Isolation of hCG and its characterization by radioimmunoassay, enzyme-immunoassay, and radio-receptor assay.

The nature of human chorionic gonadotropin (hCG) molecules present during early pregnancy of Indian women is poorly understood. Therefore, a study has been undertaken to isolate hCG and characterize different forms of hCG from urine. The hCG molecules from urine of pregnant women (45-75 days post LMP) were adsorbed onto kaolin, eluted with ammonium hydroxide, and precipitated using acetone and then lyophilized. The lyophilized extract was subjected to Sephadex G-100 chromatography followed by ion-exchange fractionation. Three major fractions of protein (i.e., Peaks I, II, and III) associated with carbohydrate activity were obtained. Peaks II and III eventually resolved into a single peak I following repeated ion exchange chromatography, which suggested the presence of aggregates of molecules. Further purification on an affinity column resolved all three peak fractions into one unadsorbed and two adsorbed (A and B) fractions. These adsorbed fractions were characterized by radioreceptor assay (RRA), radioimmunoassay (RIA), and enzyme linked immunosorbent assay (ELISA). The activity was standardized against WHO reference preparation 75/589. Peaks I (A and B) were found to have maximum at about 75% of immunologically potent hCG, followed by peaks II (40%) and III (5%). The molecular sizes of peaks I, II, and III on a Sephadex G-200 column corresponded to 27,500D, 66,000D, and 84,000D, respectively. Relative mobilities of all adsorbed fractions in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of hCG-alpha (mol. wt. 19,539D) and hCG-beta (28,870D) subunits. The presence of both subunits of hCG were also revealed by Western blot analysis. For the first time, we report the low molecular weight hCG molecule, of 27,500D by size exclusion chromatography, which has immunological and biological activity as measured by RIA, ELISA, and RRA.

Isolation of alpha- and beta-subunits of peak-I hCG and generation of highly specific polyclonal antisera.

Development of polyclonal antisera is still a choice in some hard-pressed budget laboratories. In the present study, an attempt was made to isolate alpha- and beta-subunits from peak-I hCG, generation of polyclonal antisera and their characterization. The anti-hCG-a antisera showed titres of 1: 8000 and anti-hCG-beta antisera 1: 16,000 at 50% binding to radiolabelled hCG in RIA. Studies on specificity using anti hCG-beta antiserum demonstrated no cross-reaction with several hormones tested in the present study, except for hCG-beta and hCG, thus eliciting a highly specific hCG-beta antiserum.

Rat prostatic inhibin: less than 35 kDa protein(s).

An attempt was made to purify and physicochemically characterize the inhibin-like activity of rat ventral prostate based on the technique of immunosorption. It was possible to partially purify prostatic inhibin by affinity chromatography and the molecular size of inhibin-like material was shown to be less than 35 kDa.

Mode of action of inhibin-like pineal antigonadotropin is different from melatonin during compensatory ovarian hypertrophy.

Both melatonin and pineal antigonadotropic peptides have the same end effect, i.e., prevention of the hypertrophic response when tested in the conventional compensatory ovarian hypertrophy (COH) model. The present work was undertaken to study the effect of melatonin and a melatonin- and steroid-free inhibin-like ovine pineal antigonadotropin (PI) on serum follicle stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) following hemoiovariectomy in adult Holtzman rats and also to ascertain if any similarity exists in their mode of action during COH. While melatonin prevented the transient rise in FSH at 12 hr after unilateral ovariectomy (ULO), thus retaining the basal preoperative level, PI depressed basal levels of FSH too. In addition, melatonin suppressed PRL and LH levels at 12 hr and 120 hr after ULO, respectively. PI, on the other hand, had no effect on serum LH and PRL levels. In light of our earlier in vitro results, which showed a direct inhibitory effect of PI and not of melatonin on pituitary FSH synthesis and release, the present results indicate a dichotomy in the mode of action of PI and melatonin. PI acts directly at the level of the pituitary while melatonin may act at the level of the hypothalamus or higher brain centers to suppress the FSH surge and the ensuing compensatory response.

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.

Inhibin-like "antigonadotropic" activity in the ovine pineal.

In an attempt to define the antigonadotropic activity associated with the protein/peptide extracts of ovine pineals, melatonin- and steroid-free fractions obtained after subjecting crude ovine pineal acetone powder to soft gel chromatography were tested in three bioassay systems: (1) the conventional compensatory ovarian hypertrophy inhibition assay in rats, (2) the coupled bioassay in immature mice that enables one to distinguish factors acting at the level of the pituitary from those acting at the gonadal level, and (3) the classical in vitro pituitary cell culture assay for inhibin. The large molecular weight fraction, referred to as PI, behaved like a classical antigonadotropin as it suppressed compensatory ovarian hypertrophy following unilateral ovariectomy. Further, it not only lowered the basal and gonadotropin releasing hormone (GnRH)-stimulated release of follicle stimulating hormone (FSH) by the cultured pituitary cells, but also inhibited the human chorionic gonadotropin (hCG)-induced uterine weight gain in the coupled assay, thus acting like inhibin in these two assay systems. In addition, it showed immunological cross-reactivity with ovine testicular inhibin. The present results strongly support the view that inhibin-like activity may be the major player in what has so far been referred to as antigonadotropic activity.

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.

Secretion of inhibin-like material by rat ventral prostate epithelial cells in culture.

Monolayer cultures of epithelial as well as stromal components of the rat ventral prostate were established, taking advantage of differences in the requirement of sera supplementation and also in the temporal span for attachment. These monolayers were characterized and used to identify the cellular source of prostatic inhibin. The spent medium obtained from epithelial cell monolayer alone had significant inhibin-like activity.

Further studies on the synthesis and secretion of inhibin-like material by rat ventral prostate explants in vitro.

To authenticate further the testicular androgen independent status of synthesis and secretion of inhibin-like material by the rat ventral prostate, explants of this organ were maintained for a period of 6 days in vitro, altering the kind of medium used for culture. The various spent media had significant inhibin-like activity despite the absence of sera supplement in some cases.

Isolation of luteinizing hormone receptor binding inhibitor from ovine corpora lutea.

Procedures for the isolation of luteinizing hormone receptor binding inhibitor (LHRBI) from crude extracts of ovine corpora lutea are described. Microsomal pellets recovered by differential centrifugation of homogenates of fresh corpora lutea were stored at -20 degrees C for 3 weeks and then extracted with Tris-HCl buffer (pH 7.4). On sequential filtration of the extract on Amicon UM-20, PM-10 and UM-2R filters, the corresponding retentates (UM-20R, PM-10R and UM-2R) demonstrated inhibition of radio-labeled human chorionic gonadotrophin (hCG) to ovine luteal cells. The retentate of UM-2 filter (UM-2R) was further fractionated by reverse phase high pressure liquid chromatography. The active fractions- III, IX, X and XIII, thus obtained, calculated by their ability to inhibit 125I-hCG binding in dose dependent manner, were purified 150 to 1000-fold.

Some observations on the role of LH receptor binding inhibitor of ovine corpora lutea in ovarian processes.

Preliminary studies, both in vitro and in vivo were carried out to understand the physiological significance of LH receptor binding inhibitors isolated from ovine corpora lutea. Among the four active fractions tested, while UM-2R-III administered intraperitoneally suppressed both hMG and hCG-induced stimulation of uterine weights in immature mice, UM-2R-IX reduced the hCG-induced uterine weight. Fraction UM-2R-III inhibited both FSH and LH-stimulated cyclic AMP production by rat granulosa cells. Intraperitoneal injections of UM-2R-III and IX to adult cycling rats on the day of proestrus partially blocked ovulation. Similar i.p. treatment of cycling rats in 2 doses on day 1 of diestrus, prevented implantation/or early pregnancy (on 10th day of pregnancy) in 70 to 80% of rats. As the plasma levels of progesterone were low in the treated animals compared to the control, the decrease was attributed to the inadequate development of the corpus luteum. Of these fractions, UM-2R-IX was more effective at 5 to 10 times less doses compared to UM-2R-III. Further studies are needed to assess the route and dose of administering the active LHRBR components.

Ontogeny-related changes in the peptide profiles of the germinal epithelium: analysis by sodium dodecyl sulfate gradient gel electrophoresis.

The premise that one manifestation of the nexus between Sertoli cells and germ cells may be an orderly and sequential change in their protein profiles has been examined in relation to the ontogeny of spermatogenesis in the colony-bred albino rat. Viable "Sertoli cell-germ cell associations" isolated from the testes of animals of defined postnatal age and incubated in an appropriate medium were separated into a Sertoli cell and a germ cell fraction and processed for analysis by sodium dodecyl sulfate gradient gel electrophoresis. The resulting stained bands were "mapped" and assigned relative mobility values by comparison with standard marker proteins. This enabled identification by serial number of individual bands from an overall total of 163. For purposes of detailed analysis, they were classified into high, medium-high, medium, and low molecular weight bands. Two major categories were delineated: 1) those associated uniquely with a specified day of ontogeny and 2) those appearing intermittently. Significantly enough, not one of the bands was encountered on all days examined. The relevance of the patterns observed to the possible exchange of "information" between Sertoli cells and germ cells during spermatogenesis is mooted.

Protein synthesis in the rat seminiferous epithelium during onset of spermatogenesis--an autoradiographic study.

The pattern of protein synthesis in Sertoli and germ cells during the ontogeny of spermatogenesis in the rat was followed by autoradiography using (3H)-Leucine as precursor. The results suggest that the protein synthetic potential of Sertoli cells is greater than that of germ cells at any stage of differentiation. Significantly enough, the ontogeny-related protein synthetic capabilities of the germ cell compartment find a parallel in those reported for the adult rodent. Furthermore, the data support the suggestion that proteins may be transported from Sertoli cells to germ cells.

On the identity of bovine seminal plasma inhibin.

In light of current discussions on multiple forms of inhibin, it was thought of interest to ascertain the identity of the postulated 'iso-hormones' of bull seminal plasma inhibin (Chari et al., 1978). By subjecting the biologically active fraction, obtained by Sephadex G-100 gel filtration of bull seminal plasma acetone powder, to extensive dialysis in distilled water adjusted to pH 5.8, it was possible to remove the bulk of inert protein as a precipitate. The resulting active preparation could be readily fractionated by preparative iso-electric focusing in the pH range 4.0-6.5 yielding 2 distinct homogeneous peptides, alpha and beta, capable of suppressing hCG-induced uterine weight increase in immature mice, in a 'reversed Steelman-Pohley' assay design. However, of these, alpha alone was able to suppress post-castrational serum gonadotropin rise in appropriate animal models. This peptide is highly acidic in nature (iso-electric point congruent to 2.2) and has a molecular weight (Mr) of 18200 and a Stokes radius of 1.90 nm. On the basis of currently available evidence, it is concluded that the molecule consists of a single peptide chain.