Early Germ Cell Research Articles

Analysis of a novel gene, Sdgc, reveals sex chromosome-dependent differences of medaka germ cells prior to gonad formation.

In vertebrates that have been examined to date, the sexual identity of germ cells is determined by the sex of gonadal somatic cells. In the teleost fish medaka, a sex-determination gene on the Y chromosome, DMY/dmrt1bY, is expressed in gonadal somatic cells and regulates the sexual identity of germ cells. Here, we report a novel mechanism by which sex chromosomes cell-autonomously confer sexually different characters upon germ cells prior to gonad formation in a genetically sex-determined species. We have identified a novel gene, Sdgc (sex chromosome-dependent differential expression in germ cells), whose transcripts are highly enriched in early XY germ cells. Chimeric analysis revealed that sexually different expression of Sdgc is controlled in a germ cell-autonomous manner by the number of Y chromosomes. Unexpectedly, DMY/dmrt1bY was expressed in germ cells prior to gonad formation, but knockdown and overexpression of DMY/dmrt1bY did not affect Sdgc expression. We also found that XX and XY germ cells isolated before the onset of DMY/dmrt1bY expression in gonadal somatic cells behaved differently in vitro and were affected by Sdgc. Sdgc maps close to the sex-determination locus, and recombination around the two loci appears to be repressed. Our results provide important insights into the acquisition and plasticity of sexual differences at the cellular level even prior to the developmental stage of sex determination.

Granulosa cells and retinoic acid co-treatment enrich potential germ cells from manually selected Oct4-EGFP expressing human embryonic stem cells

Differentiation of human embryonic stem (HES) cells to germ cells may become clinically useful in overcoming diseases related to germ-cell development. Niches were used to differentiate HES cell lines, NTU1 and H9 Oct4-enhanced green fluorescence protein (EGFP), including laminin, granulosa cell co-culture or conditioned medium, ovarian stromal cell co-culture or conditioned medium, retinoic acid, stem cell factor (SCF) and BMP4–BMP7–BMP8b treatment. Flow cytometry showed that granulosa cell co-culture (P < 0.001) or conditioned medium (P = 0.007) treatment for 14 days significantly increased the percentages of differentiated H9 Oct4-EGFP cells expressing early germ cell marker stage-specific embryonic antigen 1(SSEA1); sorted SSEA1[+] cells did not express higher levels of germ cell gene VASA and GDF9. Manually collected H9 Oct4-EGFP[+] cells expressed significantly higher levels of VASA (P = 0.005) and GDF9 (P = 0.001). H9 Oct4-EGFP[+] cells developed to ovarian follicle-like structures after culture for 28 days but with low efficiency. Unlike SCF and BMP4, retinoic acid co-treatment enhanced VASA, GDF9 and SCP3 expression. A protocol is recommended to enrich differentiated HES cells with germ-cell potential by culture with granulosa cells, conditioned medium or retinoic acid, manual selection of Oct4-EGFP[+] cells, and analysis of VASA, GDF9 expression, or both.

Genetic and Epigenetic landscape of Germline Stem Cells

Elucidating the critical epigenetics events involved in differentiation and reprogramming of cells to primordial germ cells (PGCs) is among the interesting issues in stem cell research. Here, I will talk about critical transcription factors and global hypomethylation in development of germ cells. Evidence strongly suggests that the earliest PGCs emerging in the E7.25 mouse embryo epiblast have a highly methylated genome, and high level of H3K9me2 in chromatin but during development, genome demethylated and patterns of histone codes changes dramatically. We designed a polycistroniclentiviral vector and overexpressed Stella, Oct4 and Nanos2 simultaneously in transduced cells; Increasing level of Prdm14, Nanog and decreasing of G9a expression is an interesting finding which might be considered as a primary step of reprogramming toward germline progenitor cells, here we propose decreasing H3K9me2 level as a consequence of G9a down regulation is a critical step which facilitated transition to different stemness state through creating a new epigenetic memory for the early germ cells. Keywords: Epigenetic, hypomethylation, Germ line Stem Cells, polycistroniclentiviral vector.

Cisplatin-induced alterations in the functional spermatogonial stem cell pool and niche in C57/BL/6J mice following a clinically relevant multi-cycle exposure

A typical clinical cis-diamminedichloroplatinum(II) (cisplatin) dosing regimen consists of repeated treatment cycles followed by a recovery period. While effective, this dosing structure results in a prolonged, often permanent, infertility in men. Spermatogonial stem cells (SSCs) are theoretically capable of repopulating the seminiferous tubules after exposure has ceased. We propose that an altered spermatogonial environment during recovery from the initial treatment cycle drives an increase in SSC mitotic cell activity, rendering the SSC pool increasingly susceptible to cisplatin-induced injury from subsequent cycles. To test this hypothesis, the undifferentiated spermatogonia population and niche of the adult mouse (C57/BL/6J) were examined during the recovery periods of a clinically-relevant cisplatin exposure paradigm. Histological examination revealed a disorganization of spermatogenesis correlating with the number of exposure cycles. Quantification of terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick end labeling (TUNEL) staining indicated an increase in apoptotic frequency following exposure. Immunohistochemical examination of Foxo1 and incorporated BrdU showed an increase in the undifferentiated spermatogonial population and mitotic activity in the recovery period in mice exposed to one cycle, but not two cycles of cisplatin. Immunohistochemical investigation of glial cell line-derived neurotrophic factor (GDNF) revealed an increase in production along the basal Sertoli cell membrane throughout the recovery period in all treatment groups. Taken together, these data establish that the impact of cisplatin exposure on the functional stem cell pool and niche correlates with: (1) the number of dosing cycles; (2) mitotic activity of early germ cells; and (3) alterations in the basal Sertoli cell GDNF expression levels after cisplatin-induced testicular injury.

Hypoxia inducible factor 2α/insulin-like growth factor receptor signal loop supports the proliferation and Oct-4 maintenance of mouse germline stem cells

Hypoxia inducible factor 2α (HIF-2α) is critical for primordial germ cell (PGC) survival as knockout of HIF-2α (HIF-2α(-/-)) decreases both expression of Oct-4 and PGC number in genital ridge. Hypoxia is known to stabilize HIF-2α protein from proteasomal degradation. However, little is known about the hypoxia-associated endocrinal signaling in HIF-2α expression. The current work demonstrates a role for an endocrine insulin-like growth factor-I receptor (IGF-IR)-PI3K/Akt-mTOR-HIF-2α regulatory loop in the proliferation and Oct-4 maintenance of PGC-like alkaline phosphatase positive mouse germline stem cells (AP(+)GSCs). We found that hypoxia greatly increased the cell proliferation and the levels of nuclear Oct-4/HIF-2α protein of AP(+)GSCs. The hypoxic-AP(+)GSCs presented stronger stemness ability for germ cell differentiation than normoxic, with expressions of c-KIT (differentiation germ cell marker), VASA (differentiation germ cell marker) and SCP3 (meiotic marker) using a renal capsule transplantation assay. Meanwhile, hypoxia significantly increased the expression levels of secreted-IGF-I and IGF-IR. The IGF-I dose dependently increased the HIF-2α expression levels in AP(+)GSCs; and, the inhibition of IGF-IR by RNA interference (shIGF-IR) or LY294002 (PI3K inhibitor)/Rapamycin (mTOR inhibitor) effectively suppressed the IGF-I- and/or hypoxia-induced HIF-2α and Oct-4 expression, suggesting that the IGF-IR and its downstream Akt/mTOR signaling are involved in the IGF-I/hypoxia effects. Additionally, knockdown of HIF-2α dramatically suppressed Oct-4 and IGF-IR protein levels in AP(+)GSC cells. In conclusion, the present study demonstrates a regulatory loop of IGF-IR-PI3K/Akt-mTOR-HIF-2α in proliferation and Oct-4 maintenance of PGC-like AP(+)GSCs under hypoxia. This finding provides insights into the niche endocrinology underlying early germ cell development.

Epigenetic regulation of oogenesis and germ stem cell maintenance by the Drosophila histone methyltransferase Eggless/dSetDB1

The Drosophila melanogaster histone lysine methyltransferase (HKMT) Eggless (Egg/dSETDB1) catalyzes methylation of Histone H3 lysine 9 (H3K9), a signature of repressive heterochromatin. Our previous studies showed that H3K9 methylation by Egg is required for oogenesis. Here we analyze a set of EMS-induced mutations in the egg gene, identify the molecular lesions of these mutations, and compare the effects on oogenesis of both strong loss-of-function and weak hypomorphic alleles. These studies show that H3K9 methylation by Egg is required for multiple stages of oogenesis. Mosaic expression experiments show that the egg gene is not required intrinsically in the germ cells for their early differentiation, but is required in the germ cells for their survival past stage 5 of oogenesis. egg is also required in germ stem cells for their maintenance, since egg− germ stem cells initially survive but are not maintained as females age. Mosaic analysis also reveals that the early egg chamber budding defects in egg− ovaries are due to an intrinsic requirement for egg in follicle stem cells and their descendents, and that egg plays a non-autonomous role in somatic cells in the germarium to influence the differentiation of early germ cells.

Changes in rRNA Transcription Influence Proliferation and Cell Fate Within a Stem Cell Lineage

Ribosome biogenesis drives cell growth and proliferation, but mechanisms that modulate this process within specific lineages remain poorly understood. Here, we identify a Drosophila RNA polymerase I (Pol I) regulatory complex composed of Under-developed (Udd), TAF1B, and a TAF1C-like factor. Disruption of udd or TAF1B results in reduced ovarian germline stem cell (GSC) proliferation. Female GSCs display high levels of ribosomal RNA (rRNA) transcription, and Udd becomes enriched in GSCs relative to their differentiating daughters. Increasing Pol I transcription delays differentiation, whereas reducing rRNA production induces both morphological changes that accompany multicellular cyst formation and specific decreased expression of the bone morphogenetic protein (BMP) pathway component Mad. These findings demonstrate that modulating rRNA synthesis fosters changes in the cell fate, growth, and proliferation of female Drosophila GSCs and their daughters.

Role of Pten deletion and BRafV600E mutation in the generation of testicular germ cell tumors

Testicular germ cell tumors (TGCT) represent the most common solid malignancy affecting males between the ages of 15 and 35, while ovarian germ cell tumours (OGCT) are a type of ovarian neoplasm principally affecting young women. Germ cell tumors (GCTs) account for about 95 % of testicular cancer cases and for only 2-3% of ovarian cancer cases (Siegel et al., 2011). Most TGCT are potentially curable, however approximately 5% of patients with TGCT develop chemoresistance and die from the disease. PTEN deletion and mutational activation of BRAF are frequent genetic alterations found in human TGCTs, suggesting that they might be directly involved in germ cell tumorigenesis. Furthermore, BRAF mutation positively correlates with the acquisition of resistence to cisplatin, the most commonly chemotherapic agent employed for the treatment of human TGCTs. We obtained heterozygous floxed Ptenloxp/+ BRafCA Spo11Cre mice showing ovarian teratomas and testicular tumors with an incidence of about 30% at 20 days post partum (dpp) . Since Spo- 11Cre is active at around 13.5 days post coitum (dpc) in female germ cells and at around 7 dpp in male germ cells (18), these results suggest that ovarian teratomas origin from early meiotic germ cells in the fetal period whereas GCT formation in males can be a postnatal event. By histological inspection, we found that cancer cells in testes showed features reminiscent of seminoma such as a diffuse, confluent multinodular pattern. However, by immunohistochemical staining, we observed that the cells within the tumor showed heterogeneous positivity for the pluripotency markers Oct4, Sox2, Nanog, Kit and Prdm14, suggesting that they can represent a mixed form of seminoma and embryonal carcinoma cells. Our results indicate that deregulated MAP and PI3 Kinase activation can lead to postnatal male germ cells transformation.

Increased Prevalence of Severe Rheumatic Heart Disease Requiring Early Valve Replacement Surgery and Fragmented Germ Cells

Background: Patients requiring valve replacement at a younger age, for rheumatic heart disease was apparently increasing after the new age practice of contraception. Methods: In 2012, data of patients referred to tertiary care hospital from two cardiology screening camps conducted in the community, were analyzed for the severity of presentation of rheumatic heart disease and associated contraception status by dividing into the three age groups—20 - 35 years, 36 - 50 years, >51 years; ~400 patients attended the camps; 31 female and 2 male patients were referred to the tertiary care hospital for further evaluation and categorized treatment, including valve replacement surgeries; among the 33 referred patients, belonging to the three age groups, the data were analyzed for [if any] correlation of contraception status with severe rheumatic heart disease. 8 randomly chosen males, of the 3 age groups, from a different community, whose life partners had undergone sterilization, were assessed for their serum testosterone levels. Results: Among 20 - 35 years, 7 female patients with contraception required valve replacement surgeries [p 51 years, 3 female and 2 male patients without contraception received medical treatment only [p

Cg-SoxE and Cg-β-catenin, two new potential actors of the sex-determining pathway in a hermaphrodite lophotrochozoan, the Pacific oyster Crassostrea gigas

Sex determination is poorly known in mollusks, lophotrochozoa and most hermaphrodites. In the oyster Crassostrea gigas, an irregular successive hermaphrodite, sex determination may occur at the end of a gametogenetic cycle to prepare the next cycle. To investigate further into these aspects we have focused on orthologs of SoxE and β-catenin, key players of the male and female gonadic differentiation respectively in mammals. Based on phylogenetic analyses, Cg-SoxE, the oyster's SoxE ortholog, is closely related to vertebrate SoxE genes while Cg-β-catenin, the oyster's β-catenin ortholog, is classified amongst lophotrochozoa. The mRNA expression measured by qPCR in the gonadic area during a gametogenetic cycle is maximal for Cg-SoxE when sex is indiscernible and for Cg-β-catenin in mature females. Both expressions are localized from early germ cells to spermatocytes and pre-vitellogenic oocytes, and potentially in somatic cells. Cg-β-catenin is also expressed in vitellogenic oocytes. These actors may be involved in early oyster gonadic differentiation, which includes sex determination. Our results enhance the understanding of sex determination in C. gigas and in mollusks and they provide additional knowledge in compared genomics of reproduction and in molecular phylogeny.

MEIOB Targets Single-Strand DNA and Is Necessary for Meiotic Recombination

Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB). This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1 −/− spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob −/− meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination.

Smurf-mediated differential proteolysis generates dynamic BMP signaling in germline stem cells during Drosophila testis development

Germline stem cells (GSCs) produce gametes throughout the reproductive life of many animals, and intensive studies have revealed critical roles of BMP signaling to maintain GSC self-renewal in Drospophila adult gonads. Here, we show that BMP signaling is downregulated as testes develop and this regulation controls testis growth, stem cell number, and the number of spermatogonia divisions. Phosphorylated Mad (pMad), the activated Drosophila Smad in germ cells, was restricted from anterior germ cells to GSCs and hub-proximal cells during early larval development. pMad levels in GSCs were then dramatically downregulated from early third larval instar (L3) to late L3, and maintained at low levels in pupal and adult GSCs. The spatial restriction and temporal down-regulation of pMad, reflecting the germ cell response to BMP signaling activity, required action in germ cells of E3 ligase activity of HECT domain protein Smurf. Analyses of Smurf mutant testes and dosage-dependent genetic interaction between Smurf and mad indicated that pMad downregulation was required for both the normal decrease in stem cell number during testis maturation in the pupal stage, and for normal limit of four rounds of spermatogonia cell division for control of germ cell numbers and testis size. Smurf protein was expressed at a constant low level in GSCs and spermatogonia during development. Rescue experiments showed that expression of exogenous Smurf protein in early germ cells promoted pMad downregulation in GSCs in a stage-dependent but concentration-independent manner, suggesting that the competence of Smurf to attenuate response to BMP signaling may be regulated during development. Taken together, our work reveals a critical role for differential attenuation of the response to BMP signaling in GSCs and early germ cells for control of germ cell number and gonad growth during development.

Caspase 3 in molluscan tissues: Localization and possible function

The presence and localization of caspase 3, a master enzyme in apoptosis, have been analyzed in tissues of four species of Molluscs: the Bivalves Callista chione and Adamussium colbecki and the Gastropods Helix pomatia and Neobuccinum eatoni. Western blotting and immunocytochemical analyses show that a 32 kDa caspase 3 is present in guts, in gill lamellae and in digestive glands during digestion but not in heart tissue or in resting digestive glands. The enzyme is also found in gonads of both sexes but there are significant differences between Bivalves and Gastropods and between male and female germ cells, especially in late stages of differentiation. In particular, caspase 3 is abundant in Bivalves oocytes and in Gastropods spermatozoa but not in Gastropods oocytes; in Callista spermatozoa it appears only after sea water activation. TUNEL staining and PCNA immunolocalization, carried out on C. chione and H. pomatia tissues, suggest that caspase 3 is primarily associated with degenerative processes in gut, digestive gland and early stage germ cells while in gills, oocytes and mature spermatozoa it is primarily correlated with cell function and/or differentiation.

Gastric Metastases From Testicular Cancer: Case Report and Review of Literature

Testicular germ cell tumors represent approximately 1 % of all cancers in man and are the most common malignancy between 15and 35-year olds [1, 2]. Testicular malignancy has a common precursor and reflects a continuum of differentiation potential of early neoplastic germ cell. They are classified into seminomas (classic and spermatocytic) and non-seminomas (embryonal carcinoma, yolk sac tumor, choriocarcinoma, and teratoma). Seminomas are characterized by a more favorable clinical outcome compared to nonseminomas [3]. Pure seminomatous cancers account for 40 % of cases, while non-seminomatous and mixed tumors represent about 60 % of cases [1]. Approximately one half of patients with non-seminomatous germ cell tumors present at stage IV [2, 4]. Generally, these tumors metastasize to the retroperitoneal lymph nodes and, less commonly, to the lungs, liver, and brain [5]. Gastrointestinal metastases are very rare and may present with bowel obstruction or, less frequently, with hemorrhage [6]. We present the case of a 44-year-old man affected by a mixed testicular cancer, who began with symptoms related to bleeding from gastric metastasis.

Phenotypic characterization and in vitro propagation and transplantation of the Nile tilapia (Oreochromis niloticus) spermatogonial stem cells

In association with in vitro culture and transplantation, isolation of spermatogonial stem cells (SSCs) is an excellent approach for investigating spermatogonial physiology in vertebrates. However, in fish, the lack of SSC molecular markers represents a great limitation to identify/purify these cells, rendering it difficult to apply several valuable biotechnologies in fish-farming. Herein, we describe potential molecular markers, which served to phenotypically characterize, cultivate and transplant Nile tilapia SSCs. Immunolocalization revealed that Gfra1 is expressed exclusively in single type A undifferentiated spermatogonia (Aund, presumptive SSCs). Likewise, the expression of Nanos2 protein was observed in Aund cells. However, Nanos2-positive spermatogonia have also been identified in cysts with two to eight germ cells that encompass type A differentiated spermatogonia (Adiff). Moreover, we also established effective primary culture conditions that allowed the Nile tilapia spermatogonia to expand their population for at least one month while conserving their original undifferentiated (stemness) characteristics. The maintenance of Aund spermatogonial phenotype was demonstrated by the expression of early germ cell specific markers and, more convincingly, by their ability to colonize and develop in the busulfan-treated adult Nile tilapia recipient testes after germ cell transplantation. In addition to advancing our knowledge on the identity and physiology of fish SSCs, these findings provide the first step in establishing a system that will allow fish SSCs expansion in vitro, representing an important progress towards the development of new biotechnologies in aquaculture, including the possibility of producing transgenic fish.

GASZ promotes germ cell derivation from embryonic stem cells

Primordial germ cells (PGCs) are the first germ-line population that forms from the proximal epiblast of the developing embryo. Despite their biological importance, the regulatory networks whereby PGCs arise, migrate, and differentiate into gametes during embryonic development remains elusive, largely due to the limited number of germ cells in the early embryo. To elucidate the molecular mechanisms that govern early germ cell development, we utilized an in vitro differentiation model of embryonic stem cells (ESCs) and screened a series of candidate genes with specific expression in the adult reproductive organs. We discovered that gain of function of Gasz, a gene previously reported to participate in meiosis of postnatal spermatocytes, led to the most robust upregulation of PGC formation from both human and murine ESCs. In contrast, Gasz deficiency resulted in pronounced reduction of germ cells during ESC differentiation and decreased expression of MVH and DAZL in genital ridges during early embryonic development. Further analyses demonstrated that GASZ interacted with DAZL, a key germ cell regulator, to synergistically promote germ cell derivation from ESCs. Thus, our data reveal a potential role of GASZ during embryonic germ cell development and provide a powerful in vitro system for dissecting the molecular pathways in early germ cell formation during embryogenesis.

Rapid Progression of a Germ Cell Tumor Encasing the Inferior Vena Cava and Aorta following a Radical Orchiectomy

Early stage testicular germ cell tumors are highly curable malignancies, but the need for close radiologic and biomarker surveillance is pivotal. Even in the setting of recurrence, rescue therapy has been successfully implemented. The present report describes a patient that had rapid and aggressive recurrence after radical orchiectomy for a testicular germ cell tumor and presented with bulky disease necessitating reconstruction of the inferior vena cava at the time of salvage retroperitoneal debulking.

Identification of nine new susceptibility loci for testicular cancer, including variants near DAZL and PRDM14

Testicular germ cell tumor (TGCT) is the most common cancer in young men and is notable for its high familial risks1,2. To date, six loci associated with TGCT have been reported3-7. From GWAS analysis of 307,291 SNPs in 986 cases and 4,946 controls, we selected for follow-up 694 SNPs, which we genotyped in a further 1,064 TGCT cases and 10,082 controls from the UK. We identified SNPs at nine new loci showing association with TGCT (P<5×10−8), at 1q22, 1q24.1, 3p24.3, 4q24, 5q31.1, 8q13.3, 16q12.1, 17q22 and 21q22.3, which together account for an additional 4-6% of the familial risk of TGCT. The loci include genes plausibly related to TGCT development. PRDM14, at 8q13.3, is essential for early germ cell specification8 whilst DAZL, at 3p24.3, is required for regulation of germ cell development9. Furthermore, PITX1, at 5q31.1 regulates TERT expression, and is the third TGCT locus implicated in telomerase regulation10.

The Regulation of Testicular Descent and the Effects of Cryptorchidism

The first half of this review examines the boundary between endocrinology and embryonic development, with the aim of highlighting the way hormones and signaling systems regulate the complex morphological changes to enable the intra-abdominal fetal testes to reach the scrotum. The genitoinguinal ligament, or gubernaculum, first enlarges to hold the testis near the groin, and then it develops limb-bud-like properties and migrates across the pubic region to reach the scrotum. Recent advances show key roles for insulin-like hormone 3 in the first step, with androgen and the genitofemoral nerve involved in the second step. The mammary line may also be involved in initiating the migration. The key events in early postnatal germ cell development are then reviewed because there is mounting evidence for this to be crucial in preventing infertility and malignancy later in life. We review the recent advances in what is known about the etiology of cryptorchidism and summarize the syndromes where a specific molecular cause has been found. Finally, we cover the recent literature on timing of surgery, the issues around acquired cryptorchidism, and the limited role of hormone therapy. We conclude with some observations about the differences between animal models and baby boys with cryptorchidism.

Mouse primordial germ cells produce cysts that partially fragment prior to meiosis

Mammalian germ cells divide mitotically and form nests of associated cells just prior to entering meiosis. At least some nests contain germline cysts that arise by synchronous, incomplete mitotic divisions, but others may form by aggregation. To systematically investigate early murine germ cell development, we lineage marked the progeny of individual, newly arrived primordial germ cells in the E10.5 gonad. All the marked germ cells initially develop into clones containing two, four or eight cells, indicating cyst formation. Surprisingly, growing cysts in both sexes partially fragment into smaller cysts prior to completion and associate with cysts from unrelated progenitors. At the time divisions cease, female clones comprise five cysts on average that eventually give rise to about six primordial follicles. Male cyst cells break apart and probably become spermatogonial stem cells. Thus, cysts are invariant units of mouse germ cell development and cyst fragmentation provides insight into the amplification of spermatogonial stem cells and the origin of primordial follicles.