{"paper_id":"7fb05b9a-0442-4fde-b945-d6a39ddde1d1","body_text":"The most important issues in human reproductive medicine are disorders of sex development (DSD) and infertility. According to data, the proportion of DSD patients ranges between 1:2000–1:4500 [ 1 ]; however, the underlying mechanisms of this congenital disease remain unclear, and genetic diagnostic cannot be performed on up to 75% of patients [ 2 ]. As for infertility, 8–12% of couples reproductive-aged are affected by this condition globally [ 3 ], which can also be caused by genetic background. In order to diagnose and treat DSD and infertility, a substantial amount of research has been devoted to sex determination, whose abnormality can result in these. In mammal, this process is governed by sex chromosomes [ 4 ] and involves the sexual differentiation of bipotential gonads and primordial germ cells (PGCs) [ 5 ]. In livestock production, the molecular mechanism of sex determination has been a research hotspot for many years because it has the potential to significantly increase production efficiency; however, a lack of theoretical knowledge prevents commercial application impossible [ 6 ]. In this review, we focused primarily on molecular, cellular, and genetic studies on early gonadal development and sexual differentiation of bipotential gonads, both with PGC formation, migration, and gender-specific differentiation. Although some studies in this field have been reviewed elsewhere [ 7 , 8 , 9 , 10 , 11 , 12 ], we summarized these review articles and combined single-cell RNA sequencing results on the basis of the original research to discuss some potential mechanistic links between bipotential gonads and PGC and find genes that play key roles in multiple stages of early gonadal development and sex determination. On the other hand, we described novel gene editing experiments that lead to sex reversal, potential gender differences before sex-specific differentiation, and the function of epigenetic regulation in these processes, with the hope of informing future studies on abnormal development of reproductive organs and infertility in humans, as well as sex manipulation technologies in livestock.\n\nIn mammal, both the testis and ovary develop from the genital ridge (GR), which first appears at approximately four-five weeks of pregnancy in humans [ 13 ] and around embryonic day (E) nine and a half in mice [ 8 ], when coelomic epithelial cells begin to proliferate on the ventromedial surface of the mesonephros [ 14 , 15 , 16 ]. Each mesonephros contains a Wolffian duct and a Müllerian duct, which give rise to the epididymis, vas deferens, and seminal vesicles [ 17 , 18 ] or the fallopian tubes, uterus, and a portion of the vagina [ 19 ]. At around 32 days of pregnancy in humans (E10.5 in mice), coelomic epithelial cells differentiate into two distinct somatic precursor lineages (supporting cell precursors and steroidogenic cell precursors) [ 20 , 21 ]. Recent single-cell sequencing research has confirmed that mammalian gonadal cells originate from the same progenitor [ 22 , 23 ]. However, little is known about the development of the gonad prior to sex determination, as the bipotential gonad contains multiple uncharacterized subpopulations lacking specific markers [ 22 ]. In the last few decades, knockout mice models and mutation data from DSD patients have been used extensively to investigate transcription factors essential for genital ridge formation. These important genes are detailed in  Table 1  and  Figure 1 . However, these genes, such as  GATA4 ,  POD1 ,  PBX1 , and  ODD1 , were also involved in the formation of various organs [ 24 ], which hinders the effectiveness of research in the field.\n\nAt approximately 6–7 weeks of pregnancy in humans (E12.5 in mice), testis cords are observed in XY gonads, indicating the start of sex differentiation in bipotential gonads [ 51 , 52 , 53 , 54 ]. Controlled by gene expression dynamics and antagonistic genetic programs, sexual differentiation begins when the antagonistic network’s balance is tilted toward males or females. Furthermore, these antagonistic genetic programs will be maintained into adulthood to preserve gonad stability and reproductive capacity [ 55 ]. The  SRY  on the Y chromosome is the “master switch” for testis determination in mammal. When  SRY  is expressed in bipotential gonads during a critical window of fetal development,  SOX9  expression and a male-promoting regulatory network are activated, resulting in testis differentiation. In contrast, ovary differentiation will be triggered when the balance is tilted towards a female-promoting regulatory network [ 56 ]. However, recent research has not identified genes with a similar function role to  SRY  in female sexual differentiation. By inhibiting  SOX9  expression,  WNT4 ,  RSPO1 , and  FOXL2  were previously involved in the ovarian-determination pathway.\nIn 1989, a 35-kb region from the Y chromosome was identified in 46,XX DSD patients, and it was believed to be the possible carrier of the testis-determining factor gene [ 57 ]. A year later, an open reading frame (ORF) encoding a new gene, later designated  SRY  (sex-determining region Y), was discovered in this Y fragment [ 58 ]. The  SRY  encodes a transcription factor with an N-terminal domain (NTD), a high conserved mobility group (HMG) domain, and a C-terminal domain (CTD) [ 59 , 60 ]. The  SRY  mutation analysis revealed that the NTD in  SRY  is associated with nuclear importation [ 61 , 62 ], whereas the CTD may contribute to the conformation and function of  SRY  [ 63 ] and be required for  SOX9  activation [ 60 ]. Most human male-to-female sex reversal syndrome cases are believed to be caused by a mutation located in the HMG domain of  SRY  [ 64 , 65 ]. Recent gene-editing research demonstrated that knockout of the HMG domain of the porcine  SRY  gene could result in male-to-female sex reversal [ 66 ]; however, additional tests are necessary to confirm the fertility of these transgenic animals. Interestingly, a study found that XX mice carrying a chimeric  SRY / SOX  construct (replacing the HMG domain of  SRY  with the HMG domain of  SOX3  or  SOX9 ) also exhibited sexual reversal [ 67 ], suggesting that  SOX3  and  SOX9  HMG domains can functionally replace  SRY  HMG domain. In addition, a two-exon  SRY  transcript was required for male testis determination, disproving the theory that  SRY  is a single-exon gene [ 68 ].\nDuring gonadal development in mice, the  SRY  is initially expressed in Sertoli cell progenitors at E10.5, reaches its maximum expression at E11.5, and then disappears at E12.5. In contrast,  SRY  expression in humans begins around 41 days of pregnancy and peaks at 44 days [ 69 ]. In contrast to mice, human  SRY  expression gradually decreases to a base level around 60 days of pregnancy and is maintained until adulthood [ 70 ]. In addition,  SRY  expression in the post-testis determination stage has been detected in goats, sheep, pigs, rabbits, and cattle [ 71 ], prompting additional research into the mechanism of the male-promoting regulatory network in mice following sex determination.  SRY  expression is non-synchronous in the gonad; the wave of  SRY  expression moves from the center to the poles between E10.5 and E12.5, limiting transcriptomics research on sex determination to some extent [ 72 , 73 ].\nThe  SOX9  expression reaches a plateau around 48 days of pregnancy in human testis [ 70 ] due to the synergistic action of  SRY  and  NR5A1  [ 74 ] (E11.5–12.5 in mice [ 75 ]), thereby activating the male-promoting regulatory network and testis determination [ 76 , 77 ], followed by suppressing  SRY  expression and binding to  NR5A1  to maintain expression [ 78 ]. In addition,  FGF9  and  PGD2  signaling pathways are activated after activation of testis-related genes, repression of anti-testis genes, inducing Supporting-to-Sertoli cell differentiation [ 79 ]. The  SOX9  was sufficient to induce testis differentiation in the absence of  SRY  in transgenic XX gonads [ 80 , 81 ], which was later confirmed in a 46,XX mosaic male patient [ 82 ]. Therefore,  SRY  may only regulate  SOX9  expression during testis development, which requires further investigation.\nThe  WNT4  is a member of the  WNT  family, essential for early embryonic development, the transition between naive and primed embryonic stem cells (ESC), and tissue homeostasis in adults [ 83 ]. The  WNT4  is initially expressed in the undifferentiated early gonad at E11.25 [ 84 ], and  WNT4  knockout translated into a significant increase in steroidogenic cells in both sexes [ 85 ]. In addition, the proliferation of coelomic epithelial cells was reported to be inhibited in the early gonads of  WNT4 - RSPO1 -double-knockout mice [ 84 ], indicating that  WNT4  may play the same role in both sexes during the early stages of gonadal development. Moreover, Müllerian duct formation failed in male and female  WNT4 −/−  mice [ 86 ]. Similarly,  WNT4  mutant 46,XX was found to have Müllerian duct abnormalities [ 87 ] and regression [ 88 ]. This suggests that  WNT4  is necessary for regulating the histogeny of the Müllerian duct in both sexes.\nThe  WNT4  is a component of the  WNT / β-catenin  signaling pathway and is essential in female sexual differentiation. The activation of the  WNT / β-catenin  signaling pathway is first detected in both sexes’ bipotential gonads at E11.5, acting as an anti-testicular agent by limiting the expression of  SOX9  [ 84 ], but is downregulated by  SRY  in males [ 89 ]. An increase in  WNT4  copies in humans was shown to result in a male-to-female sex reversal in 46,XY patients [ 90 ], while  WNT4  inactivation or mutation resulted in sex reversion–kidneys, adrenal, and lung dysgenesis (SERKAL) syndrome [ 91 ] or virilization [ 88 ]. The  WNT4  participates in accessory gland development by regulating hormone secretion, e.g., in  WNT4 −/−  XX mice, genes involved in testosterone (a hormone involved in the formation of the epididymis, vas deferens, and seminal vesicles) synthesis were found elevated [ 92 ]. In contrast, steroidogenic enzymes 3β-hydroxysteroid dehydrogenase and 17α-hydroxylase, which are required for testosterone synthesis, were expressed in ovaries [ 86 ]. However, these transgenic models do not affect steroidogenic cell differentiation [ 93 ], revealing that  WNT4  inhibits testosterone secretion by antagonizing steroidogenic cell migration rather than steroidogenic cell differentiation [ 94 ]. In humans,  WNT4  mutant 46,XX patients were reported to suffer hyperandrogenism [ 87 ].\nThe  WNT4  is required after sexual differentiation to prevent the formation of testis-specific vasculature, one of the earliest morphological changes during testicular differentiation [ 93 , 95 ]. In addition, it plays an essential role in the survival of oocytes and the maintenance of ovarian function [ 93 , 96 ]. Moreover, it is required for secreting steroid hormones in granulosa cells, which regulate normal ovarian follicle development and female fertility [ 97 ].\nThe  RSPO1  was discovered in the dorsal neural tube of mice in 2004 [ 98 ]. Since then, the  RSPO1  family has been extensively studied, and the other three family members ( RSPO2 ,  RSPO3 , and  RSPO4 ) were discovered later. In mammal, these four  RSPO1  family members have similar domain organization and are essential for embryogenesis, development, and tumorigenesis [ 98 , 99 , 100 ].\nThe  RSPO1  is mainly expressed in mice XX gonadal somatic cells during ovary determination and suppressed in mice XY gonad, with only interstitial cells having low expression [ 101 , 102 ]. Loss-of-function experiment showed that  RSPO1  knockout led to sex reversal and formation of ovotestis in XX mice [ 103 ]. In humans, the  RSPO1  mutation caused hermaphroditism [ 104 ], palmoplantar hyperkeratosis, and squamous cell carcinoma [ 101 ]. Furthermore, RSPO1 functions conservatively in various vertebrates during ovarian development. In a recent study, goat BAC clones containing the  RSPO1  gene ( gRSPO1 ) were injected into mouse oocytes, which resulted in the restoration of sex-reversal in  RSPO1  knockout XX mice [ 105 ]. Although the function of  RSPO1  in inhibiting testicular differentiation still requires further research, the result described above provides essential insights into DSD treatment.\nWith the deepening of research, RSPO1 protein has been identified as an agonist of the  WNT / β-catenin  signaling pathway [ 106 ];  RSPO1 −/−  mice demonstrated an absence of activation of  WNT4  [ 102 ]. In addition, the ovarian phenotype of  RSPO1  knockout mice recapitulated with those of  WNT4  knockout female mice [ 107 ]. The  RSPO1  mutation 46,XX ovotestis, reduced expression of β-catenin protein and  WNT4  mRNA, restricted ovarian differentiation. Transfection of  RSPO1  resulted in activation of the β-catenin responsive TOPFLASH reporter (1.8-fold maximum), whereas  RSPO1  and  CTNNB1  (encoding β-catenin) synergy resulted in a 10-folds increased activation [ 108 ]. Above all,  RSPO1  functions as an enhancer of β-catenin signaling during early ovary development. Interestingly, a novel role of  RSPO1  in steroid hormone secretion independent of WNT/β-catenin signaling was discovered. After luminal cells-specific  RSPO1  knockout,  ESR1  (estrogen receptor alpha) expression was decreased, and mammary side branches were reduced. However,  ESR1  expression was increased after luminal cell-specific knockout of  WNT4 , both with the attenuation of WNT/β-catenin signaling activities [ 109 ], revealing  RSPO1  may involve in other signaling pathways that regulate female sexual differentiation.\nThe  RSPO1  is also reported to participate in oocyte differentiation and meiosis after sex determination, as germ cell proliferation,  STRA8  (early meiotic marker) expression, and the number of germ cells entering meiosis were all reported impaired in the  RSPO1 −/−  fetal ovary [ 110 ]. However, in human disease,  RSPO1  was found to promote progression in ovarian cancer by increasing the proliferation and migration of ovarian cancer cells and reducing ovarian cancer cells’ apoptosis [ 111 ].\nThe  FOXL2  is one of the earliest markers of ovary differentiation in a mammal, which is sexual-specific and expressed in female gonads after E12.5 [ 112 ]. In  FOXL2 -knockout XX mice, granulosa cells and steroidogenic theca cells were reprogrammed into Sertoli-like cells and Leydig-like cells under the repression of  SOX9  [ 113 ]. In the in vitro system, up-expression of  NR5A1  was antagonized by  FOXL2 , and a 2-fold increase in  NR5A1  expression was detected in  FOXL2 −/−  mice relative to wild-type mutant [ 114 ]. According to findings,  FOXL2  may regulate early ovarian development by directly suppressing the expression of testis-specific genes. Although  FOXL2  plays a vital role in ovarian development in goats, it is more involved in fetal development than postnatal maintenance when compared to mice [ 115 ]. In XY transgenic mice, over-expression of  FOXL2  led to the impairment of testis tubule differentiation [ 116 ], and  RSPO1 - FOXL2 -double-knockout mice showed a similar phenotype earlier stage of sex reversal than  RSPO1  knockout mice, revealing a potential interaction between these two female sex determination genes [ 117 ].\nThe  FOXL2  becomes involved in follicle development by inducing Follicle-stimulating hormone (FSH) synthesis following sex determination [ 118 , 119 ], whose expression is regulated by ovarian hormones [ 120 , 121 ]. Furthermore,  FOXL2  plays a role in the development and maintenance of the ovary via interacting with  STAR  [ 122 ],  ESR2  [ 123 ]), and  p27  [ 124 ]. Moreover,  FOXL2  is expressed in the other components of the female reproductive tract, including the uterus, cervix, and oviduct, and plays a crucial role in postnatal uterine maturation [ 125 ]. The  FOXL2  mutations are linked to Blepharophimosis-Ptosis-Epicanthus Inversus syndrome (BPES) [ 126 , 127 , 128 ], adult ovarian granulosa-cell tumor [ 129 ], testicular adult-type granulosa cell tumors [ 130 , 131 ], ovarian Sertoli-Leydig cell tumors [ 132 ], incompletely differentiated sex cord-stromal tumors [ 131 ] and ovarian sex cord-stromal tumors [ 133 ] in human.\nWhen these sexual differentiation-related genes are taken together, they regulate the testis- and ovarian-determination network (listed in  Table 2  and  Figure 1 ) during embryonic development and throughout adulthood. They are also associated with developing other organs, reproductive capacity, and health. As a result, research aimed at developing animal models and modifying offspring sex ratios using gene-editing technology has been hampered for a long time due to organ failure. Although some research has investigated the underlying mechanisms of these sexual differentiation-related genes and has produced sexual reversal offspring, the development of offspring reproductive organs was significantly retarded. The number of available knockout offspring is lacking, limiting the study on growth performance. In addition, recent research has emphasized the importance of epigenetics in regulating sexual differentiation [ 134 ], inspiring future research in exploring the function of DNA methylation, histone modifications, non-coding RNA, and RNA methylation during sex determination gonads.\n\nThe PGCs are distinct stem cells that can give rise to other stem cell types and pass on their genome to the next generation. PGC research offers new hope for treating infertility patients by in vitro mediating PGC differentiation, even though germ cell yields remain low. As a result, research on the formation of PGCs will be an important future research direction envisaged to promote in vitro derivation of human PGCs. Furthermore, there may be gender differences during the migration and differentiation of PGCs, which could provide a theoretical foundation for manipulating offspring sex ratios in livestock production by changing the ratio of Y- and X-chromosome-bearing sperm through gene editing.\nPGCs originate from a subpopulation of cells in the proximal epiblast (PE) at around two weeks of pregnancy in humans (around E6.5 in mice) [ 157 ]. Subsequently, these cells cluster and are located in the base of incipient allantois [ 158 ]. Current research has identified that bone morphogenic proteins (BMPs) mainly induce PGCs specification signals secreted from surrounding extraembryonic ectoderm ( BMP4 ,  BMP8b ) [ 159 , 160 ] and visceral endoderm ( BMP2 ) [ 161 ]. However, BMPs signals alone could not determine PGCs fate because only a subset of PE cells can induce differentiation into germ cells. Therefore, several in vivo and in vitro studies identified positive and negative signals directing PGC fate (listed in  Table 3  and  Figure 1 ). Furthermore, significant differences in PGC formation regulatory actions have been observed between humans and mice, such as  SOX2  is required for PGC development in mice, while  SOX17  is required in humans [ 162 , 163 , 164 ]. Moreover,  KLF4  is only involved in maintaining pluripotency in human PGCs [ 165 ]. Interestingly, LncPGCAT-1 was found to positively regulate the formation of PGCs by elevating the expression of  Cvh  and  C-kit  and repressing the  NANOG  in vitro and in vivo [ 166 ], providing a new direction for research into the underlying biology of PGCs formation. In addition, recent single-cell sequencing research showed that the germline development between bovines and humans [ 167 ] and between mice and humans [ 165 ] were similar, which may provide new model organisms for the research on the development of PGCs.\nTo examine the mammalian germline’s developmental mechanism, mice PGCs were first isolated in 1982 [ 184 ]. Since then, PGCs of other species have been successfully isolated, including goats [ 185 ], rabbits [ 186 ], sheep [ 187 ], and humans [ 188 ]. However, due to the low PGCs generation rate, the current research was devoted to deriving PGCs from pluripotent cells. So far, primordial-germ-cell-like cells (PGCLCs) [ 189 ] and long-term expanded PGCLCs [ 190 ] have been developed to generate fertile mice oocytes [ 191 ] and produce offspring [ 192 ] in vitro. On the other hand, the same gene expression patterns were observed for human PGCLCs and PGCs [ 165 ], cementing the feasibility of researching PGCs formation in vitro. Several genes important in PGCs formation and maintenance, such as  TFAP2C  [ 193 , 194 ],  SSEA1  [ 195 ],  DND1  [ 196 ], and  SOX15  [ 197 ], were identified by employing sequencing technology, cell biology techniques, and genome editing technology on PGCLCs. However, there were still many challenges ahead; for instance, human PGCLCs derived in vitro could not meiosis completely during the embryonic stage [ 198 ]. Therefore, recent research is dedicated to the optimization of PGCs derivation routes.\nAccording to recent studies, niche environments are important for differentiating human PGCs from pluripotent cells. Franklin D. West et al. discovered that co-culturing with mouse embryonic fibroblasts increased the expression of germ-cell-specific genes [ 199 ]. One year later, human fetal gonadal stromal cells were used for co-culturing with human embryonic stem cells (ESCs), significantly improving PGCs generation efficiency [ 200 ].\nOn the other hand, research on optimizing cell culture medium was carried out since Niels Geijsen et al. derived PGCs from ESCs by culturing with leukemia inhibitory factor in 2004 [ 201 ]. Until now, there have been several biochemical agents used in inducing PGCLCs differentiation in vitro, such as retinoic acid promoting the differentiation of PGCLCs from skin-derived stem cells [ 202 ]; retinoic acid combined with CHIR99021 promoting the differentiation of PGCLCs from human ESCs [ 203 ]; luteinizing hormone regulating the proliferation of porcine PGCLCs through ceRNA network [ 204 ]. Furthermore, recent research has identified the role of epigenetic modification in the differentiation of PGCLCs in vitro. MIR-10B has been discovered to play a role in differentiating PGCLCs from human mesenchymal stem cells [ 205 ]. In addition, α-ketoglutarate can promote PGCLCs specialization by regulating epigenetic reprogramming [ 206 ]. Similarly, the cell adhesion microenvironment was found to contribute to the differentiation of ESCs, which provide new ideas for PGCs derivation in vitro, where mesh substrates were found to induce self-organize and differentiation of ESCs, transiting to a PGCs-like state without the addition of biochemical inducers [ 207 ]. Interestingly, sex differences were found in the associations between Bisphenol A and PGCLC proliferation, with downregulated X-linked gene expression and PGCLC proliferation inhibited in XX cells but not in XY cells [ 208 ], providing a theoretical basis for intervening in the fate of different gender PGCs.\nThe differentiation of PGCs to embryonic germ cells (EGCs) has a lot of promise in studying the mechanisms of PGC survival, proliferation, and regulation. During the conversion process from PGCs to EGCs, the whole-transcriptome analysis revealed that  BLIMP1  and  Akt  were involved in the specification and reprogramming of PGCs, respectively [ 209 ]. Further research showed that  Akt  activation promoted G1-S transition and enhanced PGCs reprogramming by downregulating H3K27me3 [ 210 ]. In addition, methylation changes at imprinting control centers (ICCs) during this conversion process were also discovered, stating that methylated ICCs are critical for PGCs derivation from ESCs [ 211 ]. Moreover, many new cell models have been developed to study factors regulating PGCs biologies, such as induced pluripotent stem cells [ 212 ] and PGCs derivation from nuclear transfer ESCs [ 213 ].\nSuccessful migration of PGCs to gonads is essential for gametogenesis in mammal, while anomalous migration of PGCs is required for the origin of endometriosis [ 214 ]. Although, with molecular biology development, the stages in PGC migration, with the underlying transcriptional regulatory network and signal pathways, have gradually been discovered and reviewed before [ 10 , 215 , 216 , 217 ], how PGCs migrates remains an important question.\nFollowing PGC specification, PGCs first move from the primitive streak to allantois, where members of the interferon-inducing transmembrane protein ( IFITM ) family play a role in PGC incorporation into the hindgut [ 218 ]. Subsequently, the hindgut elongated, and PGCs moved into the dorsal mesentery through a fragmented basement membrane and finally colonized the GRs. Jingjing Sun et al. [ 219 ] found that, in the absence of  MSX1  and  MSX2 , PGCs migration defected. The number of PGCs was reduced due to the reduction in the expression level  WNT5A ,  which promoted directional migration of PGCs [ 220 ]. With improved molecular technology, several other regulatory RNAs have been discovered in the recent years such as  NUP50  [ 221 ],  SMAD4  [ 222 ],  XVLG1  [ 223 ],  HSP70  [ 224 ],  PRDM1  [ 225 ],  Ptch2 / Gas1  and  Ptch1 / Boc  [ 226 ].\nDuring migration, the epigenome of PGCs undergoes comprehensive remodeling, including global DNA-demethylation, erasure of genomic imprints, and removal of H3K9me2; however, how they occur in PGCs is yet unknown. Anna Mallol et al. identified that  PRDM14  was involved in global and X-chromosomal reprogramming, which upregulated the repressive H3K9me2 dose dependently and removed H3K27me3 from the inactive X-chromosome [ 227 ]. In addition, the DNA methylome between human PGCs and mice PGCs was found to be roughly comparable before PGCs differentiation [ 165 ], providing a basis for the future establishment of animal models in epigenetic research. However, a recent study indicated that PGCs migration mechanisms vary among mammals. PE Høyer et al. found an association between human PGCs and autonomic nerve fibers, which suggested that PGCs might be guided by nerve fibers [ 228 ], which was confirmed by Mollgard K et al. [ 229 ]. However, in mice and a non-human primate (marmoset monkey), most PGCs maintained a minimum distance of 50 µm from the closest neuron during different stages of embryonic development. More importantly, PGCs were discovered to reach the gonads before the emergence of neurons around the gonads [ 230 ]. Above all, whether PGCs migration mechanisms in different species are diversified remains controversial.\nAnother factor that affects PGCs migration is DNA damage response (DDR) which is present at all embryonic development stages and results in apoptosis or delayed proliferation of PGCs. However, the underlying mechanisms remain partially known. Recent genetic studies showed that  FANCM  or  MCM9  deficiency reduced the number of PGCs before and after arriving in gonads. Interestingly,  FANCM- MCM9 -double-knockout mice showed an additive reduction of PGCs number [ 231 ], indicating that different DDR pathways can cause impaired PGCs migration. In another recent study, conditional knockout of  PRMT5  activated DDR inducing sterility through PIWI-interacting RNA (piRNA) pathway indicated that  PRMT5  was an important DNA protector [ 232 ]. The DDR was further studied with Ionizing radiation (IR), where, following germ cell differentiation and uncoupling of meiotic initiation in IR-treated female PGCs, gender differences were observed. In contrast, piRNA metabolism repression and transposon de-repression were detected in IR-treated male PGCs [ 233 ]. Importantly, this work provided new ideas for the research on sex manipulation by identifying genes that fit the established XX or XY germline.\nThe PGCs begin to increase during migration and continue until a global change in gene expression occurs; PGCs are ready for gametogenesis. However, the mechanisms regulating the balance between proliferation and differentiation of PGCs remained unclear. Andrea V Cantú et al. discovered that  WNT5A  involves the proliferation of PGCs in different niches by repressing  β-catenin -dependent and  ROR2 -mediated pathways [ 234 ], revealing that the tissue microenvironment regulated PGCs proliferation during migration rather than embryonic age. Another research using conditional knockout models showed that  MASTL  is vital for anaphase entry in female PGCs. Simultaneous deletion of  PPP2R1A  in  MASTL -knockout PGCs can rescue the failure of PGCs to proceed beyond the metaphase-like stage, demonstrating that  MASTL  with  PPP2A  was essential for establishing female germline by regulating PGCs proliferation through phosphatase activity [ 235 ].\nFurthermore, proteomic techniques were used to investigate PGC proliferation mechanisms, and it was reported that fatty acid degradation might play an important role in PGC proliferation. Furthermore, in vitro experiments demonstrated that when fatty acid degradation was suppressed, the number of PGCs decreased. Moreover, the expression levels of  AMPK  ( p53  activator to induce cell cycle arrest), phosphorylated  AMPK , phosphorylated  p53 , and cyclin-dependent kinase inhibitor 1 were increased, indicating that fatty acid degradation is involved in the proliferation of female PGCs via the  p53  pathway [ 236 ]. Interestingly, some genes functioned at both proliferation and differentiation. For instance,  ERK1-2  was expressed in PGCs at E8.5–E10.5 and gradually increased from E12.5–E14.5. After culturing PGCs with U0126 ( MEK -specific inhibitor),  ERK-12  expression was repressed, reducing PGCs at E8.5. Moreover, there were sex differences in controlling meiosis that only progression through meiotic prophase I of female PGCs treated with U0126 were slowed down [ 237 ]. In addition to participating in sexual differentiation of bipotential gonad,  FGF9  was dose-dependent in regulating mice XY PGCs fate. Low doses of  FGF9  (0.2 ng/mL) increased male-specific genes expression ( DNMT3L  and  NANOS2 ) in XY PGCs, while a high dose of  FGF9  (25 ng/mL) repressed the expression of male-specific genes and stimulated XY PGCs proliferation, revealing that  FGF9  regulates the balance between proliferation and differentiation of XY PGCs in a dose-dependent manner [ 238 ]. These could be used as a selective mechanism to favor male or female migrators by repressing the proliferation or differentiation of one through conditional knockouts or conditional overexpression. Interestingly,  EMX2  regulated the  FGF9  pathway in somatic cells [ 239 ], which was important for GR formation, demonstrating that sex determination occurs throughout mammals’ lives.\nBefore gametogenesis, PGCs required permission to start meiosis and sexual differentiation; however, it remained unknown whether this permission was cell-autonomous or gonad-independent. Yueh-Chiang Hu et al. built a  GATA4  (gene only expressed in somatic cells) conditional knockout model. They found that PGCs in  GATA4 -knockout embryos can migrate to the genital ridge but fail to start meiotic [ 240 ], indicating that gonad signaling is essential for gametogenesis. To fully explore the function of gonads, single-cell transcriptomics analysis was used in human fetal gonads. Four major signaling pathways ( WNT ,  NOTCH ,  TGFβ / BMP , and receptor tyrosine kinases) were found to be involved in ligand-receptor interactions between PGCs and gonadal somatic cells using the CellPhoneDB algorithm [ 241 ].  WNT  signaling pathway has been studied in depth because it is believed to be involved in sex determination throughout the life cycle. Anne-Amandine Chassot et al. found that spermatogonial proliferation was repressed and spermatocyte apoptosis increased following activation of the  WNT / β-catenin  pathway [ 242 ], which is consistent with the theory mentioned above that the  WNT  signaling pathway inhibits male-related biological processes. Another study identified  WNT  signaling as a “central gatekeeper” in female gametogenesis. PGCs maintained pluripotency or entered prematurely in the β-catenin gain- and loss-of-function models.\nAdditionally, by interacting with  POU5F1 ,  β-catenin  was involved in pluripotency maintenance, and germ cell differentiation occurred when the  WNT / β-catenin  pathway was repressed after  ZNRF3  upregulation [ 243 ]. The  FGF  signaling has been shown to regulate PGCs differentiation in two ways, i.e., by repressing female-related gene expression and activating downstream nodal/activin signaling to promote male gamete differentiation through degrading retinoic acid [ 244 ] and by activating the expression of  NANOS2  (male germ cell marker) [ 245 ], which can prevent XX PGCs meiosis and induce male-like differentiation [ 246 ]. Interestingly, Quan Wu et al. found that  SMAD2 , a putative gene downstream from nodal/activin signaling, promoted male differentiation through a retinoic acid-independent routine because retinoic acid signaling suppression did not rescue male-specific gene expression in  SMAD2  conditional knockout testes [ 247 ]. In mice PGCs proteomic research, there was no close correlation between proteomic data with published transcriptomic data using comparative analysis [ 248 ], revealing that the molecular mechanisms of gametogenesis may extend beyond the scope of the transcriptome, providing us essential inspiration for human gametogenesis research.\nPGCs undergo a wide range of epigenetic reprogramming before sex-specific differentiation. DNA methylation has been extensively studied in PGCs, associated with chromatin reorganization, genomic imprinting erasure, and X-chromosome reactivation [ 165 , 249 , 250 ]. This process is mainly achieved by repressing DNA methylation-related genes (such as  DNMT3A / B / L ) and activating  TET  proteins, though there are still many unknowns in this field. Several upstream regulatory genes have been identified using gene-editing technology such as  PRDM14  [ 251 ]. Moreover, Peter W S Hill et al. found that  TET1  was involved in the maintenance of DNA demethylation rather than activation, providing a complete understanding of the  TET  family [ 252 ]. The  SMARCB1  was discovered to have gender differences in regulating PGC epigenetic reprogramming. In  SMARCB1 -null female mice, meiosis-related genes were repressed, resulting in defects in synapse formation and DNA double-strand break repair. In contrast, in mutant male mice, the expression of genes related to growth and de novo DNA methylation was abnormal, resulting in mitotic arrest delay and hypomethylation of retrotransposons and imprinted genes [ 253 ].\nFurthermore,  DND1  was identified as a negative regulator of pluripotency and a positive regulator of epigenetic modifiers in male germ cell differentiation. In  DND1 Ter/Ter  mutant mice, genes associated with pluripotency, cell cycle, male differentiation, and chromatin regulation were repressed, translating into entering G1/G0 impairment and teratomas formation [ 254 ]. These findings supported manipulating sex-dependent differentiation of PGCs; however, the function of these genes in humans remains unknown. As a result, recent research has examined the transcriptome and DNA methylome landscapes of human PGCs, laying the groundwork for understanding the complex relationship between gene regulatory networks and DNA methylation during the global epigenetic reprogramming process of human PGCs [ 165 ]. In addition, DNA methylomes of human PGCs during epigenetic reprogramming were roughly similar to mice [ 165 , 249 ]. However, human PGCs also show a unique gene regulatory network in epigenetic modification different from mice PGCs [ 249 , 250 ]. On the other hand, recent studies have identified additional epigenetic reprogramming of PGCs before sexual differentiation, such as histone acetylation [ 255 ] and noncoding RNAs [ 256 ].\nUnder the influence of a male or female regulatory network, PGCs give rise to spermatogonial stem cells or oogonia. It is worth noting that the previously mentioned antagonistic network still determines the fate of these germ cells.  WNT4 , activated by  CTNNB1  signaling, can suppress spermatogonial stem cell activity in Sertoli cells [ 257 ], while female germ cell survival in the ovary is maintained by the  WNT4 / β-catenin  pathway [ 258 ].\n\nDSD has been a problem for humans for many years, and identifying functional variants of sex-related genes in DSD patients remains challenging. Infertility is another significant medical issue for which no effective treatments exist. The mechanisms underlying reproductive organogenesis and gametogenesis remain unknown despite considerable progress in recent years. However, it is worth mentioning that we have identified the interaction between somatic and germ cells and that signaling from somatic cells was essential for the proliferation and differentiation of PGCs. In contrast, female germ cells contributed to ovary maintenance. In addition, it was found that genes from males- and female-promoting antagonistic network primarily regulated the mammalian sex determination, which begins during embryonic development and continues throughout the life cycle. Nevertheless, several genes in this antagonistic network are also involved in the biological processes of organ maintenance and development, limiting the application of transgenic technology. In addition, transgenic efficiency remains low, and the sex-reversal trait cannot be stably transmitted to the next generation.\nAlternatively, epigenetic changes during reproductive organogenesis and gametogenesis may explain the inability to identify DSD through genetic diagnosis. In order to address the issues above, it is possible to divide further future research into three distinct areas: (i) Intercellular signaling mechanisms must be first investigated, (ii) the structure and regulatory regions of known sex-related genes, intergenic regulatory networks, and identification of novel sex-related genes should be focused on in the future, and (iii) using sequencing technology, changes in DNA methylation, histone modifications, non-coding RNA, and RNA methylation need to be identified during sex determination. With additional research, we will better understand the processes underlying the development of the gonad and germline in humans, mice, and other mammals, which will aid in diagnosing and treating DSD and human infertility. In addition, these studies can offer theoretical support for manipulating offspring sex ratios in livestock production.","source_license":"CC-BY-4.0","license_restricted":false}