Male germ cells undergo a complex sequence of developmental events throughout fetal and postnatal life that culminate in the formation of haploid gametes: the spermatozoa. Errors in these processes result in infertility and congenital abnormalities in offspring. Male germ cell development starts when pluripotent cells undergo specification to sexually uncommitted primordial germ cells, which act as precursors of both oocytes and spermatozoa. Male-specific development subsequently occurs in the fetal testes, resulting in the formation of spermatogonial stem cells: the foundational stem cells responsible for lifelong generation of spermatozoa. Although deciphering such developmental processes is challenging in humans, recent studies using various models and single-cell sequencing approaches have shed new insight into human male germ cell development. Here, we provide an overview of cellular, signaling and epigenetic cascades of events accompanying male gametogenesis, highlighting conserved features and the differences between humans and other model organisms.

Transfer of genetic information across generations in practically all multicellular species depends upon the germline: a unique cellular lineage that can be distinguished from the remaining somatic cell lineages early in development. Through a complex developmental process, the germline differentiates into either spermatozoa or oocytes. Subsequent fertilization of oocytes by spermatozoa results in the generation of totipotent zygotes that can develop into complete organisms with an intact germline, thus reinitiating the germline life cycle (Saitou and Miyauchi, 2016; Saitou and Yamaji, 2012). As the germline is essential for transmitting genetic information to offspring, any mutations or other genetic and/or epigenetic changes incurred in their genomes could have profound functional outcomes in subsequent generations. Thus, the germline has evolved to sustain the integrity of the genome and epigenome across multiple generations while still permitting the low frequency of mutations required for species evolution. Accordingly, analyses of germline development hold the key to understanding evolution, heredity and hereditary diseases.

The first cell type that develops in the germline is the primordial germ cell (PGC), a sexually uncommitted precursor for both spermatozoa and oocytes. Among multicellular organisms, two distinct mechanisms exist through which PGCs are specified: preformation and epigenesis (also known as induction). Preformation, seen in Caenorhabditis elegans, Drosophila, fish and birds establishes PGC fate through maternally (oocyte)-derived substances, including RNAs and proteins, called germ plasm, which is trapped within the oocyte cytoplasm following spatially asymmetric cell division before zygotic transcription commences (Extavour and Akam, 2003; Hansen and Pelegri, 2021; Lehmann, 2016). After zygotic cleavage, cells inheriting germ plasm are destined to a germline fate.

In the epigenesis mode of germline specification, PGCs are specified from pluripotent cells through signaling from neighboring somatic cells. This mode is considered the ancestral mechanism of germline specification through which all mammals, including humans and mice, generate PGCs. However, preformation has occurred multiple times in various taxa through convergent evolution (Extavour and Akam, 2003). Germline specification through epigenesis shares its origin with somatic cell specification, as both lineages originate from pluripotent cells. In this regard, pluripotency can be considered a unique germline state that transiently exhibits bi-potency towards both germline and somatic cells. Because of this shared origin, PGC specification and subsequent development require unique programs to suppress somatic programs and epigenetic memories established during early peri-implantation development (Saitou and Yamaji, 2012).

Origin of PGCs in humans and non-human primates

Studies on PGC specification mechanisms in humans have been hampered by both ethical and technical constraints that limit the accessibility of early post-implantation stage embryos in which such events take place (Ismaili M'Hamdi et al., 2022; Matthews et al., 2021; Rugg-Gunn et al., 2023). Additionally, the morphogenesis of post-implantation embryogenesis has diverged in mice and humans, and precludes the simple extrapolation of our knowledge of mouse germline specification pathways to that of humans (Box 1) (Molè et al., 2020; Rossant and Tam, 2022). For example, in mice, the extra-embryonic ectoderm (ExE), a derivative of the trophectoderm lineage, is juxtaposed to the proximal epiblast and provides a source of BMP4, an essential factor for mouse PGC specification (Lawson et al., 1999; Ohinata et al., 2009) (Fig. 1A). In contrast, human and most mammalian embryos do not develop the ExE (Luckett, 1978; Molè et al., 2020). Moreover, mouse embryos exhibit a unique cylindrical shape, thereby forming a proximal-distal embryonic axis that is absent in humans and most mammalian embryos, which bear a flat epiblast disc (Fig. 1) (Molè et al., 2020; Rossant and Tam, 2022). Thus, signaling principals in murine PGC specification are not directly applicable to humans and other species, highlighting the need for alternative models that recapitulate human PGC specification.

Box 1. PGC specification in mice

The germline in mice is first induced in the most proximal and posterior epiblasts at embryonic day (E) 6.0 and appears as approximately six BLIMP1+ founder PGCs. These founder PGCs consequently form clusters of ∼30-40 cells within the extra-embryonic mesoderm at around E7.25 (Ginsburg et al., 1990; Ohinata et al., 2005; Saitou et al., 2002). Specification of PGCs is mediated by inductive cues from surrounding cells, including BMP4 from the extra-embryonic ectoderm and inhibitory cues, including CER1 and DKK1 from the anterior visceral endoderm (Fig. 1A) (Lawson et al., 1999; Ohinata et al., 2009). As a result of this signaling, the three transcription factors TFAP2C, BLIMP1 and PRDM14 are induced to form PGCs with distinct characteristics (Nakaki et al., 2013; Ohinata et al., 2005; Weber et al., 2010; Yamaji et al., 2008). These factors cooperatively mediate three key events to initiate germline fate: repression of the somatic program, genome-wide epigenetic reprogramming and re-acquisition of latent pluripotency. WNT3, which is secreted from proximal posterior epiblasts and/or the posterior visceral endoderm (Liu et al., 1999; Rivera-Pérez and Magnuson, 2005), is required to render epiblasts competent to respond to BMP4-mediated inductive signals (Fig. 1A) (Ohinata et al., 2009). This process is mediated by canonical β-catenin signaling and its target mesodermal gene, T (brachyury) (Aramaki et al., 2013). Notably, this process also appears to be dependent on the Hippo-signaling mediator YAP (Kagiwada et al., 2021). Moreover, as BMP4 safeguards emerging PGCs from T-mediated mesodermal programs, BMP4 and WNT3 cooperatively regulate germline program in mice (Aramaki et al., 2013).

Fig. 1.

Germ cell specification in mice and cynomolgus monkey. (A) In mice, primordial germ cells (PGCs) are induced at the posterior and proximal end of the epiblast (EPI) in response to BMP4 signaling from extra-embryonic ectoderm (ExE), a trophectoderm lineage, that later forms a part of the placenta. WNT3 secreted from the visceral endoderm (VE) and/or EPI is also essential for the EPI cells to acquire responsiveness to BMP4 to become PGCs. CER1 and DKK1, antagonistic factors for BMP4 and WNT3 signaling, respectively, are secreted from the anterior visceral endoderm (AVE) and inhibit PGC specification at the anterior EPI. (B) Cynomolgus monkey (cy) PGCs are first identified at embryonic day (E) 11, Carnegie stage (CS) 5c in the dorsal aspect of the nascent amnion (AM), before the onset of primitive streak formation at E12. Later, cyPGCs are primarily found in posterior and proximal amnion and the VE (CS5c-6b). BMP4 is expressed in the nascent AM, which appears to exert an autocrine effect to form the cyPGCs. WNT3A, a canonical WNT-signaling ligand is expressed in the cytotrophoblast (CT) juxtaposing the dorsal AM and therefore might be involved in PGC specification. Similar to mice, CER1 and DKK1 are expressed in the AVE, which might contribute to the posteriorizing of cyPGC specification. EXMC, extra-embryonic mesenchyme.

Fig. 1.

Germ cell specification in mice and cynomolgus monkey. (A) In mice, primordial germ cells (PGCs) are induced at the posterior and proximal end of the epiblast (EPI) in response to BMP4 signaling from extra-embryonic ectoderm (ExE), a trophectoderm lineage, that later forms a part of the placenta. WNT3 secreted from the visceral endoderm (VE) and/or EPI is also essential for the EPI cells to acquire responsiveness to BMP4 to become PGCs. CER1 and DKK1, antagonistic factors for BMP4 and WNT3 signaling, respectively, are secreted from the anterior visceral endoderm (AVE) and inhibit PGC specification at the anterior EPI. (B) Cynomolgus monkey (cy) PGCs are first identified at embryonic day (E) 11, Carnegie stage (CS) 5c in the dorsal aspect of the nascent amnion (AM), before the onset of primitive streak formation at E12. Later, cyPGCs are primarily found in posterior and proximal amnion and the VE (CS5c-6b). BMP4 is expressed in the nascent AM, which appears to exert an autocrine effect to form the cyPGCs. WNT3A, a canonical WNT-signaling ligand is expressed in the cytotrophoblast (CT) juxtaposing the dorsal AM and therefore might be involved in PGC specification. Similar to mice, CER1 and DKK1 are expressed in the AVE, which might contribute to the posteriorizing of cyPGC specification. EXMC, extra-embryonic mesenchyme.

Until recently, our knowledge regarding human PGC development has been limited to morphological observational studies conducted primarily in the early-mid 20th century (Fuss, 1912; McKay et al., 1953; Politzer, 1930, 1933; W. Felix, 1911; Witschi, 1948). In these studies, human PGCs were first identified by unique cellular features in the posterior region of the yolk sac endoderm at about 3-4 weeks post-conception, corresponding to ∼E7.5 mouse embryos in which PGCs have already been specified and have migrated into the yolk sac endoderm (Box 1). Recent studies using non-human primates, in which early developmental events more closely recapitulate those used in human development, have shed new insight into the earliest stage of germline development, including specification and subsequent early PGC development (Bergmann et al., 2022; Ma et al., 2019; Sasaki et al., 2016). Cynomolgus monkeys (Macaca fascicularis), which are assigned to the genus Macaca of Old World monkeys, together with rhesus monkeys (Macaca mulatta), are the most common non-human primates used for biomedical research. Their pre- and post-implantation development processes are remarkably similar to humans, and thus serve as valuable models for embryology studies. Using both histological and single-cell RNA sequencing (scRNA-seq) approaches on a series of early post-implantation cynomolgus monkey embryos [E11-E17, Carnegie stage (CS) 5b-6b], Sasaki and colleagues demonstrated that PGCs are specified in the nascent amnion at around E11-E17 (Sasaki et al., 2016). In their study, PGCs first appear as a cluster of ∼10 TFAP2C+SOX17+BLIMP1+ cells within the dorsal end of the nascent amnion, juxtaposed to the cytotrophoblast layer (Fig. 1). Notably, the regions where PGCs localize gradually shifted towards the more proximal and posterior end of the amnion between E12 and E17. After this time, PGCs appear to break away from the basement membranes, undergo epithelial-to-mesenchymal transition-like changes and emigrate into the yolk sac endoderm (Sasaki et al., 2016). Importantly, PGC specification in cynomolgus monkeys is confined to the nascent amnion and precedes gastrulation, which starts at E12 within the posterior epiblasts, and is spatially separated from PGC-forming nascent amnion. These findings suggest that, in contrast to mice and other mammals, cynomolgus monkey PGCs are not specified within the epiblast (Kobayashi et al., 2017, 2020, 2021; Ohinata et al., 2005). This surprising finding raises two key questions: how does the nascent amnion, an extra-embryonic tissue, acquire germ cell competence and what is the source of inductive signals for PGC specification in primates?

In primates, the amnion is formed by cavitation within the center of the epiblastic knot immediately after implantation (amniogenesis by cavitation, Box 2), and thus constitutes one of the earliest lineages segregated from the epiblast. Accordingly, the nascent amnion at E11 bears a pluripotent gene expression signature (i.e. POU5F1, NANOG and SOX2), similar to the epiblast, that is gradually diminished as PGCs are established at E11-E17 (Sasaki et al., 2016). In mice, germline competence (i.e. a permissive state in which BMP4 can promote differentiation of PGCs from the pluripotent state) is conferred by canonical WNT signaling (Ohinata et al., 2009). Given that WNT3A, a WNT ligand, is primarily expressed in cytotrophoblasts that are transiently juxtaposed with the nascent amnion at E11-E12 (then separated by intercalating extra-embryonic mesenchyme after E12) and the strong expression of AXIN2, a WNT target gene, in the nascent amnion at E11, it can be speculated that cynomolgus germline competence is conferred to the pluripotent nascent amnion at its interface to the cytotrophoblasts during this narrow time window (Sasaki et al., 2016).

Box 2. Amniogenesis by cavitation

Amniogenesis by cavitation is seen in various eutherian species, including Haplorhini primates (e.g. monkeys, apes and humans), tenrecid, erinaceid Insectivora, Dermoptera and some Chiroptera, and appears to be a derived condition that has emerged multiple times as a result of convergent evolution (Luckett, 1975). Indeed, amniogenesis by folding is the primitive condition and is characteristic of all non-eutherian amniotes. In primates, the amnion in Strepsirhine primates (e.g. lemurs, lorises), which are phylogenetically more primitive than Haplorhini, is formed by folding, whereas that in haplorhini is formed by cavitation. Curiously, in haplorrhine tarsiers, the most primitive form of haplorhini, amniogenesis shows intermediate features; although the amniotic cavity initially develops by cavitation, the dorsal part of the amniotic cavity and overlying polar trophoblasts rupture at the time of implantation. These ruptures are closed by a subsequently formed definitive amnion through folding mechanisms (Luckett, 1975). Thus, amniogenesis by cavitation is the derived condition and was acquired between ∼40 and 63 million years ago, when tarsiers separated from ancestral primates. It will be of great interest to see whether the transition in the mode of PGC specification from the posterior epiblast to the amnion also accompanies this phylogeny.

With regard to the inductive signal, the nascent amnion expresses BMP4 and its downstream target genes, ID2 and MSX2, suggesting that the nascent amnion generates PGCs through an autocrine mechanism (Fig. 1B) (Sasaki et al., 2016). The number of target genes downstream of BMP4 continue to be upregulated in pre-migratory (E13-E20) cynomolgus PGCs. Interestingly, the anterior aspect of the visceral endoderm expresses CER1 and DKK1, inhibitory cytokines for BMP4 and WNT, respectively, which might inhibit PGC specification within the epiblast and the anterior region of the nascent amnion (Sasaki et al., 2016). These data suggest that PGCs in mice and cynomolgus monkeys are specified in response to the same germline competence and inductive factors, although these factors are provided by different sources.

Recently, PGC specification in pigs and rabbits has been described (Kobayashi et al., 2017, 2021; Zhu et al., 2021). These studies suggest that PGCs are specified in the posterior epiblasts (primitive streak) in response to BMP4 and WNT3, likely provided by neighboring cells in the posterior epiblasts. Notably, in contrast to primates, the amnion of these species is formed through folding of the mesoderm-derived extra-embryonic somatopleure after gastrulation commences, thus limiting their utility as a model of PGC specification in humans (Chuva De Sousa Lopes et al., 2022; Luckett, 1975). Nonetheless, these studies highlight the marked divergence of PGC specification in other eutherian species and prompt the further exploration of PGC specification.

Ex vivo culture of PGCs

Several groups have recently reported on extended embryo 3D cultures that recapitulate early post-implantation development in mice, monkeys and humans (Bedzhov et al., 2014; Deglincerti et al., 2016; Gong et al., 2023; Ma et al., 2019; Shahbazi et al., 2016; Zhai et al., 2023). Remarkably, extended culture of cynomolgus monkey embryos has confirmed the emergence of PGCs within the nascent amnion, and successfully captured the single-cell transcriptomes of emerging PGCs, which appear to bear a close transcriptomic relationship with nascent amnion cells (Ma et al., 2019). Future studies with experimental perturbation using pharmacological inhibitors or surgical separation of the amnion from other embryonic compartments will further address the molecular mechanisms of PGC specification in primates. Although PGC specification has not been recapitulated in extended human embryo 3D cultures, self-organizing amnion reconstituted from human pluripotent stem cells has revealed PGC-like cells within BMP4-expressing amnion (Zheng et al., 2019, 2022). Consistent with these findings, Jirasek identified several putative PGCs within the proximal posterior nascent amnion in Hematoxylin and Eosin images of human embryos at E13.5, although not much detail was provided (Jirásek, 2008). Although these studies support the amniotic origin of human PGCs, additional studies using extended 3D culture of human embryos or blastoids under the appropriate ethical guidance will be required to further address the origin and the mechanisms of PGC specification in humans (Rosa and Shahbazi, 2022).

Pluripotent stem cell-based model of human PGC specification

PGC specification takes place at an early post-implantation stage of human development that is essentially inaccessible for experimental purposes due to ethical and technical constraints (Rugg-Gunn et al., 2023). Thus, reconstitution of human germ cell specification using directed induction of human pluripotent stem cells is used as an alternative method to identify the genetic and epigenetic mechanisms underlying human germ cell specification, which in turn offers crucial insight into causes of human infertility. Over the past decade, there has been remarkable progress in the field of in vitro gametogenesis in mice, which culminated in the successful in vitro generation of both fertility competent sperm and oocytes through PGC-like cells (PGCLCs) induced from embryonic stem cells and/or induced pluripotent stem cells (iPSCs) (Hikabe et al., 2016; Ishikura et al., 2021; Yoshino et al., 2021) (Fig. 2A). These promising results provide hope that fertility-competent human gametes can also be derived from iPSCs.

Fig. 2.

Male in vitro gametogenesis from pluripotent stem cells in mice and humans. (A) Schematic of culture schemes for the derivation of functional male spermatozoa in mice. Ohinata et al. demonstrated that induced PGCs from epiblast cells transplanted into neonatal mouse testes can result in functional spermatozoa (Ohinata et al., 2009). In other studies, PGCLCs are induced from ESCs/iPSCs through EpiLCs. PGCLCs are subsequently matured into fertility-competent spermatozoa, either through direct orthotopic transplantation (Hayashi et al., 2011) or through reconstituted testis (rTestis) culture (Ishikura et al., 2016; Ishikura et al., 2021) followed by derivation of germline stem cell-like cells (GSCLCs), which bear spermatogonial stem cell potential. Through orthotopic transplantation or further culture in the explanted neonatal testes (ex vivo organ culture), GSCLCs are induced into haploid spermatozoa (round spermatid-like cells). Dotted arrows represents the development occurring in vivo after orthotopic transplantation. (B) Schematic of pro-spermatogonial induction from human induced pluripotent stem cells (hiPSCs). Human primordial germ cell-like cells (hPGCLCs) induced from hiPSCs via incipient mesoderm-like cells (iMeLCs) are further induced into pro-spermatogonia through xenogeneic rTestis (xrTestis) culture at the air-liquid interface (Hwang et al., 2020). EpiLCs, epiblast-like cells; ESCs, embryonic stem cells; ICM, inner cell mass; iPSCs, induced pluripotent stem cells; M, multiplying pro-spermatogonia; MLCs, M pro-spermatogonia-like cells; T1LCs, primary transitional (T1) pro-spermatogonia-like cells; TCs, transitional cells.

Fig. 2.

Male in vitro gametogenesis from pluripotent stem cells in mice and humans. (A) Schematic of culture schemes for the derivation of functional male spermatozoa in mice. Ohinata et al. demonstrated that induced PGCs from epiblast cells transplanted into neonatal mouse testes can result in functional spermatozoa (Ohinata et al., 2009). In other studies, PGCLCs are induced from ESCs/iPSCs through EpiLCs. PGCLCs are subsequently matured into fertility-competent spermatozoa, either through direct orthotopic transplantation (Hayashi et al., 2011) or through reconstituted testis (rTestis) culture (Ishikura et al., 2016; Ishikura et al., 2021) followed by derivation of germline stem cell-like cells (GSCLCs), which bear spermatogonial stem cell potential. Through orthotopic transplantation or further culture in the explanted neonatal testes (ex vivo organ culture), GSCLCs are induced into haploid spermatozoa (round spermatid-like cells). Dotted arrows represents the development occurring in vivo after orthotopic transplantation. (B) Schematic of pro-spermatogonial induction from human induced pluripotent stem cells (hiPSCs). Human primordial germ cell-like cells (hPGCLCs) induced from hiPSCs via incipient mesoderm-like cells (iMeLCs) are further induced into pro-spermatogonia through xenogeneic rTestis (xrTestis) culture at the air-liquid interface (Hwang et al., 2020). EpiLCs, epiblast-like cells; ESCs, embryonic stem cells; ICM, inner cell mass; iPSCs, induced pluripotent stem cells; M, multiplying pro-spermatogonia; MLCs, M pro-spermatogonia-like cells; T1LCs, primary transitional (T1) pro-spermatogonia-like cells; TCs, transitional cells.

Over the past two decades, attempts to derive germ cells from human pluripotent stem cells have been made in a number of laboratories (Chen et al., 2007; Clark et al., 2004; Eguizabal et al., 2011; Kee et al., 2007, 2009; West et al., 2010). Although originally plagued by poor induction efficiency and lack of in-depth validation of putative germ cells, recent studies have overcome these shortcomings and established a highly efficient induction scheme (Irie et al., 2015; Sasaki et al., 2015) (Fig. 2B). In these studies, BMP4-based cytokine cocktails similar to those used in mouse PGCLC induction successfully generated human PGCLCs that expressed a number of early PGC markers, including pluripotency-associated genes (e.g. POU5F1 and NANOG) and germ cell specifier genes [e.g. TFAP2C and BLIMP1 (PRDM1)], but were devoid of late PGC markers, such as DDX4 or DAZL, which are upregulated only after PGCs migrate into the gonads (Fig. 3). Comparison of these cells with PGCs in cynomolgus monkey embryos suggests that they represent pre-migratory stage PGCs (Sasaki et al., 2016). The successful derivation of PGCLCs from human iPSCs has been somewhat surprising because it was long thought that human iPSCs represented a primed state of pluripotency, similar to epiblast stem cells (EpiSCs) in mice (Tesar et al., 2007), which do not contribute to the germline (Hayashi et al., 2011). However, murine epiblast-like cells (EpiLCs) transiently induced from naïve pluripotent cells by activin and bFGF, represent a peri-implantation epiblast state that is competent to generate PGCLCs in response to BMP4 (Fig. 2A) (Hayashi et al., 2011). Further comparative studies have revealed that transcriptomes of human iPSCs fall in between mouse EpiLCs (∼E5.5) and mouse EpiSCs (∼E6.5), suggesting that the primed pluripotent state in humans is distinct from that in mice and bears germline competency (Nakamura et al., 2016; Sasaki et al., 2015).

Fig. 3.

Schematic of the human male germ cell development. Key developmental events, cellular state and position, marker genes and global DNA methylation levels during male germ cell development are depicted. Cell types of male germ cell lineage and their marker genes are shown. PGC, primordial germ cell; M, M pro-spermatogonia; T1/f0, T1(f0) pro-spermatogonia. The designation of Adark and Apale spermatogonia is based on studies correlating nuclear morphology and a limited number of marker gene expression or mitotic index (Clermont, 1966; Di Persio et al., 2017; Ehmcke and Schlatt, 2006; Fayomi and Orwig, 2018). Strict correlation of cell types defined by scRNA-seq (S0-S4) and by nuclear morphology (Adark and Apale) awaits future studies using a spatial omics approach.

Fig. 3.

Schematic of the human male germ cell development. Key developmental events, cellular state and position, marker genes and global DNA methylation levels during male germ cell development are depicted. Cell types of male germ cell lineage and their marker genes are shown. PGC, primordial germ cell; M, M pro-spermatogonia; T1/f0, T1(f0) pro-spermatogonia. The designation of Adark and Apale spermatogonia is based on studies correlating nuclear morphology and a limited number of marker gene expression or mitotic index (Clermont, 1966; Di Persio et al., 2017; Ehmcke and Schlatt, 2006; Fayomi and Orwig, 2018). Strict correlation of cell types defined by scRNA-seq (S0-S4) and by nuclear morphology (Adark and Apale) awaits future studies using a spatial omics approach.

Transcriptional analyses of human PGCLCs and PGCs highlight notable differences from their mouse counterparts. For example, human PGCs and/or PGCLCs persistently express SOX17 (De Jong et al., 2008; Irie et al., 2015; Sasaki et al., 2015), previously known as an endodermal marker that is expressed only transiently in early mouse PGCs (Hara et al., 2009; Yabuta et al., 2006). By contrast, SOX2, a core transcription factor for pluripotency and crucial for germ cell development in mice (Campolo et al., 2013) is not expressed in human PGCs and/or PGCLCs (Campolo et al., 2013; Irie et al., 2015; Sasaki et al., 2015). PGCLCs can be induced in a similar manner from embryonic stem cells and/or iPSCs of non-human primates (cynomolgus and rhesus macaque, and marmoset) and also exhibit transcriptional features similar to humans and distinct from mice (Kobayashi et al., 2017; Sakai et al., 2020; Seita et al., 2023). The induction platforms described above, combined with CRISPR/Cas9 technologies, have been used to dissect the genetic network required to specify human germ cell fate (Irie et al., 2015; Kobayashi et al., 2017; Kojima et al., 2017, 2021; Pierson Smela et al., 2019; Sasaki et al., 2015; Sybirna et al., 2020; Zhang et al., 2021). Specifically, recent seminal studies demonstrate that the genetic network orchestrated by TFAP2C, SOX17 and BLIMP1 is crucial for specification of hPGCLCs (Irie et al., 2015; Kojima et al., 2017, 2021; Sasaki et al., 2015). These studies have further revealed that EOMES serves as an effector of canonical WNT signaling-derived SOX17 (Kojima et al., 2017), whereas GATA2 and/or GATA3 triggered by BMP4 drives TFAP2C and SOX17, which in turn triggers BLIMP1 expression (Kojima et al., 2021). Consistently, doxycycline-induced overexpression of GATA3, TFAP2C and SOX17 sufficiently confers PGC fate with a transcriptional profile similar to that induced by cytokines (Kojima et al., 2021). In cynomolgus monkeys, these transcription factors are all expressed in the nascent amnion, further asserting that PGC specification takes place there (Kojima et al., 2021; Sasaki et al., 2016).

Comparative studies have revealed that the genetic circuit of mouse PGC specification has diverged from humans. In mice, Sox17 is dispensable for PGC specification and function in vivo (Hara et al., 2009). Crucially, however, endogenous WNT signaling triggered by BMP4 activates T, which in turn deploys core germ cell specifying transcription factors Prdm14, Blimp1 and Tfap2c in mouse PGCs and PGCLCs (Aramaki et al., 2013), whereas T is dispensable for human PGCLC specification (Kojima et al., 2017). These studies suggest that despite the conservation of signaling mechanisms (i.e. BMP4 and WNT), there is significant divergence in downstream genetic networks and hierarchies governing germ cell specification. Such findings highlight the need to study additional organisms present in the clade between humans and mice to understand how human germ cell specification pathway has been shaped during evolution.

Early studies of the histological and ultrastructural features of the pre-migratory and migratory phases of human PGCs suggest that, after specification, PGCs transiently localize to the posterior yolk sac endoderm at the orifice of the allantoic diverticulum (Funkuda, 1976; Fuss, 1912; Politzer, 1930; Witschi, 1948). PGCs subsequently migrate through the hindgut endoderm and dorsal mesentery until they reach the bipotential gonads (i.e. precursor for the ovaries and testes) at 5-6 weeks post-fertilization (wpf). This migratory behavior is overall well conserved between mice and humans. In mice, the initial phase of migration is primarily passive (Hara et al., 2009; Kanamori et al., 2019). Subsequently, active amoeboid migratory movement guided by CXCL12 and/or CXCR4, stem cell factor (SCF) and/or KIT and WNT5A and/or ROR2 signaling is promoted by niche cells along the migratory route and/or gonads (Ara et al., 2003; Buehr et al., 1993; Doitsidou et al., 2002; Laird et al., 2011; Molyneaux et al., 2003; Zama et al., 2005). In mice, cells that fail to enter the gonads by E10.5 are eliminated by apoptosis due to the lack of a SCF-dependent survival signal provided by the gonads (Runyan et al., 2006).

Similar to mouse PGCs, cynomolgus monkey PGCs appear to be less migratory immediately after specification, when they are primarily confined to the posterior yolk sac endoderm. They undergo more active movement after E24, during which time PGCs initiate unsynchronized migratory activities and can be found widely scattered across the hindgut endoderm, dorsal mesentery wall and coelomic angles at various positions along the anterior-to-posterior axis (Sasaki et al., 2016). The majority of PGCs colonize the gonads by 6 wpf in humans (∼52%) and by E36 in monkeys (∼61%) (Cheng et al., 2022; Sasaki et al., 2016). The destiny of PGCs outside the gonads by these stages remains unknown but might be linked to the formation of germ cell tumors, which are often seen in the midline alongside the route of PGC migration (Oosterhuis and Looijenga, 2019). Further studies are warranted to define the window during which PGCs continue to migrate into the gonads in these species. Until now, cellular and molecular features of human migratory PGCs have been poorly understood due to the lack of tractable in vitro model system. Culture of hPGCLCs within hindgut organoids might be a promising approach to recapitulate human PGC behaviors at the migratory stage and the interaction between PGCs and hindgut endoderm or stroma (Alves-Lopes et al., 2023).

Sex-specific gamete specification

The number of human germ cells continuously increases from ∼50 at 3 wpf, ∼600 at 4 wpf, ∼3500 at 5 wpf and 11,000 at 6 wpf, at which time the majority of PGCs migrate into the gonads (De Felici, 2013; Mamsen et al., 2011; Politzer, 1930, 1933; Witschi, 1948). Upon colonization of the gonads, germ cells upregulate germline genes, including DDX4, DAZL and PIWIL2 (Hwang et al., 2020; Li et al., 2017; Tang et al., 2015) (Fig. 3). In mice, these genes bear promoters that are regulated by methylation, and thus appear to be coupled to the global DNA demethylation that occurs during the migration phase (Hackett et al., 2012; Maatouk et al., 2006).

Around the time that the first cohort of human PGCs arrives at the gonads (∼5-6 wpf, CS15), the gonads are still sexually bipotent. Subsequent sex determination is mediated by SRY in XY gonads and WNT4/RSPO1/β-catenin in XX gonads (Cheng et al., 2022; Hanley et al., 2000; Mamsen et al., 2017; Yang et al., 2019). Accordingly, the first sign of sexual dimorphism becomes apparent at around 6-7 wpf, as evidenced by the emergence of Sertoli and granulosa cells in fetal testes and ovaries, respectively. Colonized PGCs receive sex-specific niche signals from these sexually committed gonadal somatic cells, which then guide their sexually dimorphic development. Germ cell sexual dimorphism is initially revealed by the initiation of prophase of meiosis I in female germ cells, whereas male germ cells undergo mitotic arrest without entering into meiosis (Fig. 3). In females, this results in the emergence of retinoic acid (RA)-responsive fetal germ cells at ∼11 wpf that express key targets of RA signaling (e.g. STRA8 and ZGLP1), followed by meiotic (∼14 wpf onwards) and oogenesis stages of germ cells (∼18 wpf onwards) (Li et al., 2017). In males, mitotic arrest appears to start at ∼14 wpf (Guo et al., 2021). At the transcriptional level, sexual dimorphism is evident in germ cells as early as 5 wpf, with differential expression of several sex chromosomal and autosomal genes between male and female germ cells (Li et al., 2017). These genes include the Y-chromosomal gene DDX3Y (Y-chromosomal microdeletions in which are potentially a cause of male infertility), which inevitably confers sexual dimorphism in a germ cell-intrinsic manner. Future studies will be needed to further clarify the signaling and genetic mechanisms that shape sexual dimorphism in human germ cells.

Pro-spermatogonial development

In fetal testes, PGCs undergo a complex developmental cascade to transition into spermatogonia, during which a series of distinct cell types emerge that are distinguishable by their morphology, cellular properties and gene expression (Hilscher et al., 1974). Specifically, upon colonization of the gonads, rodent male PGCs first differentiate into multiplying (M) pro-spermatogonia (∼E12) before becoming mitotically quiescent primary transitional (T1) pro-spermatogonia (E14) (Hilscher et al., 1974; McCarrey, 2013). Immediately after birth, T1 pro-spermatogonia migrate centrifugally from the center of the seminiferous cord towards the basement membrane – the site of the spermatogonial niche. During this migratory phase, T1 pro-spermatogonia differentiate into secondary transitional (T2) pro-spermatogonia, which resume mitotic activities. T2 pro-spermatogonia are regarded as the immediate precursors to spermatogonia, which are the founding population required for continuous spermatogenesis. Recent scRNA-seq analyses have successfully captured these transitions and accompanying transcriptomic dynamics in mice (Tan et al., 2020a; Zhao et al., 2021).

Similar to rodents, human male PGCs appear to transition into mitotically active M pro-spermatogonia upon colonization of the gonads (Fig. 3). This event appears to occur immediately after colonization, although further verification with an increased sample size is required (Li et al., 2017). M-to-T1 transition also occurs in humans and this transition appears to start at ∼14 wpf and continues throughout the fetal and early post-natal stages (Guo et al., 2021). As a result, fetal testes after ∼14 wpf and early post-natal testes contain a heterogeneous mixture of mitotically active M and quiescent T1 pro-spermatogonia (Guo et al., 2021; Hwang et al., 2020; Li et al., 2017). Interestingly, many T1 pro-spermatogonia in humans and monkeys localize to the periphery of the seminiferous cord, in contrast to those in mice, in which they are are exclusively located centrally (Hwang et al., 2020; McKinnell et al., 2013; Sasaki et al., 2016). This suggests the divergence of pro-spermatogonial development between rodents and primates. The causative relationship between the topological orientation of these cell types and the genetic programs governing their fates remains unknown; however, it is possible that a unique niche present at the periphery of the seminiferous cord provides an environment favorable to the survival and/or development of T1 pro-spermatogonia in humans and monkeys.

Similar to mice, the M-to-T1 transition in humans accompanies downregulation of germ cell specifier genes (e.g. SOX17, TFAP2C, BLIMP1 and NANOS3) and pluripotency-associated genes (e.g. POU5F1, NANOG and TFCP2L1). Moreover, a number of genes, previously recognized as markers for spermatogonia (e.g. NANOS2, SIX1, ID4, DCAF4L1, PLPPR3 and EGR4) (Guo et al., 2021; Hermann et al., 2018; Tan et al., 2020b; von Kopylow and Spiess, 2017), are upregulated during this transition, suggesting the close similarities of T1 pro-spermatogonia and adult spermatogonia (Fig. 3) (Guo et al., 2021; Hwang et al., 2020; Li et al., 2017).

The signaling cue driving the M-to-T1 transition in mice is mediated, at least in part, by paracrine factors, including activin and PGD2 alongside FGF9, which triggers the expression of key male germline factor, NANOS2 (Wu et al., 2013). NANOS2, cooperatively with DND1, activates a male-specific genetic program and inhibits entry into meiosis (Hirano et al., 2022; Suzuki and Saga, 2008). In contrast, CRIPTO and/or NODAL signaling, which are also triggered by FGF9, are involved in the maintenance of pluripotency and counteract the M-to-T1 transition (Spiller et al., 2012). STRA8 induced by retinoic acid (RA) is the key trigger of meiotic initiation in fetal ovaries (Anderson et al., 2008; Baltus et al., 2006). In mouse testes, degradation of endogenous RA occurs by a p450 enzyme, CYP26B1, expressed specifically in Sertoli cells, which altogether suppress the precocious meiotic entry and confers the male characteristics observed in T1 pro-spermatogonia in mice (Bowles et al., 2006). To date, niche factors involved in the M-to-T1 transition in humans and genetic networks orchestrating this process remain unknown.

During the migratory and gonadal stage of development, germ cells undergo dynamic epigenetic reprogramming, during which essentially all DNA methylation, including imprinted loci, are erased mainly through replication-coupled passive demethylation (Kagiwada et al., 2013; Kobayashi et al., 2013; Lee et al., 2014; Seisenberger et al., 2012), which occurs by E13.5 in mice. Subsequently, from T1 pro-spermatogonial differentiation at E13.5 until birth, male germ cells undergo progressive de novo methylation to acquire androgenic epigenomes, including paternal imprinted loci. De novo methylation in males is mediated by DNA methyltransferase families, DNMT3A, DNMT3B and DNMT3L, which cooperatively establish DNA methylation across genomes of the male germline through the addition of various histone marks (e.g. H3K36me2) and use of piRNA machinery (Kato et al., 2007; Kobayashi et al., 2013; Kuramochi-Miyagawa et al., 2008; Molaro et al., 2014; Shirane et al., 2020; Singh et al., 2013). In humans, PGCs exhibit substantial genome-wide DNA demethylation (∼20%) immediately after colonization of the gonad, but methylation is reduced to ∼5% by 9 wpf (Gkountela et al., 2015; Guo et al., 2015; Tang et al., 2015). Although established spermatogonia exhibit high DNA methylation similar to sperm, when and how de novo methylation commences during male germline development remains unknown (Guo et al., 2017). It will be worthwhile to explore the genetic and epigenetic machinery mediating this process in future studies. Given the marked temporal heterogeneity of male germ cell development, it will also be interesting to determine whether de novo methylation is coupled to cellular state (M versus T1).

Pro-spermatogonia to spermatogonia transition

In mice, T1 pro-spermatogonia transition into mitotically active T2 pro-spermatogonia at postnatal day (P) 2-3. This transition is characterized by the resumption of mitotic activity and centrifugal translocation from the center of the seminiferous cord to the basement membranes (McCarrey, 2013). Recent scRNA-seq analyses have successfully captured the lineage progression from T1 to T2 pro-spermatogonia and accompanying gene expression dynamics in mice. Notably, this analysis has revealed a previously unreported Elmo1- and Palld-expressing intermediary cell state [intermediate (I) pro-spermatogonia], which is characterized by a lack of mitotic activity and the expression of genes involved in cell migration and cytokinesis, consistent with their migratory activity (Tan et al., 2020a). T2 pro-spermatogonia then bifurcate to establish two distinct cell fates. In the first cell fate trajectory, T2 pro-spermatogonia progress directly into the spermatogenic differentiation pathway without an intervening self-renewal phase, contributing to the first wave of spermatogenesis that generates functional spermatozoa as early as P35 (Geyer, 2017; Yoshida et al., 2006). This apparently rodent-specific mode of spermatogenesis allows young male rodents to become fertile early in their life, thereby conferring a reproductive advantage. In the second cell fate trajectory, T2 pro-spermatogonia transition into foundational spermatogonial stem cells (SSCs) that, through sustained self-renewal and differentiation into spermatozoa, contribute to steady-state and life-long spermatogenesis (Kluin and de Rooij, 1981; McCarrey, 2013). As SSCs are the only cell type within the mammalian germline that possess stem cell activity, their emergence heralds the transition from a directional and progressive developmental phase to a homeostatic phase of the male germ cell life cycle.

A pioneering study by Kluin and colleagues demonstrated that within heterogeneous populations of neonatal pro-spermatogonia, cells destined to form foundational SSCs can be distinguished from those in a differentiation state based on histomorphological features (Kluin and de Rooij, 1981). More recently, Yoshida and colleagues have confirmed this finding by genetic lineage tracing studies that demonstrated that NGN3 pro-spermatogonia give rise to differentiating spermatogonia that contribute to the first wave of spermatogenesis, whereas NGN3+ cells contribute to self-renewing SSCs (Yoshida et al., 2006). A recent study by Law and colleagues has demonstrated that a subset of ID4-EGFP+ pro-spermatogonia (but not ID4-EGFP- pro-spermatogonia) in E16.5 mouse fetuses can colonize and establish a SSC pool in germline-ablated testes, suggesting that the SSC fate may be preprogrammed during late fetal development (Law et al., 2019). This preprogramming hypothesis is controversial, however, with some proposing that SSCs are the products of cellular-level natural selection (Nguyen and Laird, 2021; Nguyen et al., 2020). Because colonization and subsequent development of transplanted fetal germ cells in the adult testes occurs in a regenerative and unphysiological setting, future studies using pulse-chase lineage tracing of these pro-spermatogonia will be warranted to confirm that these fetal pro-spermatogonia irreversibly acquire SSC fate under physiological conditions. Moreover, the mechanisms underlying such pro-spermatogonial fate determination remain poorly understood and will require clarification in future studies.

The pro-spermatogonia-to-spermatogonial transition remains enigmatic in humans due to limited access to relevant samples and lack of genetic tools for analyses. However, recent scRNA-seq studies using fetal and neonatal human testes suggest that human T1 pro-spermatogonia (designated as the f0 state in the study) immediately transition to undifferentiated spermatogonia (designated as state 0) without an intervening T2 phase, and that T1 (f0) and state 0 undifferentiated spermatogonia bear minimal transcriptional differences (Fig. 3) (Guo et al., 2021). Moreover, first wave spermatogenesis does not appear to occur in humans (Guo et al., 2020, 2021; Sohni et al., 2019). Thus, it is likely that, in contrast to mice, all human T1 pro-spermatogonia are preprogrammed to become SSCs, thereby contributing to steady-state spermatogenesis. Those that fail to do so might be eliminated through apoptotic cell death. Notably, some M-pro-spermatogonia (or PGCs) expressing POU5F1 or TFAP2C appear to persist in early postnatal life in humans and marmoset (McKinnell et al., 2013; Mitchell et al., 2008; Sohni et al., 2019). Whether such immature germ cells present in postnatal testes undergo normal development and contribute to functional spermatogenesis remains unknown, but some suggest that the persistence of such cells might predispose testes to the development of intratubular germ cell neoplasm, the precursor for testicular germ cell tumor (Hoei-Hansen et al., 2004; Rajpert-De Meyts, 2006). Regardless of the significance, these findings exemplify the remarkable heterogeneity of male germ cell development in primates.

Steady-state spermatogenesis

Spermatogenesis is a dynamic cellular differentiation process that gives rise to mature spermatozoa within the seminiferous tubules and is therefore crucial for male reproduction. With the exception of first wave spermatogenesis in rodents, spermatogenesis is sustained by self-renewing SSCs. SSCs comprise a small subset of undifferentiated spermatogonia that reside in the basal compartment of seminiferous tubules. As the cellular and molecular mechanisms contributing to the stemness and differentiation pathways of SSCs have been extensively reviewed elsewhere (De Rooij, 2017; Kanatsu-Shinohara and Shinohara, 2013; Kubota and Brinster, 2018; Yoshida, 2018, 2020), only a brief overview will be provided here to highlight the key differences between SSCs in mice and humans. In the prevailing model for rodent spermatogenesis, which is primarily based on histomorphological observation of the clonal arrangement of cells, SSC activity resides in Asingle spermatogonia, which divide to generate interconnected syncytium of two (Apaired) or more (Aaligned) spermatogonia, that are primed to differentiate (De Rooij and Russell, 2000). Upon Sertoli cell-derived RA stimulation, Aaligned further generate differentiating spermatogonia (A1, A2, A3, A4, intermediate and B spermatogonia) (Helsel and Griswold, 2019; Kirsanov et al., 2023) that are devoid of stem cell potential and further divide to produce primary spermatocytes undergoing meiosis (De Rooij and Grootegoed, 1998; Yoshida, 2018). Recent studies using live imaging or lineage tracing in mice suggest that steady-state SSC activity resides in a subset of undifferentiated spermatogonia genetically labeled by various markers (Gfra1, Nanos2, Bmi1, Id4, Pax7 and Eomes) (Aloisio et al., 2014; Hara et al., 2014; Komai et al., 2014; Sada et al., 2009; Sun et al., 2015), and encompass As, Apaired and a small fraction of Aaligned. However, in a regenerative context, such as during recovery from busulfan treatment or post-transplantation, an otherwise differentiation-primed Ngn3+ spermatogonial population (encompassing Aaligned and a small fraction of As and Apaired) can re-express Gfra1 and serve as self-renewing SSCs. These studies highlight the plasticity and complexity of spermatogenesis (Nakagawa et al., 2007).

In humans and non-human primates, SSCs are described based on nuclear features, with Adark and Apale constituting undifferentiated spermatogonia that generate differentiating spermatogonia (B spermatogonia), which eventually form meiotic primary spermatocytes (Clermont, 1966; Clermont and Antar, 1973; Clermont and Leblond, 1959; Fayomi and Orwig, 2018). Although clonal development in the spermatogenic lineage is difficult to analyze in humans, recent scRNA-seq studies have shed new insights into the heterogeneity of SSCs and gene expression dynamics associated with human spermatogenesis (Guo et al., 2017, 2020, 2021; Hermann et al., 2018; Sohni et al., 2019; Wang et al., 2018). According to these studies, GFRA1+ undifferentiated spermatogonia (s0 state, UTF1lowGFRA1highETV5+L1TD1+FGFR3+) are preceded by a UTF1highGFRA1low state (s1 state, also characterized by PIWIL4, MAGEC2, EGR4 and ID4 expression) (Fig. 3). In contrast, a GFRA1high state is thought to represent steady-state SSCs in mice (Green et al., 2018; Hara et al., 2014; Hermann et al., 2018; Meng et al., 2000; Tan et al., 2020a). GFRA1 is a GDNF-binding subunit of the GDNF receptor complex that consists of RET receptor tyrosine kinase (Saarma and Sariola, 1999). Because a GDNF/GFRA1 interaction is essential for maintaining SSCs in mice (Meng et al., 2000), the GFRA1low state in the most undifferentiated spermatogonial population in humans raises the interesting possibility that GDNF-independent mechanisms maintain human SSCs. Notably, these undifferentiated spermatogonia express FGFR3 (Guo et al., 2018; Wang et al., 2018), one of the causative genes of thanatophoric dysplasia, a ‘paternal age effect’ disorder in which a gain-of-function mutation of FGFR3 in SSCs of aged individuals confers a growth advantage and selfish spermatogonial selection (Goriely and Wilkie, 2012). Accordingly, as in mice, FGF-mediating signaling may play a crucial role in human SSC function that warrants further investigation.

Initiation of spermatogenesis in humans

In contrast to rodents that initiate spermatogenesis immediately after the neonatal phase, humans have a protracted juvenile period, typically of 9-14 years, in which the onset of spermatogenesis is delayed by maintaining spermatogonia in a relatively quiescent phase (Chemes, 2001; Guo et al., 2020; Paniagua and Nistal, 1984). The mechanisms of such protracted dormancy of spermatogenesis remain elusive. Interestingly, however, some studies suggest that during this period, a small number of spermatogonia undergo incomplete and/or abortive spermatogenesis, during which they progress into meiotic spermatocytes that subsequently undergo apoptosis and do not generate haploid spermatids (Chemes, 2001). Upon the onset of puberty, some undifferentiated spermatogonia transition into mitotically active KIT+SYCP3+DMRT1+ differentiating spermatogonia (s2-4 state) (Guo et al., 2021; Wang et al., 2018), which subsequently become primary and secondary spermatocytes, and finally generate haploid spermatids (round and elongating spermatids) (Fig. 3). Testosterone produced from Leydig cells accelerate the maturation of Sertoli cells (Smith and Walker, 2014). Accordingly, testes from testosterone-suppressed trans-females revealed UTF1+ undifferentiated spermatogonia without progression into differentiating spermatogonia (Guo et al., 2020). The detailed hormonal and molecular cues that triggers a switch from abortive to complete spermatogenesis remain elusive.

Orthotopic spermatogonial transplantation developed by Brinster and colleagues allowed for quantitative assessment of SSC activity. Using this method, SSCs transplanted orthotopically into germ cell-depleted seminiferous tubules of recipient mice colonize and contribute to lifelong spermatogenesis (Brinster and Avarbock, 1994; Brinster and Zimmermann, 1994). Subsequent studies further revealed that if neonatal mice are used as donors, PGCs could also be engrafted and differentiated into SSCs, and can contribute to functional spermatogenesis (Chuma et al., 2005). These surprising results not only highlight the remarkable flexibility of male germline development but also allow for the functional validation of PGCs derived in vitro. Accordingly, pioneering works by Saitou and colleagues demonstrated that mouse PGCLCs induced ex vivo from mouse E6.0 epiblast cells or from embryonic stem cells through epiblast-like cells (EpiLCs) can differentiate into SSCs and yield functional spermatozoa upon orthotopic transplantation into neonatal mouse testes (Fig. 2A) (Hayashi et al., 2011; Ohinata et al., 2009). More recently, male in vitro gametogenesis methods have evolved by recapitulating environmental cues provided by testicular niche cells in vivo. Recent studies by Kanatsu-Shinohara and colleagues have revealed that a self-renewing SSC state can be captured in vitro by 2D culture of spermatogonia on mouse embryonic fibroblasts in the presence of GDNF, FGF2, EGF and LIF (Kanatsu-Shinohara et al., 2003). These cells, designated as germline stem cells (GSCs), exhibit indefinite self-renewal capacity while maintaining SSC activity, as revealed by spermatogenesis upon orthotopic transplantation. Leveraging this method, Ishikura and colleagues successfully derived GSC-like cells (GSCLCs) from PGCLCs by culturing them with testicular somatic cells obtained from E12.5 male embryos (Fig. 2A) (Ishikura et al., 2016). The resulting GSCLCs were capable of colonizing the adult testis and yielded fertile spermatozoa upon orthotopic transplantation. More recently, using the testicular organ culture method originally described by Ogawa and colleagues (Sato et al., 2011), the same authors demonstrated that the entirety of male germ cell development could be completed in vitro (Ishikura et al., 2021). With this method, GSCLCs induced in vitro from PGCLCs were transplanted into the neonatal testes, which were subsequently subjected to organ culture at the air-liquid interface. GSCLCs colonized the seminiferous tubules and underwent differentiation into haploid round-spermatid-like cells that could fertilize oocytes following a round spermatid injection (ROSI) procedure to yield healthy offspring (Ishikura et al., 2021). These studies will provide a valuable platform to decipher the molecular underpinnings of male germ cell development in mice.

Hwang and colleagues have recently established human M and T1 pro-spermatogonia-like cells from hiPSCs (Fig. 2B). In this study, hiPSCs were first induced into hPGCLCs through iMeLCs, which were then re-aggregated with mouse testicular somatic cells obtained from E12.5 embryonic testes to generate xenogeneic reconstituted testes (xrTestis) (Hwang et al., 2020). Upon a long-term air-liquid interface culture of nearly 120 days, a small proportion of the hPGCLCs differentiated into mitotically active M and quiescent T1 pro-spermatogonia-like cells, with remarkable transcriptional similarity to their in vivo counterparts. As xrTestis gradually disintegrate upon further cultivation, further optimization of the culture will be required to obtain later stages of human male germ cells in vitro. The use of human fetal testes or equivalent testicular cell types derived in vitro from hiPSCs may provide a physiological niche that is more suited to the survival and maturation of human pro-spermatogonia.

In this Review, we have summarized our recent understanding of male germline development from specification to the establishment of spermatogonia. Although most of our knowledge has been obtained using the mouse as a model organism, recent studies using single-cell analyses and in vitro gametogenesis provide new insights into cellular and molecular pathways involved in this process, and highlight the divergence of developmental programs between mice and primates. Future studies using single-cell ‘omics’ approaches on in vivo samples and further reconstitution of male pathways using hiPSCs will illuminate the unique properties of developing human male germ cells and open up new possibilities for a regenerative approach to treating male infertility.

We thank Dr Leslie King and members of the Sasaki lab for critically reading the manuscript and for constructive feedback. Figs 1 and 2 were originally created using BioRender.

Funding

The authors’ research was supported by grants from the Open Philanthropy Project (2019-197906 and 10080664) and the Health Research Formula Funds the Pennsylvania Department of Health (RFA#67-80).

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Competing interests

The authors declare no competing or financial interests.