Cell cycle regulation for meiosis in mammalian germ cells

Different Modes of Cell Cycle Regulation in Early Germ Cell Development

In mammalian germ cell development, differences in cell cycle regulation play a fundamental role in the reproductive strategy of each sex. During mouse embryogenesis, sexually bipotent germ cells are specified as primordial germ cells (PGCs) by the actions of BMP and WNT signals, which comprise approximately 50–80 cells [1]. These sexually potent PGCs migrate through the hindgut and dorsal mesentery and eventually colonize the genital ridge by E10.5 (Fig. 1). During the migratory phase, PGCs proliferate to increase the number to ~25,000 cells by E13.5 [2, 3]. Since the proliferation of germ cells during this period principally determines the number of gamete sources in gonads, an active mitotic cell cycle plays a pivotal role in ensuring reproductive capacity in life.

Initially, PGCs were sexually neutral, and their sexual development depended on the surrounding environment. Approximately E11.5, gonadal somatic cells undergo sex-specific differentiation mediated by genetic cues, and germ cells adopt the sex of the surrounding somatic cells. With respect to sex-specific differentiation of germ cells, the most noteworthy event at E13.5 onward is that while male germ cells undergo cell cycle arrest at the G0/G1 phase, those of females quit the mitotic cell cycle and enter the meiotic cell cycle (Fig. 1). Therefore, the change in the cell cycle mode leads to fundamental differences between male and female germ cell development.

External Signals for Oogenic Development and Meiotic Entry in Female Germ Cells

Meiosis occurs between males and females at different periods during germ cell development. In mouse fetal ovaries, meiotic entry is restricted within a narrow window at approximately E13.5-E14.5 (Fig. 1A). In mice, female germ cells/oogonia quit the mitotic cell cycle, enter meiosis after sex determination [4,5,6], and downregulate pluripotency genes [3]. Meiotic entry propagates in a wave from the anterior to posterior pole with radial geometry in mouse fetal ovaries [7,8,9,10]. In the fetal ovary of cynomolgus monkeys, meiotic entry is observed approximately 10–12 weeks post-fertilization (wpf), which occurs earlier in the germ cells located in the inner cortex than in the outer cortex [11]. In human females, oogonia differentiate into oocytes at approximately 10–11 weeks of gestation (gw), and the majority of oocytes show meiotic gene expression at approximately 14–17 weeks of gestation [12, 13]. Another subpopulation of oogonia continues to express pluripotency markers until approximately gw of 19–20. After gw 20, the majority of oocytes initiate meiosis and get arrested at the diplotene stage [14].

Germ cells in the fetal ovary require several external signals to become competent for meiotic entry. Upon feminization of somatic cells, pre-granulosa cells express BMP2 in response to WNT signaling. WNT signaling and the downstream transcription factor β-catenin play a role in osteogenic differentiation to allow meiotic entry in the fetal ovary in a timely manner [15, 16]. BMP signaling leads to oogenic gene activation and meiosis competency in female germ cells (Fig. 1B) [17]. SMAD4 and SMAD1/5/8 act as transducers of BMP signaling, which is assumed to mediate the activation of the oogenic program, and their absence leads to male-type differentiation even in the ovary [18]. Furthermore, under BMP signaling, ZGLP1 activates key genes involved in oogenesis, including meiosis [19]. MSX1 and MSX2 have been hypothesized to be required for meiosis in female germ cells in response to BMP signaling [20]. Thus, BMP signaling directs multiple regulatory pathways to coordinate oogonial development before meiotic entry.

Retinoic acid (RA) is one of the key signals that instruct germ cells to express meiotic genes, such as stimulated by retinoic acid gene 8 (Stra8) and ensure timely entry into meiosis in the fetal ovary [4, 5, 21, 22]. RA signaling is mediated by retinoic acid receptors (RARs; RARα, RARβ, and RARγ) and retinoid X receptors (RXRs) that are bound by RA (Fig. 1B). RARs and RXRs form heterodimers and bind to retinoic acid responsive elements (RAREs) of target genes that are required for meiosis and other oogonial development. Two sources of RA are known [21, 23]. One is the mesonephros, from which the RA diffuses towards the fetal gonad, and the other is the somatic cells in the fetal ovary (Fig. 1B). Retinaldehyde dehydrogenase ALDH1A (ALDH1A1, ALDH1A2, and ALDH1A3) synthesizes RA from the retinol [21, 23]. Since RA or RAR-agonists upregulate the expression of meiotic genes, and pan-RAR antagonists suppress their expression in embryonic ovaries [4, 5], it has been assumed that female germ cells respond to RA prior to meiotic entry. However, genetic studies of mice lacking ALDH1A families or RARs provided contrasting evidence, demonstrating that RA signaling was dispensable for meiosis [23,24,25,26]. These studies imply that RA signaling is not the sole determinant of meiotic initiation and/or that other signaling substances that are metabolized by CYP26B1 may trigger meiotic entry. Altogether, external signals by RA and BMP synergistically coordinate oogonial development and meiotic entry into the fetal ovary.

G0/G1 Cell Cycle Arrest in Fetal Male Germ Cells

In fetal testes, male germ cells exit the mitotic cell cycle and enter G0/G1 arrest at approximately E13.5, which contrasts with female germ cells entering meiosis at the corresponding time (Fig. 1A) [27,28,29,30]. During G0/G1 arrest, fetal male germ cells (gonocytes/pro-spermatogonia) undergo de novo CpG methylation, and a subset of them subsequently establish a stem cell population called spermatogonial stem cells (SSCs) shortly after birth. During puberty, male germ cells enter meiosis when differentiation into spermatocytes is initiated. Thereafter, meiosis continuously occurs in the testes throughout life.

There are cell-intrinsic factors that direct male germ cells to G0/G1 arrest rather than meiotic entry. The retinoblastoma protein family (RB, p107, and p130) is known to be a negative regulator of S phase entry in the cell cycle and plays a pivotal role in cell cycle arrest. RB binds to E2F and represses S-phase-related gene expression [31]. Phosphorylation of RB by CDK4/6 leads to its dissociation from E2F, reducing the expression of S-phase-related genes. After germ cell sex determination, the phosphorylation level of RB is high in male germ cells from E12.5-E13.5 but decreases at E14.5-P1.5, which coincides with G0/G1 arrest [30]. Disruption of RB results in the active proliferation of male germ cells at E14.5, indicating that RB is required for G0/G1 arrest. Furthermore, STRA8 and meiosis-associated genes are ectopically upregulated in RB-deficient gonocytes, suggesting that the suppression of meiotic entry is compromised in the absence of RB [30]. However, even in the absence of RB, male germ cells eventually stop proliferating at later embryonic stages and are consequently removed by apoptosis, suggesting the existence of both RB-dependent and RB-independent mechanisms that arrest the cell cycle in fetal male germ cells [28, 30]. Similar phenomena have been reported for transcription factor BCN2-deficient gonocytes, which undergo premature re-entry into the cell cycle and exhibit ectopic expression of meiotic genes [32]. These findings imply that cell cycle arrest is indispensable for male germ cell development because it blocks meiotic cell cycle entry.

Similar to embryonic females, RA is synthesized at and secreted from the mesonephros in embryonic males [5, 26]. Although female germ cells receive RA signals, male germ cells are refractory to RA. In fetal testes, cytochrome P450 enzyme CYP26B1 degrades RA, preventing male germ cells from responding to RA (Fig. 1C) [4, 5, 33, 34]. When CYP26B1 expression decreases after E14.5, male germ cells alternatively exhibit cell-intrinsic mechanisms to prevent RA signaling and ectopic entry into meiosis.

NANOS families are evolutionarily conserved RNA-binding proteins with three orthologous genes in the mammalian genome [35]. Nanos2 is specifically expressed in male germ cells and precedes G0/G1 arrest. NANOS2 forms a complex with the RNA-binding proteins DND1 and deadenylation enzyme [36] and represses numerous genes that promote cell cycle and female-type germ cell differentiation, thereby maintaining gonocytes in G0/G1 arrest. One of the critical NANOS2 targets is Dazl, which encodes an RNA-binding protein and is required for acquiring competence to enter the meiotic prophase [37]. If the degradation-resistant form of Dazl that lacks the 3′-UTR is expressed, gonocytes exit G0/G1 arrest and resume the cell cycle [38]. Furthermore, NANOS2 suppresses the ectopic expression of RA-responsive genes, such as Stra8 in fetal testes; otherwise, male germ cells would exhibit signs of activation of meiosis-related genes [39]. Altogether, abrogation of cell cycle arrest leads to ectopic signs of meiotic entry in gonocytes, due to, at least in part, the susceptibility to ectopic RA signaling.

Reciprocally, ectopic RA signaling suppressed NANOS2 expression in male germ cells (Fig. 1C). This effect is counteracted by p38 mitogen-activated protein kinase (MAPK) signaling, which is required for cell cycle arrest at the G1 phase in a variety of differentiated cells. The active phosphorylated form of p38 is present in both male and female germ cells at E11.5 but is restricted to male germ cells at later stages. Abrogation of MAPK signaling by the inhibitor SB203580 leads to the downregulation of Nanos2 and concomitant ectopic signs of meiotic entry [40]. Thus, MAPK signaling promotes the entry of male germ cells in the G0/G1 arrest phase, antagonizing the meiotic entry [40, 41].

The mTORC1 pathway promotes cell cycle progression in response to stimuli, such as growth factors, hormones, and cellular stress. NANOS2 suppresses mTORC1 activity by directly binding to Rheb mRNA, which encodes an activator of mTORC1 [42]. Although mTORC1 is typically inactive in quiescent male germ cells, it is activated in the absence of NANOS2. As the majority of mTORC1-active cells in Nanos2-KO mice express the mitotic cell cycle marker Ki67, mTORC1 suppression is one of the key mechanisms that induce G1/G0 arrest in embryonic male germ cells. In addition, NANOS2 is known to suppress mTORC1 by trapping mTOR protein onto cytoplasmic mRNP in SSCs [43]. Therefore, NANOS2 represses mTORC1 activity through both post-transcriptional and post-translational mechanisms. Thus, NANOS2 acts as a node for different cascades to initiate and maintain cell cycle arrest.

Meiotic Cell Cycle in the Germ Cell Development

In mammals, the decision to enter meiosis is one of the most critical events for sexual reproduction because meiosis leads to germ cell differentiation into sperm or oocytes. Meiosis is a specialized cell cycle that modifies the mechanism of the canonical mitotic cell cycle such that it undergoes one round of DNA replication, followed by two rounds of chromosome segregation. Meiotic entry always coincides with the “pre-meiotic S” phase, which has a longer duration than the mitotic S phase [44] (Fig. 2A). Meiotic prophase I is a specialized stage that follows the pre-meiotic S phase and is equivalent to G2 phase of the canonical mitotic cell cycle. Meiotic prophase I is substantially prolonged compared to the canonical G2 phase to ensure meiosis-specific chromosome events, such as homolog synapsis, meiotic recombination, and reductional segregation of homologous chromosomes [45,46,47,48,49]. In male mice, approximately ten days are required to complete meiotic prophase I.

Meiotic prophase I demonstrates a feature of chromosome dynamics distinct from those of mitosis. Meiotic prophase I is divided into four substages ( leptotene, zygotene, pachytene, and diplotene) according to chromosome morphology and the extent of homolog synapsis (Fig. 2B). During meiotic prophase I, sister chromatids are organized into an axis structure called an axial element (AE). Mitotic-type cohesins (RAD21) are replaced by meiotic-type cohesins (RAD21L and REC8) [49]. Accordingly, the overall chromatin organization drastically changes with the disappearance of topologically associated domains (TAD) [50,51,52]. The pre-leptotene stage is defined with the mitosis-to-meiosis transition just before the start of meiotic prophase I, where the axis structure is yet to be developed, and AE components (e.g., SYCP3) are often seen as aggregates. The cytologically defined pre-leptotene stage partly overlaps with the pre-meiotic G1/S transition of the cell cycle when pre-meiotic DNA replication begins. Leptotene is defined by a chromosome morphology with a developing axis structure. Zygotene is defined as the time when the axis structure is developed and homologous chromosomes are about to undergo synapsis. Pachytene is defined as the time at which homologous chromosomes are fully synchronized by the synaptonemal complex (SC). During pachytene, meiotic recombination generates crossovers between homologous chromosomes, yielding physical linkages known as chiasmata. Diplotene is defined as the time at which homolog synapsis is disassembled. Following meiotic prophase I, homologous chromosomes, rather than sister chromatids, are segregated toward the opposite poles of the spindle by the dissolution of chiasmata in meiotic division I. Finally, in meiotic division II, sister chromatids are segregated to produce haploid gametes.

Sexually Different Regulation of the Meiotic Cell Cycle

The meiotic cell cycle is regulated by sexually different mechanisms that coordinate with the developmental program of germ cells. In mouse fetal ovaries, germ cells enter meiosis within a narrow window around E13.5-E14.5, and subsequently undergo dictyate/diplotene arrest in the middle of the meiotic prophase around birth (Fig. 1A). Dictyate/diplotene arrest is a long-term dormant state in oocytes that persists until the resumption of meiosis [53]. In human females, the dormant state of oocytes persists for more than 40 years. During dictyate/diplotene arrest, oocytes show a large nucleus called the germinal vesicle (GV) and reside in the primordial follicles. Thus, the female reproductive lifespan is determined by the oocyte pool that has undergone meiotic initiation during this limited period in fetal ovaries. From the viewpoint of the cell cycle, dictyate/diplotene arrest occurs in the “G2 phase” and halts undergoing meiotic division I, which is remarkably different from G0/G1 arrest observed in quiescent cells of somatic tissues. The mechanism underlying how oocytes sustain long-term arrest remains elusive. In males, meiosis occurs continuously during puberty in the testis. In contrast to female meiosis, spermatocytes undergo two successive rounds of chromosome segregation.

Remarkable differences are observed in the sensitivity of checkpoint responses to chromosomal errors during the meiotic prophase between spermatocytes and oocytes. Aberrations in meiotic recombination and homolog synapsis during the meiotic prophase are more strictly monitored in spermatocytes than in oocytes. ATR kinase plays a crucial role in meiotic prophase checkpoints that monitor proper homologous synapsis. ATR phosphorylates H2AX along unsynapsed chromosomes, which induces the meiotic silencing of unsynapsed chromosomes (MSUC) /meiotic sex chromosome inactivation (MSCI) [54, 55]. In spermatocytes, most X- and Y-chromosome regions are unsynapsed owing to limited homology and are transcriptionally silenced in the heterochromatic XY body. MSCI is essential for passing the pachytene checkpoints in spermatocytes [56]. Similar sexual differences in meiotic prophase have also been observed in response to heat stress. While the ovaries reside in the abdominal cavity, the testes are always maintained at a lower temperature than that of the body core. Progression of meiotic prophase in testes is sensitive to body core temperature, and spermatocytes with aberrations in meiotic recombination and homolog synapsis under heat stress are eliminated before progressing beyond pachytene [57].

Sexual differences in the mode of the cell cycle were observed during the transition from meiosis I to meiosis II [58]. In male meiosis, there is a short period of the interphase-like stage called “interkinesis” between meiosis I and meiosis II, during which chromosomes are decondensed, and the nuclear membrane is reassembled. In females, chromosomes are persistently condensed after completion of the first meiotic division until meiosis II, without such an interphase-like stage. This is partly due to different CDK1-Cyclin B activities accompanied by the regulation of the anaphase-promoting complex (APC/c) during the meiosis I-meiosis II transition between spermatocytes and oocytes [59].

MEIOSIN and STRA8 Activate Meiotic Cell Cycle Program

Although the mechanism of meiotic initiation is fundamental, how germ cells regulate the mitosis-to-meiosis transition remains unclear. Stimulated by retinoic acid gene 8 (Stra8) was originally identified as a gene that is induced by RA in mouse P19 embryonic carcinoma cells [60]. STRA8 is transiently expressed in the pre-meiotic germ cells of embryonic ovaries and postnatal testes, and its temporal expression corresponds to the pre-leptotene stage, the earliest time point of meiotic prophase I. In females, STRA8 is expressed around E13.5-E14.5, which coincides with the meiotic entry in the embryonic ovary [8,9,10, 22]. In males, STRA8 is transiently expressed twice during germ cell development, the first of which occurs in differentiating spermatogonia, and the second of which occurs at the time of differentiation into spermatocytes [61,62,63,64] (Fig. 1A). While meiosis does not occur at the first expression of STRA8 in spermatogonia, it occurs at the second stage of STRA8 expression in spermatocytes [61,62,63,64] (Fig. 1A). It is unclear why meiosis is induced only at the second expression of STRA8 but not at the first expression of STRA8 in the testis. Since Stra8 KO germ cells fail to undergo normal meiosis in males and females [61, 65,66,67], STRA8 has been assumed to play a key role in entry into meiosis. Although the significance of STRA8 in meiotic initiation is well known, the molecular function of STRA8 remains elusive.

Our proteomic approach identified a previously uncharacterized protein, Gm4969 as an STRA8-interacting factor, which was named meiosis initiator (MEIOSIN) [68]. MEIOSIN was transiently co-expressed with STRA8 in pre-leptotene spermatocytes and oocytes. Since expression of STRA8 is still observed in Meiosin KO and vice versa, expression of Meiosin and Stra8 is regulated independently of each other. MEIOSIN/STRA8 co-expressing pre-leptotene germ cells overlap with EdU incorporation, representing de novo DNA replication. Thus, the co-expression of MEIOSIN and STRA8 coincides with the pre-meiotic S phase, the time at which meiosis is initiated (Fig. 3A). While STRA8 is observed in the cytoplasm and nuclei, MEIOSIN is exclusively localized in the nuclei of spermatocytes and oocytes. In the absence of MEIOSIN, STRA8 fails to localize to the nucleus and remains largely in the cytosol. This indicates that MEIOSIN plays a role in the nuclear translocation of STRA8.

MEIOSIN protein possesses DNA-binding domains, basic helix–loop–helix (HLH), and high mobility group (HMG) box domains. Similarly, STRA8 possesses an HLH domain and an HMG-like domain [65, 69]. MEIOSIN and STRA8 form a complex that binds to the transcriptional start sites of numerous genes in the preleptotene germ cells [68, 69] (Fig. 3B). Crucially, the target genes bound by MEIOSIN and STRA8 are associated with meiotic prophase processes, such as meiotic chromosome dynamics and meiotic recombination. These include genes encoding meiosis-specific chromosome components (Sycp2, Sycp3, Hormad1, and Hormad2), synaptonemal complex (Sycp1, Syce1, Syce2, Syce3, and Tex12), meiotic cohesin subunits (Rad21L, Smc1β, and Stag3), meiotic recombination components (Prdm9, Dmc1, Spo11, Top6BL, Meiob, Ankrd31, Hsf2BP, Brme1, and Msh5), and meiosis-specific telomere proteins (Kash5, Majin, Terb1, and Sun1). Indeed, MEIOSIN/STRA8 target meiotic genes were downregulated in Meiosin KO and Stra8 KO spermatocytes. This suggested that the MEIOSIN-STRA8 complex acts as a transcriptional activator of meiosis-related genes to initiate meiosis in germ cells. Expression of MEIOSIN and STRA8, which coincides with the pre-meiotic S phase, suggests that the MEIOSIN-STRA8 complex coordinates meiotic entry with the cell cycle by robust transcriptional activation of the target genes for the involvement of a meiotic program in the S phase.

Meiotic gene expression has been hypothesized to be activated via STRA8-dependent and STRA8-independent pathways [70, 71]. Indeed, some meiotic genes, such as Sycp3 and Rec8 are already expressed at low levels prior to meiosis in an RA-dependent manner [70,71,72,73,74]. Meiotic gene promoters are poised by the enrichment of H3K4me2 and RNA polymerase II prior to meiosis [75] and primed with basal transcription. This hypothesis is exemplified by the fact that the expression of the MEIOSIN/STRA8 target genes is still detected at residual levels in Meiosin KO and Stra8 KO [68, 69]. The MEIOSIN-STRA8 complex probably boosts the transcription of genes primed for meiotic entry, which is in line with the proposed action of STRA8, which amplifies the transcription of a broad range of meiotic genes [69].

MEIOSIN and STRA8 are Required for Switching the Cell Cycle from Mitosis to Meiosis

In the testes and embryonic ovaries of Meiosin KO and Stra8 KO mice, although germ cells fail to enter meiotic prophase and do not show any signs of homolog synapsis or meiotic recombination, mitotic metaphase cells marked by histone H3Ser10 phosphorylation accumulate [76]. Furthermore, in Meiosin KO, aberrant mitotic-like cells emerge with ectopic misexpression of mitotic Cyclin A2, which should be suppressed at meiotic prophase in the wild-type [77,78,79]. These aberrant mitotic-like cells in Meiosin KO and Stra8 KO were eliminated by apoptosis. This phenotype could be attributed in part to the downregulation of MEIOC and YTHDC2, which bind to RNA and destabilize mitotic cell cycle-associated transcripts at the post-transcriptional level [77,78,79,80,81,82]. In Meioc KO and Ythdc2 KO mice, germ cells failed to maintain meiotic prophase, showing ectopic expression of mitotic cyclin and metaphase chromosome condensation [77,78,79], which is also observed in Meiosin KO and Stra8 KO mice [61, 68]. This evidence suggests that if meiotic gene expression programs are not installed in germ cells at a proper time, the exit of the mitotic cell cycle and entry into meiosis will fail.

It should be noted that a small population of Meiosin KO ovarian germ cells differentiate into mature oocyte-like cells exhibiting germinal vesicles (GV) and zona pellucida even though they fail to undergo meiotic prophase [68]. This phenomenon has also been observed in Stra8 KO female [67]. GV oocyte-like cells isolated from Meiosin KO ovaries can progress to a metaphase-like status in the culture, similar to WT oocytes. As WT oocytes undergo meiotic recombination during meiotic prophase, 20 pairs of bivalents with chiasma are observed during metaphase I. However, Meiosin KO oocyte-like cells possess 40 sister chromatid-like mitotic cells, suggesting that they have skipped meiotic prophase. This suggests that some oocyte-like cells undergo folliculogenesis without passing through the mitosis-to-meiosis transition in Meiosin KO and Stra8 KO females. This hypothesis is further supported by evidence that the development of oocyte-like cells can be directly induced from pluripotent stem cells by overexpressing key transcription factors in vitro [83]. Therefore, meiotic gene activation is genetically independent of oocyte development. Taken together, MEIOSIN and STRA8 play a role in switching the cell cycle from mitosis to meiosis in germ cells.

Conclusion

The fate of sexually neutral germ cells is determined by the cell cycle during development. Whether fetal germ cells undergo the meiotic cell cycle or G0/G1 arrest is induced by external signals and intrinsic mechanisms after mitotic proliferation. NANOS2 and CYP26B1 are key regulators that direct male germ cells to G0/G1 arrest and prevent them from entering the meiotic cell cycle. In the fetal ovary, RA, BMP, and WNT signaling force female germ cells to become competent in the meiotic cell cycle. MEIOSIN and STRA8 ensure the establishment of the meiotic cell cycle by activating meiotic genes, such that meiotic entry coincides with the S phase. Thus, the decision of germ cell fate is determined around E13.5 -14.5 by the change in cell cycle mode from the active mitotic cell cycle.

There are several issues yet to be understood regarding the regulation of the decision to enter the meiotic cell cycle. How the expression of MEIOSIN and STRA8 is regulated by upstream pathways [84, 85], and how the activation of the meiotic program synchronizes with the S phase of the cell cycle, are critical questions that are required to understand the precise mechanism of the meiotic cell cycle. Given that non-canonical subunits of basal transcriptional initiation complex TF IID are expressed in germ cells [86,87,88], how MEIOSIN and STRA8 transactivate meiotic genes together with germ cell-type TF IID should be examined. Furthermore, how meiotic initiation and subsequent prophase are regulated in sexually distinct ways is not well understood. The sexually dimorphic mechanisms of meiotic initiation should be considered in the context of the cell cycle, which requires further investigation.

Conflict of Interests

The authors declare no conflicts of interest.