Low expression of SEMA6C accelerates the primordial follicle activation in the neonatal mouse ovary

The primordial follicle assembly, activation and the subsequent development are critical processes for female reproduction. A limited number of primordial follicles are activated to enter the growing follicle pool each wave, and the primordial follicle pool progressively diminishes over a woman's life‐time. The number of remaining primordial follicles represents the ovarian reserve. Identification and functional investigation of the factors involved in follicular initial recruitment will be of great significance to the understanding of the female reproduction process and ovarian ageing. In this study, we aimed to study whether and how semaphorin 6C (Sema6c) regulated the primordial follicle activation in the neonatal mouse ovary. The attenuation of SEMA6C expression by SiRNA accelerated the primordial follicle activation in the in vitro ovary culture system. PI3K‐AKT‐rpS6 pathway was activated when SEMA6C expression was down‐regulated. And the LY294002 could reverse the effect of low SEMA6C expression on primordial follicle activation. Our findings revealed that Sema6c was involved in the activation of primordial follicles, and the down‐regulation of SEMA6C led to massive primordial follicle activation by interacting with the PI3K‐AKT‐rpS6 pathway, which might also provide valuable information for understanding premature ovarian failure and ovarian ageing.     

Pathways coordinating oocyte attrition and abundance during mammalian ovarian reserve establishment

The mammalian ovarian reserve is comprised of a finite pool of primordial follicles, representing the lifetime reproductive capacity of females. In most mammals, the reserve is produced during embryonic and early postnatal development with oocyte numbers peaking during mid‐to‐late gestation, and then experiencing a dramatic decline continuing until shortly after birth. Oocytes remaining after the bulk of this attrition are subsequently surrounded by a layer of somatic pre‐granulosa cells with these units then referred to as “primordial follicles.” The complex and varied cell death mechanisms intrinsic to this process are not only characteristic of, but also essential for, the proper formation of this pool of follicles, and as a result must be immaculately balanced to ensure long‐term fertility and reproductive health. Too few follicles can lead to Primary Ovarian Insufficiency, resulting in fertility loss and other features of aging, such as an overall shorter lifespan. On the other hand, whereas an excess of follicles might extend reproductive lifespan, this might also be the underlying etiology of other ovarian pathologies. The last decade, in particular, has vastly expanded our understanding of oocyte attrition and determinants of ovarian reserve abundance. By continuing to decipher the intricacies underlying the cell death processes and development of the initial primordial follicle pool, we may be in a much better position to understand idiopathic cases of premature follicle depletion and improve ovarian health in reproductive‐age women.     

Proliferating cell nuclear antigen (PCNA) regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries

Primordial follicles, providing all the oocytes available to a female throughout her reproductive life, assemble in perinatal ovaries with individual oocytes surrounded by granulosa cells. In mammals including the mouse, most oocytes die by apoptosis during primordial follicle assembly, but factors that regulate oocyte death remain largely unknown. Proliferating cell nuclear antigen (PCNA), a key regulator in many essential cellular processes, was shown to be differentially expressed during these processes in mouse ovaries using 2D-PAGE and MALDI-TOF/TOF methodology. A V-shaped expression pattern of PCNA in both oocytes and somatic cells was observed during the development of fetal and neonatal mouse ovaries, decreasing from 13.5 to 18.5 dpc and increasing from 18.5 dpc to 5 dpp. This was closely correlated with the meiotic prophase I progression from pre-leptotene to pachytene and from pachytene to diplotene when primordial follicles started to assemble. Inhibition of the increase of PCNA expression by RNA interference in cultured 18.5 dpc mouse ovaries strikingly reduced the apoptosis of oocytes, accompanied by down-regulation of known pro-apoptotic genes, e.g. Bax, caspase-3, and TNFα and TNFR2, and up-regulation of Bcl-2, a known anti-apoptotic gene. Moreover, reduced expression of PCNA was observed to significantly increase primordial follicle assembly, but these primordial follicles contained fewer granulosa cells. Similar results were obtained after down-regulation by RNA interference of Ing1b, a PCNA-binding protein in the UV-induced apoptosis regulation. Thus, our results demonstrate that PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries.     

Regulation of the ovarian reserve by members of the transforming growth factor beta family

Genetic or environmental factors that affect the endowment of oocytes, their assembly into primordial follicles, or their subsequent entry into the growing follicle pool can disrupt reproductive function and may underlie disorders such as primary ovarian insufficiency. Mouse models have been instrumental in identifying genes important in ovarian development, and a number of genes now associated with ovarian dysfunction in women were first identified as causing reproductive defects in knockout mice. The transforming growth factor beta (TGFB) family consists of developmentally important growth factors that include the TGFBs, anti‐Müllerian hormone (AMH), activins, bone morphogenetic proteins (BMPs), and growth and differentiation factor 9 (GDF9). The ovarian primordial follicle pool is the source of oocytes in adults. Development of this pool can be grossly divided into three key processes: (1) establishment of oocytes during embryogenesis followed by (2) assembly and (3) activation of the primordial follicle. Disruptions in any of these processes may cause reproductive dysfunction. Most members of the TGFB family show pivotal roles in each of these areas. Understanding the phenotypes of various mouse models for this protein family will be directly relevant to understanding how disruptions in TGFB family signaling result in reproductive diseases in women and will present new areas for development of tailored diagnostics and interventions for infertility. Mol. Reprod. Dev. 79: 666–679, 2012. © 2012 Wiley Periodicals, Inc.     

KIT signaling regulates primordial follicle formation in the neonatal mouse ovary

The pool of primordial follicles determines the reproductive lifespan of the mammalian female, and its establishment is highly dependent upon proper oocyte cyst breakdown and regulation of germ cell numbers. The mechanisms controlling these processes remain a mystery. We hypothesized that KIT signaling might play a role in perinatal oocyte cyst breakdown, determination of oocyte numbers and the assembly of primordial follicles. We began by examining the expression of both KIT and KIT ligand in fetal and neonatal ovaries. KIT was expressed only in oocytes during cyst breakdown, but KIT ligand was present in both oocytes and somatic cells as primordial follicles formed. To test whether KIT signaling plays a role in cyst breakdown and primordial follicle formation, we used ovary organ culture to inhibit and activate KIT signaling during the time when these processes occur in the ovary. We found that when KIT was inhibited, there was a reduction in cyst breakdown and an increase in oocyte numbers. Subsequent studies using TUNEL analysis showed that when KIT was inhibited, cell death was reduced. Conversely, when KIT was activated, cyst breakdown was promoted and oocyte numbers decreased. Using Western blotting, we found increased levels of phosphorylated MAP Kinase when KIT ligand was added to culture. Taken together, these results demonstrate a role for KIT signaling in perinatal oocyte cyst breakdown that may be mediated by MAP Kinase downstream of KIT.     

Follicular expression of c-Kit/SCF and inhibin-alpha in mouse ovary during development.

The mechanism of development of the ovarian follicles has been largely unknown. We performed an immunohistochemical (IHC) study to determine the follicular expressions of c-kit, SCF, and inhibin-alpha at different developmental stages in mouse ovary. Ovaries were obtained from 14 and 16 days post coitum and 2, 7, and 21 days post partum (dpp) mice. IHC for c-kit, SCF, and inhibin-alpha was carried out. c-Kit and SCF were expressed on oogonia regardless of the developmental stage. Immunoreactive c-kit and SCF antigens were expressed on oocytes of primordial and primary follicles of neonate mouse ovaries. In 21 dpp mouse ovary, the expression of c-kit/SCF in oocytes gradually decreased as the follicles developed. c-Kit/SCF was expressed strongly in oocytes of preantral follicles and weakly in granulosa and thecal cells. Inhibin-alpha was mainly expressed on granulosa cells of preantral and early antral follicles of the 21 dpp mouse ovaries. These findings suggest that the IHC expression of c-kit/SCF proteins is specific in all developmental stages of ovarian follicles and is decreased after the follicle starts to grow. The expression of inhibin-alpha is negatively correlated with the expression of c-kit/SCF in the ovarian follicles in mice.     

Oocyte regulation of anti-Müllerian hormone expression in granulosa cells during ovarian follicle development in mice

In the ovarian follicle, anti-Müllerian hormone (Amh) mRNA is expressed in granulosa cells from primary to preovulatory stages but becomes restricted to cumulus cells following antrum formation. Anti-Müllerian hormone regulates follicle development by attenuating the effects of follicle stimulating hormone on follicle growth and inhibiting primordial follicle recruitment. To examine the role of the oocyte in regulating granulosa cell Amh expression in the mouse, isolated oocytes and granulosa cells were co-cultured and Amh mRNA levels were analysed by real-time RT-PCR. Expression in freshly isolated granulosa cells increased with preantral follicle development but was low in the cumulus and virtually absent in the mural granulosa cells of preovulatory follicles. When preantral granulosa cells were co-cultured with oocytes from early preantral, late preantral or preovulatory follicles, and when oocytes from preovulatory follicles were co-cultured with cumulus granulosa cells, Amh expression was increased at least 2-fold compared with granulosa cells cultured alone. With oocytes from preantral but not preovulatory follicles, this was a short-range effect only observed with granulosa cells in close apposition to oocytes. We conclude that stage-specific oocyte regulation of Amh expression may play a role in intra- and inter-follicular coordination of follicle development.     

Effects of BMP4/SMAD signaling pathway on mouse primordial follicle growth and survival via up‐regulation of Sohlh2 and c‐kit

Bone morphogenetic protein 4 (BMP4) is essential for the development of primordial follicles, although its underlying mechanism remains largely unknown. By using cultured ovaries, the effects of BMP4 and the potential signal transduction pathways were investigated. Ovaries from 3‐day‐old female mouse pups were maintained in organ culture in the absence (control) or presence of BMP4 (100 ng/ml). At different culture time, the effects of BMP4 on primordial follicle growth and survival were assayed by follicle count and TUNEL labeling. The expression of phospho‐SMAD1/5/8, Sohlh2, and c‐kit were measured by immunohistochemistry, RT‐PCR, and Western blotting. Immunohistochemistry was also performed to determine the expression pattern of BMP4, pSMAD1/5/8, Sohlh2, and c‐kit in vivo during ovarian development. The results showed treatments of ovaries with BMP4 resulted in a significant (P < 0.05) increase on the primordial‐to‐primary follicle transition. The oocytes of primordial follicles treated with BMP4 were also less likely to undergo apoptosis. BMP4 enhanced the phosphorylation of SMAD1/5/8 and up‐regulated the expression of Sohlh2 and c‐kit in primordial follicles. During ovarian development in vivo, Sohlh2, and c‐kit exhibited similar expression patterns to BMP4 and pSMAD1/5/8 in primordial follicles. The present studies suggest that BMP4/SMAD signaling pathway initiate primordial follicle growth and prevented oocyte apoptosis via up‐regulation of Sohlh2 and c‐kit. Mol. Reprod. Dev. 80: 70–78, 2013. © 2012 Wiley Periodicals, Inc.     

中国荷斯坦奶牛卵巢卵泡细胞的免疫组织化学研究

目的和方法应用七种特异性抗体(sGCα、sGCβ、nNOS、FOXO1、PDE4、PKB/Akt和Inhibinα)对中国荷斯坦奶牛卵巢卵泡细胞进行免疫组织化学定位。结果表明PKB主要存在于原始卵泡、腔前卵泡和有腔卵泡颗粒细胞,黄体细胞中没有检测到;FoxO1主要位于原始卵泡、腔前卵泡和有腔卵泡颗粒细胞;PDE4仅位于部分有腔卵泡的颗粒细胞;抑制素α、nNOS、sGCα和β存在于各级卵泡的颗粒细胞层,其中sGCα和β主要存在于原始卵泡和腔前卵泡的颗粒细胞。结论这七种蛋白的阶段和细胞特异性表达表明它们与中国荷斯坦卵巢卵泡的发育、闭锁和黄体化过程有着密切的关系。     

Effect of holding medium, temperature and time on structural integrity of equine ovarian follicles during the non-breeding season

The objective was to evaluate the efficiency of phosphate-buffered saline (PBS) and Minimum Essential Medium (MEM) during the transport of equine preantral and antral follicles at various temperatures and incubation interval. Equine ovaries (n = 10) from an abattoir were cut into 19 fragments; one was immediately fixed in Bouin's solution (control) and the other fragments were placed in PBS or MEM solution at 4, 20, or 39 °C for 4, 12, or 24 h. After the respective incubation periods, all fragments were fixed in Bouin's solution for 24 h and then submitted to standard histologic analysis. In total, 2567 ovarian follicles were analyzed, including 1752 primordial, 764 primary, 34 secondary and seven antral follicles. Relative to the control group, the transport of equine ovarian fragments in both solutions significantly reduced the percentage of morphologically normal follicles with increasing time and temperature. At 4 °C for 4 h, considering primordial and developing follicles, PBS had a higher (P < 0.05) rate (98.9%) of morphologically normal follicles than MEM, 48.7%. At 39 °C for 12 h, all follicles in both solutions were degenerated. Regarding the stage of follicular development, primordial follicles were less (P < 0.05) affected by preservation than primary and secondary follicles in all media, times and temperatures tested, except at 4 °C for 12 h in PBS, in which the primary and secondary follicles were less (P < 0.05) affected. Overall, 43% of antral follicles were morphologically normal when maintained in MEM at 4 °C for 4 h. In conclusion, equine follicles were successfully preserved in ovarian fragments at 4 °C in phosphate-buffered saline for up to 4 h.     

Newborn pig ovarian tissue xenografted into Severe Combined Immunodeficient (SCID) mice acquires limited responsiveness to gonadotropins

In the pig ovary, the transition from primordial to primary and secondary ovarian follicles begins before birth, but antral follicles can be observed, for the first time, at ∼60-90 d of age. At approximately the same time, secondary follicles become responsive to gonadotropins, leading to the formation of antral follicles. Placing pieces of ovarian tissue under the kidney capsule of immunodeficient (SCID) mice allows the requirements for follicular recruitment and development to be studied. The objective of this study was to investigate if primordial follicles contained in ovarian fragments isolated from newborn piglets (36 ± 12 h old) and immediately transplanted under the kidney capsule of SCID mice, are able to become responsive to gonadotropins after 60 d (as in an unaltered animal). Ovarian fragments were transplanted under the kidney capsule of three groups of four female and four male SCID mice. The first group did not receive any hormonal treatment for 12 wk. The second group was treated from the 9th week with 1 IU of FSH/LH on alternating days for 3 wk, and the third group was treated with 5 IU Pregnant Mare Serum Ganadotropin (PMSG) 48 h before euthanasia. Primordial follicles contained in ovarian fragments isolated from newborn piglets developed only to the secondary stage. Therefore, development of gonadotropin responsiveness in ovarian fragments xenotransplanted in SCID mice was delayed compared to what occurs in the unaltered animal, and there was minimal response to exogenous gonadotropins.     

TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary

Although it is well established that both follicular assembly and the initiation of follicle growth in the mammalian ovary occur independently of pituitary hormone support, the factors controlling these processes remain poorly understood. We now report that neurotrophins (NTs) signaling via TrkB receptors are required for the growth of newly formed follicles. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF), the preferred TrkB ligands, are expressed in the infantile mouse ovary. Initially, they are present in oocytes, but this site of expression switches to granulosa cells after the newly assembled primordial follicles develop into growing primary follicles. Full-length kinase domain-containing TrkB receptors are expressed at low and seemingly unchanging levels in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes, where it is targeted to the cell membrane as primary follicles initiate growth. Using gene-targeted mice lacking all TrkB isoforms, we show that the ovaries of these mice or those lacking both NT-4 and BDNF suffer a stage-selective deficiency in early follicular development that compromises the ability of follicles to grow beyond the primary stage. Proliferation of granulosa cells-required for this transition-and expression of FSH receptors (FSHR), which reflects the degree of biochemical differentiation of growing follicles, are reduced in trkB-null mice. Ovaries from these animals grafted under the kidney capsule of wild-type mice fail to sustain follicular growth and show a striking loss of follicular organization, preceded by massive oocyte death. These results indicate that TrkB receptors are required for the early growth of ovarian follicles and that they exert this function by primarily supporting oocyte development as well as providing granulosa cells with a proliferative signal that requires oocyte-somatic cell bidirectional communication. The predominance of truncated TrkB receptors in oocytes and their developmental pattern of subcellular expression suggest that a significant number of NT-4/BDNF actions in the developing mammalian ovary are mediated by these receptors.     

Estrogen actions on follicle formation and early follicle development

Estradiol-17beta (E(2)) affects late follicular development, whereas primordial follicle differentiation and early activation are believed to be independent of E(2). To test this hypothesis we compared numbers of primordial and primary follicles in wild-type and E(2)-deficient, aromatase knockout (ArKO) mice, and the immunohistochemical staining or mRNA expression of Mullerian inhibiting substance (MIS), Wilms tumor 1 (WT-1), and growth differentiation factor (GDF9), which are known to effect early follicular differentiation. Proliferating cell nuclear antigen (PCNA) staining was a marker of proliferative index. The effects of E(2) replacement for 3 wk in 7-wk-old ArKO and wild-type mice on these parameters were also tested. ArKO mice had reduced numbers of primordial and primary follicles compared with wild-type mice (63%, P < 0.001 and 60%, P = 0.062, respectively). This reduction was not corrected by E(2) treatment, suggesting that E(2) affects the initial formation or activation of primordial follicles. There was a significant increase in the diameters of the oocytes in primordial follicles of ArKO mice compared with mice of the wild type. There were no differences in the immunostaining of MIS, WT-1, and PCNA in primordial and primary follicles between wild-type and ArKO mice. The only difference was as a consequence of Sertoli and Leydig cells that develop in ovaries of ArKO mice. GDF9 mRNA expression was markedly increased in ArKO ovaries. E(2) treatment restored the ovarian follicular morphology in ArKO mice, and consequently the immunostaining patterns, but had no effect on early follicle numbers. In conclusion, E(2) has a role in controlling the size of the oocyte and primordial follicle pool in mice.     

Expression and localization of P-, K-, and OB-cadherin in the prepubertal rat ovary.

Classical and atypical cadherins mediate calcium-dependent cell adhesion and play an important role in morphogenetic processes. We have shown, previously, N- and E-cadherin expression in the rat ovary. This expression, however, was not associated with specific follicle-restructuring events such as antrum formation and segregation of mural from cumulus granulosa cells suggesting that other cadherins may serve this function. In this study, RT-PCR and immunostaining techniques showed that three other cadherins are expressed throughout prepubertal ovarian development in the rat: one classical (P-) cadherin, and two atypical (K- and OB-) cadherins. RT-PCR analysis of isolated ovarian tissue compartments (granulosa cells and the residual ovarian tissue) agreed with the immunostaining results. Immunostaining showed P- and K-cadherin expression by granulosa, as well as thecal/interstitial cells, and also in oocytes of primordial follicles. P-cadherin expression was absent in oocytes of follicles in later stages of development compared to K-cadherin, which was found in oocytes at all stages of folliculogenesis. P-, K-, and OB-cadherin were expressed by the ovarian surface epithelial cells of neonatal animals but only P- and OB-cadherin expression were maintained in these cells in 25 day-old animals. Cellular OB-cadherin staining was absent in follicles at all stages of development and its expression was restricted to the ovarian hilar region and portions of the stroma. In summary, cadherin expression and distribution profiles changed during ovarian growth and folliculogenesis suggesting a role for cadherins in organizational and morphogenetic processes within the developing rat ovary.     

Cell-specific expression and regulation of soluble guanylyl cyclase alpha 1 and beta 1 subunits in the rat ovary

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and carbon monoxide, resulting in cGMP production. Recent studies indicate that NO and cGMP influence ovarian functions. However, little information is available regarding the ovarian expression of sGC. The present study examined sGC alpha(1) and beta(1) subunit protein levels in the ovary during postnatal development, gonadotropin-induced follicle growth, ovulation, and luteinization as well as in cultured rat granulosa cells. In postnatal rats, sGC alpha(1) subunit immunoreactivity was high in granulosa cells of primordial and primary follicles on Day 5 but low in granulosa cells of larger follicles on Days 10 and 19. Theca cells of developing follicles, but not stromal cells, also demonstrated moderate sGC alpha(1) immunoreactivity. In gonadotropin- treated immature rats, intense sGC alpha(1) subunit staining was similarly observed in granulosa cells of primordial and primary follicles, but such staining was low in granulosa cells of small antral follicles and undetectable in granulosa cells of large antral and preovulatory follicles. Following ovulation, corpora lutea expressed moderate sGC alpha(1) immunoreactivity. Similar ovarian localization and expression patterns were seen for sGC beta(1), indicating regulated coexpression of sGC subunits. Immunoblot analysis revealed no change in total ovarian sGC alpha(1) and beta(1) subunit protein levels during gonadotropin treatment. Similarly, no effect of FSH on sGC subunit protein levels was apparent in cultured granulosa cells. These findings indicate regulated, cell- specific patterns of sGC expression in the ovary and are consistent with roles for cGMP in modulating ovarian functions.