Secretion of IGF-1 by ovine granulosa cells: effects of growth hormone and follicle stimulating hormone

Insulin-like growth factor-1 (IGF-1) is implicated in follicle development and is considered to mediate the actions of growth hormone (GH) and gonadotrophins at the ovarian level. However, the expression and secretion of IGF-1 by the ovary are controversial, partly because of species and cell-type specificity. The present study investigated whether IGF-1 is produced by ovine granulosa cells and whether its production is regulated by GH and follicle stimulating hormone (FSH). Follicles (>/=4.0 mm) were obtained from ewes during seasonal anoestrus. Granulosa cells were cultured for a total period of 96 h in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium supplemented with BSA (0.1%, w:v), transferrin (0.5 microg/ml) and testosterone (100 ng/ml). In the first set of experiments, cells were incubated in the presence of bovine calf serum (BCS) (2.5%) for the initial 48 h of culture. The cells were then cultured for the next 48 h in medium without BCS, but containing either GH (0, 2, 20, and 200 ng/ml) or FSH (0, 20, 200, and 2000 ng/ml). The medium was assayed for oestradiol (E), progesterone (P) and IGF-1. There were six wells per treatment and the experiment was carried out four times. Control granulosa cells maintained both IGF-1 and E secretion, with only low levels of progesterone output. In all experiments, both GH and FSH produced significant (P<0.001) dose-related increases in E, IGF-1 and P secretion into the medium. The maximum responses to GH (20 or 200 ng/ml) were 402% for E and 528% for IGF-1 compared with controls. The maximum responses to FSH (200 or 2000 ng/ml) were 460% for E and 514% for IGF-1. The objective of the second set of experiments was to determine the effect of the progestogenic status of cells on IGF-1 production. Granulosa cells were cultured both in the presence and absence of BCS (2.5% in the medium) during the initial 48 h of culture. For the next 48 h, cells were cultured in serum-free medium. Addition of BCS to the medium during the initial 48 h of culture stimulated progesterone production. However, it did not affect either IGF-1 or oestradiol secretion between 49 and 96 h of culture, or the cell numbers at the end of culture. In conclusion, (1) IGF-1 is secreted by granulosa cells irrespective of their progestogenic status and (2) concomitant increases in E and IGF-1 production by granulosa cells as a result of GH and/or FSH treatment suggest a role for GH and FSH in the regulation of ovarian function.     

Biological characteristics of a factor suppressing follicle stimulating hormone (inhibin) from ovine testes

Inhibin (follicle stimulating hormone suppressing factor) isolated from ovine testes has been characterized for its biological activity using a variety of tests. The bioassay used — inhibition of the human chorionic gonadotropin induced increment in the mouse uterine weight-demonstrates that there is a significant increment in specific activity (approx. 300-fold) with the progress of purification. Eventhough the final product has not been obtained in a homogenous state it has been possible to show that(a) [¹²⁵I]-labelled inhibin is preferentially taken up and retained by the pituitary, pretreatment of rats with testosterone facilitating this uptake;(b) it is able to suppress specifically the levels of follicle stimulating hormone in castrated as well as immature intact rats and (c) treatment of immature male rats with inhibin preparation for ten days results in impairment of testicular function as judged by³H-thymidine incorporation into testicular DNA and testicular hyaluronidase activity.     

Sperm production and fertility of bonnet monkeys (Macaca radiata) following immunization with ovine follicle stimulating hormone.

Adult male bonnet monkeys were rendered oligospermic but not azoospermic following active immunization with ovine follicle stimulating hormone. The percentage of sperms in the semen having good motility was reduced with a concomitant increase in the sperm ATPase activity. Eight out of 10 immunized monkeys failed to impregnate females of proven fertility after mating for consecutive three cycles while the remaining two impregnated the cohabitated females during the third cycle at a time when the antibody titer was reduced. Active immunization with ovine follicle stimulating hormone may not produce complete azoospermia but renders adult male monkeys infertile provided sufficient antibody titer is maintained.     

Neuroendocrine regulation of luteinizing hormone and follicle stimulating hormone: a review

Since the pioneering studies of Everett, Sawyer and Markee (1) it has been generally accepted that the central nervous system (CNS) regulates the secretion of the pituitary gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH). However, great gaps still exist in our understanding of the neural mechanisms that regulate the secretion of these hormones. The purpose of this review is to provide the reader with a concise overview of this topic. Gaps, inconsistencies and future directions of this area of research are also presented.     

Early pituitary follicle stimulating hormone response to gonadotrophin releasing hormone in ewes

The object of our experiments was to characterize the response of plasma follicle stimulating hormone (FSH) within minutes of an i.v. injection of high or low doses of gonadotrophin releasing hormone (GnRH), especially in relation to contemporary changes in luteinizing hormone (LH) concentrations. In the deep anoestrous period (June), three intact ewes and two ovariectomized ewes were injected with 1 mug synthetic GnRH followed 2 h later by a second identical injection. A week later, the same regimen was repeated with the same sheep but with 50 mug GnRH after an interval of 5 h 20 min. Blood samples were collected every 15 sec for 15 min after each injection (early release), then at longer intervals (main release) till the next treatment, followed by sampling for a further 6-h period after the second treatment. FSH was released as soon as the second minute after GnRH injection in all ewes. The mean pituitary FSH response, during this early release, in intact and ovariectomized ewes was similar after either 1 or 50 mug GnRH. However, the main release was less pronounced in the ovariectomized sheep and was not stimulated after the second treatment in all sheep. Three other ewes were injected with 40 mug GnRH and sampled every 15 sec for seven, 6-min periods during the period of release to compare FSH and LH secretion. The profiles reflected a similarity in sensitivity and responsiveness to GnRH, especially soon after GnRH injection. Increases in both hormones were formed by several grouped associated spikes. It is suggested that a readily releasable pool of FSH exists in the ewe. There are probably differences in the mechanisms of synthesis and/or release between pituitary FSH and LH.     

Preparation of highly substituted gamma-lactam follicle stimulating hormone receptor agonists

An unusual combination of Weinreb amidation and Mitsunobu lactam formation was used to prepare highly substituted gamma-lactam analogues of a thiazolidinone follicle stimulating hormone receptor agonist. The analogue synthesis was stereoselective and the final products were chemically stable. Biological properties of the target molecules were nearly identical to those of the lead compound.     

重组人卵泡刺激素在昆虫细胞中的表达

为了研究在昆虫细胞中表达重组人卵泡刺激素,我们以人胎盘组织提取的染色体DNA为模板,利用重叠PCR方法扩增出hFSHβ亚基的cDNA的编码区。将此cDNA克隆入核型多角体病毒(AcNPV)非融合蛋白基因表达载体pVLl393,我们得到了表达载体pVLl393-hFSHβ,然后与BaculoGold^TM线性杆状病毒DNA共转染昆虫细胞SF9,经多次扩增后获得高滴度的重组病毒AcNPV-hFSHβ。将此重组病毒感染昆虫细胞,我们得到了在胞浆中表达的hFSHβ亚基,Western blot显示分子量大约为21kDa。以重组病毒AcNPV-hFSHβ与AcNPV-hCGoL一同感染昆虫细胞得到了具有分泌性的重组hFSH异二聚体,在非还原的条件下Western blot显示分子量大约为33kDa。