Signaling Roadmap Modulating Chicken Primordial Germ Cells Proliferation and Self-Renewal

Iranian Journal of Applied Animal Science

دوره 13، شماره 1، خرداد 2023، صفحه 1-10   

نوع مقاله: Review Article

نویسندگان

M. Zare¹؛ S.Z. Mirhoseini^{* 2}؛ S.N. Hassani^{* 3}؛ S. Ghovvati¹

¹Department of Animal Science, Faculty of Agricultural Science, University of Guilan, Rasht, Iran

²Department of Animal Science, Faculty of Agricultural Science, University of Guilan,Guilan, Rasht, Iran

³Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran

چکیده

In the life science technologies, the chicken Primordial Germ Cells (PGCs) are recognized as the most practical cells compared to the other chicken stem cells. For this purpose, the isolation and long-term culture of these cells are of great importance for the production of therapeutic proteins, such as monoclonal antibodies, vaccines, endangered species protection, and chimeric bird production. However, one of the major challenges of working with these cells is their lack of proliferation and self-renewal ability in the laboratory environment. Recent researches on chick PGCs have shown that the pluripotency-related signaling pathways active in these cells are highly effective in their in vitro proliferation and self-renewal. Therefore in this review, we tried to summarize and evaluate the mechanisms of the most important pluripotency-related signaling pathways in chicken PGCs, which may result in achieving the reproducible line for these cell types. Studies have shown that one method to induce pluripotency in PGCs is to manipulate signaling pathways, including TGF-β and Wnt/β-catenin, using growth factors and small molecules. Along with activation of signaling pathways involved in self-renewal, improving culture conditions can be an effective way to achieve chicken PGC cell line. It could be concluded that providing a defined culture condition and activating specific signaling pathways can lead to induction of proliferation in chick PGCs.

کلیدواژه‌ها

pluripotency؛ primordial germ cell؛ self-renewal؛ signaling pathway؛ small mole-cule

اصل مقاله

INTRODUCTION

Primordial germ cells (PGCs) are precursor germ cells and play a central role in the continuation of the species by the transmission of genetic information from one generation to the next. In comparison to the other chicken stem cells, PGCs have unique developmental characteristics (Han, 2009) and numerous signaling pathways play important roles in the development of their self-regulation (De Felici et al. 2009). Due to the lack of proper avian PGCs derivation methods during the early stages of embryonic development in the last 15 years, there is still much to investigate regarding PGCs. However, due to the complicated regulatory mechanism of PGCs, only insignificant progress was observed. It has also been shown that complex regulatory networks in PGCs are controlled by various external factors that lead to the activation of intracellular signaling pathways (De Felici et al. 2009). For instance, efficient production of cultured PGCs with increased cell survival and proliferation rate can be enriched by numerous factors and mechanisms. The most important of these factors and mechanisms include fibroblast growth factor (FGF), retinoic acid (RA), stem cell factor (SCF), leukemia inhibitory factor (LIF), and bone morphogenetic protein 4 (BMP4) (Matsui et al. 1992; Resnick et al. 1992; Tang et al. 2006). Although the signaling pathways involved in chicken PGCs pluripotency are unclear, many recent studies have focused on TGF-β signaling pathways (Whyte et al. 2015), Wnt/Gsk3/β-catenin (Lee et al. 2016), BMP4 (Whyte et al. 2015), and basic FGF (bFGF) (Choi et al. 2010; Macdonald et al. 2010; Miyahara et al. 2014). The ones mentioned above are the best-studied signaling pathways in controling chicken PGCs' survival and proliferation. Recently, three different signaling pathways, including transforming growth factor beta (TGFβ), FGF, and insulin, have been reported to be required for PGC growth in chickens, suggesting the existence of a combined effect of multiple signaling pathways (Whyte et al. 2015). Moreover, many researches have proved the crosstalk between Wnt/β-catenin and FGF signaling (Lee et al. 2016). Therefore, it is necessary to study the regulatory mechanisms of PGCs growth. This can be achieved by investigating the role of endogenous and exogenous factors and finding out their roles in controlling the signaling pathways, which subsequently leads to the recognition of the key genes in the pathway. Despite numerous studies focusing on PGCs extraction and different techniques of long-term PGCs maintenance in the culture medium in the last decades, reproducibility, economic values, and long term maintenance of chicken PGCs are still remaining uncertain (Whyte et al. 2015). In this review, we discuss the signaling systems that control the regulation of long-term maintenance and self-renewal of chicken PGCs. The new findings from the signaling pathways and critical regulator genes of the chicken PGCs could provide insights into the biology of chicken pluripotent stem cell (PSC) lines and using them for the creation of recombinant proteins and vaccines.

 

Factors and mechanisms involved in chicken PGCs proliferation

Wnt/Gsk3/β-catenin signaling

It is shown that Wnt/Gsk3/β-catenin signaling can act as a key player in maintenance of PSC pluripotency and self-renewal. The Wnt is a member of the secretory protein family and signaling molecules that regulate numerous pathological and physiological processes during the development processes in all animal cells (Logan and Nusse, 2004). Also, it is shown that Wnt can act as a key player in promotion of proliferative capacity in both cancer and stem cells (Sato et al. 2004; Polakis, 2012). In the canonical Wnt pathway, Wnt ligands bind to frizzled (Fzd)-lipoprotein related protein (Lrp) 5/6 receptor complexes and activate Dishevelled (Dvl) proteins, leading to the phosphorylation and inactivation of GSK3β and accumulation of β-catenin in the cytoplasm (Figuer 1). Upon suppression of GSK3β, β-catenin enters the nucleus and creates heterodimers with T-cell factor (TCF) transcription factors and regulates various target genes (Fu et al. 2011; Valenta et al. 2012).

 

Figure 1 Wnt/β-catenin and LIF/STAT3 signaling pathways in chicken PGC self-renewal

 

Actually, studies accomplished by the activation of Wnt ligands showed improvement of self-renewal in mouse PGCs. Consistent with our results, recent studies on PGC development in mice have revealed the roles of several Wnt ligands including Wnt3 and Wnt3a in the specification (Ohinata et al. 2009; Bialecka et al. 2012; Aramaki et al. 2013; Tanaka et al. 2013), Wnt4 ligand in female sexual differentiation of these cells (Vainio et al. 1999; Chassot et al. 2012), and Wnt5a ligand and its receptor Ror2 in migration (Laird et al. 2011; Chawengsaksophak et al. 2012). Also, some experiments has demonstrated that self-renewal and proliferation of avian PGCs, similar to mammalian PGCs require Wnt/β-catenin signaling (Kimura et al. 2006; Ohinata et al. 2009; Chawengsaksophak et al. 2012) and Wnt/β-catenin signaling is considered to be an important pathway in chicken PGCs by governing their pluripotency. Compensatory pluripotency-related signaling is possible description of this observation, that begins after culturing PGCs under culture conditions (Hassani et al. 2014). To examine the importance of the Wnt/β-catenin pathway in chicken PGCs, in 2016, researchers applied JW74 and BIO, inhibitors of β-catenin and GSK3-β, respectively (Lee et al. 2016). The results of their study suggested that inhibition of Wnt/β-catenin signaling causes cell cycle arrest through downregulation of CDK6 and CCND1 (Sun et al. 2008), therefore, it showed that Wnt/β-catenin signaling is needed for the maintenance of PGC proliferation. Since BIO and JW74 could activate and inactivate β-catenin, respectively, in cultured chicken PGCs trhough modulating its active form level. Lee et al. (2016) used Wnt ligands or BIO to activate the Wnt signaling pathway in chicken PGCs. The outcomes of their proliferation assay proposed that the Wnt/β-catenin signaling activation caused in vitro proliferation of chicken PGCs, whereas JW74 inhibitrd this. These outcomes suggested that Wnt/β-catenin signaling is needed for in vitro proliferation of chicken PGCs (Lee et al. 2016). In the other hands, studies showed that β-catenin is a target gene of FGF signaling using RAS/ ERK or PI3K/AKT cascades (Israsena et al. 2004; Ding et al. 2010). Notably, if bFGF is not exist, β-catenin will be only located in the membrane with E-cadherin, where it accumulates and in the presence of bFGF is translocated to the nucleus to activate downstream genes (Ding et al. 2010; Valenta et al. 2011). Also, some experiments have demonstrated that bFGF increases β-catenin signaling in cells via increasing the level of β-catenin mRNA, nuclear translocation of β-catenin, and phosphorylation of GSK-3-β (Otero et al. 2004). Thus, β-catenin through the overexpression of bFGF lead to in vitro expansion of stem cells, while its absence enhances differentiation (Israsena et al. 2004). Also, due to the β-catenin role in the chicken PGCs proliferation that was previously reported (Ge et al. 2012), Lee et al. (2016) suggested that bFGF signaling develop the chicken PGCs proliferation through the β-catenin activation and the downstream signaling. Overall, activition of Wnt/β-catenin signaling was found to be necessary for proliferation of chicken PGCs.

 

FGF and MAPK/Erk signaling               

The FGFs are a family of cell-signaling proteins that are classified into six different FGF sub-families (Itoh, 2007). FGF2, which is also known as bFGF encodes FGF-β which is a signaling protein in the FGF family (Dionne et al. 1990; Kim, 1998). Like other members of FGF family, bFGF possesses cell survival characteristics and broad mitogenic and is involved in various biological processes such as cell proliferation regulation, embryonic development, migration, differentiation, and cell growth (Feldman et al. 1995; Delaune et al. 2005). Also, bFGF is a potent mitogen in mammalian PGCs medium culture (Resnick et al. 1998), and FGF signaling is vital to PGCs migration and controls the number of germ cells in mice (Takeuchi et al. 2005). In chickens, bFGF is among the factors supporting preblastodermal cells (Park et al. 2006), embryonic germ cells (EGCs) (Park and Han, 2000), and PGCs proliferation (van de Lavoir et al. 2006). Choi et al. (2010) and Macdonald et al. (2010) identified bFGF as an fundemental factor for in vitro proliferation of chicken PGCs. In PGCs of chicken, FGF2 activates the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), that has anti-apoptosic features and promotes the cell cycle. In the FGF/MAPK/Erk pathway, the FGF ligand that binds to FGFR, a family of receptor tyrosine kinases (RTKs), causes receptor dimerization and then leading to phosphorylation RTKs. Phosphorylated RTKs activate important signal pathways, such as the MAPK/Erk and the PI3K/Akt signaling pathways (Figure 2).

 

Figure 2 FGF and GFs signaling pathway in chicken PGC self-renewal

 

When MEK and FGF2 receptors are prohibited by PD0325901 and PD173074, respectively, ERK activation does not happen. It should be noted that when MEK/ ERK signaling is not activated, PGCs started degeneration and failed to proliferate (Choi et al. 2010; Macdonald et al. 2010). It can be concluded that bFGF via MEK/ERK signaling plays a crucial role in the long‑term proliferation and survival of chicken PGCs (Choi et al. 2010). The data demonstrated that MEK/ERK is a downstream target of bFGF, which activates a wide range of second messengers and supports cell proliferation in PGCs of chicken (Choi et al. 2010). In the other hands, FGF9 and FGF8 are other FGFs family members, which involved in a variety of biological processes including differentiation, and cell growth. Among FGFs, Ulu et al.) 2017) showed that FGF9 regulates PGCs in a dose-dependent manner. FGF9 promotes differentiation into SSCs at low concentration (0.2 ng/mL), while it prohibits differentiation and facilitates PGCs self-renewal at high concentration (25 ng/mL) (Ulu et al. 2017). It has been showed that the FGF family such as FGF8 can cause in vitro proliferation of mouse PGCs (Kawase et al. 2004) and stimulate PGCs self-renewal through regulating the MAPK/ERK signal (Dolci et al. 2001; Choi et al. 2010). It was found that adding FGF8 to the mouse embryos promotes PGCs proliferation (Kawase et al. 2004). Another study showed that this was obtained through activating ERK1/2 in MAPK/ERK signaling pathway (Choi et al. 2010). Also, FGF8 was differentially expressed in the chicken SSCs, PGCs, and ESCs. It has been demonstrated that it participated in cell differentiation regulation and embryogenesis. Wang et al. (2018), found that FGF8 knockdown promotes formation of SSCs whereas its overexpression can promote chicken PGCs self-renewal and prohibit SSCs formation. In conformation with the earlier studies, researchers found the positive correlation between MAPK/ERK signaling pathway and the FGF8 expression level (Astick, 2012). The FGF8 knockdown can prohibit the MAPK/ERK signaling pathway, whereas FGF8 overexpression can enhance it. There are pieces of literature that report the differentiation of other types of germ cells that are controlled by FGFs. Kurimoto et al. (2014) revealed that ERK signaling negative regulators such as Spry2 and Dusp6, are explicitly up-regulated in PGC precursors and nascent PGCs during PGCs specification. Kimura et al. (2014) showed that FGF-ERK signaling inhibition causes induction of PGC-like cells from mouse ESCs. Significantly, overexpression of FGF8 can promote proliferation of PGCs and also prevent its differentiation into SSCs (Kimura et al. 2014). In general, the FGFs family members by activation the FGF/MAPK/Erk signaling pathway plays an important role in PGCs proliferation and consequently, it is necessary that the addition of bFGF to the cell culture medium.

 

PI3K/Akt signaling

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is another factor and mechanism involved in proliferation and survival in PSCs (Brazil et al. 2002). Indeed, the PI3K signaling, has belonged to the family of lipid kinases, exerts its biological effects through the activation of various downstream molecules such as AKT (Brazil et al. 2002; Cantley, 2002). The PI3K/AKT signaling pathway activated by growth factors such as LIF, FGF, SCF and RA through phosphorylation of the 3-hydroxyl group of the plasma membrane cell. Interestingly, activation of the PI3K/Akt signaling is performed by the interaction between Receptor tyrosine kinase (c-Kit) and its ligand (KL) (Figuer 2). In other cell types, KL binding induces autosphorylation of the c-Kit receptor, and the activated receptor, in turn, phosphorylates various substrates, thus activating cascades or distinct signaling pathways such as the Janus kinase (JAK)_signal transducer and activator of transcription (STAT), the PI3K_AKT_mTOR_p70S6K, Ras_mitogen-activated protein kinase kinase (MEK)_mitogen-activated protein kinase (MAPK), and the Src signaling pathways (Blume-Jensen et al. 1993; Rameh and Cantley, 1999; Ueda et al. 2002). So, the Akt serine/threonine kinase (also known as protein kinase B) activation is necessary for the proliferation and survival of mammalian endogenous PGCs and the in vitro establishment of EG cells (Kimura et al. 2008; Matsui et al. 1992). A number of studies suggest that PI3K/Akt signaling promotes the dedifferentiation of mice PGCs (Kimura et al. 2006). However, in chicken PGCs, the inhibition of PI3K/Akt signaling arrests in vitro proliferation and promotes apoptosis (Macdonald et al. 2010; Whyte et al. 2015). On the other hand, growth factors such as SCF, as the c-Kit ligand, activate PI3K/Akt signaling. Thus, c-Kit and its ligand are the key regulators of PGCs growth and survival (Besmer, 1993), and interaction between c-KIT (In mice, c-KIT is expressed on the surface of PGCs) and SCF is needed for migration, inhibition of apoptosis, and germ cell proliferation (Dolci et al. 1991; De Felici et al. 1999; Gu et al. 2009). These data propose that SCF has been apploed as a support factor for in vitro proliferation and survival of mouse PGCs (Dolci et al. 1991; Matsui et al. 1992; De Felici et al. 1999). Mouse SCF1 ability for the survival of PGCs culture is limited, and it is not involved in its proliferation (Dolci et al. 1991). Meanwhile, PGCs proliferation in vitro is enhanced by SCF2 and can be used for the long-term cultivation of murine PGCs (Dolci et al. 1991; De Felici et al. 1999). In various studies, SCF has also been used for the culture of PGCs in chickens (van de Lavoir et al. 2006; Choi et al. 2010; Macdonald et al. 2010; Naito et al. 2012). Some studies have suggested that administration of chicken SCF (SCF) may positively affect in vitro proliferation of chicken PGCs (Karagenç and Petitte, 2000; Glover and McGrew, 2012). However, chicken SCF2 can significantly improve the proliferation of chicken PGCs in vitro by activating the PI3K/Akt signaling pathway (Miyahara et al. 2016). RA is another important factor in the activation of the PI3K/Akt signaling pathway (Koshimizu et al. 1995; Yu et al. 2011). RA treatment can activate PI3K in cultured PGCs and subsequently induce Akt phosphorylation. These outcomes peopose that RA stimulates NF-kB activation as a downstream target modulator of PI3K/Akt signaling (Yu et al. 2011). Activated Akt induces NF-kB nuclear import and thus gives access to target gene promoters (Paling et al. 2004; Chen and Khillan, 2010; Han et al. 2010). Some of the signals that act on PGCs to initiate proliferation are produced by targeted cell cycle gene products (Ge et al. 2007; Yu et al. 2011). Inhibition of PI3K/Akt and NF-kB signaling causes the down-regulation of cell cycle regulatory genes and suppresses cell proliferation in RA-treated PGCs. RA remarkably increases the number of migratory phase PGCs and attenuates the depletion of gonadal PGCs in vitro (Matsui et al. 1992; Koshimizu et al. 1995). Yu et al. (2012) revealed that RA significantly promotes colony formation in PGCs and enhances their survival and mitotic activity. Accordingly, the results showed that the addition of RA stimulates the survival and proliferation of chicken PGCs through activation of the PI3K/Akt pathway, which includes the NF-kB signaling cascade. This suggests that activation of PI3K/Akt signaling is necessary for the proliferation and survival of chicken PGCs.

 

LIF/Stat3 signaling

Leukemia inhibitory factor (LIF) is a member of the cytokine family which contains cardiotrophin, oncostatin M, IL-11, and IL-6. LIF acts on various types of cell, such as neurons, adipocytes, hepatocytes,osteoblasts, and embryonic stem (ES) cells (Nicola and Hilton, 1997; Taga and Kishimoto, 1997). Initially, LIF was detected as an inducer of M1 leukemia cell differentiation (Tomida et al. 1984) and rediscovered as an inhibitor of mouse ES cell differentiation (Smith et al. 1988). Activation of the LIF/Stat3 signaling pathway begins when LIF binds to the cell surface via two transmembrane proteins, the LIF receptor beta (LIFRβ) and gp130. LIF uses the membrane protein gp130 to form the heterodimer receptors (Figuer1). This heterodimer then phosphorylates and activates a gp130-associated cytoplasmic tyrosine kinase - Janus kinase (Jak) - which then activates three intracellular signaling pathways: the Jak/signal transducer and activator of transcription 3 (Stat3) pathway, the PI3K/protein kinase Akt pathway, and the MAPK/ Erk pathway (Hassani et al. 2014). LIF/Jak/Stat3 are the first pluripotency-related signal transductions in mouse ES cells. Activated Jak can phosphorylate tyrosine residues on gp130 and provide a docking site for the proteins that include src homology 2 (SH2) domains such as Stat3. Furthermore, Jak directly phosphorylates Stat3 on tyrosine 705 (Stat3pTyr705), thereby leading to its homodimerization. Dimerized Stat3 enters the nucleus and regulates the expression of target genes (Hassani et al. 2014). Some research suggests a role for the JAK-STAT signaling pathway in the growth of male germ cells in Drosophila and mice (Sheng et al. 2009; Oatley et al. 2010). In mammalian PGCs, short-term PGC culture experiments and genetic knockout models have involved in the growth factor LIF in proliferationand early survival (Dolci et al. 1991; Matsui et al. 1992; Dolci et al. 1993; Farini et al. 2005). Besides, Horiuchi et al. (2004) showed that in the undifferentiated state, chicken LIF could maintain chicken ES cell cultures (Horiuhci et al. 2004). Moreover, Zhang et al. (2017) showed that the JAK-STAT pathway is involved in the proliferation, differentiation, and signal transduction of chicken PGCs. Furthermore, Zhang et al. (2017) proposed that JAK-STAT effectively promoted the PGCs formation in the genital ridge during early embryogenesis in vivo and played a positive role in the regulation of ESC differentiation into SSC in vitro, with STAT3 and JAK2 function as pivotal factors for intracellular signal transduction (Zhang et al. 2017). Thus, mammalian cytokines such as human and mouse LIF (Petitte et al. 2004) as well as avian cytokines such as LIF are able to maintain self-renewal state in chicken stem cells (Nakano et al. 2011). In general, LIF activation of the JAK/STAT pathway can lead to long-term culture of chicken stem cells (Petitte et al. 2004).

 

TGFβ, Activin and Nodal/Smad2/3 signaling

Recent studies have shown that the signal transduction through TGF-β/Activin/SMAD2/3 has been shown to be essential for the proliferation and self-renewal of PSCs. It is suggested that TGF/β signaling inhibiting mouse PGCs proliferation in culture (Godin and Wylie, 1991). While some experiments has demonstrated that self-renewal of avian PGCs (Whyte et al. 2015), similar to mammalian epiblast stem cells (epiSCs), human embryonic stem cells (hESC) and human iPSC require a TGF/β signaling for self-renewal. TGF/β ligands such as Activin A and Nodal lead to phosphorylate regulatory Smad (R-Smad) proteins through heterodimers of TGF-βR 1 and 2. R-Smads bind to co-Smad (Smad4) following phosphorylation and translocate into the nucleus and eventually lead to hundreds of genes up or down regulated within 1-2 hours (Figuer 3). This phosphorylation was ablated by the addition of one of the type II receptor chemical inhibitors SB431542, which selectively suppresses ALK1/5/7 type I receptors. Furthermore, the addition of SB431542 resulted in abrupt exit from the pluripotent state and self-renewal, and as well the activation of lineage specific genes (Yakhkeshi et al. 2018). Thus, the TGF-β/Activin and Nodal/Smad2, 3 signaling are known to perform pivotal functions in the differentiation, proliferation, migration, and apoptosis of reproductive cells. Furthermore, TGF/β has an inhibitory effect on the proliferation of at least two stem cell populations: hemopoietic stem cells and intestinal crypt cells in the mice. Many studies have suggested that TGF/β inhibiting mouse PGC proliferation in culture may be a general mechanism in the complicated process of stem cell proliferation (Godin and Wylie, 1991). Although several studies have identified TGFβ as an active signaling pathway in PGCs, the role of TGFβ in chicken PGCs remains unclear. Recently, it is represented that TGF-β/Activin/Nodal signaling pathways are active in chicken PGCs both in vivo and in vitro. It can support the self-renewal of germ-line competent PGCs in combination with insulin and FGF2 (Whyte et al. 2015). Moreover, Yakhkeshi et al. (2018) showed that through the activation of TGF-β signaling pathways by SMAD2/3 phosphorylation, Activin A leads to in vitro expansion of quail gonadal-derived PGCs. Therefore, their findings suggested that Smad2/3 signaling is a critical pathway in quail PGCs proliferation (Yakhkeshi et al. 2018). Furthermore, it has been showed that signal transduction by TGF-β/Activin/Nodal is crucial for chicken PGC proliferation (Macdonald et al. 2010; Whyte et al. 2015; Lee et al. 2016).

 

Figure 3 BMP/SMAD and Activin/Nodal Signaling Pathway in Chicken PGC Self-renewal

 

BMP4/Smad1/5/8 signaling

Bmp4 is a multifunctional cytokine that belongs to the TGFβ superfamily of intercellular signaling proteins. As other members of the TGFβ superfamily, BMPs transduce their signals by binding and inducing the dimerization of different type I and type II serine/threonine kinase receptors, and cause phosphorylation of the type I receptors (BMP receptor 1A (BMPR1A) or BMP receptor1B (BMPR1B)). The activation of BMP receptors causes phosphorylation of SMAD1/5/8 (R-Smads ) proteins (Figuer 3). Afterwards, R-Smads bind to co-Smad (Smad4) and translocate into the nucleus, where they act as transcription factors and regulate the expression of target genes (Kawabata et al. 1998). The functional significance of BMP4 and its signal transduction pathway components in PGCs differentiation during embryonic gonad development has been documented through gene knockout mouse models. For instance, Bmp4-null mutant embryos are deficient in the development of PGCs (Lawson et al .1999), and other studies have shown that BMPR1 (de Sousa Lopes et al. 2004) and its signal transducers Smad1 (Tremblay et al. 2001; Hayashi et al. 2002) and Smad5 (Chang and and Matzuk, 2001) are also essential for PGCs formation. Researches in the mouse (Lawson et al. 1999; Ying and Zhao, 2000; Ying and Zhao, 2001) and the axolotl (Johnson et al. 2003; Chatfield et al. 2014) showed that BMP signal transduction is essential for specifying PGCs through induction and for the initial formation of the germ cell lineage and PGC survival (Lawson et al. 1999; Ying and Zhao, 2001; Farini et al. 2005). While few studies have concentrated on the specific role of BMP signaling in the PGC formation in chickens, some researches show that this pathway promotes PGCs proliferation in the chicken, and their migration in embryos may be regulated by this pathway (Whyte et al. 2015). Recently Zuo et al. (2019) showed that BMP4 and RA signaling play a different role in PGC formations (Zuo et al. 2019). RA is the most usual reagent applied during in vitro germ cell differentiation of pluripotent cells (Imamura et al. 2014). Among genes influenced by RA treatment, there are members of the BMPs family including BMP2, BMP4, and BMP8b which activate Smad1/5 in the PGCs. Moreover, the BMP-Smad1/5 signaling pathway is necessary for PGCs proliferation and specification (Tremblay et al. 2001; Chang et al. 2012; Whyte et al. 2015). As a result, the BMP4 and RA signaling pathways play opposite roles in the formation of germ cell driven by epigenetic processes including histone acetylation, phosphorylation, and ubiquitination. Also, Smurf and Smad5 are crucial to the interaction between the two pathways (Zuo et al. 2019).

 

CONCLUSION

Maintaining a well-balanced network for transmitting signals has been shown to play an important role in self-renewal and cell line production in stem cell cultures. Wnt/Gsk3/β-catenin, TGF-β/Activin/Nodal and FGF/MAPK/Erk signal pathways are considered to be the most vital pathways for self-renewal and long-term culture of PCGs. Interestingly, the signaling pathways that support PGC pluripotency and proliferation exhibit diverse functions in different species. For example, the TGF-β/Activin pathways in mouse PGCs lead to inhibition of self- renewal, which are thought to be prominent signaling pathways regarding maintenance of pluripotency and long-term culture of chicken PGCs. TGF-β signaling pathway appears to be the only signaling pathway which is essential in maintaining pluripotency in chicken PGCs. However, a closer look reveals that the connections of all signaling pathways lead to the production of cell lines. Overall, in vivo and in vitro evidence suggest that chicken PGCs are quite different from mammalian PGCs due to their unique development in terms of functional behavior and signal transduction mechanisms. Although some recent studies have shown the production of PGCs, the demonstration of all pluripotent properties in long-term culture has not yet been achieved. It appears the pluripotency to be a unique phenomenon, and the production of PGC lines requires further efforts to identify interactions between underlying molecular mechanisms and signaling pathways. To use PGCs for biotechnology purposes, it is essential to obtain evidence of how to stabilize pluripotency in culture.

 

ACKNOWLEDGEMENT

The authors deeply appreciate the efforts by their collaborators at Department of Stem Cells and Developmental Biology at Royan Institute.

مراجع

Aramaki S., Hayashi K., Kurimoto K., Ohta H., Yabuta Y., Iwanari H., Mochizuki Y., Hamakubo T., Kato Y., Shirahige K. and Saitou M. (2013). A mesodermal factor, T, specifies mouse germ cell fate by directly activating germline determinants. Dev. Cell. 27, 516-529.

Astick M.R. (2012). Fgf regulated cadherin expression in cranial nucleogenesis. PhD Thesis. University College London, London, United Kingdom.

Besmer P. (1993). The kit- ligand (steel factor) and its receptor c-kit/W: Pleiotropic roles in gametogenesis and melanogenesis. Development. 1, 125-137.

Bialecka M., Young T., de Sousa Lopes S.C., ten Berge D., Sanders A., Beck F. and Deschamps J. (2012). Cdx2 contributes to the expansion of the early primordial germ cell population in the mouse. Dev. Biol. 371, 227-234.

Blume-Jensen P., Siegbahn A., Stabel S., Heldin C.H. and Rönnstrand L. (1993). Increased Kit/SCF receptor induced mitogenicity but abolished cell motility after inhibition of protein kinase C. EMBO J. 12, 4199-4209.

Brazil D.P., Park J. and Hemmings B.A. (2002). PKB binding proteins. Getting in on the Akt cell. J. Cell. 111, 293-303.

Cantley L.C. (2002). The phosphoinositide 3-kinase pathway. Science. 296, 1655-1657.

Chang H. and Matzuk M.M. (2001). Smad5 is required for mouse primordial germ cell development. Mech. Dev. 104, 61-7.

Chang J.S., Fernand V., Zhang Y., Shin J., Jun H.J., Joshi Y. and Gettys T.W. (2012). NT-PGC-1α protein is sufficient to link β3-adrenergic receptor activation to transcriptional and physiological components of adaptive thermogenesis. J. Biol. Chem. 287, 9100-9111.

Chassot A.A., Bradford S.T., Auguste A., Gregoire E.P., Pailhoux E., De Rooij D.G., Schedl A. and Chaboissier M.C. (2012). WNT4 and RSPO1 together are required for cell proliferation in the early mouse gonad. Development. 139, 4461-4472.

Chatfield J., O'Reilly M.A., Bachvarova R.F., Ferjentsik Z., Redwood C., Walmsley M., Patient R., Loose M. and Johnson A.D. (2014). Stochastic specification of primordial germ cells from mesoderm precursors in axolotl embryos. Development. 141, 2429-2440.

Chawengsaksophak K., Svingen T., Ng E.T., Epp T., Spiller C.M., Clark C., Cooper H. and Koopman P. (2012). Loss of Wnt5a disrupts primordial germ cell migration and male sexual development in mice. Biol. Rep. 86, 4-11.

Chen L. and Khillan J.S. (2010). A novel signaling by vitamin A/retinol promotes self renewal of mouse embryonic stem cells by activating PI3K/Akt signaling pathway via insulin-like growth factor-1 receptor. Stem Cells. 28, 57-63.

Choi J.W., Kim S., Kim T.M., Kim Y.M., Seo H.W., Park T.S., Jeong J.W., Song G. and Han J.Y. (2010). Basic fibroblast growth factor activates MEK/ERK cell signaling pathway and stimulates the proliferation of chicken primordial germ cells. PLoS One . 5, e12968.

De Felici M., Carlo A.D., Pesce M., Iona S., Farrace M.G. and Piacentini M. (1999). Bcl-2 and Bax regulation of apoptosis in germ cells during prenatal oogenesis in the mouse embryo. Cell Death Differ. 6, 908-915.

De Felici M., Farini D. and Dolci S. (2009). In or out stemness: comparing growth factor signalling in mouse embryonic stem cells and primordial germ cells. Curr. Stem. Cell. Res. Ther. 4, 87-97.

De Sousa Lopes S.M., Roelen B.A., Monteiro R.M., Emmens R., Lin H.Y., Li E., Lawson K.A. and Mummery C.L. (2004). BMP signaling mediated by ALK2 in the visceral endoderm is necessary for the generation of primordial germ cells in the mouse embryo. Genes Dev. 18, 1838-1849.

Delaune E., Lemaire P. and Kodjabachian L. (2005). Neural induction in Xenopus requires early FGF signalling in addition to BMP inhibition. Development. 132, 299-310.

Ding V.M., Ling L., Natarajan S., Yap M.G., Cool S.M. and Choo A.B. (2010). FGF-2 modulates Wnt signaling in undifferentiated hESC and iPS cells through activated PI3‐K/GSK3β signaling. J. Cell. Physiol. 225, 417-428.

Dionne C.A., Crumley G., Bellot F., Kaplow J.M., Searfoss G., Ruta M., Burgess W.H., Jaye M. and Schlessinger J. (1990). Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors. EMBO J. 9, 2685-2692.

Dolci S., Williams D.E., Ernst M.K., Resnick J.L., Brannan C.I., Lock L.F., Lyman S.D., Boswell H.S. and Donovan P.J. (1991). Requirement for mast cell growth factor for primordial germ cell survival in culture. Nature. 352, 809-811.

Dolci S., Pesce M. and De Felici M. (1993). Combined action of stem cell factor, leukemia inhibitory factor, and cAMP on in vitro proliferation of mouse primordial germ cells. Mol. Reprod. Dev. 35, 134-139.

Dolci S., Pellegrini M., Di A.S., Geremia R. and Rossi P. (2001). Signaling through extracellular signal-regulated kinase is required for spermatogonial proliferative response to stem cell factor. J. Biol. Chem. 276, 40225-40233.

Farini D., Scaldaferri M.L., Iona S., La Sala G. and De Felici M. (2005). Growth factors sustain primordial germ cell survival, proliferation and entering into meiosis in the absence of somatic cells. Dev. Biol. 285, 49-56.

Feldman B., Poueymirou W., Papaioannou V.E., DeChiara T.M. and Goldfarb M. (1995). Requirement of FGF-4 for postimplantationmouse development. Science. 267, 246-249.

Fu Y., Zheng S., An N., Athanasopoulos T., Popplewell L., Liang A., Li K., Hu C. and Zhu Y. (2011). Β-Catenin as a potential key target for tumor suppression. Int. J. Cancer. 129, 1541-1551.

Ge S., Yang C.H., Hsu K.S., Ming G.L. and Song H. (2007). A critical period for enhanced synaptic plasticity in newly generated neurons of the adult brain. Neuron. 54, 559-566.

Ge C., Yu M. and Zhang C. (2012). G protein-coupled receptor 30 mediates estrogen-induced proliferation of primordial germ cells via EGFR/Akt/β-catenin signaling pathway. Endocrinology. 153, 3504-3516.

Glover J.D. and McGrew M.J. (2012). Primordial germ cells technologies for avian germplasm cryopreservation and investigating germ cell development. J. Poult. Sci. 49, 155-162.

Godin I. and Wylie C.C. (1991). TGF beta 1 inhibits proliferation and has a chemotropic effect on mouse primordial germ cells in culture. Development. 113, 1451-1457.

Gu Y., Runyan C., Shoemaker A., Surani A. and Wylie C. (2009). Steel factor controls primordial germ cell survival and motility from the time of their specification in the allantois, and provides a continuous niche throughout their migration. J. Dev. Sci. 136, 1295-1303.

Han J.Y. (2009). Germ cells and transgenesis in chickens. Comp. Immunol. Microbiol. Infect. Dis. 32, 61-80.

Han L., Yang Y., Yue X., Huang K., Liu X., Pu P., Jiang H., Yan W., Jiang T. and Kang C. (2010). Inactivation of PI3K/AKT signaling inhibits glioma cell growth through modulation of β-catenin-mediated transcription. Brain Res. 1366, 9-17.

Hassani S.N., Totonchi M., Sharifi-Zarchi A., Mollamohammadi S., Pakzad M., Moradi S., Samadian A., Masoudi N., Mirshahvaladi S., Farrokhi A. and Greber B. (2014). Inhibition of TGFβ signaling promotes ground state pluripotency. Stem Cell Rev. Rep. 10, 16-30.

Hayashi K., Kobayashi T., Umino T., Goitsuka R., Matsui Y. and Kitamura D. (2002). SMAD1 signaling is critical for initial commitment of germ cell lineage from mouse epiblast. Mech. Dev. 118, 99-109.

Horiuchi H., Tategaki A., Yamashita Y., Hisamatsu H., Ogawa M., Noguchi T., Aosasa M., Kawashima T., Akita S., Nishimichi N. and Mitsui N. (2004). Chicken leukemia inhibitory factor maintains chicken embryonic stem cells in the undifferentiated state. J. Biol. Chem. 279, 24514-24520.

Imamura M., Hikabe O., Lin Z.Y.C. and Okano H. (2014). Generation of germ cells in vitro in the era of induced pluripotent stem cells. Mol. Reprod. Dev. 81, 2-19.

Israsena N., Hu M., Fu W., Kan L. and Kessler J.A. (2004). The presence of FGF2 signaling determines whether beta-catenin exerts effects on proliferation or neuronal differentiation of neural stem cells. Dev. Biol. 268, 220-231.

Itoh N. (2007). The Fgf families in humans, mice, and zebrafish: their evolutional processes and roles in development, metabolism, and disease. Biol. Pharm. Bull. 30, 1819-1825.

Johnson A.D., Crother B., White M.E., Patient R., Bachvarova R.F., Drum M. and Masi T. (2003). Regulative germ cell specification in axolotl embryos: a primitive trait conserved in the mammalian lineage. Biol. Sci. 358, 1371-1379.

Karagenç L. and Petitte J.N. (2000). Soluble factors and the emergence of chick primordial germ cells in vitro. Poult. Sci. 79, 80-85.

Kawabata M., Inoue H., Hanyu A., Imamura T. and Miyazono K. (1998). Smad proteins exist as monomers in vivo and undergo homo-and hetero-oligomerization upon activation by serine/threonine kinase receptors. EMBO J. 17, 4056-4065.

Kawase E., Hashimoto K. and Pedersen R.A. (2004). Autocrine and paracrine mechanisms regulating primordial germ cell proliferation. Mol. Reprod. Dev. 68, 5-16.

Kim H.S. (1998). Assignment1 of the human basic fibroblast growth factor gene FGF2 to chromosome 4 band q26 by radiation hybrid mapping . Cytogenet. Cell. Genet. 83, 73-81.

Kimura T., Nakamura T., Murayama K., Umehara H.. Yamano N., Watanabe S., Taketo M.M. and Nakano T. (2006). The stabilization of β-catenin leads to impaired primordial germ cell development via aberrant cell cycle progression. Dev. Biol. 300, 545-553.

Kimura T., Tomooka M., Yamano N., Murayama K., Matoba S., Umehara H., Kanai Y. and Nakano T. (2008). AKT signaling promotes derivation of embryonic germ cells from primordial germ cells. Development. 135, 869-879.

Kimura T., Kaga Y., Ohta H., Odamoto M., Sekita Y., Li K., Yamano N., Fujikawa K., Isotani A. and Sasaki N. (2014). Induction of primordial germ cell-like cells from mouse embryonic stem cells by ERK signal inhibition. Stem Cells. 32, 2668-2672.

Koshimizu U., Watanabe M. and Nakatsuji N. (1995). Retinoic acid is a potent growth activator of mouse primordial germ cells in vitro. Dev. Biol. 168, 683-685.

Kurimoto S., Jung J., Tapadia M., Waterman M., Mozaffar T. and Gupta R. (2014). Targeting the Wnt/ß-Catenin signaling pathway after traumatic nerve injury to improve functional recovery: N/A-Not a clinical study. J. Hand Surg. Am. 39, 13-17.

Laird D.J., Altshuler-Keylin S., Kissner M.D., Zhou X. and Anderson K.V. (2011). Ror2 enhances polarity and directional migration of primordial germ cells. PLoS Genet. 7, e1002428.

Lawson K.A., Dunn N.R., Roelen B.A., Zeinstra L.M., Davis A.M., Wright C.V., Korving J.P. and Hogan B.L. (1999). Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev. 13, 424-36. 

Lee H.C., Lim S. and Han J.Y. (2016). Wnt/β-catenin signaling pathway activation is required for proliferation of chicken primordial germ cells in vitro. Sci. Rep. 6, 1-8.

Logan C.Y. and Nusse R. (2004). The Wnt signaling pathway in development and disease. Annu. Rev. Cell Dev. Biol. 20, 781-810.

Macdonald J., Glover J.D., Taylor L., Sang H.M. and McGrew M.J. (2010). Characterisation and germline transmission of cultured avian primordial germ cells. PLoS One. 5, e15518.

Matsui Y., Zsebo K. and Hogan B.L. (1992). Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell. 70, 841-847.

Miyahara D., Mori T., Makino R., Nakamura Y., Oishi I., Ono T., Nirasawa K., Tagami T. and Kagami H. (2014). Culture conditions for maintain propagation, long-term survival and germline transmission of chicken primordial germ cell-like cells. J. Poult. Sci. 51, 87-95.

Miyahara D., Oishi I., Makino R., Kurumisawa N., Nakaya R., Ono T., Kagami H. and Tagami T. (2016). Chicken stem cell factor enhances primordial germ cell proliferation cooperatively with fibroblast growth factor 2. J. Rep. Dev. 62, 143-149.

Naito M., Harumi T. and Kuwana T. (2012). Expression of GFP gene in cultured PGCs isolated from embryonic blood and incorporation into gonads of recipient embryos. J. Poult. Sci. 2, 116-121.

Nakano M., Arisawa K., Yokoyama S., Nishimoto. M., Yamashita Y., Sakashita M., Ezaki R., Matsuda H., Furusawa S. and Horiuchi H. (2011). Characteristics of novel chicken embryonic stem cells established using chicken leukemia inhibitory factor. J. Poult. Sci. 48, 64-72.

Nicola N.A. and Hilton D.J. (1997). Growth Factors and Cytokines in Health and Disease. JAI Press, Greenwich, United Kingdom.

Oatley J.M., Kaucher A.V., Avarbock M.R. and Brinster R.L. (2010). Regulation of mouse spermatogonial stem cell differentiation by STAT3 signaling. Biol. Rep. 83, 427-433.

Ohinata Y., Ohta H., Shigeta M., Yamanaka K., Wakayama T. and Saitou M. (2009). A signaling principle for the specification of the germ cell lineage in mice. Cell. 137, 571-584.

Otero J.J., Fu W., Kan L., Cuadra A.E. and Kessler J.A. (2004). β-Catenin signaling is required for neural differentiation of embryonic stem cells. Development. 131, 3545-3557

Paling N.R.D., Wheadon H., Bone H.K. and Welham M.J. (2004). Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. J. Biol. Chem. 279, 48063-48070.

Park H.J., Park T.S., Kim T.M., Kim J.N., Shin S.S., Lim J.M. and Han J.Y. (2006). Establishment of an in vitro culture system for chicken preblastodermal cells. Mol. Reprod. Dev. 73, 452-461.

Park T.S. and Han J.Y. (2000). Derivation and characterization of pluripotent embryonic germ cells in chicken. Mol. Reprod. Dev. 56, 475-482 .

Petitte J., Liu G. and Yang Z. (2004). Avian pluripotent stem cells. Mech. Dev. 121, 1159-1168.

Polakis P. (2012). Wnt signaling in cancer. Cold Spring Harb. Perspect. Biol. 4, a008052.

Rameh L.E. and Cantley L.C. (1999). The role of phosphoinositide 3-kinase lipid products in cell function. J. Biol. Chem. 274, 8347-8350.

Resnick J.L., Bixler L.S., Cheng L. and Donovan P.J. (1992). Long-term proliferation of mouse primordial germ cells in culture. Nature. 359, 550-551.

Resnick J.L., Ortiz M., Keller J.R. and Donovan P.J. (1998). Role of fibroblast growth factors and their receptors in mouse primordial germ cell growth. Biol. Reprod. 59, 1224-1229.

Sato N., Meijer L., Skaltsounis L., Greengard P. and Brivanlou A.H. (2004). Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat. Med. 10, 55-63.

Sheng X.R., Posenau T., Gumulak-Smith J.J., Matunis E., Van Doren M. and Wawersik M. (2009). Jak–STAT regulation of male germline stem cell establishment during Drosophila embryogenesis. Dev. Biol. 334, 335-344.

Smith A.G., Heath J.K., Donaldson D.D., Wong G.G., Moreau J., Stahl M. and Rogers D. (1988). Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature. 336, 688-690.

Sun F., Fu H., Liu Q., Tie Y., Zhu J., Xing R., Sun Z. and Zheng X. (2008). Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest. FEBS Lett. 582, 1564-1568.

Taga T. and Kishimoto T. (1997). Gp130 and the interleukin-6 family of cytokines. Annu. Rev. Immunol. 15, 797-805.

Takeuchi Y., Molyneaux K., Runyan C., Schaible K. and Wylie C. (2005). The roles of FGF signaling in germ cell migration in the mouse. Development. 132, 5399-5409.

Tanaka S.S., Nakane A., Yamaguchi Y.L., Terabayashi T., Abe T., Nakao K., Asashima M., Steiner K.A., Tam P.P. and Nishinakamura R. (2013). Dullard/Ctdnep1 modulates WNT signalling activity for the formation of primordial germ cells in the mouse embryo. PLoS One. 8, e57428.

Tang X., Zhang C., Zeng W., Mi Y. and Liu H. (2006). Proliferating effects of the flavonoids daidzein and quercetin on cultured chicken primordial germ cells through antioxidant action. Cell Biol. Int. 30, 445-451.

Tomida M., Yamamoto-Yamaguchi Y. and Hozumi M. (1984). Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells. J. Biol. Chem. 259, 10978-10982.

Tremblay K.D., Dunn N.R. and Robertson E.J. (2001). Mouse embryos lacking Smad1 signals display defects in extra-embryonic tissues and germ cell formation. Development. 128, 3609-3621.

Ueda S., Mizuki M., Ikeda H., Tsujimura T., Matsumura I., Nakano K., Daino H., Hondaz I.Z., Sonoyama J., Shibayama H. and Sugahara H. (2002). Critical roles of c-Kit tyrosine residues 567 and 719 in stem cell factor–induced chemotaxis: contribution of src family kinase and PI3-kinase on calcium mobilization and cell migration. Blood. 99, 3342-3349.

Ulu F., Kim S.M., Yokoyama T. and Yamazaki Y. (2017). Dose-dependent functions of fibroblast growth factor 9 regulate the fate of murine XY primordial germ cells. Biol. Reprod. 96, 122-129.

Vainio S., Heikkilä M., Kispert A., Chin N. and McMahon A.P. (1999). Female development in mammals is regulated by Wnt-4 signalling. Nature. 397, 405-409.

Valenta T., Gay M., Steiner S., Draganova K., Zemke M., Hoffmans R., Cinelli P., Aguet M., Sommer L. and Basler K. (2011). Probing transcription-specific outputs of β-catenin in vivo. Genes Dev. 25, 2631-2643.

Valenta T.. Hausmann G. and Basler K. (2012). The many faces and functions of β-catenin. EMBO J. 31, 2714-2736.

Van de Lavoir M.C., Diamond J.H., Leighton P.A., Mather‑Love C., Heyer B.S., Bradshaw R., Kerchner A., Hooi L.T., Gessaro T.M., Swanberg S.E. and Delany M.E. (2006). Germline transmission of genetically modified primordial germ cells. Nature. 441, 766-769.

Wang M., Zhang C., Huang C., Cheng S., He N., Wang Y., Ahmed M.F., Zhao R., Jin J., Zuo Q. and Zhang Y. (2018). Regulation of fibroblast growth factor 8 (FGF8) in chicken embryonic stem cells differentiation into spermatogonial stem cells. J. Cell. Biochem. 119, 2396-2407.

Whyte J., Glover J.D., Woodcock M., Brzeszczynska J., Taylor L., Sherman A., Kaiser P. and McGrew M.J. (2015). FGF, insulin, and SMAD signaling cooperate for avian primordial germ cell self-renewal. Stem Cell Rep. 5, 1-12.

Yakhkeshi S., Rahimi S., Sharafi M., Hassani S.N., Taleahmad S., Shahverdi A. and Baharvand H. (2018). In vitro improvement of quail primordial germ cell expansion through activation of TGF-beta signaling pathway. J. Cell. Biochem. 119, 4309-4319.

Ying Y. and Zhao G.Q. (2000). Detection of multiple bone morphogenetic protein messenger ribonucleic acids and their signal transducer, Smad1, during mouse decidualization. Biol. Rep. 63, 1781-1786.

Ying Y. and Zhao G.Q. (2001). Cooperation of endoderm-derived BMP2 and extraembryonic ectoderm-derived BMP4 in primordial germ cell generation in the mouse. Dev. Biol. 232, 484-492.

Yu X., Shen N., Zhang M.L., Pan F.Y., Wang C., Jia W.P., Liu C., Gao Q., Gao X., Xue B. and Li C.J. (2011). Egr‐1 decreases adipocyte insulin sensitivity by tilting PI3K/Akt and MAPK signal balance in mice. EMBO J. 30, 3754-3765.

Zhang Y., Zhang L., Zuo Q., Wang Y., Zhang Y., Xu Q., Li B. and Chen G. (2017). JAK-STAT signaling regulation of chicken embryonic stem cell differentiation into male germ cells. In vitro Cell. Dev. Biol. 53, 728-743.

Zuo Q., Jin J., Jin K., Sun C., Song J., Zhang Y., Chen G. and Li B. (2019). Distinct roles of retinoic acid and BMP4 pathways in the formation of chicken primordial germ cells and spermatogonial stem cells. Food Funct. 10, 7152-7163.