on bull sperm reported that within two different sperm populations separated by density gradient centrifugation (DGC), there were 31 proteins more abundant in low motile spermatozoa, while 80 proteins were more abundant in the high motile population (D'Amours et al., 2018). Following discontinuous gradient centrifugation of spermatozoa from normozoospermic men, Jenkins et al. (2014) proved the existence of differentially methylated regions in the DNA between high-quality and low-quality fractions. Immunocytological studies on the location of opsins in human and mouse spermatozoa revealed heterogeneous staining patterns for each of the studied proteins involved in the thermotaxis response (Pérez-Cerezales et al., 2015) and reported that a specific subpopulation (∼15% of the ejaculate) showing rhodopsin at a specific cellular location is the one with the capability of migrating toward the higher temperature within a gradient (Pérez-Cerezales et al., 2018).

The heterogeneity of the spermatozoa is also reflected at the genetic and epigenetic levels. Thus, single cell analysis of DNA fragmentation by the Comet assay reveals high intra-sample heterogeneity in human spermatozoa, as described by Simon et al. (2017a). We have observed as well that the level of DNA fragmentation of individual spermatozoa ranged from 0 to 70% in normozoospermic men and from 0 to 65% in epididymal mouse spermatozoa (Pérez-Cerezales et al., 2018). The sperm sample also shows various subpopulations at the chromatin packaging level, as evaluated by the sperm chromatin structure assay (SCSA). This assay discriminates subpopulations with high and low chromatin compaction, with different degrees among individual spermatozoids (Evenson, 2016). Moreover, about 9% of the human spermatozoa port some chromosomic alteration (Martin, 2008) and a high percentage of them carries punctual mutations (Wang et al., 2012). In addition, single cell analysis of the telomere length of human spermatozoa also revealed intra-sample heterogeneity with no differences between normal or abnormal spermatozoa (Antunes et al., 2015). These new findings included in the –omics fields reveal the high level of complexity of the ejaculate and the intimate association between these features and male infertility (for a comprehensive review on sperm –omics, we direct the reader to Sinha et al., 2017). The deep study of the genome, epigenome, transcriptome, proteome, and metabolome at the single cell level or in specific sperm subpopulations is expected to reveal higher levels of complexity than the currently known. This type of analyses could help to identify and correlate the presence/absence of specific sperm subpopulations with fertility, and these subpopulations could also be selected for improving ARTs outcome.

In conclusion, it is widely accepted that not all the spermatozoa from an ejaculate are equally good for achieving fertilization in vivo or in vitro (Holt and Fazeli, 2015). Due to the heterogeneity of the ejaculate, sperm selection prior to ARTs has been considered an important step for ensuring a successful outcome and a strategy to improve ARTs efficiency.

#### ROUTINE PREPARATION OF SEMEN FOR ARTs

Sperm selection/preparation techniques should be operatively simple and economic in order to fit in the routines of the human and veterinary clinics. Also, they always must ensure the enrichment of the sample in high-quality spermatozoa in the shortest time possible. Besides removing low quality spermatozoa, including those immotile, sperm preparation techniques should allow to eliminate other cells such as leukocytes and bacteria, as well as toxic or bioactive substances like reactive oxygen species (ROS) (Henkel and Schill, 2003).

Because they satisfy all these requirements, swim-up (SU) and DGC (**Figure 1**) are the most extended techniques for the preparation of spermatozoa (Henkel and Schill, 2003). SU was first described by Mahadevan and Baker (1984) and the general principle is the recovery of motile spermatozoa that migrate toward a cells-free medium usually placed above the sperm sample. Different variants of SU are available involving centrifugation (Mahadevan and Baker, 1984), straight migration from unprocessed semen (Aitken and Clarkson, 1988), recovery of spermatozoa from non-resuspended pellet (Carreras et al., 1990), or spermatozoa sedimentation by gravity prior to SU [migration sedimentation (MS) method] (Kiratli et al., 2018). On the other hand, DGC is based on the capacity of motile spermatozoa to progress through a gradient of density constituted by colloidal particles during centrifugation (Henkel and Schill, 2003). Variants of the method consist in different types of gradient: continuous or discontinuous (Henkel and Schill, 2003) and substances used to generate the gradient of density: ficoll (Harrison, 1976), PVP-coated silica particles or Percoll, or the current commercial variants such as IxaPrep R (MediCult, Copenhagen, Denmark), SilSelect R (FertiPro N.V., Beernem, Belgium), PureSperm R (NidaCon Laboratories AB, Gothenburg, Sweden), or ISolate R (Irvine Scientific, Santa Ana, CA, United States) (Henkel and Schill, 2003) used in human clinics to substitute Percoll due to its toxicity (Mousset-Siméon et al., 2004) and side effects on the sperm function (Strehler et al., 1998). In all the SU and DGC variants, it has been shown that the recovered sample is enriched in motile spermatozoa showing normal morphology. It has even been observed that both techniques select those spermatozoa with the longest telomeres (Zhao et al., 2016), this being an indicator of correct spermatogenesis (Rocca et al., 2016).

A number of studies have tried to clarify which of these two methods is more efficient; however, the results available in the literature are multiple and contradictory. In a first study approaching sperm selection for ICSI in patients with cryptozoospermia or severe oligoasthenoteratozoospermia (OAT), Sanchez et al. (1996) showed that the MS method (a type of SU) yielded a sperm fraction with better motility, vitality,

morphology, and chromatin condensation than when using minipercoll (a type of DGC), achieving 39.7% of pregnancies (n = 159 cycles). Also, Zini et al. (2000) showed that DGC is less efficient than SU for the separation out of spermatozoa with less DNA integrity. These results were contradictory to those reported by Sakkas et al. (2000) indicating the opposite. Amiri et al. (2012) reported that spermatozoa from normozoospermic donors selected by DGC showed less DNA fragmentation, higher normal morphology, and higher motility than when selected by SU (24 vs 32%, 28 vs 14%, and 76 vs 52%, respectively). Supporting these results, Wang et al. (2014) proved that DGC reduces DNA fragmentation in patients with oligozoospermia and astenozoospermia. Karamahmutoglu et al. (2014) reported that the use of spermatozoa of subfertile patients selected by DCG for intrauterine insemination significantly improved the pregnancy rate compared to the use of SU (18 vs 7%). In contrast, Oguz et al. (2018) reported that SU enriched the sample in spermatozoa with less DNA fragmentation in patients with mild or idiopathic male factor, while DGC did not allow such enrichment in those same patients. A variant of SU, the direct micro-SU, has shown comparable fertilization percentages by ICSI to those of DCG but higher blastocyst development in vitro and pregnancy rates (42 vs 26%, respectively), also reducing the abortion rate (13 vs 29%, respectively) (Palini et al., 2017).

In livestock species, SU and DGC are also the most used techniques for sperm selection (Arias et al., 2017) and although Percoll is currently avoided in human ART because of safety issues, as above indicated, it is still a method of choice for animal ART (Cesari et al., 2006). In rams, it has been reported that SU gives better results in terms of reducing the presence of apoptotic spermatozoa vs DGC with Percoll (67 vs 72%, respectively). However, DGC with Percoll delivered a higher percentage of capacitated spermatozoa than the SU method (Martí et al., 2006). In bovine, when comparing DGC (BoviPureTM) (Nidacon, Sweden) vs SU for selecting frozenthawed spermatozoa, BoviPureTM resulted in spermatozoa showing a greater progressive motility and viability, as well as a higher blastocyst yield (31.79 vs 21.91%, respectively) (Samardzija et al., 2006). Arias et al. (2017) reported that Percoll gradient recovered more spermatozoa with intact plasma and acrosomal membranes (89.8 and 87.5%) than BoviPure gradient (83.3 and 80.4%), but ROS levels were higher with Percoll separation. In addition, Percoll gradient resulted in lower blastocyst yield in comparison to BoviPureTM (∼20 vs 30%, respectively) using frozen-thawed semen (Lee et al., 2009). Thus, BoviPureTM should be considered the best frozen-thawed sperm selection technique for in vitro production of bovine embryos.

Both SU and DGC are regularly used in laboratories around the world prior to IVF and ICSI but, as discussed in the section "Introduction," the success rates of both techniques should be improved in both human and veterinary practices. Both methodologies for sperm selection are exclusively based on the motile capacity of sperm, which does not mean that all motile sperm are of the highest quality. For instance, it has been pointed that both SU and DGC are not efficient methods to select spermatozoa in terms of apoptosis, DNA integrity, membrane maturation, and sperm ultrastructure (Said and Land, 2011). Furthermore, centrifugation steps inherent to both techniques generate ROS, which have a detrimental effect on sperm quality (Aitken and Clarkson, 1988; Henkel and Schill, 2003). Therefore, new sperm selection methods should be based on sperm characteristics that are better connected to fertilization ability and quality of the spermatozoa, as well as on their contribution to support embryo development to term.

#### DIRECT SELECTION OF IMMOTILE SPERM

It is important to note that samples in which spermatozoa are immotile, or where the percentage of motile spermatozoa is low, are not suitable for SU and DGC methodologies. This is the case of sperm samples obtained by testicular sperm extraction (TESE) in azoospermic (AO) patients. Nowadays, in the routine practice of fertility clinics, only vague and subjective morphological

criteria are followed to select the immotile spermatozoa prior to ICSI, like the identification of spermatozoa with normal head and tail. However, in the last decades, a number of efforts have been made in order to find a selection method for this type of sample, aiming to discriminate viable spermatozoa directly under the micromanipulator irrespectively of their motility.

Marques de Oliveira et al. (2004) proposed the mechanical touch technique (MTT). According to these authors, MTT allows to identify immotile but viable spermatozoa because their tail is flexible when applying a lateral force to the flagella with the ICSI micropipette. In contrast, flagella of non-viable spermatozoa remain rigid to the same force. These authors conducted the only published clinical trial of MTT and found no differences in the ICSI outcome, including deliveries, between motile spermatozoa, and those immotile selected by MTT, in both fresh or frozen samples (Marques de Oliveira et al., 2004). Unfortunately, this study was never corroborated and completed by others. It would be desirable to report as well the comparison between the use of viable vs non-viable spermatozoa selected by MTT for ICSI. Without this information, it is difficult to envisage the actual utility of this technique.

Another sperm selection method that has been investigated is the hipo-osmotic swelling (HOS) test. This technique is based on the fundament that the tails of viable spermatozoa swell or curl under hypo-osmotic conditions due to normal membrane function, thus allowing their identification and recovery under the microscope, potentially for their use for ICSI (Verheyen et al., 1997). Sallam et al. (2005) conducted a randomized study with 79 couples following TESE-ICSI. In this study, where both frozen and fresh samples with total absence of sperm motility were used, pregnancy rates were 20.5% for the 44 couples following HOS and 2.9% for the 35 couples following morphological evaluation (normal head and tail) prior to ICSI. Fertilization and grade I and II embryos rates were also significantly higher in the HOS group. However, when data were analyzed separately, considering the type of sperm sample (fresh or frozen), pregnancy results were not significantly different.

Under polarized light microscopy, the head of viable spermatozoa is birefringent. On this basis, Baccetti (2004) proposed to use this property as a parameter for sperm selection. Indeed, the selection of immotile spermatozoa with birefringent head for ICSI has shown to increase clinical pregnancy and implantation rates compared to control immotile spermatozoa (58 vs 9% and 42 vs 12%, respectively) (Gianaroli et al., 2008) and to increase clinical pregnancy when compared to those selected by HOS (45 vs 11%, respectively) (Ghosh et al., 2012).

The use of chemical inducers of sperm motility has also been investigated. Thus, phosphodiesterase inhibitors of the xanthine family, such as pentoxifylline (PTF), demethylxanthine theophylline (TPF), and papverine have shown to activate the motility in a fraction of immotile testicular sperm (Ta¸sdemir et al., 1998; Ebner et al., 2011; Terriou et al., 2015). The use of this motile fraction, whether using PTF, TPF, or papverine, has allowed to achieve normal fertilization, pregnancy, and birth after ICSI (Terriou et al., 2000; Kovaci ˇ c et al., 2006 ˇ ; Amer et al., 2013; Sandi-Monroy et al., 2019). Furthermore, Mangoli et al. (2011) showed that the use of PTF compared with HOS selected spermatozoa, yielded higher fertilization rate, and doubled clinical pregnancies (32 and 16%, respectively, n = 25). Interestingly, the successful treatment of male infertility associated to the Kartagener's syndrome has been reported with both PTF and HOS, achieving the delivery of healthy babies in ICSI cycles (Hattori et al., 2011; Montjean et al., 2015). These findings show the therapeutic potential of both methodologies that could be useful for the treatment of other male infertility cases.

Motility of immotile but vital spermatozoa can also be induced with a single laser shot to the tip of the flagellum (Aktan et al., 2004). Thus, the laser-assisted immotile sperm selection (LAISS) has been proposed as an alternative to the use of chemicals like xanthines, avoiding in this way their potential toxic effects. This method has shown to significantly increase cleavage and birth rates after ICSI using both testicular and ejaculated sperm when compared to control groups (Aktan et al., 2004; Nordhoff et al., 2013). Moreover, LAISS has shown its utility to restore fertility following ICSI in specific cases of male patients showing primary cilia dyskinesia (Gerber et al., 2008) and the Kartagener's syndrome (Ozkavukcu et al., 2018). Despite the potential of this technique, its complexity and cost have possibly prevented its use in clinical routines and explain the scarce number of published studies.

In addition to these specific procedures, the methodologies described in the following sections, that do not require sperm motility, in principle are also suitable for sperm selection in immotile samples, although their efficiency for this type of samples should be further explored.

#### SPERM SELECTION BASED ON MEMBRANE CHARACTERISTICS

The outer membrane of the spermatozoa is crucial for their functionality as it takes part in a number of basic processes such as cell metabolism, capacitation, ova binding, acrosome reaction, etc. Its accessibility and relation with sperm vitality and quality make this organelle, its integrity, and variable characteristics, a logic target for the development of selection methods of highquality spermatozoa for their use in ARTs.

## Annexin V Magnetic Activated Cell Sorting (AV-MACS)

Magnetic activated cell sorting (MACS) is a method that allows the separation of cell populations based on their surface antigens (Plouffe et al., 2015). Coating magnetic nanoparticles with a molecule with affinity for these antigens allows the trapping of the desired cell subpopulation and its separation within a column subject to a strong magnetic field. Thus, cells expressing the antigen stay in the column while other cells flow through for downstream applications. The loss of membrane integrity is an early event of the apoptotic response

and leads to the externalization of phosphatidylserine (Elmore, 2007). On this basis, magnetic nanoparticles coated with Annexin V, a molecule showing high affinity for phosphatidylserine (Vermes et al., 1995), bind to apoptotic spermatozoa that are retained in the MACS column, allowing the recovery of non-apoptotic spermatozoa for their use in ARTs (Gil et al., 2013) (**Figure 2A**).

The use of Annexin V MACS (AV-MACS) for sperm selection in the human clinical practice was first reported by Grunewald et al. (2001), demonstrating the utility of this technique for the enrichment of the sample with non-apoptotic spermatozoa retaining membrane integrity. Since then, a number of studies have shown the enrichment of high-quality spermatozoa with reduced levels of DNA fragmentation using AV-MACS (**Table 1**). Said et al. (2006) employed AV-MACS for the selection of semen from 35 healthy donors, reporting an enrichment of spermatozoa with lower DNA fragmentation and higher oocyte penetration capacity. Using this method, Zahedi et al. (2013) also reported an enrichment of spermatozoa with lower DNA fragmentation in both fertile and infertile (terato- and asthenozoospermic) patients. Additionally, Lee et al. (2010) and Degheidy et al. (2015) proved that spermatozoa selected by AV-MACS from idiopathic infertile patients and patients diagnosed with varicocele showed lower DNA fragmentation than the original sample and kept intact sperm motility. However, a number of studies comparing AV-MACS with SU and DGC question the actual utility of this technique for the enrichment in spermatozoa with higher quality. Therefore, Tavalaee et al. (2012) found that combined AV-MACS followed by DGC on semen from infertile patients with different etiologies was more efficient at enriching the sample in spermatozoa with lower DNA fragmentation than both procedures autonomously or DGC followed by AV-MACS. Nadalini et al. (2014) obtained better results with DGC followed by SU than with DGC followed by AV-MACS in terms of sperm quality as determined by motility, morphology, and DNA fragmentation. Cakar et al. (2016) did not find significant differences in any of the analysis of sperm quality, including DNA fragmentation, by using AV-MACS, SU, DG, SU/AV-MACS, and DG/AV-MACS, perhaps due to the low sample size of the study. In contrast, Berteli et al. (2017) found a better quality in terms of motility and DNA fragmentation in spermatozoa selected by AV-MACS followed by DCG than with the other combinations tested: DCG and AV-MACS alone or DCG followed by AV-MACS. Similar to these results, Zhang et al. (2018) reported that the combination of DCG followed by AV-MACS in immotile sperm samples resulted in the recovery of a sperm population with lower DNA fragmentation compared to DGC alone. In agreement, Esbert et al. (2017) reported that DGC followed by AV-MACS reduced the number of spermatozoa porting chromosomal abnormalities.

Regarding the use of the spermatozoa selected by AV-MACS for ARTs, reported results are scarce and unclear (**Table 2**). An early study employing 196 oligoasthenozoospermic patients reported an increase of cleavage and pregnancy rates when AV-MACS was applied prior to ICSI in comparison to DGC (Dirican et al., 2008). However, another study registering 237 infertile couples following ICSI with donated oocytes did not find significant differences in terms of embryo quality and in the ratios of fertilization, implantation, pregnancy, and live birth when the semen was cryopreserved and then selected by SU or SU followed by AV-MACS (Romany et al., 2014). García-Ferreyra et al. (2014) reported higher, but not statically significant pregnancy and implantation rates when semen showing basal high DNA fragmentation was selected by AV-MACS followed by DGC, compared to DGC alone in ICSI treatments. These results are supported by those reported by Stimpfel et al. (2018) in couples of teratozoospermic patients and women over 30 years old undergoing ICSI, showing higher quality blastocysts when

#### TABLE 1 | DNA fragmentation of spermatozoa selected by AV-MACS.

DNA fragmentation was analyzed in all cases by TUNEL. Values of % DNA fragmentation are expressed as % ± SEM, SD or (interquartile range). ∗P < 0.05, ∗∗∗P < 0.001 and different letters indicate differences between groups (P < 0.05).

spermatozoa were selected by DGC/SU followed by AV-MACS, compared to DGC/SU. Recently, Ziarati et al. (2019) reported a significant increase in the percentage of high-quality embryos and clinical pregnancies employing DGC followed by MACS in 80 infertile couples showing male factor and undergoing ICSI when compared to DGC itself.

Studies conducted in livestock species using MACS are missing. In rabbits, AV-MACS did not show a clear enrichment in non-apoptotic spermatozoa and did not affect the reproductive outcome when the selected semen was used for AI (Vasicek et al., 2014).

These studies collectively show that the combination of DGC and MACS might provide slight benefits in patients with male factor undergoing ICSI in terms of clinical pregnancy. However, it is not clear to which extent live birth rate is actually improved. Thus, the type of donor or patient and the combination of AV-MACS with other sperm preparation methods could be determinant for improving ARTs. More experiments, registering larger number of patients, and exploring the different variables involved, are needed in order to certificate the utility of AV-MACS for the human clinical practice.

#### Hyaluronic Acid (HA) Binding

The hyaluronic acid (HA) is one of the main components of the extracellular matrix surrounding the cumulus-oocyte complex (COC) (Dandekar et al., 1992), and those spermatozoa that follow an adequate spermatogenesis and maturation exhibit binding sites to it (Cayli et al., 2003; Huszar et al., 2006). Therefore, two methodologies of sperm selection have been developed based on the spermatozoa-HA interaction: (i) recovering those spermatozoa trapped on the surface of HA coated dishes (Huszar et al., 2006) (**Figure 2B**) and (ii) picking up those spermatozoa moving slow when swimming in a medium containing HA in solution (Barak et al., 2001).

According to published data, both methods have shown their capacity to select spermatozoa with lower DNA fragmentation (**Table 3**). In an early work, Nasr-Esfahani et al. (2008), employing HA-coated dishes, reported a significant inverse correlation among the percentage of HA-bounded spermatozoa and protamine deficiency, DNA fragmentation, and abnormal sperm morphology in the original sample. While Razavi et al. (2010) reported that spermatozoa recovered from HA-coated dishes show the same level of DNA fragmentation than the unselected ones, other laboratories reported higher DNA integrity in spermatozoa selected by HA-binding methods (Parmegiani et al., 2010a; Yagci et al., 2010; Mongkolchaipak and Vutyavanich, 2013) Furthermore, Parmegiani et al. (2010a) and Huang et al. (2015) showed lower DNA fragmentation in spermatozoa selected in HA solution compared to SU, and by HA coated dishes compared to DGC, respectively. However, both studies reported similar levels of DNA fragmentation when compared to spermatozoa selected under the microscope regarding motility and morphological features. Similarly, Mongkolchaipak and Vutyavanich (2013) did not find any difference between DGC/HA coated dishes and DGC/intracytoplasmic morphologically selected sperm injection

TABLE 2 | Reproductiveoutcomes of ARTs employing spermatozoa selected by AV-MACS.

All the reported cases followedICSI. Values are expressed as % ± w/o, SEM, SD, or (interquartile range). ∗P<0.05, ∗∗P<0.01.

(IMSI) respect to sperm DNA fragmentation. These results question the utility of sperm selection based on HA-binding.

Although the use of HA was first conceived for substituting the routinely used PVP as an agent to decrease sperm motion prior ICSI (Sbracia et al., 1997; Barak et al., 2001), the potential as a selective agent was soon explored (Cayli et al., 2003). Thus, Park et al. (2005) used HA solution to select boar spermatozoa and found a significant increase of embryos with normal chromosomal counts and less chromosome abnormalities when compared to conventional ICSI. Few months later, similar results were reported in humans showing that sperm selection by HA led to a reduction of chromosomal disomy frequencies, diploidy, and sex chromosome disomy; all abnormalities associated to embryos produced by ICSI (Jakab et al., 2005). All these publications did not report clear effects on ICSI outcomes; however, this was possibly not carefully studied until 2008. Since then, a number of publications have specifically checked the capability of sperm selection by HA-binding for improving ICSI outcome (**Table 4**). In a first attempt, Nasr-Esfahani et al. (2008) found an increase of the fertilization rate using HA-coated dishes but there was no detectable effect on pregnancy or implantation rates. Parmegiani et al. (2010b) studied the reproductive outcome in 331 patients undergoing ICSI using HA solution (293 patients) or conventional ICSI (86), finding no differences in clinical pregnancies or live birth ratios but an improvement in embryo quality and implantation rate. In another study, the same authors reported comparable results employing 206 oligozoospermic patients (Parmegiani et al., 2010a). Similar results were reported in subsequent studies using either HA-coated dishes or HA solution (Choe et al., 2012; Parmegiani et al., 2012; Majumdar and Majumdar, 2013; Mokánszki et al., 2014). In contrast, Worrilow et al. (2013) reported that the use of HA-coated dishes significantly increased pregnancy (Worrilow et al., 2013; Mokánszki et al., 2014) and live birth rate (Mokánszki et al., 2014) for those patients undergoing ICSI which semen showed a HA-binding capacity ≤ 65%, as measured by the Hyaluronan binding assay. This effect proves that the method could be useful only in specific cases and indicates the need of a preliminary semen analysis for determining the suitability of the technique. Therefore, Erberelli et al. (2017) reported a significant improvement of ICSI using HA-coated dishes in patients with various categories of male factor. Their results also suggested that the benefit of this method could be higher for teratozoospermic patients. In a randomized study employing a large number of patients (2772 couples), Miller et al. (2019) did not find any significant benefit of HA-coated dishes compared to conventional ICSI and either detected an association between results of Hyaluronan binding assay and ICSI outcomes, contradicting previous works above described. These results collectively show a poor capacity of HA binding methods for improving ICSI; however, more studies are needed to identify the types of patients that could benefit from this procedure.

#### Zeta Method

Sperm membrane is negatively charged (Engelmann et al., 1988). On this basis, methods for separating X- and Y-bearing

Analysis of DNA fragmentation was conducted by Sperm Chromatin Dispersion (SCD) test, Acridin Orange fluorescence (AOF), or TUNEL. Values of % of DNA fragmentation are expressed as % ± SEM. ∗∗P < 0.01 and different letters indicate differences between groups (P < 0.05).

#### TABLE 4 | Reproductiveoutcomes of ARTs spermatozoa selected by hyaluronic acid.

All the reported cases followedICSI. Values are expressed as %±w/o, SEM, SD, or (interquartile range). ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001.

Novel Sperm Selection for ARTs

fcell-07-00298 November 29, 2019 Time: 17:0 # 11

spermatozoa (Engelmann et al., 1988) and for the separation of pure sperm heads from disintegrated mammalian spermatozoa (Chaudhuri and Datta, 1994) were developed decades ago. Later, the methods developed by Chan et al. (2006) and Ainsworth et al. (2005) (**Figure 2C**) allowed to collect the charged spermatozoa adhered to the wall of a centrifuge tube or migrating within an electric field, respectively. With both methodologies, downstream analysis of the selected spermatozoa revealed an increased percentage of spermatozoa with higher quality and porting highintegrity DNA (Kheirollahi-Kouhestani et al., 2009; Razavi et al., 2010; Zarei-Kheirabadi et al., 2012; Zahedi et al., 2013) (**Table 5**). This procedure, known as Zeta method, has allowed the selection of spermatozoa with lower DNA fragmentation compared to the HA-coated dish selection (Razavi et al., 2010). Also, sperm DNA fragmentation has been proven to be lower using DGC-Zeta than DGC by itself (Zarei-Kheirabadi et al., 2012) or MACS-DGZ (Zahedi et al., 2013). However, despite these promising results, only one randomized study has been published using spermatozoa selected by Zeta method in patients undergoing ICSI (Esfahani et al., 2016). In this research, significant increases in top quality embryos [45.83 ± 3.11% vs 35.38 ± 4.64% (P = 0.04)] and pregnancy rates [39.2 vs 21.8% (P = 0.009)]

were obtained with DGC/Zeta compared to DGC, respectively. However, further analysis is needed to know the potential of Zeta method for improving ARTs.

#### Future of Sperm Selection Based on Membrane Characteristics

As exposed above, sperm selection by AV-MACS and HA does not seem to contribute in general terms to a significant improvement of ARTs outcome, and Zeta method needs to proof its efficiency in larger studies. However, the use of the properties of the plasma membrane for sperm selection could really improve ARTs if markers with higher potential for discriminating highquality spermatozoa are discovered. Thus, new antigens could be used as targets for selection by MACS, by coated plates or even by fluorescence-activated cell sorting (Funaro et al., 2013). However, this research direction is currently poorly explored, possibly because of the lack of basic knowledge about markers discriminating the various sperm populations contained in the ejaculate. Heidari et al. (2018) explored three surface proteins, HSPA2, Dj-1, and serum amyloid P, as biomarkers of sperm DNA integrity and found that the abundance of these three

Analysis of DNA fragmentation was conducted by Sperm Chromatin Dispersion (SCD) test, Acridin Orange fluorescence (AOF), or TUNEL. Values of % of DNA fragmentation are expressed as % ± SEM or SD. ∗P < 0.05 and different letters indicate differences between groups (P < 0.05).

proteins in the semen was directly correlated to sperm quality, DNA integrity, and embryo quality. Mostek et al. (2018) found that high-quality bull ejaculates showed higher abundance of extracellular sperm surface proteins and that sperm proteins in low-quality ejaculates are characterized by high carbonylation levels. All these membrane features and new ones yet to be discovered could be used with the aim of improving ARTs through sperm selection.

#### SPERM SELECTION BASED ON SPERM MORPHOLOGY – INTRACYTOPLASMIC MORPHOLOGICALLY SELECTED SPERM INJECTION (IMSI)

The morphometric evaluation of spermatozoa is widely applied to analyze sperm quality due to its correlation to fertility (Yániz et al., 2015; Soler et al., 2016). Furthermore, the introduction of computer-enhanced digital microscopy has enabled the analysis and quantification of detailed features of the cell that applied to motile spermatozoa configure the "Motile Sperm Organelle Morphology Examination" (MSOME) (Bartoov et al., 2002). The use of MSOME for selecting spermatozoa for ICSI is known as IMSI. This technique consists in the selection and direct capture of those spermatozoa with a low number of vacuoles and a nucleus of normal morphology under a microscope equipped with a micromanipulation system and a magnification system of 6300x (Bartoov et al., 2002) (**Figure 3**). The power of selecting spermatozoa with higher DNA integrity has been proved by different authors, although contradictory results are also present in the literature (**Table 6**). Using this methodology, Franco et al. (2008) revealed that the presence of large vacuoles in the sperm nucleus is associated with a higher DNA fragmentation compared to those spermatozoa with normal nucleus. The correlation between the presence of vacuoles, as detected by MSOME, and DNA fragmentation was later confirmed by Wilding et al. (2011). However, Leandri et al. (2013) did not find significant differences in this respect between the spermatozoa selected by IMSI and those selected as conventionally done during regular ICSI. Other authors have suggested that IMSI could be useful only in specific cases of male infertility. Accordingly, when spermatozoa from infertile donors showing more than 13% of DNA fragmentation were selected by IMSI, those showing normal morphology under high magnification delivered less DNA fragmentation than those classified as motile and normal using conventional magnification of 200x (Hammoud et al., 2013). Moreover, the authors reported in the same work that those selected under lower magnification showed the same DNA fragmentation than the unsorted spermatozoa. Results reported by Lavolpe et al. (2015) suggest that in patients whose semen scored ≤ 4% according to the strict morphology index, the presence of vacuoles in the nucleus of the spermatozoa was less related to DNA fragmentation and chromatin compaction than in those patients with strict morphology index ≥ 14%.

Recently, in the largest study reported to date where 873 sperm samples were analyzed by MSOSE, the presence of head vacuoles

was not associated to sperm DNA fragmentation and live birth rate (Fortunato et al., 2016). Authors claim that vacuoles are physiological features that do not alter sperm functionality. The controversy about the convenience of IMSI for improving ARTs exists from the first moment the method was described. We address here the reader to an excellent systematic review on the literature published between 2001 and 2013, where the authors conclude that IMSI was only proven to improve reproductive outcome in cases of recurrent implantation failure following ICSI (Boitrelle et al., 2014). The same year, other authors published a meta-analysis on 13 publications confronting IMSI vs conventional ICSI in cases with previous ICSI failures and when the male factor was the cause of infertility, and concluded that in both cases, IMSI improved the reproductive outcome (Setti et al., 2014). However, authors pointed to the lack of randomized studies to corroborate their conclusions. Since then, together with the study of Fortunato et al. (2016), others have shown the failure of IMSI for improving ARTs (**Table 7**). Thus, Leandri et al. (2013), having examined 458 couples, did not find significant differences between IMSI or ICSI in the reproductive outcome, regardless of the initial characteristics of semen regarding DNA fragmentation, chromatin compaction, morphology, or motility. Setti et al. (2015) showed no benefits of IMSI for couples with poor ovarian response. In contrast, Shalom-Paz et al. (2015) reported that IMSI significantly increased implantation and clinical pregnancy rates in patients with repeated IVF-ICSI failure. However, Gatimel et al. (2016) failed in confirming these results. Kim et al. (2014) and Goswami et al. (2018) showed that IMSI applied to OAT patients of various severities resulted in significantly higher implantation, pregnancy rates, and live birth rates when compared to a previous ICSI cycle performed to the same patients, suggesting an actual utility of IMSI for these specific cases. Also, in another study on 170 patients,

TABLE 6 | DNA fragmentation of spermatozoa selected by IMSI.

Analyses of DNA fragmentation were conducted by, Acridin Orange fluorescence (AOF) or TUNEL. Values of % of DNA fragmentation are expressed as % ± SEM or SD. Values separated by double slash indicate correspondence to the employed method of DNA fragmentation in that column. In Lavolpe et al. (2015) % of DNA fragmentation are intervals according to different patterns of vacuole number, morphology, and distribution. ∗P < 0.05, ∗∗∗P < 0.001 and different letters indicate differences between groups (P < 0.05).

the results suggest that IMSI can improve pregnancy rates in patients with severe sperm pathologies affecting various sperm parameters (Schachter- Safrai et al., 2019). Overall, although the literature dissuades the routine use of IMSI, it seems that it could be highly indicated for the treatment of severe cases of male factor. Larger head-to-head randomized studies discriminating seminal parameters in infertile pathologies are still needed in order to elucidate under which circumstances IMSI could benefit reproductive outcome in the human clinical practice.

#### SPERM SELECTION BASED ON GUIDANCE MECHANISMS

As we discussed in the section "Introduction," guidance within the female genital tract has been suggested as a physiological mechanism for the selection of those spermatozoa able to fertilize the egg and ensuring the ulterior embryo development to term (Pérez-Cerezales et al., 2017). Hence, a strategy to develop new sperm selection methodologies is to employ in vitro the same principles that govern the sperm selection operating within the female genital tract. Consequently, there are three known mechanisms that have been proposed to guide the spermatozoa within the oviduct and they have been tested accordingly for their capacity to select spermatozoa with the aim of improving ARTs.

#### Rheotaxis

Miki and Clapham (2013) proved for the first time that both human and mice spermatozoa orientate their swimming against a fluid flow, process known as rheotaxis. Latter, El-Sherry et al. (2014) and Romero-Aguirregomezcorta et al. (2018) confirmed the occurrence of this phenomenon also in bovine, stallion, and ram spermatozoa, suggesting rheotaxis as a conserved feature of mammalian spermatozoa. Furthermore, the existence in mice of oviductal flow toward the uterus intensified after copula supports rheotaxis as a long-distance guidance mechanism within the oviduct (Miki and Clapham, 2013).

De Martin et al. (2017) conducted a first study employing rheotaxis to select spermatozoa from normozoospermic donors and reported an enrichment in spermatozoa with higher chromatin compaction (99%) compared to the sample before selection (71%) and to spermatozoa selected by DCG (83%). Zaferani et al. (2018) reported the design of a microfluidic device for selecting motile spermatozoa on the basis of rheotaxis; however, these authors did not

Novel Sperm Selection for ARTs

fcell-07-00298 November 29, 2019 Time: 17:0 # 15

#### TABLE 7 | Reproductiveoutcomes of ARTs spermatozoa selected by IMSI.

Values are expressed as%±w/o, SEM, or SD.∗P<0.05,∗∗P<0.01,∗∗∗P<0.001.

analyze the quality of the selected spermatozoa. Nagata et al. (2018) used another microfluidic device to select frozen bull spermatozoa by rheotaxis (**Figure 4A**). These authors reported a reduction of the level of DNA fragmentation (0.37%) in the spermatozoa selected by rheotaxis when compared to unselected semen (7%). The use of these selected bull spermatozoa for artificial insemination delivered pregnancy results similar to semen without selection, but using a 20 times lower dose (1 million sperm/insemination against 20 million sperm/insemination, respectively).

Thus, the results reported to date are promising, but the passive nature of rheotaxis, which is based on the hydrodynamics of the motile spermatozoa (Zhang et al., 2016), indicates that this mechanism has poor selective potential beyond the separation of those spermatozoa with a correct swimming behavior. As a matter of fact, half of the spermatozoa orient their swimming by rheotaxis, independently of being or not capacitated when directly observed under the microscope (Miki and Clapham, 2013). Although this is the in vitro situation, the in vivo picture could be drastically different. Moreover, it has been proposed that the planar move of non-capacitated spermatozoa would make them easier to stick to the epithelium preventing their migration by rheotaxis (Miki and Clapham, 2013) under the physiological conditions of viscosity. In contrast, the rotation along their longitudinal axis that determines the swimming of capacitated spermatozoa under a viscous medium could allow their detachment from this epithelium, allowing their free swimming against the fluid current (Miki and Clapham, 2013). In this way, rheotaxis would be part of a selection system of capacitated spermatozoa allowing only this subpopulation to migrate toward the in vivo fertilization site. In vitro developed procedures could test this hypothesis for sperm selection by rheotaxis considering these physiological conditions and then configure an effective way of selecting capacitated spermatozoa for improving ARTs.

#### Chemotaxis

Chemotaxis is the mechanism of navigation that the spermatozoa use in the proximity of the oocyte to orient their swimming in a gradient of chemical substances released by the COC where progesterone (P4) seems to be the main chemoattractant (Oren-Benaroya et al., 2008). It should be noted that only capacitated spermatozoa respond by chemotaxis, so this property could allow the selection of this specific subpopulation (Oren-Benaroya et al., 2008). Thus, Gatica et al. (2013) selected spermatozoa from both normozoospermic donors and subfertile patients by employing a simple device named "Sperm Selection Assay" (SSA) in which a P4 gradient was established (**Figure 4B**). In this work, it was found that in both cases, the selected spermatozoa were three times more capacitated, showed less DNA fragmentation and less oxidative stress than the semen before the selection. Li et al. (2018) also reported an improvement in the sperm quality from normozoospermic donors in terms of normal morphology, lower DNA fragmentation, and lower percentage of apoptotic spermatozoa when using another device composed of a system of microfluidic channels. Dominguez et al. (2018) proved that the use of the SSA device allows the selection of higher quality bull spermatozoa, improving cleavage rates using both sexed and unsexed semen for IVF. However, the effectiveness of sperm selection by chemotaxis in the improvement of ARTs in clinical practice has not been studied to date.

## Thermotaxis

Sperm thermotaxis consists in the orientation of the spermatozoa movement toward the highest temperature in a gradient (Bahat et al., 2003). Evidence indicates that this mechanism allows sperm to orient itself in the fallopian tubes to ascend to the ampulla (Pérez-Cerezales et al., 2017). As in chemotaxis, only capacitated spermatozoa respond to thermotactic stimuli, so based on it, this subpopulation could be selected (Bahat et al.,

2003). In a recent publication, employing a simple method (**Figure 4C**), we showed in mice and in normozoospermic patients that spermatozoa selected by SU/thermotaxis possess higher DNA integrity compared to the seminal sample prior to selection and after selection by SU (Pérez-Cerezales et al., 2018). In addition, the use of these selected spermatozoa for ICSI in mice increased cleavage rates and blastocyst production, as well as implantation and live birth rate as opposed to the use of spermatozoa selected by SU. Although this is the only work related to the use of spermatozoa selected by thermotaxis in ARTs, a recent study carried out in bulls has shown that seminal samples delivering high pregnancy rates after artificial insemination show a greater response to thermotaxis (Mondal et al., 2016). However, as in the case of rheotaxis and chemotaxis, there are still no available publications showing the capability of sperm selection by thermotaxis to improve the ARTs efficiency in the human clinic.

#### SPERM SELECTION BASED ON SPERM MOTILITY (MICROFLUIDICS)

As we have seen in some of the previous methodologies, microfluidic systems are becoming transversal devices for sperm selection based on several fundaments. Therefore, microfluidic systems have been tested for sperm selection by rheotaxis (Zaferani et al., 2018), chemotaxis (Ko et al., 2018; Nagata et al., 2018), and thermotaxis (Ko et al., 2018), as well as by other physical properties that operate at small scale, allowing the selection of motile spermatozoa (**Table 8**). Nosrati et al. (2014) reported the improvement of human spermatozoa selected by a microfluidic device composed of 500 parallel microchannels in terms of reducing more than 80% DNA fragmentation. Similar results were reported by Kishi et al. (2015), showing a significant reduction of DNA fragmentation in spermatozoa selected by a commercially available device based on microfluidics and flow dynamics (Sperm Sorter Qualis R , Menicon, Kasugai, Japan) when compared to SU or unselected spermatozoa. Using the same device, Shirota et al. (2016) also reported the selection of spermatozoa with lower DNA fragmentation compared to spermatozoa selected by DGC followed by SU in healthy donors. In addition, Nagata et al. (2018) reported a robust selection of human spermatozoa porting low DNA fragmentation by utilizing a diffuser-type microfluidic sperm sorter (DMSS). Quinn et al. (2018) employed another commercially available device, a singleuse chip with an inlet sample chamber connected to an outlet collection chamber by a microfluidic channel (FERTILE, Zymot, DxNow Inc., Gaithersburg, MD, United States), and showed a very effective selection of spermatozoa with low fragmented DNA compared to unselected spermatozoa or those selected by DGC-SU.

Due to the novelty of these methodologies, there is only one study that employs the selected spermatozoa in ARTs. In a recent publication in which spermatozoa were selected from 122 patients with infertility of unknown etiology using a commercial microfluidic device (61 patients) (Fertile Chip R , KOEK Biotechnology, Turkey) or by SU (61 patients), no differences were found in terms of fertilization, pregnancy, and live birth rates during ICSI between both sperm selection methods (Yetkinel et al., 2019). However, these authors observed

Analyses of DNA fragmentation were conducted by sperm chromatin structure assay (SCSA), sperm chromatin dispersion (SCD) test, or TUNEL. Values of % of DNA fragmentation are expressed as % ± SEM or SD. In Nosrati et al. (2014), DNA fragmentation separated by slash corresponds to the microchannel lengths 6/7.5/9 mm, respectively. Negative linear correlation between microchannel lengths was reported (R2 = 0.99). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Different letters indicate differences between groups (P < 0.005).

a higher quality in those embryos produced with spermatozoa selected by Fertile Chip R . Further studies are then necessary to determine under which conditions these devices are suitable for improving ARTs outcomes.

#### CONCLUSION

The efficiency of ARTs has a margin for improvement. Sperm selection may be an important factor for achieving higher live birth rates in ARTs, especially in infertility cases where the male factor is present. However, the methodologies developed to date have not proven to be useful for their routine application in the clinical practice and seem to be effective only in specific cases of male infertility. Some novel methods described here based on the physiological selection operating in vivo and on microfluidic environments have delivered promising results yet to be confirmed in large studies in the context of clinical practice. These studies should be randomized and strict in the confrontation of results with sperm samples of different qualities minimizing the female factor. It may even be necessary to combine several sperm selection methodologies to increase the efficiency of the ARTs.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

IO-L and SR-D conducted literature review and wrote the manuscript. PR-I wrote the manuscript. SP-C defined the topic, conducted literature review, and structured and wrote the manuscript.

#### FUNDING

This work was supported by Grant RTI2018-096736-A-I00 from the Spanish Ministry of Science, Innovation, and Universities. IO-L and SR-D were supported by CONACYT fellowship of the Mexican Government (283833) and "doctorados industriales 2018," fellowship of Comunidad of Madrid (IND2018/BIO-9610). PR-I was supported by a Talent Attraction Fellowship from Comunidad de Madrid. SP-C was supported by a Ramón y Cajal contract from the Spanish Ministry of Science, Innovation, and Universities (RYC-2016-20147).

#### ACKNOWLEDGMENTS

We thank Anna Riabzev for making the drawing.

over a wide range. PLoS One 7:e41915. doi: 10.1371/journal.pone.00 41915

functional human sperm even in subfertile samples. Mol. Hum. Reprod. 19, 559–569. doi: 10.1093/molehr/gat037

sperm injection. Curr. Opin. Obstet. Gynecol. 18, 260–267. doi: 10.1097/01.gco.<PHONE_NUMBER>.98061.2f

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Oseguera-López, Ruiz-Díaz, Ramos-Ibeas and Pérez-Cerezales. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Successful ICSI in Mice Using Caput Epididymal Spermatozoa

Raúl Fernández-González\*, Ricardo Laguna, Priscila Ramos-Ibeas, Eva Pericuesta, Víctor Alcalde-Lopez, Serafín Perez-Cerezales and Alfonso Gutierrez-Adan

Departamento de Reproducción Animal, INIA, Madrid, Spain

Spermatozoa undergo their last phase of spermiogenesis, known as maturation, as they pass through the epididymis. A recent report indicates that mouse immature spermatozoa retrieved from the caput epididymis for intracytoplasmic sperm injection (ICSI) give rise to embryos with multiple developmental defects. Further, these embryos were unable to develop to term after their transfer to surrogate mothers. Herein, we examined the potential of mouse caput spermatozoa to produce normal embryos by comparing the use of caput vs. cauda epididymal spermatozoa for in vitro fertilization (IVF) or ICSI. Two methods for the separation of sperm heads prior to ICSI were also compared: freezing/thawing or drawing through a syringe. We found that in contrast to caudal spermatozoa, caput spermatozoa failed to produce embryos via IVF, confirming their immature state. However, regardless of the method employed for the separation of sperm heads, similar efficiencies of blastocyst production in vitro and development to term after transfer to surrogate mothers were observed following ICSI using both caput and cauda epididymal spermatozoa. It therefore seems that mice spermatozoa recovered from the caput epididymis are as valid as caudal spermatozoa for the production of embryos and offspring by ICSI.

Keywords: ICSI, caput, epididymis, mice, sperm

## INTRODUCTION

Spermatozoa acquire their final mature state during passage through the epididymis. Mammalian immature sperm produced in the testis move from the ducts of the testis into the caput epididymis, where they are completely immobile and unable to fertilize an egg cell; then they are transported through the epididymis to the cauda region as a result of rhythmic contractions of this organ, where they undergo complex modifications, acquire motility and become fertilization-competent, denominating then mature sperm (Gervasi and Visconti, 2017). Thus, in theory, ejaculated sperm should have a better fertilizing capacity than sperm recovered from the testis as they have transited through the epididymis, where the final steps of maturation take place. These steps include compaction of sperm chromatin, formation of disulfide bridges between protamines and epigenetic remodeling (Ariel et al., 1994; Haidl et al., 1994; Perez-Cerezales et al., 2012). Chromatin maturation is required to fully stabilize and protect DNA from damage induced by endogenous nucleases or reactive oxygen species during sperm passage through the epididymis (Irvine et al., 2000; Perez-Cerezales et al., 2012). The importance of sperm maturation in the epididymis has also been confirmed by in vitro fertilization (IVF) assays performed with spermatozoa collected from different sites of the epididymis. In these assays, sperm retrieved from the distal part acquire a higher motility

#### Edited by:

Xin Zhiguo Li, University of Rochester, United States

#### Reviewed by:

Giuliano Callaini, University of Siena, Italy Duancheng Wen, Weill Cornell Medicine, Cornell University, United States

#### \*Correspondence:

Raúl Fernández-González<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cell Growth and Division, a section of the journal Frontiers in Cell and Developmental Biology Received: 29 July 2019 Accepted: 03 December 2019 Published: 17 December 2019

#### Citation:

Fernández-González R, Laguna R, Ramos-Ibeas P, Pericuesta E, Alcalde-Lopez V, Perez-Cerezales S and Gutierrez-Adan A (2019) Successful ICSI in Mice Using Caput Epididymal Spermatozoa. Front. Cell Dev. Biol. 7:346. doi: 10.3389/fcell.2019.00346

and ability to fertilize the oocyte (Sullivan and Mieusset, 2016). The epididymis is also able to eliminate defective or immature germ cells (Ramos-Ibeas et al., 2013).

Recently, it has been reported that small RNAs are trafficked to mammalian sperm during the process of post-testicular maturation in the epididymis (Sharma et al., 2018). It has been also described that 2-cell embryos generated via intracytoplasmic sperm injection (ICSI) using sperm obtained from the proximal (caput) versus distal (cauda) epididymis show similar RNA expression, but as development progresses, their gene expression profiles diverge (Conine et al., 2018). Further, embryos arising from caput spermatozoa (caput embryos) have been shown to have multiple developmental defects, and when transferred to surrogate mothers were unable to reach term (Conine et al., 2018). However, the injection of cauda- specific microRNAs (miRNAs) into caput zygotes was observed to suppress the specific gene expression profile of caput embryos and rescue their potential for development. These results indicate that dynamic small RNAs are essential for sperm maturation and also necessary for successful development and were taken as evidence that sperm-borne small RNAs derived from near the cauda region are critical for fetal development (Conine et al., 2018; Sharma et al., 2018). However, both testis spermatids and sperm support full-term development when injected into oocytes (Kimura and Yanagimachi, 1995; Moreira et al., 2016). In this context, the findings of Conine et al. (2018) suggest that the ability of sperm to support full-term embryo development after ICSI is acquired in the testis, then lost in the caput epididymis, and re-acquired during epididymal transit prior to arriving at the cauda. However, this hypothesis contradicts the results reported by others showing that ICSI produces normal offspring using caput sperm (Suganuma et al., 2005; Zhou et al., 2019).

Our hypothesis is that the developmental potential of caput and cauda fertilized embryo is similar and that the discrepancies of the results reported by Conine et al. (2018) respect to the literature could be due to differences in ICSI protocols, mainly in the technique used to separate the head from the tail of the sperm, since the different techniques can produce different degrees of alterations in the sperm (Tateno, 2008). Mouse spermatozoa are particularly sensitive to mechanical damage (Mazur et al., 2000), and these damages can be more decisive for the sperm of the caput, since it has been shown that there are more DNA fragmentation and less chromatin compaction in the caput than in the cauda epididymal spermatozoa (Perez-Cerezales et al., 2012), and that sperm obtained from the caput are more susceptible to nuclease-dependent DNA damage than sperm obtained from other epididymal regions (Yamauchi et al., 2007). Moreover, it has been reported that ICSI in mice using DNAfragmented sperm has been linked to altered gene expression at the preimplantation stage, reduction of fetal development, an increased risk of genetic and epigenetic abnormalities both in embryos and offspring (Ramos-Ibeas et al., 2014). Separation of the sperm heads from the tail prior to ICSI is commonly conducted by sonication, by fine needle shearing, by unprotected freezing, by piezo pulses, or by detergent treatment, and all these protocols may produce different level of chromosome aberrations in mouse (Martin et al., 1988; Tateno, 2008).

Our study addresses the reproductive potential of caput spermatozoa used for IVF or ICSI. As a positive control, we used caudal spermatozoa, regularly used for these procedures in mice. Our objective was to test the reported inability of caput spermatozoa to produce viable ICSI-derived mice by injecting into oocytes caput or cauda epididymal sperm recovered from standard B6D2F1 hybrid and inbred B6 mouse strains using similar protocols to previously described (Conine et al., 2018).

### MATERIALS AND METHODS

#### Handling of Mice and Sperm Samples

Experiments in mice were carried out in strict accordance with recommendations of the guidelines of the European Community Council Directive 86/609/EEC. The study protocol was approved by the Committee on the Ethics of Animal Experiments of the INIA (2016 permit number CEEA 2014/025). Spermatozoa were collected from the caput and caudal epididymis of 20-weekold B6D2F1 males (C57/BL/6JxDBA/2J) and B6 (C57BL/6NHsd) mice (**Figure 1**). Sperm were released by making an incision in the epididymal tissue and then squeezing to release its contents. The sperm were then suspended in M2 medium (Sigma–Aldrich, Madrid, Spain) for the ICSI experiments, or in human tubal fluid medium (HTF) supplemented with 1% BSA for IVF tests. To capacitate the sperm for the IVF experiments, samples were incubated for 30 min under an atmosphere of 5% CO2 at 37◦C.

#### Intracytoplasmic Sperm Injection (ICSI) and in vitro Fertilization (IVF)

For our ICSI experiments, the caput and caudal epididymis of the same animal were dissected separately, and sperm collected in M2 medium (6 adult individuals) as described previously (Perez-Cerezales et al., 2012). The sperm was mixed with five volumes of a 10% solution of polyvinyl-pyrrolidone in M2. ICSI was performed in M2 medium at room temperature (Ramos-Ibeas et al., 2014). Sperm heads were decapitated by the freezingthawing method (Moreira et al., 2003) or by drawing the sperm repeatedly through a fine-gauge needle (25G) into a 1 mL syringe 20–30 times (Conine et al., 2018). In parallel, oocytes were collected at metaphase II from B6D2F1 8-week-old females, which were superovulated by standard intraperitoneal injection (Moreira et al., 2005), and incubated with hyaluronidase (300 µg/mL) to remove cumulus cells. After allowing their recovery for 15 min, sperm were injected into the oocytes in M2 at room temperature. Surviving zygotes were cultured until the blastocyst stage to check their development or until the 2-cell stage for the embryo transfer experiments.

For the IVF experiments, oocytes were obtained from superovulated female mice as described above. The method used has been described elsewhere (Hourcade et al., 2010). Briefly, 2.5– 10.0 µL of fresh caput or cauda epididymal sperm were added to each fertilization drop to achieve a final concentration of ∼1– 2 × 106 spermatozoa/mL. Four hours after oocyte and sperm co-incubation at 37◦C in a humidified atmosphere of 5% CO2 in air, putative zygotes were washed and cultured in KSOM.

Statistical differences between caput- and cauda- derived embryos and offspring were assessed using an unpaired t-test and ANOVA (SigmaStat package), respectively. Significance was set at p < 0.05.

#### RESULTS

Sperm from the cauda and caput epididymis were collected from six 20-week-old sexually mature B6D2F1 and four inbred B6 male mice (Perez-Cerezales et al., 2012) and prepared according to requirements for IVF or ICSI (**Figure 1**). For IVF, the sperm were capacitated for 30 min and for ICSI the sperm were frozen without a cryoprotectant (Moreira et al., 2003) or drawn repeatedly through a fine-gauge needle into a 1 mL syringe 20–30 times (Conine et al., 2018) to obtain tail-free sperm heads. Our IVF experiments revealed that only caudal sperm had the capacity to fertilize oocytes. Thus, using caudal sperm, 96.29 ± 3.70% of fertilized oocytes (n = 48) underwent 2-cell cleavage and all of these embryos developed to blastocysts. In contrast, no fertilization was produced with sperm obtained from the caput epididymis: only 4 out of 128 (3.25 ± 3.25%) fertilized oocytes developed to the 2-cell stage, and none developed into blastocysts. This result could be explained by a lack of motility of caput sperm.

We next microinjected caput and caudal sperm (heads decapitated by freezing-thawing) of B6D2 mice into oocytes and found that embryos produced with both sperm types developed in vitro to the blastocyst stage at similar efficiencies (**Figure 2**). Given that caput-derived embryos were perfectly capable of developing to the blastocyst stage in vitro (57% of blastocysts produced by caput sperm vs. 63% by caudal sperm, four replicates and more than 100 embryos produced in each group), we then generated both caput- and caudaderived embryos by ICSI using paired sperm samples from the same animal, and surgically transferred 15–20 2-cell embryos into pseudopregnant surrogate mothers to analyze their in vivo developmental capacity. First, we used our classic method of fast freezing/thawing to separate sperm heads. As we show in **Table 1**, we found similar percentages of cleaved embryos and live offspring using caput or caudal sperm. We then used the drawing through a syringe method to decapitate sperm (Conine et al., 2018), and using both B6D2 and B6 mice, we also observed similar percentages of 2-cell embryos and live offspring derived from caput or caudal sperm. Interestingly, we noted a small but non-significant reduction in the number of live offspring when sperm were treated with the syringe, and also a non-significant reduction in the percentage of live offspring generated from caput sperm. In total, 20 and 30 pups were obtained by both procedures from caput and cauda sperm, respectively. Birth weights and blastocyst development were similar between the caput and cauda groups and both males and females were fertile (n = 8 per group).

## DISCUSSION

A recent report describes that embryos generated via ICSI using immature sperm derived from the caput epididymis show multiple defects during the peri-implantation period and quickly fail to undergo implantation (Conine et al., 2018). The authors

TABLE 1 | Development of oocytes injected with sperm from caput vs. caudal epididymis.

Four independent experiments were conducted. B6D2 = C57/BL/6 JxDBA/2 J; B6 = C57BL/6NHsd; DWG T syringe = Drawing through syringe. aAll recipients were pregnant. P > 0.05 Chi-square analysis.

of this paper also observed that these defects could be rescued by the injection of small RNAs derived from extracellular vesicles (epididymosomes), which deliver their small RNA repertoire to mature sperm, suggesting that small RNAs in mature sperm play major roles during early mouse embryogenesis. Here, we report that similar embryo development and offspring production results for ICSI using immature sperm from the caput epididymis or mature sperm from the caudal epididymis.

In mice, ICSI is performed with isolated sperm heads. However, sperm heads can be obtained by different protocols and this could have an appreciable impact on the sperm. In our hands, both protocols used to generate individual sperm heads (freezing/thawing, the standard method used in our laboratory, and drawing through a syringe, the method employed by Conine et al. (2018) produced healthy offspring from immature caput sperm. However, the number of live offspring generated with caput sperm was slightly lower than that generated with caudal sperm. In effect, this could be due to differences in maturation and sperm chromatin status between caput and caudal sperm. We earlier reported that the immaturity of mouse spermatozoa in the caput epididymis was revealed by a higher permeability of membranes to propidium iodide (PI) (Perez-Cerezales et al., 2012). Sperm chromatin undergoes the formation of disulfide bridges between protamines during the transit through the epididymis, increasing compaction of the genetic material (Golan et al., 1996). Also, using the comet assay and sperm chromatin structure assay (SCSA), we previously described different maturation stages of spermatozoa along the epididymis consisting of more DNA fragmentation and less chromatin compaction in the caput- than cauda epididymal spermatozoa (Perez-Cerezales et al., 2012). These differences in chromatin structure could make caput sperm more sensitive to decapitation procedures. Perhaps, a more aggressive technique will lead to more severe chromatin alterations in spermatozoa from the caput epididymis, or leave DNA more exposed and thus susceptible to degradation by endogenous or exogenous nucleases. Oocytes can support the preimplantation development of sperm with damaged chromatin, but major developmental defects appear later, after implantation (Fernandez-Gonzalez et al., 2008). This is in line with clinical observations that ejaculated sperm with damaged DNA go through normal preimplantation embryonic stages but fail to develop beyond this stage (Tesarik et al., 2004). Further, another two procedures designed to separate the sperm head from tail have been used to produce normal offspring from caput sperm (Suganuma et al., 2005; Zhou et al., 2019). The sperm head can be separated from the midpiece and tail by applying one or a few piezo pulses (Suganuma et al., 2005) or through sonication or the use of detergents like Triton X-100 or 3-[(3-cholamidopropyl) dimethylammonio]-1 propanesulfonate (Zhou et al., 2019).

There are several methodological differences between Conine et al. (2018) and previous studies (Suganuma et al., 2005; Zhou et al., 2019) which could potentially account for the discrepancy about the ability of caput-derived embryos to implant. In our experiments we used similar protocols to Conine et al. (2018) with the exception of the mouse strain background (we use B6D2 hybrids and inbred B6, and Conine et al. (2018) use inbred FVB). Further, other inbred mice have been also used, like 129Sc (Suganuma et al., 2005), showing to produce normal caput-derived mice. Although we cannot eliminate the possibility that the ability of caput-derived embryos to implant could be affected by strain background our results rule out the general character of the findings reported by Conine et al. (2018) for the mouse species.

In summary, sperm from the caput region of the epididymis are incapable of IVF but, contrary to recent findings (Conine et al., 2018), we here confirm that healthy fertile mice can be produced by ICSI from caput sperm.

#### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/supplementary material.

#### ETHICS STATEMENT

The animal study was reviewed and approved by the Committee on the Ethics of Animal Experiments of the INIA (2016 permit number CEEA 2014/025).

#### AUTHOR CONTRIBUTIONS

RF-G and RL produced the ICSI mice and collaborated in the phenotyping. PR-I, EP, VA-L, and SP-C performed all the experiments and co-wrote the manuscript. AG-A conceived the experiments and co-wrote the manuscript.

## FUNDING

This work was supported by Grant RTI2018-093548-B-I00 from the Spanish Ministry of Science, Innovation and

#### REFERENCES

Universities. PR-I was funded by a Talent Attraction Fellowship from the Madrid Community (2017-T2/BIO-5182). SP-C was supported by the Ramón y Cajal Programme (RYC-2016-20147).

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Fernández-González, Laguna, Ramos-Ibeas, Pericuesta, Alcalde-Lopez, Perez-Cerezales and Gutierrez-Adan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Signaling Enzymes Required for Sperm Maturation and Fertilization in Mammals

Souvik Dey, Cameron Brothag and Srinivasan Vijayaraghavan\*

Department of Biological Sciences, Kent State University, Kent, OH, United States

#### Edited by:

Zhibing Zhang, Virginia Commonwealth University, United States

#### Reviewed by:

Andrew Burgess, Anzac Research Institute, Australia Haim Breitbart, Bar-Ilan University, Israel Ana Maria Salicioni, University of Massachusetts Amherst, United States

> \*Correspondence: Srinivasan Vijayaraghavan<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cell Growth and Division, a section of the journal Frontiers in Cell and Developmental Biology

Received: 30 June 2019 Accepted: 03 December 2019 Published: 18 December 2019

#### Citation:

Dey S, Brothag C and Vijayaraghavan S (2019) Signaling Enzymes Required for Sperm Maturation and Fertilization in Mammals. Front. Cell Dev. Biol. 7:341. doi: 10.3389/fcell.2019.00341 In mammals, motility and fertilizing ability of spermatozoa develop during their passage through the epididymis. After ejaculation, sperm undergo capacitation and hyperactivation in the female reproductive tract – a motility transition that is required for sperm penetration of the egg. Both epididymal initiation of sperm motility and hyperactivation are essential for male fertility. Motility initiation in the epididymis and sperm hyperactivation involve changes in metabolism, cAMP (cyclic adenosine monophosphate), calcium and pH acting through protein kinases and phosphatases. Despite this knowledge, we still do not understand, in biochemical terms, how sperm acquire motility in the epididymis and how motility is altered in the female reproductive tract. Recent data show that the sperm specific protein phosphatase PP1γ2, glycogen synthase kinase 3 (GSK3), and the calcium regulated phosphatase calcineurin (PP2B), are involved in epididymal sperm maturation. The protein phosphatase PP1γ2 is present only in testis and sperm in mammals. PP1γ2 has a isoform-specific requirement for normal function of mammalian sperm. Sperm PP1γ2 is regulated by three proteins – inhibitor 2, inhibitor 3 and SDS22. Changes in phosphorylation of these three inhibitors and their binding to PP1γ2 are involved in initiation and activation of sperm motility. The inhibitors are phosphorylated by protein kinases, one of which is GSK3. The isoform GSK3α is essential for epididymal sperm maturation and fertility. Calcium levels dramatically decrease during sperm maturation and initiation of motility suggesting that the calcium activated sperm phosphatase (PP2B) activity also decreases. Loss of PP2B results in male infertility due to impaired sperm maturation in the epididymis. Thus the three signaling enzymes PP1γ2, GSK3, and PP2B along with the documented PKA (protein kinase A) have key roles in sperm maturation and hyperactivation. Significantly, all these four signaling enzymes are present as specific isoforms only in placental mammals, a testimony to their essential roles in the unique aspects of sperm function in mammals. These findings should lead to a better biochemical understanding of the basis of male infertility and should lead to novel approaches to a male contraception and managed reproduction.

Keywords: hyperactivation, fertility, PP1γ2, GSK3α, epididymal sperm maturation, PP2B, PKA, contraception

## INTRODUCTION

fcell-07-00341 December 16, 2019 Time: 15:39 # 2

Testicular spermatozoa in mammals are immotile and lack the ability to fertilize eggs. Motility initiation and fertilizing ability develop during their passage through the epididymis (Cooper, 1986; Bedford and Hoskins, 1990). Epididymal maturation is an absolute necessity to produce fertile spermatozoa. Sperm undergo hyperactivation in the female reproductive tract before penetration of the egg. Motile sperm failing to undergo hyperactivation cannot fertilize (Marquez and Suarez, 2008). Understanding the biochemical basis for how motility is attained and modified prior to fertilization is essential for understanding male fertility and infertility.

Sperm flagellar activity and also ciliary motility of somatic cells are known to be regulated by the intracellular mediators cAMP, calcium and pH. Capacitation is operationaly defined as functional modifications that enable sperm to fertilize eggs. Sperm undergo various changes during capacitation: (a) removal of membrane cholesterol followed leading to a decrease in cholesterol to phospholipid ratio (b) membrane hyperpolarization, rise in intra-sperm pH and cAMP levels, (c) increase in calcium uptake, and (d) an increase in protein tyrosine phosphorylation possibily due to increased protein kinase A (PKA) activity (Arcelay et al., 2008; Bailey, 2010; Alvau et al., 2016; Jin and Yang, 2017; Molina et al., 2018). It is generally accepted that these changes during capacitation lead to: (a) the ability of the sperm to bind the oocyte's extracellular matrix, the zona pellucida (ZP) (Si and Olds-Clarke, 1999; Topper et al., 1999) and subsequently undergo the acrosome reaction, (b) hyperactivation, a whiplash flagellar motion required to penetrate the egg (Ho and Suarez, 2001), and (c) the capacity to fuse with the oocyte (Evans and Florman, 2002). Hyperactivation, however, is regulated by similar yet distinct signaling events that distinguish it from capacitation (Marquez and Suarez, 2004).

Considerable progress has been made in understanding changes in sperm cAMP, calcium, and pH during sperm activation and hyperactivation. The proteins responsible for the generation of cAMP, calcium influx and changes in pHi (intracellular pH) have been identified and their functions elucidated by biochemical and genetic approaches. Despite this information, we still do not understand, in biochemical terms, how these second messengers enable sperm to acquire motility in the epididymis and how motility is altered in the female reproductive tract. Our understanding is incomplete because other signaling enzymes can profoundly alter the action and effects of second messengers in sperm. These signaling enzymes, protein phosphatase 1 (PP1), glycogen synthase kinase 3 (GSK3) and the calcium activated phosphatase (PP2B), affect sperm function in the epididymis and in the female reproductive tract. Several reviews detail the roles of sperm cAMP-PKA and calcium metabolism (Visconti et al., 2002; Burton and McKnight, 2007; Buffone et al., 2014; Stival et al., 2016; Freitas et al., 2017; Balbach et al., 2018; Leemans et al., 2019; Stewart and Davis, 2019). Following a brief summary of the actions of cAMP-PKA, calcium and pH, the remainder of the review will be focused largely devoted to examination of the key roles of PP1, GSK3, and calcium activated protein phosphatase, PP2B (PPP3R2/CC) in mediating the actions of the second messengers during sperm maturation and fertilization.

## ROLES OF cAMP AND PROTEIN KINASE A IN SPERM FUNCTION

Motility can be induced in demembranated testicular and caput epididymal sperm in the presence of ATP and cAMP, as well as appropriate calcium levels and pH (Lindemann, 1978; Mohri and Yanagimachi, 1980; Yeung, 1984; Lindemann et al., 1987; Bedford and Hoskins, 1990; Lesich et al., 2008). Early studies on the motility effects of phosphodiesterase inhibitors (such as caffeine, theophylline, and IBMX), on intact sperm led to the discovery of the role of cAMP in sperm motility regulation (Dougherty et al., 1976; Jiang et al., 1984). Phosphodiesterase inhibitors or cell permeable cAMP analogs can initiate motility in immotile spermatozoa and stimulate increased motility in motile sperm. Motility activation of demembranated sperm is abrogated in the presence of protein phosphatases in the reactivation medium, suggesting that cAMP mediates its motility effect through protein phosphorylation (Takahashi et al., 1985; Murofushi et al., 1986). An elevation of intra-sperm cAMP is also thought to be involved in motility initiation in the epididymis (Hoskins et al., 1974; Amann et al., 1982; Vijayaraghavan et al., 1985). A unique, hormone insensitive but bicarbonate sensitive, soluble adenylyl cyclase (sAC) is responsible for the synthesis of cAMP in sperm. Sperm cAMP levels should also be regulated by phosphodiesterases (PDEs) enzymes that degrade cAMP. Of the eleven PDEs, PDE1, PDE4, PDE8, PDE10, and PDE11 are present in testis and sperm (Omori and Kotera, 2007; Keravis and Lugnier, 2012).

It is well known that cAMP acts through a protein kinase (PKA). Knockout of the sperm sAC or PKA leads to infertility due to impaired sperm motility and the inability of sperm to undergo hyperactivation (Hess et al., 2005; Xie et al., 2006). Mature sperm contain PKA which is composed of regulatory subunit RIIα and a sperm specific catalytic subunit Cα2. Targeted knock-out of sperm-specific Cα2 results in male infertility. Spermatogenesis in these mutant mice is normal, while kinetic vigor and beat amplitude of epididymal sperm were markedly reduced. Mutant sperm are unable to undergo bicarbonate induced motility stimulation, and hyperactivation. It is known that PKA in cells owes it specificity and function to its localization through anchoring proteins known as AKAPs (Colledge and Scott, 1999). Sperm contain at least two PKA anchoring proteins AKAP 3 and 4 (also known as AKAP110 and 82, respectively) (Lin et al., 1995; Vijayaraghavan et al., 1999). Targeted deletion of AKAP 3 or 4 results in sperm dysfunction and male infertility (Jackson Labs Mouse Repository) (Miki et al., 2002). AKAP82 has been shown to play significant roles mediating the PKA action in murine and human sperm (Moss et al., 1999; Turner et al., 1999).

One of the downstream effects of PKA has been suggested to involve activation of protein tyrosine kinases (PTKs): proline−rich tyrosine kinase 2 (PYK2), ABL (Abelson murine leukemia viral oncogene homolog 1), SRC, and FER

(Baker et al., 2006, 2009; Alvau et al., 2016; Brukman et al., 2019). However, targeted disruption of these PTKs did not impair male fertility. Thus the exact role, of protein tyrosine phosphorylation in sperm function is still not well understood. PKA has also been suggested to regulate cAMP-phosphodiesterase activity. Phosphorylation by PKA has been shown to increase catalytic activities of PDE4 and PDE11 (Sette and Conti, 1996; Yuasa et al., 2000) suggesting a feedback regulation of sperm cAMP levels. Hyper-activated motility in sperm capacitation may also include phospholipase D-dependent actin polymerization (Itach et al., 2012). Thus while it is known that cAMP and PKA are essential in sperm the exact downstream biochemical actions of PKA are still unknown.

### THE ROLES OF pHi, CALCIUM AND CatSper CHANNELS IN SPERM FUNCTION

The role of pH in regulating flagellar motion was first recognized in the mechanism underlying activation of sea urchin sperm in sea water, which occurs due to increased pHi mediated by a sodium – proton exchange (Lee et al., 1983). Several studies have now shown that increasing intracellular pH activates motility of sperm in a number of species (Vijayaraghavan et al., 1985; Hamamah and Gatti, 1998; Carr and Acott, 2005; Nishigaki et al., 2014). A change in pHi is also thought to accompany sperm maturation in the epididymis (Vijayaraghavan et al., 1985). Acidic pH and the high concentration of lactate which acts as a membrane permeable proton carrier render sperm immotile in the luminal fluid of the cauda epididymis (Carr et al., 1985; Vijayaraghavan and Hoskins, 1988). Dilution of the quiescent caudal sperm, during ejaculation or in a buffer in vitro, results in initiation of vigorous motility. More recently, elevation of pHi has also been recognized to be a key event required for initiation of calcium influx during sperm hyperactivation. Knockout of channels, Slo3, NHE1 (Slca9c) or SLC26A3, responsible for changes in pH renders sperm infertile (Wang et al., 2007; Chen et al., 2009; Santi et al., 2010; Zeng et al., 2011; Chavez et al., 2014; Toure, 2019). A significant advance in understanding hyperactivation came from the discovery of calcium channels (CatSper) as essential mediators of sperm calcium influx. The calcium channel is composed of several essential subunits (Qi et al., 2007; Chang and Suarez, 2011; Singh and Rajender, 2015; Williams et al., 2015; Sun et al., 2017). Alkalinization of the sperm cytosol followed by activation of calcium channels (CatSper) triggers hyperactivation (Carlson et al., 2003; Qi et al., 2007; Lishko and Kirichok, 2010; Chung et al., 2011; Chavez et al., 2014; Lishko and Mannowetz, 2018; Orta et al., 2018). Prevention of the increase in pHi or loss of CatSper leads to infertility. A recent study shows that CatSper channels also mediate Zn2+-dependent stimulation of sperm hyperactivation (Allouche-Fitoussi et al., 2018). A model for sperm hyperactivation incorporating the functions of cAMP-PKA, calcium, and pHi is shown in **Figure 1**. This model is based on those presented in reviews noted earlier on the mechanisms regulating sperm function. Absent in theses models are the essential functions of the signaling enzymes PP1γ2, GSK3α, and PP2B required for sperm motility initiation and fertility.

## PROTEIN PHOSPHATASE ISOFORM, PP1γ2 IN SPERM FUNCTION

The presence of a protein kinase in a cell requires a corresponding protein phosphatase. The phosphorylation status of a protein is the result of the opposing activities of protein kinases and protein phosphatases. Based on the observation that protein phosphatases prevent motility initiation of demembranated spermatozoa and also enzyme activity measurements in sperm extracts (Swarup and Garbers, 1982; Takahashi et al., 1985; Murofushi et al., 1986), it was long suspected that a protein phosphatase regulates flagellar motility. However, the identity of the phosphatase and details of its regulation were not known. Research on protein phosphatases was boosted by the discovery of compounds, calyculin A, okadaic acid, and microcystin, isolated from marine organisms that are potent inhibitors of protein phosphatases (Cohen, 1990; Cohen et al., 1990; Fernandez et al., 2002). The inhibitors display distinct inhibition profiles against the serine/threonine phosphatases PP1, PP2A, and PP2B enabling their identification in cellular extracts (da Cruz e Silva et al., 1995). The protein phosphatase inhibitors, calyculin A and okadaic acid, were among the most potent in initiating and stimulating motility of sperm (Smith et al., 1996, 1999; Vijayaraghavan et al., 1996). The inhibitors initiated and stimulated epididymal sperm motility at nanomolar and micromolar concentrations, respectively. The inhibition profile of enzyme activity in sperm extracts suggested that the predominant phosphatase in sperm was protein phosphatase 1 (PP1) along with measurable levels of PP2A. There are four isoforms of PP1, PP1α, PP1β, PP1γ1, and PP1γ2, encoded by three genes (Okano et al., 1997; Lin et al., 1999). The amino acid sequences of all four proteins are essentially identical except at their C-termini. The two PP1 isoforms, PP1γ1 and PP1γ2, are alternate transcripts from a single gene, Ppp1c (**Figure 2A**). Based on enzyme activity profiles and western blot analysis, we found that the predominant serine/threonine protein phosphatase in spermatozoa is PP1γ2. High PP1γ2 activity is associated with low sperm motility, while low PP1γ2 activity is associated with vigorous motility (Smith et al., 1996, 1999; Vijayaraghavan et al., 1996). A decline in PP1γ2 activity occurs during epididymal sperm maturation, due to a decrease in its catalytic activity.  The enzyme PP1γ2 is present in spermatozoa of a wide range of mammalian species including human and non-human primates (Chakrabarti et al., 2007a; Vijayaraghavan et al., 2007).

In most tissues and cells, the loss of any one of the PP1 isoforms is compensated by one of the other isoforms. In yeast the loss of its endogenous protein phosphatase (GLC7) can be functionally replaced by one of the four mammalian PP1

isoforms, highlighting their functional equivalence (Gibbons et al., 2007). Because PP1γ2 was implicated in sperm motility it was of interest to see how its loss would affect sperm function. Loss of Ppp1cc leads to defects in spermiogenesis and lack of sperm in the epididymis (Varmuza et al., 1999; Chakrabarti et al., 2007b). The enzyme PP1γ2 has a dual role, one, during spermatogenesis and the other in sperm after their exit from the seminiferous tubules. This dual role of PP1γ2 is intriguing because loss of the enzymes of cAMP metabolism and action, sAC or PKA in testis, does not impair sperm morphogenesis or sperm formation. It is likely that other serine/threonine protein kinases along with PP1γ2 are responsible for regulation of protein phosphorylation during spermatogenesis (Kawa et al., 2006; Xu et al., 2007; MacLeod et al., 2014; Cruz et al., 2019). Due to its requirement in spermatogenesis, it is not possible to obtain sperm lacking PP1γ2, which is a limitation for the study of the enzyme in mature sperm.

The only phenotype resulting from the knockout of Ppp1cc is male infertility. Females lacking Ppp1cc are normal and fertile (**Table 1**). Conditional knockout in post-meiotic developing germ cells also has the same phenotype as the global loss of Ppp1cc showing the requirement of Ppp1cc only in differentiating germ cells in testis (Sinha et al., 2013). It should be noted that the Ppp1cc gene is responsible for expression of both the PP1 isoforms: PP1γ1 and PP1γ2. Since, PP1γ2 is the predominant isoform in testis, it strongly suggests, but does not prove that the reason for male infertility in mice lacking Ppp1cc is likely due to the absence of only PP1γ2 in differentiating spermatogenic cells. It was later confirmed that, despite the global absence of PP1γ1, transgenic expression of PP1γ2 driven by the PGK2 promoter in spermatocytes and spermatids of Ppp1cc null mice restored spermatogenesis, sperm function, and fertility (Sinha et al., 2012). These data provide compelling evidence that the PP1γ2 isoform expressed only in developing germ cells is sufficient for normal sperm function and fertility.

The phosphatase isoform, PP1γ2, is present only in eutherian mammals. Sperm from non-mammalian species and invertebrates contain one of the three PP1 isoforms – PP1α,

retained as an extended exon leading to the eight amino acid C-terminus of PP1γ1 (note that "exon 8" is part of its 30UTR). The PP1γ1 encodes a protein containing 323 amino acids derived from the seven exons along with the 8 amino acid C-terminus from the extended exon 7. In the post-meiotic germ cells in testis the intron 7 is spliced out, thus, producing a shorter Pp1γ2 transcript of approximately 1.7 kb. Exon 8 codes for the 22 amino acid C-terminus in PP1γ2. Thus, the amino acid sequences of PP1γ1 and PP1γ2 are identical in all respects except for their extreme C-termini. (B) Constructs for generating transgenic PP1γ1 mice. Rescues I–III constructs contain the entire or a portion of intron 7 which is part of the 30UTR of the messenger RNA for PP1γ1. There was little or no transgenic expression of PP1γ1 in testis of mice generated from these constructs. The last rescue construct (Rescue IV) lacks the 0.9 kb region of intron 7 following the stop codon in PP1γ1 mRNA. Transcript from this construct will resemble PP1γ2 mRNA except that PP1γ1 protein will be produced. The transgenic mice produced from this construct expressed high testis levels of transgenic PP1γ1 and rescued spermatogenesis but not sperm fertility.

PP1β, or PP1γ1 – which is able to support sperm motility and fertility in these species. The fact that PP1γ2 alone is sufficient for male fertility does not necessarily suggest that the other PP1 isoforms would be unable to functionally replace PP1γ2. Is the requirement for PP1γ2 an evolutionary accident or is there an isoform specific function for it in mammalian sperm? Can expressing the PP1γ1 isoform in testis, restore spermatogenesis and fertility of Ppp1cc null mice? Employing the same strategy used in the PP1γ2 transgenic rescue approach, the PGK2 promoter was used to drive transgenic expression of PP1γ1. This approach which was successful for PP1γ2 expression (Sinha et al., 2012), failed to transgenically express PP1γ1 (Dudiki et al., 2019) in testis. The transgene construct in all these failed attempts contained portions of the intron preceding exon 8, which is part of the 30UTR of the mRNA for PP1γ1 (**Figure 2B**). In the fourth attempt, removal of this entire portion of the 30UTR in the cDNA in the transgene construct (**Figure 2B**) led to robust expression of PP1γ1 in developing spermatocytes and spermatids (Dudiki et al., 2019). Transgenic expression of PP1γ1 in testis of Ppp1cc null mice was able to fully restore spermatogenesis. However, sperm function and fertility were severely compromised in these PP1γ1 rescue mice. Motility of PP1γ1-bearing sperm was diminished and their flagellar beat amplitude was severely dampened (Dudiki et al., 2019). Fertility defects in the rescue mice were most likely due to the inability of sperm bearing PP1γ1 to undergo hyperactivation. Thus PP1γ2 is essential in sperm for its normal function and fertility.

A specific isoform requirement of a protein is usually thought to arise due to isoform specific binding partners for that protein or due to its unique biochemical activity. An isoform specific function could also be due to the restricted spatiotemporal expression of the protein isoform during cell or tissue development. Despite the knowledge that PP1γ2 is the only PP1 isoform expressed in developing spermatocytes and spermatids (Chakrabarti et al., 2007b). it was anticipated that specific binding proteins for PP1γ2 exist in testis and sperm. However, binding partners of sperm PP1γ2 identified so far are ubiquitous in tissues and organisms and are known to bind to all PP1 isoforms (Goswami et al., 2019). That is, these proteins would bind to any PP1 isoform if present in sperm, just as they do in other cells and tissues. The three protein regulators of PP1γ2 identified are PPP1R2 (inhibitor I2), PPP1R7 (SDS22), and PPP1R11 (inhibitor I3).  Thus, loss of any one them is likely to cause embryonic lethality. It is intriguing that the sperm specific isoform, PP1γ2, is regulated by these ancient, ubiquitous, and essential PP1 binding proteins. The three regulators share localization with PP1γ2 in the head and the principal piece of sperm. The association of inhibitors to PP1γ2 changes during epididymal sperm maturation. In immotile caput epididymal sperm, PPP1R2 and PPP1R7 are not bound to PP1γ2, whereas in motile caudal sperm, all three inhibitors are bound as hetero-dimers or hetero-trimers (Goswami et al., 2019) (**Table 2** and **Figures 3A,B**). In caudal sperm from male mice lacking sAC and GSK3 (see below), where motility and TABLE 2 | The binding profile of the regulators with PP1γ2 in caput and caudal epididymal sperm is summarized in the table along with data with caudal sperm from Gsk3α knockout, PKA Cα2 knockout and sAC knockout mice where the binding status of sds22 resembles that in wild type caput sperm.

Thus, epididymal sperm maturation appears to be altered in these knockout mice.

fertility are impaired, the association of PP1γ2 to the inhibitors resembles immature caput sperm. In sperm containing PP1γ1 the association of these inhibitors are altered resembling that of PP1γ2 in immotile caput epididymal sperm (Goswami et al., 2019) (**Table 2**). It is known that binding of inhibitor 2 to PP1 is regulated by GSK3 (**Figure 3B**). It is likely that binding of the other two inhibitors, PPP1R7 and PPP1R2, to PP1γ2 are also regulated by phosphorylation. Changes in the associations of the regulators with PP1γ2, are likely part of biochemical mechanisms responsible for the development of motility and fertilizing ability of sperm.

A recently identified potential binding protein for PP1γ2 is CCDC181 (Schwarz et al., 2017). It is likely that CCDC181 binds to PP1γ1 and PP1γ2 with differing affinities leading to preferential localization of PP1γ2 to the flagellum. Determination of the protein targets of PP1γ2 in the flagellum and how CCDC181 regulates PP1γ2 is under active investigation.

#### GLYCOGEN SYNTHASE KINASE 3α, GSK3α, IN SPERM

The enzyme GSK3, a serine/threonine protein kinase, was named GSK-3 because it was discovered after PKA and phosphorylase kinase (GSK-1 and GSK-2). Two other GS kinases, GSK-4 and -5, named based on their relative elution profiles in phosphocellulose chromatography of muscle extracts, were later renamed as casein kinase 1 and 2, respectively (Parker et al., 1982). The enzyme GSK3, retained its original name even though it was later found to be a key signaling component of a large number of cellular processes (Kaidanovich-Beilin and Woodgett, 2011; Medina and Wandosell, 2011). An array of functions attributed to GSK3 include insulin action, regulation of cell survival, apoptosis, embryonic development, Wnt/β-catenin and hedgehog signaling, and growth factor action. It is also a target for drug development in several clinical disorders including cancer (Jope et al., 2007).

In mammals, GSK3 is ubiquitous and is expressed as two isoforms, GSK3α and GSK3β, encoded by different genes. The catalytic domains of the two isoforms are 98% identical while

GSK3α, PKA Cα2, PP1γ2 and PP2B (PPP3R2/CC) during epididymal sperm maturation and sperm hyperactivation in female reproductive tract. Curved orange

arrow(s) indicate relative degree of activities of the enzymes; straight black arrow(s) denote relative level of the ion/protein inside the cell.

their N- and C-termini are distinctive (Woodgett, 1990). While there are reports ascribing distinct roles for each of the isoforms (McNeill and Woodgett, 2010) under most circumstances the two isoforms are redundant and functionally interchangeable. Knockout of Gsk3β in mice causes late embryonic lethality (Hoeflich et al., 2000). The inability of GSK3α to substitute for GSK3β in the developing embryo may be due to the non-overlapping expression of the two isoforms. Conditional knockout of the floxed Gsk3β alleles on a Gsk3α null background show that complete loss of both isoforms impairs signaling and tissue function. However, one allele of Gsk3β or Gsk3α on a Gsk3α or Gsk3β null background, respectively, is sufficient to maintain normal Wnt signaling and tissue function (Doble et al., 2007; McNeill and Woodgett, 2010) highlighting the functional redundancy of the two isoforms in most tissues and cell types.

The protein, GSK3 was first discovered as an enzyme responsible for activation of PP1γ2 in bovine sperm (Vijayaraghavan et al., 1996). Both α and β isoforms of GSK3 are present in sperm. Immotile caput sperm contain four-fold higher GSK3 activity than motile caudal epididymal sperm. Both tyrosine phosphorylation (which stimulates catalytic activity) and serine phosphorylation of GSK3 (an inhibitory mechanism) increase significantly in sperm during their passage through the epididymis (Somanath et al., 2004). Incubation of motile or immotile sperm with compounds that activate PKA (e.g., dbcAMP) or inhibit protein phosphatase (e.g., calyculin A) is accompanied by increases in GSK3 serine phosphorylation and motility stimulation. GSK3 tyrosine phosphorylation which is believed to be autoregulatory, remains unchanged during capacitation, while only GSK3α ser21 phosphorylation is altered during this event (Dey et al., 2019a,b).

It was recently shown that Gsk3α null mice exhibit male infertility (Bhattacharjee et al., 2015, 2018). Knockout of GSK3α in post-meiotic testicular germ cells, using the Cre-Lox strategy, also results in male infertility. Mice with a testis knockout of GSK3β are normal and fertile. Thus, GSK3α has an isoform specific function in sperm. Analysis of sperm lacking GSK3α showed that adenine nucleotide levels, energy metabolism,

and protein phosphatase and kinase activities were affected suggesting impaired sperm maturation in the epididymis. A recent report also documents the role for GSK3 and a noncanonical Wnt signaling during epididymal sperm maturation: loss of Wnt signaling in sperm results in male infertility (Koch et al., 2015). The activity of GSK3α isoform has also been correlated with human sperm motility (Freitas et al., 2019). The inability of GSK3β to replace GSK3α, only in testis and sperm, is surprising given the fact that the two isoforms are functionally interchangeable in most cellular contexts and in tissues. Thus, despite the presence of both GSK3 isoforms, mammalian sperm are unique in their requirement for the GSK3α isoform. Taken together, these studies, support the notion that GSK3α is essential for epididymal sperm maturation, motility, and fertilization.

## CALCINEURIN IN SPERM FUNCTION

Calcineurin (also known as PP2B or PPP3C) is a serine/threonine phosphatase regulated by calcium. In response to an elevation of cellular calcium, calmodulin binds to a calmodulin binding region of the catalytic subunit PPP3C. This binding causes an auto-inhibitory arm of calcineurin to move away from the substrate binding site thus activating the enzyme by enabling its access to substrates (Rusnak and Mertz, 2000; Parra and Rothermel, 2017). The catalytic activity of the enzyme is also regulated by calcium binding to a regulatory subunit (PPP3R2). Regulation and function of calcineurin in several cell types has been extensively studied (Rusnak and Mertz, 2000; Parra and Rothermel, 2017). More than two decades ago a role for a calcium regulated protein phosphatase was proposed in the regulation of sperm motility (Tash et al., 1988; Tash and Bracho, 1994). In non-mammalian sperm, calcineurin has been shown to have role in activation of progressive motility and egg activation (Levasseur et al., 2013; Krapf et al., 2014). The catalytic and regulatory subunits of calcineurin are present as testis-specific isoforms, PPP3CC and PPP3R2. It was shown by super resolution microscopy that the catalytic subunit of calcineurin, PPP3CC, is localized near the quadrilateral structures of CatSper in the axoneme (Chung et al., 2014). In CatSper1-deficient spermatozoa, PPP3CC can be seen localized mostly to the axoneme but disappears from the quadrilateral structures. Another report showed that pharmacological inhibition of calmodulin affects protein tyrosine phosphorylation seen during sperm capacitation (Navarrete et al., 2015). In another study, micromolar amounts of FK506 has been demonstrated to prevent sperm acrosomal exocytosis (Castillo Bennett et al., 2010). A recent report now shows that knockout of either Ppp3CC or Ppp3R2 present only in testis and sperm resulted in male infertility (Miyata et al., 2015). Sperm numbers and testis weights in these knockout mice are normal; but sperm motility is impaired with a stiffened mid-piece. The Ppp3CC or Ppp3R2 knockout mice are infertile in vivo. Sperm from these knockout mice also cannot fertilize eggs in vitro. Surprisingly, wild type sperm treated with the calcineurin inhibitors, FK506 and cyclosporine, did not affect in vitro fertilization. Thus, infertility was thought to be due to impaired sperm function in the male reproductive tract. Calcineurin inhibitors injected into mice resulted in reversible male infertility. The investigators concluded that calcineurin was required for epididymal sperm maturation: genetic disruption or pharmacological inhibition in vivo affected sperm maturation causing infertility (Miyata et al., 2015).

The questions of how calcineurin may act and how it is activated during epididymal sperm maturation were not addressed. Recent data show that calcineurin and GSK3 are interrelated in their roles in epididymal sperm maturation and absence of calcineurin increases GSK3 phosphorylation resulting in its lower catalytic activity (Dey et al., 2019b). It is suspected that calcineurin regulates mitochondrial energization directly and glycolysis indirectly through its effect on GSK3. It is likely that high calcium levels in immature sperm (Vijayaraghavan and Hoskins, 1989, 1990) activates calcineurin.

#### INTERRELATIONSHIP BETWEEN PKA, PP1, GSK3α, AND PP2B

As described earlier the requirement and the roles of cAMP and the kinase activated by it, PKA, in sperm are well known. The relationship between PKA and GSK3 in sperm was indicated by the fact that cAMP analogs increased GSK3-α/β phosphorylation, without any isoform specificity. Phosphorylation of both GSK3 α and β isoforms were reduced and its catalytic activity increased in sperm with diminished cAMP (using KH7, a sAC inhibitor), or due to knock out of sAC. That is, GSK3 is a target of PKA phosphorylation. This relationship between GSK3 and PKA was further validated in GSK3α knockout mice. Loss of GSK3α or β by targeted disruption or pharmacological inhibition of the enzyme significantly reduced sperm cAMP levels (Dey et al., 2018). The decrease in cAMP levels was attributed to increased phosphodiesterase activity. Together these data support the possibility that GSK3 and cAMP form an interrelated regulatory loop (Dey et al., 2018).

Inhibition of the predominant sperm protein phosphatase PP1γ2 by calyculin A significantly increased phosphorylation of both GSK3 isoforms (Ser21/9) in caudal epididymal sperm and a concomitant decrease in its catalytic activity. Conversely, increased GSK3 activity is associated with increased PP1γ2 activity. One of the ways by which GSK3 regulates PP1 activity is by its phosphorylation of the inhibitor I2 as discussed earlier. Phosphorylated I2 dissociates from PP1 leading to its activation (**Figure 3B**). The roles of binding and dissociation of PP1γ2 binding proteins due to their reversible phosphorylation is a feature of the regulation of the phosphatase in sperm (Goswami et al., 2019). Sperm PP2A also targets GSK3, without any isoform specificity; however, its role in regulating GSK3 has not been investigated in detail (Dudiki et al., 2015).

New information shows that not only is GSK3 a target of PP1γ2, but it is also regulated by calcineurin. While PP1γ2 acts on both isoforms of GSK3 (Somanath et al., 2004),

calcineurin preferentially dephosphorylates only the GSK3α isoform (Dey et al., 2019a,b). Phosphorylation of the GSK3α (Ser21) isoform is elevated in sperm lacking calcineurin (Dey et al., 2019a,b). Following a decrease in their catalytic activities in caudal epididymal sperm during motility initiation, surprisingly the catalytic activities of both GSK3α and calcineurin increase during sperm capacitation and hyperactivation, recapitulating the situation in caput sperm (**Figure 3C**) (Dey et al., 2019a,b). Increased calcineurin activity, presumably following increased sperm Ca2+ during hyperactivation is responsible for decreased phosphorylation and increased catalytic activity of GSK3α. Pharmacologic inhibition of calcineurin during capacitation abrogated this decrease in phosphorylation and the increase in GSK3 activity. Increased activities of GSK3α and PP2B appear to be characteristics of not only capacitated sperm, but also, paradoxically, of immature caput epididymal sperm. Thus, phosphatases (PP1γ2 and PP2B) and the kinases (PKA and GSK3) are mechanistically interrelated during epididymal initiation of motility and also during fertilization of the egg (summarized in **Figure 3C**).

#### THE PROTEINS PP1γ2, GSK3α, PKA Cα2, AND PPP3R2/CC PRESENT ONLY IN SPERM ARE CONSERVED IN MAMMALS

As noted earlier the PP1γ2 isoform in sperm, is present only in mammals. Examination of the available annotated genome databases shows that monotremes and other non-mammalian vertebrates contain the Ppp1cc gene but the PP1γ2 isoform cannot form due to the absence of splice sites at exon 7 in the gene. Also absent is a region corresponding to exon 8, which is highly conserved and present in the Ppp1cc in all mammals (**Figure 4A**). This PP1γ2 isoform derived from exon 8 has a

conserved in all mammals only a portion of the N-terminus sequence of PPP3R2 is shown.

unique 22 amino acid C-terminus present and conserved in all mammals (**Figure 4A**). The somatic isoform, PP1γ1 has a six amino acid C-terminus derived from the extended exon 7. Aside from these differences in the C-termini, the rest of 315 amino acid sequences of the both PP1γ1 and PP1γ2 are identical. Why mammalian sperm contain only PP1γ2 is unknown.

It is known that knockout of the enzyme synthesizing cAMP in sperm, sAC, and that of PKA result in male infertility. It is intriguing that the catalytic subunit of PKA is also expressed as a sperm specific isoform (PrkaCA also known as PKA Cα2) with a unique six amino acid N-terminus due to expression of an alternate exon, exon 1b (Agustin et al., 2000; Desseyn et al., 2000; San Agustin and Witman, 2001) (**Figure 4B**). The exon 1a codes for 12 amino acids of the N-terminus of the ubiquitous and the somatic cell version of the catalytic subunit. Aside from this difference in their N-termini the primary sequences of the rest of the catalytic subunits are identical. The reason this different N-terminus is required in testis and sperm remains puzzling because both sperm and somatic cell forms of the PKA catalytic subunits have identical biochemical properties (Vetter et al., 2011). Removal of exon 1b leading to the loss of PKA in sperm, renders males infertile (Nolan et al., 2004). Whether replacement

of the sperm form of the enzyme with the somatic form of the PKA catalytic subunit would sustain normal sperm function is not known. However, it is intriguing that exon 1b, the sperm specific isoform of PKA, is present only in mammals (Soberg et al., 2017). Non-mammalian species only contain the isoform derived from utilization of exon 1a (**Figure 4B**).

A germ cell–enriched protein, viz. sperm PKA interacting factor (SPIF), was found to be co-expressed and co-regulated with PKACα2 and with t-complex protein (TCP)-11 (Stanger et al., 2016). These three proteins constitute part of a novel trimeric complex in murine spermatozoa. During capacitation, the SPIF undergoes phosphorylation leading to a molecular rearrangement that brings PKACα2 and TCP11 into close proximity of each other. These results could explain how PKA Cα2 functions as a specific isoform complexed with SPIF and TCP11 during capacitation and fertilization.

The GSK3α isoform is essential in mammalian sperm despite the fact both GSK3α and β isoforms are interchangeable in other cells and tissues. GSK3α arose in vertebrates presumably by gene duplication of GSK3β which is the only isoform in invertebrates. Among vertebrates the GSK3α isoform is absent in birds (Alon et al., 2011). Sequence comparison of the extended glycine rich N-terminus present only in GSK3α shows that this sequence segment is highly conserved only in placental mammals. This extended N-terminus is present, but not conserved in non-mammalian vertebrates (**Figure 4C**). We predict that sperm from non-mammalian vertebrates contain only GSK3β and not GSK3α. In fact, we have shown that Xenopus sperm contains only GSK3β despite the fact both GSK3 isoforms are present in the genome of this species. These observations are compatible with the possibility that mammalian sperm contain a GSK3α-specific binding protein. Efforts to identify a isoform-specific binding protein using a two-hybrid approach with testis cDNA yielded a number of GSK3 binding proteins, but these GSK3 interactors were not isoform specific (Freitas et al., 2019). However, a yeast two-hybrid approach using human fetal cDNA yielded four GSK3α-specific binding proteins one of which appears to play a role in regulating circadian rhythm (Zeidner et al., 2011). One of these four GSK3α binding proteins is CENPV which is highly expressed in testis (NCBI, mouse ENCODE transcriptome data). CENPV also binds to tubulin (Honda et al., 2009) and is therefore, expected to be localized along the length of the flagellum. Thus, GSK3α in the flagellum is likely to orchestrate phosphorylation of proteins involved in regulating sperm motility and hyperactivation. The requirement of sperm GSK3α with its conserved of N-terminus suggests a role for it in maturation and fertilization events unique to mammals.

A relatively recent addition to the list of signaling enzymes regulating sperm function is the calcium regulated phosphatase, PP2B. Catalytic activity of PP2B is required in sperm during their passage through the epididymis as is the case with GSK3α. Sperm and testis express specific isoforms of the catalytic and regulatory calcineurin, PPP3CC and PPP3R2, respectively. Examination of the genomic sequences of several species shows that PPP3R2 is present only in eutherian mammalians and its amino acid sequence is remarkably conserved in the 121 mammals for which annotated genomic databases exist (**Figure 4D**).

Several predominantly testis-expressed proteins present in mammalian sperm have been identified as mammal-specific. These include proteins involved in DNA binding, sperm egg binding or those required for required for the unique structural features of mammalian sperm, such as protamine 3, SMCP, and ADAM proteins (Cho, 2012; Luis Villanueva-Canas et al., 2017). However, the observation that the four signaling enzymes suggested to be mechanistically interrelated are mammal specific isoforms is significant, suggesting a unique function in male gametes (**Figure 3C**). The specific isoforms PP1γ2, GSK3, PKA and PP2B play key roles in regulation sperm motility and hyperactivation, a phenomenon unique only to mammals (**Figure 5**). It would appear that their roles in these physiological functions in mammals arise due to their location in the flagellum. **Figure 5** shows the intrasperm localization of these enzymes. It would be interesting to determine if one or more of CatSper complexes located along the flagellum are mammal specific. The two possible binding proteins for PP1γ2 in the flagellum are CCDC181 (Schwarz et al., 2017) and PPP1R32 (Cifuentes et al., 2018), both of which are expressed predominantly or exclusively in the testis. As described above, the GSK3α binding protein CENPV should be present bound to the flagellum because CENPV also binds to tubulin, which in sperm is only present in tail (Honda et al., 2009). Sperm calcineurin has been shown by high resolution microscopy to be localized along the flagellum (Chung et al., 2014). The nature of the binding protein for calcineurin in sperm is not known. Finally, PKA is known to bind AKAP3, which is present along the sperm flagellum (Vijayaraghavan et al., 1999). How the unique N-terminus of the sperm specific PKA catalytic subunit affects this localization is not known. Thus, all the four signaling proteins localized in the flagellum (PP1γ2, GSK3α, PP2B, and PKA) are likely to determine the phosphorylation status of proteins orchestrating motility and metabolism required for normal sperm function (**Figure 3C**).

In summary, considerable data show that the enzymes PP1γ2, GSK3 and PP2B, along with PKA, are mechanistically interrelated in regulating the two physiological processes unique to mammals: epididymal sperm maturation and sperm hyperactivation preceding fertilization (**Figure 5**).

#### AUTHOR CONTRIBUTIONS

SV and SD contributed to the conception and outline of the review. SD and CB organized the database. SV wrote the first draft of the manuscript. SD wrote the sections of the manuscript. All authors contributed to the manuscript reading and revisions.

#### FUNDING

This work was funded by the National Institute of Health (SV): R03HD096176 and R21HD086839.

## REFERENCES

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signaling shown by using an allelic series of embryonic stem cell lines. Dev. Cell 12, 957–971. doi: 10.1016/j.devcel.2007.04.001

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fertility and sperm cell hyperactivated motility. Proc. Natl. Acad. Sci. U.S.A. 104, 1219–1223. doi: 10.1073/pnas.0610286104

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a novel, sperm-specific protein kinase A-anchoring protein. Mol. Endocrinol. 13, 705–717. doi: 10.1210/mend.13.5.0278

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Dey, Brothag and Vijayaraghavan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# A Kinase Anchor Protein 4 Is Vulnerable to Oxidative Adduction in Male Germ Cells

Brett Nixon1,2 \*, Ilana R. Bernstein1,2, Shenae L. Cafe1,2, Maryse Delehedde3 , Nicolas Sergeant3,4, Amanda L. Anderson1,2, Natalie A. Trigg1,2, Andrew L. Eamens1,2 , Tessa Lord1,2, Matthew D. Dun5,6, Geoffry N. De Iuliis1,2 and Elizabeth G. Bromfield1,2,7

1 Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, Discipline of Biological Sciences, The University of Newcastle, Callaghan, NSW, Australia, 2 Pregnancy and Reproduction Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia, 3 SPQI – 4BioDx-Breeding Section, Lille, France, 4 University of Lille, INSERM UMRS, Lille, France, 5 Cancer Signalling Research Group, School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, Callaghan, NSW, Australia, 6 Priority Research Centre for Cancer Research Innovation and Translation, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia, 7 Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands

#### Edited by:

Xin Zhiguo Li, University of Rochester, United States

#### Reviewed by:

Woo-Sung Kwon, Kyungpook National University, South Korea Philip Chi Ngong Chiu, The University of Hong Kong, Hong Kong

\*Correspondence: Brett Nixon<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cell Growth and Division, a section of the journal Frontiers in Cell and Developmental Biology

Received: 11 July 2019 Accepted: 20 November 2019 Published: 20 December 2019

#### Citation:

Nixon B, Bernstein IR, Cafe SL, Delehedde M, Sergeant N, Anderson AL, Trigg NA, Eamens AL, Lord T, Dun MD, De Iuliis GN and Bromfield EG (2019) A Kinase Anchor Protein 4 Is Vulnerable to Oxidative Adduction in Male Germ Cells. Front. Cell Dev. Biol. 7:319. doi: 10.3389/fcell.2019.00319 Oxidative stress is a leading causative agent in the defective sperm function associated with male infertility. Such stress commonly manifests via the accumulation of pathological levels of the electrophilic aldehyde, 4-hydroxynonenal (4HNE), generated as a result of lipid peroxidation. This highly reactive lipid aldehyde elicits a spectrum of cytotoxic lesions owing to its propensity to form stable adducts with biomolecules. Notably however, not all elements of the sperm proteome appear to display an equivalent vulnerability to 4HNE modification, with only a small number of putative targets having been identified to date. Here, we validate one such target of 4HNE adduction, A-Kinase Anchor Protein 4 (AKAP4); a major component of the sperm fibrous sheath responsible for regulating the signal transduction and metabolic pathways that support sperm motility and capacitation. Our data confirm that both the precursor (proAKAP4), and mature form of AKAP4, are conserved targets of 4HNE adduction in primary cultures of post-meiotic male germ cells (round spermatids) and in mature mouse and human spermatozoa. We further demonstrate that 4HNE treatment of round spermatids and mature spermatozoa results in a substantial reduction in the levels of both proAKAP4 and AKAP4 proteins. This response proved refractory to pharmacological inhibition of proteolysis, but coincided with an apparent increase in the degree of protein aggregation. Further, we demonstrate that 4HNE-mediated protein degradation and/or aggregation culminates in reduced levels of capacitation-associated phosphorylation in mature human spermatozoa, possibly due to dysregulation of the signaling framework assembled around the AKAP4 scaffold. Together, these findings suggest that AKAP4 plays an important role in the pathophysiological responses to 4HNE, thus strengthening the importance of AKAP4 as a biomarker of sperm quality, and providing the impetus for the design of an efficacious antioxidant-based intervention strategy to alleviate sperm dysfunction.

Keywords: 4-hydroxynonenal, A-kinase anchor protein 4, male germ cells, oxidative stress, spermatozoa, sperm capacitation, sperm motility

## INTRODUCTION

fcell-07-00319 December 19, 2019 Time: 16:47 # 2

A male factor is implicated in approximately half of all cases of infertility (Dohle et al., 2005), and in the majority of these individuals, the underlying etiology is attributed to dysregulation of sperm function (Aitken, 2018). Among the myriad of causative agents, oxidative stress has repeatedly been implicated as a key contributor to the loss of sperm developmental and functional competence (Tremellen, 2008; Aitken et al., 2010). Indeed, notwithstanding the important physiological role that reactive oxygen species (ROS) serve to drive the latter phases of sperm maturation (Aitken and Nixon, 2013), exposure to even modest levels of ROS has the potential to induce a state of oxidative stress that deleteriously affects the unique physiology of the male gamete (Aitken et al., 2016). Notably, the legion of pathologic impacts resulting from elevated ROS vary considerably depending on the timing of exposure, with recent evidence suggesting that the round spermatid stage of development is particularly sensitive to oxidative attack. Specifically, post-ROS exposure, round spermatids rapidly succumb to a novel form of regulated cell death, termed ferroptosis (Bromfield et al., 2019). By contrast, equivalent levels of ROS elicit functional lesions in the mature spermatozoon, which compromise their fertilization potential, but does not negatively impact their viability (Bromfield et al., 2015). Such differential pathogenesis may be attributed to the highly specialized architecture of the male germ cell, which depending on their stage of differentiation, features an abundance of substrates for free radical attack, minimal antioxidant defense enzymes, and limited capacity for self-repair when oxidative damage is sustained (Walters et al., 2018b).

Irrespective of the timing of oxidative stress exposure, a virtually ubiquitous response to this insult is lipid peroxidation, a process whereby hydroxyl radicals attack the unsaturated fatty acyl chains of membrane phospholipids (Hauck and Bernlohr, 2016). The ensuing peroxidation reactions generate appreciable levels of highly reactive short chain carbonyl compounds. Among the most abundant and cytotoxic of these secondary oxidation products is 4-hydroxynonenal (4HNE) (Petersen and Doorn, 2004); an aldehyde that has become a major focus in the field due to the pathophysiological impact it exerts on both male (Aitken et al., 2012; Baker et al., 2015; Bromfield et al., 2017b) and female germ cells (Lord et al., 2015; Mihalas et al., 2017, 2018). Under normal physiological conditions, aldehyde-metabolizing enzymes function to detoxify 4HNE, and to abrogate its cellular accumulation, thus limiting its ability to propagate oxidative cellular damage (Hauck and Bernlohr, 2016). However, when challenged with either chronic or acute oxidative stress, 4HNE can accrue within the cellular environment, and, owing to its inherent stability and rapid diffusion, direct the formation of Michael adducts with nucleophilic sites in DNA, lipids and proteins (LoPachin et al., 2009). Curiously, despite 4HNE being capable of disseminating across large functional boundaries, not all elements of the cellular proteome display equivalent susceptibility to 4HNE carbonylation, with only a relatively small number of putative targets identified in spermatozoa to date (Aitken et al., 2012).