Transcript expression profiling of Rhopr5HTR2b in various tissues of fifth instar stage R. prolixus revealed greatest enrichment in MTs and salivary glands. Pharmacological analyses in the heterologous system suggests this receptor could be the target of serotonin acting as a diuretic hormone enabling increased solute and water transport by the MTs following a blood meal. This notion is supported by previous studies that found ketanserin and spiperone are potent antagonists of serotonin-stimulated fluid secretion by MTs (Maddrell et al., 1991; Te Brugge et al., 2001), which were also the most active antagonists on the receptor we identified in this study. Interestingly, relative to the unfed animal, it appears there is no change in receptor transcript abundance over the first 24 h after blood meal engorgement, suggesting the Malpighian tubule serotonin receptor that responds to haemolymph-borne serotonin is available in advance of feeding to ensure a prompt response to initiate the rapid post-feeding diuresis.

The pharmacological characteristics of the serotonin receptor type on R. prolixus salivary glands are not known. In Calliphora vicina salivary glands, different serotonin receptor types may regulate salivation since ketanserin is not effective at blocking serotonin-stimulated fluid secretion by isolated salivary glands (Maddrell et al., 1991), although serotoninstimulated salivation in C. vicina is significantly inhibited by gramine (Berridge, 1972; Trimmer, 1985). Gramine was not effective at inhibiting the activation of the Rhopr5HTR2b receptor functionally analyzed here. Two receptors have been recently identified in blowfly salivary glands belonging to the serotonin receptor type-2a and type-7 classes (Roser et al., 2012), although both receptors displayed sensitivity to ketanserin (Roser et al., 2012). A different receptor subtype could be expressed in the R. prolixus salivary glands where serotonin is delivered in the nerve supply and induces a dose-dependent increase in the frequency and amplitude of phasic muscle contractions (Orchard and Te Brugge, 2002). Serotonin has also been reported to dose-dependently stimulate secretion of saliva (Orchard, 2006).

Rhopr5HTR2b transcript expression was also detected in the CNS, dorsal vessel, foregut and hindgut of fifth instar R. prolixus. In the nervous system of insects, serotonin is known to play a variety of roles that includes regulation of feeding (Falibene et al., 2012; French et al., 2014), motor output and locomotion (Claassen and Kammer, 1986; Silva et al., 2014), control of clock neurons (Hamasaka and Nassel, 2006; Kolodziejczyk and Nassel, 2011), and olfactory function (Mercer and Menzel, 1982; Kloppenburg et al., 1999; Dacks et al., 2006a; Tsuji et al., 2007; Kloppenburg and Mercer, 2008; Siju et al., 2008; Zhao and Berg, 2009; Watanabe et al., 2014). In R. prolixus, serotonin's distribution in the CNS is extensive, with approximately 150 neurons identified, whose projections and arborizations suggest that this neurochemical has a multitude of central as well as peripheral roles (Lange et al., 1988). A subset of the peripheral serotonin-like immunoreactive projections in R. prolixus are associated with regions of the gut including the foregut and hindgut (Orchard et al., 1988; Orchard, 2006), These regions of the gut also demonstrated Rhopr5HTR2b transcript enrichment, suggesting serotonin's roles could be mediated by this receptor in these tissues. In contrast, serotonin's action on the anterior midgut as a regulator of transepithelial transport (Farmer et al., 1981) and muscle contraction (Te Brugge et al., 2009) may not involve the identified receptor since no transcript was detected in this tissue. It is possible, however, that a closely related receptor (e.g., type 2a receptor class) may control these serotonergic actions in the anterior midgut since cAMP responsiveness to

## References

serotonin showed a similar pharmacological profile, including sensitivity to the agonist α-methyl serotonin and inhibition by antagonists including ketanserin, mianserin and cyproheptadine, while the serotonin-induced increase of cAMP in the anterior midgut was not affected by spiperone (Barrett et al., 1993), one of the more active antagonists effective on Rhopr5HTR2b. Spiperone may be useful in discriminating between these closely related receptor subtypes.

The dorsal vessel is the primary circulatory organ in insects and is regulated by a variety of cardioactive factors, such as serotonin (Chiang et al., 1992; Zornik et al., 1999; Koladich et al., 2002; Dasari and Cooper, 2006; Feliciano et al., 2011). As previously reported (Chiang et al., 1992; Orchard, 2006), serotonin stimulates increases in heart rate in the low nanomolar range (threshold between 1 and 10 nM). In contrast to the pharmacological sensitivity of Rhopr5HTR2b as determined through the heterologous functional assay, the cardioacceleratory activity of serotonin had a unique pharmacological profile. High doses of the type 2 agonist, alpha-methyl serotonin, were not effective in significantly modifying the frequency of heart contractions. In addition, the dorsal vessel bioassay revealed insensitivity to ketanserin and mianserin. Taken together, these results suggest that at least two distinct serotonin receptors control critical physiological functions in this blood-feeding insect, one of which is Rhopr5HTR2b, involved in fluid secretion by the MTs. Other serotonin receptors appear to be involved in absorption by the anterior midgut and cardioacceleratory effects on the dorsal vessel.

## Acknowledgments

This research was supported through individual Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grants to IO, AL, and JP.

distribution, physiology and pharmacological profile. J. Insect Physiol. 49, 1073–1082. doi: 10.1016/j.jinsphys.2003.08.004

**Conflict of Interest Statement:** 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 © 2015 Paluzzi, Bhatt, Wang, Zandawala, Lange and Orchard. 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) or licensor 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.

## Tweaking the structure to radically change the function: the evolution of transthyretin from 5-hydroxyisourate hydrolase to triiodothyronine distributor to thyroxine distributor

## **Samantha J. Richardson\***

School of Medical Sciences, RMIT University, Bundoora, VIC, Australia

### **Edited by:**

Hubert Vaudry, University of Rouen, France

### **Reviewed by:**

Stacia A. Sower, University of New Hampshire, USA Barbara Anne Demeneix, Museum Nationale d'Histoire Naturelle, France

### **\*Correspondence:**

Samantha J. Richardson, School of Medical Sciences, RMIT University, PO Box 71 Bundoora, VIC 3083, Australia e-mail: samantha.richardson@rmit. edu.au

Often, we elucidate evolutionary processes backwards, starting with eutherian mammals and gradually climbing down the evolutionary tree to those species who have survived since long before mammals evolved. This is also true for elucidating the evolution of specific proteins, in this case, the protein currently known as "transthyretin" (TTR). TTR was first described in eutherian mammals and was known as a thyroxine (T4) binding protein. However, mammals are the exception among vertebrates in respect to the function ofTTR, as in teleost fish, amphibians, reptiles and birds TTR preferentially binds triiodothyronine (T3), which is the active form of thyroid hormone (TH). The TTR gene possibly arose as a duplication of the transthyretin-like protein (TLP) gene, around the stage of the agnathans. Some vertebrate species have both the TTR and TLP genes, while others have "lost" the TLP gene.TLP genes have been found in all kingdoms.TheTLPs analyzed to date do not bind THs or their analogs, but are enzymes involved in uric acid metabolism; specifically, they are 5-hydroxyisourate hydrolases. A SalmonellaTLP knock-out strain demonstrated thatTLP was essential for the bacteria's survival in the high uric acid environment of the chicken alimentary tract. Many other TLPs are yet to be characterized for their function although several have been confirmed as 5-hydroxyisourate hydrolases. This review describes the evolution of TLP/TTR and how subtle changes in gene structure or amino acid substitution can drastically change the function of this protein, without altering its overall 3D conformation.

**Keywords: 5-hydroxyisourate hydrolase, mechanism, prealbumin, protein evolution, transthyretin, transthyretinlike protein, triiodothyronine, thyroxine**

### **THE ROLE OF TRANSTHYRETIN IN THYROID HORMONE DISTRIBUTION**

Thyroid hormones (THs) are involved in the regulation of growth, development and metabolism. There are two main forms of THs: 3 0 ,50 ,3,5-tetraiodo-l-thyronine (thyroxine, T4) and 50 ,3,5-triiodol-thyronine (T3) (**Figure 1**). The only site of TH synthesis is the thyroid gland, which secretes the THs (predominantly as T4) into the blood. THs are lipophilic and preferably partition into the lipid phase rather than the aqueous phase (1). To prevent the avid partitioning of THs into the membranes of the first cells they encounter, there are specific proteins in the blood that bind and distribute THs, thereby creating a circulating pool of sufficient size to distribute THs from their site of synthesis (the thyroid gland) via the aqueous environment of the blood stream to their sites of action, i.e., cells throughout the body (2, 3). In humans, there are three TH distributor proteins in the blood: albumin, transthyretin (TTR), and thyroxine-binding globulin. Of these three proteins, albumin is present in highest abundance but binds

THs with lowest affinity, thyroxine-binding globulin is present in lowest abundance but binds THs with highest affinity, and TTR is present in intermediate abundance and binds with intermediate affinity. Taking into consideration the affinities these proteins have for THs and the capillary transit times through tissues, albumin binds so weakly that the amount of TH that it delivers is extremely low; thyroxine-binding globulin binds THs so tightly, that the amount of TH it delivers is also low; whereas TTR binds THs with an intermediate affinity rendering it the most significant in terms of TH delivery to tissues [for a detailed quantitative analysis, see Richardson (4)]. This can be seen as analogous to the situation for Goldilocks and the Three Bears.

Once THs are bound to a TH distributor protein, with the affinity determined by the on and off rates, the TH can dissociate and enter a cell, either by diffusion (1) or via a membrane-bound TH transporter (5). Inside the cell, the TH can then be activated or inactivated by a family of enzymes called deiodinases (6). T4 is known as the "transport form" of TH, as it is the predominant form present in blood, whereas T3 is known as the "active form" of TH as it has higher affinity for the TH receptors (7), which are nuclear transcription factors. Thus, deiodinases can activate T4 to T3, or can inactivate T4 to reverse T3 (rT3) or

**Abbreviations:** TTR transthyretin, TLP transthyretin-like protein, 5-HIUase 5 hydroxyisourate hydrolase.

inactivate T3 to T2 (6). T3 can bind to cytosolic proteins and also to the thyroid hormone receptors (TRs), which can translocate into the nucleus, dimerize, recruit co-modulator proteins, and regulate transcription of specific genes. Many such genes are involved in growth, development, and metabolism, a spectacular example being metamorphosis of a tadpole (aquatic, herbivorous, gills, tail for locomotion) to a frog (terrestrial,carnivorous,lungs,four limbs

thyroid hormone that binds to the nuclear thyroid hormone receptors.

for locomotion) [see Shi (8)]. Thus, TTR is a member of one of the five known classes of TH-binding proteins (TH distributor proteins; TH transporter proteins; deiodinases; cytosolic proteins; nuclear receptors) (**Figure 2**).

Transthyretin is a homo-tetramer, held together by noncovalent interactions, without post-translational modifications. Each subunit comprises eight β-strands that form two β-sheets and a short region of α-helix (**Figure 3**). The holo-protein has a central channel, which has two TH-binding sites (9); however, under physiological conditions only one site is occupied, due to negative co-operativity (10).

## **TTR NULL MICE HAVE A SUBTLY ALTERED PHENOTYPE, BUT HUMANS LACKING TTR HAVE NOT BEEN DESCRIBED**

Mice in which the TTR gene has been inactivated [TTR null mice; (11)] have delayed development of several TH-regulated events including central nervous system development, growth of long bones, suckling-to-weaning transition (12), and adult TTR null mice have a hypothyroid phenotype in the central nervous system (13). Thus, the role of TTR in TH distribution in eutherians has been demonstrated, despite having additional TH distributor proteins (albumin, TTR and thyroxine-binding globulin

**FIGURE 2 |The five classes ofTH-binding proteins**. T4 is the predominant form of TH secreted by the thyroid gland in mammals. T4 is the predominant form of TH in mammalian blood and T3 is also present in significant quantities, but for simplicity T4 is the only form of TH shown in blood in this schematic. More than 99% of TH (both T4 and T3) in blood is bound to a TH distributor protein (1) e.g. albumin, transthyretin (TTR) or thyroxine-binding globulin. TH can dissociate from its distributor protein

and enter cells via either TH transporters (2) or by diffusion. Inside the cell, THs can be deiodinated by a family of deiodinases (3), which can either activate (e.g. T4 to T3) or inactive (e.g. T3 to T2) the TH. THs can bind to cytosolic proteins (4). Predominantly T3 binds to TH nuclear receptors (5), which bind to specific regulatory regions of TH-regulated genes and protein complexes to either promote or repress transcription of those genes (4).

in the blood are all synthesized by the liver and secreted into the blood). Humans lacking TTR have not been documented, although humans lacking albumin and thyroxine-binding globulin have been reported [see Harms et al. (14)]. Possibly, this is because TTR is the only TH distributor protein synthesized in the central nervous system: in the choroid plexus, which forms the blood–cerebrospinal fluid barrier. This TTR has been implicated in moving TH from the blood into the cerebrospinal fluid (1, 15, 16).

### **SHORTENING OF THE N-TERMINAL REGION OF TTR RESULTED IN CHANGING THE LIGAND FROM T3 TO T4**

The amino acid sequence has been determined or derived from cDNA sequences for TTRs from more than 20 vertebrate species including teleost fish, amphibians, reptiles, birds, and mammals. The amino acid sequence has been highly conserved throughout vertebrate evolution, in particular, the amino acids which correspond to regions involved in monomer–monomer interactions, dimer–dimer interactions and those involved in TH binding [see Prapunpoj et al. (17)]. The region of TTR which has changed the most during vertebrate evolution is the N-terminal region, which has changed from longer (e.g. in amphibians) to shorter (e.g. in eutherian mammals). This has occurred in a step-wise manner, which implies a specific and persistent selection pressure acting on TTR during vertebrate evolution (**Figure 4**). Comparison of the cDNA with the genomic DNA in this region for each species revealed that the exon 1–exon 2 border was in the region of the gene corresponding to the N-terminal region of the protein subunit. Further analyses revealed that the position of the exon 1–intron 1 border did not change during vertebrate evolution. However, the position of the intron 1–exon 2 border appears to have shifted in the 30 direction, in a step-wise manner, due to a series of single base changes in the gene. Thus, the mechanism for the shortening of the N-terminal regions of TTRs can be explained by a series of

changes in the gene that "moved" an increasing number of bases from exon 2 into intron 1 (18) (**Figure 5**).

The effect of moving a series of bases of the TTR gene from exon 2 to intron 1 shortened the N-termini of the TTR tetramer but, most importantly, changed the function of TTR. Perhaps, this was the driving selection pressure for the movement of the intron 1–exon 2 splice site in the 30 direction.

Transthyretins with longer N-terminal regions (including those from teleost fish, amphibians, reptiles and birds) have higher affinity for T3 than for T4 (17, 19–22), whereas TTRs with shorter N-terminal regions (including those from marsupials and eutherians) have higher affinity for T4 (21) (**Table 1**). Thus, mammalian TTRs are the exception binding T4 > T3, as TTRs from all other classes of vertebrates bind T3 > T4. The hypothesis that binding of T3 or T4 was due to the N-terminal regions of TTRs was tested by two separate studies. The first study involved the purification of TTR from chicken blood followed by elucidation of the X-ray crystal structure of chicken TTR, to determine if there were structural changes in the TH-binding site between human TTR (which preferentially binds T4) and a TTR that preferentially bound T3. The structure of the TH-binding site in chicken TTR did not differ to that of human TTR (23). Thus, a different region of the molecule must be responsible and the best candidates were the N-terminal regions, which move freely in solution around the entrances to the channel containing the TH-binding sites. The second study involved a set of recombinant TTRs, including chimeric TTRs whose N-terminal regions had been swapped (e.g. N-terminal region of crocodile TTR attached to the "body" of human TTR and vice versa). These TTRs were analyzed for their affinities to T3 and T4. Indeed, the structure of the N-terminal regions did influence the affinity and preference of ligand binding (24).

Thus, by shifting the position of the intron 1–exon 2 border, TTR was able to change from being a T3 distributor to a T4 distributor. What could the selection pressure have been for changing the ligand of TTR from T3 to T4? T3 is the active form of the hormone whereas T4 is the pro-hormone. Perhaps, distributing the pro-hormone could be considered safer than distributing the active form of the hormone, requiring an additional level of activation of the pro-hormone by tissue-specific deiodinases, which are very tightly regulated in terms of developmental and tissuespecificity. In particular, this could be important in the central nervous system, as in (for example) the rat brain, the proportion of T3 generated by local deiodination of T4 is specific to the region e.g. 65% in the cortex, 51% in the cerebellum, 35% in the pons, 32% in the hypothalamus, 30% in the medulla oblongata and 22% in the spinal cord (25). Such tight regulation might not be possible if T3 were the predominant form of TH in the blood and cerebrospinal fluid.

To put this into context, we should consider the total T4 and total T3 levels in blood from various classes of vertebrates. A comprehensive review by Hulbert (26) has tabulated the concentrations of T3 and T4 in the blood of more than 80 vertebrate species (including several life stages for several species). In mammals, birds and reptiles, the circulating levels of T4 are higher than those of T3. However, in some amphibians and teleost fish, the levels for circulating T3 and T4 are similar, while in other species the levels of T4 are higher than those of T3. Of particular interest,

those animals undergoing metamorphosis or smolting have the characteristic peak in TH concentrations in their blood. In some species, this has been correlated with a transient expression of TTR (teleost fish; amphibian; reptile; polyprotodont marsupial) or TBG (diprotodont marsupial) (27). Speculation as to the driving force for greater control of T3 availability in specific brain areas in mammals, as opposed to other vertebrates is very tempting, but extremely speculative. Key TH-related features that distinguish mammalian brains from those of other vertebrates include (i) the corpus callosum and its extensive myelination and (ii) the highly developed cerebral cortex. Furthermore, mammalian fetuses have low levels of circulating T3 and maternal T4 (not T3) has a crucial role in brain development (28).

### **THE TTR GENE AROSE AS A DUPLICATION OF THE TLP GENE**

As mentioned earlier, the amino acid sequence of TTR has been highly conserved during vertebrate evolution, to the extent that the gene most likely arose prior to the divergence of vertebrates from non-vertebrates. Therefore, open reading frames which would code for transthyretin-like proteins (TLPs) were searched for and identified (17). Subsequently, as increasing numbers of genomes were sequenced, genes coding for more than 80 potential TLPs have been identified (and verified to be full-length and not to contain in-frame stop codons, etc.) in all kingdoms (29). Phylogenetic analysis revealed that TLP sequences generally clustered according to organism groups. Vertebrate TLPs clustered together, close to TTRs. TTRs were only found in vertebrate species, whereas TLP sequences were found in both vertebrate and non-vertebrate species (29). Analyses of the TLP gene sequences by signal-peptide prediction programs revealed that TLPs could be divided into three groups: (1) those without signal peptides were predicted to be cytoplasmic, which included most bacterial TLPs; (2) those with periplasmic localization signals, which were the enterobacteria; (3) those with peroxisomal

(PTS2) signal peptides, which included TLPs from plants and most metazoans (29) (**Figure 6**). Several bacterial species have more than one copy of the TLP gene, often one copy that codes for a cytoplasmic TLP and another copy coding for a periplasmic TLP (29). A neighbor-joining tree analyzing the relationship between cytoplasmic and periplasmic TLPs showed that within a given species, cytoplasmic TLPs clustered separately to periplasmic TLPs. For example, all periplasmic TLPs group together. This suggested that periplasmic TLP sequences probably evolved along a separate evolutionary pathway to cytoplasmic TLPs (30). Thus, TLPs appear to have evolved different functions, depending on their sub-cellular localization, rendering them very versatile proteins. These characteristics of TLPs which result from the variety of sub-cellular localizations contrast with TTRs, which are secreted.

### **SUBTLE CHANGES IN THE ACTIVE SITE CHANGED TLPs (ENZYMES INVOLVED IN URIC ACID OXIDATION) INTO TTRs (THYROID HORMONE DISTRIBUTORS)**

It was revealed by PCR that the TLP genes from a plant (*A. thaliana*), a worm (*C. elegans*) and bacteria (*E. coli* and *S. dublin*) were expressed in their respective species i.e. these open reading frames were genes that were expressed in nature. The respective cDNAs were cloned, sequenced and recombinant TLPs from these species were synthesized and found to be tetramers, similarly to TTR. However, these TLPs did not bind THs or TH analogs (29).

**Table 1 | Comparison of affinities ofTTRs forT4 andT3 (4)**.

Toad 508.0 248.0 0.49

The X-ray crystal structure of recombinant *Salmonella dublin* TLP was determined (**Figure 7A**) and was completely superimposable over those of vertebrate TTRs (31). The only differences were subtle changes in the region equivalent to the TH-binding site. Whereas in TTRs the binding site is deep and negatively charged, the equivalent position in TLP was shallow, elongated and positively charged, thereby preventing binding of THs (**Figure 7B**). Careful analysis of the operons within which several TLP genes were situated, allowed the identification of TLP in (at least several) bacteria as a 5-hydroxyisourate hydrolase (5-HIUase), involved in the oxidation of uric acid to allantoin (**Figure 8A**). This was confirmed experimentally for *S. dublin* TLP, including identification of some of the amino acids required for catalysis (**Figure 8B**) (31) and has also been confirmed for TLP in *Bacillus subtilis* (32), zebra fish (33), mouse (34), *E. coli* (35), *Arabidopsis thaliana* (36), *Klebsiella pneumonia* (37), Coelacanth (38), amphioxus (39) and rainbow trout (40).

An insightful paper by Cendron and colleagues (41) identified two amino acid substitutions that were most probably fundamentally critical for the modification of the TLP active site ablating enzymatic activity and allowing opening up of the central channel to allow binding of THs. These are Ile to Ala at position 16 of zebrafish TLP (corresponding to position 19 of human TTR)

overall structure is almost identical to that of human TTR. **(B)**. The thyroid hormone binding site in human TTR is deep and negatively charged, whereas the equivalent site in S. dublin TLP is shallow and positively charged (31).

and Tyr to Thr at position 116 of zebrafish TLP (corresponding to position 119 of human TTR). Li and colleagues demonstrated that in amphioxus (protochordate, close relative of vertebrates) TLP, the point mutation Tyr to Thr at position 156 (corresponding to position 116 in human TTR) was required for abolishing 5-HIUase activity and enabling T4 binding (39). While these mutations were probably instrumental in changing the landscape of the binding site from a shallow catalytic site to a deep channel, further minor mutations were probably required to optimize the channel for tighter binding of T3.

Interestingly, there are three splice variants of *Arabidopsis thaliana* TLP: two are cytoplasmic and one is located in the peroxisome (36). The peroxisomal isoform has a 5-HIUase domain

and an OHCU decarboxylase domain i.e. both enzymes occur in the one transcript resulting in a bi-functional enzyme. There is an internal peroxisomal signal peptide between the two domains (N-terminal to the OHCU decarboxylase domain), presumably targeting the bi-functional TLP to the peroxisome, where uric acid degradation occurs. The function(s) of the cytoplasmic TLPs are not yet known. Furthermore, teleost fish, whose genomes have undergone an additional whole genome duplication, have two forms of 5-HIUase. One form contains the peroxisomal signal peptide and the other does not (40). Thus, in organisms where there have been further TLP gene duplications, such as *Arabidopsis* and (at least some) teleost fish, there is further scope for neo-functionalization of the TLP gene products i.e. the protein resulting from the duplicated TLP gene could acquire a different function by acceptance of point mutations in the duplicated gene while the original TLP gene/protein remains unchanged.

### **IN SALMONELLA, TLP IS REQUIRED FOR SURVIVAL IN HIGH URIC ACID ENVIRONMENTS**

To demonstrate the function of *Salmonella* TLP in an animal model, a *Salmonella typhimurium* TLP knock-out strain was generated and its survival was compared with that of wildtype *Salmonella typhimurium*. The absence of TLP did not affect *Salmonella* survival in mice, whether the *Salmonella* were injected into the tail vein (and monitored for weight loss, development of enteric fever and bacterial load in liver or spleen) or infected orally (and bacterial load determined in Peyer's Patches, mesenteric lymph nodes, liver and spleen) (30). Consequently, it was reasoned that if TLP was a 5-HIUase located in the periplasm of the *Salmonella*, then it would be important for the survival of the *Salmonella* in high uric acid environments such as the gastrointestinal tract of birds and reptiles (uric acid is present in high amounts in feces of reptiles and birds). Thus, mice might have been an inappropriate model animal for testing the effect of TLP on *Salmonella* survival.

Hens were inoculated with either the wildtype or the TLP knock-out strain of *Salmonella*. The feces of hens inoculated with the TLP knock-out strain of *Salmonella* contained significantly less live *Salmonella* than the feces of the hens inoculated with the wildtype *Salmonella* (**Figure 9**) (30). This demonstrated that TLP was important for the survival of *Salmonella* in high uric acid environments. Thus, not only is it important to choose the most appropriate animal model for such experiments, but this clearly showed that TLP is important in the survival of *Salmonella* in high uric acid environments.

### **MICE LACKING TLP SUFFER TOXICITY FROM URIC ACID OXIDATION INTERMEDIATES**

Mice lacking TLP were generated and found to have increased thrombopoietin synthesis by the liver and enlarged livers (hepatomegaly) resulting in increased platelet counts in the blood (thrombocytosis). Most mice lacking TLP also developed hepatocellular carcinoma (42). It was concluded that this phenotype was due to the toxic uric acid oxidation intermediates resulting from the lack of 5-HIUase in the cytoplasm of the mouse hepatocytes.

**FIGURE 9 |TLP is required for survival of Salmonella typhimurium in high uric acid environments such as chicken feces**. **(A)**. Numbers of viable bacteria in the spleen, liver and cecum of hens 7 days after oral inoculation with either wildtype Salmonella (open circles) or TLP null Salmonella (closed circles). Horizontal lines: average number of bacteria per organ; dotted line: lower detection limit of experiment. **(B)**. Numbers of viable bacteria in the feces of hens up to 21 days following oral inoculation with either wildtype Salmonella (open circles) or TLP null Salmonella (closed circles). Horizontal lines: average number of bacteria per gram feces; dotted line: lower detection limit of experiment. There was a significantly lower number of TLP null Salmonella than wildtype Salmonella in the feces and each time point measured (30).

### **CONCLUSION**

TLP/TTR is an excellent model for the study of protein evolution. Notably because (i) it is found in all kingdoms, (ii) it has a stable structure, (iii) it lacks post-translational modification, (iv) it can be directed to many sites within a cell or secreted, (v) modification of just a few amino acids in the active site changed its function from a 5-HIUase to a T3 distributor, (vi) successive shifts in the position of the intron 1–exon 2 splice site changed TTR from a T3 distributor to a T4 distributor. Thus, apparently two different molecular mechanisms have resulted in changes in the functions of TLP/TTR. The functions of TLPs in plants and other organisms are likely to be different to those in *Salmonella* or mice. The differing signal peptides for the various groups of TLPs could be interrogated in conjunction with RNAseq analyses to gain insights into the functions of TLPs in various organisms. The suggestion that cytoplasmic and periplasmic TLPs within a species evolved in separate pathways suggests divergence of functions between these groups of compartmentally distinct TLPs. Presumably, additional evolutionary mechanisms have also been used to modify the functions of TLPs in each compartment in these species. These mechanisms remain to be investigated.

### **ACKNOWLEDGMENTS**

This research was funded by the Australian Research Council.

### **REFERENCES**

(shifting the 30 splice site of intron 1)[erratum appears in Eur J Biochem 1998 Mar 15;252(3):612]. *Eur J Biochem* (1997) **246**:401–9. doi:10.1111/j.1432-1033. 1997.t01-1-00401.x

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

*Received: 10 November 2014; accepted: 23 December 2014; published online: 11 February 2015.*

*Citation: Richardson SJ (2015) Tweaking the structure to radically change the function: the evolution of transthyretin from 5-hydroxyisourate hydrolase to triiodothyronine distributor to thyroxine distributor. Front. Endocrinol. 5:245. doi: 10.3389/fendo.2014.00245*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology.*

*Copyright © 2015 Richardson. 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) or licensor 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.*

# Parasites and steroid hormones: corticosteroid and sex steroid synthesis, their role in the parasite physiology and development

### Marta C. Romano1 \*, Pedro Jiménez 2 , Carolina Miranda-Brito1 and Ricardo A. Valdez 1

1 Departamento de Fisiología, Biofísica y Neurociencias, CINVESTAV del IPN, Mexico city, Mexico, 2 Centro de Investigación en Reproducción Animal, CINVESTAV-UAT, Tlaxcala, Mexico

### Edited by:

Hubert Vaudry, University of Rouen, France

### Reviewed by:

Rafael Vazquez-Martinez, University of Cordoba, Spain Yves Tremblay, Laval University and Centre Hospitalier Universitaire de Québec, Canada

### \*Correspondence:

Marta C. Romano, Departamento de Fisiología, Biofísica y Neurociencias, CINVESTAV del IPN, Apdo. Postal 14-745, 07360 Mexico city, Mexico<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience

Received: 11 December 2014 Accepted: 08 June 2015 Published: 30 June 2015

### Citation:

Romano MC, Jiménez P, Miranda-Brito C and Valdez RA (2015) Parasites and steroid hormones: corticosteroid and sex steroid synthesis, their role in the parasite physiology and development. Front. Neurosci. 9:224. doi: 10.3389/fnins.2015.00224 In many cases parasites display highly complex life cycles that include the penetration and permanence of the larva or adults within host organs, but even in those that only have one host, reciprocal, intricate interactions occur. Evidence indicates that steroid hormones have an influence on the development and course of parasitic infections. The host gender's susceptibility to infection, and the related differences in the immune response are good examples of the host-parasite interplay. However, the capacity of these organisms to synthesize their own steroidogenic hormones still has more questions than answers. It is now well-known that many parasites synthesize ecdysteroids, but limited information is available on sex steroid and corticosteroid synthesis. This review intends to summarize some of the existing information in the field. In most, but not all parasitosis the host's hormonal environment determines the susceptibility, the course, and severity of parasite infections. In most cases the infection disturbs the host environment, and activates immune responses that end up affecting the endocrine system. Furthermore, sex steroids and corticosteroids may also directly modify the parasite reproduction and molting. Available information indicates that parasites synthesize some steroid hormones, such as ecdysteroids and sex steroids, and the presence and activity of related enzymes have been demonstrated. More recently, the synthesis of corticosteroid-like compounds has been shown in Taenia solium cysticerci and tapeworms, and in Taenia crassiceps WFU cysticerci. In-depth knowledge of the parasite's endocrine properties will contribute to understand their reproduction and reciprocal interactions with the host, and may also help designing tools to combat the infection in some clinical situations.

Keywords: parasites, steroid synthesis, sex steroids, corticosteroids, steroidogenic enzymes, Taenias, cysticerci

## Parasites and Host Interplay: Steroid Hormones Influence Parasite Development and Survival

Progesterone, androgens, and estrogens are present and have critical roles in the vertebrate reproduction and metabolism, but the influence and occurrence of steroid hormones in invertebrates had received less attention (Lafont, 2000) the interplay between the parasite and the host defines the intensity of parasite infections. In many cases, the presence of parasites in the host changes its endocrine equilibrium due to the activation of the immune system response, which finally affects the endocrine system through the influence of cytokines and growth factors released by the immune cells. It is now widely accepted that corticosteroids and sex-related hormones influence the immune response (Roberts et al., 2001; Coutinho and Chapman, 2011; Reyes-Hernandez et al., 2013), thereafter any endocrine perturbation initiated by an infection will change the neuroendocrine equilibrium. These hormonal changes resulting from a spontaneous or experimental infection, affect the parasitic charge, the course of the infection and the parasite's survival (Barthelemy et al., 2004). On the other hand, some parasite infections disrupt the host endocrine system, in a noteworthy example, the dromedary bull, Camelus dromedarius, parasitized with Tripanosoma evansi, presents changes in the sexual steroid plasmatic concentrations, as well as in the semen characteristics (Al Qarawi, 2005). Furthermore, the male Fence Lizard (Sceloporus occidentalis) infected with the malarial parasite Plasmodium mexicanum shows several reproductive pathologies, such as fewer courtship and decreased testosterone levels (Dunlap and Schall, 1995) and the Toxoplasma gondee infection enhances testicular steroidogenesis in rats (Lim et al., 2013). Interestingly, it has been shown that the host hormonal environment determines the susceptibility, the course, and severity of many parasite infections, and therefore a clear dichotomy in infection susceptibility between males and females had been observed (Morales-Montor et al., 2004). A rich estrogen environment facilitates Taenia crassiceps cysticerci proliferation, blocking thus the P450-aromatase with fadrozole decreased parasite load (Morales-Montor et al., 2002). Parasites may also alter the host's reproductive behavior (Thompson and Kavaliers, 1994) as had been also shown in Taenia crassiceps ORF infected male mice (Morales et al., 1996).

Steroids and steroid synthesis inhibitors influence the fertility of Schistosoma mansoni in vitro (Morrison et al., 1986) while progesterone, makisterone, and ecdysone increased the length of the larvae Ascaris suum, a roundworm swine parasite (Fleming, 1985), while progesterone, 17β-estradiol, and testosterone added to the culture medium enhanced the number of Plasmodium falciparum gametocytes (Lingnau et al., 1993). Progesterone, but not testosterone decreased the in vitro molting process of Trichinella spiralis(Hernández-Bello et al., 2011) and T. crassiceps ORF cysticerci cell proliferation was increased by physiological concentrations of testosterone, and 17β-estradiol added to the culture media (Romano et al., 2003), while high concentrations inhibited its reproduction (Escobedo et al., 2004).

## The Role of Corticosteroids in the Host-parasite Interplay

It is well-known that non-physiological stress situations, such as social isolation, infections, persecution, etc., increase serum corticosteroids levels with the consequent impairment of the immune response. The interplay host-parasite is not the exception, for example social stress caused by female isolation increased blood Typanosoma cruzi infection in the wild mouse Calomys callosus, which showed body weight loss and impaired immune response (Santos et al., 2008), whereas the hypothalamus-pituitary-interregnal axis of rainbow trout was altered by the parasite haemoflagelate Cryptobia infection (Madison et al., 2013). In addition, the infection with Anguillicola novaezelandiae affects cortisol levels in the European Eel (Dangel et al., 2014). Frequently, the host and the parasites are affected in the course of infection, as in Bluegill Sunfish (Lepomis macrochirus) infected with Utterbackia imbecillis, which results in plasma cortisol increment in the host, favoring thus the parasite juvenile metamorphosis (Dubansky et al., 2011). On the other hand, in vivo treatment of rats with cortisol increased the growth rate of the protozoan parasite Toxoplasma gondii in isolated peritoneal macrophages (Wang et al., 2014).

## Direct Effects of Glucocorticoids on Parasite Growth

Aside from the influence of corticosteroids in the course of parasitic infections, it had been shown that these hormones directly influence parasite's growth. For instance, cortisol and dexamethasone increase the in vitro multiplication of the haemoflagellate Cryptobia salmonistica, possible by their interaction with glucocorticoid receptor-like protein (Li et al., 2013, 2014). In this regard, dehydroepiandrosterone (DHEA) addition to the culture media decreased, while cortisol increased the in vitro growth and viability of Entamoeba histolytica (Carrero et al., 2006). We had shown that corticosterone and dexamethasone increase the capacity of T. crassiceps WFU cysticerci to synthesize androgens and estrogens, hormones that favor the parasite reproduction (Hinojosa et al., 2011).

## Parasites Synthesize Steroid Hormones

## Lipids and Steroid Hormones

Lipids, and particularly cholesterol and their metabolites, are required and synthesized by some parasites. Bansal et al. (2005) reviewed the requirement for lipids, particularly cholesterol, by pathogens like protozoa (Leishmaniosis, Malaria, and Toxoplasmosis). It has recently been stated that cholesterol exerts many of its functions by maintaining a specialized type of membrane domain called "lipid rafts" in a functional state. These domains are rich in cholesterol and sphingolipids and could be involved in signal transduction and in the entry of pathogens to the host cells (Simons and Toomre, 2000).

The incorporation and utilization of arachidonic acid, linoleic acid, 3-sn-phosphatidycholine, tripalmitylglycerol,and cholesterol by adult Schistosoma mansoni was demonstrated by Rumjanek and Simpson (1980). These parasites exchange cholesterol and other metabolites during reproduction (Popiel and Basch, 1986; Silveria et al., 1986), while cholesterol is absorbed by the hydatid cysts of Echinococcus granulosus (Bahr et al., 1979). On the other hand, parasites like Trypanosome cruzi and some species of Leishmania cannot use cholesterol but they synthesize ergosterol and related 24-alkylated sterols. Furthermore, Leishmania has a strict requirement for ergosterol for their survival and growth (Urbina et al., 2002; Magaraci et al., 2003; Bazin et al., 2006).

## Ecdysteroids

Several parasites synthesize ecdysteroids, steroid hormones that are essential for arthropod molting; the capacity to synthesize these hormones has been used to classify cestodes (de Loof and Huybrechts, 1998). However, the role of these steroids in parasites remains obscure. Ecdysterone production has been reported in a variety of helminth species, among them, the nematodes Dirofilaria immitis, Brugia pahangi, Ascaris suum, and Anisakis simplex, the cestodes: Moniezia expansa, Echinococcus granulosus, and Hymenolepis diminuta and the trematodes: Fasciola hepatica and Schistosoma mansoni (Mendis et al., 1983, 1984; Fleming, 1985; Cleator et al., 1987; Evershed et al., 1987; Mercer et al., 1987a,b, 1990; Foster et al., 1992). In male and female, Dirofilaria immitis, and in female, Ascaris suum ecdysteroids were concentrated in the reproductive system, whereas the presence of ecdysteroids in the eggs of Schistosoma mansoni suggests a regulatory role in embryogenesis (Mercer, 1985). Ecdysteroids have also been found in cestodes like the sheep parasite Moniezia expansa and in the rat tapeworm Hymenolepis diminuta (Mercer et al., 1987b). Ecdysterone can be detected in the sera of infected hosts parasitized by Schistosoma, a trematode that synthesizes and releases the steroid to the host's circulation (Mercer, 1985). Although the role of ecdysteroids during insect metamorphosis had been widely demonstrated, the function of these steroids in parasites remains obscure.

## Sex Steroids

Evidence for the steroidogenic capacity of Schistosoma mansoni was provided years ago by data showing the conversion of steroid precursors to its metabolites by the parasite homogenate (Briggs, 1972). We had shown that cultured Trypanosoma cruzi trypomastigotes synthesize androgens and estrogens from androstenedione and DHEA (Vacchina et al., 2008). We had also investigated the steroidogenic capacity of the cysticerci and tapeworm from Taenia solium and Taenia crassiceps WFU. The adult worm of Taenia solium and crassiceps WFU tapeworms are attached to the host gut with hooks that surrounds their head, and develop reproductive units called proglottids, where testis and ovaries gradually differentiate, and finally contain spermatocytes and infective eggs (Willms et al., 2003). Taenia solium cysticerci is the larval stage of the parasite and is found in the brain or muscle of humans and pigs, whereas Taenia crassiceps WFU cysticerci constitute a useful laboratory model due to their reproduction by budding in the peritoneal cavity of mice. We had been exploring the capacity of cysticerci to synthesize sex steroids in vitro and found that T. solium and T. crassiceps ORF cysticerci transform steroid precursors such as progesterone, dehydroepiandrostenedione, and androstenedione to androgens and estrogens (Gómez et al., 2000; Jiménez et al., 2006; Valdez et al., 2006). Other experiments showed that drugs that block steroidogenic enzymes interfered the in vitro steroid synthesis, suggesting the existence of this enzymes in the parasites (Aceves-Ramos et al., 2013).

Because of the presence of testis and ovaries in tapeworm proglottids, we had investigated the in vitro steroidogenic capacity of experimental Taenia crassiceps WFU and solium tapeworms obtained from the intestine of infected hamsters. Taenia crassiceps WFU tapeworms were incubated in the presence of 3H-DHEA and tritiated androstenediol and 17β-estradiol where recovered from the culture media, which strongly suggest the presence and activity of enzymes from the 15 steroid pathway in these tapeworms (Fernández Presas et al., 2008). Taenia solium tapeworms kept in culture also synthesized sex steroids (Valdez et al., 2014).

## Corticosteroids

Since the above mentioned literature indicated that Taenia solium and Taenia crassiceps cysticerci and tapeworms synthesized sex steroid hormones, we thought these organisms could also synthesize corticosteroids. Thereafter, we had incubated Taenia crassiceps cysticerci in the presence of 3H-progesterone and found an important transformation into deoxycorticosterone (Valdez et al., 2012), a steroid that has mineralocorticoid functions in vertebrates and also display some glucocorticoid properties. In addition, the parasites synthesized corticosterone, which was measured by radioimmunoassay in the culture media. More recently, we found corticosteroid-like synthesis in Taenia solium tapeworms (Valdez et al., 2014). It had been shown that in the adrenal reticular zone of mammals an excess of progesterone can be inactivated by 20HSD activity (Pelletier et al., 2005) and that cortisol and corticosterone can be inactivated by 11β-hydroxylase in the adrenal glomerulose zone (for a review see Odermatt and Atanasov, 2009) but to our knowledge these enzymes had not been investigated in Taenia solium or crassiceps WFU organisms. Besides their effects on the own parasite development and differentiation, the cysticerci, and tapeworm's steroidogenic capacity might play a role in the permanence of the parasites in muscle and brain tissues and in the host intestine.

## Parasites Express Steroidogenic Enzymes

Some steroidogenic enzymes had been described in parasites. For instance, sterol-sterifying enzymes were found in Toxoplasma gondee, a protozoan incapable of cholesterol de novo synthesis (Lige et al., 2013). The presence of 3βhydroxysteroid dehydrogenase in Sarcocystis spp had been shown by immunohistochemistry (Yarim et al., 2004); the enzyme is also present in Taenia solium cysticerci and tapeworms (Fernández Presas et al., 2008). Genes from the cholesterol synthesis pathway have been found and expressed in Giardia intestinalis (Hernandez and Wasserman, 2006). The parasite flatworm Schistosoma japonicum has a type 12 17-HSD that metabolizes estrone to estradiol (Zhou et al., 2009).

We have recently shown that Taenia solium cysticerci express the enzyme 17β-HSD that belongs to the short chain dehydrogenases/reductase family (Aceves-Ramos et al., 2014). Transient transfection of HEK293T cells with Tsol17β-HSD-pcDNA3.1 (+) induced expression of Tsol17β-HSD that transformed 3H-androstenedione into testosterone. In contrast, 3H-estrone was not significantly transformed into estradiol. Therefore, Taenia solium cysticerci express a 17β-HSD that catalyzes the androgen reduction and belongs to the short chain dehydrogenases/reductase (SDR) protein superfamily (Aceves-Ramos et al., 2014). Recently, a sequence (EmW000624600 which is available at: www.genedb.org/Homepage/Emultilocularis) with an identity of 84% with Tsol-17βHSD and a total coverage has been described for E. multilocularis, suggesting the presence of 17 HSD enzymes in this Taeniid family (Tsai et al., 2012). However, the expression level and enzyme activity has not been yet investigated.

## References

## Concluding Remarks

The knowledge of parasite endocrinology will contribute to our understanding of parasite biology and their interactions with the host. Sex steroids and corticosteroids are important hormones for the growth, differentiation, and performance in many species. Therefore, the synthesis of these hormones by parasites themselves may be critical for their own development and viability. In addition, it was shown that the production of steroids by parasites is regulated by corticosteroids and affected by steroidogenic inhibitors, which may be used as tools to combat the infection in some clinical situations. Furthermore, steroid synthesis by parasites may contribute to defend them from the attack of immune cells and therefore facilitate their survival in the host tissues.

**Conflict of Interest Statement:** 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 © 2015 Romano, Jiménez, Miranda-Brito and Valdez. This is an openaccess 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) or licensor 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.

## Bisphenol A and phthalate endocrine disruption of parental and social behaviors

## *Cheryl S. Rosenfeld\**

*Bond Life Sciences Center, Genetics Area Program, Biomedical Sciences, University of Missouri, Columbia, MO, USA*

### *Edited by:*

*Hubert Vaudry, University of Rouen, France*

### *Reviewed by:*

*John Vandenbergh, Duke University, USA Marie-chantal Canivenc-lavier, French National Institute for Agricultural Research, France*

### *\*Correspondence:*

*Cheryl S. Rosenfeld, Bond Life Sciences Center, Genetics Area Program, Biomedical Sciences, University of Missouri, 1201 E. Rollins Rd., Columbia, MO 65211, USA e-mail<EMAIL_ADDRESS>Perinatal exposure to endocrine disrupting chemicals (EDCs) can induce promiscuous neurobehavioral disturbances. Bisphenol A and phthalates are two widely prevalent and persistent EDCs reported to lead to such effects. Parental and social behaviors are especially vulnerable to endocrine disruption, as these traits are programmed by the organizational-activational effects of testosterone and estrogen. Exposure to BPA and other EDCs disrupts normal maternal care provided by rodents and non-human primates, such as nursing, time she spends hunched over and in the nest, and grooming her pups. Paternal care may also be affected by BPA. No long-term study has linked perinatal exposure to BPA or other EDC and later parental behavioral deficits in humans. The fact that the same brain regions and neural hormone substrates govern parental behaviors in animal models and humans suggests that this suite of behaviors may also be vulnerable in the latter. Social behaviors, such as communication, mate choice, pair bonding, social inquisitiveness and recognition, play behavior, social grooming, copulation, and aggression, are compromised in animal models exposed to BPA, phthalates, and other EDCs. Early contact to these chemicals is also correlated with maladaptive social behaviors in children. These behavioral disturbances may originate by altering the fetal or adult gonadal production of testosterone or estrogen, expression of ESR1, ESR2, and AR in the brain regions governing these behaviors, neuropeptide/protein hormone (oxytocin, vasopressin, and prolactin) and their cognate neural receptors, and/or through epimutations. Robust evidence exists for all of these EDC-induced changes. Concern also exists for transgenerational persistence of such neurobehavioral disruptions. In sum, evidence for social and parental deficits induced by BPA, phthalates, and related chemicals is strongly mounting, and such effects may ultimately compromise the overall social fitness of populations to come.

### **Keywords: EDC, bisphenol A, phthalate, xenoestrogen, rodent models, brain development, epigenetics, neuropeptides**

## **INTRODUCTION**

The perinatal environment can dramatically shape later adult behaviors, and these disruptions can be propagated through transgenerational transmission to future generations (Rosenfeld, 2014). The impact of varying parental investment on offspring brain development is also gaining interest (Dulac et al., 2014; Rilling and Young, 2014). Disturbances in parental behaviors, such as nursing/feeding, huddling over, and grooming the neonates can have dramatic epigenetic and phenotypic consequences that persist for generations to come (Weaver et al., 2006).

Due to underlying neural disruptions, parental and social behaviors can be impacted by developmental exposure to endocrine disrupting chemicals (EDCs). Strong conservation exists in brain development and function across species spanning from rodents to humans (Rice and Barone, 2000; Howdeshell, 2002). In animal models and humans, neurobehavioral development is vulnerable to EDCs, because of the organizationalactivational of endogenous steroidogenic hormones.

 This early brain programming is termed the "organizational effects" of these steroid hormones. However, full elaboration of many sex-dependent behaviors requires a later surge of testosterone in adult males ("activational effects"). The organizational-activational effects of testosterone in many brain regions are due to aromatization to estrogen (Watson and Adkins-Regan, 1989; Konkle and McCarthy, 2011). Exposure to EDCs that disrupt these normal steroidogenic effects may thus result in neurobehavioral deficits, including social and parental behavioral deficits.

The two most common EDCs associated with parental and social behavioral disturbances are bisphenol A (BPA) and phthalates. Therefore, these two will be the primary focus, although, where appropriate, effects of other EDCs on these traits will be discussed. BPA is one of the most widely prevalent EDCs with production currently reported to be in excess of 15 billion pounds per year (Vandenberg et al., 2013; Grandviewresearch, 2014). BPA is present in a wide variety of commonly used products and applications, including polycarbonate plastics, the lining of metal food cans, certain dental sealants, thermal receipt paper, and many other items that are currently not required to be labeled that they contain BPA. The abundance of BPA has ensured extensive and longstanding exposure of animals and humans, including pregnant women (Environment Canada, 2008; Vandenberg et al., 2013). There is strong evidence that BPA is a neuroendocrine disruptor (Leon-Olea et al., 2014).

Di(2-ethylhexyl) phthalate (DEHP) is another ubiquitous EDC present in plastic products personal care products, paints, pharmaceuticals, food products, and textiles (Latini et al., 2003). Similar to BPA, this chemical can leach out of plastic products when heated. Its primary metabolite, mono-ethylhexyl-phthalate (MEHP) can also result in neuroendocrine disruption (Leon-Olea et al., 2014).

This review will first consider the evidence that BPA, phthalates, and other EDCs can alter parental care provided by the mother and the scant evidence to date by the father. Next, we will consider the impact of developmental exposure to these chemicals on social behaviors in animal models and linkages in humans. Finally, the potential mechanisms by which EDCs may affect these behavioral patterns will be explored.

## **EVIDENCE OF ENDOCRINE DISRUPTION OF PARENTAL BEHAVIORS**

There have been no long-term studies examining for potential linkage of early exposure to these chemicals and later parental behavior deficits in humans. Therefore, this section will focus on the existing evidence from rodent and other animal models (Summarized in **Table 1**). A handful of rodent studies have reported that early exposure to EDCs can result in later effects on maternal care. General maternal behaviors assessed in these collective studies include the amount of time the female spends nursing, hunched over and in the nest, grooming, and latency to retrieve her pups.

CD-1 female mice exposed orally during the fetal and/or adult period to BPA (10μg/kg body weight (bw)/day for gestational days 14–18) spend less time nursing their pups and huddling over them while in the nest (Palanza et al., 2002). Acute exposure of adult female Sprague-Dawley rats exposed to BPA (40μg/kg bw, orally per day) from mating to weaning of their pups disrupts both active and passive maternal care, manifested by decreased time licking and grooming the pups, a trend to less ano-genital licking, and less time assuming an arched-back posture to allow the pups to suckle (Della Seta et al., 2005). Similarly, Wistar female rats treated with 5μg BPA/kg bw/day from the first day of gestation through lactation (postnatal day, PND 21) decreased the amount of time they nursed and attended to their pups (Boudalia et al., 2014). Another study tested the effects of gestational exposure (0–19 days) to three oral doses of BPA (2, 20, and 200μg/kg bw/day) on maternal care provided by BALB/c mice. In contrast to the prior studies, the highest dose of BPA tested increased the amount of time females spent licking and grooming and nursing their pups in an arched back position (Kundakovic et al., 2013).

These conflicting rodent model findings may be attributed to various factors. First, all four of these above studies used rats or mice of different strains, and there is evidence of strain-specificity and phenotypic differences in response to BPA and other estrogenic EDC (Spearow et al., 1999, 2001; Kendziorski et al., 2012). The administered dose and timing, duration of exposure (perinatal vs. adult), and corresponding generational differences (F0 vs. F1) may also be potential factors. Finally, differences in animal husbandry (composition of the cages and water bottles, dietphytoestrogen or non-phytoestrogen free, and shavings-corn cob or aspen) might account for the disparate rodent results.

Infants may influence, through vocalizations, direct contact, and other forms of communication, the amount of parental care provided to them with males tending to initiate and receive more parental investment (Della Seta et al., 2005; Hao et al., 2011). Early EDC exposure though may alter an infant's ability to stimulate maternal care provided to them. For instance, the behavioral patterns of Cynomolgus monkeys (*Macaca fascicularis*) male infants prenatally (gestational day 20 through term) exposed to BPA (10μg/kg bw/day via osmotic pump) were more reminiscent of females, and these mothers nursed their "feminized" sons less than those rearing control males (Nakagami et al., 2009).

While there are no studies to date on whether acute and developmental exposure to phthalates affects maternal care, other EDCs have been reported to disrupt these behavioral patterns. Long-Evans (LE) rat pups prenatally exposed through their biological dam to the environmental chemical 3, 4, 3- , 4- tetrachlorobiphenyl (PCB 77, 2000μg/kg bw/day on gestational days 6 through 18 via daily subcutaneous injections) resulted in their foster dams spending an increased frequency nursing them and allogrooming (Cummings et al., 2005). This cross-foster approach further revealed the complex interactions exist between maternal and fetal exposure to PCB 77 and amount of time foster dams spent in the nest and grooming the pups, along with decreased duration engaged in high-crouch nursing. Perinatal exposure (either 3 days after the dam was paired with a male or 3 days after parturition of a previous litter through weaning of pups) via daily maternal oral dosing to diethylstilbestrol (DES, 0.2μg/kg bw/day) or methoxychlor (MXC, 2000 mg/kg bw/day) of monogamous female pine voles did not however affect their later maternal care behaviors (Engell et al., 2006).

The impact of EDCs on later paternal care provided is largely unknown, probably because few mammalian species exhibit biparental care (Clutton-Brock, 1989). Yet, disturbances in paternal care may have dramatic epigenetic and phenotypic consequences that persist for several generations (Bredy et al., 2007; Braun and Champagne, 2014; McGhee and Bell, 2014). Even so, there have been no published rodent studies to date on how early exposure to EDCs affects paternal care. However, in polygynous sand gobies (*Pomatoschistus minutus*), where the male is responsible for tending the eggs, endocrine disruption of paternal care behaviors has been reported (Saaristo et al., 2010). Polygynous sand goby males compete for females and then assume responsibility for nest building and attendance. However, adult males

**Table 1 | Animal model and human epidemiological studies linking bisphenol A (BPA) and phthalates exposure to parental and social behavioral changes.**

### **Table 1 | Continued**

*(Continued)*

### **Table 1 | Continued**

### **Table 1 | Continued**

*(Continued)*

**Table 1 | Continued**

exposed for 1–4 weeks to 11 ng/L water (36.7 pM) of 17α-ethinyl estradiol (EE2, estrogen in birth control pills) showed suppressed nest building activity, along with decreased courtship behaviors. These combined deficiencies thus likely effect successful fry rearing by exposed males. Future work is needed in monogamous and biparental rodent and other animal models to determine how developmental exposure to EDC affects paternal care and parenting providing by his partner.

### **EVIDENCE THAT ENDOCRINE DISRUPTORS AFFECT SOCIAL BEHAVIORS**

Extensive data from retrospective human studies and animal models links perinatal exposure to EDCs and later social deficits (**Table 1**). The evidence in animal models will first be considered. Social behaviors to be considered include any that involve an interaction between difference members of the same species, such as various forms of communication, mate choice, pair bonding, social inquisitiveness and recognition, play behavior, social grooming, copulation, and aggression.

### **ANIMAL MODELS**

Animal communication assumes various forms besides direct vocalizations. In California mice (*Peromyscus californicus*), males use territorial marking to communicate and protect their home range and mate from intruders. Males pericoceptionally through perinatally exposed (2 weeks prior to breeding of the dam through weaning at PND 30) to BPA (50 mg/kg feed weight in the maternal diet) show reduction of this behavior when a control male is present in the testing arena (Williams et al., 2013). BPA exposure (1280μg/L water, 5.6μM for 14 days) of native blacktail shiner fish (*Cyprinella venusta*) and introduced red shiner fish (*C. venusta*) disrupts normal visual communication signals and abolishes the species-dependent sexually selected behavioral traits with the net potential for hybridization to occur between these two otherwise isolated species (Ward and Blum, 2012). Sprague-Dawley female rats prenatally exposed from gestational days 16 to 18 via daily intraperitoneal injection to the PCB mixture Aroclor (A) 1221 (100μg/kg, 1000μg/kg, and 10,000μg/kg bw/day) have later vocalization deficits, as well as changes in mating patterns, movement and likelihood to mate (Steinberg et al., 2007).

Mate choice and pair-bonding formation are vulnerable to endocrine disruption. In mate choice trials, control and BPAexposed female deer mice (*P. maniculatus bairdii*) selectively reject males developmentally exposed to BPA (50 mg/kg feed weight through the maternal diet) (Jasarevic et al., 2011). Likewise, female F3 Sprague-Dawley rats prefer F3 males whose ancestors were not prenatally exposed to the anti-androgenic EDC, vinclozolin (100 mg/kg bw/day from gestational days 8 to 14 via daily intraperitoneal injection) (Crews et al., 2007).  Pair-bonded monogamous Mongolian gerbil (*Meriones unguiculatus*) females treated for a 3 week duration (from pairing to day 21 of cohabituation) with daily oral administration of either BPA (2 or 20μg/kg bw/day) or EE2 (0.04μg/kg bw/day) exhibit heightened investigation of their partner and less exploration than untreated controls (Razzoli et al., 2005).

Introduction of new animals into the habitat results in social inquisitiveness and in time recognition of the new member with reduced bouts investigating this individual, i.e. habituation. Developmental and transgenerational exposure to BPA (5000μg/kg feed weight through the F0 maternal diet 7–10 days prior to being paired with a breeder male and for 2 weeks thereafter) prolonged the duration of time juvenile F1 and F3 C57BL/6 mice, respectively, spend investigating a novel animal (Wolstenholme et al., 2013). Further, F3 females, whose ancestors were exposed to BPA, persisted in investigating novel females longer than controls, suggestive of impaired dishabituation.

Exposure to BPA may affect other socio-sexual behaviors, including play, social investigation, copulation, and aggression. Decrease amount of play with males and social grooming were evident in female Sprague-Dawley rats perinatally exposed to BPA (4000μg/kg bw/day to the dams via daily oral administration from mating through weaning at PND 21), which indicates that this chemical can defeminize select female social behaviors (Porrini et al., 2005). Sprague-Dawley rats exposed perinatally to BPA (40 mg/kg bw/day from conception to weaning or 400 mg/kg/day from gestational day 14 to postnatal day 6 via daily maternal oral dosing) demonstrated masculinization of female social behaviors with such females selectively preferring to play with and engage in socio-sexual exploration of other females (Dessi-Fulgheri et al., 2002). Another study though with C57Bl6J mice suggests that gestational exposure to 1250μg BPA/kg feed weight increased play solicitations and approaches by both sexes (Wolstenholme et al., 2011b).

Sexual behaviors are also vulnerable to the early effects of BPA. Male LE rats, who received a "*low dose*" BPA (50μg/kg bw/day through maternal oral administration spanning gestation and lactation (gestational day 7 through PND 14), were sexually incompetent as adults, but no deficits in female sexual behavior were noted in this study (Jones et al., 2011). In contrast, another report found that neonatal exposure of inbred Wistar-derived female rats to BPA (50μg/kg bw/day or 20,000μg/kg bw/day via daily subcutaneous injections to the pups) had later reductions in proceptive behaviors (Monje et al., 2009). While BPA exposure (50, 100, and 200μg per egg) did overtly effect male Japanese quail (*Coturnix japonica*) copulatory behaviors, *in ovo* treatment with other estrogenic chemicals, including estradiol benzoate (10 or 25μg per egg), DES (700 ng per egg), and the soy phytoestrogen, genistein (1000μg per egg), abolished this behavior in pubertal males (Panzica et al., 2005).

Current data demonstrate that phthalates can also affect varying social behaviors in rodents and fish. Benzyl butyl phthalate (BBP) exposure through the water (100μg/L, 0.32μM) altered the shoaling behaviors (collection of fish for social purposes) in threespine stickleback (*Gastrosteus aculeatus*), who were treated each day for 26 days, and mummichog, a small killifish, (*Fundulus heteroclitus*), who were treated daily for 4 weeks (Wibe et al., 2002; Kaplan et al., 2013). BPP-treated threespine stickleback fish tended to aggregate in a single shoal that remained at the bottom of the test aquarium; whereas mummichog exposed fish preferred to shoal with smaller size fish. Male Sprague-Dawley rats exposed from adolescence to adulthood (5–6 weeks of age to 20–21 weeks of age) to BPP through the drinking water (5000μg/L and 10, 000μg/L or 0.016 and 0.032μM, respectively) displayed aberrant social behaviors (Betz et al., 2013). Male and female Wistar-Imamichi rats developmentally exposed through the maternal diet from gestational day 15 through weaning (PND 21) to various phthalate chemicals (di-n-butyl phthalate, DBP-20, 200, 2000, and 10,000 mg/kg bw/day; diisononyl phthalate, DINP- 40, 400, 4000, and 20,000 mg/kg bw/day; di-2-ethylhexyl adipate, DEHA- 480, 2400, and 12,000 mg/kg bw/day) exhibited sexual behavioral deficits (Lee et al., 2006). Copulatory behavior was diminished in male rats exposed to the varying doses of these chemicals. Similarly, the lordosis quotient was reduced in all treatment group females.

## **HUMAN EPIDEMIOLOGICAL STUDIES**

Associations between early BPA exposure and social behavioral disruptions in children are generally based on urinary BPA concentrations in the mother or child (**Table 1**). An African-American and Dominican women and their children population cohort that included 87 boys and 111 girls spanning 3–5 years of age showed higher exposure of BPA correlated with increased aggressive behaviors in boys but decreased aggression in girls (Perera et al., 2012). A more recent study with 153 six to ten year old children (77 boys and 76 girls) also demonstrated similar sex differences in vulnerability with increased maternal urinary BPA concentrations linked with greater externalizing and aggressive behaviors in boys (Evans et al., 2014). However, a study examining 249 mothers and their 2-year old children reported elevated prenatal BPA exposure associated with greater externalizing (aggression and hyperactivity) in girls but not boys (Braun et al., 2009).

Perinatal exposure to phthalates is also correlated with social behavioral disturbances. A study population of 404 mothers and their 7–9 years of age children pairs linked early contact to this chemical and later childhood social impairments, including social cognition, communication, and awareness (Miodovnik et al., 2011). This study however did not find any association between BPA exposure and effects on the examined social behaviors or sex-dependent differences. Examination of 122 mother-8 year old child pairs from Taiwan showed positive correlation of delinquent and aggressive behavioral scores and early exposure to different phthalate chemicals, as evidenced by maternal urinary concentrations (Lien et al., 2014). However, this study did not examine for potential sex-dependent differences. Another study with 153 mothers and their 6–10 year old children indicates that prenatal phthalate exposure is associated with later sex-dependent behavioral disturbances (Kobrosly et al., 2014). Boys of mothers with higher urinary monoisobutyl phthalate concentrations were more likely to show higher scores for inattention, rulebreaking behavior, aggression, and conduct problems; whereas increased concentration of monobenzyl phthalate was correlated with conduct problems in boys but reduced anxiety scores in girls.

### **MECHANISMS BY WHICH ENDOCRINE DISRUPTORS MAY AFFECT PARENTAL AND SOCIAL BEHAVIORS DISRUPTION OF STEROID HORMONE PRODUCTION OR AT THE STEROID RECEPTOR LEVEL**

As detailed above, full elaboration of many social and parental behaviors is dependent on the organizational and activational effects of testosterone and estrogen (Phoenix et al., 1959; Arnold and Breedlove, 1985; Morris et al., 2004). **Figure 1** illustrates the primary factors and enzymes required for normal testosterone and estrogen production. Both BPA and phthalates can affect steroidogenesis at several points in the pathway for males and females. Steroidogenic acute regulatory protein (StAR, STARD1)

is an essential mitochondrial protein for transporting cholesterol into the cell and is thus considered the rate-limiting step for steroid hormone production. BPA suppresses *StAR* mRNA expression in male and female gonads of rodents and fish (D'Cruz et al., 2012; Horstman et al., 2012; Liu et al., 2012; Peretz and Flaws, 2013; Savchuk et al., 2013). Expression of gonadal steroidogenic enzymes, including *Cyp11*, *Hsd3b*, *Hsd17b*, *Cyp17*, *Cyp19* is also generally inhibited by BPA exposure (D'Cruz et al., 2012; Liu et al., 2012; Nanjappa et al., 2012; Peretz and Flaws, 2013; Savchuk et al., 2013).

Most animal model studies report BPA exposure decreases production of testosterone (T) and estrogen (E2) by male and female gonads (Akingbemi et al., 2004b; Peretz et al., 2011; D'Cruz et al., 2012; Nanjappa et al., 2012). However, a study with isolated porcine ovarian follicles suggests potential dosedependent effects with lower dose BPA concentrations (0.1 mM) elevating E2; whereas large doses (1 and 10 mm) suppressing E2 levels (Grasselli et al., 2010). *In ovo* BPA treatment (0.01 or 1.4 ppm) of *Caiman latirostris* eggs elevated E2 and lowered T concentrations and concomitantly reversed the normal temperature sex determination mechanisms with a predominance of females occurring in these groups (Stoker et al., 2008). In girls with precocious puberty, elevated urinary BPA concentrations were associated with elevated T, E2, and pregnenolone levels (Lee et al., 2014).  In contrast, one report identifying disrupted social interactions in phthalate-treated rats indicated that this chemical increased E2 concentrations (Betz et al., 2013).

BPA and phthalates may also disrupt normal organizational/activational steroidogenic effects by altering the expression of the cognate receptors, ESRs and AR, in the neural regions governing social and parental behaviors, namely the hypothalamus. In this region, developmental exposure to BPA has been shown to alter the expression of ESR1 and ESR2 in rodent and ovine models, although the directionality is seemingly dependent upon sub-region, dose, time of assessment, and possibly species (Ramos et al., 2003; Ceccarelli et al., 2007; Monje et al., 2007; Mahoney and Padmanabhan, 2010; Cao et al., 2012, 2013, 2014; Patisaul et al., 2012; Kundakovic et al., 2013). Scant information exists on how phthalates may affect neural expression of ESRs and AR. The aforementioned rat phthalate study above also showed increased ESR1 expression in the amygdala of rats treated with 5 mg/L (0.16μM) BBP (Betz et al., 2013). Combined treatment of 4 wk old rats with BPA (285.4 mg/kg) and DBP (285.4 mg/kg) in the feed increased AR expression in the brain (Zhang et al., 2013), which might render these animals more sensitive to endogenous and exogenous androgens.

### **DISRUPTION OF THE HYPOTHALAMIC-PITUITARY-ADRENAL (HPA) AXIS**

BPA, phthalates, and other EDC exposure may disturb parental and social behaviors through disruptions in the classic stressassociated adrenocortical axis. One rat study showed that early BPA exposure (oral administration of 40μg/kg bw/day to the dam throughout gestation and lactation) led to sex dependent effects on circulating corticosterone concentrations (elevated in BPA-exposed females) and neural (hippocampal expression) of *Gr* (lower expression in BPA-exposed males) (Poimenova et al., 2010). Further, BPA-treated female rats developmentally exposed to this same dosing regimen exhibited increased basal corticosterone and suppressed hypothalamic *Gr* expression (Panagiotidou et al., 2014).

Another study in rats where dams were subcutaneously injected with 2μg BPA/kg bw/day from gestational day 10 to lactational day 7 showed sex-dependent alterations in the adreno-cortical axis (Chen et al., 2014). BPA-exposed male rats had increased basal concentrations of serum corticosterone and adrenocorticotropin (ACTH) and corticotropin-releasing hormone (*Crh*) mRNA expression, but these changes were not evident in exposed females. Following subsequent exposure to a mild stressor, corticosterone and ACTH concentrations increased further in BPA males; whereas, these hormones decreased in BPA females. In non-stressed animals, *Gr* mRNA was increased in the hippocampus and hypothalamic paraventricular nucleus of BPA females. In contrast, these transcripts decreased in BPA males. The collective results suggest that BPA exposure might affect the HPA axis in males and females but the specific effects are sex-dependent.

Phthalates also appear to disrupt the HPA axis. Oral exposure to DEHP (500 mg/kg bw/day for 4 days) increased concentrations of ACTH and corticosterone in treated male rats at 20 and 40 days of age; whereas no effects were observed in adult (60 days of age) males (Supornsilchai et al., 2007). Isolated adrenocortical cells from the 20 and 40 day old treated rats were more sensitized to the effects of ACTH, dibutyrl cAMP, and 22R-hydroxylase, as evidenced by increased production of corticosterone and ACTH treatment stimulated greater transport of endogenous cholesterol into the mitochondria.

### **DISRUPTION OF NEUROPEPTIDE/PROTEIN HORMONES AND THEIR COGNATE RECEPTORS**

In the mouse and rat, central oxytocin receptors (OXTR) are obligatory for the expression of maternal behavior, and variation in OXTR levels in the medial preoptic area of the hypothalamus (MPOA) are functionally linked to differences in degree of maternal care (Champagne et al., 2006; Weaver, 2007). California mice fathers are known to exhibit increased oxytocin immunoreactivity compared to non-paternal males (Lambert et al., 2011). Increased signaling through the OXTR is correlated with high degree of maternal behavior, such as pup licking and grooming, while ESR1 is an important regulator of OXTR expression (Champagne et al., 2006). Prolactin, oxytocin, and vasopressin have been implicated in paternal care in California mice, monogamous prairie voles (*Microtus ochrogaster*), and humans (Gubernick and Nelson, 1989; Parker and Lee, 2001; Bales et al., 2004; Wynne-Edwards and Timonin, 2007; Gordon et al., 2010). Social behaviors are also dependent upon oxytocin, vasopressin, and protein binding to their cognate receptors in the brain (Carlson et al., 2006; Benarroch, 2013; Walker and McGlone, 2013; Wudarczyk et al., 2013; Babb et al., 2014; Lieberwirth and Wang, 2014).

Brains from 18.5 days post-coitus (dpc) mice exposed to BPA express less oxytocin, OXTR, and vasopressin than control males (Wolstenholme et al., 2011a, 2012).  Another study, however, suggests that in the paraventricular nucleus (PVN), oxytocinimmunnoreactive neurons increase in neonatally exposed BPA female Long Evans rats (Adewale et al., 2011), but non-pregnant, non-lactating females were examined. A recent study with juvenile prairie voles showed neonatal exposure to BPA increased AVP-immunoreactive neurons in the anterior PVN but decreased OT-immunoreactive neurons in this same region (Sullivan et al., 2014). *In vivo* and *in vitro* studies suggest that BPA increases PRL levels in male and female rats (Steinmetz et al., 1997; Goloubkova et al., 2000; Ramos et al., 2003; Delclos et al., 2014). Likewise, phthalate exposure is associated with elevated prolactin production in rats of both sexes and men (Lee et al., 2004; Li et al., 2011). No previous study has assessed the effects of BPA or phthalate-exposure on PRL in parenting males and females.

### **EPIMUTATIONS**

Epimutations are the most plausible mechanisms by which early exposure to BPA, phthalates, and other EDCs may lead to later neurobehavioral disturbances. Such changes may include alteration in DNA methylation, histone proteins, non-coding RNA, or chromatin arrangement. It is now apparent from a variety of animal model studies that BPA, phthlates, and other EDCs can lead to DNA methylation and corresponding gene expression changes in a variety of tissues, including the brain (Yaoi et al., 2008; Jang et al., 2012; Tang et al., 2012; Kundakovic et al., 2014; Zhao et al., 2014; Martinez-Arguelles and Papadopoulos, 2015).

Only two studies to date have shown BPA-induced DNA methylation changes in the brain. Developmental exposure to BPA alters the DNA methylation promoter state in many genes of the mouse forebrain (Yaoi et al., 2008). Persistent DNA methylation changes are evident in one of the promoters of *Bdnf* in the hippocampus and cord blood in female and male mice subjected to prenatal exposure to BPA with hypemethylation apparent in females but hypomethylation observed in males (Kundakovic et al., 2014). DNA methyl transferase (DNMT) and methyl CPG binding proteins (MECP) guide global DNA methylation changes.

BPA may simultaneously effect DNA methylation and histone protein modifications. For instance, exposure to BPA suppresses rat brain cortical expression of the ion transporter (*Kcc2*) through both DNA methylation via MECP2 and histone protein (H3K9) binding to this gene (Yeo et al., 2013). Phthalates can also induce histone protein modifications in neuronal cells, as evidenced by phthalate-induced Sp3 suppression associated with deacetylation (via HDAC4) and ensuing polyubiquination in neuroblastoma cells (Guida et al., 2014).

The expression of *Dnmt1*, *Dntm3a*, *Dnmt3b*, and *Mecp2* is altered in various brain regions, including basolateral amygdala, cortex, and hypothalamus, in BPA-treated rodent models (Kundakovic et al., 2013; Warita et al., 2013; Zhou et al., 2013). The histone protein modification enzyme (HDAC2) is upregulated in the hippocampus of adult males exposed to BPA (Zhang et al., 2014a). The histone methyltransferase enzyme (EZH2) and histone H3 trimethylation is upregulated in the mammary gland of mice exposed to BPA *in utero* and MCF-7 cells treated with this chemical (Doherty et al., 2010).

While no study has determined whether BPA exposure affects non-coding (nc) RNAs (including microRNAs, miRNAs) in the brain, there is evidence that this chemical can affect expression of these biomolecules in other cells, including MCF7 (Tilghman et al., 2012), ovarian (Veiga-Lopez et al., 2013), and placental cells (Avissar-Whiting et al., 2010). No study to date has assessed whether phthalate exposure affects ncRNAs in the brain or other regions.

## **CONCLUSIONS**

Strong evidence exists that parental and social behaviors in a wide variety of species, including by translation humans, are vulnerable to perinatal exposure to EDCs, including BPA and phthalates. These effects are likely multifactorial in origin, but the net result is presumably that these chemicals disrupt normal organizational and activational programming of the brain. EDC-induced disruptions on neural programming may occur as a result of alterations in fetal or adult steroid hormone production, steroid receptor expression in the brain regions governing these traits, neuropeptide/protein hormones and their cognate receptors, and/or through epimutations. It remains to be determined though whether BPA, generally considered a "*weak estrogen,*" results in similar mechanistic disruptions as phthalates with their predominantly anti-androgenic effects. Both chemicals can potentially disrupt all of the above pathways. Further, it is now apparent that both chemicals may lead to pleiotropic disturbances through steroidogenic and non-steroidogenic pathways (Leon-Olea et al., 2014).

EDC-induced parental and social behavioral deficits could affect the general livelihood, social well-being, ability to attract mate(s), reproduction, and likelihood of successfully rearing young. Thus, early exposure of animal and humans to these widely prevalent chemicals may lead to insidious behavioral effects. Moreover, there is ample evidence in animal models that EDCs (and humans in the case of DES) can induce transgenerational effects (Newbold et al., 2000, 2006; Klip et al., 2002; Titus-Ernstoff et al., 2006; Wolstenholme et al., 2012, 2013; Doyle et al., 2013; Manikkam et al., 2013; Schneider et al., 2013; Zhang et al., 2014b). Therefore, these chemicals might already be exhuming a toll on the social lives of unborn generations. While a call to action to legislate further these chemicals seems a reasonable course of action based on the existing data and precautionary principle, identifying the underpinning mechanisms leading to these behavioral disturbances might provide the incentive for policymakers to act. Long-term studies examining for linkages in parental deficiencies and transgenerational effects in human populations exposed to these EDCs may regrettably be needed before the production of such chemicals is minimized or outright banned.

### **ACKNOWLEDGMENT**

The studies were supported by NIH Grant 5R21ES023150-02 (to C.S.R.).

### **REFERENCES**

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of rat offspring after maternal exposure during the period from late gestation through lactation. *Toxicology* 203, 221–238. doi: 10.1016/j.tox.2004. 06.013

in male descendants of mice exposed developmentally to diethylstilbestrol. *Carcinogenesis* 21, 1355–1363. doi: 10.1093/carcin/21.7.1355

in basolateral amygdala accounts for anxiety in rat offspring exposed perinatally to low-dose bisphenol A. *J. Psychiatr. Res.* 47, 1535–1544. doi: 10.1016/j.jpsychires.2013.05.013

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

*Received: 21 November 2014; accepted: 09 February 2015; published online: 03 March 2015.*

*Citation: Rosenfeld CS (2015) Bisphenol A and phthalate endocrine disruption of parental and social behaviors. Front. Neurosci. 9:57. doi: 10.3389/fnins.2015.00057 This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience.*

*Copyright © 2015 Rosenfeld. 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) or licensor 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 multi-oscillatory circadian system times female reproduction

### *Valérie Simonneaux1 \* and Thibault Bahougne1,2*

*1 Institut des Neurosciences Cellulaires et Intégratives, CNRS (UPR 3212), Strasbourg, France, 2Service d'Endocrinologie et Diabète, Hôpital Civil, Hôpitaux Universitaires de Strasbourg, Strasbourg, France*

Rhythms in female reproduction are critical to insure that timing of ovulation coincides with oocyte maturation and optimal sexual arousal. This fine tuning of female reproduction involves both the estradiol feedback as an indicator of oocyte maturation, and the master circadian clock of the suprachiasmatic nuclei (SCN) as an indicator of the time of the day. Herein, we are providing an overview of the state of knowledge regarding the differential inhibitory and stimulatory effects of estradiol at different stages of the reproductive axis, and the mechanisms through which the two main neurotransmitters of the SCN, arginine vasopressin, and vasoactive intestinal peptide, convey daily time cues to the reproductive axis. In addition, we will report the most recent findings on the putative functions of peripheral clocks located throughout the reproductive axis [kisspeptin (Kp) neurons, gonadotropin-releasing hormone neurons, gonadotropic cells, the ovary, and the uterus]. This review will point to the critical position of the Kp neurons of the anteroventral periventricular nucleus, which integrate both the stimulatory estradiol signal, and the daily arginine vasopressinergic signal, while displaying a circadian clock. Finally, given the critical role of the light/dark cycle in the synchronization of female reproduction, we will discuss the impact of circadian disruptions observed during shift-work conditions on female reproductive performance and fertility in both animal model and humans.

# Keywords: female reproduction, circadian clock, suprachiasmatic nuclei, kisspeptin, GnRH, LH, estradiol,

## Introduction

shift-work

Ovulation in female mammals is a complex process, which is exquisitely regulated by a number of environmental (time of day, time of year, food resources, and stress level) and internal (development stage, hormonal milieu, and metabolic rate) factors. Indeed, female reproduction is a long-term, demanding process and therefore, it is important that a limiting critical status is reached to ensure successful reproductive outcome. In adult females where all these criteria are attained, there are still two important cues that time ovulation: the circulating level of gonadal hormones, specifically estradiol, which is an indicator of oocyte maturation, and the time of day arising from biological clocks. This dual regulation ensures that the timing of ovulation coincides with the period of maximal activity and sexual motivation. Most mechanistic studies aimed at understanding this subtle timing of ovulation have been performed in laboratory rodents, but ovulation in humans is also gated by similar hormonal and circadian inputs. Hence, this review will not only focus on the mechanisms regulating the timing of reproduction in female rodents, but will also discuss human female fertility, including the desynchronization associated with modern life styles (shift work, jet lag, and sleep alteration).

### *Edited by:*

*Hubert Vaudry, University of Rouen, France*

### *Reviewed by:*

*Yoshitaka Oka, University of Tokyo, Japan Vance Trudeau, University of Ottawa, Canada*

### *\*Correspondence:*

 *Valérie Simonneaux, Institut des Neurosciences Cellulaires et Intégratives, CNRS (UPR 3212), 5 rue Blaise Pascal, Strasbourg 67084, France<EMAIL_ADDRESS>
### *Specialty section:*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology*

*Received: 14 June 2015 Accepted: 21 September 2015 Published: 20 October 2015*

### *Citation:*

*Simonneaux V and Bahougne T (2015) A multi-oscillatory circadian system times female reproduction. Front. Endocrinol. 6:157. doi: 10.3389/fendo.2015.00157*

## Female Reproduction is Rhythmic

Reproductive activity in female mammals displays a regular cycle (menstrual cycles in women, estrous cycles in rodents) driven by a complex interaction of the circadian system, hypothalamic neuropeptides, gonadotropins [luteinizing hormone (LH) and follicle-stimulating hormone (FSH), both secreted by the pituitary gonadotroph cells], and sex steroid hormones produced by the ovaries. The final output of this regulatory process is to combine the production of a mature oocyte (ovulation) with a receptive reproductive tract, which will ensure the embryo's development.

During the first part of the reproductive cycle (follicular phase in women; metestrus–diestrus in rodents), gonadotrophs produce more FSH than LH. This relative FSH preponderance contributes to the recruitment and development of ovarian follicles. FSH promotes follicular growth leading to a progressive increase of the sex steroid hormone, estradiol, and increases LH receptor expression in granulosa cells (1). During this early phase, LH pulses occur with a high frequency (period of 1–2 h in women, 20 min in rodents) and uniform amplitude, and the pulse frequency tends to increase toward the end of the phase. The second part of the reproductive cycle (luteal phase in women; proestrus–estrus in rodents) begins with a marked and transient secretion of LH (**Figures 1A,D**). The LH surge has three functions: (1) induction of ovulation of mature follicles, (2) resumption of oocyte meiosis, and (3) arrest of granulosa cell proliferation and luteum induction. After the LH surge, ovulation generally follows a few hours later in rodents, and 24–48 h in women. The preovulatory LH surge takes place approximately every 4–5 days in rodents and every 28 days in women and its occurrence depends on high circulating estradiol levels (2, 3). Additionally, the LH surge requires a daily signal since it arises at a very specific time of day, usually at the end of the resting period, thus in the late afternoon in nocturnal rodents and the end of the night/early morning in the diurnal rodent Arvicanthis (4) and in humans (5–7). Indeed, in 80% of women, the LH surge occurs around 8 a.m. At the end of the reproductive cycle, LH pulse frequency decreases significantly down to a pulse interval of 2–6 h with variable amplitude (8).

The secretion of both LH and FSH is under the control of a hypothalamic neurohormone, gonadotropin-releasing hormone (GnRH), which is synthesized in neurons scattered throughout the preoptic area (POA) and the organum vasculosum laminae terminalis. These neurons project to the median eminence where they release GnRH in the portal circulation in a pulsatile manner. GnRH activates specific receptors located on pituitary gonadotrophs inducing the synthesis and release of LH and FSH. GnRH is essential for reproduction as mutations in the gene coding for GnRH (10) and GnRH receptor (11) are proposed to be responsible for idiopathic hypogonadotropic hypogonadism (IHH), characterized by delayed puberty and infertility (11). The pulsatility of GnRH release is critical to induce proper gonadotropin secretion, and there is a tight correlation between GnRH and LH pulsatilities. Pulsatile administration of exogenous GnRH (one pulse per hour) is capable of restoring the preovulatory surge, ovulation, and normal menstrual cycles in patients suffering from Kallmann syndrome (12, 13). In contrast, continuous GnRH administration induces a reversible blockage of the pituitary gonadotroph cells' secretion (14).

Various (neuro)transmitters had been proposed to regulate GnRH neuronal activity until the neuropeptide kisspeptin (Kp) was discovered as a potent activator of GnRH release. In 1996, the *Kiss1* gene was discovered and reported to encode a peptide called metastin, because of its anti-metastatic property on malignant melanoma cells (15). However, the receptor of this peptide, GPR54, was later found to play a critical role in reproductive physiology when two groups reported that mutation of the *GPR54* receptor results in IHH in humans, with an identical phenotype observed in mice with a targeted deletion in this receptor (16, 17). The *Kiss1* gene was shown to encode a family of Kps from an initial 145 amino acid propeptide, Kp-145, which is cleaved into peptides of different sizes from Kp-54 (previously named metastin) to Kp-10. The discovery of Kp's role in reproductive function has been a milestone in the field of reproductive biology, and numerous studies now indicate that Kps are critical regulators of sexual differentiation and maturation as well as of normal adult reproductive functioning across mammalian species, including humans (18). Kp neurons are localized within two hypothalamic areas, in the arcuate nucleus (ARN) and the rostral periventricular nucleus of the third ventricle, also called anteroventral periventricular nucleus (AVPV), or the preotic area (according to species). They send projections mainly to the GnRH neuron cell bodies (AVPV Kp neurons) and nerve terminals [ARN Kp neurons (19–22)] (**Figure 1A**). The AVPV presents a marked sexual dimorphism, with more Kp neurons in females as compared to males (20, 23). The AVPV Kp neurons are the main drivers of the preovulatory GnRH/LH surge (24). In contrast, the ARN Kp neurons are not sexually dimorphic (20, 23). The Kp receptor, Kiss1R (formerly GPR54), is highly expressed in GnRH neurons but also in other brain areas (25, 26) and in most endocrine tissues like the pituitary gland, ovary, and placenta (27). Kp has a very potent stimulatory action on GnRH release and, therefore, gonadotropin secretion in all mammalian species investigated so far (18, 19, 28, 29). Central injection of doses as low as 0.1–1 pmol Kp10 is indeed sufficient to evoke robust LH secretion in rats and monkeys (28, 30). Kp injections must be short and at least 2 h apart to induce the LH peak since the repeated administration of Kp induces Kiss1R desensitization (31, 32). Notably, Kp release in the stalk-median eminence is pulsatile (33), and pulsatile Kp drives LH secretion in juvenile monkeys (34). A recent study reported that pulsatile administration of Kp was able to evoke dramatic synchronous activation of *GnRH* gene transcription with robust stimulation of GnRH secretion in murine-cultured hypothalamic explants (35). The preeminent phenotypes of impaired reproduction (abnormal sexual maturation, small uterus, ovaries without mature follicles, no estrous cycles) often arise from mutations in *Kiss1* (36, 37) and *Kiss1R* (16, 38, 39), which suggest that the Kiss1/Kiss1R complex is essential for the central regulation of the gonadotropic axis.

Other classical neurotransmitters and neuropeptides have been reported to regulate GnRH neuron activity albeit not to the same extent as Kp. GABA and glutamate fibers are found close to GnRH perikarya in the POA and axons in the median eminence. Both neurotransmitters have been shown to play a role in the regulation of GnRH release. Glutamate stimulates *Gnrh* gene

FIGURE 1 | The multi-oscillatory network of the female hypothalamic–pituitary gonadal axis in rodents. (A) Schematic representation of the neuroendocrine pathway timing female reproduction: the master circadian clock located in the hypothalamic suprachiasmatic nucleus (SCN) is synchronized to the daily cycle mostly via the light/dark cycle and to a less extend by other time cues (food intake and sleep/wake activity); two SCN peptidergic transmitters forward the daily information to the reproductive axis: arginine vasopressin (AVP) is the most essential daily transmitter projecting to the kisspeptin (Kp) neurons of the anteroventral periventricular nuclei (AVPV), which in turn strongly activate the GnRH neurons located in the preoptic area (POA); vasoactive intestinal peptide (VIP) directly modulates GnRH neuron activity and possibly indirectly via neurons of the dorsomedial hypothalamus (DMH), which express RF-related peptide (RFRP); GnRH released in the portal blood system activates the synthesis and release of the gonadotropins luteinizing hormone (LH) and folliculo-stimulating hormone (FSH), which in turn regulate oocyte maturation, estradiol synthesis, and finally ovulation triggered by the LH surge; throughout the estrous cycle, estradiol feeds back onto different levels of the reproductive axis principally via the nuclear estrogen receptor α (ERα) and to a less extend via ERβ. Kp neurons are the main estradiol targets with an inhibitory effect of low estradiol on Kp neurons of the arcuate nucleus (ARN) and a stimulatory action of high estradiol on AVPV Kp neurons. In addition to the master SCN clock, peripheral clocks are located in the AVPV Kp neurons, GnRH neurons, pituitary gonadotrophs, and the different cell types of the ovary. (B) Representative pictures showing PER1 immunoreactivity (green) in Kp immunoreactive neurons (red) in the AVPV of female mice sampled at zeitgeber time 20 (8 h after lights off on a 12 h light/12 h dark schedule) on the day of proestrus [adapted from Ref. (9)]. (C) Representative bioluminescence traces from isolated AVPV explants of PER2:LUCIFERASE female mice, in absence (0 E2, light gray line) or in presence of estradiol (1 μM E2, black line) in the culture medium; τ indicates the period of the circadian AVPV oscillations in both conditions [adapted from Ref. (9)]. (D) Schematic representation of the mouse estrus cycle including the four stages: metestrus, diestrus, proestrus (the stage at which the LH concentration raises as an LH surge), and estrus; during the first part of the reproductive cycle, low circulating level of estradiol displays a negative feedback, whereas at proestrus, high estradiol levels exert a positive feedback, causing a synchronized activation of GnRH neurons leading to the preovulatory surge of LH; notably, the LH surge occurs at the end of the resting period, which is late day in nocturnal species.

expression and GnRH release during the LH surge, whereas a glutamate antagonist blocks *Gnrh* gene expression and the LH surge when administered in the morning (40–42). Administration of an AMPA agonist enhances the *in vivo* LH secretion in OVX rats only with estradiol substitution, whereas glutamate stimulates *in vitro* GnRH secretion in a estradiol-independent matter (43). The role of GABA on GnRH neuronal activity is debated since inhibitory and stimulatory effects have been observed depending on the protocols used, the presence of sex steroid treatment, the timing in the estrus cycle and the hypothalamic region studied (44, 45). Fibers containing the orexigenic neuropeptide Y contact a majority of GnRH neurons, which express neuropeptide Y receptors. This neuropeptide has been reported to exert variable effects depending on the metabolic and reproductive status of the animal, but most of the studies describe an inhibitory effect of neuropeptide Y on GnRH neurons (46–48). Recent studies indicate that another neuropeptide belonging to the same RF-amide peptide family as Kp, RFRP-3 (the mammalian homolog of avian gonadotropin-inhibitory hormone), regulates GnRH neuron activity [for review, see Ref. (49–51)]. Unlike Kp, RFRP-3 can activate or inhibit the reproductive axis according to gender, species, and environmental conditions (26, 51–53). In female rodents RFRP neuronal activity is decreased at the time of the LH surge, possibly to relieve the inhibitory effect of RFRP-3 on GnRH neurons (54). However, mice bearing a null mutation of GPR147, the RFRP-3 receptor, present only a mild reproductive phenotype (55).

## Estrogenic Regulation

Estradiol produced by the ovaries exerts both positive and negative feedback upstream of the reproductive axis, modulating GnRH neuron activity and anterior pituitary gonadotroph cells. During the first part of the reproductive cycle, estradiol induces a negative feedback, whereas when estradiol concentration is the highest (at the end of the follicular phase in humans or proestrus in rodents) the feedback becomes positive, causing a synchronized activation of GnRH neurons leading to GnRH release in the hypophyseal portal blood and finally the preovulatory LH surge (56) (**Figures 1A,D**). The effect of estradiol is mediated via two types of nuclear estrogen receptors (ERs), which induce long lasting genomic action, ERα and ERβ (57, 58), but it can also have a rapid action via membrane bound estradiol receptors, including the GPR30 (59, 60).

### GnRH Neurons

Gonadotropin-releasing hormone neurons contain few, if any, ERα (61–63), but do express ERβ (64–66) and GPR30 (67). Estradiol application to cultured primate GnRH neurons induces a rapid increase in action potential firing frequency (68) and intracellular calcium oscillations (69). Similar effects have been reported in the mouse GnRH neurons (70). This rapid effect of estradiol is proposed to be mediated via GPR30 in primates (67) and ERβ in mice (70, 71). Using an *in vitro* GnRH neuronal model, the GT1-7 cells, it was reported that *Kiss1R* expression is estradiol dependant, with a Kp-induced GnRH increase only in cells treated with estradiol (72, 73). A primary effect of estradiol on GnRH neurons has been hypothesized to upregulate expression of channel transcripts (TRPC4 channels and HCN1 channels) that orchestrate the downstream signaling of Kiss1R in GnRH neurons (74, 75). Therefore, estradiol could be a potent regulator of ion channel and receptor expression in GnRH cells, hence controlling the sensitivity of GnRH neurons to Kp. However, a recent study reported that mutant mice with a GnRH neuron-selective deletion of ERβ exhibit normal cycles and negative feedback, leaving the critical role for ERβ in GnRH neuron activity still an open question (76).

### Kisspeptin Neurons

In contrast to GnRH, Kp neurons have a high density of ERα and are, therefore, considered as the intermediate node for the estradiol feedback on GnRH neurons (19, 20, 32, 61, 77–79) (**Figure 1A**). Interestingly, the estradiol feedback in rodents depends on the Kp neuron localization as estradiol stimulates *Kiss1* expression in the AVPV and inhibits *Kiss1* expression in the ARN (77, 80–82). In non-rodent mammals, a similar differential regulation by estradiol is also observed with a stimulatory effect in the rostral periventricular/POA and an inhibitory effect in the ARN (83, 84). The mechanism underlying these differences in the regulation of *Kiss1* expression by ERα in both structures is not yet fully understood. AVPV *Kiss1* activation requires an estrogen response element (ERE)-dependent pathway, whereas inhibition of *Kiss1* expression in the ARN involves ERE-independent mechanisms (80, 85). Recent studies have reported that estradiol additionally modulates daily activity of AVPV Kp neurons. Hence, the daily variation in c-Fos activation, *Kiss1* mRNA and peptide content observed in proestrus or in ovariectomized estradioltreated rodents is abolished or strongly reduced in diestrus or in ovariectomized animals (9, 86–88). Furthermore, estradiol has been reported to increase the number of arginine vasopressin (AVP) synaptic contacts onto Kp neurons (89), regulate AVP1a receptor expression by Kp neurons (88) and be permissive for the AVP-induced electrical activation of Kp neurons (90). Altogether these observations indicate that high circulating levels of estradiol gate the action of AVP onto AVPV Kp neurons (see Role of the Suprachiasmatic Nuclei for the role of AVPV on Kp neurons).

### Circadian System

Estradiol is also suggested to influence daily functions since shifts are observed during the pubertal period, pregnancy, menopause, and throughout the reproductive cycle (91–93). In rodents, ERα and ERβ are expressed in the retina, the retino-hypothalamic tract, the geniculohypothalamic tract and raphe nuclei-derived serotonergic inputs, all major inputs to the suprachiasmatic nuclei (SCN), which contain the master circadian clock (94). In mice, the second half of the proestrus night is often, but not consistently, characterized by increased motor activity compared to the remaining nights of the estrous cycle (95, 96). Furthermore, ovariectomy reduces total motor activity, and estradiol reverses this effect, while also shortening the length of the free running period and advancing the onset of wheel running activity (96). Estradiol may act directly on SCN clock gene oscillation since estradiol treatment in ovariectomized rats decreased *Cry2* mRNA levels (97) and estradiol application to SCN slices increased the spontaneous firing frequency and depolarized cell membranes of the SCN neurons (98). However, other studies reported that estradiol treatment of SCN explants from PER2:LUCIFERASE mice has no effect on the period and amplitude of the circadian oscillations (9, 99). Alternatively, estradiol may alter rhythms in running activity via indirect effects on the medial POA or striatum (100, 101).

### Pituitary

Pituitary gonadotrophs express ERα, and estradiol has been proposed to exert a direct negative feedback effect on gonadotropin secretion (102–104). Chronic treatment with estradiol induces negative feedback effects on gonadotrophin secretion after GnRH supraphysiologic stimulation in ewes (105) or humans (106). Interestingly, a recent study reported that mice with a selective deletion of ERα in pituitary gonadotroph cells had elevated serum LH and estradiol values, and displayed irregular estrous cycles punctuated by prolonged periods of disorganized cycling, pointing to ERα participation in the estradiol negative feedback at the pituitary level (107). It is worth mentioning that this phenotype was much less severe than the one observed after a total ERα deletion. Although the role of Kiss/Kiss1R at the pituitary is still the subject of debate, it is interesting to note that the activation of pituitary ERα up-regulates *Kiss1* expression, whereas chronic exposure to estradiol down regulates *Kiss1R* expression on pituitary gonadotrophs (108).

### Uterus

The uterus expresses high levels of ERα, and is an important site for the estrogenic control of reproductive physiology (109, 110). Estradiol, together with progesterone, regulates uterine growth and differentiation, which in turn control embryo-endometrial interactions during early pregnancy (110). Furthermore, estradiol treatment also shortens the period of the circadian clock in the uterus (99).

## Daily and Circadian Regulations

## Role of the Suprachiasmatic Nuclei The Hypothalamic Suprachiasmatic Nuclei Host the Master Biological Clock

Most biological functions, including female reproduction, are synchronized to the daily variation of environmental factors. Among these factors, the recurring light/dark cycle is the most predictable environmental cue used by mammals to adjust their behavior and physiology appropriately. The mechanisms by which light and dark synchronize biological functions involve the master biological clock located in the hypothalamic SCN and a retino-hypothalamic tract, which forwards the non-visual light to the SCN.

The demonstration that a biological clock located in the basal hypothalamus was driving circadian rhythms came from experiments showing that SCN lesions in rodents abolished circadian rhythms in locomotor activity, which were restored following exogenous SCN implants (111, 112). The circadian activity of SCN neurons relies on a complex molecular system cycling endogenously with a period of about 1 day (*circa dies*). This molecular clockwork is composed of transcription–translation loops, which are now well described (113, 114). Dimers of the CLOCK and BMAL1 proteins bind to a specific E-box promoter to induce the transcription of four clock genes *Per1*, *Per2*, *Cry1*, and *Cry2*, which after translation produce proteins which form dimers to repress their own transcription by competing with the CLOCK/BMAL1 binding. Following degradation of the inhibitory proteins, the transcription–translation loop starts over for another circadian cycle. When SCN explants or dissociated cells are kept *in vitro*, the endogenous circadian oscillations continue for weeks or months, providing that the culture medium gets enough nutrients and oxygen for the cell metabolism. A very interesting animal model used to demonstrate these sustained endogenous oscillations is the PER2:LUCIFERASE mice, where the expression of the *luciferase* gene is driven by the *Per2* promoter (115, 116). When explants or dissociated cells of PER2:LUCIFERASE mouse SCN are placed in a culture medium containing luciferine, the rhythmic expression of PER2 drives a rhythmic expression of luciferase, which by oxidizing luciferin causes the emission of a bioluminescent signal with a circadian period.

In order to achieve its role in adjusting biological functions with the astronomical daily cycles, the SCN circadian activity has to be synchronized with the time of day and transmit this timing information to the rest of the body. Light has long been known to be the main synchronizer of the SCN circadian clock, but interestingly it uses a specific non-visual pathway, which includes melanopsin-containing intrinsically photosensitive retinal ganglion cells projecting directly to the SCN (117, 118). Upon light activation, these ganglion cell terminals release glutamate and pituitary activating cAMP peptide, which change the phase (synchronizes) of the circadian clock. The synchronizing property of light depends on the time of application during the day, and the characteristics of the phase responses depend on species. The astronomical light/dark alternation synchronizes the circadian clock in order to attain a daily rhythm of a precise 24-h period. The CLOCK/BMAL1 dimers not only activate canonical clock gene expression, but other clock-controlled genes whose promoters display E-boxes and therefore undergo rhythmic expression. This mechanism was first demonstrated for the gene encoding AVP, an important output of the SCN clock (119). Levels of SCN AVP mRNA are markedly higher during the day than at night, but in *Clock* KO mice the SCN AVP rhythm is strongly dampened (120).

## SCN Lesion or Clock Gene Mutations Alter the Reproductive Cycle

Various experiments aiming at impairing clock function were performed to delineate whether functional SCN neurons are required for the daily timing of the LH surge, mostly in female rodents. Early experiments of SCN lesions (121) or SCN-POA neuronal connection cut (122) resulted in an impaired LH surge and estrous cyclicity in female rats. Furthermore, female mice carrying mutations of *Clock* or *Bmal1* displayed disrupted estrous cycles (123–127). Clock−/− mutant mice, for example, have extended and disrupted estrous cycles under both a light/ dark cycle and during continuous darkness. In humans, it was reported that women with single-nucleotide polymorphisms in the ARNTL (*Bmal1*) have more miscarriages and less pregnancies than those without (128). Although these experiments have pointed to a crucial role of the SCN in the proper timing of estrous cyclicity, reproductive impairment following clock gene mutations could as well result from peripheral clock desynchronization (see Other Clocks in the Reproductive System). Interestingly, the reproductive phenotypes of young Clock-, Bmal1-, or Per1/Per2-mutated mice resemble that of middle-aged (over 10-month old) wild-type mice, with increased length and decreased frequency of estrous cycles (129, 130). These observations indicate that alterations in central or peripheral clocks may lead to advanced reproductive senescence (130).

The preovulatory LH surge is initiated by a SCN-derived stimulatory signal, at a time closely preceding general activity onset. However, this signal is effective at stimulating GnRH neurons to produce the LH surge only when estradiol concentrations have reached a critical threshold. Prior to the day of proestrus, the developing ovarian follicles secrete insufficient estradiol to fulfill this criteria and therefore, the SCN signal does not trigger the LH surge (56). The occurrence of the daily stimulatory SCN signal can be unmasked by implanting female rodents with estradiol capsules that result in proestrus concentrations of this hormone; in this case, a LH surge occurs every day (3, 56, 131).

### SCN Neuropeptides Involved in the Timing of the GnRH/LH Surge

Transplant of fetal SCN tissue into bilaterally SCN-lesioned hamsters restores locomotor, but not endocrine rhythms in the absence of neural outgrowth, suggesting that intact neural connections are required for endocrine rhythmicity, whereas behavioral rhythms can be supported by a diffusible signal (132). Neuroanatomical studies have pointed to two putative SCN neural outputs signaling daily information to the reproductive axis, AVP and vasoactive intestinal peptide (VIP). Early experiments have identified SCN-originating, VIP-containing fibers contacting GnRH neurons (133, 134), which express the VIP receptor VPAC2 (135). However, more recent studies indicate that the SCN signals the time of day to GnRH neurons indirectly via AVP fibers projecting to the Kp neurons of the AVPV (78, 89).

Anterograde tracing studies show that a number of AVPcontaining axons originating in the SCN make appositions to Kp neurons, whereas very few or no VIP terminals were found apposed to Kp neurons (87, 89). Furthermore, AVP is released with a peak coinciding with the onset of the LH surge (136) and AVPV Kp neurons express V1a receptors (87). Interestingly, the AVP input to Kp neurons is sensitive to estradiol since estradiol treatment significantly increases the number of AVP terminal appositions on individual Kp neurons (89) and circadian expression of V1a mRNA is abolished in ovariectomized animals (88). Furthermore, AVP signaling onto Kp neurons is critically dependent on circulating estradiol as AVP no longer activates Kp neurons in ovariectomized mice, an effect that is fully restored by estradiol treatment (90). Altogether, these results are consistent with the hypothesis that Kp neurons located in the rodent AVPV receive daily information from the SCN via an AVPergic monosynaptic pathway, a signal which is modulated (gated) by circulating estradiol (**Figure 1A**).

In line with these neuroanatomical observations, earlier physiological experiments pointed to a functional role of AVP in the GnRH/LH surge timing, even though the importance of Kp neurons was not yet known. Inhibition of AVP signaling with a V1a antagonist resulted in a reduction in the estradiol-induced LH surge (137), while intracerebroventricular infusion of AVP in SCN-lesioned, ovarectomized, and estradiol-treated rats was able to induce an LH surge (138). Furthermore, in co-cultures of POA and SCN, the GnRH surge was coordinated with the rhythm in AVP, but not VIP, and administration of AVP, not VIP, to preoptic explants in the presence of estradiol significantly increased GnRH release, providing further evidence for an important role of AVP in the LH surge generation (139). Finally, a recent study reported that intracerebroventricular administration of AVP in female Syrian hamsters activates Kp neurons similarly in the early or late part of the day, while in the same animals GnRH neurons are activated only late in the day (87). This observation was further confirmed by *in vitro* electrophysiological recordings of Kp-GFP neurons showing that AVP increases the firing rate of most Kp neurons during proestrus, independently of the time of day (90). Altogether these findings indicate that AVP activates Kp neurons every day, and the daytime gating of the GnRH/LH surge does not take place through SCN AVP–AVPV Kp signaling but rather downstream at the AVPV Kp–POA GnRH signaling.

A significant role of the SCN-derived VIP output in female reproduction should not be excluded (**Figure 1A**). Indeed, VIP afferents on GnRH neurons are sexually dimorphic, with female rats exhibiting higher VIPergic innervation than males (134). Furthermore, central administration of VIP antiserum reduces the LH surge (140), while central infusion of VIP is able to rescue the LH surge in middle-aged female rats (141). Finally, blocking the VPAC2 receptor attenuates GnRH neuronal cell firing during the afternoon surge in female, estradiol-treated mice (142). A recent study reported that the SCN-derived VIP neurons project to RFRP-3 neurons and central administration of VIP markedly suppresses RFRP-3 cellular activity in the evening, but not the morning, therefore indicating a specific role of VIP on neurons expressing RFRP-3, a neuropeptide thought to participate in the circadian-timed removal of estradiol negative feedback (143).

### Other Clocks in the Reproductive System

It has long been thought that the SCN-driven outputs are the sole source forwarding circadian signals to the female reproductive system. However, a growing body of evidence now suggests that structures and organs that are part of the gonadotropic axis might also play an intrinsic role in the timing of female reproduction.

In mammals, the timing system is now described as a multioscillator hierarchy of coordinated and synchronized cell and tissue clocks (144). The use of *Per1-luc* transgenic rats (145) and Per2:LUCIFERASE transgenic mice (115), where the *Per1* or *Per2* promoter drives expression of the *luciferase* gene, was decisive for the demonstration that non-SCN central structures and peripheral organs can sustain endogenous circadian oscillations. Thus, central structures (e.g., olfactory bulb, ARN, and retrochiasmatic area) and peripheral organs (e.g., liver, lung, heart, and kidney) are able to display endogenous-sustained circadian rhythmicity. The phase and the period of these "peripheral" clocks are tissue characteristic and different from those of the "central" clock of the SCN. The strength of their endogenous oscillations is often lesser than that of the SCN since according to tissues (and transgenic mice) the oscillations last from 2 to 20 cycles on average, whereas those of SCN can last several months. The oscillations generated by the peripheral clocks are independent of the SCN activity (they persist in SCN-lesioned animals) but their rhythms appear synchronized by the master clock, which is sometimes referred to as the conductor of the organism's multi-oscillatory network. In the context of such a complex circadian network, recent evidence now suggests that the hypothalamo-pituitary gonadal axis is also a functional multi-oscillatory axis. Indeed, reproductive tissues from hypothalamic Kp and GnRH neurons down to the ovaries and the uterus display endogenous circadian oscillations of clock genes, as explained below. However, the functional role of these reproductive clocks with regards to the timing of reproduction (ovulation, implantation, and parturition) has yet to be determined.

### Kisspeptin Neurons

Daily and circadian activities of Kp neurons in the AVPV area have been investigated in female rodents because of their strategic position between the integration of SCN-derived AVP input on one hand (87, 89) and the triggering of the preovulatory GnRH/ LH surge on the other (19). Additionally, the modulatory effect of circulating estradiol on the daily activity of Kp neurons has also been investigated because of the potent effect of estradiol on Kp synthesis (77). Under high circulating estradiol levels, either in proestrus or in ovariectomized + estradiol-supplemented rodents, Kp neuronal activity (as seen by c-FOS activation) and Kiss1 mRNA are significantly increased about 3 h before lights off, thus 2 h before the LH surge (9, 86–88). Furthermore, we recently reported that Kp immunoreactivity is markedly but transiently decreased at the same time (9). In contrast, in low circulating estradiol conditions, in diestrus or ovariectomized animals, the daily variation in neuronal activity, Kiss1 mRNA and Kp immunoreactivty is abolished or strongly dampened (9, 86, 88). The daily activation of Kp neurons is triggered by the SCN AVP input since central injection of AVP induces c-FOS in Kp neurons and increases Kiss1 mRNA (87). Although AVP is released from the SCN neurons in the afternoon (146), AVP can activate Kp neurons in the morning or in the afternoon, indicating that the daily control of the LH surge is not gated by the AVPV Kp neurons (87). A recent study recording Kp neuron electrical activity confirmed that Kp neuron responsiveness to AVP depends on the concentrations of estradiol (90). Altogether these data indicate that under high circulating estradiol (when oocytes are mature enough to be released), Kp neurons can be activated by the SCN-derived AVP to increase Kp synthesis and release in order to induce GnRH neuronal activation and the downstream LH surge. In addition to the AVP activation of Kp neurons, we recently reported that these neurons host an intrinsic circadian clock, named Kiss-Clock (9). A preliminary study reported that the clock genes *Per1* and *Bmal1* are expressed in the rat AVPV, but their cellular localization was not established (88). We further demonstrated that virtually all AVPV Kp neurons express the PER1 protein (**Figure 1B**) with a daily rhythm both in proestrus and diestrus, but with a phase delay of about 3 h in diestrus as compared to proestrus (9). Furthermore, we reported that isolated Kp-expressing AVPV explants from PER2:LUCIFERASE mice display endogenous circadian oscillations with a period of 23.2 h (thus 1 h shorter than the SCN circadian period of the same mice), confirming the presence of an intrinsic circadian oscillator in AVPV Kp neurons (9) (**Figure 1C**). Remarkably, the period of this circadian clock is increased by 1 h in the presence of estradiol in the culture medium (**Figure 1C**), which is in line with the observed phase difference in PER1 expression according to the estrous stage. In contrast, the period of the SCN clock is not altered by environmental estradiol either *in vivo* or *in vitro* (9). The role of this Kiss-Clock has yet to be established. However, according to previous studies one might hypothesize that it could time sensitivity to estradiol since *ERα* gene expression can be regulated by the BMAL1/CLOCK dimer (147), or it could also impact *Kiss1* gene expression since a circadian transcriptional factor, albumin D-site binding protein (Dbp), was reported to trigger *Kiss1* transcription via the D-box (148).

### GnRH Neurons