Radioimmunoassay of polypeptide hormones using immunochemically coated plastic tubes

A method has been developed for immobilisation of antisera on fresh plastic tubes through an immunochemical bridge. This type of immobilisation has been shown to be more consistent than direct adsorption on plastic. Such immunochemically coated antisera on plastic tube has been used in the development of a noncentrifugation radioimmunoassay. This assay system has been found to be technically as sound as the conventional method.     

Relative ability of ovine follicle stimulating hormone and itsβ-subunit to generate antibodies having bioneutralization potential in nonhuman primates

The relative ability of ovine follicle stimulating hormone and itsβ-subunit, two potential candidates for male contraceptive vaccine, to generate antibodies in monkeys capable of bioneutralizing follicle stimulating hormone was assessed usingin vitro model systems. Antiserum against native ovine follicle stimulating hormone was found to be highly specific to the intact form with no cross-reactivity with either of the two subunits while the antiserum againstβ-subunit of follicle stimulating hormone could bind to theβ-subunit in its free form as well as when it is combined withα-subunit to form the intact hormone. Both antisera could block the binding of the hormone to the receptor if the hormone was preincubated with the antibody. However, the follicle stimulating hormoneβ-antisera could only inhibit the binding of the hormone partially (33% inhibition) if the antibody and receptor were mixed prior to the addition of the hormone, while antisera to the native follicle stimulating hormone could block the binding completely (100% inhibition) in the same experiment. Similarly antisera to the native follicle stimulating hormone was significantly effective in blocking (100%) response to follicle stimulating hormone but not theβ-subunit antisera (0%) as checked using anin vitro granulosa cell system. Thus the probability of obtaining antibodies of greater bioneutralization potential is much higher if intact hormone is used as an antigen rather than itsβ-subunit as a vaccine. Majority of the work reported here was carried out during the tenure of Visiting Scientist fellowship awarded by the MRC Canada to the first author.     

Mechanism of down regulation of luteinizing hormone receptors and steroidogenesis in corpora lutea

The mechanism of ‘down regulation’ of luteinizing hormone receptors was investigated in pseudopregnant rats using a modified radioimmunoassay capable of measuring endogenous tissue-bound hormone. Treatment of pseudopregnant animals with a desensitizing dose (desensitization treatment) of human chorionic gonadotropin resulted in a decrease in receptor concentration. This decrease was prevented if the animals were treated prior to the desensitization treatment with indomethacin, an inhibitor of prostaglandin biosynthesis, suggesting a role for prostaglandins in down regulation. The desensitization treatment resulted in a time-dependent decrease in subsequent responsiveness of the tissue to luteinizing hormone. Basal progesterone production rate was also decreased following desensitization. Total tissue cholesterol was found to be decreased following desensitization treatment, without any change in the ratio of free to esterified cholesterol. Mitochondrial cholesterol was significantly reduced and pregnenolone production by the mitochondria of desensitized corpora lutea was also markedly reduced. However, when cholesterol was added to the mitochondria of desensitized corpora lutea, pregnenolone production was increased, reaching values almost equal to that shown by the control mitochondria. These results show that decrease in the responsiveness following desensitization treatment is due to, besides receptor loss, decrease in tissue cholesterol, in particular mitochondrial cholesterol. The cholesterol side chain cleavage activity, although low, appears to be functionally intact; the low activity could be attributed to low levels of mitochondrial cholesterol.     

Studies on metabolism of vitamin A. The effect of hormones on gestation in retinoate-fed female rats

1. The preparation of cell suspensions by treatment of chick embryo hearts with collagenase at various stages of development is described. 2. Measurements of oxygen consumption, incorporation of labelled leucine into protein and accumulation of labelled alpha-aminoisobutyric acid against a concentration gradient indicated a long-lasting viability of the isolated heart cells in vitro; a satisfactory preservation of subcellular structures, including plasma membrane, was assessed by electron-microscopic examination. 3. The rate of alpha-aminoisobutyric acid accumulation by cardiac cells isolated from hearts at different stages of embryological development decreased with aging; insulin stimulated the intracellular accumulation of this amino acid analogue. 4. Insulin increased the uptake by isolated heart cells of several (14)C-labelled naturally occurring amino acids; however, the fraction of amino acid taken up by the cells that was recovered free intracellularly, and therefore the concentration ratio (between intracellular water and medium), was enhanced by the hormone only with glycine, proline, serine, threonine, histidine and methionine. When isolated heart cells were incubated in the presence of a mixture of labelled amino acids, the addition of insulin increased the disappearance of radioactivity from the medium. 5. The general pattern of amino acid transport (in the absence and in the presence of insulin) in isolated cardiac cells was similar to that found in intact hearts, suggesting that the biological preparation described in this paper might be useful for studies of cell permeability and insulin action.     

Studies on regulation of chorionic gonadotropin secretion in primates

The regulation of secretion of chorionic gonadotropin in primates has been studied using bothin vivo andin vitro models.In vivo studies using the pregnant bonnet monkey revealed that at the doses tested, the administration of progesterone or estradiol 17Β in combination or alone did not result in any appreciable change in the duration or magnitude of serum chorionic gonadotropin levels. However, administration of lutropin-releasing hormone by intravenous route resulted in significant increase in chorionic gonadotropin levels within 30–60 min and the extent of stimulation seemed to depend on the state of pregnancy. Forin vitro studies, explants or cells prepared from first trimester human placenta has been used. The functional integrity of these cells has been established by demonstrating the binding of [¹²⁵I]-labelled human chorionic gonadotropin antibody to the cells as well as the synthesis of [³H]-labelled human chorionic gonadotropin.In vitro studies using the cells revealed that addition of lutropin-releasing hormone caused a significant increase in chorionic gonadotropin and estradiol 17Β secreted into the medium. Thus bothin vivo andin vitro results suggest that lutropin-releasing hormone could be one of the factors involved in regulation of chorionic gonadotropin secretion in primates.     

Effects of a highly purified antiserum to FSH on testicular function in immature rats

Effects of highly purified antiserum (AS) to follicle stimulating hormone (FSH) on testicular function was studied in immature rats. Treatment with FSHAS for 10 days, from 25-34, decreased weights of the testis (p .001) and increased weights of the epididymis (p .05). Numbers of the cell types in the seminiferous epithelium, particularly Type A spermatogonia pachytene spermatocytes and spermatids, were markedly reduced, possibly due to: 1) decreased division of the initial stem cells, 2) impairment of division of Type B spermatogonia and their transformation to pachytene spermatocytes, and 3) desquamation and degeneration of pachytene spermatocytes and spermatids. FSHAS also affected the sertoli cell function which was reflected in the decreased binding of androgens to supernatant fraction of the testis and epididymides. Treatment with luteinizing hormone-AS for 5 days did not affect testicular function but the binding of androgens to the supernatants of the caput and cauda epididymides and ventral prostate was significantly reduced (p .001). These data indicate that FSH is necessary for the maintenance of the cellular integrity of the seminiferous epithelium during the completion of the 1st wave of spermatogenesis.     

Studies on follicular growth in the immature rat and hamster: Effect of a single injection of gonadotropin or estrogen on the rate of3H-thymidine incorporation into ovarian DNAin vitro

Initiation of follicular growth by specific hormonal stimuli in ovaries of immature rats and hamsters was studied by determining the rate of incorporation of³H-thymidine into ovarian DNAin vitro. Incorporation was considered as an index of DNA synthesis and cell multiplication. A single injection of pregnant mare serum gonadotropin could thus maximally stimulate by 18 hr³H-thymidine incorporation into DNA of the ovary of immature hamsters. Neutralization of pregnant mare serum gonadotropin by an antiserum to ovine follicle stimulating hormone only during the initial 8–10 hr and not later could inhibit the increase in³H-thymidine incorporationin vitro observed at 18 hr, suggesting that the continued presence of gonadotropin stimulus was not necessary for this response. The other indices of follicular growth monitored such as ovarian weight, serum estradiol and uterine weight showed discernible increase at periods only after the above initial event. A single injection of estrogen (diethyl stilbesterol or estradiol-l7β) could similarly cause 18 hr later, a stimulation in the rate of incorporation of³H-thymidine into DNAin vitro in ovaries of immature rats. The presence of endogenous gonadotropins, however, was obligatory for observing this response to estrogen. Evidence in support of the above was two-fold: (i) administration of antiserum to follicle stimulating hormone or luteinizing hormone along with estrogen completely inhibited the increase in³H-thymidine incorporation into ovarian DNAin vitro; (ii) a radioimmunological measurement revealed following estrogen treatment, the presence of a higher concentration of endogenous follicle stimulating hormone in the ovary. Finally, administration of varying doses of ovine follicle stimulating hormone along with a constant dose of estrogen to immature rats produced a dose-dependent increment in the incorporation of³H-thymidine into ovarian DNAin vitro. These observations suggested the potentiality of this system for developing a sensitive bioassay for follicle stimulating hormone.     

Effects of cadmium salt on phosphomonoesterases activity and fertilizing ability of fowl spermatozoa.

Daily administration of cadmium salt for 25 days (2.5 mg per Kg body weight) in the male domestic fowl caused the end of treatment period. An increased incidences of concentration. Fertility dropped to zero at the end of the treatment period. Activity of acid and alkaline phosphomonoesterases were also drastically reduced by the end of treatment period. An increased incidences of morphological abnormalities of spermatozoa were noticed in the treated birds. After 46 days cessation of the treatment, full recovery of the above measures was found. These alterations suggest the reversible type of effect of cadmium chloride on the spermatozoa of male domestic fowl.     

The influence of prepubertal partial sinistral ovariectomy on the subsequent reproductive function of domestic hens (Gallus domesticus )

Each of 20 White Leghorn hens of 13 to 14 weeks were subjected to partial sinistral ovariectomy and sham-operations. In half of the hens from each group, the percentage of egg production and clutch size were noted until 50 weeks of age. The growing pattern of normal ovarian follicles was also recorded at 26 weeks of age in a rest half ofthe hens in the two groups. The percentage of egg production and the mean and variance of clutch size did not differ significantly (P / 0.05) between the partially ovariectomized and sham-operated groups. The growing yellow follicles (>8 mm) in the rapidly developing phase in these two groups did not vary, although the smaller follicles (4 to 8 mm in diameter) remained significantly (P / 0.01) more in the shamoperated control group than in the partially ovariectomized group. This observation indicates that smaller follicles (4 to 8 mm) developed in the larger (>8 mm) follicles more efficiently in partially ovariectomized hens than in the sham-operated (control) hens. In a second experiment, one group of hens had all the yellow follicles (>8 mm) removed, while a second group of hens was left untreated. On the 3rd and 6th day post treatment, the hens were examined for the presence of ovarian follicles. No significant (P / 0.05) difference in the growing pattern of subsequent follicles (2 to 4 or 4 to 8 mm) was detected due to treatment. These data demonstrate that the mechanisms that regulate follicular growth and atresia are adjust to maintain normal ovulation following partial ovariectomy.     

Invitro responsiveness of hamster corpora lutea undergoing luteolysis to luteinizing hormone

Corpora lutea removed from pregnant hamster deprived of endogenous luteinizing hormone for varying periods were compared for their responsiveness to externally added luteinizing hormone. The corpora lutea removed on the 8th day of pregnancy exhibited a dose-dependent increase in progesterone production in response to added luteinizing hormone upto a concentration of 2.5 Μg/ml. The total progesterone synthesised by the corpora lutea decreased with increase in the duration ofin vivo luteinizing hormone deprivation. However, the hormone deprivation had to be for a minimum period of 24 h before a marked reduction in thein vitro responsiveness could be seen. Neutralisation of endogenous luteinizing hormone increased the luteal cholesterol ester concentration, whilein vitro incubation of such tissue with luteinizing hormone resulted in a marked reduction in cholesterol ester levels. Corpora lutea removed from hamsters on day 8, 15 and 16 of pregnancy when compared for their responsivenessin vitro to added luteinizing hormone showed that the luteal tissue of day 8 produced more progesterone relative to those of day 15/16. In contrast, depletion of free and esterified cholesterol increased with the increase in age of corpora lutea (from 15% on day 8 to 67% on day 16).