Crossbred recipient goats can be selected from those exhibiting estrus 48–72 hours prior to the scheduled embryo transfer. Nuclear transfer embryos are transferred into oviduct's recipients. For the embryo transfer procedure, we used the same general anesthesia protocol as for oocyte collection. Likewise, goats are placed in dorsal recumbence for midventral laparotomy procedure. The ovaries are then examined for evidence of ovulation; thereafter, the uterus is exteriorized, and embryos are transferred into the oviduct (2-day embryos) or the uterus (7 day embryos) ipsilateral to the ovary with the most ovulation points. Embryos (5–10/recipient)

of culture into a microwell (400×). F: Transgenic cloned blastocyst on day 7 of development (600×).

**4. Embryo transfer and pregnancy diagnosis**

cloned transgenic goat embryos [39].

78 Insights from Animal Reproduction

The need for early diagnosis of pregnancy in goats, especially for valuable embryos, as in the case of transgenesis and cloning, lead to the common application of transrectal or abdominal real-time B-mode ultrasonography (US) for pregnancy examination. The distension of the uterus, the evidence of embryonic vesicles or the embryo, the fetal heartbeat, and placentomes are positive signals for pregnancy. Ideal time for abdominal scanning is between 40 and 60 days. Prior to 40–45 days, the transducer may have to be placed higher in the inguinal region, but if transrectal US is used the diagnosis is anticipated up to 25–30 days. In our studies, transrectal US examinations of recipients begin on day 25 of gestation, whereas additional US assessments are performed at intervals of 7 and 10 days until unequivocal pregnancy diagno‐ sis. The fetal heartbeat is normally used to diagnose pregnancy and fetus viability. Goats with pregnancy confirmed are monitored at 2-week intervals until day 90, when they are separated from the others and placed in groups (two or three) per pen. Monitoring of pregnant recipients past day 45 is performed through abdominal US (Figure 7).

**Figure 7.** Pregnancy diagnosis in recipient goats. A: Real-time ultrasonography (US) equipment and its probes for transrectal and abdominal examination. Pregnant recipients at 25 (B) and 45 days (C) after embryo transfer.

Real-time US is also currently used for other kind of assessments, such as to verify and monitor the ovarian follicular dynamics, the time of ovulation, and to accurately estimate the number of corpora lutea at days 6–7 after induced superovulation in goats. Nevertheless, studies focusing the superovulation response in goats, particularly, to verify early ovulatory response (*corpora haemorraghica* (CH)), are very limited, and the accuracy of the method with this objective has not been assessed. Thus, our group proposed to assess the efficiency of real-time US as a noninvasive method to estimate the ovulation ratio after superovulation in goats by counting the number of CH's in the ovaries. The sensitivity, specificity, and total efficiency of the ultrasonographic estimation of ovulations in a goat were 100.0% [40]. Thus, transrectal realtime US is a valuable method for assessing the ovulatory response and can be used to select donor goats in a transgenesis program or to estimate embryo yield after superovulation.

#### **5. Conclusions**

Research in producing transgenic animals currently uses goats for its lower cost and precise genomic alterations. This statement is reinforced by the approval and the commercialization of the first human biological drug from the milk of transgenic goats.

Nuclear transfer protocols have evolved, which make it possible to obtain transgenic goats efficiently. Also, it seems that goats generated by SCNT do not suffer from the health problems reported in sheep and cattle. Moreover, the HMC technique does not require the use of micromanipulators and accessories. Also, the HMC procedure does not request micromani‐ pulators or related equipment, making the method accessible to laboratories with limited funding.

#### **Acknowledgements**

Technical information in this chapter was derived from experiments funded by the following Brazilian funding agencies: CNPq (Brasília), CAPES (Brasília), and FUNCAP (Fortaleza) for providing the conditions for the experiments. The authors would like to thank the staff of the Laboratory of Physiology and Control of Reproduction for the technical assistance and animal care.

#### **Author details**

Vicente J. F. Freitas1\*, Luciana M. Melo1 , Dárcio I.A. Teixeira1 , Maajid H. Bhat1 , Irina A. Serova2 , Lyudmila E. Andreeva3 and Oleg L. Serov2

\*Address all correspondence to<EMAIL_ADDRESS>
1 Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará, Fortaleza, Brazil

2 Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk, Russia

3 Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia

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the ultrasonographic estimation of ovulations in a goat were 100.0% [40]. Thus, transrectal realtime US is a valuable method for assessing the ovulatory response and can be used to select donor goats in a transgenesis program or to estimate embryo yield after superovulation.

Research in producing transgenic animals currently uses goats for its lower cost and precise genomic alterations. This statement is reinforced by the approval and the commercialization

Nuclear transfer protocols have evolved, which make it possible to obtain transgenic goats efficiently. Also, it seems that goats generated by SCNT do not suffer from the health problems reported in sheep and cattle. Moreover, the HMC technique does not require the use of micromanipulators and accessories. Also, the HMC procedure does not request micromani‐ pulators or related equipment, making the method accessible to laboratories with limited

Technical information in this chapter was derived from experiments funded by the following Brazilian funding agencies: CNPq (Brasília), CAPES (Brasília), and FUNCAP (Fortaleza) for providing the conditions for the experiments. The authors would like to thank the staff of the Laboratory of Physiology and Control of Reproduction for the technical assistance and animal

, Dárcio I.A. Teixeira1

1 Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State

2 Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk, Russia

3 Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia

, Maajid H. Bhat1

, Irina A. Serova2

,

of the first human biological drug from the milk of transgenic goats.

**5. Conclusions**

80 Insights from Animal Reproduction

funding.

care.

**Author details**

Lyudmila E. Andreeva3

Vicente J. F. Freitas1\*, Luciana M. Melo1

University of Ceará, Fortaleza, Brazil

and Oleg L. Serov2

\*Address all correspondence to<EMAIL_ADDRESS>
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[20] Freitas VJF, Melo LM, Batista RITP, Souza-Fabjan JMG, Teixeira DIA, Serova IA, An‐ dreeva LE, Burkov IA, Serov OL. Goats (*Capra hircus*) as bioreactors for production of recombinant proteins interesting to pharmaceutical industry. Clon Transgen.

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[40] Pereira AF, Feltrin C, Almeida KC, Carneiro IS, Avelar SRG, Alcântara Neto AS, Sou‐ sa FC, Melo CHS, Moura RR, Teixeira DIA, Bertolini LR, Freitas VJF, Bertolini M. Analysis of factors contributing to the efficiency of the in vitro production of trans‐ genic goat embryos (*Capra hircus*) by handmade cloning (HMC). Small Rum Res.

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84 Insights from Animal Reproduction

### **Major Components in Limiting Litter Size**

#### María-José Argente

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60829

#### **Abstract**

The litter size is an important trait in prolific species such as rabbits and pigs. However, selection on litter size has had limited success in these species because of its low heritabil‐ ity and sex-limited expression. The litter size is a complex physiological trait in prolific species, affected by several components that are expressed sequentially, for example, ov‐ ulation, fertilization, embryo development, and fetal survival. The selection for ovulation rate or/and prenatal survival has been proposed to improve litter size indirectly. Howev‐ er, these alternative methods have not reached the expected response rate. Implantation is also a critical point in successful gestation, one-third to one-half of prenatal mortality occurring during peri-implantation. The uterus must provide an adequate microenviro‐ ment for the growth and development of embryo and for receptivity to implantation. There are multitudes of cellular events involved in crosstalk between embryo and mater‐ nal uterus during peri-implantation. A better understanding of molecular mechanisms af‐ fecting the implantation process could help to propose new strategies for litter size improvement in prolific species.

**Keywords:** Ovulation rate, embryonic survival, litter size, candidate gene, quantitative trait loci (QTL)

#### **1. Introduction**

Litter size is a complex physiological trait in prolific species, being affected by several com‐ ponent traits representing sequential events, e.g., ovulation, fertilization, embryo develop‐ ment, and fetal survival. Fertilization rate is usually high, exceeding 90 to 95% in rabbit [1], pigs [2], and mice [3]. Therefore, prenatal survival is considered a limiting factor of litter size. Uterine capacity is an important component in prenatal survival [4; for a review address to 5]. Whenever the ovulation is not a limiting factor, this trait depicts the ability of the uterus to support embryo development through gestation [6]. Approximately 30 to 40% of ova shed do not result in fetuses at term in prolific species such as rabbits, pigs, and mice (see review [7])

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

due to potentially viable embryos exceeds uterine capacity. One-third to one-half of these losses occurs during peri-implantation in rabbits [8], pigs [9], and mice [10]. Preimplantation embryo losses are mainly associated with embryonic viability [4], including chromosomal abnormalities [11] and oviductal or uterine environment, particularly in relation with the suitability of oviductal or uterine secretions [12]. The oviduct must provide an adequate milieu for sperm capacitation, gamete fertilization, and the first embryo cleavages until the embryo enters the uterus [13]. Many proteins that may contribute to these functions have been identified in the oviduct, including the insulin-like growth factor 1 (IGF1) [14], oviductin (OVGP1, known also as MUC9) [15], tissue inhibitor of metalloproteinase (TIMP1) [16], plasminogen activator inhibitor 1 (PAI1) [17], uteroglobin [18], and leptin [19]. A progesteroneprimed uterus coordinates the embryo survival and receptivity. The asynchrony between embryo development and uterine environment increases the number of dead embryos in the peri-implantation gestational stage [20, 21].

In rabbits, the period between pregnancy days 8 to 17 is critical for fetal survival, corresponding to the stage when the hemochorial placenta of rabbit is established and the control of the fetal nutrition is transferred to the placenta [22]. A secondary critical period for fetal survival occurs between pregnancy days 17 and 24, accompanying the uterine enlargement, the increased tension exerted on the spherical conceptus, and the reduction of blood flowing through the maternal vessels of the uterus [23]. In this moment, the placenta should compensate these limiting factors and increase its surface area for fetomaternal exchanges [24] and stimulate the development of an adequate vascular network [25, 26]. In pigs, the distribution of mortality after implantation is slightly different throughout pregnancy [9], the first peak occurring between days 30 and 40 of gestation (10-15%) while the second peak is observed in the last two weeks of gestation (5-10%). Despite the existing species differences, it is now accepted that each embryo requires a minimum space in uterus to attach, survive, and develop. Thus, a decrease in the availability of uterine space increases prenatal mortality in pluriparous species, such as rabbits [4] or pigs [27], despite that the factors involved in the process may differ among species. In pigs, the area of attachment between the placenta and endometrium is a limiting factor of uterine capacity, due to its noninvasive placentation [9]. Thus, it was proposed that uterine capacity in pigs could be defined more correctly as the total amount of placental mass or surface area that a dam can support to term [28]. Earlier studies in pigs indicate that the limiting influence of the uterine capacity is generally exerted after the day 30 of gestation and that the effects of moderate crowding of embryos before day 30 could be compensated by increased placental efficiency later in gestation (see review [29]).

#### **2. Selection experiments for components of litter size**

Litter size is an important economic trait in prolific species such as rabbits or pigs. However, direct selection for litter size has not presented the success expected for these species (see review [7, 30]), which may be due to the fact that it is a female sex-limited trait with low heritability. The selection response for litter size has been established around 0.1 young per generation in rabbits [31, 32] and pigs [33-36]. This response has been much lower than that reported in mice: 0.15 to 0.20 young per generation [37-40]. Recently, in pigs, a selection experiment for litter size at day 5 after farrowing obtained a selection response around to 0.25 young per generation [41]. However, additional information on following generations will be required to confirm this trend.

due to potentially viable embryos exceeds uterine capacity. One-third to one-half of these losses occurs during peri-implantation in rabbits [8], pigs [9], and mice [10]. Preimplantation embryo losses are mainly associated with embryonic viability [4], including chromosomal abnormalities [11] and oviductal or uterine environment, particularly in relation with the suitability of oviductal or uterine secretions [12]. The oviduct must provide an adequate milieu for sperm capacitation, gamete fertilization, and the first embryo cleavages until the embryo enters the uterus [13]. Many proteins that may contribute to these functions have been identified in the oviduct, including the insulin-like growth factor 1 (IGF1) [14], oviductin (OVGP1, known also as MUC9) [15], tissue inhibitor of metalloproteinase (TIMP1) [16], plasminogen activator inhibitor 1 (PAI1) [17], uteroglobin [18], and leptin [19]. A progesteroneprimed uterus coordinates the embryo survival and receptivity. The asynchrony between embryo development and uterine environment increases the number of dead embryos in the

In rabbits, the period between pregnancy days 8 to 17 is critical for fetal survival, corresponding to the stage when the hemochorial placenta of rabbit is established and the control of the fetal nutrition is transferred to the placenta [22]. A secondary critical period for fetal survival occurs between pregnancy days 17 and 24, accompanying the uterine enlargement, the increased tension exerted on the spherical conceptus, and the reduction of blood flowing through the maternal vessels of the uterus [23]. In this moment, the placenta should compensate these limiting factors and increase its surface area for fetomaternal exchanges [24] and stimulate the development of an adequate vascular network [25, 26]. In pigs, the distribution of mortality after implantation is slightly different throughout pregnancy [9], the first peak occurring between days 30 and 40 of gestation (10-15%) while the second peak is observed in the last two weeks of gestation (5-10%). Despite the existing species differences, it is now accepted that each embryo requires a minimum space in uterus to attach, survive, and develop. Thus, a decrease in the availability of uterine space increases prenatal mortality in pluriparous species, such as rabbits [4] or pigs [27], despite that the factors involved in the process may differ among species. In pigs, the area of attachment between the placenta and endometrium is a limiting factor of uterine capacity, due to its noninvasive placentation [9]. Thus, it was proposed that uterine capacity in pigs could be defined more correctly as the total amount of placental mass or surface area that a dam can support to term [28]. Earlier studies in pigs indicate that the limiting influence of the uterine capacity is generally exerted after the day 30 of gestation and that the effects of moderate crowding of embryos before day 30 could be compensated by

peri-implantation gestational stage [20, 21].

88 Insights from Animal Reproduction

increased placental efficiency later in gestation (see review [29]).

**2. Selection experiments for components of litter size**

Litter size is an important economic trait in prolific species such as rabbits or pigs. However, direct selection for litter size has not presented the success expected for these species (see review [7, 30]), which may be due to the fact that it is a female sex-limited trait with low heritability. The selection response for litter size has been established around 0.1 young per generation in rabbits [31, 32] and pigs [33-36]. This response has been much lower than that The leading components of litter size are the ovulation rate and the prenatal survival; these parameters are also the limiting factors for the litter size improvement. For this reason, selection for ovulation rate and prenatal survival has been proposed as an indirect approach for increasing litter size. Selection for ovulation rate in prolific species, namely rabbits [42], pigs [43-45], and mice [40, 46], were in fact successful to improve the ovulation rate, but it was not convoyed by a corresponding increase in litter size in either pigs or rabbits, which was attributed to an increase in prenatal loss.

The selection for prenatal survival in pigs [45] and mice [40] allowed to increase litter size in both pigs and mice, but it was not more advantageous compared with the direct selection for litter size. Besides, prenatal survival might be limited by uterine capacity, defined as the maximum number of fetuses that a dam can support at birth when ovulation rate is not a limiting factor [6]. However, the ability to establish this trait is not easily performed across species, as it is dependent on the species physiology. In pigs, litter size in unilaterally ovariohysterectomized females may be considered an indirect measure of the uterine capacity in pigs [6], since the remaining ovary nearly doubled its ovulation rate originating the fetal over‐ crowding in the ipsilateral uterine horn. Conversely, in unilaterally ovariectomized female rabbits and mice, which possess a duplex uterus that impedes intercornual transmigration, the number of total fetuses would represent their uterine capacity [47, 48].

Selection experiments for increased uterine capacity failed to obtain the expected success on litter size, in rabbits [8, 49], pigs [44], or mice [50]. Considering that uterine capacity in pigs would be more appropriately measured using the total amount of placental mass or the surface area that a dam can support to term as variables [28], a divergent selection experiment for placental efficiency developed in this species achieved success [51]. However, selection for increased placental efficiency will unlikely result in correlated increase in litter size [52].

A joint selection for ovulation rate and prenatal survival using an index would expectably show a greater response on litter size, since these parameters are optimally weighted [53, 54]. In pigs [55] and mice [56], the use of this joint selection successfully increased litter size, but the gain was lower than expected, probably due to a low precision of the estimated genetic correlations or the use of inappropriate economic weights [57]. Alternatively, a two-stage selection was proposed, which would be less affected by the precision of the genetic parame‐ ters; its application to rabbits [58] and pigs [59] obtained greater response on litter size than the observed in the other experiments of selection for litter size [31-36].

Selection for the environmental variability in litter size has been recently proposed as an alternative method for increased litter size. A reduction in litter size environmental variability would increase litter size heritability, and consequently its response to selection [60]. In rabbits, the environmental variance of a doe was estimated as within-doe variance of litter size. Selection for environmental variance of litter size was successful [61], and as a consequence of reducing litter size variability the litter size was increased due to higher embryo survival [61].

#### **3. Genetic control before implantation**

The maternal genome controls virtually all aspects of early embryo development, through several maternal gene products such as mRNA and protein, which are loaded into the egg during oogenesis. As development proceeds, two processes subsequently lead to the maternalto-zygotic transition (MZT) during which developmental control is transferred to the zygotic genome: first, a subset of the maternal mRNAs is degraded; second, the embryonic genome is transcriptionally activated. These maternal gene products play an important role in the regulation of the first cleavages until embryonic genome is activated [62]. Zygotic genome activation (ZGA) is a critical event determining the transition from maternal to embryonic control of development. Disruption of these critical events by specific chemicals or environ‐ mental factors results in irreversible arrest of embryo development [63]. ZGA has been shown to be a species-specific phenomenon, occurring at 2-cell stage in mice [64], 4-cell stage in pigs [65], and 8- to 16- cell stages in sheep, cows, or rabbits [66]. Many maternal-effect genes have been identified initially in mouse during the MZT, and several of whom have been detected posteriorly in rabbits and pigs.

Genome-wide gene activation in the zygote (ZGA) is regarded as crucial for preimplantation embryonic development. Multiple maternal factors were identified on the regulation of ZGA, which are listed in Table 1. Ablation of the gene encoding for these proteins results in embry‐ onic arrest at cleavage-stage development.

These factors play critical roles in the regulation of embryo preimplantation development. For example, DICER1 enzyme is required for completion of oocyte mitotic maturation, and oocytes are arrested in metaphase of meiosis II when DICER1 gene is deleted [67, 68]. HSF1 protein is required for oocyte maturation. Embryos produced in knockout females for this gene are unable to proceed into the 2-cell stage after fertilization, possibly due to mitochondrial damage and altered redox homeostasis [69, 70]. UCHL1 is an import factor in blocking polyspermy [71, 72]. AGO2 is involved in the destruction of maternally inherited transcripts and activation of zygotic gene expression; knockout female for this factor is infertile because embryos fail to undergo the first cleavage [73, 74]. Moreover, embryos from MATER, ZAR1, PADI6, and SEBOX knockout females do not develop beyond the 2-cell stage embryo [75-82], and embryos lacking SMARCA4, DNMT1, DNMT3A, TET, and KLF4 are unable to reach the 8-cell stage [83-91]. BCLXL, HDAC1, and C-MYC exhibit maximum expression in 8-cell rabbit embryos coinciding with start of ZGA [90]. Hence the peak expression of transcripts at ZGA might be a requirement for embryo development. OCT4, NANOG, and SOX2 were co-expressed in epiblasts, and the combinatorial expression of these three genes is critical for the embryo development [92, 93].

**Table 1.** Maternal genes acting on early fetal embryogenesis.

Selection for environmental variance of litter size was successful [61], and as a consequence of reducing litter size variability the litter size was increased due to higher embryo survival [61].

The maternal genome controls virtually all aspects of early embryo development, through several maternal gene products such as mRNA and protein, which are loaded into the egg during oogenesis. As development proceeds, two processes subsequently lead to the maternalto-zygotic transition (MZT) during which developmental control is transferred to the zygotic genome: first, a subset of the maternal mRNAs is degraded; second, the embryonic genome is transcriptionally activated. These maternal gene products play an important role in the regulation of the first cleavages until embryonic genome is activated [62]. Zygotic genome activation (ZGA) is a critical event determining the transition from maternal to embryonic control of development. Disruption of these critical events by specific chemicals or environ‐ mental factors results in irreversible arrest of embryo development [63]. ZGA has been shown to be a species-specific phenomenon, occurring at 2-cell stage in mice [64], 4-cell stage in pigs [65], and 8- to 16- cell stages in sheep, cows, or rabbits [66]. Many maternal-effect genes have been identified initially in mouse during the MZT, and several of whom have been detected

Genome-wide gene activation in the zygote (ZGA) is regarded as crucial for preimplantation embryonic development. Multiple maternal factors were identified on the regulation of ZGA, which are listed in Table 1. Ablation of the gene encoding for these proteins results in embry‐

These factors play critical roles in the regulation of embryo preimplantation development. For example, DICER1 enzyme is required for completion of oocyte mitotic maturation, and oocytes are arrested in metaphase of meiosis II when DICER1 gene is deleted [67, 68]. HSF1 protein is required for oocyte maturation. Embryos produced in knockout females for this gene are unable to proceed into the 2-cell stage after fertilization, possibly due to mitochondrial damage and altered redox homeostasis [69, 70]. UCHL1 is an import factor in blocking polyspermy [71, 72]. AGO2 is involved in the destruction of maternally inherited transcripts and activation of zygotic gene expression; knockout female for this factor is infertile because embryos fail to undergo the first cleavage [73, 74]. Moreover, embryos from MATER, ZAR1, PADI6, and SEBOX knockout females do not develop beyond the 2-cell stage embryo [75-82], and embryos lacking SMARCA4, DNMT1, DNMT3A, TET, and KLF4 are unable to reach the 8-cell stage [83-91]. BCLXL, HDAC1, and C-MYC exhibit maximum expression in 8-cell rabbit embryos coinciding with start of ZGA [90]. Hence the peak expression of transcripts at ZGA might be a requirement for embryo development. OCT4, NANOG, and SOX2 were co-expressed in epiblasts, and the combinatorial expression of these three genes is critical for the embryo

**3. Genetic control before implantation**

90 Insights from Animal Reproduction

posteriorly in rabbits and pigs.

development [92, 93].

onic arrest at cleavage-stage development.

#### **4. Genetic control during implantation**

Implantation requires a complex interaction among the developing embryo, decidualizing endometrium and developing maternal immune tolerance. For the successful implantation, it is of upmost importance the synchronization between the acquisition of implantation compe‐ tency by the blastocyst and a receptive state in the uterine endometrium, for which the concurrence of the ovarian steroid hormones dynamics is crucial. The ovarian hormones determine a complex interplay of locally produced molecules in the endometrium, including cytokines, growth factors, homeobox transcription factors, lipid mediators, and morphogen genes that are involved in the complex process of implantation. The crosstalk between the blastocyst and the uterus is limited in most mammal species for a brief period, named as *window of implantation*. During this short period, in response to a viable embryo, the endometrium responds through species-specific transformation of the superficial tissue architecture, a process known as decidualization, allowing the organ to accommodate embryonic growth and placentation. The decidua will function like a barrier, protecting the embryo against the maternal immune system [see review 94].

#### *Steroid hormones*

The progesterone receptor (PR) was identified as one of the molecules that genetic polymor‐ phisms were associated with the risk of implantation failure. The PR is encoded by *PGR* gene, and has two isoforms, PRA and PRB, both of them are expressed in the uterus [94]. Studies in mice showed that a deletion on PRA provokes severe abnormalities in ovarian and uterine function and impairs implantation [94]. A study in rabbits reported that favorable allele of PRG had an additive effect of 0.25 for implanted embryos and kits for litter size [95].

Estrogen receptor (ER) was also implied in uterine receptivity for embryo implantation. ER presents two isoforms, known as ERα (encoded by *ESR1* gene) and ERβ (encoded by *ESR2* gene). In mice, knocking out the ERα gene leads to unsuccessful implantation [94]. In pigs, favorable allele of ESR shows an additive effect between 0.45 and 0.75 piglets for litter size [96, 97]. In another hand, it has been shown that the ERBB receptor feedback inhibitor 1 (*ERRFI1*) gene is involved with successful implantation, which was associated with its suppression of *ESR1* activity in the uterine epithelium, a crucial event for embryo implantation. Despite that the ovaries of ERRFI1 knockout female mice show a normal morphology and steroidogenesis function, its uterine horns do not develop an implantation site [98]. The steroid receptor coactivators (SRC1 and SRC2) present distinct physiological functions in the female repro‐ ductive system. For example, female mice lacking either *SCR1* or *SCR2* show progesterone resistance and compromised decidualization, whereas deletion of both *SCR1* and *SCR2* genes provoke infertility in female due to a complete blockage of decidualization [99], suggesting that one of the steroid receptor coactivators may, at least in part, compensate the absence of the other. Also the blockage of the repressor of estrogen receptor activity (REA), a significant modulator of estrogen responsiveness, was reported to induce implantation failures [100].

Moreover, prostaglandins (PGs) play an important role in various reproductive processes, including ovulation and implantation [101]. Cyclo-oxygenases (COX-1 and COX-2) are crucial enzymes in the synthesis of various PGs. Females lacking COX-1 and COX-2 are infertile, due to abnormalities in ovulation, fertilization, implantation, and decidualization [102]. A study in pigs showed that favorable allele of COX-2 had an additive effect of 0.3 piglets for litter size [103].

#### *Cytokines*

During the embryo implantation, the endometrium undergoes a dramatic transformation into a specialized transitory tissue known as the decidua in species with invasive hemochorial placenta, such as rodents and lagomorphs. In other species with no invasive placenta as pigs, the changes in endometrium ought to allow the trophoblast and its supporting layer of extraembryonic mesoderm to contact successfully with the uterine epithelium. The placenta surrounding the developing embryo facilitates the nutrient transfer and limits trophoblast invasion. The endometrium is recognized as an important site of production of cytokines and their receptors, which are also potential regulators of the phenotype and activation status of the uterine-resident leukocytes. Leukocytes infiltrated in the endometrium are required for the immunotolerance pathways that allow the maternal organism to accommodate the conceptus during implantation and placental development. Several cytokines, such as the macrophage colony-stimulating factor 1 (CSF1, also known as M-CSF), granulocyte colonystimulating factor (CSF3, also known as G-CSF), and granulocyte-macrophage colonystimulating factor (CSF2, also known as GM-CSF), are implicated in the recruitment and phenotypic regulation of the abundant populations of endometrial macrophages, granulo‐ cytes, and dendritic cells. These cytokines appear to be related to the successful embryo implantation and placental growth [104]. Also leukemia inhibitory factor (LIF), interleukin-6 (IL-6) and interleukin-11 (IL-11), and its receptor (IL-11Ra) have unquestionable roles in the implantation process [105-107]. Studies in pig have reported an association between certain polymorphisms in LIF gene with the number of piglets [108-111].

#### *Growth factors*

cytokines, growth factors, homeobox transcription factors, lipid mediators, and morphogen genes that are involved in the complex process of implantation. The crosstalk between the blastocyst and the uterus is limited in most mammal species for a brief period, named as *window of implantation*. During this short period, in response to a viable embryo, the endometrium responds through species-specific transformation of the superficial tissue architecture, a process known as decidualization, allowing the organ to accommodate embryonic growth and placentation. The decidua will function like a barrier, protecting the embryo against the

The progesterone receptor (PR) was identified as one of the molecules that genetic polymor‐ phisms were associated with the risk of implantation failure. The PR is encoded by *PGR* gene, and has two isoforms, PRA and PRB, both of them are expressed in the uterus [94]. Studies in mice showed that a deletion on PRA provokes severe abnormalities in ovarian and uterine function and impairs implantation [94]. A study in rabbits reported that favorable allele of

Estrogen receptor (ER) was also implied in uterine receptivity for embryo implantation. ER presents two isoforms, known as ERα (encoded by *ESR1* gene) and ERβ (encoded by *ESR2* gene). In mice, knocking out the ERα gene leads to unsuccessful implantation [94]. In pigs, favorable allele of ESR shows an additive effect between 0.45 and 0.75 piglets for litter size [96, 97]. In another hand, it has been shown that the ERBB receptor feedback inhibitor 1 (*ERRFI1*) gene is involved with successful implantation, which was associated with its suppression of *ESR1* activity in the uterine epithelium, a crucial event for embryo implantation. Despite that the ovaries of ERRFI1 knockout female mice show a normal morphology and steroidogenesis function, its uterine horns do not develop an implantation site [98]. The steroid receptor coactivators (SRC1 and SRC2) present distinct physiological functions in the female repro‐ ductive system. For example, female mice lacking either *SCR1* or *SCR2* show progesterone resistance and compromised decidualization, whereas deletion of both *SCR1* and *SCR2* genes provoke infertility in female due to a complete blockage of decidualization [99], suggesting that one of the steroid receptor coactivators may, at least in part, compensate the absence of the other. Also the blockage of the repressor of estrogen receptor activity (REA), a significant modulator of estrogen responsiveness, was reported to induce implantation failures [100]. Moreover, prostaglandins (PGs) play an important role in various reproductive processes, including ovulation and implantation [101]. Cyclo-oxygenases (COX-1 and COX-2) are crucial enzymes in the synthesis of various PGs. Females lacking COX-1 and COX-2 are infertile, due to abnormalities in ovulation, fertilization, implantation, and decidualization [102]. A study in pigs showed that favorable allele of COX-2 had an additive effect of 0.3 piglets for

During the embryo implantation, the endometrium undergoes a dramatic transformation into a specialized transitory tissue known as the decidua in species with invasive hemochorial placenta, such as rodents and lagomorphs. In other species with no invasive placenta as pigs, the changes in endometrium ought to allow the trophoblast and its supporting layer of

PRG had an additive effect of 0.25 for implanted embryos and kits for litter size [95].

maternal immune system [see review 94].

*Steroid hormones*

92 Insights from Animal Reproduction

litter size [103].

*Cytokines*

The epidermal growth factor (EGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), the vascular endothelial growth factors (VEGFs), the IGF-I and IGF-II as well as the IGF-binding protein-1 (IGFBP-1) are important factors for implantation. It was shown that EGF plays a critical role in trophoblast invasion, differentiation, and proliferation. EGF deficiency during pregnancy causes intrauterine growth retardation or abortion [112], while the deletion of epidermal growth factor receptor (*EGFR* or *ERB1)* gene causes failure in the embryo development and the placenta formation [113]. Likewise, other factors involved in the implantation process, such as HB-EGF, are expressed in endometrial stromal and epithelial cells. It has been demonstrated that HB-EGF regulates endometrial cell proliferation, glandular epithelial secretion, and decidual transformation [113]. Gene knockout studies reveal that deletion of *HBEGF* reduces litter size [114].

VEGF is an endothelial-cell specific mitogen *in vitro*, and it is the main factor responsible for de novo blood vessel formation (vasculogenesis) and angiogenesis in vivo [115]. Proper level of VEGF expression is required for implantation [116]. Many critical cell responses, including mitogenesis, proliferation, growth, differentiation, and angiogenesis, are mediated by IGF-I and IGF-II [117]. Both IGF-I and IGF-II are necessary to maintain normal embryonic growth rates [117]. In addition, higher expression of *IGF1* mRNA has been observed during the periimplantation period in mouse uterus [118]. Also in rabbit, it has been observed that IGF-II receptor plays an important role in embryo development and its implantation [119].

#### *Transcription factors*

*Homeobox* genes are transcriptional regulators evolutionarily conserved that control embryonic morphogenesis and differentiation [120]. *Homeobox A* (*HOXA10* and *HOXA11*) and *H6 homeobox 3* (*HMX3*) genes are expressed in uterine stromal cells during the receptivity period, and upregulated upon decidualization in response to steroid hormone stimulus. Ablation of *HOXA10*, *HOXA11*, or *HMX3* genes leads to implantation defects [121-123]. *MSX1* (also known as *HOX7.1*) and *MSX2* also belong to *homeobox* genes, and deletion of both genes results in female infertility due to altered uterine polarity and integrity of the surface epithelium [124]. The Kruppel-like transcription factors (*KLFs*) are implicated in diverse cellular processes, including proliferation, differentiation, and apoptosis. The Kruppel-like factor 5 (*KLF5*), one of these transcription factors, is essential for the establishment of uterine receptivity [125], and its depletion induces implantation failure [126]. *KLF9* is another KLF that plays an important role in blastocyst attachment; its loss reduces female fertility due to defective implantation [127]. The transcription factor named heart and neural crest derivatives-expressed transcript 2 (*HAND2*) and its protein are present in endometrial stromal cells adjacent to the surface epithelium in the uterus prior to the onset of implantation [128], suggesting that they may play a key role in uterine receptivity in mice [129]. Forkhead box protein A2 (FOXA2) is only expressed in the glandular epithelium of the uterus, and FOXA2 deficiency affect the endo‐ metrial gland formation and decidualization [130]. Deletion of chicken ovalbumin upstream promoter transcription factor II (*COUP-TFII*), which is mainly expressed in uterine stromal cells, originates implantation failure due to disrupted uterine receptivity associated with high estrogen activity [131].

#### *Morphogen genes, lipid mediatiors, integrins, mucins, and others molecules*

Proteins belonging to the transforming growth factor beta superfamily (TGF-β), Wingless (WNT), Hedgehog, and Notch have been identified as morphogens. Morphogens act directly on angiogenesis, cell growth, pattern formation, embryo development, metabolic regulation, cell migration, and tissue repair, while also presenting neurotropic effects. Five TGF-βs have been identified, of which TGF-β1, β2, β3 are abundant in mammals. However, only TFG-β1 appears to limit the number of implanted embryos [132]. Activins are also members of the TGF-β superfamily that participate in the regulation of several biological processes, including cell differentiation and proliferation, apoptosis, and the immune response [133]. Activin A plays an important role in the implantation of embryos in rabbits and mice, promoting decidualization and preventing the activation of T cells [134]. Among all bone morphogenetic proteins expressed in the uterus, only BMP2 shows intense expression in the stromal cells surrounding the implanted embryo under response to progesterone [135]. *In vitro* studies in undifferentiated stromal cells demonstrated that silencing the expression of BMP2 efficiently blocks the decidualization [136]. Deletion of *NODAL* gene was accompanied by severe malformation of the maternal decidua basalis during placentation and increasing fetal losses before birth [137]. Several components of WNTs signaling pathway are spatiotemporally regulated in the peri-implantation uterus and are crucial to implantation. The absence of WNT4, WNT5a, and WNT7a in the uterus induces defective embryo implantation and subsequent decidualization failure [138-140], while deletion of *WNT7b* gene provokes fetal losses during mid-gestation, due to failure of chorioallantoic fusion [141]. Indian hedgehog (IHH) is a member of the Hedgehog family. It has been reported that conditional deletion IHH protein in the uterus results in implantation failure [142]. Activation of smoothened (SOM), another member of the Hedgehog family, provokes hypertrophy in uterus along and conse‐ quently failure to decidual response [143]. The *NOTCH1* is responsible for cell survival, cellto-cell communication, differentiation, and all fundamental processes for successful decidualization [144].

The blastocyst has a significant number of cannabinoid receptors (CB1) that are activated by the anandamide (AEA) produced in the uterus. It has been found that the levels of AEA are lower in the receptive uterus and at implantation sites than in the non-receptive uterus or at inter-implantation sites. These findings suggest the need for low AEA levels to activate uterine receptivity [145].

The integrin family of cell adhesion molecules is a major class of receptors for the extracellular matrix. They have many functions in cellular processes including differentiation, apoptosis, and attachment [146]. Previous studies have demonstrated that integrins exhibit distinctive expression patterns in different phases of uterine receptivity. Both α4β1 and αvβ3 integrins are present in uterus at the time of implantation, and intrauterine inhibition of these two molecules results in defective implantation [147, 148].

**Table 2.** Genes considered as critical to implantation.

female infertility due to altered uterine polarity and integrity of the surface epithelium [124]. The Kruppel-like transcription factors (*KLFs*) are implicated in diverse cellular processes, including proliferation, differentiation, and apoptosis. The Kruppel-like factor 5 (*KLF5*), one of these transcription factors, is essential for the establishment of uterine receptivity [125], and its depletion induces implantation failure [126]. *KLF9* is another KLF that plays an important role in blastocyst attachment; its loss reduces female fertility due to defective implantation [127]. The transcription factor named heart and neural crest derivatives-expressed transcript 2 (*HAND2*) and its protein are present in endometrial stromal cells adjacent to the surface epithelium in the uterus prior to the onset of implantation [128], suggesting that they may play a key role in uterine receptivity in mice [129]. Forkhead box protein A2 (FOXA2) is only expressed in the glandular epithelium of the uterus, and FOXA2 deficiency affect the endo‐ metrial gland formation and decidualization [130]. Deletion of chicken ovalbumin upstream promoter transcription factor II (*COUP-TFII*), which is mainly expressed in uterine stromal cells, originates implantation failure due to disrupted uterine receptivity associated with high

Proteins belonging to the transforming growth factor beta superfamily (TGF-β), Wingless (WNT), Hedgehog, and Notch have been identified as morphogens. Morphogens act directly on angiogenesis, cell growth, pattern formation, embryo development, metabolic regulation, cell migration, and tissue repair, while also presenting neurotropic effects. Five TGF-βs have been identified, of which TGF-β1, β2, β3 are abundant in mammals. However, only TFG-β1 appears to limit the number of implanted embryos [132]. Activins are also members of the TGF-β superfamily that participate in the regulation of several biological processes, including cell differentiation and proliferation, apoptosis, and the immune response [133]. Activin A plays an important role in the implantation of embryos in rabbits and mice, promoting decidualization and preventing the activation of T cells [134]. Among all bone morphogenetic proteins expressed in the uterus, only BMP2 shows intense expression in the stromal cells surrounding the implanted embryo under response to progesterone [135]. *In vitro* studies in undifferentiated stromal cells demonstrated that silencing the expression of BMP2 efficiently blocks the decidualization [136]. Deletion of *NODAL* gene was accompanied by severe malformation of the maternal decidua basalis during placentation and increasing fetal losses before birth [137]. Several components of WNTs signaling pathway are spatiotemporally regulated in the peri-implantation uterus and are crucial to implantation. The absence of WNT4, WNT5a, and WNT7a in the uterus induces defective embryo implantation and subsequent decidualization failure [138-140], while deletion of *WNT7b* gene provokes fetal losses during mid-gestation, due to failure of chorioallantoic fusion [141]. Indian hedgehog (IHH) is a member of the Hedgehog family. It has been reported that conditional deletion IHH protein in the uterus results in implantation failure [142]. Activation of smoothened (SOM), another member of the Hedgehog family, provokes hypertrophy in uterus along and conse‐ quently failure to decidual response [143]. The *NOTCH1* is responsible for cell survival, cellto-cell communication, differentiation, and all fundamental processes for successful

The blastocyst has a significant number of cannabinoid receptors (CB1) that are activated by the anandamide (AEA) produced in the uterus. It has been found that the levels of AEA are

*Morphogen genes, lipid mediatiors, integrins, mucins, and others molecules*

estrogen activity [131].

94 Insights from Animal Reproduction

decidualization [144].

Mucins also participate in the decidualization process. For example, mucin (MUC1) has been identified as an effective barrier that prevents embryo attachment to the uterine epithelium. Uterine MUC1 expression declines to undetectable levels prior to blastocyst attachment, reinforcing the impression that loss of MUC1 contributes to the establishment of a receptive uterus [149-151]. Other oviductal glycoprotein, as OVGP1 or MUC9, seems to affect the differentiation of endometrial and fetal cells by paracrine pathway, inhibiting the implantation and fetal development [152]. The active beta-catenin (CTNNB1) is only detected in morula and early blastocyst stages, its signal disappearing as soon as the blastocyst hatches from the zona pellucida [153]. Furthermore, deletion of *CTNNB1* gene provokes severe gastrulation defects that results in embryonic lethality [154]. Connexin 43 (CX43, also known as GJA1) is a major gap junction protein that is markedly expressed in the uterine stromal cells surrounding the implanted embryo during early pregnancy. Deletion of *CX43* gene leads to aberrant differen‐ tiation of uterine stromal cells, preventing the secretion of angiogenic factors, such as the VEGF. As consequence, the development of new blood vessels within the uterine stromal compart‐ ment suffers a striking impairment, resulting in the arrest of embryo growth and early pregnancy loss [155]. All these critical genes on successful implantation are listed on Table 2.

#### **5. Approaches for identifying genes in pigs and rabbits**

In pigs and rabbits, genetic markers associated with reproductive traits have been identified through two complementary approaches. The first approach has been performed through unbiased genome scans with anonymous DNA markers, such as microsatellites and more recently with thousands of single nucleotide polymorphisms (SNPs), which have been used to identify quantitative trait loci (QTL) with effects on reproductive traits. Genome-wide scanning usually proceeds without any presuppositions regarding the importance of specific functional features of the investigated traits. Until now, a total of 28 suggestive QTL have been reported on pig chromosomes (SSC) 2, 6, 7, 8, 11, 12, 14, 15, 16, 17, and 18 for litter size [156-165], on SSC 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, and 18 for ovulation rate [165-171], and on SSC8 for the uterine capacity and prenatal loss [159, 166, 168].

In the second approach, the physiological role of candidate gene is known, and the gene is scanned for polymorphisms and associations to variations within the trait. Numerous genes have been evaluated as candidate genes affecting litter size in pigs, such as the estrogen receptor (*ESR*) [95-97], retinol binding protein 4 (*RBP4*) [172], gonadotrophin-releasing hormone receptor (*GNRHR*) [173], osteopontin (*OPN*) [174], folate-binding protein (*FBP*) [175], mitogen-activated protein kinase 3 (*MAP3K3*) [176], vascular endothelial growth factor receptor (*KDR*) [176], ERBB2 interacting protein (*ERBB2IP)* [176], and peroxisome proliferatoractivated receptor delta (*PPARD*) [176]. Another candidate genes have been found for progesterone receptor (*PRG*), *TIMP1*, oviductal glycoprotein 1 (*OVPG1*), hydroxysteroid-17 beta- dehydrogenase 4 (*HSD17B4*), endoplasmic oxidoreductin-1-like protein (*ERO1L*), and octamer-binding transcription factor 4 (*OCT4*) in rabbits [177-181]. However, associations of these genes with litter size are always population specific, and the causative mutations underlying litter size remain unexplored.

DNA microarray is a new powerful tool for studying the molecular basis of interactions on a scale that is impossible using conventional analysis, making possible to examine the expression of thousands of genes simultaneously. In order to expand the understanding of the biological processes involved in the success of female reproduction, several studies in gene expression were developed in pigs targeting to identify the changes in ovaries [182, 183] and the endo‐ metrium at implantation [184-191]. For example, after selecting for 11 generations using an index of ovulation rate and embryonic survival, followed by 7 generations of selection for litter size, a total of 71 differentially expressed genes were identified in ovarian tissues of the selected and control lines at days 2–6 of the follicular stage of the estrous cycle [182]. Many of these genes had not been previously associated with reproduction. From these genes, 59 were homologous to genes of known function, 5 had no known matches in GenBank, and 7 were homologous to sequences of unknown function. Among the differentially expressed genes identified were those associated with the transport of cholesterol in ovarian follicles and the synthesis of steroids, such as collagen type I receptor (*CD36L1*, also known as scavenger receptor class B type I). The experiment also showed the importance of studying the expression of all these genes at different times of estrous cycle. For instance, genes of steroidogenic acute regulatory protein (*STAR*), 3-β-hydroxysteroid dehydrogenase (*3βHSD*), were overexpressed in higher producing pig ovaries at day 2 of analysis, while were underexpressed at day 3. In contrast, plasminogen activator inhibitor 1 (*PAI1*) and cytochrome P450 17-α-hydroxylase (*CYP17*) were overexpressed at day 3.

Mucins also participate in the decidualization process. For example, mucin (MUC1) has been identified as an effective barrier that prevents embryo attachment to the uterine epithelium. Uterine MUC1 expression declines to undetectable levels prior to blastocyst attachment, reinforcing the impression that loss of MUC1 contributes to the establishment of a receptive uterus [149-151]. Other oviductal glycoprotein, as OVGP1 or MUC9, seems to affect the differentiation of endometrial and fetal cells by paracrine pathway, inhibiting the implantation and fetal development [152]. The active beta-catenin (CTNNB1) is only detected in morula and early blastocyst stages, its signal disappearing as soon as the blastocyst hatches from the zona pellucida [153]. Furthermore, deletion of *CTNNB1* gene provokes severe gastrulation defects that results in embryonic lethality [154]. Connexin 43 (CX43, also known as GJA1) is a major gap junction protein that is markedly expressed in the uterine stromal cells surrounding the implanted embryo during early pregnancy. Deletion of *CX43* gene leads to aberrant differen‐ tiation of uterine stromal cells, preventing the secretion of angiogenic factors, such as the VEGF. As consequence, the development of new blood vessels within the uterine stromal compart‐ ment suffers a striking impairment, resulting in the arrest of embryo growth and early pregnancy loss [155]. All these critical genes on successful implantation are listed on Table 2.

In pigs and rabbits, genetic markers associated with reproductive traits have been identified through two complementary approaches. The first approach has been performed through unbiased genome scans with anonymous DNA markers, such as microsatellites and more recently with thousands of single nucleotide polymorphisms (SNPs), which have been used to identify quantitative trait loci (QTL) with effects on reproductive traits. Genome-wide scanning usually proceeds without any presuppositions regarding the importance of specific functional features of the investigated traits. Until now, a total of 28 suggestive QTL have been reported on pig chromosomes (SSC) 2, 6, 7, 8, 11, 12, 14, 15, 16, 17, and 18 for litter size [156-165], on SSC 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, and 18 for ovulation rate [165-171],

In the second approach, the physiological role of candidate gene is known, and the gene is scanned for polymorphisms and associations to variations within the trait. Numerous genes have been evaluated as candidate genes affecting litter size in pigs, such as the estrogen receptor (*ESR*) [95-97], retinol binding protein 4 (*RBP4*) [172], gonadotrophin-releasing hormone receptor (*GNRHR*) [173], osteopontin (*OPN*) [174], folate-binding protein (*FBP*) [175], mitogen-activated protein kinase 3 (*MAP3K3*) [176], vascular endothelial growth factor receptor (*KDR*) [176], ERBB2 interacting protein (*ERBB2IP)* [176], and peroxisome proliferatoractivated receptor delta (*PPARD*) [176]. Another candidate genes have been found for progesterone receptor (*PRG*), *TIMP1*, oviductal glycoprotein 1 (*OVPG1*), hydroxysteroid-17 beta- dehydrogenase 4 (*HSD17B4*), endoplasmic oxidoreductin-1-like protein (*ERO1L*), and octamer-binding transcription factor 4 (*OCT4*) in rabbits [177-181]. However, associations of these genes with litter size are always population specific, and the causative mutations

**5. Approaches for identifying genes in pigs and rabbits**

96 Insights from Animal Reproduction

and on SSC8 for the uterine capacity and prenatal loss [159, 166, 168].

underlying litter size remain unexplored.

In a different study, 189 genes were found to be differentially expressed in the ovaries of pregnant pigs with high and low prolificacy, of which 72 were overexpressed in the high prolificacy group, while 133 of them were overexpressed in the low prolificacy group [183]. These genes appear to cluster in three main biological processes: the first group would be related to the immune system response activation against external stimulus, the second group included integrated genes that regulate maternal homeostasis by complement and coagulation cascades, and the third was involved in lipid and fatty acid enzymes of metabolic processes of the steroidogenesis pathway. Among validated genes, 2-5-oligoadenylate synthetase 1 (*OAS1*) was found overexpressed in high prolificacy females, while a family with sequence similarity with 46 member C (*FAM46C*), secreted phosphoprotein 1 (*SPP1*), thiosulfate sulfurtransferase (*TST*), and vitronectin (*VTN*), were reported overexpressed in low prolificacy females.

Recent microarray analysis revealed more than 2000 differentially expressed genes in endo‐ metrium between pregnant and cyclic pigs at the time of implantation, i.e., on days 12 [186], 13 [191], 14 [187], 15 [188], 16 [192], 18 [191], or 24 [191] of gestation. Most genes were involved in cell motility as well as apoptosis, transporter activity, calcium ion binding, lipid metabolic processes, hormone activity, vascular development and proteolysis, immune response. The identified and validated genes that are upregulated included ADAM metallopeptidase with thrombospondin type 1 motif 20 (A*DAMTS20*), mucin 4 (*MUC4*), leukemia inhibitory factor receptor alpha (*LIFR*), interleukin 6 receptor (*IL6R*), interferon regulatory factor 1 (*IRF1*), immunoresponsive 1 homolog (IRG1), secreted phosphoprotein 1 (*SPP1*), osterocrin (*OSTN*), nuclear receptor interacting protein 1 (*NRIP1*), proteolipid protein 1 (*PLP1*), signal transducer and activator of transcription 1 (*STAT1*), serpin peptidase inhibitor, clade B (ovalbumin), member 7 (*SERPINB7*), s100 calcium binding protein A 9 (*S100A9*), Erb-B2 receptor tyrosine kinase 3 (*ERBB3*), and fibroblast growth factor 9 (*FGF9*).  Litter size is controlled by a large number of genes.

Improvement in litter size has become one main objective of selection in pig and rabbit breeding programs. However, litter size is a complex trait, because it is controlled by numerous genes in complicated physiological networks such as those affecting ovulation rate, embryo survival, and uterine capacity. The genomic approaches, both QTL mapping and candidate gene analysis, have helped increase understanding in genetic control of litter size. Moreover newly developed tools based on DNA microarray techniques appear to be useful for in-depth understanding of the genetics of litter size in pigs and rabbits. A better understanding of genetic mechanisms controlling litter size could help to design more efficient selection strategies in improvement of this trait.

#### **Author details**

María-José Argente\*

Address all correspondence to<EMAIL_ADDRESS>
Departamento de Tecnología Agroalimentaria, Universidad Miguel Hernández de Elche, Orihuela, Alicante, Spain

#### **References**

[5] Ford SP. Embryonic and fetal development in different genotypes in pigs. Journal of Reproduction and Fertility. Supplement. 1997; 52: 165–176.

and activator of transcription 1 (*STAT1*), serpin peptidase inhibitor, clade B (ovalbumin), member 7 (*SERPINB7*), s100 calcium binding protein A 9 (*S100A9*), Erb-B2 receptor tyrosine kinase 3 (*ERBB3*), and fibroblast growth factor 9 (*FGF9*). Contrasting, mucin 5AC, oligomeric mucus/gel-forming (*MUC5AC*), interleukin 11 receptor alpha (*IL11RA*), interleukin 24 (*IL24*), brain and acute leukemia cytoplasmic (*BAALC)*, defensin beta 1 (*PBD-2*), defensin beta 1 (*PBD-2*), cadherin 17 li (*CDH17*), FXYD domain containing ion transport regulator 4 (*FXYD4*), G protein-coupled receptor 83 (*CPR83*), and fibroblast growth factor receptor 3 (*FGFR3*) are

Improvement in litter size has become one main objective of selection in pig and rabbit breeding programs. However, litter size is a complex trait, because it is controlled by numerous genes in complicated physiological networks such as those affecting ovulation rate, embryo survival, and uterine capacity. The genomic approaches, both QTL mapping and candidate gene analysis, have helped increase understanding in genetic control of litter size. Moreover newly developed tools based on DNA microarray techniques appear to be useful for in-depth understanding of the genetics of litter size in pigs and rabbits. A better understanding of genetic mechanisms controlling litter size could help to design more efficient selection

Departamento de Tecnología Agroalimentaria, Universidad Miguel Hernández de Elche,

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downregulated [182-187, 191]. Litter size is controlled by a large number of genes.

strategies in improvement of this trait.

Address all correspondence to<EMAIL_ADDRESS>
Journal of Animal Science. 2002; 80: E54–E65.

**Author details**

98 Insights from Animal Reproduction

María-José Argente\*

Orihuela, Alicante, Spain

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### **Germ Cell Determinant Transmission, Segregation, and Function in the Zebrafish Embryo**

Celeste Eno and Francisco Pelegri

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62207

#### **Abstract**

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Animals specify primordial germ cells (PGCs) in two alternate modes: preformation and epigenesis. Epigenesis relies on signal transduction from the surrounding tissues to in‐ struct a group of cells to acquire PGC identity. Preformation, thought to be the more de‐ rived PGC specification mode, is instead based on the maternal inheritance of germ celldetermining factors. We use the zebrafish as a model system, in which PGCs are specified through maternal inheritance of germ plasm, to study this process in vertebrates. In ze‐ brafish, maternally inherited germ plasm ribonucleoparticles (RNPs) have co-opted the cytoskeletal machinery to reach progressive levels of multimerization, resulting in the formation of four large masses of aggregated germ plasm RNPs. At later stages, germ plasm masses continue to use components of the cell division machinery, such as the spindles, centrosomes, and/or subcellular organelles to segregate asymmetrically during cell division and subsequently induce germ cell fate. This chapter discusses the current knowledge of germ cell specification focusing on the zebrafish as a model system. We al‐ so provide a comparative analysis of the mechanism for germ plasm RNP segregation in zebrafish versus other known vertebrate systems of germ cell preformation, such as in amphibian and avian models.

**Keywords:** Germ cells, germ plasm, zebrafish, cell division, cytoskeleton, RNP segrega‐ tion

#### **1. Introduction**

#### **1.1. Fundamentals of germ cell specification: Epigenesis vs. preformation**

One of the most fundamental early cell fate decisions in animal embryos is the specification of primordial germ cells (PGCs) from the somatic tissue. PGCs are the precursors to gametes and thus hold the information to recreate the species in each generation. Consequently, individuals

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

from every population must employ a robust mechanism for PGC induction. Specification occurs by one of two mechanistic modes: *epigenesis* or *preformation* (Figure 1). Epigenesis refers to a mechanism by which certain cells receive PGC-inducing signals from surrounding tissues. Epigenesis is found in mammals, as well as species in many other clades. Preformation describes a cell autonomous specification of germ cells from maternally inherited germ plasm deposits that specify the PGCs. This mechanism of preformation is used in a large number of organisms distributed in many animal lineages, including major model systems for biological research, such as the fruit fly *Drosophila melanogaster* and the nematode *Caenorhabditis elegans*, amphibians, such as *Xenopus laevis* and *X. tropicalis*, and the chick.

**Figure 1.** Mechanisms of primordial germ cell specification. Species employing preformation deposit germ plasm in oocytes. Throughout development, germ plasm localizes into select cells that eventually become the germ cells. In spe‐ cies employing epigenesis, cells receive inductive signals from neighboring tissue to become the primordial germ cells.

When comparing epigenesis and preformation as PGC-determining mechanisms, it was originally pointed out that epigenetic germ cell determination is an exception, and most animals use germ plasm [1, 2]. Subsequently, the analysis of distribution of these mechanisms within the evolutionary tree has led to the hypothesis that epigenesis is an ancestral PGCdetermining mechanism and that preformation has arisen multiple times from this basal mode. Many clades employ both mechanisms, for example, within amphibians axolotls (urodeles) [3] use epigenesis and frogs (anurans) employ preformation [4]. Similarly, species in the group reptilia, such as turtles, use epigenesis [5], and species of the related group aves (birds) use preformation [6].

When protein-coding sequences of vertebrate species using epigenesis are compared with sister taxa employing preformation, genes of the latter evolve more rapidly. For example, when comparing protein-coding sequences from species in the amphibian and actinopterygian (rayfinned fishes) taxons, in all cases, species using preformation grouped further away from mammals than those using epigenesis. Evans et al. [7] found that no biological factor, including genome size, longevity, and generation time, correlates with sequence rate changes as optimally as the mode of PGC induction. This has led to the suggestion that the use of germ plasm relieves constraints on somatic development by dissociating PGC development from somatic development during early embryogenesis. This is because in animals using prefor‐ mation mechanisms, changes in gene networks involved in somatic tissue development can occur without causing deleterious effects on the germ line, which would cause sterility and would therefore be selected against. This developmental flexibility allows for a faster rate of evolution of developmental programs. For example, ancestral gene regulatory networks (GRNs) for pluripotency and mesoderm specification found in axolotls are conserved through the evolution of mammals, whereas in frogs, pluripotency GRNs have been lost and the number of key regulators (i.e. multiple copies of Nodal and Mix) have increased in the mesoderm specification GRN [2]. Since axolotls specify PGCs through epigenesis within the mesoderm, the GRN is under constraints preventing variation—constraints that are not present in frogs. However, frogs carry preformed germ plasm, whose direct role in PGC induction allows the embryo to undergo drastic changes to the mesoderm specification GRN, such as Nodal expansion [8]. Consistent with this hypothesis, the use of germ plasm coincides with increased morphological variation and enhanced speciation within a clade [7].

from every population must employ a robust mechanism for PGC induction. Specification occurs by one of two mechanistic modes: *epigenesis* or *preformation* (Figure 1). Epigenesis refers to a mechanism by which certain cells receive PGC-inducing signals from surrounding tissues. Epigenesis is found in mammals, as well as species in many other clades. Preformation describes a cell autonomous specification of germ cells from maternally inherited germ plasm deposits that specify the PGCs. This mechanism of preformation is used in a large number of organisms distributed in many animal lineages, including major model systems for biological research, such as the fruit fly *Drosophila melanogaster* and the nematode *Caenorhabditis elegans*,

**Figure 1.** Mechanisms of primordial germ cell specification. Species employing preformation deposit germ plasm in oocytes. Throughout development, germ plasm localizes into select cells that eventually become the germ cells. In spe‐ cies employing epigenesis, cells receive inductive signals from neighboring tissue to become the primordial germ cells.

When comparing epigenesis and preformation as PGC-determining mechanisms, it was originally pointed out that epigenetic germ cell determination is an exception, and most animals use germ plasm [1, 2]. Subsequently, the analysis of distribution of these mechanisms within the evolutionary tree has led to the hypothesis that epigenesis is an ancestral PGCdetermining mechanism and that preformation has arisen multiple times from this basal mode. Many clades employ both mechanisms, for example, within amphibians axolotls (urodeles) [3] use epigenesis and frogs (anurans) employ preformation [4]. Similarly, species in the group reptilia, such as turtles, use epigenesis [5], and species of the related group aves (birds) use

preformation [6].

amphibians, such as *Xenopus laevis* and *X. tropicalis*, and the chick.

116 Insights from Animal Reproduction

Although these two PGC-determining mechanisms differ, many of the factors involved in PGC development are highly conserved regardless of the mechanism employed to differen‐ tiate PGCs. For example, the gene product of *vasa* is found in germ cells of a wide range of organisms from planaria [9] to humans [10]. Importantly, both mechanisms, epigenesis and preformation, in addition to promoting germ cell fate in PGCs, must also block somatic fate in this cell type [11].

#### **1.1. Epigenesis, a mechanism by which cells receive signals from their surroundings**

Most animal clades include lineages that use epigenesis to specify PGCs. Within the amphibian clade, axolotl (salamander) PGC specification occurs in primitive ectoderm (animal cap) cells in response to ventral mesoderm-inducing signals [12], including fibroblast growth factor (FGF) and BMP4 [13]. PGC precursors arise in the ventral marginal zone and migrate over the blastopore during gastrulation and by tailbud stage are detected in the posterior of the dorsal– lateral mesoderm expressing PGC-specific genes, such as *dazl* [3] and *vasa* [14]*.*

All mammals specify PGCs through inductive signaling, and this mechanism is most well studied in mouse. In these organisms, extraembryonic tissue signals the most proximal region of the postimplantation epiblast to become the PGCs, whereas cells that do not receive the signals give rise to somatic tissue [15]. The mouse PGC precursors are detected just prior to primitive streak formation, and by the end of primitive streak formation, a population of around 40 PGCs are generated. Key instructive signals are the bone morphogenic proteins (BMP2, BMP4, and BMP8B), which act through SMAD1 and SMAD5 [16, 17,18]. The popula‐ tion of PGCs is heavily reliant on BMP dosage as loss of BMP4 reduces their induction [18]. Some RNAs present in the germ plasm in organisms with a preformative mechanism, such as *nanos*, *dead-end* (*dnd*), and *dazl*, also have roles in germ cell development in the mouse [19]. For example, mouse Dazl protein has a complex role in PGC development by targeting mRNAs for translational inhibition, thus preventing somatic differentiation, pluripotency, and apoptotic death [20]. In addition, mouse Dazl also has a role at later stages of germ cell development, during spermatogonia differentiation [21]. This latter role is likely conserved in humans, having led to the original identification of this gene (DAZ or "deleted in azoosper‐ mia") as being affected in 13% of human male infertility cases [22, 23].

#### **1.2. Preformation: Germ plasm deposits determine the germ cell fate**

Preformation describes a mechanism whereby differentiation as germ cells is decided by the acquisition of maternally inherited determinants following fertilization [24]. The common term to describe such determinants is germ plasm, which is considered as a specialized cytoplasm enriched for factors that function in PGC determination. Germ plasm contains RNAs (both coding and noncoding) and proteins, which in many organisms appear to assemble together in punctate structures known as ribonucleoparticles (RNPs). In many organisms, germ plasm closely associates with the cytoskeleton and/or mitochondria. Proposed functions of germ plasm include the translational regulation of germ plasm RNAs, the establishment of a partially repressive chromatin in the germ line and the prevention of activation of somatic development by repressing mRNA transcription (reviewed in [25, 26]).

In *Drosophila*, arguably one of the most well-characterized species that use germ plasm as a means of PGC determination, the progenitors of the germ line are four to five pole cells found at the posterior pole of the embryo before blastoderm formation [27]. Pole cells acquire PGC identity through inheritance of the germ plasm (reviewed in [28]) called pole plasm in this organism [29, 30]. Polar granules appear at the posterior tip during mid-oogenesis, coincident with the time when vitellogenesis occurs. Toward the end of oogenesis, the polar granules associate with other germ plasm-associated structures (i.e. mitochondria) [31]. Germ plasm components are released from cortical actin at the posterior of the oocyte, and are transported via dynein motors on astral microtubules to centrosomes associated with nuclei in posterior regions of the early embryo [32]. These posterior nuclei subsequently separate from the syncytial mass through cell cleavage to form the PGC precursors or pole cells (reviewed in [28]). Embryological manipulations confirm that pole plasm is a true germ cell determinant as it can recreate pole cells when transplanted ectopically, for example, in the anterior region of the embryo. Interestingly, pole cell number relies on the dosage of a single RNA, *oskar* [33], which is required for proper induction of germ cells. However, the function of *oskar* in PGC determination is restricted to dipterans [34], so that other factors must take its role in other organisms.

P-granules are another type of RNA-rich cytoplasm structures involved in the specification of the *C. elegans* germ line. These granules are scattered evenly throughout the cytoplasm before and just after fertilization [35]. At a time coincident with pronuclear fusion, under the influence of cell polarity factors (PAR factors) and implemented by microfilament-dependent cytoplas‐ mic streaming [36, 37], P-granules translocate to and accumulate at the posterior of the early embryo. As a result of asymmetric positioning of the spindle during cell divisions, each division generates daughters of unequal size, with the smaller daughter inheriting most of the P-granules asymmetrically until the generation of the P4 cell, after four cell divisions. However, the minority of P-granules remaining at the anterior end or in non-P4 cells is degraded [38, 39]. Interestingly, P-granule localization during these early divisions (which generate P2, P3, and P4 cells) relies on attachment to the nuclear periphery coupled to centrosome rotation, which leads to asymmetric placement of the P-granules with respect to the nucleus, specifically at the ventral side of the P cell [38]. Although "local gathering mechanisms" that each of the P cells use to deliver P-granules to the proper location appear to vary, asymmetric cell division and directional P-granule segregation occur through the first four cell cycles, generating Pgranule-containing cell P4. This cell type, whose nucleus is surrounded by large, coalesced Pgranules, is the first cell whose descendants are restricted to the germ line [40, 41] and whose ablation results in sterility [42]. The P4 cell divides symmetrically during embryogenesis resulting in Z2 and Z3, which after the first larval stage continue to divide symmetrically to produce about 1000 germ cells in the adult gonad [43]. P-granules continue to associate with the nuclear envelope of germ cells throughout embryonic and larval divisions and only detach from this structure during gamete maturation. However, the significance of this dynamic association remains poorly understood.

Although research on PGC specification is sparse for colonial ascidians, studies of a single species, *Botryllus schlosseri*, describe aggregation and ventral localization of maternally inherited germ plasm RNAs, in particular *vasa* mRNA [44]. The solitary ascidian (*Ciona intestinalis*) also uses a specialized cytoplasm (myoplasm) containing maternally inherited determinants of germ cells [45]. The first pre-PGCs form at the posterior end of the early embryo [46] and express conserved germ line gene, such as *vasa* [47]. Interestingly, when the PGCs are ablated, a new collection of *vasa*-expressing cells appears in the juvenile after metamorphosis. Because these cells did not inherit myoplasm, this suggests an alternative, compensatory mechanism for PGC specification in this organism, possibly similar to an inductive mode of PGC specification [48], which may be occurring as a safe–fail mechanism in this organism. Other well-studied systems that use germ plasm to specify PGCs, now within vertebrate lineages, include zebrafish, which is the focus of this chapter, *Xenopus* and chicken (see below).

#### **2. Germ cell determinant segregation in zebrafish**

#### **2.1. Maternal inheritance of germ plasm RNPs**

(BMP2, BMP4, and BMP8B), which act through SMAD1 and SMAD5 [16, 17,18]. The popula‐ tion of PGCs is heavily reliant on BMP dosage as loss of BMP4 reduces their induction [18]. Some RNAs present in the germ plasm in organisms with a preformative mechanism, such as *nanos*, *dead-end* (*dnd*), and *dazl*, also have roles in germ cell development in the mouse [19]. For example, mouse Dazl protein has a complex role in PGC development by targeting mRNAs for translational inhibition, thus preventing somatic differentiation, pluripotency, and apoptotic death [20]. In addition, mouse Dazl also has a role at later stages of germ cell development, during spermatogonia differentiation [21]. This latter role is likely conserved in humans, having led to the original identification of this gene (DAZ or "deleted in azoosper‐

Preformation describes a mechanism whereby differentiation as germ cells is decided by the acquisition of maternally inherited determinants following fertilization [24]. The common term to describe such determinants is germ plasm, which is considered as a specialized cytoplasm enriched for factors that function in PGC determination. Germ plasm contains RNAs (both coding and noncoding) and proteins, which in many organisms appear to assemble together in punctate structures known as ribonucleoparticles (RNPs). In many organisms, germ plasm closely associates with the cytoskeleton and/or mitochondria. Proposed functions of germ plasm include the translational regulation of germ plasm RNAs, the establishment of a partially repressive chromatin in the germ line and the prevention of activation of somatic

In *Drosophila*, arguably one of the most well-characterized species that use germ plasm as a means of PGC determination, the progenitors of the germ line are four to five pole cells found at the posterior pole of the embryo before blastoderm formation [27]. Pole cells acquire PGC identity through inheritance of the germ plasm (reviewed in [28]) called pole plasm in this organism [29, 30]. Polar granules appear at the posterior tip during mid-oogenesis, coincident with the time when vitellogenesis occurs. Toward the end of oogenesis, the polar granules associate with other germ plasm-associated structures (i.e. mitochondria) [31]. Germ plasm components are released from cortical actin at the posterior of the oocyte, and are transported via dynein motors on astral microtubules to centrosomes associated with nuclei in posterior regions of the early embryo [32]. These posterior nuclei subsequently separate from the syncytial mass through cell cleavage to form the PGC precursors or pole cells (reviewed in [28]). Embryological manipulations confirm that pole plasm is a true germ cell determinant as it can recreate pole cells when transplanted ectopically, for example, in the anterior region of the embryo. Interestingly, pole cell number relies on the dosage of a single RNA, *oskar* [33], which is required for proper induction of germ cells. However, the function of *oskar* in PGC determination is restricted to dipterans [34], so that other factors must take its role in other

P-granules are another type of RNA-rich cytoplasm structures involved in the specification of the *C. elegans* germ line. These granules are scattered evenly throughout the cytoplasm before and just after fertilization [35]. At a time coincident with pronuclear fusion, under the influence

mia") as being affected in 13% of human male infertility cases [22, 23].

118 Insights from Animal Reproduction

**1.2. Preformation: Germ plasm deposits determine the germ cell fate**

development by repressing mRNA transcription (reviewed in [25, 26]).

organisms.

The fish model, *Danio rerio*, employs the preformative model of germ cell specification wherein the mother deposits factors important for PGC specification into the oocytes, which are transmitted to the embryo. RNPs containing RNAs for all known germ plasm factors in this organism originally localize to the same structure during oogenesis. This is the mitochondrial cloud or Balbiani body (Bb), a large aggregate of organelles found in the oocytes of many species, that translocates from a location near the nucleus to the vegetal pole during oogenesis. Germ plasm RNA and mitochondrial enrichment to the Bb depend on the functional product of *buckyball* (*buc*), a novel vertebrate-specific protein that is the first gene identified as being required for the formation of this structure [49]. The Bb disassembles by stage II oogenesis, when the germ plasm RNAs localize to the vegetal pole via a pathway similar to the messenger transport organizer (METRO) pathway originally described in *Xenopus.* Subsequently, different germ plasm RNAs undergo different patterns of distribution in the oocyte: *vasa* becomes redistributed along the cortex, *nanos* becomes evenly distributed in the oocyte and *deleted in azoospermia* (*dazl*) remains localized to the vegetal pole (reviewed in [50]; see also [51]). These various patterns of redistribution during oogenesis result in the creation of two types of localized germ plasm RNPs, those present in the forming blastodisc at the animal region (animal germ plasm RNPs) and those remaining in the vegetal region (vegetal germ plasm RNPs).

#### *2.1.1. Animal region germ plasm factors*

Known animal region germ plasm factors include *vasa,* a gene whose RNA or gene product is one of the most widely used markers for germ cells or germ plasm in metazoans [52]. As mentioned above, *vasa-*containing germ plasm RNPs localize along the cortical region of the oocyte at stage II and after egg activation are rapidly transported to the animal pole, where they are found dispersed evenly around the blastodisc. The protein product for this gene is a DEAD-box ATP-dependent helicase involved in RNA metabolism that facilitates interactions between RNA–RNA and RNA–protein [53]. Loss of function mutations in *vasa* lead to reduction in number or functionality of germ cells in almost all organisms [54–58]. In zebrafish, *vasa* morphant embryos (knock down of protein using a morpholino-conjugated oligonucleo‐ tide (MO)) do not exhibit defects in the establishment of the germ line [59]. Moreover, injection of *vasa* RNA into one-cell zebrafish embryos does not lead to an increase in PGCs; instead this RNA is degraded, suggesting that endogenous *vasa* RNA levels are carefully controlled by an intrinsic cellular mechanism [60]. These studies suggest that *vasa* is generally required, but is not sufficient, for germ cell development. However, a more rigorous set of tests using a knockdown or ectopic expression initiated during oogenesis, or *vasa/ddx4* maternal mutants, has not yet been reported.

Another animal germ plasm RNA is that of the gene *nanos*. Before egg activation, *nanos* mRNA is not localized; however, shortly after activation *nanos* is found to colocalize with *vasa* RNA. *nanos* RNA and/or gene product are also commonly associated with germ cell development and present in germ plasm. First described in *Drosophila*, *nanos* encodes a RNA-binding zinc finger protein. In *Drosophila*, *nanos* is not generally required for PGC formation; however, *Drosophila* PGCs deficient in *nanos* activity have abnormal development, including failure to migrate to the gonad, reduction in egg number, early activation of germ cell genes, deregulated expression of somatic mRNAs, and irregular morphology [61–63]. In zebrafish, knockdown of *nanos1* using antisense morpholinos injected at the one-cell stage proves that it is essential for germ cell development [64]. In these morphants, PGCs are specified, yet these undergo aberrant migration and exhibit reduced number.

Other germline-specific RNAs found at the animal pole include *dnd*, *askopos*(*kop*), and *TDRD7*. *dnd* encodes a RNA-binding protein that blocks negative regulation through mRNA target degradation caused by binding of several microRNAs (miRNAs). Dnd protein achieves this protective function against miRNA-mediated mRNA degradation by binding mRNAs at miRNA target sites contained within these transcripts, thus blocking miRNA binding. This mechanism is used in the zebrafish germ line by PGC factor Dnd1 to protect several mRNAs, including *nanos* and *TDRD7*, from miR-430 repression, which is achieved through Dnd binding to U-rich regions in mRNA targets [65, 66]. Similar to *nanos1*, knockdown knockdown of *dnd* in morphants results in defects in PGC migration exhibits defects in PGC migration and viability [67].

*kop* mRNA was found to localize to zebrafish germ plasm and encodes a novel nuclear protein [68] that is not yet well studied. Some tudor domain-containing genes are expressed in germ cells, *RNF17*, *TDRD1*, *TDRD6*, and *TDRD7*. The latter (*TDRD7*) also localizes to germ plasm as it aggregates to the forming furrows, and TDRD7 protein colocalizes with Vasa in PGCs [69]. In *Drosophila*, homologous tudor proteins have been proposed to serve as a platform for polar granule assembly [70], a function that is consistent with the presence of TDRD7 protein in the zebrafish germ plasm. Zebrafish *TDRD7* morphants lack normal germ cell granule integrity at late somite stage [69]. Interestingly, TDRD7 is highly expressed in the developing lens, and mutations of TDRD7 in human, mouse, and chick lead to cataracts [71].

For tested animal germ plasm RNAs (*vasa*, *nanos*, and *dnd*), animal RNPs appear to colocalize and exhibit a similar size [72]. These observations suggest that germ plasm RNPs may be composites containing a set of germ plasm factors essential for germ plasm determination.

#### *2.1.2. Vegetal region germ plasm factors*

cloud or Balbiani body (Bb), a large aggregate of organelles found in the oocytes of many species, that translocates from a location near the nucleus to the vegetal pole during oogenesis. Germ plasm RNA and mitochondrial enrichment to the Bb depend on the functional product of *buckyball* (*buc*), a novel vertebrate-specific protein that is the first gene identified as being required for the formation of this structure [49]. The Bb disassembles by stage II oogenesis, when the germ plasm RNAs localize to the vegetal pole via a pathway similar to the messenger transport organizer (METRO) pathway originally described in *Xenopus.* Subsequently, different germ plasm RNAs undergo different patterns of distribution in the oocyte: *vasa* becomes redistributed along the cortex, *nanos* becomes evenly distributed in the oocyte and *deleted in azoospermia* (*dazl*) remains localized to the vegetal pole (reviewed in [50]; see also [51]). These various patterns of redistribution during oogenesis result in the creation of two types of localized germ plasm RNPs, those present in the forming blastodisc at the animal region (animal germ plasm RNPs) and those remaining in the vegetal region (vegetal germ plasm

Known animal region germ plasm factors include *vasa,* a gene whose RNA or gene product is one of the most widely used markers for germ cells or germ plasm in metazoans [52]. As mentioned above, *vasa-*containing germ plasm RNPs localize along the cortical region of the oocyte at stage II and after egg activation are rapidly transported to the animal pole, where they are found dispersed evenly around the blastodisc. The protein product for this gene is a DEAD-box ATP-dependent helicase involved in RNA metabolism that facilitates interactions between RNA–RNA and RNA–protein [53]. Loss of function mutations in *vasa* lead to reduction in number or functionality of germ cells in almost all organisms [54–58]. In zebrafish, *vasa* morphant embryos (knock down of protein using a morpholino-conjugated oligonucleo‐ tide (MO)) do not exhibit defects in the establishment of the germ line [59]. Moreover, injection of *vasa* RNA into one-cell zebrafish embryos does not lead to an increase in PGCs; instead this RNA is degraded, suggesting that endogenous *vasa* RNA levels are carefully controlled by an intrinsic cellular mechanism [60]. These studies suggest that *vasa* is generally required, but is not sufficient, for germ cell development. However, a more rigorous set of tests using a knockdown or ectopic expression initiated during oogenesis, or *vasa/ddx4* maternal mutants,

Another animal germ plasm RNA is that of the gene *nanos*. Before egg activation, *nanos* mRNA is not localized; however, shortly after activation *nanos* is found to colocalize with *vasa* RNA. *nanos* RNA and/or gene product are also commonly associated with germ cell development and present in germ plasm. First described in *Drosophila*, *nanos* encodes a RNA-binding zinc finger protein. In *Drosophila*, *nanos* is not generally required for PGC formation; however, *Drosophila* PGCs deficient in *nanos* activity have abnormal development, including failure to migrate to the gonad, reduction in egg number, early activation of germ cell genes, deregulated expression of somatic mRNAs, and irregular morphology [61–63]. In zebrafish, knockdown of *nanos1* using antisense morpholinos injected at the one-cell stage proves that it is essential for

RNPs).

120 Insights from Animal Reproduction

*2.1.1. Animal region germ plasm factors*

has not yet been reported.

Vegetally localized germ plasm RNPs contain RNAs for the genes *deleted in azoospermia-like* (*dazl*) and *bruno-like* (*brul*). These germ plasm RNPs are initially localized to the vegetal pole of the egg and upon egg activation translocate toward the blastomeres forming at the animal pole [73–75]. Both *dazl* and *brul* encode RNA-binding proteins present in germ plasm across a wide variety of animal lineages. Although Dazl protein is not on its own sufficient to promote translation, this factor promotes RNA polyadenylation to enhance the stability of PGC transcripts, such as those for *TDRD7* and *dazl*. Thus, Dnd1 and Dazl may work additively in this fashion to protect RNAs from miRNA repression [76]. Studies focusing on *dazl* RNA aggregates show that these are restricted to the vegetal cortex in the mature egg and move animally after egg activation, so that 45 minutes post fertilization *dazl* RNP aggregates begin to be observed at the site of germ plasm accumulation at the first cleavage furrow [75, 77]. During animally directed movement, aggregates do not appear to be of uniform size and may translocate using two pathways: an ooplasmic streaming pathway mediated through axial streamers in internal regions of the zygote and a cortical pathway along meridional streamers in more superficial regions [73, 75, 77]. During furrow formation, vegetally derived germ plasm RNPs associate with large germ plasm masses of animal germ plasm RNPs forming at the furrows, in a cortical location near the distal-most end of the furrow and close to the yolk membrane cortex (see below). This location suggests that the animally directed cortical pathway may be the primary pathway used by vegetal germ plasm RNPs fated to become a part of a reconstituted and functional germ plasm mass.

Recent studies have shown that vegetal RNPs appear to be differentially distributed in the vegetal cortex, specifically at a cortical depth that is deeper than that of factors that also localize to the animal pole but are required for axis induction [78]. This differential distribu‐ tion along cortical depth, with germ plasm RNPs at deeper levels and dorsal factors at more superficial ones, likely facilitates the symmetric animally directed movement of germ plasm RNPs [75, 79] in spite of the asymmetric movement of the bulk of the cortex required for axis specification [80, 81].

#### **2.2. Repackaging germ plasm: Gradual multimerization and recruitment**

As mentioned above, after egg activation and prior to the first cell division, animally localized RNPs are present in single particles, which we refer to as singletons, spread throughout the developing blastodisc. Germ plasm RNP singletons aggregate in a wave-like fashion, where the wave of aggregation emanates from the center of the blastodisc outwards, toward the edge of the blastodisc.

Germ plasm RNP multimerization at this stage depends on the interplay between microtubule and microfilament networks during blastomere cell division (Figure 2A). After fertilization in zebrafish, paternally derived centrioles act as a template to reconstruct the centrosome using maternally derived components. This newly formed centrosome nucleates a sperm aster or monoaster prior to initiation of the first cell division cycle. In this structure, plus ends of the astral microtubules interact with germ plasm RNPs at the cortex and help direct germ plasm RNP multimerization in a process of **pre-aggregation** (the prefix of this term indicates that this process occurs prior to furrow formation). The growing tips of astral microtubules thus generate an aggregation front of multimerizing RNPs, initiating near the center of the blasto‐ disc and moving outward. The inner rim of RNP aggregates remains surrounded by a field of RNPs that have not yet been influenced by astral microtubules and therefore remains as singletons until the front arrives [72, 75]. As this process ensues, central regions of the blastodisc become depleted of germ plasm RNPs, generating an outwardly expanding RNPfree zone (Figure 2B). When embryos are treated with the microtubule inhibitor nocodazole, germ plasm RNPs do not undergo multimerization and instead remain dispersed throughout the cortex. This effect is also observed in embryos deficient for the Chromosomal Passenger Complex protein, Birc5b/Survivin, encoded by the gene *motley* [72]. In wild-type embryos, Birc5b protein localizes to the tips of astral microtubules (plus ends) and interacts with germ plasm RNPs. In motley-mutant embryos, mutated Birc5b/Survivin product localizes to germ plasm RNPs but not to microtubule plus ends, demonstrating Birc5b is an important linker between the two and explaining the *motley*-mutant phenotype. The fact that in these mutants germ plasm RNPs do not multimerize indicates that this linkage is necessary for the aggrega‐ tion process [72].

Germ Cell Determinant Transmission, Segregation, and Function in the Zebrafish Embryo http://dx.doi.org/10.5772/62207 123

furrows, in a cortical location near the distal-most end of the furrow and close to the yolk membrane cortex (see below). This location suggests that the animally directed cortical pathway may be the primary pathway used by vegetal germ plasm RNPs fated to become a

Recent studies have shown that vegetal RNPs appear to be differentially distributed in the vegetal cortex, specifically at a cortical depth that is deeper than that of factors that also localize to the animal pole but are required for axis induction [78]. This differential distribu‐ tion along cortical depth, with germ plasm RNPs at deeper levels and dorsal factors at more superficial ones, likely facilitates the symmetric animally directed movement of germ plasm RNPs [75, 79] in spite of the asymmetric movement of the bulk of the cortex required for axis

As mentioned above, after egg activation and prior to the first cell division, animally localized RNPs are present in single particles, which we refer to as singletons, spread throughout the developing blastodisc. Germ plasm RNP singletons aggregate in a wave-like fashion, where the wave of aggregation emanates from the center of the blastodisc outwards, toward the edge

Germ plasm RNP multimerization at this stage depends on the interplay between microtubule and microfilament networks during blastomere cell division (Figure 2A). After fertilization in zebrafish, paternally derived centrioles act as a template to reconstruct the centrosome using maternally derived components. This newly formed centrosome nucleates a sperm aster or monoaster prior to initiation of the first cell division cycle. In this structure, plus ends of the astral microtubules interact with germ plasm RNPs at the cortex and help direct germ plasm RNP multimerization in a process of **pre-aggregation** (the prefix of this term indicates that this process occurs prior to furrow formation). The growing tips of astral microtubules thus generate an aggregation front of multimerizing RNPs, initiating near the center of the blasto‐ disc and moving outward. The inner rim of RNP aggregates remains surrounded by a field of RNPs that have not yet been influenced by astral microtubules and therefore remains as singletons until the front arrives [72, 75]. As this process ensues, central regions of the blastodisc become depleted of germ plasm RNPs, generating an outwardly expanding RNPfree zone (Figure 2B). When embryos are treated with the microtubule inhibitor nocodazole, germ plasm RNPs do not undergo multimerization and instead remain dispersed throughout the cortex. This effect is also observed in embryos deficient for the Chromosomal Passenger Complex protein, Birc5b/Survivin, encoded by the gene *motley* [72]. In wild-type embryos, Birc5b protein localizes to the tips of astral microtubules (plus ends) and interacts with germ plasm RNPs. In motley-mutant embryos, mutated Birc5b/Survivin product localizes to germ plasm RNPs but not to microtubule plus ends, demonstrating Birc5b is an important linker between the two and explaining the *motley*-mutant phenotype. The fact that in these mutants germ plasm RNPs do not multimerize indicates that this linkage is necessary for the aggrega‐

**2.2. Repackaging germ plasm: Gradual multimerization and recruitment**

part of a reconstituted and functional germ plasm mass.

specification [80, 81].

122 Insights from Animal Reproduction

of the blastodisc.

tion process [72].

**Figure 2.** Germ plasm accumulation during the first zebrafish cell cycles. A) In the egg, animal pole (AP) germ plasm RNPs are dispersed at the blastodisc cortex, whereas vegetal pole (VP) germ plasm is localized at the vegetal pole. B) Prior to the initiation of cell cycling, during pronuclear fusion, astral microtubule growth moves AP germ plasm RNPs outward, whereas VP germ plasm begins to travel toward the AP. During the next several cell cycles, aggregated RNPs are collected in the (C) first furrow, (D) second furrow and (E) third furrow. F) Germ plasm RNPs in the first and sec‐ ond furrows are stabilized, whereas all other RNPs are cleared. Figure adapted from [86].

Another maternal factor important for germ plasm RNP recruitment during these early stages is Buc. In addition to a role for Buc to assemble the Bb during oogenesis, this factor is also required for recruitment of germ plasm during early embryogenesis, and its overexpression in the early embryo leads to an increase in PGCs [49]. Recently, a microtubule motor protein, Kif5Ba (kinesin), was found to bind to Buc and mediate its recruitment (thereby recruitment of other germ plasm molecules) to the cleavage furrow. Germ plasm in *kif5Ba* mutants is spread throughout the blastomeres, and Kif5Ba is required for the excess of PGCs that form when Buc is overexpressed [82]. This is reminiscent of the role of kinesins in germ plasm movement in *Xenopus*, where Xklp1 (kinesin-like protein) has also been shown to be required for germ plasm localization, and depletion of Xklp1 leads to arrest of germ plasm aggregation [83]. In *Xenopus*, another kinesin has been shown to have a role in later PGC development: KIF13B functions in migration and polarity of germ cells, as manipulations of xKIF13B result in erroneous PGC migration and reduced numbers [84].