Number Of Germ Cells Research Articles

Fate of the germ cells in mammalian ovary: A review

Ovary has a fix number of germ cells during fetal life in mammals. The germ cells are depleted rapidly and a large number of germ cells (≥99%) are eliminated from the cohort of ovary through follicular atresia during prepubertal life. The various cell death pathways including apoptosis, autophagy, necrosis, and necroptosis are involved in follicular atresia. Hence, <1% of germ cells are culminated into oocytes that are available for meiotic maturation and ovulation during entire reproductive life. These oocytes are arrested at diplotene stage of meiotic prophase-I and remain arrested for few months to several years during entire reproductive life. Resumption from diplotene arrest in follicular oocytes starts in response to gonadotropins surge and progresses through metaphase-I to metaphase-II stage that extrudes first polar body at the time of ovulation. Surprisingly, oocytes do not wait for fertilizing spermatozoa and quickly undergo abortive spontaneous oocyte activation (SOA) in few mammalian species including humans. The abortive SOA makes oocyte unfit for fertilization and limits assisted reproductive technologies outcome. Indeed, majority of germ cells and oocytes are eliminated from the cohort of ovary and only few oocyte that are of good quality get selectively recruited to become right gamete after ovulation during entire reproductive life span in mammals.

Germ cell nests in adult ovaries and an unusually large ovarian reserve in the naked mole-rat.

The naked mole-rat (NMR, Heterocephalus glaber) is renowned for its eusociality and exceptionally long lifespan (> 30 y) relative to its small body size (35-40 g). A NMR phenomenon that has received far less attention is that females show no decline in fertility or fecundity into their third decade of life. The age of onset of reproductive decline in many mammalian species is closely associated with the number of germ cells remaining at the age of sexual maturity. We quantified ovarian reserve size in NMRs at the youngest age (6 months) when subordinate females can begin to ovulate after removal from the queen's suppression. We then compared the NMR ovarian reserve size to values for 19 other mammalian species that were previously reported. The NMR ovarian reserve at 6 months of age is exceptionally large at 108,588 ± 69,890 primordial follicles, which is more than 10-fold larger than in mammals of a comparable size. We also observed germ cell nests in ovaries from 6-month-old NMRs, which is highly unusual since breakdown of germ cell nests and the formation of primordial follicles is generally complete by early postnatal life in other mammals. Additionally, we found germ cell nests in young adult NMRs between 1.25 and 3.75 years of age, in both reproductively activated and suppressed females. The unusually large NMR ovarian reserve provides one mechanism to account for this species' protracted fertility. Whether germ cell nests in adult ovaries contribute to the NMR's long reproductive lifespan remains to be determined.

Effect of adjunctive hormonal therapy on testicular descent and spermatogenic function among children with cryptorchidism: a systematic review and meta-analysis

To systematically evaluate the effect of adjunctive hormonal therapy on testicular descent and spermatogenic function among children with cryptorchidism. A comprehensive literature search of electronic databases up to February 21, 2019 was performed. Continuous data of fertility indices were evaluated using weighted mean difference (WMD) with 95% confidence interval (CI), while nominal data of fertility indices and the complete descent rates were analyzed by risk ratio (RR) with 95% CI. A fixed effect or random effect model was used. A total of 27 previous studies were included for the current analysis. Hormonal therapy increased complete testicular descent rate, reaching statistical significance (RR = 3.74; 95% CI, 2.78-5.04; P < 0.001). The success rate was 22.43%. A similar result was found in subgroup analysis of hormonal category and effect on unilateral or bilateral cryptorchidism. Studies reporting primary outcome as continuous data showed that cryptorchid males have significantly increased germ cell numbers per tubule (WMD = 0.10; 95% CI, 0.01-0.20, P = 0.032) after hormonal therapy. The nominal data of pooled studies showed no significant difference (RR = 1.62; 95% CI, 0.65-4.00, P = 0.298). In addition, a significant result was noted in the luteinizing hormone-releasing hormone (LHRH) therapy group but not in those undergoing human chorionic gonadotropin (hCG) treatment. Our findings have demonstrated that hormonal therapy can effectively increase the success rate of complete testicular descent, while some boys may benefit as regards improvement of the fertility index.

DETECTION OF DISSEMINATED TUMOR CELLS IN THE BONE MARROW OF PATIENTS WITH NON-SMALL CELL LUNG CANCER

Introduction. There is evidence that disseminated tumor cells (DOCs) in the bone marrow (BM) are precursors of subsequent distant metastases. The detection of DOCs in non-small cell lung cancer (NSCLC) will provide important information about the features of metastasis, as well as the possibilities of identifying new targets for the treatment of NSCLC. Purpose of the study is to evaluate the possibility of detection DOCs in BM and to identify the frequency of BM involvement in patients with NSCLC. Materials and methods . Morphological and immunological methods studied 62 BM samples from patients with NSCLC. DOCs analysis was performed using flow cytometry (FACSCanto II, USA, Kaluza Analysis v 2.1 software), monoclonal antibodies to CD45, cytokeratins directly labeled with various fluorochromes were used. Results . DOCs (EPCAM+CD45–) in the BM were found in 43.5 % of patients (threshold level: 1 cell per 10 million myelocaricytes). The presence of DOCs did not correlate with tumor size, lymph node status, stage of the tumor process. The highest detection rates of DOCs were observed at stages IA and IIA: 60.7 % (3/5) and 58.3 % (7/12) respectively. BM involvement in adenocarcinoma was observed in 45 % (15/33) cases, in squamous cell carcinoma – in 37 % (10/27) samples (p = 0.501). It was found that DOCs are more often detected in more differentiated tumors (p = 0.023). Significant correlations between the presence of DOCs in the BM and myelogram parameters have not been established. A decrease in the number of granulocyte germ cells was observed in 4 % of BM involvement (p = 0.036). Conclusion. The possibility of detecting DOCs in the BM of NSCLC patients has been established. BM involvement was 43.5 %. DOCs are detected even in the early stages of NSCLC. Relationship between BM involvement and the degree of tumor differentiation was found. More frequent BM involvement was observed in adenocarcinoma compared with squamous cell carcinoma of the lung. Compliance with patient rights and principles of bioethics The study protocol was approved by the biomedical ethics committee of N.N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of Russia. All patients gave written informed consent to participate in the study.

Apoptosis during folliculogenesis in pigs

The number of female germ cells in pig fetuses decreases by 70% between day 50 after mating and day 300 after birth. Approximately 55% of antral follicles undergo degeneration (atresia) except during the 3 days before oestrus, when only 15% of the follicles survive to ovulate. Apoptosis, a form of programmed cell death, is recognized as the mechanism of germ cell death and follicle atresia at all stages of folliculogenesis. The internucleosomal cleavage of genomic DNA caused by caspase-induced deoxyribonuclease activity was measured in pig granulosa cells by DNA fluorescence flow cytometry, densitometry of fluorescently labelled internucleosomal DNA fragments and immunohistochemical analysis of the 3' end labelling of deoxyribonuclease- nicked DNA on frozen tissue sections. Follicular atresia during the 3 days before oestrus is associated with a 60-70% decrease in the secretion of FSH. In granulosa cells, apoptosis is associated with decreased cell proliferation and reduced production of oestradiol and inhibin. In cultured pig granulosa cells, FSH and IGF-I are anti-apoptotic and a caspase inhibitor blocked apoptosis, thereby providing evidence of caspase activity. Oocytes in most follicles have resumed meiotic maturation; therefore, one role for apoptosis and follicle atresia may be to act as a barrier to ovulation of oocytes that have not remained in meiotic arrest.

Parental Acceptance Rate of Testicular Tissue Cryopreservation in Danish Boys with Cryptorchidism

Despite orchidopexy within the first year of life, 20-25% of boys with nonsyndromic cryptorchidism may risk infertility according to histological and hormonal data obtained during surgery. The aim of this study was to evaluate the acceptance rate of testicular tissue cryopreservation among parents of prepubertal boys with cryptorchidism. Fourteen boys with cryptorchidism and high infertility risk were offered cryopreservation as an additional procedure after orchidopexy based on abnormal histopathological findings at primary surgery, whereas 27 boys with bilateral cryptorchidism were offered cryopreservation at the initial orchidopexy. A total of 90% of parents (37/41, 13/14, and 24/27) gave consent to perform cryopreservation, despite being well-informed that the procedural efficacy is largely unproven and may only be needed in about 20% of cases. The number of germ cells per tubule cross-section was 0.03-1.70 (median 0.37) and 22 boys (54%, 22/41) had a value below the lower range. Twelve boys (29%, 12/41) had no type A dark spermatogonia in their biopsy. Cryopreservation of testicular tissue is the first step to introduce spermatogonial stem cell-based therapy into clinical male infertility treatment. At the time of orchidopexy, a testicular biopsy can be collected to ascertain the infertility risk, and it may be an option for boys with bilateral cryptorchidism to have spermatogonial stem cells frozen as a fertility reserve.

PRMT7 is involved in regulation of germ cell proliferation during embryonic stage

Arginine methylation is one of the most important post-translational modifications which is catalyzed by protein arginine methyltransferases (PRMTs). Previous studies have demonstrated that Prmt5 plays important role in germ cell development. Prmt7 is the only family member responsible for mono-methylation of arginine residue. However, whether Prmt7 is also involved in germ cell development remains unclear. In this study, we find that PRMT7 is abundantly expressed in the male germ cells during embryonic stage (from E10.5). Depletion of Prmt7 results in the defect of germ cell proliferation during embryonic stage and the number of primordial germ cells is significantly reduced in Prmt7−/− mice at E11.5. We also find that the size of testes is reduced in Prmt7−/− mice at P5 with reduced germ cell number and the diameter of seminiferous tubules. Further study reveals that the expression of BMPs and TGF-β singling pathway is significantly changed in germ cells of Prmt7−/− mice at E12.5. However, no defect of testes development is observed in adult Prmt7-/flox; Mvh-Cre mice. Collectively, this study demonstrates that Prmt7 plays roles in male germ cell proliferation during embryonic stages and it is not required for germ cell development postnatally.

Effects of gonadotropins on testis cell subpopulations of newly hatched chicks treated during embryonic development

The aim of the present study was to evaluate histological and stereological changes, as well as the variations in the number and size of cells from diverse cell subpopulations in testes of newly hatched chicks treated with follicle stimulating hormone (FSH) and luteinizing hormone (LH) during embryonic development. Stereological results indicated that in FSH-treated chicks total volume of the tubular compartment constitutes most of the testis. In contrast, the total volume of interstitial tissue constitutes most of the testis of LH-treated chicks. Results indicate that the number of germ and Sertoli cells increases as a result of FSH and LH treatment, but in FSH-treated testis, Sertoli cells were the most numerous cell type in seminiferous tubules; whereas germ cells were the most numerous cell type in testis of LH-treated chicks. Results also indicate there was a larger total volume of Leydig cells in the testes of FSH- and LH-treated chicks. The larger volume of Leydig cells in FSH-treated chicks is due to a larger cellular volume of these cells, and not due to the number, which remains constant. In contrast, in testes of LH-treated chicks, there is a larger number and volume of Leydig cells. These results indicate the testes of chick embryos respond to FSH and LH treatment, with there being modifications in the seminiferous tubules and interstitial tissue, but these changes differ markedly, indicating that FSH and LH have differential effects on chick testes.

Fertility Potential is Impaired in Boys with Bilateral Ascending Testes.

Ascending testes have been documented to be descended in the scrotum within the first year of life and then reascended. The aim of this study was to investigate to what extent the fertility potential was impaired in boys with such testes compared to the fertility potential of boys with late referral congenital cryptorchidism. A total of 153 consecutive boys underwent bilateral orchiopexy at age 2 to 7 years (median 3.9) between 2011 and 2018. Of the patients 67 were diagnosed with bilateral ascended testes and 86 with late referral bilateral congenital cryptorchidism. We assessed serum levels of inhibin B and gonadotropins and histological parameters, number of germ cells per tubule cross-section and number of type A dark (Ad) spermatogonia per tubule cross-section. All values were compared to our normal material. Number of germ cells per tubule cross-section of boys with ascended testes (median 0.50, range 0 to 2.29) was not significantly higher compared to boys with congenital cryptorchidism (median 0.37, range 0 to 2.57; p=0.11). Mean number of germ cells per tubule cross-section was below normal range in 40 boys with ascending testes (60%) vs 57 boys with late referral congenital cryptorchidism (66%, p=0.40). Biopsies absent of Ad spermatogonia were noted in 31% of boys with ascending testes (21 of 67) vs 34% of boys with congenital cryptorchidism (29 of 86, p=0.76). Serum levels of inhibin B and gonadotropins did not differ between the 2 groups. The fertility potential of boys with bilateral ascended testes was impaired to almost the same level as that of boys with bilateral congenital cryptorchidism and should therefore be surgically corrected as soon as the diagnosis of ascended testes is settled.

Deleterious effects of Pfaffia glomerata (Spreng.) Pedersen hydroalcoholic extract on the seminiferous epithelium of adult Balb/c mice.

Several plant species such as Pfaffia glomerata are widely used in traditional Brazilian medicine as stimulants and aphrodisiacs. In this regard, the aim of our study was to explore the effects of the long-term intake of the hydro-alcoholic root extract of Pglomerata on the germ and somatic cells within the seminiferous tubules in adult Balb/c mice. The experimental groups were placed as: controls (water and DMSO), and treated with 300 and 400mg/kg of the root extract. The number of germ and somatic cells, the proportion of pathological seminiferous tubules, and the germ cell apoptotic levels were evaluated. The volume and proportion of the seminiferous epithelium was decreased after the extract intake due to the increased germ cell apoptotic levels. Vacuolization of Sertoli cell cytoplasm was observed widely in pathological tubules, along with fully disorganized epithelia, showing multinucleated cells, which lead to decreased daily sperm production. Taken together, our results indicate that long-term intake of the Pglomerata caused deleterious effects on spermatogenesis by inducing apoptosis and altering the seminiferous tubule's epithelial dynamics.

Male reproductive toxicity involved in spermatogenesis induced by perfluorooctane sulfonate and perfluorooctanoic acid in Caenorhabditis elegans.

As a persistent organic pollutant, perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) have gained increasing research attention over recent years because of their potential risk to humans and the environment. In this paper, we investigated the reproductive toxicity of these pollutants using a C. elegans model to evaluate spermatogenesis throughout the entire developmental cycle of him-5 mutant by exposing to 0.001, 0.01, and 0.1mmol/L PFOS or PFOA for 48h. Experimental results suggested that PFOS and PFOA exposure led to reductions in brood size, germ cell number, spermatid size, and motility, and increases in rate of malformation spermatids. Analysis of variance (ANOVA) showed that exposure to PFOS resulted in higher levels of damage than PFOA in germ cells only in 0.001mmol/L exposure group. RT-qPCR was used to further investigate the expression of genes associated with different stages of spermatogenesis, such as mitosis and meiosis, fibrous body-membranous organelles (FB-MOs), and sperm activation. The expression levels of wee-1.3, spe-4, spe-6, and spe-17 genes were increased, while those of puf-8, spe-10, fer-1, swm-1, try-5, and spe-15 genes were decreased. Our results suggesting that PFOS or PFOA may cause spermatogenesis damage by disrupting the mitotic proliferation, meiotic entry, formation of the MOs, fusion of the MOs and plasma membrane (PM), and pseudopods. Loss-of-function studies using puf-8 and spe-10 mutants revealed spe-10 gene was specifically involved in PFOS- or PFOA-induced reproductive toxicity via regulating one or more critical palmitoylation events, while puf-8 gene was not direct target of PFOS and PFOA, and PFOS and PFOA may act on the upstream gene of puf-8, thus affecting reproductive ability. Taken together, these results demonstrate the potential adverse impact of PFOS and PFOA exposure on spermatogenesis and provide valuable data for PFC risk assessment. Grapical abstract.

Epididymal contribution to male infertility: An overlooked problem.

The diagnosis and treatment of male infertility, excluding assisted conception, are limited because of, but not limited to, poor understanding of sperm post-testicular development and storage. Many may think that sperm dysfunction is only self-contained in the sperm cell itself as a result of defective spermatogenesis. However, it can also be a consequence of inadequate epididymal maturation following disorders of the epididymis. Improper epididymal functions can disturb semen parameters and sperm DNA integrity, result in high leucocyte concentrations and high numbers of immature germ cells and debris or even cause idiopathic infertility. To date, the data are limited regarding critical markers of sperm maturation and studies that can identify such markers for diagnosis and managing epididymal dysfunction are scarce. Therefore, this article aims to draw attention to recognise a disturbed epididymal environment as a potential cause of male infertility.

Quantitative analysis of PAH compounds in DWH crude oil and their effects on Caenorhabditis elegans germ cell apoptosis, associated with CYP450s upregulation

The Deepwater Horizon (DWH) oil spill marked the largest environmental oil spill in human history, where it was estimated a large amount of the polycyclic aromatic hydrocarbons (PAHs) were released with crude oil into the environment. In this study, common PAH compounds were quantitatively determined in crude oil from the DWH spill by gas chromatography-mass spectroscopy (GC–MS). Twelve PAH compounds were identified and quantified from a 100× dilution of DWH crude oil: naphthalene (7800 ng/mL), acenaphthylene (590 ng/mL), acenaphtehen (540 ng/mL), fluorene (2550 ng/mL), phenanthrene (2910 ng/mL), anthracene (840 ng/mL), fluoranthene (490 ng/mL), pyrene (290 ng/mL), benzo(k) fluoranthene (1050 ng/mL), benzo(b)fluoranthene (1360 ng/mL), dibenz(a,h)anthracene (2560 ng/mL), and benzo(g, h, i) perylene (630 ng/mL). Toxicity assays using the nematode, Caenorhabditis elegans (C. elegans), indicated a single PAH compound naphthalene, exposure increased C. elegans germ cell apoptosis which may adversely affect progeny reproduction. The number of apoptotic germ cells significantly increased from 1.4 to 2.5 when worms were treated with 10 μg/mL of naphthalene and from 1.3 to 2.5 and 3.5 cells in presence of 1 μg/mL and 5 μg/mL of benzo(a)pyrene, respectively. Five CYP450 genes (CYP14A3, CYP35A1, CYP35A2, CYP35A5, and CYP35C1) were significantly upregulated following 500× dilution of dispersed crude oil exposure (p < 0.05). These results suggest that CYP450s may play a role in bioactivation of PAHs in crude oil, resulting in DNA damage related germ cell apoptosis.

Stem Cell Factor Receptor Immunoexpression in Adolescent Varicocele.

Stem cell factor receptor (c-kit) plays a crucial role in regulating proliferation and survival of germ cells. The aim of this study was to find the correlation between the number of c-kit positive germ cells, testicular asymmetry and histological grade in varicocele affected testis samples of adolescents. Twenty testicular biopsy samples of adolescents affected by varicocele and eight normal control testes were included. The relationship between percentage of testicular asymmetry, number of tubular c-kit positive germ cells and severity of spermatogenic failure was assessed. The mean (SD; median) histological grade for spermatogenic failure in controls was 1.37(0.52; 1), while in the varicocele group, it was 2.70(1.08; 3) (P = .0052). Mean(SD; median) number c-kit positive germ cells in the control group were 20.1(2.52; 20), while in the varicocele group it was 12.35(7.16; 12.5) (P = .0059). Spearman test documented a significant positive correlation between percentage of hypotrophy and histological grade of spermatogenic failure (r = 0.5544 , 95% CI: 0.1345 to 0.8055, P = .0112) but a negative correlation with the number of c-kit positive cells (r = - 0.5871, 95% CI: - 0.8219 to -0.1817, P = .0065). Moreover, a significant negative correlation was found between grade of histological changes and number of c-kit positive germ cells (P < .0001). A significant correlation between hypotrophy, histological lesions and c-kit positive germ cells exists in varicocele testes. This finding suggests a possible role for c-kit in the pathogenesis of germ cell impairment in varicocele. Histological changes and lack of c-kit germ cells were also noted in testes not displaying hypotrophy. We believe that reliable markers should be found as better predictors of testicular function in adolescent with varicocele.

Chestnut polysaccharides benefit spermatogenesis through improvement in the expression of important genes.

Recently there has been a continuing worldwide decrease in the quality of human spermatozoa, especially in spermatozoa motility and concentration. Many factors are involved in this decline, and great efforts have been made to rescue spermatogenesis; however, there has been little progress in the improvement of sperm quality. Chestnuts are used in traditional Chinese medicine; their major active components are chestnut polysaccharides (CPs). CPs have many biological activities but their effects on spermatogenesis are unknown. The current investigation was designed to explore the impact of CPs on spermatogenesis and the underlying mechanisms. We demonstrated that CPs significantly increased sperm motility and concentration (4-fold and 12-fold, respectively), and improved seminiferous tubule development by increasing the number of germ cells after busulfan treatment. CPs dramatically rescued the expression of important genes and proteins (STRA8, DAZL, SYCP1, SYCP3, TNP1 etc.) in spermatogenesis. Furthermore, CPs increased the levels of hormone synthesis proteins such as CYP17A1 and HSD17β1. All the data suggested that CPs improved the testicular microenvironment to rescue spermatogenesis. With CPs being natural products, they may be an attractive alternative for treating infertile patients in the future. At the same time, the deep underlying mechanisms of their action need to be explored.

Bcl2-Associated X (BAX) Knockout in Mice Resulted in Persistence of Neonatal Testicular Germ Cells (Gonocytes)

During early testicular development, neonatal gonocytes transform into spermatogonial stem cells (SSC), and any untransformed gonocytes are thought to undergo apoptosis. In human cryptorchidism, persisting gonocytes may lead to seminoma. Using Bcl2-associated X knockout (BAXKO) mice, we investigated apoptosis in gonocyte development during mouse minipuberty. Testes from BAXKO, heterozygous (HET), and wild-type (WT) littermates were collected on postnatal days 1, 3, 6, and 9 (n = 6/group), labelled with antibodies against mouse vasa homologue (MVH, germ cell marker) or promyelocytic leukaemia zinc-factor (PLZF, SSC marker) and imaged for cell counting. Total germ cells/tubule, i.e., the number of germ cells on and off the basement membrane (BM), were counted using Image J followed by 2-way ANOVA analysis with Prism. Total PLZF+ germ cells/tubule, PLZF+ germ cells/tubule off BM, total MVH+ germ cells/tubule, and MVH+ germ cells off BM/tubule were significantly higher at day 9 in BAXKO compared to WT and HET mice (p < 0.01). In conclusion, knockout of BAX in mouse leads to gonocytes persisting at the centre of the tubules after minipuberty, which failed to migrate and transform into SSC, indicating the important role of apoptosis is to eliminate undifferentiated gonocytes during transformation. Failed apoptosis in gonocytes may be the cause of malignancy in humans with cryptorchidism.

Amh dominant expression in Sertoli cells during the testicular differentiation and development stages in the olive flounder Paralichthys olivaceus.

The olive flounder Paralichthys olivaceus, an important marine fish, shows gender differences in growth. The mechanism on its gonadal differentiation direction affected with exogenous factors still needs to be clarified. The anti-Müllerian hormone (amh) gene is involved in fish testicular differentiation and maintenance. The aim of this study was to investigate the expression of the flounder amh in tissues and the gonads. The quantitative expression analysis results showed that it was highly expressed in the testis, especially in the testis at stages I - IV (P<0.05). Also, amh was detected in Sertoli cells surrounding spermatogonia and peripheral seminiferous lobule of the testis with in situ hybridization (ISH) and immunohistochemistry (IHC). During the differentiation period, the amh expression in the testis of the tamoxifen treatment group (100ppm) was higher than that in the ovary of the 17β-estradiol (E2, 5ppm) group, and the expression levels of amh during process of the male differentiation in the tamoxifen group were higher than those in the 17ɑ-methyltestosterone (MT, 5ppm) group (P<0.05). ISH results also exhibited that amh was expressed in the somatic cells that surrounded the germ cells of juvenile flounder similar to adult ones. Furthermore, the flounder gonads in the tamoxifen group maintained more germ cells and somatic cells than those in the MT group from 20 to 80mm total length (TL). Especially, at 60 and 80mm TL, the numbers of germ and somatic cells in the tamoxifen group were significantly higher than those in the MT group (P<0.05). In summary, amh might initiate the process of testicular differentiation, and is involved in the early development and maintenance of testis.

Reduced retinoic acid synthesis accelerates prophase I and follicle activation.

In female mammals, reproductive potential is determined during fetal life by the formation of a non-renewable pool of primordial follicles. Initiation of meiosis is one of the defining features of germ cell differentiation and is well established to commence in response to retinoic acid. WIN 18,446 inhibits the conversion of retinol to retinoic acid, and therefore it was used to explore the impact of reduced retinoic acid synthesis on meiotic progression and thus germ cell development and subsequent primordial follicle formation. e13.5 mouse fetal ovaries were cultured in vitro and treated with WIN 18,446 for the first 3 days of a total of up to 12 days. Doses as low as 0.01 µM reduced transcript levels of the retinoic acid response genes Stra8 and Rarβ without affecting germ cell number. Higher doses resulted in germ cell loss, rescued with the addition of retinoic acid. WIN 18,446 significantly accelerated the progression of prophase I; this was seen as early as 48 h post treatment using meiotic chromosome spreads and was still evident after 12 days of culture using Tra98/Msy2 immunostaining. Furthermore, ovaries treated with WIN 18,446 at e13.5 but not at P0 had a higher proportion of growing follicles compared to vehicle controls, thus showing evidence of increased follicle activation. These data therefore indicate that retinoic acid is not necessary for meiotic progression but may have a role in the regulation of its progression and germ cell survival at that time and provide evidence for a link between meiotic arrest and follicle growth initiation.

MON-032 Excessive Ovarian Sympathetic Activity Impairs Embryonic Development and Causes Reproductive and Metabolic Dysfunction

Nerve growth factor is a member of the neurotrophin family and within the ovary, it plays an important role in sympathetic innervation and in the development and maintenance of folliculogenesis. Despite its critical role, excessive levels of ovarian NGF may lead to ovarian pathology and to the development of features of polycystic ovary syndrome (PCOS), which is the most common endocrine disorder among women of reproductive age. Here, using a transgenic mouse model overexpressing NGF selectively in the ovary (17NF mice), we studied how ovarian sympathetic hyperactivity affects embryonic development and reproductive and metabolic function in adulthood. Firstly, we showed that ovarian NGF excess caused growth restriction in the developing female fetuses, which was driven by defects in the placenta function. Moreover, the 17NF fetuses experienced a reduction of germ cell number along with delayed gonocyte and primary oocyte maturation. The adult 17NF mice displayed irregular cyclicity and aberrant ovarian expression of steroidogenic genes and epigenetic markers. The ovarian sympathetic hyperactivity also led to increased systemic sympathetic outflow, indicated by increased circulating dopamine levels, and to metabolic dysfunction in adulthood. The 17NF mice had increased adiposity, impaired glucose metabolism and decreased energy expenditure. The subcutaneous and parametrial fat depots displayed impaired function due to ovarian NGF excess, wherein the subcutaneous fat increased mass by enhanced preadipocyte differentiation and enlarged adipocyte size, while the parametrial fat had smaller adipocyte size and a modest increase in stimulated lipolysis. These defects also led to hepatic steatosis. Overall, our findings indicate that ovarian sympathetic hyperactivity has deleterious effects on whole-body homeostasis and leads to impaired embryonic development and to reproductive and metabolic defects in adult life.

A novel pattern of germ cell divisions in the production of hymenopteran insect eggs.

Egg development is a defining process of reproduction in higher eukaryotes. In the fruit fly, Drosophila melanogaster, this process begins with four mitotic divisions starting from a single germ cell, producing a cyst of 16 cystocytes; one of these cells will become the oocyte and the others supporting nurse cells. These mitotic divisions are exceptional because cytokinesis is incomplete, resulting in the formation of cytoplasmic bridges known as ring canals that interconnect the cystocytes. This organization allows all cystocytes to divide synchronously during each mitotic round, resulting in a final, power-of-2 number of germ cells. Given that numerous insects obey this power-of-2 rule, we investigated if strict cell doubling is a universal, underlying cause. Using confocal microscopy, we found striking departures from this paradigm in three different power-of-2 insects belonging to the Apocrita suborder (ants, bees and wasps). In these insects, the earliest-formed cystocytes cease to divide during the latter mitotic cycles while their descendants undergo further division, thereby producing a 'radial' direction of division activity. Such cystocyte division patterns that depart from strict cell doubling may be 'fine-tuned' in order to maintain a final, power-of-2 germ cell number.