Steady‐state level of kit ligand mRNA in goat ovaries and the role of kit ligand in preantral follicle survival and growth in vitro

The aims of this study were to investigate steady‐state level of Kit Ligand (KL) mRNA and its effects on in vitro survival and growth of caprine preantral follicles. RT‐PCR was used to analyze caprine steady‐state level of KL mRNA in primordial, primary, and secondary follicles, and in small (1–3 mm) and large (3–6 mm) antral follicles. Furthermore, ovarian fragments were cultured for 1 or 7 days in Minimal Essential Medium (MEM⁺) supplemented with KL (0, 1, 10, 50, 100, or 200 ng/ml). Noncultured (control) and cultured fragments were processed for histology and transmission electron microscopy (TEM). RT‐PCR demonstrated an increase in steady‐state level of KL mRNA during the transition from primary to secondary follicles. Small antral follicles had higher steady‐state levels of KL mRNA in granulosa and theca cells than large follicles. After 7 days, only 50 ng/ml of KL had maintained the percentage of normal follicles similar to control. After 1 day, all KL concentrations reduced the percentage of primordial follicles and increased the percentage of growing follicles. KL at 10, 50, 100, or 200 ng/ml increased primary follicles, compared to MEM⁺ after 7 days. An increase in oocyte and follicular diameter was observed at 50 ng/ml of KL. TEM confirmed ultrastructural integrity of follicles after 7 days at 50 ng/ml of KL. In conclusion, the KL mRNAs were detected in all follicular categories. Furthermore, 50 ng/ml of KL maintained the integrity of caprine preantral follicle cultured for 7 days and stimulated primordial follicle activation and follicle growth. Mol. Reprod. Dev. 77: 231–240, 2010. © 2009 Wiley‐Liss, Inc.     

BMPRIB and BMPRII mRNA expression levels in goat ovarian follicles and the in vitro effects of BMP-15 on preantral follicle development

This study evaluated the levels of bone morphogenetic protein receptors BMPRIB and BMPRII mRNA in goat follicles and the effects of bone morphogenetic protein-15 (BMP-15) on the in vitro development of cultured preantral follicles. Real-time polymerase chain reaction (PCR) was used to analyze the levels of BMPRIB and BMPRII mRNA in caprine preantral follicles and in small and large antral follicles. Preantral follicles (≥150 μm) were also isolated from goat ovaries and cultured for 18 days in α-MEM(+) supplemented with or without BMP-15 (10, 50, or 100 ng/ml). At the end of culture, some follicles were fixed for ultrastructural evaluation. Real-time PCR showed a reduction in BMPRII mRNA levels from the primary to secondary follicles. Higher levels of BMPRIB mRNA were observed in granulosa/theca cells from large antral follicles compared with small antral follicles. Moreover, BMPRII mRNA was expressed to a greater extent in cumulus-oocyte complexes from large antral follicles than in their respective granulosa/theca cells. In culture, 50 ng/ml BMP-15 positively influenced antral cavity formation and follicle growth after 18 days and also maintained follicular integrity. Thus, BMPRIB and BMPRII mRNAs are present in all follicular categories. BMP-15 (50 ng/ml) stimulates growth, antrum formation and the ultrastructural integrity of isolated caprine preantral follicles after 18 days of culture.     

In vitro production of a caprine embryo from a preantral follicle cultured in media supplemented with growth hormone

The objective was to evaluate the effects of growth hormone (GH) on the survival, growth, maturation, and fertilization of oocytes derived from caprine preantral ovarian follicles cultured in vitro. Preantral follicles were isolated from the cortex of caprine ovaries and individually cultured for 18 d in the absence (control) or presence of bovine GH at concentrations of 10 or 50 ng/mL (GH10 and GH50, respectively). Follicle development was evaluated on the basis of survival, antral cavity formation, diameter increase, and the presence of healthy cumulus-oocyte complexes and mature oocytes. After culture, oocytes were subjected to in vitro maturation (IVM) and in vitro fertilization (IVF). The rate of antrum formation after Day 6 of culture was higher in both GH10 and GH50 than in the control (81.0, 92.7, and 47.6%, respectively, P < 0.05). Percentages of grown oocytes that were acceptable for IVM were also higher (P < 0.05) in GH-treated groups than in the control (54.8, 48.8, and 11.9% for GH10, GH50, and Control). A higher percentage of oocytes in the GH50 treatment underwent meiotic resumption (50.0%), produced mature oocytes, and enabled production of an embryo after IVF than in the control group (0.0%; P < 0.05). In conclusion, GH promoted in vitro growth and maturation of goat preantral follicle oocytes and enabled production of an embryo. Furthermore, this study was apparently the first to produce a caprine embryo by in vitro fertilization of oocytes derived from preantral follicles grown in vitro.     

IGF-Ⅰ及其受体、IGF结合蛋白-2和LH受体mRNA在卵泡中的表达

利用原位杂交和原位DNA－3’末端标记的方法研究了胰岛素样生长因子河（IG－I）、IGF－I受体、IGF结合蛋白－2、和促性腺激素受体的信使核糖核酸（mRNA）在不同生长与闭锁阶段的大鼠卵巢卵泡中表达的变化。结果表明：IGF－I主要在正常生长的初级卵泡、窦前卵泡和小窦状卵泡中表达。在各生长与成熟阶段的卵泡中都检测到IGF－I受体mRNA,闭锁卵泡的IGF－I受体表达降低。窦前与窦状的生长和闭锁卵泡均表达IGFBP－2。促卵泡激素（FSH）受体在窦前和小窦状卵泡的表达水平比其在大卵泡中的高。窦前与小窦状卵泡仅在膜细胞中表达黄体生成素（LH）受体mRNA,大卵泡的膜细胞与颗粒细胞均表达LH受体,在闭锁卵泡中仅在膜细胞中观察到LH受体的信号。综上结果,提示IGF－I,IGF－I受体和FSH受体在窦前和小窦状卵泡中的协同表达对卵泡的早期发育有重要作用。LH受体mRNA特异地在大卵泡的颗粒细胞中表达可能与优势卵泡选择相关。     

Spatiotemporal expression of epidermal growth factor receptor messenger RNA and protein in the hamster ovary: follicle stage-specific differential modulation by follicle-stimulating hormone,luteinizing hormone,estradiol, and progesterone

Spatiotemporal expression, endocrine regulation, and activation of epidermal growth factor receptor (EGFR) in the hamster ovary were evaluated by immunofluorescence and in situ hybridization localization. Whereas granulosa cells (GC) of primordial through large preantral (stage 6, 7-8 layers GC) follicles had low immunoreactivity, granulosa cells of antral follicles, theca, and interstitial cells had intense EGFR immunoreactivity. EGFR expression in GC of primordial and small preantral follicles increased progressively from estrous through proestrous, but a significant increase occurred in mural GC of antral follicles following the gonadotropin surge. Interstitial cells around small preantral follicles had strong immunofluorescence, and the intensity increased significantly in fully differentiated thecal cells. Distinct EGFR protein was localized in the nucleus of the oocytes and granulosa cells. FSH significantly stimulated EGFR expression in the GC, especially the mural GC, theca, and interstitial cells in hypophysectomized hamster. Estrogen stimulated EGFR expression in preantral GC as well as in interstitial cells. Progesterone and hCG effect was limited to theca and interstitial cells. EGFR expression correlated well with EGFR activation following endogenous or exogenous gonadotropin exposure. Receptor mRNA expression closely followed the protein expression, with increased mRNA expression in mural GC of antral follicles. These results suggest that low levels of EGF signal as a consequence of low levels of receptors in preantral GC may be critical for cell proliferation, but higher receptor density may evoke increased signal intensity due to activation of other intracellular signal pathways, which activate cellular processes related to granulosa, theca, and interstitial cell differentiation. The spatiotemporal cell type and follicle stage-specific expression of receptor mRNA and protein and EGFR activation is critically regulated by gonadotropins and ovarian steroids, primarily estradiol.     

Identification and gene expression analyses of natriuretic peptide system in the ovary of goat (Capra hircus)

Natriuretic peptides (NPs) are involved in maintaining cardiovascular and fluid homeostasis, regulating reproductive processes and bone growth, and other numerous functions. To better understand the role of NPs in goat (Capra hircus), in the present study, full-length cDNAs of goat Nppa (natriuretic peptide precursor A), Nppb (natriuretic peptide precursor B) and Nppc (natriuretic peptide precursor C), respectively encoding ANP, BNP and CNP, were cloned from adult goat heart and ovary. The putative prepropeptide ANP (prepro-ANP) and prepro-CNP share a high amino acid sequence identity with other species. Real-time PCR showed that Nppa, Nppb and Nppc were widely expressed in adult goat tissues. The mRNA expression of Nppa and Nppb in the heart was extremely higher compared with other tissues. Nppc mRNA expression in the lung and uterus was also higher than in other tissues. The expression of Nppa, Nppb and Nppc genes was examined at different ovarian follicle stages using RT-PCR. The mRNAs of Nppa and Nppb were detected in secondary follicles as well as in COCs (cumulus–oocyte-complexes) and granulosa cells of antral follicles. However, the mRNA expression of Nppc was observed throughout ovarian follicle development, and it was especially higher in granulosa cells of antral follicles. In vitro, stimulating goat granulosa cells with FSH led to an increase in the expression of Nppc by dose- and time-dependent manners and a rapid decline was induced by LH stimulation, but the expression of Nppa and Nppb did not change after FSH or LH treatment. These results suggest that Nppc is a gonadotropin-induced gene in granulosa cells of goat ovary and CNP may be involved in the regulation of ovarian follicle development and oocyte maturation.     

New approach to in situ quantification of ovarian gene expression in rat using a laser microdissection technique: relationship between follicle types and regulation of inhibin-α and cytochrome P450aromatase genes in the rat ovary

The recently developed laser microdissection (LMD) technique makes it possible to quantify local gene expression in the target cells of various tissues. Using the LMD technique, this study aimed at comparing the amounts of mRNAs encoding the inhibin-α subunit and cytochrome P450 aromatase (P450_arom) in granulosa cells between preantral and antral follicles in immature rat ovaries. Serial frozen sections of the ovaries from 24-day-old female Wistar rats were made and 30 healthy preantral (100–200 μm maximum diameter) and ten healthy antral ( > 300 μm maximum diameter) follicles were selected in each ovary based on morphological examinations, including immunohistochemistry for inhibin-α, in sections adjacent to those used for LMD. The amounts of mRNAs encoding inhibin-α subunit and P450_arom were quantified by real-time polymerase chain reaction (PCR). While the amount of P450_arom mRNA in the granulosa cell layers from the antral follicles was about 12-times higher than that in the preantral follicles, no difference in the amount of inhibin-α mRNA was found between these two types of follicles. Thus, the LMD technique allowed the in situ quantification of gene expression in the ovary and revealed that each granulosa cell expresses a stable amount of inhibin-α subunit mRNA independently of antral formation in immature rat ovaries.     

Expression of retinol-binding protein and cellular retinol-binding protein in the bovine ovary

Retinol (vitamin A) is essential for reproduction, and retinoids have been suggested to play a role in ovarian steroidogenesis, oocyte maturation, and early embryonic development. Retinol is transported systemically and intercellularly by retinol-binding protein (RBP). Within the cell, cellular retinol-binding protein (CRBP) functions in retinol accumulation and metabolism. Since the actions of retinoids are mediated, in part, by retinoid-binding proteins, the objective of this study was to investigate cell-specific expression of RBP and CRBP in the bovine ovary. Immunocytochemical analysis (ICC) localized RBP to the thecal and granulosa cell layers of antral and preantral follicles with the most intense staining in the cells of large, healthy follicles. The tunica adventitia of arterial blood vessels also exhibited RBP staining. Immunostaining of CRBP was most intense in the granulosa cells of preantral follicles and present, but diminished, in thecal and granulosa cells of antral follicles. Within the corpus luteum, both proteins were observed in large luteal cells, but only RBP was observed in small luteal cells. Northern blot analysis demonstrated that thecal and granulosa cells from antral follicles and luteal tissue expressed RBP and CRBP mRNA. Synthesis and secretion of RBP by thecal cells, granulosa cells, and luteal cells were demonstrated by immune-complex precipitation of radiolabeled RBP from the medium of cultured cells or explants, followed by SDS-PAGE and fluorography. Follicular fluid was collected from small (<5 mm) and large (8-14 mm) follicles, pooled according to follicular size, and analyzed for retinol, RBP, estradiol-17beta, and progesterone. Concentrations of retinol, RBP, and estradiol were greater in the fluid of large follicles. Results demonstrate retinoid-binding protein expression by bovine ovaries and provide physical evidence that supports the concept that retinoids play a role in ovarian function.     

Cryopreservation of ovarian tissues temporarily suppresses the proliferation of granulosa cells in mouse preantral follicles

Cryopreservation of ovarian tissue has been reported to delay the development of preantral follicles during in vitro culture, but the mechanism causing this impairment has not been brought to light. In order to elucidate the underlying mechanism of delayed follicular development, we evaluated the effects of cryopreservation on the proliferation of granulosa cells during culture of mouse preantral follicles, as a sufficient population of granulosa cells is critical for normal follicular development. Additionally the initial cell death of granulosa cells was estimated immediately after cryopreservation. The ovarian tissues obtained from 12-day-old female mice were cryopreservation by vitrification. The granulosa cell proliferation was evaluated by measuring the PCNA expression and the expression of cell cycle regulators such as cyclin D2, CDK4, cyclin E and CDK2 in preantral follicles isolated from fresh and cryopreserved ovarian tissues that were cultured for 48 h. The viability of granulosa cells was evaluated by measuring the proportion of necrotic areas. The granulosa cell proliferation of the cryopreserved preantral follicles was decreased significantly compared to that of the fresh controls at 0 and 24 h after culture (P < 0.05), and this was increased to the control levels after 48 h of culture. The expressions of cyclin D2, Cdk 4, cyclin E and Cdk2 were also decreased in the cryopreserved ovarian tissues at 0 and 24 h after culture (P < 0.05), but they were increased to the control levels after 48 h of culture. The proportion of the necrotic area was significantly higher in cryopreserved preantral follicles compared to that of the fresh preantral follicles (P < 0.05). This suggests that cryopreservation of ovarian tissues may delay the preantral follicle development by temporary suppressing the granulosa cell proliferation through the cell cycle regulators (cyclin D2, Cdk4, cyclin E and Cdk2) and by granulosa cell death immediately after warming.     

Effects of methoxychlor on IGF-I signaling pathway in rat ovary

Follicular development and other ovarian functions are regulated by growth factors that can be affected by exogenous agents. Methoxychlor (MXC) is an organochloride pesticide that causes female infertility. We investigated how MXC affects the distribution of developing ovarian follicles in adult rats after treatment between embryonic day (E) 18 and postnatal day (PND) 7. We also measured insulin-like growth factor-I (IGF-I) and its receptor, IGF-IR, expressions in ovarian follicles and investigated whether MXC changed the levels of IGF-I and IGF-IR in the ovary. Using immunohistochemical (IHC) staining, we detected IGF-I expression in oocytes and granulosa cells of the follicles, luteal cells, interstitial cells, theca externa and theca interna, and the smooth muscle of ovarian vessels. IGF-IR was co-localized with IGF-I in the ovary except for the theca externa. IGF-I expression was decreased in granulosa cells of preantral and antral follicles after treatment with MXC compared to granulosa cells of preantral and antral follicles of the control group. We also observed that oocytes of secondary follicles and granulosa cells of secondary and preantral follicles of the MXC treated groups showed increased IGF-IR expression compared to oocytes of secondary follicles and granulosa cells of secondary and preantral follicles of the control group. We also detected more secondary and preantral follicles, and fewer primordial and antral follicles after MXC administration compared to controls. Therefore, the IGF signaling pathway may participate in MXC induced ovary dysfunction and female infertility.     

Vitrification of goat preantral follicles enclosed in ovarian tissue by using conventional and solid-surface vitrification methods

Caprine preantral follicles within ovarian fragments were exposed to or vitrified in the presence of sucrose, dimethyl sulfoxide (DMSO), ethylene glycol (EG), or various combinations thereof. The fragments were cryopreserved by using either a conventional (CV) or a solid-surface vitrification (SSV) protocol, and the cryoprotectants were removed by equilibrating vitrified ovarian fragments in “warming solution” consisting of minimum essential medium and heat-inactivated fetal calf serum (MEM⁺) followed by washes in MEM⁺ with or without sucrose. Histological analysis of follicle integrity showed that the percentages of normal follicles in ovarian fragments vitrified in sucrose mixed with EG and/or DMSO (CV method) or mixed with EG or DMSO (SSV method) followed by washes in MEM⁺ plus sucrose were similar to those of controls (ovarian fragments fixed without previous vitrification). Unlike for MEM⁺ (supplemented or unsupplemented by sucrose) and DMSO followed by washes in the absence of sucrose, the percentages of normal follicles found after exposure to cryoprotectant did not significantly differ from that found after vitrification, indicating that follicular degeneration was attributable to a toxic effect of cryoprotectants and not to the vitrification procedure. The viability of preantral follicles after the CV and SSV procedures was investigated by using calcein-AM and the ethidium-homodimer as “live” and “dead” markers, respectively. In both tested vitrification procedures, the highest percentages of viable follicles were observed when a mixture of sucrose and EG (70.3% for CV and 72.4% for SSV) was used. Preantral follicles were also vitrified (either by CV or SSV) in sucrose and EG and then cultured for 24 h, after which their viability was compared with that of cultured fresh and uncultured vitrified follicles. The viability of these follicles was maintained after SSV, but not after CV. Thus, the viability of caprine preantral follicles can be best preserved after SSV in a mixture of sucrose and EG, followed by washes in medium containing sucrose.CAPES/Brazil supported this work. Regiane Rodrigues dos Santos is a recipient of a grant from CAPES/Brazil.     

Expression and regulation of mRNAs for insulin-like growth factor (IGF-I), IGF-binding protein-2, and LH receptor in the process of follicular atresia

Expression of mRNAs for IGF-I, IGF-binding protein-2 (IGFBP-2), and LH receptor (LHR) as well as their regulations during induced follicular atresia was determined. 26-day-old female rats received 15 IU pregnant mare serum gonadotropins (PMSG). Through detection, it was demonstrated that apoptosis occurred in some small antral follicles after 48 h of PMSG treatment. At 96 h, apoptosis occurred in preovulatory follicles. At 120 h, numerous apoptotic cells appeared in preovulatory follicles. IGF-I was mainly expressed in preantral and small antral follicles from 48 to 120 h. At 48 and 96 h, the theca cells of preantral and antral follicles expressed high level of IGFBP-2 mRNA. At 48 h, there were strong signals of LHR mRNA in granulosa cells, but the LHR signals in granulosa cells significantly decreased at 96 and 120 h (p<0.001). Both epidermal growth factor (EGF) and IGF-I inhibited apoptosis in preantral and antral follicles. Meanwhile, it was observed that EGF promoted IGF-I mRNA expression, and in preovulatory follicles, IGF-I stimulated LHR mRNA expression. These results show that the interaction between ECF and IGF-I may be involved in the regulation of atresia of follicles at different stages of development.     

Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.     

Estrogen receptor protein and mRNA expression in the ovary of sheep

Using immunohistochemistry and in situ hybridization, we attempted to identify the estrogen receptor (ER) protein and messenger RNA (mRNA) in sheep ovaries during the follicular phase of the estrous cycle. Monoclonal anti-ER antibodies H222 and 1D5 were used for localizing estrogen receptor on ovarian cryo-sections. Labeling for ER was found over the nuclei of surface epithelium, interstitial tissue, and granulosa cells of small as well as large ovarian follicles. In the preantral and small antral follicles, intense nuclear ER labeling was observed in mural granulosa cells and particularly in cumulus/granulosa cells surrounding the oocyte. In the large healthy looking follicles, greater diversity in labeling for ER was observed, which is characterized by mixed populations of granulosa cells expressing positive and more or less negative nuclear labeling. Such a pattern of labeling was particularly evident in follicles showing the signs of atresia. Generally, more intense nuclear staining was localized in granulosa cells proximal to basal membrane. In situ hybridization studies revealed the presence of ER mRNA in ovarian tissue. Autoradiographic visualization localized ER mRNA expression over the granulosa cells of healthy follicles of all sizes. Level of hybridization signal was comparable in mural and cumulus granulosa cells. In atretic follicles, the level of hybridization signal in granulosa cells was comparable to that of healthy follicles. A relatively weaker level of labeling was observed in granulosa cells dispersed in follicular antrum in follicles with advanced atretic lesions. Theca cells expressed a lower level of labeling than granulosa cells. Specificity of labeling for both ER protein and mRNA in ovary was proved by parallel probing the ovine uterus. Ovine ER recognition by both H222 and 1D5 antibodies was also proved by immunoblotting. These studies demonstrate the presence of the estrogen receptor and its messenger RNA in the sheep ovary and suggest an autocrine/paracrine role of estradiol and its receptor in the regulation of ovarian follicle development in sheep. Mol. Reprod. Dev. 48:53–62, 1997. © 1997 Wiley-Liss, Inc.     

Expression and regulation of mRNAs for insulin-like growth factor (IGF-I), IGF-binding protein-2, and LH receptor in the process of follicular atresia

Expression of mRNAs for IGF-I, IGF-binding protein-2 (IGFBP-2), and LH receptor (LHR) as well as their regulations during induced follicular atresia was determined. 26-day-old female rats received 15 IU pregnant mare serum gonadotropins (PMSG). Through detection, it was demonstrated that apoptosis occurred in some small antral follicles after 48 h of PMSG treatment. At 96 h, apoptosis occurred in preovulatory follicles. At 120 h, numerous apoptotic cells appeared in preovulatory follicles. IGF-I was mainly expressed in preantral and small antral follicles from 48 to 120 h. At 48 and 96 h, the theca cells of preantral and antral follicles expressed high level of IGFBP-2 mRNA. At 48 h, there were strong signals of LHR mRNA in granulosa cells, but the LHR signals in granulosa cells significantly decreased at 96 and 120 h (p<0.001). Both epidermal growth factor (EGF) and IGF-I inhibited apoptosis in preantral and antral follicles. Meanwhile, it was observed that EGF promoted IGF-I mRNA expression, and in pr