Primordial Follicle Development Research Articles

Comparative proteome profile of immature rat ovary during primordial follicle assembly and development

The assembly of primordial follicles early in ovarian development and subsequent transition to primary follicles are critical processes in ovarian biology. Inappropriate coordination of these processes contributes to ovarian pathologies such as premature ovarian failure and infertility. To better understand the molecular mechanisms involved in primordial follicle assembly and development, 2-D PAGE and MALDI-TOF/TOF technologies were used to construct a comparative proteome profile of the immature rat ovary at specific time-points (0, 24, 48, and 72 h postpartum). A total of 154 differential protein spots corresponding to 134 different proteins were definitively identified between any two time-points. Further cluster analysis showed four expression patterns, and each pattern correlated with specific cell processes that occur during early ovarian development. Seven proteins were randomly selected to verify expression patterns using Western blotting, and subsequently immunohistochemistry was performed to further investigate their cellular localization. Additionally, detailed functional analyses of these differentially expressed proteins were performed. Elucidation of how these changes in protein expression level coordinate primordial follicles assembly and development is intended to provide a better understanding of these critical biological processes early in ovarian development and will provide potential therapeutic molecular targets to regulate ovarian function and treat ovarian disease.

GPR30 Signaling in Neonatal Hamster Ovary Involves PI3 Kinase (PI3K) and ERK-1/2 Pathways.

We have shown that estradiol (E2) stimulates the formation and development of hamster primordial follicles in vivo and in vitro. A part of the E2 action is mediated by GPR30, but the mechanisms are not known. The objective of this study was to investigate whether GPR30 signaling during primordial follicle formation involved PI3 kinase or ERK-1/2 pathway. Wortmannin or U0126 suppressed the stimulatory effect E2-BSA on primordial follicle formation. PI3 kinase protein was detected in the oocytes of 13-day old fetus (E13), but the enzyme appeared in somatic cells on postnatal day 6 (P6). The intensity of PI3K immunosignal increased in the granulosa cells of newly formed primordial follicles on P8 and primary follicles on P9. PI3K expression correlated well with the formation of primordial follicles, with a higher expression on P8, when the first cohort of primordial follicles appear. The levels of phosphoAkt (pAkt) also increased from P7 and reached a peak on P9, suggesting that PI3K action via Akt might be involved in primordial follicle formation. pAkt immunosignal was located mainly in the oocytes and in adjacent somatic cells before the formation of primordial follicles. After follicle formation, pAkt immunosignal shifted to the granulosa cells of primordial and primary follicles. Similarly, higher ERK-1/2 immunosignal was present in the oocytes and adjacent somatic cells, and increased ERK1/2 phosphorylation correlated with the appearance of the first cohort of primordial follicles. Treatment of cultured P6 ovarian cells with 10 nM E2 significantly increased the levels of pAkt and pERK-1/2 within 10 min, while 1 µM E2 had no effect. 10 nM E2 also stimulated the phosphorylation of cRaf and MEK. Whereas Wortmannin blocked E2-induced Akt phosphorylation without affecting ERK-1/2 phosphorylation, U0126 blocked E2-induced ERK1/2 phosphorylation without affecting Akt phosphorylation. Similar phosphorylation patterns were observed when cells were exposed to G1, a GPR30 specific ligand. siRNA knockdown of GPR30 significantly reduced E2- or G1-induced Akt and ERK1/2 phosphorylation, thus providing additional evidence for a GPR30-mediated action. These results together with our previous findings suggest that the mechanisms of E2-regulated primordial follicle formation may involve two parallel signaling modalities, and GPR30 mediates a part of the E2 action during primordial follicle formation. (platform)

The effect of nerve growth factor on the development of human primordial follicles in vitro culture

OBJECTIVE: To investigate the actions of NGF on human early follicle development. DESIGN: Case-controlled experiment. MATERIALS AND METHODS: Ovarian biopsies from 21 patients were cut into ovarian pieces(opt) of lmm thickness which distributed into 5 groups: groupI (non-culture group),groupII (NGF group),groupIII (basic culture group),groupIV (anti-NGF antibody group),groupV (control IgG group). After culture, all the tissue samples were fixed for histological analysis, immunohistochemistry analysis,TUNEL test. RESULTS: Here were 33.83%±31.73%, 53.51%±39.41%, 39.30±31.73% primordial follicles among groupII, groupIII and groupIV,significantly lower than the proportion of primordial of groupI,which was 82.16%±13.63%, P<0.01; the percentage of developing follicles in groupII was 54.14%±26.16%, significantly higher than that of groupI, groupIII and groupIV, which were 14.63%±15.00%, 23.39%±28.66%, 31.44±32.24% respectively, P<0.01. The percentage of abnormal follicles in groupI was 3.22%±7.48%, significantly lower than that of groupIII, groupII, groupIV, which were 23.1%±30.94%, 12.03%±11.75%, 29.26±28.61% respectively, P<0.05. The Positive Unit value of groupI, groupIII, groupII, groupIV were 6.63 ±3.77, 5.66 ± 4.74, 12.48 ± 6.61, 6.67±4.58, 6.92±6.66 respectively, the PU value of groupII was significantly higher than that of the other groups, p<0.05. The PCNA positive follicles in groupI, groupIII, groupII, groupIV, groupV were 28.45%, 37.25%, 59.79%, 22.72%, 33.33% respectively, the rate of positive follicles in groupII was higher than that of groupIII and groupIV, p<0.005. The apoptotic index of stroma cells of groupII, groupIII, groupIV, groupV were 0.06±0.06, 0.12±0.07, 0.14±0.09, 0.12±0.09 repectively. The apoptotic index of stroma cell in groupII was significantly lower than that of groupIII, groupIV, p<0.01. CONCLUSIONS: NGF can help to promote the transition of primordial-to-primary follicles and their development in vitro culture of human ovarian tissues; By decreasing the apoptosis and increasing proliferation of stroma cells, NGF can help to promote the survival and development of early follicles in vitro culture of human ovarian tissues.

Effects of basic fibroblast growth factor on in vitro development of human ovarian primordial follicles

To evaluate whether basic fibroblast growth factor (FGF) benefits the in vitro development of human primordial follicles. Human ovarian tissue was placed in organ culture for 4 weeks with basic FGF and either fetal calf serum or a serum-free combination. Control groups were cultured with a neutralizing antibody against basic FGF. Major tertiary care and referral academic centers. Fourteen women/girls undergoing various gynecological operations and two fetuses from women undergoing pregnancy terminations. None. Follicular counts, immunohistochemistry for proliferating cell nuclear antigen and bromodeoxyuridine incorporation and measurement of 17beta E(2) production. Only in the serum-free culture system was the number of developing follicles in samples cultured with basic FGF significantly higher than in uncultured specimens. The E(2) production increased significantly in the second week, and there was a significant reduction in E(2) secretion with the addition of the neutralizing antibody against basic FGF. The percentage of granulosa cells (GCs) that stained for proliferating cell nuclear antigen or bromodeoxyuridine was significantly higher in developing follicles than in primordial follicles, regardless of treatment. Basic FGF apparently plays a role in the E(2) production of early follicles. High doses of basic FGF enhanced follicular development in serum-free media.

Influence of different concentrations of LH and FSH on in vitro caprine primordial ovarian follicle development

The roles of gonadotrophins in the regulation of primordial ovarian follicular development remains unclear. The aims of the present study were to investigate the effects of LH alone or in combination with FSH on the survival, activation and growth of in vitro caprine primordial follicles, using histology and transmission electron microscopy (TEM). To this end, samples of the caprine ovarian cortex were cultured for 1 or 7 days (39 °C), in an atmosphere of 5% CO 2, in minimum essential medium (MEM—control medium) supplemented with different concentrations of LH (0, 1, 5, 10, 50 or 100 ng/ml—Experiment 1). In Experiment 2, the control medium was supplemented with FSH (50 ng/ml) and the different concentrations of LH (0, 1, 5, 10 or 50 ng/ml also used). Small samples of non-cultured ovarian tissue, as well as those cultured for 1 or 7 days in a specific medium were processed for classical histological evaluation and TEM to evaluate the follicular integrity and to calculate the percentage of normal follicles. Additionally, the effects of FSH on the oocyte and follicle diameters of the cultured follicles were evaluated. Results showed that after 7 days of culture, the presence of LH (10, 50 or 100 ng/ml) in the culture media significantly increases the percentage of developing follicles ( P < 0.05). In addition, fragments cultured in media supplemented with FSH alone or FSH plus LH had a higher percentage of developing follicles after 1 and 7 days of culture, compared to the control ( P < 0.05). At the end of culture period, 1 and 5 ng/ml LH increased the follicular diameter, while the addition of 1, 5 or 10 ng/ml LH increased the oocyte diameter ( P < 0.05). A combination of FSH with 1 and 5 ng/ml LH increased the follicular diameter. With regard to follicle survival, low doses of LH (1 ng/ml) maintained follicle viability—similar to day 0 and in the control medium after 7 days of culture. However, higher concentrations of LH (5, 10, 50 and 100 ng/ml) induced atresia in the goat follicles. Culture of the ovarian cortex for 7 days in medium supplemented with FSH alone or FSH plus 1 ng/ml LH kept the ultrastructural characteristics of follicles similar to that of day 0. In conclusion, it can be said that, the addition of low concentrations of LH (1 ng/ml) combined with or without FSH maintained the goat follicular ultrastructural integrity, but LH in doses higher than 5 ng/ml induced atresia in in vitro goat preantral follicles.

G Protein-Coupled Receptor 30 Expression Is Required for Estrogen Stimulation of Primordial Follicle Formation in the Hamster Ovary

Estradiol-17beta (E2) plays an important role in the formation and development of primordial follicles, but the mechanisms remain unclear. G protein-coupled receptor 30 (GPR30) can mediate a rapid and transcription-independent E2 signaling in various cells. The objectives of this study were to examine whether GPR30 was expressed in the neonatal hamster ovary and whether it could mediate estrogen action during the formation of primordial follicles. GPR30 mRNA levels decreased from the 13th day of gestation (E13) through the second day of postnatal (P2) life, followed by steady increases from P3 through P6. Consistent with the changes in mRNA levels, GPR30 protein expression decreased from E13 to P2 followed by a significant increase by P7, the day before the first appearance of primordial follicles in the hamster ovary. GPR30 was expressed both in the oocytes and somatic cells, although the expression in the oocytes was low. GPR30 protein was located primarily in the perinuclear endoplasmic reticulum, which was also the site of E2-BSA-FITC (E2-BSA-fluorescein isothiocyanate) binding. E2 or E2-BSA increased intracellular calcium in neonatal hamster ovary cells in vitro. Exposure to GPR30 small interfering RNA in vitro significantly reduced GPR30 mRNA and protein levels in cultured hamster ovaries, attenuated E-BSA binding to cultured P6 ovarian cells, and markedly suppressed estrogen-stimulated primordial follicle formation. These results suggest that a membrane estrogen receptor, GPR30, is expressed in the ovary during perinatal development and mediates E2 action on primordial follicle formation.

Zona pellucida components are present in human fetal ovary before follicle formation

The zona pellucida is a glycoprotein matrix surrounding oocytes and early-stage embryos in mammals. To elucidate the roles of the zona pellucida glycoproteins ZP1 and ZP3 and their key regulatory factor FIGLA in human ovarian development and folliculogenesis, their expression and localization was studied in human fetal and adult ovaries. FIGLA mRNA and ZP3 mRNA/protein were localized mainly in the oocytes, and during fetal development their maximal expression was observed around the 20th week, the time of follicle formation. The expression of ZP1 mRNA was low both in fetal and adult ovaries. Present findings demonstrate that ZP3 and FIGLA transcripts are expressed in the oocytes from early ovarian development. The function of ZP proteins during early fetal life is not clear, but the simultaneous expression of FIGLA and ZP3 suggests, that they may have a role in the development of primordial follicle before zona pellucida formation.

Glial-derived neurotrophic factor promotes ovarian primordial follicle development and cell–cell interactions during folliculogenesis

Female fertility is determined in part by the size and development of the primordial follicle pool. The current study investigates the role of glial cell-line-derived neurotrophic factor (GDNF) in the regulation of primordial follicle development in the ovary. Ovaries from 4-day-old female rat pups were maintained in organ culture for 10 days in the absence (control) or presence of GDNF or kit ligand (KL)/stem cell factor. Ovaries treated with GDNF contained a significant increase in developing follicles, similar to that observed with KL treatment previously shown to promote follicle development. The actions of GDNF on the ovarian transcriptome were investigated with a microarray analysis. Immunohistochemical studies demonstrated that GDNF is localized to oocyte cytoplasm in follicles of all developmental stages, as well as to cumulus granulosa cells and theca cells in antral follicles. GDNF receptor alpha1 (GFRalpha1) staining was localized to oocyte cytoplasm of primordial and primary follicles, and at reduced levels in the oocytes of antral follicles. GFRalpha1 was present in mural granulosa cells of antral follicles, theca cells, and ovarian surface epithelium. The localization studies were confirmed with molecular analysis. Microarray analysis was used to identify changes in the ovarian transcriptome and further elucidate the signaling network regulating early follicle development. Observations indicate that GDNF promotes primordial follicle development and mediates autocrine and paracrine cell-cell interactions required during folliculogenesis. In contrast to the testis, ovarian GDNF is predominantly produced by germ cells (oocytes) rather than somatic cells.

Actions of anti-Müllerian hormone on the ovarian transcriptome to inhibit primordial to primary follicle transition

The oocytes found within the primordial follicles of mammalian ovaries remain quiescent for months to years until they receive the appropriate signals to undergo the primordial to primary follicle transition and initiate folliculogenesis. The molecular mechanisms and extracellular signaling factors that regulate this process remain to be fully elucidated. The current study investigates the mechanisms utilized by anti-Müllerian hormone (AMH; i.e. Müllerian inhibitory substance) to inhibit the primordial to primary follicle transition. Ovaries from 4-day-old rats were placed into organ culture and incubated in the absence or presence of AMH, either alone or in combination with known stimulators of follicle transition, including basic fibroblast growth factor (bFGF), kit ligand (KITL), or keratinocyte growth factor (KGF). Following 10 days of culture, the ovaries were sectioned, stained, and morphologically evaluated to determine the percentage of primordial versus developing follicles. As previously demonstrated, AMH treatment decreased primordial to primary follicle transition. Interestingly, AMH inhibited the stimulatory actions of KITL, bFGF, and KGF. Therefore, AMH can inhibit the basal and stimulated development of primordial follicles. To investigate the mechanism of AMH actions, the influence AMH has on the ovarian transcriptome was analyzed. AMH treatment when compared with controls was found to alter the expression of 707 genes. The overall effect of AMH exposure is to decrease the expression of stimulatory factors, increase the expression of inhibitory factors, and regulate cellular pathways (e.g. transforming growth factor beta signaling pathway) that result in the inhibition of primordial follicle development. Analysis of the regulatory factors and cellular pathways altered by AMH provides a better understanding of the molecular control of primordial follicle development.

Development of Primordial Follicles in the Hamster: Role of Estradiol-17β

The role of E2 on primordial follicle formation was examined by treating neonatal hamsters with 1 or 2 microg estradiol cypionate (ECP) at age postnatal d 1 (P1) and P4 or by in vitro culture of embryonic d 15 (E15) ovaries with 1, 5, or 10 ng/ml estradiol-17beta (E2). The specificity of E2 action was examined by ICI 182,780. One microgram of ECP maintained serum levels of E2 within the physiological range, significantly reduced apoptosis, and stimulated the formation and development of primordial follicles. In contrast, 2 microg ECP increased serum E2 levels to 400 pg/ml and had significantly less influence on primordial follicle formation. In vivo, ICI 182,780 significantly increased apoptosis and caused a modest reduction in primordial follicle formation. The formation and development of primordial follicles in vitro increased markedly with 1 ng/ml E2, and the effect was blocked by ICI 182,780. Higher doses of E2 had no effect on primordial follicle formation but significantly up-regulated apoptosis, which was blocked by ICI 182,780. CYP19A1 mRNA expression occurred by E13 and increased with the formation of primordial follicles. P4 ovaries synthesized E2 from testosterone, which increased further by FSH. Both testosterone and FSH maintained ovarian CYP19A1 mRNA, but FSH up-regulated the expression. These results suggest that neonatal hamster ovaries produce E2 under FSH control and that E2 action is essential for the survival and differentiation of somatic cells and the oocytes leading to the formation and development of primordial follicles. This supportive action of E2 is lost when hormone levels increase above a threshold.

Follicle stimulating hormone and fibroblast growth factor-2 interact and promote goat primordial follicle development in vitro

The aims of the present study were to investigate the effects of the interaction between follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on survival, follicular growth initiation and further growth of caprine preantral follicles. Pieces of caprine ovarian cortex were cultured for 1 or 7 days in minimum essential medium (MEM) supplemented with FSH, FGF-2 or FSH + FGF-2. Small fragments from non-cultured ovarian tissue and from those cultured for 1 or 7 days were processed for classical histology and transmission electron microscopy (TEM) to verify follicular morphology and growth. The results showed that, after 7 days culture, the highest percentages of normal follicles were observed in medium supplemented with FSH. After 7 days culture, the interaction between FSH and FGF-2 was most effective to promote the initiation of primordial follicles growth and oocyte growth. TEM showed ultrastructural integrity of follicles after 1 day of culture in MEM and after 7 days in all treatments, except in those follicles cultured for 7 days in MEM. In conclusion, this study demonstrated that the interaction between FSH and FGF-2 stimulates the initiation of primordial follicles growth and the subsequent growth of developing follicles. Furthermore, these data showed that FSH is important to maintain follicular integrity after 7 days culture.

Cryopreservation of the mouse ovary inhibits the onset of primordial follicle development

The cryopreservation of ovarian tissue has been reported to affect the development of preantral follicles. However, the effect of cryopreservation of ovarian tissue on the development of primordial follicles remains to be elucidated. This study was conducted to evaluate the effect of cryopreservation on the development of frozen-thawed mouse primordial follicles. One-day-old mouse ovaries were cryopreserved by either slow-freezing or a vitrification method. The development of primordial follicles was evaluated histologically and also with markers for follicle development such as: GDF-9, inhibin-α subunit and ZP3 in fresh and frozen-thawed ovaries cultured for five days. The proportion of apoptotic and necrotic areas was analyzed in fresh and frozen-thawed ovaries at one and five days after culture, in order to examine the viability of ovarian cells that influence primordial follicle development. The development rate of primordial follicles was significantly lower in slow-frozen and vitrified ovaries than the fresh controls after five days of in vitro culture ( P < 0.05). The mRNA expression for all developmental markers was slightly decreased in the frozen-thawed ovaries; this difference was not significant. The proportion of apoptosis was significantly increased in the slow-frozen and vitrified ovaries compared to the fresh ovaries at one day ( P < 0.05); however, there was no difference at five days after culture. The proportion of the area of necrosis was significantly higher in slow-frozen and vitrified ovaries compared to the fresh ovaries at one and five days after culture ( P < 0.05). Our preliminary data suggest that ovarian tissue cryopreservation using slow-freezing and vitrification methods inhibits development of primordial follicles. This may be caused by the death of ovarian cells through apoptosis and necrosis after cryopreservation.

Platelet-derived growth factor modulates the primordial to primary follicle transition

Primordial follicles steadily leave the arrested pool and undergo a primordial to primary follicle transition during the female reproductive lifespan. When the available pool of primordial follicles is depleted reproduction ceases and humans enter menopause. The present study was designed to investigate the actions of several growth factors previously identified as candidate regulatory factors for the primordial to primary follicle transition with a microarray analysis. Ovaries from 4-day-old rats were placed into culture and treated for 2 weeks with platelet-derived growth factor (PDGF), anti-PDGF neutralizing antibody, vascular endothelial growth factor (VEGF), neuregulin (NRG), or kit ligand (KITL) as a positive control. PDGF-treatment resulted in a significant decrease in the percentage of primordial follicles and a concomitant increase in the percentage of developing primary follicles compared to controls. In contrast, ovaries treated with an anti-PDGF neutralizing antibody had a significant increase in the percentage of primordial follicles demonstrating an inhibition of endogenous follicle development. Ovaries incubated in the presence of VEGF or NRG had no change in follicle development. Observations indicate that PDGF, but not VEGF or NRG, promotes the primordial to primary follicle transition. Immunohistochemical localization indicated that the PDGF protein was present in the oocytes of both primordial and developing follicles. PDGF-treatment of cultured ovaries resulted in an increase in KITL mRNA expression. KITL has been previously shown to promote the primordial to primary follicle transition. KITL-treatment of ovaries had no effect on expression of Pdgf or any PDGF homologs or receptors. Therefore, PDGF appears to be produced by the oocyte and acts as one of several extracellular signaling factors that regulate the primordial to primary follicle transition. These observations provide insight into the cell-cell interactions involved in the regulation of primordial follicle development and can be used in the future development of therapies for some forms of infertility.

The activin-follistatin system and in vitro early follicle development in goats

The aim of the present study was to investigate the effects of activin-A and follistatin on in vitro primordial and primary follicle development in goats. To study primordial follicle development (experiment 1), pieces of ovarian cortex were cultured in vitro for 5 days in minimal essential medium (MEM) supplemented with activin-A (0, 10 or 100 ng/ml), follistatin (0, 10 or 100 ng/ml) or combinations of the two. After culture, the numbers of primordial follicles and more advanced follicle stages were calculated and compared with those in non-cultured tissue. Protein and mRNA expression of activin-A, follistatin, Kit ligand (KL), growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) in non-cultured and cultured follicles were studied by immunohistochemistry and PCR. To evaluate primary follicle growth (experiment 2), freshly isolated follicles were cultured for 6 days in MEM plus 100 ng/ml activin-A, 100 ng/ml follistatin or 100 ng/ml activin-A plus 200 ng/ml follistatin. Morphology, follicle and oocyte diameters in cultured tissue and isolated follicles before and after culture were assessed. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) reactions were performed to study DNA fragmentation in follicles. In experiment 1, it was found that goat primordial follicles were activated to develop into more advanced stages, i.e. intermediate and primary follicles, during in vitro culture, but neither activin-A nor follistatin affected the number of primordial follicles that entered the growth phase. Activin-A treatment enhanced the number of morphologically normal follicles and stimulated their growth during cortical tissue culture. The effects were, however, not counteracted by follistatin. The follicles in cultured goat tissue maintained their expression of proteins and mRNA for activin-A, follistatin, KL, GDF-9 and BMP-15. Fewer than 30% of the atretic follicles in cultured cortical tissue had TUNEL-positive (oocyte or granulosa) cells. Activin-A did not affect the occurrence of TUNEL-positive cells in follicles within cortical tissue. In experiment 2, addition of activin-A to cultured isolated primary follicles significantly stimulated their growth, the effect being counteracted by follistatin. Absence of such a neutralizing effect of follistatin in the cultures with ovarian cortical tissue can be due to lower dose of follistatin used and incomplete blockage of activin in these experiments. In contrast to cortical enclosed atretic follicles, all atretic follicles that had arisen in cultures with isolated primary follicles had TUNEL-positive cells, which points to differences between isolated and ovarian tissue-enclosed follicles with regard to the followed pathways leading to their degeneration. In summary, this in vitro study has demonstrated that cultured goat primordial follicles are activated to grow and develop into intermediate and primary follicles. During in vitro culture, the follicles maintain their ability to express activin-A, follistatin, KL, GDF-9 and BMP-15. The in vitro growth and survival of activated follicles enclosed in cortical tissue and the in vitro growth of isolated primary follicles are stimulated by activin-A.

Intrauterine environment and later disease development: Infertility treatment and the risk of diabetes in offspring

The phenotype of an individual, including their susceptibility to disease, is governed by several factors including parental genes and intrauterine environment. Thus, the risk of developing Type 2 diabetes is modulated by the inheritance of specific genetic variants that are slowly being characterised by the techniques of linkage analysis and population association studies using either a candidate gene or genome-wide scan approach. At the same time, evidence has accrued that alterations in the nutritional status of the developing foetus also increase the risk of diabetes in later life. Restricting protein intake in pregnant dams or interfering with placental function increases the risk of diabetes in offspring and light weight babies are more likely to develop Type 2 diabetes as adults than heavier ones. The oocyte plays a key role, since it contains not only the maternal haplotype but other information such as mitochondrial DNA and factors that modulate the expression of genes in the developing foetus. Although the ovaries contain a huge number of primordial follicles, generally each month only one oocyte matures to ovulation. Little is known about the processes that control this phenomenon. Certainly, primordial follicles and oocytes are not all the same, differing especially in mitochondrial DNA content. As women age, the oocytes released are more likely to contain genetic errors, explaining the increased risk of Trisomy 21 with maternal age. It is generally assumed that primordial follicle development and the selection of a single ooctye for ovulation is a random process. This paper suggests that this may not be the case but that a carefully controlled system may allow the mother to release an oocyte that is best suited to the prevailing environment. This would represent an important mechanism for species adaptation. Many human infertility treatments involve pharmacological superovulation, egg harvesting and culture prior to in vitro fertilisation and reimplantation. These will bypass any system of controlled ovulation and therefore might alter the risk of diseases such as Type 2 diabetes mellitus in later life. Although the offspring of human infertility treatments are generally born healthy, it is important to note that the oldest "test-tube" baby is still less than 30-years old, so the risk of late-onset diseases is still unknown.

Expression of Growth Differentiation Factor 9 in the Oocytes Is Essential for the Development of Primordial Follicles in the Hamster Ovary

Postnatal growth differentiation factor 9 (GDF-9) expression in the hamster oocytes precedes the formation of primordial follicles. We examined the functional significance of GDF-9 in primordial folliculogenesis in the hamster ovary using RNA interference knockdown of GDF-9 mRNA and protein expression. Fifteen-day-old fetal ovaries were cultured for 9 d with or without 1 ng FSH, 1 microl Metafectane, 100 nM control nontargeting small interfering RNA (siRNA), GDF-9 siRNA, or GDF-9 siRNA + FSH, and the development of primordial follicles examined. The efficiency of siRNA transfecting ovarian cells in the organ culture was tested by culturing ovaries with siGlo, a nontargeting control siRNA labeled with Cy3. More than 90% of cells in the ovary were siGlo positive, and neither the Metafectane nor the siRNA-induced cellular apoptosis. Control siRNA did not affect the basal levels of GDF-9 mRNA, but GDF-9 siRNA slightly but significantly reduced the level. FSH markedly up-regulated the levels of GDF-9 mRNA and protein, and the effect was completely suppressed by GDF-9 siRNA. However, GDF-9 siRNA did not affect the levels of bone morphogenetic protein receptor IA or beta-actin mRNA. GDF-9 siRNA alone also reduced GDF-9 protein expression. Concurrent with GDF-9 expression, FSH significantly augmented primordial follicle formation, but the effect was abolished by GDF-9 siRNA. These results suggest that endogenous GDF-9 plays an important role in somatic cell differentiation and the formation of primordial follicles. Furthermore, FSH, by virtue of regulating GDF-9 expression, modulates oocyte regulation of primordial follicles formation.

Regulation of primordial follicle assembly and development

The assembly of the primordial follicles early in ovarian development and the subsequent development and transition of the primordial follicle to the primary follicle are critical processes in ovarian biology. These processes directly affect the number of oocytes available to a female throughout her reproductive life. Once the pool of primordial follicles is depleted a series of physiological changes known as menopause occur. The inappropriate coordination of these processes contributes to ovarian pathologies such as premature ovarian failure (POF) and infertility. Primordial follicle assembly and development are coordinated by locally produced paracrine and autocrine growth factors. Endocrine factors such as progesterone have also been identified that influence follicular assembly. Locally produced factors that promote the primordial to primary follicle transition include growth factors such as kit ligand (KL), leukaemia inhibitory factor (LIF), bone morphogenic proteins (BMP's), keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF). Factors mediating both precursor theca-granulosa cell interactions and granulosa-oocyte interactions have been identified. A factor produced by preantral and antral follicles, Müllerian inhibitory substance, can act to inhibit the primordial to primary follicle transition. Observations suggest that a complex network of cell-cell interactions is required to control the primordial to primary follicle transition. Elucidation of the molecular and cellular control of primordial follicle assembly and the primordial to primary follicle transition provides therapeutic targets to regulate ovarian function and treat ovarian disease.

Irx3 is differentially up-regulated in female gonads during sex determination

Irx3 is a member of the Iroquois homeobox gene family that encodes a protein known for its essential role in spinal cord development. Transcript screening of male and female gonads during the critical period of sex determination (E12–13.5) revealed a sexually dimorphic expression pattern for Irx3 with female gonads exhibiting a sixfold increase in expression over time. Whole mount in situ hybridization confirmed the sexually dimorphic nature of Irx3 expression and immunohistochemical analysis of gonads at E13.5 determined that IRX3 and GATA4 proteins co-localized to somatic cells of XX gonads. The Irx3 signal persisted in germ cell-depleted XX gonads resulting from Busulfan treatment suggesting that its expression was independent of germ cell regulation. Quantitative real-time PCR analysis over an extended time course determined that Irx3 message was low initially and then increased in XX gonads until E13.5, remained elevated until birth, diminished shortly after birth, and remained low in the adult ovary. In contrast, Irx3 message was 50% lower in male compared to female gonads at the initial time point, and continued to decrease over time. Further analysis of adult ovaries suggested that IRX3 expression is not present in any subpopulations of cells of the differentiated ovary. Together, these results suggest that the Irx3 signal is restricted to the somatic cell component of XX gonads and is present at a discreet period of ovarian development that ends abruptly at birth. This timing coincides with the transition of female primordial germ cells from mitotic proliferation to meiotic division, and the organization of germ cell cysts prior to primordial follicle development at birth.

Stem cell factor modulates the expression of steroidogenesis related proteins and FSHR during ovarian follicular development

Stem cell factor (SCF) is essential for the development of primordial follicles. By using cultured ovaries from neonatal rats, the effect of SCF on early follicular development was investigated. Steroidogenesis is a hallmark of follicular development. Expressions of three key protein factors in steroidogenesis, SF-1, StAR, and P450arom, were investigated using immunohistochemistry and in situ hybridization. SF-1 and StAR proteins were expressed in all ovarian cells. P450arom mRNA was localized exclusively in oocytes implying that estrogen might be synthesized by oocytes at this stage. SCF up-regulated the mRNA and protein expression of these proteins, suggesting SCF might promote the production of estrogen during this period of time. To study the differentiation status of follicular cells, the expression of FSHR and its response to SCF treatment was examined by using semi-quantitative RT-PCR. The results showed that SCF inhibited the expression of FSHR mRNA. It was also observed that SCF stimulated the expression of basic fibroblast growth factor (bFGF) in oocytes. Inactivation of bFGF by its neutralizing antibody resulted in a reversal of the inhibitory effect of SCF on the expression of FSHR. Therefore, the FSHR inhibitory effect of SCF could be mediated by bFGF. In summary, it seems that, at the early stages of follicular development, SCF might stimulate oocytes to produce estrogen while it inhibits the differentiation of granulosa cells that are the major sources of estrogen at the late stages of follicular development.

228. In vivo evidence for a role of bone morphogenetic protein-4 in ovarian function

Bone morphogenetic proteins (BMPs) were first identified on the basis of their bone inducing capacity, and later shown to be members of the transforming growth factor β (TGF β) super family. Nilsson et al.1 studied the effect of BMP-4 on follicular development in rat ovaries and found that the addition of BMP-4 to whole ovary cultures led to more numbers of developing primary follicles but less numbers of primordial follicles. Their studies indicate that BMP-4 acts as a transition factor for the conversion of primordial follicles to primary follicles. To test this hypothesis in-vivo, we conducted passive immunization studies against BMP-4 in prepubertal female mice. The mice were divided in to four groups (n = 5), and given daily SC injections of the following treatment: anti BMP-4 (50μg), PMSG (10 IU) (pregnant mare serum gonadotropin) with and without anti BMP-4 (0.5 mg/mL) and PBS for 3 days. All experimentation was approved by animal ethics committee, University of New England, Armidale, NSW. On the fourth day the mice were killed and the ovaries removed and weighed. The mice treated with anti BMP-4 had significantly smaller ovaries (4.1 ± 0.4 mg) than the control group (8.6 ± 0.9 mg). PMSG stimulated ovarian weight (21.0 ± 1.2 mg) but anti BMP-4 (23.2 ± 1.3 mg) did not significantly affect the weight of the stimulated ovaries. This data confirms BMP-4 is important in ovarian function; however, it is unclear whether this effect is on the ovary directly or via FSH. (1)Nilsson, E. E., Skinner, M.K. (2003). Bone morphogenetic protein-4 acts as an ovarian follicle survival factor and promotes primordial follicle development. Biology of Reproduction 69, 1265–1272.