In situ analysis of fetal, prepuberal and adult XX----XY chimaeric mouse testes: Sertoli cells are predominantly, but not exclusively, XY.

The testes of fetal, prepuberal and adult XX----XY chimaeras were examined using in situ hybridisation to identify the beta-globin transgenic marker contained in one component of each chimaera. This enabled the proportion of XX and XY cells contributing to the major cell lineages of the testis to be estimated from sectioned and air-dried material. A few XX Sertoli cells were found in all three age groups, but the XX contribution was always much lower than in other somatic cell types. Significantly, in fetal XX----XY testes, Sertoli cells were the only cell type to show a bias in favour of the XY component. This strengthens the view that Tdy acts solely in the lineage that gives rise to Sertoli cells. However, the finding of some fetal XX Sertoli cells means that one of the steps in the Tdy-initiated process of Sertoli cell determination is capable of locally recruiting XX cells.     

Protandric hermaphrodite peculiarities in Amphiprion frenatus Brevoort (Teleostei, Pomacentridae)

Electron microscopy of the male phase of the ovotestis of Amphiprion frenatus , a protandric hermaphrodite, showed no connective tissue between male and female areas and, as the basal lamina was lacking both along the seminiferous tubules and round the previtellogenic oocytes, the male and female germ cells were only separated by their respective surrounding somatic cells (Sertoli and follicle cells). Besides previtellogenic oocytes, oocytes in meiotic prophase and very small (young) previtellogenic oocytes, were detected in the ovarian part, as spermatogenesis proceeded, revealing oogenetic activity. Degeneration of some previtellogenic oocytes and their follicle cells was discernible.     

Degeneration of germ line cells in amphibian ovary

Ogielska, M., Rozenblut, B., Augustyńska, R., Kotusz, A. 2010. Degeneration of germ line cells in amphibian ovary. —Acta Zoologica (Stockholm) 91 : 319–327 We studied the morphology of degenerating ovarian follicles in juvenile and adult frogs Rana temporaria, Rana lessonae and Rana ridibunda. Degeneration of primordial germ cells was never observed and was extremely rare in oogonia and early oocytes in a cyst phase in juveniles. Previtellogenic oocytes were rarely affected. Three main types of atresia were identified. In type I (subdivided into stages A–D), vitellogenic oocytes are digested by proliferating follicle cells that hypertrophy and become phagocytic. A – germinal vesicle shrinks, nucleoli fuse, oocyte envelope interrupts, and follicular cells hypertrophy; B – follicular cells multiply and invade the oocyte; C – entire vesicle is filled by phagocytic cells; D – degenerating phagocytes accumulate black pigment. Type II is rare and resembles breakdown of follicles and release of ooplasm. In type III, observed in previtellogenic and early vitellogenic oocytes, ooplasm and germinal vesicle shrink, follicle cells do not invade the vesicle, and condensed ooplasm becomes fragmented. The residual oogonia in adult ovaries (germ patches) multiply, but soon degenerate.     

Development and fertility of ovaries in the B6.YDOM sex-reversed female mouse

When the Y chromosome of Mus musculus domesticus (YDOM) was introduced onto the C57BL/6 (B6) mouse background, half of the XY progeny (B6.YDOM) developed bilateral ovaries and female internal and external genitalia. We examined the fertility of the B6.YDOM sex-reversed female mouse. The chromosomal sex of the individual mouse was identified by dot hybridization with mouse Y chromosome-specific DNA probes. The results indicated that all XY females lacked regular estrous cyclicity although most were able to mate and ovulate after treatment with gonadotropins. When they had been ovariectomized and grafted with ovaries from the XX female litter mate, they initiated estrous cyclicity. Reciprocally, the XX female that had received XY ovarian grafts did not resume estrous cyclicity. Development of the XY ovary was morphologically comparable to the XX ovary until 16 day of gestation (d.g.), when most germ cells had reached the zygotene or pachytene stage of meiotic prophase. However, by the day of delivery (19 or 20 d.g.), no oocyte remained in the medullary cords of the XY ovary. In the control XX ovary, the first generation of follicles developed in the medullary region, and 5 delta-3 beta-hydroxysteroid dehydrogenase (3 beta-HSDH) activity appeared first in the stromal cells around growing follicles by 10 days after birth. In contrast, in the XY ovary, follicles were not formed in the medullary region, and 3 beta-HSDH activity appeared in epithelial cells of the oocyte-free medullary cords. Primordial follicles in the cortex region continued development in both the XX and XY ovaries. These results suggest that the XY female is infertile due to a defect inside the XY ovary. The prenatal loss of oocytes in the medullary cords may be a key event leading to abnormal endocrine function, and thereby, the absence of estrous cyclicity.     

Expression of glucose-phosphate isomerase in relation to growth of the mouse oocyte in vivo and in vitro

The activity of the enzyme glucose-phosphate isomerase (GPI-1) in mouse oocytes is subject to regulation by the cis-acting gene Gpi-lt^a. Electrophoretic analysis of oocytes from 9- and 10-day-old mice showed that oocyte-specific regulation of GPI-1 is not observed in germ cells that have not started to grow (20 μm diameter) but appears as soon as oocyte growth begins (30 μm or larger). Three in vitro culture systems were used to examine the relation of GPI-1 expression to oocyte growth: culture of intact neonatal ovaries, and co-culture of dissociated oocytes and somatic cells from neonatal and from 13-day foetal ovaries. In all three systems modification of GPI-1 expression always occurred when oocyte growth began, showing that the presence of a normal follicle is not necessary for the expression of the gene Gpi-lt^a.     

Stage-specific germ-somatic cell interaction directs the primordial folliculogenesis in mouse fetal ovaries

The mechanism regulating primordial follicle formation remains largely unexplored because of the developmental particularity of female germ cells and their ultimate functional structure as follicles. Using an in vitro follicle reconstitution culture model, we explored, in the present study, the possibility of producing transgenetic follicles in vitro. We found that mouse fetal ovarian germ cells progressively lose the flexibility for gene manipulation with their oogonia-oocyte transformation upon entering meiosis, the borderline of which was at around embryonic age of 13.5 days post coitus (dpc). Interestingly, we further observed that fetal ovarian cells, only at this age or beyond achieve the capacity to reform the follicles in culture. Screening of well-known marker gene (Zp1-3, Figalpha, and Cx43) expression in cultured fetal ovarian cells of various developmental ages revealed that Figalpha is one of the determining factors for normal primordial follicle formation. By conducting reciprocal follicle reconstitution experiments, we provided further evidence that a synchronized germ-somatic cell interaction determines the normal duration of primordial folliculogenesis. Besides uncovering a potentially important regulatory mechanism for normal oocyte differentiation and follicle formation, this observation offers an alternative approach to produce transgenic oocytes/follicles, and thus animal models.     

Requirement for Cell-Proliferation Control Genes in Drosophila Oogenesis

Genes that are required for cell proliferation control in Drosophila imaginal discs were tested for function in the female germ-line and follicle cells. Chimeras and mosaics were produced in which developing oocytes and nurse cells were mutant at one of five imaginal disc overgrowth loci (fat, lgd, lgl, c43 and dco) while the enveloping follicle cells were normal. The chimeras were produced by transplantation of pole cells and the mosaics were produced by X-ray-induced mitotic recombination using the dominant female-sterile technique. The results show that each of the genes tested plays an essential role in the development or function of the female germ line. The fat, lgl and c43 homozygous germ-line clones fail to produce eggs, indicating a germ-line requirement for the corresponding genes. Perdurance of the fat+ gene product in mitotic recombination clones allows the formation of a few infertile eggs from fat homozygous germ-line cells. The lgd homozygous germ-line clones give rise to a few eggs with abnormal chorionic appendages, a defect thought to result from defective cell communication between the mutant germ-line and the nonmutant follicle cells. One allele of dco (dcole88) prevents egg development when homozygous in the germ line, whereas the dco18 allele has no effect on germ-line development. Fs(2)Ugra, a recently described follicle cell-dependent dominant female-sterile mutation, allowed the analysis of egg primordia in which fat, lgd or lgl homozygous mutant follicle cells surrounded normal oocytes. The results show that the fat and lgd genes are not required for follicle cell functions, while absence of lgl function in follicles prevents egg development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fog-1, a Regulatory Gene Required for Specification of Spermatogenesis in the Germ Line of Caenorhabditis Elegans

In wild-type Caenorhabditis elegans, the XO male germ line makes only sperm and the XX hermaphrodite germ line makes sperm and then oocytes. In contrast, the germ line of either a male or a hermaphrodite carrying a mutation of the fog-1 (feminization of the germ line) locus is sexually transformed: cells that would normally make sperm differentiate as oocytes. However, the somatic tissues of fog-1 mutants remain unaffected. All fog-1 alleles identified confer the same phenotype. The fog-1 mutations appear to reduce fog-1 function, indicating that the wild-type fog-1 product is required for specification of a germ cell as a spermatocyte. Two lines of evidence indicate that a germ cell is determined for sex at about the same time that it enters meiosis. These include the fog-1 temperature sensitive period, which coincides in each sex with first entry into meiosis, and the phenotype of a fog-1; glp-1 double mutant. Experiments with double mutants show that fog-1 is epistatic to mutations in all other sex-determining genes tested. These results lead to the conclusion that fog-1 acts at the same level as the fem genes at the end of the sex determination pathway to specify germ cells as sperm.     

Genetic control of sex determination in the germ line of Caenorhabditis elegans

The nematode Caenorhabditis elegans normally exists as one of two sexes: self-fertilizing hermaphrodite or male. Development as hermaphrodite or male requires the differentiation of each tissue in a sex-specific way. In this review, I discuss the genetic control of sex determination in a single tissue of C. elegans: the germ line. Sex determination in the germ line depends on the action of two types of genes:--those that act globally in all tissues to direct male or female development and those that act only in the germ line to specify either spermatogenesis or oogenesis. First, I consider a tissue-specific sex-determining gene, fog-1, which promotes spermatogenesis in the germ line. Second, I consider the regulation of the hermaphrodite pattern of germ-line gametogenesis where first sperm and then oocytes are produced.     

The potential significance of binovular follicles and binucleate giant oocytes for the development of genetic abnormalities

Normal development of a fertilizable female gamete emanates from a follicle containing only one oocyte that becomes haploid after first meiotic division. Binovular follicles including two oocytes and binucleate giant oocytes that are diploid after first meiosis constitute notable exceptions from this rule. Data provided by programmes of human-assisted reproduction on the occurrence of both phenomena have been reviewed to evaluate possible implications for the formation of genetic abnormalities. To exclude confusion with oocytes aspirated from two adjacent individual follicles, true binovularity has been defined as inclusion of two oocytes within a common zona pellucida or their fusion in the zonal region. A total of 18 conjoined oocytes have been reported and one of the oocyte was normally fertilized in seven cases. Simultaneous fertilization of both female gametes occurred only once. No pregnancy was achieved after transfer of an embryo from a binovular follicle. Binucleate giant oocytes have been observed sporadically but a few reports suggest an incidence of up to 0.3% of all gametes retrieved. Extensive studies performed by two independent centres demonstrated that giant oocytes are diploid at metaphase II, can undergo fertilization in vitro with formation of two or three pronuclei and develop into triploid zygotes and triploid or triploid/mosaic embryos. In summary, giant binucleate oocytes may be responsible for the development of digynic triploidy whereas the currently available data do not support a role of conjoined oocytes in producing dizygotic twins, mosaicism, chimaeras or tetraploidy. However, more information on the maturity and fertilizability of oocytes from binovular follicles is needed. Future studies should also evaluate a possible impact of pharmaceutical and environmental oestrogens on the formation of multiovular follicles.     

Differentiation potential of germ line stem cells derived from the postnatal mouse ovary

General belief in reproductive biology is that in most mammals female germ line stem cells are differentiated to primary oocytes during fetal development and oogenesis starts from a pool of primordial follicles after birth. This idea has been challenged previously by using follicle kinetics studies and demonstration of mitotically active germ cells in the postnatal mouse ovary (Johnson et al., 2004; Kerr et al., 2006; Zhang et al., 2008). However, the existence of a population of self-renewing ovarian germ line stem cells in postnatal mammals is still controversial (Eggan et al., 2006; Telfer et al., 2005; Gosden, 2004). Recently, production of offspring from a germ line stem cell line derived from the neonatal mouse ovary was reported (Zou et al., 2009). This report strongly supports the existence of germ line stem cells and their ability to expand in vitro. Recently, using a transgenic mouse model in which GFP is expressed under a germ cell-specific Oct-4 promoter, we isolated and generated multipotent cell lines from male germ line stem cells (Izadyar et al., 2008). Using the same strategy we isolated and derived cell lines from postnatal mouse ovary. Interestingly, ovarian germ line stem cells expanded in the same culture conditions as the male suggesting that they have similar requirements for their self-renewal. After 1 year of culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. As standard protocol for differentiation induction, these cells were aggregated and their ability to form embryoid bodies (EBs) was investigated. EBs generated in the presence of growth factors showed classical morphology and expressed specific markers for three germ layers. However, in the absence of growth promoting factors EBs were smaller and large cells with the morphological and molecular characteristics of oocytes were formed. This study shows the existence of a population of germ line stem cell in postnatal mouse ovary with multipotent characteristics.     

Consequences of fetal irradiation on follicle histogenesis and early follicle development in rat ovaries

Follicle histogenesis, in which follicles arise from fragmenting ovigerous cords, is a poorly understood mechanism that is strictly dependent upon the presence of germ cells. Our previous studies have shown that severely germ cell-depleted rat ovaries after fetal gamma-irradiation display modifications of follicular endowment and dynamics during the immature period. The primordial follicle stock was absent and the follicles with primary appearance remained quiescent longer than in control ovaries during the neonatal period. The aim of the present work was to analyze the initial steps of follicle histogenesis, and to investigate the etiology of the alterations observed in the development of irradiated ovaries. Just after birth, we observed, in addition to sterile ovigerous cords, the emergence of the first follicles which exhibited several abnormal features as compared to those of control ovaries. Most of the follicles appeared as primary follicles, as they were composed of a layer of cuboidal-shaped granulosa cells surrounding an enlarged oocyte. Interestingly, the granulosa cells of these primary-like follicles did not proliferate and did not express the genes for anti-Müllerian hormone (Amh) or bone morphogenetic protein receptor type II (Bmpr2), both of which are normally expressed from the primary stage onwards. In contrast, the oocytes strongly expressed the gene for growth and differentiation factor 9 (Gdf9), which is normally upregulated from the primary follicle stage onwards, which suggests an uncoupling of granulosa cell development from oocyte development. In addition, irradiated ovaries displayed a higher frequency of follicles that contained 2 or 3 oocytes, which are also referred to as multi-oocyte follicles (MOFs). Examination at the time of follicle histogenesis indicated that MOFs arise from incomplete ovigerous cord breakdown. Taken together, the results of this study indicate that severe perturbations of follicular histogenesis take place following irradiation and massive germ cell depletion during fetal life. In addition to the classically described sterile cords, we have pointed out the differentiation of MOFs and primary-like quiescent follicles, which finally evolve into growing follicles and participate in ovarian function. We propose that these phenotypes are closely correlated to the proportion of granulosa cells to oocytes at the time of neonatal follicle histogenesis.     

CXCR4/SDF1 interaction inhibits the primordial to primary follicle transition in the neonatal mouse ovary

The molecular mechanisms behind the entry of the primordial follicle into the growing follicle pool remain poorly understood. To investigate this process further, a microarray-based comparison was undertaken between 2-day postpartum mouse ovaries consisting of primordial follicles/naked oocytes only and those with both primordial follicles and newly activated follicles (7-day postpartum). Gene candidates identified included the chemoattractive cytokine stromal derived factor-1 (SDF1) and its receptor CXCR4. SDF1 and CXCR4 have been implicated in a variety of physiological processes including the migration of embryonic germ cells to the gonads. SDF1-alpha expression increased with the developmental stage of the follicle. Embryonic expression was found to be dichotomous post-germ cell migration, with low expression in the female. Immunohistochemical studies nonetheless indicate that the autocrine pattern of expression ligand and receptor begins during embryonic life. Addition of recombinant SDF1-alpha to neonatal mouse ovaries in vitro resulted in significantly higher follicle densities than for control ovaries. TUNEL analysis indicated no detectable difference in populations of apoptotic cells of treated or control ovaries. Treated ovaries also contained a significantly lower percentage of activated follicles as determined by measurement of oocyte diameter and morphological analysis. Treatment of cultured ovaries with an inhibitor of SDF1-alpha, AMD3100, ablated the effect of SDF1-alpha. By retaining follicles in an unactivated state, SDF1/CXCR4 signaling may play an important role in maintaining the size and longevity of the primordial follicle pool.     

Developmental and ultrastructual characteristics of mouse oocytes grown <Emphasis Type="Italic">in Vitro</Emphasis> from primordial germ cells

The aim of this study was to clarify the developmental and ultrastructual characteristics of oocytes grown in vitro from primordial germ cells. The female genital ridges at 12.5 days post coitus were cultured for 18 days on an insert membrane in Waymouth's MB752/1 medium, supplemented with 15% fetal bovine serum and 1 mM sodium pyruvate; subsequently, the follicles isolated from the tissue were cultured for eight days in Waymouth's medium supplemented with 5 microg/ml insulin, 5 microg/ml transferrin, 5 ng/ml selenium, 10 mIU/ml follicle stimulating hormone, and 100 ng/ml stem cell factor. The primordial germ cells developed in vitro into oocytes of more than 60 microm in diameter. The transmission electron microscopic analysis indicated that the oocytes, which developed in vitro, showed no obvious abnormality in their ultrastructure and had organelles appropriate for the oocyte size. However, a delay in the progressive changes of morphology in some of the organelles during oocyte growth was often found when comparing them to oocytes grown in vivo.     

The spatial arrangement of germ line cells in ovarian follicles of the mutantdicephalic inDrosophila melanogaster

Summary The pattern of intercellular connections between germ line cells has been studied in follicles of the mutantdicephalic (dic), which possess nurse cell clusters at both poles. Staining of follicles with a fluorescent rhodamine conjugate of phalloidin reveals ring canals and cell membranes and thus allows us to reconstruct the spatial organization of the follicle. Each germ line cell can be identified by the pattern of cell-cell connections which reflect the mitotic history of individual cells in the 16-cell cluster. The results indicate that in both wild-type anddicephalic cystocyte clusters one of the two cells with four ring canals normally becomes the pro-oocyte. However, in some follicles (dicephalic and wild-type) oocytes were found with fewer or more than four ring canals. Indic follicles, one or several nurse cells may become disconnected from the other cells during oocyte growth at stage 9–10. Such disconnected cells cannot later on empty their cytoplasm into the oocyte. This, in turn, might be of consequence for the determination of axial polarity of the embryo.     

Identification of cells migrating from the thecal layer of ovarian follicles

In the mammalian ovary, oocytes are contained within ovarian follicles. These consist in an oocyte surrounded by supporting cells: an inner layer of granulosa cells and an outer layer of thecal cells separated by a basal lamina. At any one time, a developing cohort of follicles exists, from which only a small species-specific number are selected for continued development towards ovulation, with the remainder dying by follicular atresia. Here, we use in vitro methods to study interactions between two follicles in culture (follicle co-cultures). We show that, when two individual follicles are grown together in culture, cells and cellular processes migrate from the outer thecal layer of one follicle to the thecal layer of the other co-cultured follicle. These cells are identified as a mixed population containing primarily endothelial but also neuronal cells. Both are able to migrate through the ovarian interstitum, making contact with the basal lamina of other follicles and with similar cells from these other follicles. Networks of such cells might be involved in interfollicular communication and in the coordination of follicle selection for ovulation.     

Marker Chromosome Analysis of Tetraparental AKR↔CBA-T6 Mouse Chimaeras

Marker chromosome analysis of 18 tetraparental AKR↔CBA/H-T6 chimaeras revealed a great excess of AKR mitoses over CBA mitoses in direct preparations from lymphomyeloid tissues, corneal epithelium, intestinal epithelium and skin (Table 1). The degree of AKR dominance was strongly influenced by anatomical site (Tables 2 and 3). The testes of three out of four XY/XY males contained a marked excess of AKR germ cells and produced a parallel excess of functional AKR gametes (Table 4). Mitotic spreads in mitogen-stimulated cultures of tail blood were overwhelmingly of AKR type in 1972, but less so in 1973 (Table 5).
The coat phenotypes, and breeding results from known or presumptive XX/XX females, suggest that AKR and CBA cells were numerically balanced when melanoblasts and oocytes were formed during embryonic development, and therefore that the striking deviations from equality observed in mitotic populations of adult chimaeras arose later by differential proliferation or survival of AKR cells, or both.
The low frequency of lymphomas, compared with normal AKR mice, previously reported in these chimaeras cannot therefore be accounted for by insufficiency of AKR cells in the thymus or elsewhere in the lymphomyeloid tissues. One of three lymphomas studied was CBA type. This suggests that the high risk of lymphomatous transformation is not an autonomous property of AKR cells.     

From primordial germ cell to primordial follicle: mammalian female germ cell development

In mammals, the final number of oocytes available for reproduction of the next generation is defined at birth. Establishment of this oocyte pool is essential for fertility. Mammalian primordial germ cells form and migrate to the gonad during embryonic development. After arriving at the gonad, the germ cells are called oogonia and develop in clusters of cells called germ line cysts or oocyte nests. Subsequently, the oogonia enter meiosis and become oocytes. The oocyte nests break apart into individual cells and become packaged into primordial follicles. During this time, only a subset of oocytes ultimately survive and the remaining immature eggs die by programmed cell death. This phase of oocyte differentiation is poorly understood but molecules and mechanisms that regulate oocyte development are beginning to be identified. This review focuses on these early stages of female germ cell development.     

PAR6, A Potential Marker for the Germ Cells Selected to Form Primordial Follicles in Mouse Ovary

Partitioning-defective proteins (PAR) are detected to express mainly in the cytoplast, and play an important role in cell polarity. However, we showed here that PAR6, one kind of PAR protein, was localized in the nuclei of mouse oocytes that formed primordial follicles during the perinatal period, suggesting a new role of PAR protein. It is the first time we found that, in mouse fetal ovaries, PAR6 appeared in somatic cell cytoplasm and fell weak when somatic cells invaded germ cell cysts at 17.5 days post coitus (dpc). Meanwhile, the expression of PAR6 was observed in cysts, and became strong in the nuclei of some germ cells at 19.5 dpc and all primordial follicular oocytes at 3 day post parturition (dpp), and then obviously declined when the primordial follicles entered the folliculogenic growth phase. During the primordial follicle pool foundation, the number of PAR6 positive germ cells remained steady and was consistent with that of formed follicles at 3 dpp. There were no TUNEL (apoptosis examination) positive germ cells stained with PAR6 at any time studied. The number of follicles significantly declined when 15.5 dpc ovaries were treated with the anti-PAR6 antibody and PAR6 RNA interference. Carbenoxolone (CBX, a known blocker of gap junctions) inhibited the expression of PAR6 in germ cells and the formation of follicles. Our results suggest that PAR6 could be used as a potential marker of germ cells for the primordial follicle formation, and the expression of PAR6 by a gap junction-dependent process may contribute to the formation of primordial follicles and the maintenance of oocytes at the diplotene stage.