Role of follicle stimulating hormone in the induction of hyperplasia during compensatory ovarian hypertrophy.

In adult rats, removal of one ovary leads to an acute albeit transient rise in serum follicle stimulating hormone and an increase in the weight of the remaining ovary. In an attempt to correlate the high titre of endogenous follicle stimulating hormone with the changes taking place at the macromolecular level, the phenomenon of compensatory ovarian hypertrophy was studied for one cycle after hemiovariectomy at metoestrus in the adult, cycling female rats derived from the Holtzman strain. The significant finding with respect to hormonal changes was an acute follicle stimulating hormone surge commencing 6h post-unilateral ovariectomy, reaching a maximum at 12 h and declining thereafter, hitherto not reported in the Holtzman strain. Serum luteinizing hormone, prolactin, oestradiol-17ß and testosterone remained unaltered while progesterone showed a decline at 6 h after surgery. There was an increase in the number of healthy class III (> 350 μm) follicles with a concomitant drop in atretic class III follicles 24 h post-unilateral ovariectomy. Analysis for DNA, RNA and protein content showed that all three constituents registered a continuous rise in the hypertrophying ovary up to 120h after surgery. When expressed as μg/mg ovarian weight, the increase in DNA reached a maximum at 24 h and declined thereafter. The kinetics of DNA synthesis was followed by pulse labelling with [3H] thymidine at 18, 24, 36 and 48 h after unilateral ovariectomy. Maximum incorporation occurred at 36 h. Autoradiographic studies showed that the granulosa cells of healthy follicles preferentially incorporated the label. In an extension of this study, it was found that labelling index registered a significant increase following ovariectomy, the maximum being reached at 24 h especially in class III follicles. The results clearly point out the crucial role of hyperplasia in the response of the contralateral ovary to the surgery and implicate the rise in follicle stimulating hormone as the primary signal for initiation of such a response. This raises the question whether in compensatory ovarian hypertrophy follicle stimulating hormone has a mitogenic role.

Interrelationship between ovine follicular fluid inhibin and serum albumin.

Ovine follicular fluid inhibin (oFF-I) as isolated in this laboratory, proved to be a monomeric protein (M(r).65 kDa). It was found to share very many of the physico-chemical characteristics of ovine serum albumin (oSA)-such as molecular size, iso-electric point, N-terminal aminoacid, finger-print patterns following enzymatic or cyanogen bromide cleavage, as well as binding of estradiol-17 beta and tryptophan. Furthermore, an antiserum containing polyclonal antibodies to oSA showed perfect cross-reaction with oFF-I. Nevertheless, oFF-I is distinct and different from oSA, as would be evident from the data reported here. Of the two proteins, oFF-I alone is capable of suppressing pituitary FSH output in a dose-dependent manner. Secondly, an antiserum containing polyclonal antibodies against Fraction-S2, a partially purified, biologically active fragment (M(r): 30-40 kDa)-derived from oFF-I, cross-reacted with the 65 kDa inhibin, but did not recognize oSA. Finally, the CD-spectra of the two proteins, when examined as a function of pH, show characteristic differences.

On multiple forms of bovine seminal plasma inhibin.

Out of a possible minimum of four, three distinct molecular species of bovine seminal plasma inhibin-differing either in Mr or in pI--have been purified to homogeneity. All three molecules exhibit the same proportion of alpha-helicity and beta-form when examined for their CD-spectra in a non-aqueous solvent medium. The implication of this finding for an induced conformation at the receptor-binding site for these hormonal peptides is briefly discussed.

Validation of coupled bioassay for inhibin by pituitary cell culture assay.

Studies on the characterization of inhibin and inhibin-like factors have depended for the most part on the classical in vitro pituitary cell culture assay. A major drawback with this assay is the turn-around time which is in the order of two weeks and consequently slows down purification efforts. The 24 h bioassay for inhibin has been found to be sufficiently sensitive and also statistically valid. Unfortunately, based as it is on a secondary response, ambiguities arise in interpreting the results. By including a parallel assay in which the mice are primed with human menopausal gonadotropin rather than human chorionic gonadotropin, it was possible to device the coupled bioassay. This enables distinguishing inhibin-like factors acting to suppress pituitary follicle stimulating hormone output from those acting at the level of gonads. In this study the coupled assay for inhibin has been compared with the classical pituitary cell culture assay in order to assess its biological and statistical validity. The data validates the bioassay on both the above counts and when considered in conjunction with the short turn-around time suggests that this assay can be highly useful in studies on isolation of inhibin from various. sources.

Biological characteristics of the peptides a and β isolated from bovine seminal plasma.

The bull seminal plasma peptides a and β have been examined for their biological properties. While both the peptides were able to inhibit the human chorionic gonadotropin-dependent uterine response in the mouse, a alone exhibits the property of suppressing post-castrational rise in gonadotropin in appropriate animal models. This suggests that the peptide β must be acting directly on the ovary to suppress estrogen production and, consequently, the uterine weight increase. Such a possibility was confirmed when a and β were examined by the coupled bioassay which is capable of discriminating between pituitary feedback factors and those acting directly on the gonad. In a test system designed to examine chronic effects, both a and β showed evidence of acting directly on the ovary to inhibit human menopausal gonadotropin-induced estrogen production. Such a direct action could not be correlated with the relative potencies of these peptides when examined for their follicle stimulating hormone-receptor binding inhibitor and lutinizing hormonereceptor binding inhibitor activities.

A method for the isolation of intact Sertoli cell-germ cell associations from rat seminiferous tubules and their further partition into Sertoli cell and germ cell fractions.

A technique is described for obtaining a Sertoli cell-enriched and a germ cell-enriched fraction from immature rat testes. Sertoli cell-germ cell associations were obtained by incubating washed seminiferous tubule fragments with Collagenase and Pancreatin. They were then manually dissociated into a suspension comprising Sertoli cells as well as the various germ cell types characteristic for a given day of ontogeny. Fractionation into a Sertoli cell-enriched fraction and a germ cell-enriched fraction was effected by centrifugation following layering over a stepwise gradient of Ficoll-400. While the time-span compares favourably with other procedures reported in the literature, it is believed this is the first time a method is described that enables the simultaneous recovery of both the Sertoli cells and the germ cells.

Extra-gonadal secretion of an inhibin-like substance in the laboratory albino rat.

Acetone powder preparations of ventral prostates of adult albino rat exhibit an inhibin-like activity. In vitro cultured ventral prostate explants secrete a substance possessing similar activity which appears to be independent of testicular androgens for its elaboration.