Expression of steroidogenic acute regulatory protein and its regulation by interferon-gamma in rat corpus luteum

The steroidogenic acute regulatory protein (StAR) is the key regulatory protein of steroidogenesis.De novo synthesis of StAR protein is required for intramitochondrial translocation of cholesterol to the cytochrome P450 side chain cleavage enzyme which is located on the matrix side of the inner mitochondrial membrane. This is the rate-limiting step of steroid biosynthesis. Usingin situ hybridization and immunohistochemistry we studied StAR expression in various stages of the corpora luteal and its regulation by interferon-gamma (IFNγ) in the adult pseudopregnant rat. The results indicated that expression of StAR in the corpora luteal was correlated with progesteron production and IFNγ was capable of inhibiting its expression.     

Localization of orphan receptor TR3 mRNA in early developmental follicles in rat

Byin situ hybridization, the localization of orphan receptor TR3 mRNA has been observed in early developmental follicles. TR3 mRNA is first expressed in the ovarian interstitial cells on day 2 after birth, and then in granulosa cells (GC) in primary follicles on day 4. The expression level of TR3 mRNA in GC increases following the follicular development. Its higher expression can be observed in the outer layer of GC and inner layer of theca cells (TC) on day 6, where the cells present active proliferation and differentiation. The expression of TR3 is in an increasing manner until the large antral follicles on day 30. The mRNA is only expressed in the healthy, but not atretic follicles in adult rat ovaries. Injection of epidermal growth factor (EGF) has dramatically enhanced its expression in the early stage of developmental follicles. It is therefore suggested that TR3 may play a role in regulating growth and differentiation of ovarian somatic cells in the early stage, and its expression is regulated by EGF.     

不同发育时期小鼠卵巢腔前卵泡的分布及形态学研究

通过石蜡切片-HE染色技术,系统研究小鼠生后不同发育时期即3,5,7,10,15,20,30,45 d卵巢的卵泡分布、形态结构及发育规律.研究结果表明,随着小鼠卵巢的发育,由最初的无皮髓质之分到皮髓明显可见,卵泡从只见聚集呈条索状到发育成原始卵泡、初级卵泡、次级卵泡,直至有腔的形成.从数量上看,原始卵泡逐渐减少,初次级卵泡逐渐增多,有腔卵泡数量也从无到有从少到多.卵巢由最初的无卵泡发育到有许多大小不等、处于不同发育时期的卵泡、大部分有腔样结构.在此发育过程中会伴有卵泡的闭锁.小鼠卵巢中各级腔前卵泡的分布具有区域性.     

Abnormal sexual development in transgenic mice chronically expressing müllerian inhibiting substance

Müllerian inhibiting substance (MIS), also known as anti-Müllerian hormone, is a glycoprotein normally secreted by the Sertoli cells of the fetal and adult testis and by granulosa cells of the postnatal ovary. The production of MIS in the male fetus brings about the regression of the Müllerian ducts, the anlagen of the uterus, oviducts, and upper vagina. In addition, purified MIS induces the formation of seminiferous cord-like structures in fetal rat ovaries cultured in vitro, suggesting that MIS may influence testicular differentiation. We have produced transgenic mice chronically expressing human MIS under the control of the mouse metallothionein-1 promoter to investigate its role during sexual development. In females, chronic expression led to the inhibition of Müllerian duct differentiation, resulting in a blind vagina and no uterus or oviducts. At birth the ovaries had fewer germ cells than normal; during the next two weeks germ cells were lost and the somatic cells became organized into structures resembling seminiferous tubules. Apparently, these structures degenerate as they are undetectable in adult females. The majority of transgenic males developed normally. But in two lines with the highest levels of MIS expression, some males showed feminization of the external genitalia, impairment of Wolffian duct development, and undescended testes. These results suggest that MIS has several distinct roles in mammalian sexual development.     

Developmental study on the immunocytochemical localization of NOV protein-containing neurons in the rat brain

The localization and development of nephroblastoma overexpressed gene (nov) protein-immunoreactive neurons in the brain of E8-P300 rats have been studied using immunocytochemistry and image analysis. Results are as follows: No NOV protein-immunoreactive cells were detected in the rat brain during prenatal development. A few of positive cells were detected at the early postnatal stage. However, the number and the immunoreactivity of these cells increased gradually at later stages. NOV-immunoreactive cells were widely distributed in the rat brain during P30–P60. The number of immunoreactive cells and their intensity also peaked within this stage. The number and staining intensity of NOV-positive cells decreased gradually with age. The positive cells were mainly located in cingulate cortex, striatum, hippocampus, hypothalamus, cerebellum and brain stem. The present results indicate thatnov may play an important role in the development and differentiation of brain as well as maintaining the function of brain.     

Follicles were reconstituted from dissociated mouse fetal ovarian cellsin vitro

Early folliculogenesis involved in the interaction of germ cells and somatic cells is a complicated physiological event. Female germ cells are committed to differentiate into oocytes and finish complete development in the functional units of follicles. Thus there will be great significance in basal research and practices to evaluate the possibility of ovarian cells to reconstitute into follicles in vitro. In the present research, 12-16 dpc (days post coitum) mouse fetal ovarian cells were respectively isolated using collagenase digestion and cultured in droplets in vitro. The results revealed that the fetal ovarian cells of 12-16 dpc appeared to form multiple cell aggregates and tissue-like pieces in vitro. However, 12-13 dpc ovarian cells failed to form the follicles. 14-15 dpc ovarian cells were competent to form a few follicle-like complexes. Furthermore many small typical follicles were reconstituted from 16 dpc ovarian cells in vitro. The results showed for the first time that mouse embryonic ovarian cells were able to form the follicles in vitro. It was a gradual progression for the female germ cells to achieve the ability to induce somatic cells differentiation and reconstitu-tion into follicles, which may directly lead to the success in reorganization and transplantation of genetically modified ovary in vitro.     

Germline stem cells and follicular renewal in the postnatal mammalian ovary

A basic doctrine of reproductive biology is that most mammalian females lose the capacity for germ-cell renewal during fetal life, such that a fixed reserve of germ cells (oocytes) enclosed within follicles is endowed at birth. Here we show that juvenile and adult mouse ovaries possess mitotically active germ cells that, based on rates of oocyte degeneration (atresia) and clearance, are needed to continuously replenish the follicle pool. Consistent with this, treatment of prepubertal female mice with the mitotic germ-cell toxicant busulphan eliminates the primordial follicle reserve by early adulthood without inducing atresia. Furthermore, we demonstrate cells expressing the meiotic entry marker synaptonemal complex protein 3 in juvenile and adult mouse ovaries. Wild-type ovaries grafted into transgenic female mice with ubiquitous expression of green fluorescent protein (GFP) become infiltrated with GFP-positive germ cells that form follicles. Collectively, these data establish the existence of proliferative germ cells that sustain oocyte and follicle production in the postnatal mammalian ovary.     

Gonadotropin-releasing hormone analogue binds to luteal cells and inhibits progesterone production.

Although gonadotropin-releasing hormone (GnRH) is believed to mediate the hypothalamic control of pituitary gonadotropin secretion, continuous or repeated administration of GnRH or its agonist analogues has been shown to cause paradoxical antifertility effects in several species, including primates. GnRH-induced interruption of reproductive cycles and pregnancy is associated with diminished progesterone production, implying defective function of the corpus luteum. These luteolytic effects have been attributed to the well recognized desensitising actions of elevated luteinising hormone (LH) levels on ovarian LH receptors and steroidogenesis, subsequent to GnRH-induced gonadotropin release from the anterior pituitary. However, treatment with high doses of exogenous LH did not cause suppression of serum progesterone levels during early pregnancy in rats, whereas a highly active GnRH analogue was effective in this regard. These observations suggested that GnRH and its agonist analogues, given in high or sustained doses, can exert a direct action on the ovary that is independent of the pituitary. This hypothesis was further supported by the ability of GnRH and its agonists to inhibit human chorionic gonadotropin (HCG)-induced ovarian and uterine weight gain in hypophysectomised rats and to delay the onset of puberty in intact female rats. Also, GnRH and its agonist analogues have recently been shown to inhibit steroidogenesis induced by follicle-stimulating hormone (FSH) in cultured granulosa cells, confirming the direct action of such peptides on the ovarian follicle. The marked inhibitory effects of GnRH and its agonists on corpus luteum function suggest that these compounds could exert direct actions by binding to specific receptors on luteal cells. The present experiments, which examine the effects of GnRH agonists on luteal steroidogenesis, demonstrate the existence of such actions and their mediation by specific high-affinity receptor sites present in luteal cell membranes.     

c-fos和CYP17对多囊卵巢综合征(PCOS)患者卵巢颗粒细胞雄激素分泌过多的影响及机制

 为研究多囊卵巢综合征(PCOS)患者卵巢颗粒细胞中c-fos 及CYP17 基因表达的变化,探讨c-fos 及CYP17 基因对PCOS 卵巢颗粒细胞雄激素分泌的影响及可能的作用机制,对拟行IVF/ICSI-ET 的PCOS 患者及非PCOS 患者分为对照组(非PCOS 患者)和PCOS 组(PCOS 患者),两组患者的卵巢颗粒细胞进行体外培养48 h,利用放射免疫法检测细胞上清液中的雌二醇(E2)、孕酮(P)、睾酮(T)的水平;利用2-NBDG 标记葡萄糖检测颗粒细胞的葡萄糖摄取能力;采用免疫蛋白印迹(WesternBlot)法分别评估颗粒细胞中c-fos 及CYP17 基因的表达变化。结果发现,PCOS 组患者与对照组患者相比,颗粒细胞培养上清液中睾酮水平升高(P<0.05)、孕酮水平降低(P<0.05)、雌二醇水平无明显变化(P>0.05),且颗粒细胞对2-NBDG 标记葡萄糖摄取明显降低(P<0.05),提示PCOS 患者卵巢颗粒细胞雄激素分泌异常增高,并存在糖代谢异常。与对照组患者相比,PCOS组患者颗粒细胞CYP17 基因表达升高(P<0.05),c-fos 基因表达降低(P<0.05),提示颗粒细胞c-fos 基因异常低表达,可能降低了对CYP17 基因的抑制作用,使CYP17 高表达,可能是卵巢颗粒细胞雄激素分泌增加的原因之一。     

小檗碱和葛根素对卵巢颗粒细胞胰岛素抵抗的调控

 建立了体外的卵巢胰岛素抵抗模型。从卵巢内分子水平探讨胰岛素信号传导和促性腺激素信号传导的相互关系,揭示多囊卵巢综合征患者糖代谢异常和生殖功能障碍的关系;通过药物筛选得出两味中药增敏剂,与西药增敏剂进行了对照研究,探讨了其对卵巢颗粒细胞功能的影响。     

大熊猫（Ailuropoda Melanoleuca）卵巢的年龄变化

对比观察了5只不同年龄大熊猫(包括青年、中年和老年)的卵巢皮质石蜡切片。可见皮质结构有较大的年龄变化。主要表现在:性成熟后,卵巢皮质的厚度随年龄的增长有减薄的趋势。成年的各级正常卵泡数量较少,初级和次级的更少;闭锁卵饱和间质腺较多,并呈现出随年龄增长而增多的动向。老年卵巢结构变化明显,皮质纤维化,卵泡全部消失,仅有较多囊泡,小动脉管壁增厚,管腔变小。老年卵巢的外形、体积和重量等方面与成年之间未见明显年龄变化。     

Immunohistochemical localization of gonadotropin-releasing hormone receptors (GnRHR) in the nervous system, Hatschek’s pit and gonads of amphioxus,Branchiostoma belcheri

Using gonadotropin-releasing hormone (GnRH) anti-idiotypic antibodies and APA immunohistochemical method, the immunoreactivity of GnRHR in the nervous system, Hatschek’s pit and gonads of amphioxus has been located. It is found for the first time that the immunoreactivity of GnRHR exists in the nerve cells and fibers in the amphioxus’s brain and nerve tube and the epithelial cells of Hatschek’s pit at the different stages of gonadal development. At the same time, it is also found that GnRHR also exists in the ovary and testis of different developed stages. These findings provide morphological new proof for the informative transfer and regulation between brain and Hatschek’s pit mediation by GnRHR, and for the understanding of the mechanism of action on the reproductive endocrine control axis among brain-Hatschek’s pit-gonads.     

Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding

The mammalian hair follicle is a complex 'mini-organ' thought to form only during development; loss of an adult follicle is considered permanent. However, the possibility that hair follicles develop de novo following wounding was raised in studies on rabbits, mice and even humans fifty years ago. Subsequently, these observations were generally discounted because definitive evidence for follicular neogenesis was not presented. Here we show that, after wounding, hair follicles form de novo in genetically normal adult mice. The regenerated hair follicles establish a stem cell population, express known molecular markers of follicle differentiation, produce a hair shaft and progress through all stages of the hair follicle cycle. Lineage analysis demonstrated that the nascent follicles arise from epithelial cells outside of the hair follicle stem cell niche, suggesting that epidermal cells in the wound assume a hair follicle stem cell phenotype. Inhibition of Wnt signalling after re-epithelialization completely abrogates this wounding-induced folliculogenesis, whereas overexpression of Wnt ligand in the epidermis increases the number of regenerated hair follicles. These remarkable regenerative capabilities of the adult support the notion that wounding induces an embryonic phenotype in skin, and that this provides a window for manipulation of hair follicle neogenesis by Wnt proteins. These findings suggest treatments for wounds, hair loss and other degenerative skin disorders.     

Localization and possible role of membrane type metallo-proteinase and tissue inhibitors of metalloproteinase-1 in early stages of placentation

Human placental tissues from the first and second trimesters of gestation have been investigated using riboprobein situ hybridisation of mRNA sequences coding for membrane type metalloproteinase (MT-1-MMP) and tissue inhibitors of metalloproteinase-1 (TIMP-1). Results show that (i) both mRNAs express at a relatively high level in the chorion laeve trophoblast cells and the adjacent decidual cells of fetal membrane; (ii) the most abundant expression of the two mRNAs was found in the extravillous trophoblast between Rohrs and Nitabuch striae of basal plate, trophoblast shell and gland cells of the decidua; (iii) isolated or small groups of cytotrophoblast cells in the chorionic villi and in the cells lining arterioles in decidua and stem villi also expressed both MT-1-MMP and TIMP-1 at defferent extents. The data suggest that the coordinated expression of the MT-MMP and its inhibitor TIMP in defferent cells of the placental tissue may play an essential role in trophoblast invasion and angiogenesis related to placentation in the first two trimesters of gestation. They may also have an ability to effect separation of fetal from material tissue at a favorable junctional site during parturition.     

Expression and regulation of orphan receptor TR2 mRNA in germ cells of cryptorchid testis in rat and rhesus monkey

Expression and cellular localization of orphan receptor TR2 mRNA in relation to germ cell apoptosis in cryptorchid testes of rat and rhesus monkey have been studied by usingin situ hybridization andin situ 3′-end labeling of DNA fragments (TUNEL). The results show that: (i) TR2 mRNA is specifically expressed in the germ cells, mainly in the spermatocytes, round and elongated spermatids. The expression level of TR2 mRNA varies with the seminiferous cycle, (ii) In the rat cryptorchid testes on days 3 and 5 after the surgery, the germ cells began to undergo apoptosis with no evident decrease in TR2 mRNA level. On day 7.5, however, most germ cells underwent apoptosis, while the expression level of TR2 mRNA declined markedly, and TR2 mRNA was rarely expressed on day 10 thereafter, (iii) On days 15 and 20 of the cryptorchid testes of rhesus monkey, TR2 mRNA was only expressed in a few of primary spermatocytes and the mRNA was almost undetectable on days 30, 45, 60. These results suggest that TR2 mRNA probably plays an important role in spermatogenesis and germ cell apoptosis.     

Identification of integrin-like in guard cells ofVicia faba

We addressed the existence and localization of integrin-like in guard cells ofVicia faba by using a probe of polyclonal antibody against the human integrin (α_vβ₃/β₅). Western blot results showed that three integrins-like of about 47.3, 43.7 and 41.1 ku were detected from the preparation of membrane fragments of purified guard cell protoplasts. Further research with immunofluorescent scanning microscopy indicated that those integrins-like were localized on plasma membrane of guard cells, most nearing the dorsal wall, which is consistent with the reception of signals from epidermal cells to guard cells. Thus our results indicate, for the first time, that integrins-like are present at guard cell plasma membrane ofVicia faba.     

Follicular growth,differentiation and atresia

Only limited numbers of primordial follicles in mammalian ovary grow and differentiate to reach the stage of dominate follicles and ovulate. 99% of the follicles in the ovary undergo atresia at various stages of development.Regulation of follicular growth, development and atresia is a complex process and involves interactions between endocrine factors and intraovarian regulators. This review summarized: i ) FSH may not be a survival factor in regulating slow-growing preantral follicles. Some locally produced growth factors, activin and orphan receptors might play a more important role at this stage, ii ) Estrogen, activin/inhibin and follistatin coordinate with FSH to regulate and control follicle differentiation, iii) There are two types of follicular atresia induced by apoptosis which originates from GC or oocyte, respectively. Early translation of tPA mRNA into tPA protein in oocyte may be associated with oocytea apoptosis.     

中华蟾蜍卵巢性类固醇激素受体的免疫细胞化学研究

为探讨两栖动物卵巢内不同性类固醇激素受体在卵子发育中的调控作用.采用免疫细胞化学方法,对中华蟾蜍不同发育时期卵泡中的雌激素受体、孕激素受体和雄激素受体进行了定位检测.结果发现,三种受体在不同发育时期的卵泡中均有阳性反应:雌激素受体在卵黄合成期和卵黄合成后期的滤泡细胞表达为强阳性,在卵母细胞胞质和核膜中呈弱阳性反应;孕激素受体在卵黄合成早期的滤泡细胞和卵母细胞质、核膜中表达均较弱,在卵黄合成后期表达均增强;雄激素受体在卵黄合成早期的滤泡细胞和卵母细胞胞质、核膜中表达也均较弱,在卵黄合成后期卵母细胞胞质和核膜中表达略有增强.