Early preantral follicles of the domestic cat express gonadotropin and sex steroid signaling potential.

Key biomolecular processes, which regulate primordial ovarian follicle dormancy and early folliculogenesis in mammalian ovaries, are not fully understood. The domestic cat is a useful model to study ovarian folliculogenesis and is the most relevant for developing in vitro growth methods to be implemented in wild felid conservation breeding programs. Previously, RNA-sequencing of primordial (PrF), primary (PF), and secondary follicle (SF) samples from domestic cat implicated ovarian steroidogenesis and steroid reception during follicle development. Here, we aimed to identify which sex steroid biosynthesis and metabolism enzymes, gonadotropin receptors, and sex steroid receptors are present and may be potential regulators. Differential gene expression, functional annotation, and enrichment analyses were employed and protein localization was studied too. Gene transcripts for PGR, PGRMC1, AR (steroid receptors), CYP11A1, CYP17A1, HSD17B1 and HSD17B17 (steroidogenic enzymes), and STS (steroid metabolizing enzyme) were significantly differentially expressed (Q values of ≤0.05). Differential gene expression increased in all transcripts during follicle transitions apart from AR which decreased by the secondary stage. Immunohistochemistry localized FSHR and LHCGR to oocytes at each stage. PGRMC1 immunostaining was strongest in granulosa cells, whereas AR was strongest in oocytes throughout each stage. Protein signals for steroidogenic enzymes were only detectable in SFs. Products of these significantly differentially expressed genes may regulate domestic cat preantral folliculogenesis. In vitro growth could be optimized as all early follicles express gonadotropin and steroid receptors meaning hormone interaction and response may be possible. Protein expression analyses of early SFs supported its potential for producing sex steroids.

Altered vitamin D metabolic system in follicular cysts of sows.

This study aimed to examine 25OHD3 concentration in the fluid of follicular and follicular lutein cysts of sows in comparison with preovulatory follicles as well as immunolocalize vitamin D metabolic enzymes (CYP27B1 and CYP24A1) and determine their protein abundances in the cyst wall. We have shown for the first time that 25OHD3 level in the fluid of both cyst types was significantly lower than in preovulatory follicles. Furthermore, we have demonstrated CYP27B1 and CYP24A1 protein immunolocalization and abundance in follicular and follicular lutein cysts. The abundance of protein for both metabolic enzymes was decreased in ovarian cysts when compared to preovulatory follicles. We propose that altered VD metabolism in ovarian cyst might associate with their formation in sows.

Hypoxia-inducible factor-1alpha and nitric oxide synthases in bovine follicles close to ovulation and early luteal angiogenesis.

The objective of the study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A), inducible nitric oxide synthase (iNOS) and endothelial (eNOS) isoforms in time-defined follicle classes before and after GnRH application in the cow. Ovaries containing pre-ovulatory follicles or corpora lutea were collected by transvaginal ovariectomy (n = 5 cows/group) as follow: (I) before GnRH administration; (II) 4h after GnRH; (III) 10h after GnRH; (IV) 20h after GnRH; (V) 25h after GnRH; and (VI) 60h after GnRH (early corpus luteum). The mRNA abundance of HIF1A in the follicle group before GnRH was high, followed by a significant down regulation afterwards with a minimum level 25h after GnRH (close to ovulation) and significant increase only after ovulation. The mRNA abundance of iNOS before GnRH was high, decreased significantly during LH surge, with minimum levels afterwards. In contrast, the mRNA of eNOS decreased in the follicle group 20h after GnRH, followed by a rapid and significant upregulation just after ovulation. Immunohistochemically, the granulosa cells of antral follicles and the eosinophils of the theca tissue as well of the early corpus luteum showed a strong staining for HIF1A. The location of the eosinophils could be clearly demonstrated by immunostaining with an eosinophil-specific antibody (EMBP) and transmission electron microscopy. In conclusion, the parallel and acute regulated expression patterns of HIF1A and NOS isoforms, specifically during the interval between the LH surge and ovulation, indicate that these paracrine factors are involved in the local mechanisms, regulating final follicle maturation, ovulation and early luteal angiogenesis.

Expression of sirtuins in ovarian follicles of postnatal mice.

Mammalian ovarian follicular development is an intricate, elaborate, and well-organized phenomenon regulated by various signaling pathways; however, the underlying mechanism remains unclear. Mammalian sirtuins (sirtuin 1 to sirtuin 7) are a group of NAD+ -dependent deacetylases implicated in various physiological processes including cell proliferation, apoptosis, cell cycle progression, and insulin signaling. Mammalian ovarian sirtuins have been studied using adult and aged bovine, porcine, and murine models. However, limited information is available regarding their precise expression patterns and the localization of follicle development in mice. This study aimed to assess the dynamic expression and localization of all seven sirtuins in early postnatal mouse ovaries through real-time polymerase chain reaction analysis and immunohistochemistry, respectively. During postnatal ovarian follicle development, sirtuin 1, sirtuin 4, and sirtuin 6 were downregulated compared with those in 1-day postnatal mouse ovaries (p < .05), indicating that these three sirtuin genes may be markers of follicular development. Combining their localization in granulosa cells through immunohistochemical studies, sirtuin 1, sirtuin 4, and sirtuin 6 are suggested to play negative regulatory roles in mammal ovarian follicular granulosa cell development. Furthermore, we found that sirtuin 2 (p < .05) and sirtuin 7 (p < .05) mRNA were constantly upregulated relative to sirtuin 1, although limited information is available regarding sirtuin 7. Among all sirtuins in mouse ovaries, sirtuin 1 was relatively and steadily downregulated. Upon sirtuin 1 overexpression in 1-day postnatal mouse ovaries via sirtuin 1-harboring adenoviruses in vitro, the emergence of primary follicles was delayed, as was the emergence of secondary follicles in 4-day postnatal ovaries. Further studies on KGN cell lines reported that interfering with sirtuin 1 expression in granulosa cell significantly affected granulosa cell proliferation and the expression of mitochondrial genes. This study presents the first systemic analysis of dynamic patterns of sirtuin family expression in early postnatal mice ovaries, laying the foundation for further studies on less discussed sirtuin subtypes, such as sirtuin 5 and sirtuin 7.

A Hyperandrogenic Environment Causes Intrinsic Defects That Are Detrimental to Follicular Dynamics in a PCOS Mouse Model.

Polycystic ovary syndrome (PCOS) is a common cause of female infertility. Hyperandrogenism is both a major symptom and key diagnostic trait of PCOS; however, the direct impact of this androgen excess on ovarian dynamics is unclear. By combining a DHT-induced PCOS mouse model with an ex vivo follicle culture system, we investigated the impact of hyperandrogenism on ovarian function. Ovaries from PCOS mice exhibited the characteristic polycystic ovary morphology with numerous large cystic follicles and no corpora lutea present. Isolation and individual culture of preantral and antral follicles from PCOS mice resulted in slower growth rates during 5 days compared with the follicles isolated from control mice (P < 0.01). In contrast, preovulatory follicles from PCOS mice exhibited a significant increase in growth rate compared with controls (P < 0.01). Preantral follicles from PCOS ovaries maintained comparable follicular health as control follicles, but antral and preovulatory PCOS follicles exhibited reduced follicle health (P < 0.01) and survival rates (P < 0.01). Compared with controls, PCOS females also exhibited a poorer response to hyperstimulation (P < 0.01), impaired oocyte function evident by increased levels of reactive oxygen species (P < 0.01), and a reduction in on-time embryo development (P < 0.01). These results demonstrate that prolonged exposure to androgen excess leads to aberrant follicle development, which persists even after removal from the hyperandrogenic environment, causing perturbed follicular developmental trajectories. These findings indicate that an in vivo hyperandrogenic environment in patients with PCOS may intrinsically induce detrimental effects on follicles and oocytes.

Hippo pathway functions as a downstream effector of AKT signaling to regulate the activation of primordial follicles in mice.

Clarifying the molecular mechanisms by which primordial follicles are initiated is crucial for the prevention and treatment of female infertility and ovarian dysfunction. The Hippo pathway has been proven to have a spatiotemporal correlation with the size of the primordial follicle pool in mice in our previous work. But the role and underlying mechanisms of the Hippo pathway in primordial follicle activation remain unclear. Here, the localization and expression of the core components were examined in primordial follicles before and after activation. And the effects of the Hippo pathway on primordial follicle activation were determined by genetically manipulating yes-associated protein 1 (Yap1), the key transcriptional effector. Furthermore, an AKT specific inhibitor (MK2206) was added to determine the interaction between the Hippo pathway and AKT, an important signaling regulator of ovarian function. Results showed that the core components of the Hippo pathway were localized in both primordial and primary follicles and the expression levels of them changed significantly during the initiation of primordial follicles. Yap1 knockdown suppressed primordial follicle activation, while its overexpression led to the opposite trend. MK2206 downregulated the ratio of P-MST/MST1 and upregulated the ratio of P-YAP1/YAP1 significantly, whereas Yap1-treatment had no influence on AKT. In addition, YAP1 upregulation partially rescued the suppression of the primordial follicle activation induced by MK2206. Our findings revealed that the Hippo-YAP1 regulates primordial follicular activation, which is mediated by AKT signaling in mice, thus providing direct and new evidence to highlight the role of Hippo signaling in regulating ovarian follicles development.

Molecular mechanism of mercury-induced reproductive impairments in banded gourami, Trichogaster fasciata.

Mercury is one of the key pollutants responsible for the degradation of natural aquatic ecosystems. Among the different forms of mercury that exist in the environment, mercuric chloride (HgCl2) is the dominant pollutant for freshwater environments as it is used as an ingredient in antiseptics, disinfectants and preservatives, insecticides, batteries and in metallurgical and photographic operations. Pollutant may exert their action on organisms or populations by affecting their normal endocrine function as well as reproduction. Thus, the present study tried to understand the effect of mercuric chloride (HgCl2) on reproductive function and to decipher the molecular mechanism of Hg-induced reproductive impairments of female Trichogaster fasciata. Both in vivo and in vitro experiments were performed by using ecologically relevant doses of HgCl2 and the resulting effects on follicular development, steroidogenic potentiality, aromatase activity, aromatase gene expression and steroidogenic factor-1 (SF-1) expression pattern were analysed. In vivo exposure to HgCl2 caused reproductive impairments as shown by the inhibitory role of HgCl2 on follicular development, steroid biosynthesis and SF-1 activity. In vitro experiments revealed that aromatase activity, steroidogenesis, aromatase and SF-1 expression were blocked by HgCl2. The results obtained from this study contribute to understand the molecular mechanism of HgCl2-induced reproductive impairment of T. fasciata.

Regulation of follicle growth through hormonal factors and mechanical cues mediated by Hippo signaling pathway.

The ovary is an interesting organ that shows major structural changes within a short period of time during each reproductive cycle. Follicle development is controlled by local paracrine and systemic endocrine factors. Many hormonal and molecular analyses have been conducted to find the mechanisms underlying structural changes in ovaries, However, exact mechanisms still remain to be determined. Recent development of mechanobiology facilitates the understanding on the contribution of physical forces and changes in the mechanical properties of cells and tissues to physiology and pathophysiology. The Hippo signaling pathway is one of the key players in mechanotransduction, providing an understanding of the molecular mechanisms by which cells sense and respond to mechanical signals to regulate cell proliferation and apoptosis for maintaining optimal organ sizes. Our group recently demonstrated the involvement of the Hippo signaling pathway in the regulation of ovarian follicle development. Fragmentation of ovarian cortex into small cubes changed cytoskeletal actin dynamics and induced disruption of the Hippo signaling pathway, leading to the production of CCN growth factors and anti-apoptotic BIRC. These factors, in turn, stimulated secondary follicle growth in vitro and in vivo. In this review, we summarized hormonal regulation of follicular structural changes and further focused on the role of Hippo signaling in the regulation of follicle development. We also suggest a new strategy of infertility treatments in patients with polycystic ovary syndrome and primary ovarian insufficiency based on mechanobiology.

Stimulation of ovarian follicle growth after AMPK inhibition.

Previous studies showed that the protein kinase B (Akt)-mammalian target of rapamycin (mTOR) and Hippo signaling Yes-associated protein (YAP) pathways play important roles in promoting follicle growth. Additionally, other studies demonstrated that 5' adenosine monophosphate-activated protein kinase (AMPK) is an upstream regulatory element of mTOR and YAP. Here, we used AMPK inhibitor (Compound C) to in vitro cultured ovaries from 10-day-old mice followed by in vivo grafting into adult hosts or to in situ treated ovaries of 3-week-old mice by intrabursal injection followed by gonadotropin stimulation. We found that the phosphorylation of ovarian mTOR and downstream proteins (ribosomal protein S6 (S6) and eukaryotic translation initiation factor 4B (eIF4B)) was upregulated following Compound C administration, whereas tuberous sclerosis complex 2 (TSC2) phosphorylation was downregulated. Additionally, treatment with Compound C increased hypoxia-inducible factor 1-alpha (Hif1a), vascular endothelial growth factor A (Vegfa), VEGF receptor 2 (Vegfr2) and connective tissue growth factor (Ctgf) mRNA levels. Furthermore, treatment of 10-day-old mice with Compound C promoted the growth of preantral and antral follicles accompanied by enhanced angiogenesis. In situ intrabursal injection with Compound C, followed by controlled ovarian hyperstimulation, increased the number of ovulated oocytes in 3-week-old mice, and these oocytes could be successfully fertilized, leading to the delivery of healthy pups. Our results demonstrated that treatment with AMPK inhibitor resulted in the activation of the mTOR signaling pathway, increases in Ctgf expression in mouse ovaries, stimulation of follicle development and promotion of ovarian angiogenesis for ovary growth.

Hypothyroidism Reduces the Size of Ovarian Follicles and Promotes Hypertrophy of Periovarian Fat with Infiltration of Macrophages in Adult Rabbits.

Ovarian failure is related to dyslipidemias and inflammation, as well as to hypertrophy and dysfunction of the visceral adipose tissue (VAT). Although hypothyroidism has been associated with obesity, dyslipidemias, and inflammation in humans and animals, its influence on the characteristics of ovarian follicles in adulthood is scarcely known. Control and hypothyroid rabbits were used to analyze the ovarian follicles, expression of aromatase in the ovary, serum concentration of lipids, leptin, and uric acid, size of adipocytes, and infiltration of macrophages in the periovarian VAT. Hypothyroidism did not affect the percentage of functional or atretic follicles. However, it reduced the size of primary, secondary, and tertiary follicles considered as large and the expression of aromatase in the ovary. This effect was associated with high serum concentrations of total cholesterol and low-density lipoprotein cholesterol (LDL-C). In addition, hypothyroidism induced hypertrophy of adipocytes and a major infiltration of CD68+ macrophages into the periovarian VAT. Our results suggest that the reduced size of ovarian follicles promoted by hypothyroidism could be associated with dyslipidemias, hypertrophy, and inflammation of the periovarian VAT. Present findings may be useful to understand the influence of hypothyroidism in the ovary function in adulthood.

Constitutive Activation of PI3K in Oocyte Induces Ovarian Granulosa Cell Tumors.

Cell-cell interactions play crucial roles in the maintenance of tissue homeostasis, a loss of which often leads to varying diseases, including cancer. Here, we report that uncontrolled PI3K activity within oocytes irreversibly transforms granulosa cells (GC), causing GC tumors (GCT) through perturbed local cell communication. Previously, we reported reproductive phenotypes of transgenic mice, in which expression of constitutively active mutant PI3K was induced in primordial oocytes by Gdf9-iCre. The transgenic mice (Cre(+)) demonstrated severe ovarian phenotypes, including the overgrowth of excess ovarian follicles and anovulation. Surprisingly, the Cre(+) mice became cachectic by postnatal day 80 due to bilateral GCT. Although GCT cells proliferated independently of oocytes, local interactions with mutant PI3K-positive oocytes during early folliculogenesis were essential for the GC transformation. Growing GCT cells expressed high levels of inhibin ßA and nuclear SMAD3, and the proliferation rate was positively correlated with a high activin A to inhibin A ratio. These results suggested that the tumor cells stimulated their growth through an activin A autocrine signaling pathway, a hypothesis confirmed by activin A secretion in cultured GCT cells, which proliferated in response. Although communication between the oocyte and surrounding somatic cells is critical for the normal development of ovarian follicles, perturbations in oocyte-GC communication during early folliculogenesis can induce GCT by activating an autocrine growth circuit program in GC. Cancer Res; 76(13); 3851-61. ©2016 AACR.

The effect of FSH and activin A on Akt and MAPK1/3 phosphorylation in cultured bovine ovarian cortical strips.

BACKGROUND: rhFSH and rhActA have been used in mammalian ovarian follicle culture systems for activation of follicular growth in vitro and suggested to be responsible for primordial follicle survival through MAPK and Akt pathways. The aim of our study was to determine the effects of rhFSH and rhActA on Akt, pAkt, MAPK1/3 and pMAPK1/3 protein levels in bovine ovarian cortical strips cultured in vitro. METHODS: Ovarian cortical strips from heifers were cultured in the presence of rhFSH (50 ng/mL), rhActA (100 ng/mL) or combination of these factors for 6 days. The strips were embedded in paraffin for histological observations and homogenized for western blot to determine Akt, pAkt, MAPK1/3 and pMAPK1/3 protein levels after the culture. Determination of primordial, primary and secondary follicle proportions at the end of culture as well as comparison of healthy follicle for each developmental stage after the culture was performed to quantify follicle survival and activation. RESULTS: pAkt protein levels were significantly lower in rhFSH + rhActA group among the other groups, whereas pMAPK1/3 levels were not significantly changed. Follicular activation and survival was measured to be significantly lower in rhFSH + rhActA group. Percentage of healthy primordial follicles was higher in control group whereas healthy secondary follicle proportion was higher in both rhActA and rhFSH groups. rhActA alone had a better impact on follicular activation, since the percentage of the secondary follicles was significantly higher than other treatment groups. CONCLUSIONS: The use of rhActA and rhFSH alone or in the combined form results in differential levels of Akt and MAPK proteins. Both rhActA and rhFSH alone has a remarkable contribution in survival and activation of the follicles in accordance with higher levels of these proteins. Thus, the manipulation of Akt and MAPK pathways with appropriate activators might contribute to proper activation and development of ovarian follicles in vitro.

The Interplay between Myc and CTP Synthase in Drosophila.

CTP synthase (CTPsyn) is essential for the biosynthesis of pyrimidine nucleotides. It has been shown that CTPsyn is incorporated into a novel cytoplasmic structure which has been termed the cytoophidium. Here, we report that Myc regulates cytoophidium formation during Drosophila oogenesis. We have found that Myc protein levels correlate with cytoophidium abundance in follicle epithelia. Reducing Myc levels results in cytoophidium loss and small nuclear size in follicle cells, while overexpression of Myc increases the length of cytoophidia and the nuclear size of follicle cells. Ectopic expression of Myc induces cytoophidium formation in late stage follicle cells. Furthermore, knock-down of CTPsyn is sufficient to suppress the overgrowth phenotype induced by Myc overexpression, suggesting CTPsyn acts downstream of Myc and is required for Myc-mediated cell size control. Taken together, our data suggest a functional link between Myc, a renowned oncogene, and the essential nucleotide biosynthetic enzyme CTPsyn.

Pro-nerve growth factor in the ovary and human granulosa cells.

BACKGROUND: Pro-nerve growth factor must be cleaved to generate mature NGF, which was suggested to be a factor involved in ovarian physiology and pathology. Extracellular proNGF can induce cell death in many tissues. Whether extracellular proNGF exists in the ovary and may play a role in the death of follicular cells or atresia was unknown. MATERIALS AND METHODS: Immunohistochemistry of human and rhesus monkey ovarian sections was performed. IVF-derived follicular fluid and human granulosa cells were studied by RT-PCR, qPCR, Western blotting, ATP- and caspase-assays. RESULTS AND CONCLUSION: Immunohistochemistry of ovarian sections identified proNGF in granulosa cells and Western blotting of human isolated granulosa cells confirmed the presence of proNGF. Ovarian granulosa cells thus produce proNGF. Recombinant human proNGF even at high concentrations did not affect the levels of ATP or the activity of caspase 3/7, indicating that in granulosa cells proNGF does not induce death. In contrast, mature NGF, which was detected previously in follicular fluid, may be a trophic molecule for granulosa cells with unexpected functions. We found that in contrast to proNGF, NGF increased the levels of the transcription factor early growth response 1 and of the enzyme choline acetyl-transferase. A mechanism for the generation of mature NGF from proNGF in the follicular fluid may be extracellular enzymatic cleavage. The enzyme MMP7 is known to cleave proNGF and was identified in follicular fluid and as a product of granulosa cells. Thus the generation of NGF in the ovarian follicle may depend on MMP7.

Pregnancy-associated plasma protein A in human ovarian follicles and its association with intrafollicular hormone levels.

OBJECTIVE: To evaluate follicular fluid (FF) levels of pregnancy-associated plasma protein A (PAPP-A) in relation to levels of intrafollicular hormones. Furthermore, immunostaining of human follicles of varying diameters was studied for PAPP-A, antimüllerian hormone (AMH), and aromatase, and the biological activity of PAPP-A in FF was evaluated. DESIGN: Laboratory investigation. SETTING: University hospital. PATIENT(S): A total of 43 women with a total of 80 samples were obtained from three different size-groups of antral follicles collected before and after the LH surge. INTERVENTION(S): ELISA measurement of steroids, PAPP-A, and AMH, immunohistochemistry of PAPP-A, AMH, and aromatase on follicles of different diameter, and proteolytic activity of PAPP-A toward insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4). MAIN OUTCOME MEASURE(S): Association between FF levels of PAPP-A and measured ovarian hormones, PAPP-A activity in FF, localization of PAPP-A, AMH, and aromatase in antral follicles. RESULT(S): A highly significant association between FF levels of PAPP-A and all measured hormones were obtained with positive associations toward E2 and P, whereas AMH, T, and A showed strong negative associations. PAPP-A proteolytic activity toward IGFBP-4 was detected in human FF. PAPP-A immunostaining shifted from being primarily present in theca cells of small antral follicles to being expressed in granulosa cells (GCs) of preovulatory follicles. In contrast, AMH expression became reduced with increasing follicular diameter. Aromatase expression was highly specifically localized to GCs of preovulatory follicles. CONCLUSION(S): The results suggest that PAPP-A is specifically involved in the regulation of steroidogenesis in human antral follicles. Local regulation of IGF-II activity may represent a mechanism by which PAPP-A exerts this function and highlights the importance of IGF signaling during follicular development.

Hypoxia induces miR-210, leading to anti-apoptosis in ovarian follicular cells of marine medaka Oryzias melastigma.

Hypoxia is a major global problem that impairs reproductive functions and reduces the quality and quantity of gametes and the fertilization success of marine fish. Nevertheless, the detailed molecular mechanism underlying hypoxia-induced female reproductive impairment remains largely unknown. There is increasing evidence that miRNA is vital in regulating ovarian functions and is closely associated with female fertility in humans. Certain miRNAs that regulate apoptotic genes can be induced by hypoxia, resulting in cell apoptosis. Using primary ovarian follicular cells of the marine medaka, Oryzias melastigma, as a model, we investigated the response of miR-210 to hypoxic stress in ovarian tissues to see if it would interrupt reproductive functions. A significant induction of miR-210 was found in primary ovarian follicular cells exposed to hypoxia, and gene ontology analysis further highlighted the potential roles of miR-210 in cell proliferation, cell differentiation, and cell apoptosis. A number of miR-210 target apoptotic genes, including Deleted in liver cancer 1 protein (DLC1), STE20-like serine/threonine-protein kinase (SLK), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), RNA binding motif protein 25 (RBM25), and Ubiquitin-specific-processing protease 7 (USP7), were identified. We further showed that ectopic expression of miR-210 would result in down-regulation of these apoptotic genes. On the other hand, the inhibition of miR-210 promoted apoptotic cell death and the expression of apoptotic marker - caspase 3 in follicular cells under hypoxic treatment, supporting the regulatory role of miR-210 in ovarian cell apoptosis. This study provides new insights on how hypoxia induces miR-210, leading to anti-apoptosis in ovarian follicular cells in fish, which is fundamentally important in environmental sciences and reproductive biology.

Sohlh2 inhibits the apoptosis of mouse primordial follicle oocytes via C-kit/PI3K/Akt/Foxo3a signalling pathway.

We previously reported that bone morphogenetic protein 4/drosophila mothers against decapentaplegic protein (BMP4/Smad) signalling pathway initiated primordial follicle growth and prevented oocyte apoptosis via up-regulation of Sohlh2 and receptor for kit ligand (c-kit). The mechanism underlying this process was not fully elucidated. In the present study, primary oocyte cultures were established from ovaries of 3-day-old female mouse pups by two-step enzyme digestion. Cultures were divided into Sohlh2 small interference RNA (SiRNA) group, negative SiRNA group, Sohlh2 overexpression plasmid group and pCAG-puro group. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay was carried out to detect the oocyte apoptosis; immunocytochemical staining and quantitative real time-polymerase chain reaction detected the expression of c-kit and Forkhead box O3a (Foxo3a); Western blot was performed to detect the expression of Sohlh2, C-kit, saerine/threonine kinases (Akt1) and Foxo3a. The results showed that Sohlh2 inhibited oocyte apoptosis and upregulated c-kit expression; Sohlh2 decreased the endonuclear Foxo3a via the upregulation of phosphorylated Akt1 (P-Akt1) and phosphorylated Foxo3a (P-Foxo3a) but not total Akt1 (T-Akt1) or total Foxo3a (T-Foxo3a); Sohlh2 increased P-Akt1 but not T-Akt1; the PI3K (phosphotidylinsitol-3-kinase) inhibitor LY294002 ameliorated the role of Sohlh2 on phosphorylation of Akt1 and Foxo3a. Sohlh2 may inhibit oocyte apoptosis via c-kit/PI3K/Akt/Foxo3a signalling pathway.

Changes in the expression of DNA double strand break repair genes in primordial follicles from immature and aged rats.

Oocytes present at birth undergo a progressive process of apoptosis in humans and other mammals as they age. Accepted opinion is that no fresh oocytes are produced other than those present at the time of birth. Studies have shown that DNA repair genes in oocytes of mice and women decline with age, and lack of these genes show higher DNA breaks and increased oocyte death rates. In contrast to the ethical problems associated with monitoring the changes in DNA double-strand breaks in oocytes from young and old humans, it is relatively easy to carry out such a study using a rodent model. In this study, the mRNA levels of DNA repair genes are compared with protein products of some of the genes in the primordial follicles isolated from immature (18-20 days) and aged (400-450 days) female rats. Results revealed a significant decline in mRNA levels of BRAC1 (P < 0.01), RAD51 (P < 0.05), ERCC2 (P < 0.05), and H2AX (P < 0.01) of DNA repair genes and phospho-protein levels of BRAC1 (P < 0.01) and H2AX (P < 0.05) in primordial follicles of aged rats. Impaired DNA repair is confirmed as a mechanism of oocyte ageing.

Caloric restriction promotes the reserve of follicle pool in adult female rats by inhibiting the activation of mammalian target of rapamycin signaling.

Caloric restriction (CR) is known to increase the number of primordial follicles and prolong the reproductive life span. However, how CR modulates follicular development is not well understood. In the present study, we examined the effects of CR on follicular development in rats and investigated the underlying mechanism. After 10 weeks of CR or high-fat diet, ovarian follicles at different developmental stages were examined by histological analysis. Plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estrogen (ESG) were measured, and the levels of mammalian target of rapamycin (mTOR), p70S6 kinase (p70S6K), and phosphorylated p70S6K in the ovary were detected by Western blot. The results showed that the reserve of follicle pool in CR rats was increased, accompanied by decreased level of phosphorylated p70S6K in the ovary, and decreased serum LH, FSH, and ESG levels. Taken together, these results suggest that CR may suppress ovarian follicular development and enhance the follicle pool reserve by inhibiting mTOR signaling.

A Pak-regulated cell intercalation event leading to a novel radial cell polarity is involved in positioning of the follicle stem cell niche in the Drosophila ovary.

In the germarium of the Drosophila ovary, germline cysts are encapsulated one at a time by a follicular epithelium derived from two follicle stem cells (FSCs). Ovaries in flies mutant for the serine/threonine kinase Pak exhibit a novel phenotype, in which two side-by-side cysts are encapsulated at a time, generating paired egg chambers. This striking phenotype originates in the pupal ovary, where the developing germarium is shaped by the basal stalk, a stack of cells formed by cell intercalation. The process of basal stalk formation is not well understood, and we provide evidence that the cell intercalation is driven by actomyosin contractility of DE-Cadherin-adhered cells, leading to a column of disk-shaped cells exhibiting a novel radial cell polarity. Cell intercalation fails in Pak mutant ovaries, leading to abnormally wide basal stalks and consequently wide germaria with side-by-side cysts. We present evidence that Pak mutant germaria have extra FSCs, and we propose that contact of a germline cyst with the basal stalk in the pupal ovary contributes to FSC niche formation. The wide basal stalk in Pak mutants enables the formation of extra FSC niches which are mispositioned and yet functional, indicating that the FSC niche can be established in diverse locations.