胰岛素样生长因子-Ⅰ(IGF-Ⅰ)、IGF结合蛋白-2和LH受体mRNA在卵泡闭锁过程中的表达及调节

研究了IGF-Ⅰ、IGF结合蛋白-2(IGFBP-2)和促黄体激素受体(LHR)mRNA在卵泡闭锁过程中的表达及调节.给26日龄大鼠注射15 IU PMSG,经检测,证实PMSG处理48 h后,一些小窦状卵泡的颗粒细胞已发生凋亡;96 h在排卵前卵泡中已可检测到凋亡细胞;120 h大多数的排卵前卵泡中均出现大量的凋亡细胞.48～120 h IGF-Ⅰ主要在窦前卵泡和小窦状卵泡表达;48与96 h,窦前与窦状卵泡的膜细胞均表达高水平的IGFBP-2.在48 h,颗粒细胞中有LHR的强信号,但在96和120 h,颗粒细胞的LHR表达减弱(P＜0.001).表皮生长因子(EGF)和IGF-Ⅰ均抑制窦前和窦状卵泡颗粒细胞凋亡.同时观察到EGF促进IGF-Ⅰ mRNA表达,IGF-Ⅰ刺激排卵前卵泡表达LHR mRNA.上述结果表明,各级卵泡的闭锁可能均受EGF和IGF-Ⅰ相互作用的调节.     

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－Ｉ（ＩＧＦ－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     

卵泡闭锁与其tPA，LH受体及抑制素 α，βA亚基基因表达的关系

用DNA 3′-末端标记、免疫组化和原位杂交方法,通过连续切片比较研究了相同卵泡颗粒细胞抑制素亚基和LH受体（LHR）与卵细胞tPA表达和卵泡闭锁的关系.实验结果表明（1） 卵泡闭锁伴随卵细胞tPA活性明显增加;（2） 颗粒细胞抑制素的产生调节卵细胞tPA活性的表达并与卵泡发育状态密切相关;（3） 卵泡闭锁时,颗粒细胞几乎不表达LHR和抑制素亚基.上述结果提示卵细胞的tPA在闭锁卵泡中可能参与卵细胞的自我瓦解和清除过程;颗粒细胞表达的抑制素可能是tPA mRNA翻译的一种抑制因子,如其表达受阻,可导致卵细胞tPA蛋白活性增加引起卵泡闭锁.     

活化素和卵泡抑素对绍鸭卵泡颗粒细胞FSH受体mRNA表达作用的研究

分别用活化素（Activin）、卵泡抑素（FSP）及其组合（Activin FSP）来处理培养的鸭未成熟卵泡颗粒细胞,发现在FSH存在与不存在的情况下,Activin均能促进FSH受体mRNA的表达,且随着Activin浓度的增大,其刺激作用增强。FSP自身对FSH受体产生无显著作用,但能中和Activin对该受体产生的促进作用。这说明FSP和Activin对颗粒细胞具有自分泌作用,二者通过调节FSH受体mRNA的表达而在卵泡的生长发育过程中起着重要作用。     

GH受体、IGF-I型受体、FSH受体和LH受体mRNA在绍鸭各级卵泡颗粒层和膜层中的表达

采用相对定量反转录多聚酶链式反应(RT-PCR)的方法,以β-actin为内标,测定绍鸭排卵前卵泡F1、F3、F5及大白卵泡(LWF)颗粒层与膜层中GH受体(GHR)、IGF-I型受体(IGF-IR)和FSH受体(FSHR)、LH受体(LHR)mRNA表达水平,以分析生长轴和生殖轴激素对绍鸭卵泡发育的协同调节作用。结果表明：在所测定的各级卵泡中,GHR mRNA在膜层的表达水平均显著高于颗粒层,膜层中大白卵泡表达量最高,而颗粒层中GHR mRNA水平在各级卵泡之间没有明显差异;相反,IGF-IR mRNA在同级卵泡颗粒层的表达水平显著高于膜层,颗粒层和膜层中IGF-IR mRNA表达在各级卵泡之间的差异均不显著,只是大白卵泡的膜层与其他等级卵泡的膜层相比,IGF-IR的表达量呈现较高的趋势;FSHR mRNA表达的变化趋势类似于IGE-IR,同级卵泡颗粒层中的表达量高于膜层;LMR mRNA在各级卵泡膜层中表达没有明显差异,而颗粒层中IMR mRNA随着卵泡发育成熟逐级显著增加,且F5和LWF卵泡膜层的IMR mRNA显著高于颗粒层,与GHR mRNA的表达相似。结果提示,GH和IGF-I调节卵巢功能的优势作用位点和机制不尽相同;GHR和LHR协同表达与IGF-IR和FSHR协同表达可能对卵泡发育起到精细的调节作用;而IMR在卵泡颗粒层中表达的逐级显著增加可能与卵泡等级的建立和排卵有关。     

甾体激素合成快速调节蛋白mRNA在卵泡发育和闭锁过程的表达

以孕马血清促性腺激素（ＰＭＳＧ）激发的ＳＤ大鼠卵巢为模型,用３′－末端原位标记和形态健康卵泡和闭锁卵泡,用原侠杂交方法研究了在卵泡发育和闭锁过程中甾体激素合成快速调节蛋白（ＳｔＡＲ）ｍＲＮＡ的表达规律。发现ＳｔＡＲｍＲＮＡ在膜－间质细胞和黄体化颗粒细胞中表达,未黄体化颗粒细胞和卵母细胞均无表达。ＰＭＳＧ注射后１２小时卵巢内已有ＳｔＡＲｍＲＮＡ的表达,２４小时表达量升高,７２小时表达量达高峰。ＰＭＳ     

奶牛卵泡超数同步发育及其卵母细胞的发育潜能

目的建立促奶牛卵泡超数同步发育的方法,并利用体细胞核移植和分子技术评价来自这些卵泡的卵母细胞发育潜能。方法用E2-P4对淘汰的高产奶牛进行联合处理后,分别对其实施不同组合的外源促性腺激素处理[FSH12h组,FSH48h组,FSH48h-LH6h组（简称LH6h组）,FSH48h-LH26h组（简称LH26h组）]以促进其卵泡超数同步发育;然后从卵泡中回收COCs进行体外成熟,排放第一极体的卵供作优质种牛的体细胞核转移（nucleartransfer,NT）受体,以评价卵的发育潜能。结果E2-P4能成功地诱导奶牛卵巢中产生一个新的卵泡起始波,并能保证卵泡在外源促性腺激素作用下实现超数同步发育;上述四组不同组合外源促性腺激素处理的卵母细胞衍生的NT重构胚经体内培养7d后,后3组的囊胚发育率显著高于FSH12h组和对照组（P〈0.01）;将这些克隆囊胚移植同步发情的受体牛子宫后,仅LH26h组的卵所获得的克隆胚能实现全程发育（直至克隆小牛出生）。经RT-PCR分析颗粒细胞中FSHr、LHr和连接蛋白43（Connexin43,Cx43）等mRNA含量变化,以及Western印迹分析其Bcl-2和Bax蛋白表达水平的变化,证实该组卵巢提供的同步发育卵泡为早期闭锁卵泡。结论E2-P4-FSH48h-LH26h组合处理可人为调控牛卵泡的同步发育;处于早期闭锁状态卵泡的卵母细胞具备较高的发育潜能。     

卵泡闭锁与其tPA,LH受体及抑制素α,βA亚基基因表达的关系

用DNA 3′-末端标记、免疫组化和原位杂交方法,通过连续切片比较研究了相同卵泡颗粒细胞抑制素亚基和LH受体(LHR)与卵细胞tPA表达和卵泡闭锁的关系.实验结果表明:(1) 卵泡闭锁伴随卵细胞tPA活性明显增加;(2) 颗粒细胞抑制素的产生调节卵细胞tPA活性的表达并与卵泡发育状态密切相关;(3) 卵泡闭锁时,颗粒细胞几乎不表达LHR和抑制素亚基.上述结果提示:卵细胞的tPA在闭锁卵泡中可能参与卵细胞的自我瓦解和清除过程;颗粒细胞表达的抑制素可能是tPA mRNA翻译的一种抑制因子,如其表达受阻,可导致卵细胞tPA蛋白活性增加引起卵泡闭锁.     

血管内皮生长因子在妊娠及产后大鼠卵巢中的表达

研究了血管内皮生长因子(VEGF)在妊娠大鼠发育卵泡和妊娠不同阶段及产后黄体中的表达, 同时观察了肿瘤坏死因子-α(TNF-α)对妊娠早期黄体中VEGF表达的影响. VEGF在窦前卵泡的颗粒细胞层表达, 在小窦状卵泡的颗粒细胞和膜细胞中也有较强表达, 但在大窦状卵泡中检测不到. 妊娠各个时期及产后黄体中均有VEGF mRNA的转录, 在妊娠第7天达到最高, 第9~18天仍维持在一定水平, 第18天后明显降低, 产后降至最低水平. 用Western blot在大鼠黄体中检测到分子量约为23 ku的VEGF蛋白, VEGF蛋白与mRNA水平的变化趋势基本一致. 在大鼠妊娠第4天注射TNF-α (3000 IU/ kg)可提高黄体中VEGF的表达量. 以上结果提示, VEGF在大窦状卵泡中表达的下调可能是导致妊娠卵泡不排卵的一个因素. VEGF可能参与大鼠妊娠黄体的形成和功能维持, 其作用受细胞因子的调节.     

Annexin I在PCOS大鼠颗粒细胞中的表达及意义

目的：探讨膜联蛋白I（AnnexinI）在多囊卵巢综合征（PCOS）大鼠卵泡颗粒细胞的表达及生物学意义。方法：采用免疫组织化学方法及灰度值测定AnnexinI在PCOS组和对照组的卵泡颗粒细胞中的表达。结果：AnnexinI在两组中的各级卵泡颗粒细胞中均有表达,PCOS组AnnexinI在窦状卵泡中表达显著高于对照组（P〈0．05）。结论：PCOS组AnnexinI在窦状卵泡颗粒细胞的表达上调,且PCOS中窦状卵泡颗粒细胞的凋亡增加,说明AnnexinI参与了卵巢颗粒细胞的凋亡过程,并且发挥了重要作用。     

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.     

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     

Ontogeny and cellular localization of 125I-labeled insulin-like growth factor-I, 125I-labeled follicle-stimulating hormone, and 125I-labeled human chorionic gonadotropin binding sites in ovaries from bovine fetuses and neonatal calves.

In a previous study we reported that ovaries from bovine fetuses, which consist mainly of preantral follicles with few antral follicles, are weakly responsive to gonadotropins (FSH and LH). Insulin-like growth factor-I (IGF-I) is known to enhance gonadotropin responsiveness in vitro, but there is a lack of consistent data on the involvement of IGF-I, FSH, and LH during early stages of folliculogenesis in cattle. In the study reported here, we assessed autoradiographically the ontogeny of 125I-gonadotropin and 125I-IGF-I binding activities during preantral and early antral stages in cattle. Follicular growth was initiated around Day 180 of gestation in fetuses. The density of 125I-FSH binding was high in granulosa cells from primary (mean +/- SEM 10.5 +/- 0.7 grains/cell, 0.05-mm diam.) and secondary follicles (10.8 +/- 0.8 to 13.6 +/- 1.2 grains/cell, 0.06-0.15 mm) but increased significantly (p < 0.05) in early antral follicles (18.2 +/- 1.1 grains/cell, 0.16-3.0 mm). Specific 125I-IGF-I binding levels were low in granulosa cells from preantral follicles, averaging 2.5 +/- 0.6-3.1 +/- 0.9 grains/cell. However, after antrum formation, the density of 125I-IGF-I binding increased significantly (p < 0.05) with follicular diameter in granulosa cells and was 5.7 +/- 0.7 and 9.1 +/- 0.6 grains/cell for antral I (0.16-0.5 mm) and antral II (0.6-3.0 mm) follicles, respectively. 125I-FSH and 125I-IGF-I binding densities were low in theca cells from preantral and early antral follicles as well as in the interstitial tissue and granulosa cells from atretic follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Apelin and APJ receptor expression in granulosa and theca cells during different stages of follicular development in the bovine ovary: Involvement of apoptosis and hormonal regulation

In the mammalian ovary, the microvasculature in the thecal layer of follicles is associated with follicular development. Apelin and its receptor, APJ, are expressed in the tissues and organs which include the vasculature. The aims of the present study were to examine the mRNA expression of apelin and the APJ receptor in granulosa cells and theca tissue of bovine follicles and the effects of steroid hormone and gonadotrophins on the expression of these genes in cultured granulosa cells and theca cells. The expression of apelin mRNA was not found in the granulosa cells of bovine follicles. The expression of the APJ gene was increased in granulosa cells of estrogen-inactive dominant follicles. The expression of apelin mRNA increased in theca tissues of estrogen-inactive dominant follicles. APJ expression in theca tissues increased with follicle growth. Progesterone stimulated the expression of APJ mRNA in the cultured granulosa cells. FSH stimulated the expression of APJ mRNA in the cultured granulosa cells. LH induced the expression of apelin and APJ receptor mRNAs in cultured theca cells. Taken together, our data indicate that the APJ receptor in granulosa cells and both apelin and the APJ receptor in theca tissues are expressed in bovine ovary, that APJ in granulosa cells may be involved in the appearance of the cell apoptosis, and that LH stimulates the expression of apelin and APJ genes in theca cells.     

Ovarian expression of messenger RNA encoding the receptors for luteinizing hormone and follicle-stimulating hormone in a marsupial, the brushtail possum (Trichosurus vulpecula)

Both LH and FSH play a central role in controlling ovarian function in mammals. However, little is known about the type of ovarian cells that are responsive to LH and FSH in marsupials. We determined, using in situ hybridization, the localization of mRNA encoding the receptors (R) for LH and FSH in ovaries of brushtail possums. The mRNA encoding FSH-R was observed in granulosa cells of healthy follicles containing at least two complete layers of cells. The mRNA encoding LH-R was first observed in granulosa cells at the time of antrum formation. Cells of the theca interna expressed LH-R mRNA but not FSH-R mRNA. Neither FSH-R nor LH-R mRNA was detected in atretic follicles. Both FSH-R and LH-R mRNAs were observed in luteal tissue, but only LH-R mRNA was observed in interstitial cells. Granulosa cells from follicles of various sizes (0.5 to >2 mm in diameter) responded to LH and FSH treatment with an increase in cAMP synthesis. In contrast, luteal tissue did not respond to either FSH or LH treatment. In conclusion, expression of FSH-R in the brushtail possum ovary was similar to that observed in many eutherian mammals. However, active LH-R was expressed in granulosa cells much earlier in follicular development than has been previously observed. In addition, although mRNAs for both FSH-R and LH-R were observed, neither FSH nor LH treatment stimulated cAMP synthesis in luteal tissue.     

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.     

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.