Meiotic Entry Research Articles

OsSPL regulates meiotic fate acquisition in rice.

In angiosperms, the key step in sexual reproduction is successful acquisition of meiotic fate. However, the molecular mechanism determining meiotic fate remains largely unknown. Here, we report that OsSPOROCYTELESS (OsSPL) is critical for meiotic entry in rice (Oryza sativa). We performed a large-scale genetic screen of rice sterile mutants aimed to identify genes regulating meiotic entry and identified OsSPL using map-based cloning. We showed that meiosis-specific callose deposition, chromatin organization, and centromere-specific histone H3 loading were altered in the cells corresponding to pollen mother cells in Osspl anthers. Global transcriptome analysis showed that the enriched differentially expressed genes in Osspl were mainly related to redox status, meiotic process, and parietal cell development. OsSPL might form homodimers and interact with TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factor OsTCP5 via the SPL dimerization and TCP interaction domain. OsSPL also interacts with TPL (TOPLESS) corepressors, OsTPL2 and OsTPL3, via the EAR motif. Our results suggest that the OsSPL-mediated signaling pathway plays a crucial role in rice meiotic entry, which appears to be a conserved regulatory mechanism for meiotic fate acquisition in angiosperms.

Computational modelling of meiotic entry and commitment

In response to developmental and environmental conditions, cells exit the mitotic cell cycle and enter the meiosis program to generate haploid gametes from diploid germ cells. Once cells decide to enter the meiosis program they become irreversibly committed to the completion of meiosis irrespective of the presence of cue signals. How meiotic entry and commitment occur due to the dynamics of the regulatory network is not well understood. Therefore, we constructed a mathematical model of the regulatory network that controls the transition from mitosis to meiosis in Schizosaccharomyces pombe. Upon nitrogen starvation, yeast cells exit mitosis and undergo conjugation and meiotic entry. The model includes the regulation of Mei2, an RNA binding protein required for conjugation and meiotic entry, by multiple feedback loops involving Pat1, a kinase that keeps cells in mitosis, and Ste11, a transcription activator required for the sexual differentiation. The model accounts for various experimental observations and demonstrates that the activation of Mei2 is bistable, which ensures the irreversible commitment to meiosis. Further, we show by integrating the meiosis-specific regulation with a cell cycle model, the dynamics of cell cycle exit, G1 arrest and entry into meiosis under nitrogen starvation.

Stage-Specific Expression of CYP26B1 in the Adult Testis is Responsible for Pulsatile Retinoic Acid Signaling in Spermatogenesis

Retinoic acid (RA) plays important roles in spermatogenesis process; however, mechanisms underlying pulsatile RA signaling in meiotic initiation has not been understood yet. We studied expression pattern of RA-degrading enzyme CYP26B1 during development of rhesus monkey testis using qPCR and IHC. In developing monkey testis, the CYP26B1 protein was detected in cytoplasm of undifferentiated spermatogonia. Expression level of CYP26B1 mRNA was down-regulated during testis development which is consistent with initiation of meiosis in adult testis. In adult testes, a heterogeneous pattern of CYP26B1 expression was observed along different stages of seminiferous epithelium, presumably indicates expression pattern of protein is stage specific. Highest level of CYP26B1 protein expression was coincided with onset of meiosis and observed in early meiotic spermatocytes within seminiferous epithelial stages X-XII. Whereas, lowest level of CYP26B1 expression was observed in stages VI-IX of seminiferous epithelium, where undifferentiated Type A spermatogonia divide and differentiate to Type B spermatogonia, meiosis initiates and spermiogenesis occurs. This observations led us to suggest CYP26B1 might be responsible for pulsatile RA signaling in spermatogenesis. Findings presumably support that elevated amount of RA in undifferentiated Type A spermatogonia during stages VI-IX of the seminiferous epithelium of adult monkey testis may be responsible for differentiation of spermatogonia and their meiotic entry

The Conserved RNA Exonuclease Rexo5 Is Required for 3′ End Maturation of 28S rRNA, 5S rRNA, and snoRNAs

Non-coding RNA biogenesis in higher eukaryotes has not been fully characterized. Here, we studied the Drosophila melanogaster Rexo5 (CG8368) protein, a metazoan-specific member of the DEDDh 3'-5' single-stranded RNA exonucleases, by genetic, biochemical, and RNA-sequencing approaches. Rexo5 is required for small nucleolar RNA (snoRNA)and rRNA biogenesis and is essential in D.melanogaster. Loss-of-function mutants accumulate improperly 3' end-trimmed 28S rRNA, 5S rRNA, and snoRNA precursors invivo. Rexo5 is ubiquitously expressed at low levels in somatic metazoan cells but extremely elevated in male and female germ cells. Loss of Rexo5 leads to increased nucleolar size, genomic instability, defective ribosome subunit export, and larval death. Loss of germline expression compromises gonadal growth and meiotic entry during germline development.

Overactive type 2 cannabinoid receptor induces meiosis in fetal gonads and impairs ovarian reserve

Type 2 cannabinoid receptor (CB2R) has been proposed to promote in vitro meiotic entry of postnatal male germ cells and to maintain the temporal progression of spermatogenesis in vivo. However, no information is presently available on the role played by CB2R in male and female fetal gonads. Here we show that in vitro pharmacological stimulation with JWH133, a CB2R agonist, induced activation of the meiotic program in both male and female fetal gonads. Upon stimulation, gonocytes initiated the meiotic program but became arrested at early stages of prophase I, while oocytes showed an increased rate of meiotic entry and progression toward more advanced stage of meiosis. Acceleration of meiosis in oocytes was accompanied by a strong increase in the percentage of γ-H2AX-positive pachytene and diplotene cells, paralleled by an increase of TUNEL-positive cells, suggesting that DNA double-strand breaks were not correctly repaired during meiosis, leading to oocyte apoptosis. Interestingly, in vivo pharmacological stimulation of CB2R in fetal germ cells through JWH133 administration to pregnant females caused a significant reduction of primordial and primary follicles in the ovaries of newborns with a consequent depletion of ovarian reserve and reduced fertility in adult life, while no alterations of spermatogenesis in the testis of the offspring were detected. Altogether our findings highlight a pro-meiotic role of CB2R in male and female germ cells and suggest that the use of cannabis in pregnant female might represent a risk for fertility and reproductive lifespan in female offspring.

Diethylstilbestrol impaired oogenesis of yellow catfish juveniles through disrupting hypothalamic-pituitary-gonadal axis and germ cell development.

Diethylstilbestrol (DES), a non-steroidal estrogen, has been found to cause altered germ cell development and disordered ovarian development in fish females. However, the mechanisms that might be involved are poorly understood. In this study, female juveniles of yellow catfish (Pelteobagrus fulvidraco) (120days post-hatching) were exposed to two doses (10 and 100ng l-1 ) of DES for 28days. After the endpoint of exposure, decreased ovary weight and gonadosomatic index, as well as various ovarian impairments were observed in response to DES. Besides, DES elevated the mRNA levels of vitellogenin 1 (vtg 1) and estrogen receptor 1 (esr 1) in liver and decreased 17β-estradiol level in plasma. Correspondingly, suppressed mRNA levels of the key genes in the hypothalamic-pituitary-gonadal axis (such as cyp19a1b, gnrh-II, fshβ and lhβ in brain and fshr, lhr and cyp19a1a in ovary) after DES exposure were also observed. The declined level of plasma 17β-estradiol and altered gene expressions of genes in the hypothalamic-pituitary-gonadal axis were thus supposed to be closely related to the disrupted oogenesis in DES-treated fish. Analyses further demonstrated that, higher concentration of DES elevated the expression ratio of bax/bcl-2, indicating the enhanced apoptosis occurred in ovary. Moreover, DES upregulated the expressions of genes involved in proliferation (cyclin d1 and pcna), meiotic entry (cyp26a1 and scp3) and meiotic maintenance (dmc1), resulting in arrested oogenesis in catfish. The present study greatly extended our understanding on the mechanisms underlying of reproductive toxicity of DES on fish oogenesis.

Bone morphogenetic protein and retinoic acid synergistically specify female germ-cell fate in mice.

The mechanism for sex determination in mammalian germ cells remains unclear. Here, we reconstitute the female sex determination in mouse germ cells invitro under a defined condition without the use of gonadal somatic cells. We show that retinoic acid (RA) and its key effector, STRA8, are not sufficient to induce the female germ-cell fate. In contrast, bone morphogenetic protein (BMP) and RA synergistically induce primordial germ cells (PGCs)/PGC-like cells (PGCLCs) derived from embryonic stem cells (ESCs) into fetal primary oocytes. The induction is characterized by entry into the meiotic prophase, occurs synchronously and recapitulates cytological and transcriptome progression invivo faithfully. Importantly, the female germ-cell induction necessitates a proper cellular competence-most typically, DNA demethylation of relevant genes-which is observed in appropriately propagated PGCs/PGCLCs, but not in PGCs/PGCLCs immediately after induction. This provides an explanation for the differential function of BMP signaling between PGC specification and female germ-cell induction. Our findings represent a framework for a comprehensive delineation of the sex-determination pathway in mammalian germ cells, including humans.

The impact of Zearalenone on the meiotic progression and primordial follicle assembly during early oogenesis

Zearalenone (ZEA) is a mycotoxin produced by fusarium graminearum. It can cause abnormal reproductive function by acting as an environmental estrogen. Research has traditionally focused on acute and chronic injury on mammalian reproductive capacity after ZEA treatment. Little research has been done studying the effects of ZEA exposure on early oogenesis. In this study, we investigate the effects of ZEA exposure on meiotic entry, DNA double-strand breaks (DSBs), and primordial follicle assembly during murine early oogenesis. The results show that ZEA exposure significantly decreased the percentage of diplotene stage germ cells, and made more germ cells remain at zygotene or pachytene stages. Moreover, the mRNA expression level of meiosis-related genes was significantly reduced after ZEA treatment. ZEA exposure significantly increased DNA-DSBs at the diplotene stage. Meanwhile, DNA damage repair genes such as RAD51 and BRCA1 were activated. Furthermore, maternal exposure to ZEA significantly decreased the number of primordial follicles in newborn mouse ovaries. In conclusion, ZEA exposure impairs mouse female germ cell meiotic progression, DNA-DSBs, and primordial follicle assembly.

The epigenetic modifications and the anterior to posterior characterization of meiotic entry during mouse oogenesis.

The meiotic initiation of mammalian oogonia is a critical step during the development of primordial germ cells (PGCs)to mature oocytes. In this study, a systematic investigation of epigenetic modifications and DAZL gene expression during oogonia meiotic entry were performed. We found that the expression of DAZL was epigenetically regulated by DNA methylation of CpG islands within its promoter region. During meiotic entry, a continuously increasing level of 5hmC, a stable epigenetic marker usually associated with the activation of gene expression, was observed from 11.5 to 16.5 dpc (days post coitum). Meanwhile trimethylation of lysine 27 on histone3 (H3K27me3), usually associated with repression of gene expression, had a sustainable increase from 12.5 to 16.5 dpc. Finally, by equally dividing the ovaries into three regions representing the anterior, the middle, and the posterior of the ovary and performing immunofluorescence and qRT-PCR on the individual regions, we provided further evidences that the meiotic entry and progression of female germ cells is in an anterior to posterior pattern.

Does MAX open up a new avenue for meiotic research?

Meiosis is a central event of sexual reproduction. Like somatic cells, germ cells conduct mitosis to increase their cell number, but unlike somatic cells, germ cells switch their cell division mode from mitosis to meiosis at a certain point in gametogenesis. However, the molecular basis of this switch remains elusive. In this review article, we give an overview of the onset of mammalian meiosis, including our recent finding that MYC Associated Factor X (MAX) prevents ectopic and precocious meiosis in embryonic stem cells (ESCs) and germ cells, respectively. We present a hypothetical model of a MAX-centered molecular network that regulates meiotic entry in mammals and propose that inducible Max knockout ESCs provide an excellent platform for exploring the molecular mechanisms of meiosis initiation, while excluding other aspects of gametogenesis.

Human foetal ovary shares meiotic preventing factors with the developing testis.

How can pre-meiotic germ cells persist in the human foetal ovary? Numerous oogonia escaping meiotic entry were retrieved throughout human ovarian development simultaneously with the expression of signalling pathways preventing meiosis, typically described in the rodent embryonic testis. The transition from mitosis to meiosis is a key event in female germ cells that remains poorly documented in research on the human ovary. Previous reports described a strikingly asynchronous differentiation in the human female germ line during development, with the persistence of oogonia among oocytes and follicles during the second and third trimesters. The possible mechanisms allowing some cells to escape meiosis remain elusive. In order to document the extent of this phenomenon, we detailed the expression profile of germ cell differentiation markers using 73 ovaries ranging from 6.4 to 35 weeks post-fertilization. Pre-meiotic markers were detected by immunohistochemistry or qRT-PCR. The expression of the main meiosis-preventing factors identified in mice was analysed, and their functionality assessed using organ cultures. Oogonia stained for AP2γ could be traced from the first trimester until the end of the third trimester. Female germ cell differentiation is organized both in time and space in a centripetal manner in the foetal human ovary. Unexpectedly, some features usually ascribed to rodent pre-spermatogonia could be observed in human foetal ovaries, such as NANOS2 expression and quiescence in some germ cells. The two main somatic signals known to inhibit meiosis in the mouse embryonic testis, CYP26B1 and FGF9, were detected in the human ovary and act simultaneously to repress STRA8 and meiosis in human foetal female germ cells. N/A. Our conclusions relied partly on in vitro experiments. Germ cells were not systematically identified with immunostaining and some may have thus escaped analysis. We found evidence that a robust repression of meiotic entry is taking place in the human foetal ovary, possibly explaining the exceptional long-lasting presence of pre-meiotic germ cells until late gestational age. This result calls for a redefinition of the markers known as classical male markers, which may in fact characterize mammalian developing gonads irrespectively of their sex. This research was supported by the Université Paris Diderot-Paris 7 and Université Paris-Sud, CEA, INSERM, and Agence de la Biomédecine. The authors declare no conflict of interest.

Avian Primordial Germ Cells.

Germ cells transmit genetic information to the next generation through gametogenesis. Primordial germ cells (PGCs) are the first germ-cell population established during development, and are the common origins of both oocytes and spermatogonia. Unlike in other species, PGCs in birds undergo blood circulation to migrate toward the genital ridge, and are one of the major biological properties of avian PGCs. Germ cells enter meiosis and arrest at prophase I during embryogenesis in females, whereas in males they enter mitotic arrest during embryogenesis and enter meiosis only after birth. In chicken, gonadal sex differentiation occurs as early as embryonic day 6, but meiotic initiation of female germ cells starts from a relatively late stage (embryonic day 15.5). Retinoic acid controls meiotic entry in developing chicken gonads through the expressions of retinaldehyde dehydrogenase 2, a major retinoic acid synthesizing enzyme, and cytochrome P450 family 26, subfamily B member 1, a major retinoic acid-degrading enzyme. The other major biological property of avian PGCs is that they can be propagated in vitro for the long term, and this technique is useful for investigating proliferation mechanisms. The main factor involved in chicken PGC proliferation is fibroblast growth factor 2, which activates the signaling of MEK/ERK and thus promotes the cell cycle and anti-apoptosis. Furthermore, the activation of PI3K/Akt signaling is indispensable for the proliferation and survival of chicken PGCs.

Germ cell-specific sustained activation of Wnt signalling perturbs spermatogenesis in aged mice, possibly through non-coding RNAs.

Dysregulated Wnt signalling is associated with human infertility and testicular cancer. However, the role of Wnt signalling in male germ cells remains poorly understood. In this study, we first confirmed the activity of Wnt signalling in mouse, dog and human testes. To determine the physiological importance of the Wnt pathway, we developed a mouse model with germ cell-specific constitutive activation of βcatenin. In young mutants, similar to controls, germ cell development was normal. However, with age, mutant testes showed defective spermatogenesis, progressive germ cell loss, and flawed meiotic entry of spermatogonial cells. Flow sorting confirmed reduced germ cell populations at the leptotene/zygotene stages of meiosis in mutant group. Using thymidine analogues-based DNA double labelling technique, we further established decline in germ cell proliferation and differentiation. Overactivation of Wnt/βcatenin signalling in a spermatogonial cell line resulted in reduced cell proliferation, viability and colony formation. RNA sequencing analysis of testes revealed significant alterations in the non-coding regions of mutant mouse genome. One of the novel non-coding RNAs was switched on in mutant testes compared to controls. QPCR analysis confirmed upregulation of this unique non-coding RNA in mutant testis. In summary, our results highlight the significance of Wnt signalling in male germ cells.

Loss of MAX results in meiotic entry in mouse embryonic and germline stem cells.

Meiosis is a unique process that allows the generation of reproductive cells. It remains largely unknown how meiosis is initiated in germ cells and why non-germline cells do not undergo meiosis. We previously demonstrated that knockdown of Max expression, a gene encoding a partner of MYC family proteins, strongly activates expression of germ cell-related genes in ESCs. Here we find that complete ablation of Max expression in ESCs results in profound cytological changes reminiscent of cells undergoing meiotic cell division. Furthermore, our analyses uncovers that Max expression is transiently attenuated in germ cells undergoing meiosis in vivo and its forced reduction induces meiosis-like cytological changes in cultured germline stem cells. Mechanistically, Max depletion alterations are, in part, due to impairment of the function of an atypical PRC1 complex (PRC1.6), in which MAX is one of the components. Our data highlight MAX as a new regulator of meiotic onset.

The Mechanism of Germline Sex Determination in Vertebrates.

Germ cells are the common cells of origin for the two different types of gametes, sperm and eggs. In vertebrates so far examined, the sex of germ cells is determined by gonadal somatic cells. However, influenced by the somatic cells, how germ cells adopt their sexual fates by intrinsic factors has long been unclear in vertebrates. We recently identified forkhead box L3 (FOXL3) as a germ cell-intrinsic factor involved in the sperm-egg fate decision in the teleost fish medaka (Oryzias latipes). On the basis of the results obtained by the analysis of foxl3/FOXL3 expression and loss-of-function mutants, we review when and how germ cell sex is regulated non-cell-autonomously and cell-autonomously. We then discuss the fact that the germline sex determination pathway is genetically distinct from other essential gametogenic pathways such as meiotic entry and the establishment of germline stem cells. Another extraordinary finding in the foxl3 mutant is that functional sperm can be produced in the ovary, which provides a new notion that gametogenesis can proceed regardless of the sex of the surrounding somatic cells once the sexual identity of germ cells is established in medaka.

Loss of the histone chaperone ASF1B reduces female reproductive capacity in mice.

Anti-silencing function 1 (ASF1) is an evolutionarily conserved histone H3-H4 chaperone involved in the assembly/disassembly of nucleosome and histone modification. Two paralogous genes, Asf1a and Asf1b, exist in the mouse genome. Asf1a is ubiquitously expressed and its loss causes embryonic lethality. Conversely, Asf1b expression is more restricted and has been less studied. To determine the in vivo function of Asf1b, we generated a Asf1b-deficient mouse line (Asf1b(GT(ROSA-βgeo)437)) in which expression of the lacZ reporter gene is driven by the Asf1b promoter. Analysis of β-galactosidase activity at early embryonic stages indicated a correlation between Asf1b expression and cell differentiation potential. In the gonads of both male and female, Asf1b expression was specifically detected in the germ cell lineage with a peak expression correlated with meiosis. The viability of Asf1b-null mice suggests that Asf1b is dispensable for mouse development. However, these mice showed reduced reproductive capacity compared with wild-type controls. We present evidence that the timing of meiotic entry and the subsequent gonad development are affected more severely in Asf1b-null female mice than in male mice. In female mice, in addition to subfertility related to altered gamete formation, variable defects compromising the development and/or survival of their offspring were also observed. Altogether, our data indicate the importance of Asf1b expression at the time of meiotic entry, suggesting that chromatin modifications may play a central role in this process.

On the development of extragonadal and gonadal human germ cells.

ABSTRACTHuman germ cells originate in an extragonadal location and have to migrate to colonize the gonadal primordia at around seven weeks of gestation (W7, or five weeks post conception). Many germ cells are lost along the way and should enter apoptosis, but some escape and can give rise to extragonadal germ cell tumors. Due to the common somatic origin of gonads and adrenal cortex, we investigated whether ectopic germ cells were present in the human adrenals. Germ cells expressing DDX4 and/or POU5F1 were present in male and female human adrenals in the first and second trimester. However, in contrast to what has been described in mice, where ‘adrenal’ and ‘ovarian’ germ cells seem to enter meiosis in synchrony, we were unable to observe meiotic entry in human ‘adrenal’ germ cells until W22. By contrast, ‘ovarian’ germ cells at W22 showed a pronounced asynchronous meiotic entry. Interestingly, we observed that immature POU5F1+ germ cells in both first and second trimester ovaries still expressed the neural crest marker TUBB3, reminiscent of their migratory phase. Our findings highlight species-specific differences in early gametogenesis between mice and humans. We report the presence of a population of ectopic germ cells in the human adrenals during development.

Analgesic exposure in pregnant rats affects fetal germ cell development with inter-generational reproductive consequences.

Analgesics which affect prostaglandin (PG) pathways are used by most pregnant women. As germ cells (GC) undergo developmental and epigenetic changes in fetal life and are PG targets, we investigated if exposure of pregnant rats to analgesics (indomethacin or acetaminophen) affected GC development and reproductive function in resulting offspring (F1) or in the F2 generation. Exposure to either analgesic reduced F1 fetal GC number in both sexes and altered the tempo of fetal GC development sex-dependently, with delayed meiotic entry in oogonia but accelerated GC differentiation in males. These effects persisted in adult F1 females as reduced ovarian and litter size, whereas F1 males recovered normal GC numbers and fertility by adulthood. F2 offspring deriving from an analgesic-exposed F1 parent also exhibited sex-specific changes. F2 males exhibited normal reproductive development whereas F2 females had smaller ovaries and reduced follicle numbers during puberty/adulthood; as similar changes were found for F2 offspring of analgesic-exposed F1 fathers or mothers, we interpret this as potentially indicating an analgesic-induced change to GC in F1. Assuming our results are translatable to humans, they raise concerns that analgesic use in pregnancy could potentially affect fertility of resulting daughters and grand-daughters.

Germline Stem Cell Differentiation Entails Regional Control of Cell Fate Regulator GLD-1 in Caenorhabditis elegans

Germline stem cell differentiation in Caenorhabditis elegans is controlled by glp-1 Notch signaling. Cell fate regulator GLD-1 is sufficient to induce meiotic entry and expressed at a high level during meiotic prophase, inhibiting mitotic gene activity. glp-1 signaling and other regulators control GLD-1 levels post-transcriptionally (low in stem cells, high in meiotic prophase), but many aspects of GLD-1 regulation are uncharacterized, including the link between glp-1-mediated transcriptional control and post-transcriptional GLD-1 regulation. We established a sensitive assay to quantify GLD-1 levels across an ∼35-cell diameter field, where distal germline stem cells differentiate proximally into meiotic prophase cells in the adult C. elegans hermaphrodite, and applied the approach to mutants in known or proposed GLD-1 regulators. In wild-type GLD-1 levels elevated ∼20-fold in a sigmoidal pattern. We found that two direct transcriptional targets of glp-1 signaling, lst-1 and sygl-1, were individually required for repression of GLD-1. We determined that lst-1 and sygl-1 act in the same genetic pathway as known GLD-1 translational repressor fbf-1, while lst-1 also acts in parallel to fbf-1, linking glp-1-mediated transcriptional control and post-transcriptional GLD-1 repression. Additionally, we estimated the position in wild-type gonads where germ cells irreversibly commit to meiotic development based on GLD-1 levels in worms where glp-1 activity was manipulated to cause an irreversible fate switch. Analysis of known repressors and activators, as well as modeling the sigmoidal accumulation pattern, indicated that regulation of GLD-1 levels is largely regional, which we integrated with the current view of germline stem cell differentiation.

Meiotic onset is reliant on spatial distribution but independent of germ cell number in the mouse ovary.

Mouse ovarian germ cells enter meiosis in a wave that propagates from anterior to posterior, but little is known about contribution of germ cells to initiation or propagation of meiosis. In a Ror2 mutant with diminished germ cell number and migration, we find that overall timing of meiotic initiation is delayed at the population level. We use chemotherapeutic depletion to exclude a profoundly reduced number of germ cells as a cause for meiotic delay. We rule out sex reversal or failure to specify somatic support cells as contributors to the meiotic phenotype. Instead, we find that anomalies in the distribution of germ cells as well as gonad shape in mutants contribute to aberrant initiation of meiosis. Our analysis supports a model of meiotic initiation via diffusible signal(s), excludes a role for germ cells in commencing the meiotic wave and furnishes the first phenotypic demonstration of the wave of meiotic entry. Finally, our studies underscore the importance of considering germ cell migration defects while studying meiosis to discern secondary effects resulting from positioning versus primary meiotic entry phenotypes.