Studies on follicular atresia: role of tropic hormone and steroids in regulating cathepsin-D activity of preantral follicles of the immature rat

The ovary of the immature female rat is comprised of primary and medium-sized preantral follicles. Upon stimulation with FSH or PMSG, the cathepsin-D activity, a representative lysosomal enzyme of granulosa cells, is reduced by 50% (P < 0.01). 17β-Estradiol at the doses tried was unable to mimic this effect. Blockade of steroidogenesis with cyanoketone also had no effect on the cathepsin-D activity of isolated granulosa cells. Dihydrotestosterone (DHT), however, at a dose of 1 mg/rat was able to inhibit PMSG's tropic action. It brought about an increase in cathepsin-D activity and reduction in steroidogenic activity of isolated granulosa cells. The atretogenic activity of DHT could be relieved by supplementation with exogenous FSH. DHT was observed to significantly reduce (P < 0.01) endogenous FSH and LH levels within 12–18 h of its injection suggesting that its atretic effect was due to its action at the pituitary rather than the gonad. In addition to the above the ability of 15 IU of PMSG to reduce cathepsin-D activity of granulosa cells was also significantly reduced (P < 0.01) if endogenous FSH was neutralized by a specific FSH antiserum. The present study suggests that as far as small and medium-sized primary and preantral follicles are concerned, FSH lack is the essential signal for onset of atresia.

Mechanism of glycine uptake by the silk glands of the silkworm Bombyx mori L

The transport of glycine in vitro into the silk glands of the silkworm has been studied. Glycine accumulates inside the tissue to a concentration higher than that present outside, indicating an active transport mechanism. The kinetics of uptake show a biphasic curve and two apparent Km values for accumulation, 0.33 mM and 5.00 mM. The effect of inhibitors on the energy metabolism of glycine transport is inconclusive. Exchange studies indicate the existence of two pools inside the gland, one that is easily removed by exchange and osmotic shock, and the other which is not. The results obtained conform with the carrier model of Britten and McClure concerning the amino-acid pool in E. coli.

Termination of pregnancy in macaques (Macaca radiata) using monkey antiserum to ovine luteinizing hormone

The ability of a monkey antiserum to ovine LH to interrupt gestation in monkeys has been established. The antiserum has been shown to neutralize monkey pituitary LH by a number of criteria. The significant increase in serum progesterone level on day 23 of the cycle shown by mated monkeys has been used as an index of pregnancy. Injection of LH antiserum during the first week of missed menses (day 29–31 of cycle or day 18–20 of gestation) causes significant reduction in serum levels of progesterone followed by onset of bleeding which is interpreted as the termination of gestation. The same dose of non-immune serum given to monkeys during the same period does not have any deleterious effect on the progress of pregnancy. The antiserum-treated animals after the termination of gestation, resume cyclicity. Injection of antiserum after day 25 of gestation does not bring about termination of pregnancy. It is suggested that by using antisera raised in humans to ovine LH, this method may be developed as a fertility control measure in humans.

The onset of oestrus and ovulation in lactating rats

Lactation delays the re-initiation of oestrous cyclicity in rats, resulting in physiological sterility for the duration of suckling. During this phase, the secretion of pituitary gonadotrophins is suppressed by an unknown mechanism. Continued application of the suckling stimulus by litter replacement (Bruce, 1958; Nicoll & Meites, 1959), or injections of prolactin (Meites & Nicoll, 1959), have been shown to prolong lactation considerably beyond the usual period. The present study aimed to demonstrate the role of prolactin in inhibiting the gonadotrophin secretion necessary for the re-establishment of oestrous cyclicity during lactation. Pregnant rats weighing approximately 300 g were obtained from the Institute colony and housed in individual cages. At parturition, the number of young in the litter was adjusted to eight, two or one as required. The day following the post-partum oestrus was regarded.

The role of fsh and lh in the initiation of ovulation in rats and hamsters: a study using rabbit antisera to ovine fsh and lh

The relative rôles of FSH and LH in ovulation induction in immature and adult cycling rats and hamsters have been evaluated. Both heterologous purified pituitary hormones and homologous crude pituitary extracts have been used as ovulatory stimuli in immature animals primed with PMSG. Well-characterized FSH and LH antisera have been used in the above model systems to achieve specific neutralization of FSH and LH. The present study revealed that LH is the physiological trigger needed for induction of ovulation in both rats and hamsters and FSH cannot, by itself, induce ovulation in the total absence of LH.

Prolactin suppresses release of luteinising hormone during lactation in the monkey

STUDIES with rats have shown that during lactation there is an inhibition of luteinising hormone (LH)-dependent physiological events, such as implantation1, and a return to oestrus cyclicity2. This inhibition has been shown to occur only during the intense suckling phase and it has been correlated with the high levels of prolactin present in the circulation at this time. Although exogenous prolactin could substitute for the effects of intense suckling, it could do so only under the permissive influence of minimal suckling stimulus. We have shown that there is, in these conditions, a lowering of LH levels, and that this is due to interference by prolactin with the pituitary responsiveness to LH-releasing hormone (LHRH) (K. Muralidhar, R. M. and N. R. M., unpublished). Using the lactating monkey, we have now demonstrated a similar inhibitory effect of prolactin on pituitary responsiveness to LHRH, suggesting a mechanism by which amenorrhoeic conditions are maintained during lactation.

Characterisation of the DNA-polymerase-alpha-primase complex from the silk glands of Bombyx mori

Silk gland cells ofBombyx mori undergo chromosomal endoduplication throughout larval development. The DNA content of both posterior and middle silk gland nuclei increased by 300000 times the haploid genomic content, amounting to 18 rounds of replication. The DNA doubling time is approximately 48 h and 24 h during the fourth and fifth instars of larval development. However, DNA content does not change during the interim moult. Concomitant with DNA content, DNA polymerase activity also increases as development progressed. Enzyme activity is predominantly due to DNA polymerase with no detectable level of polymerase . DNA polymerase from silk gland extracts was purified to homogeneity (using a series of columns involving ionexchange, gel-filtration and affintiy chromatography), resulting in a 4000-fold increase in specific activity. The enzyme is a heterogeneous multimer of high molecular mass, and the catalytic (polymerase) activity is resident in the 180-kDa subunit. The enzyme shows a PI of 6.2 and theKm values for the dNTP vary over 5-16 . The polymerase is tightly associated with primase activity and initiates primer synthesis in the presence of ribonucleoside triphosphates on a single-stranded DNA template. The primase activity is resident in the 45-kDa subunit. The enzyme is devoid of any detectable exonuclease activity. The abundance of DNA polymerase α in silk glands and its strong association with the nuclear matrix suggest a role in the DNA endoduplication process.

Changes in testicular function following specific deprivation of LH in the adult male rabbit

Sexually mature male rabbits actively immunized against highly purified ovine LH (oLH) were used as a model system to study the effects of endogenous LH deprivation (and therefore testosterone) on spermatogenesis as well as pituitary FSH secretion. Immunization against oLH generated antibody titres capable of cross-reacting and neutralizing rabbit LH and this resulted in a significant reduction (P<0.01) in serum testosterone levels by 2-4 weeks of immunization. A significant increase in circulating FSH concentration (from a basal level of similar to 1 ng to 60-100 ng/ml; P<0.01) was observed within 4-6 weeks of immunization, perhaps a consequence of the negative feedback effect of the lack of testosterone. The effect of LH deprivation on spermatogenesis assessed by DNA flow cytometry and histological analyses of testicular biopsy tissue revealed that lack of testosterone primarily results in a rapid reduction and complete absence of round (1C) and elongated (HC) spermatids. The immediate effect of LH/testosterone deprivation thus appears to be at the step of meiotic transformation of primary spermatocytes (4C) to 1C. A significant reduction (>80%; P<0.01) in the 4C population and a relative accumulation (>90%; P<0.01) in spermatogonia (2C) was also observed, suggesting a need for testosterone during the transformation of 2C to 1C. In all but one of the rabbits, both qualitative and quantitative recovery in spermatogenesis occurred during the recovery phase, even at a time when only a marginal increase in serum testosterone (compared with the preimmunization) levels was observed as a result of a rapid decline in the cross-reactive antibody titres. These results clearly show that LH/testosterone deprivation in addition to primarily affecting the meiotic step also regulates the conversion of 2C to 4C during spermatogenesis.

Regulation of Reproduction in the Primitively Eusocial Wasp Ropalidia marginata: on the Trail of the Queen Pheromone

Queens and workers are not morphologically differentiated in the primitively eusocial wasp, Ropalidia marginata. Upon removal of the queen, one of the workers becomes extremely aggressive, but immediately drops her aggression if the queen is returned. If the queen is not returned, this hyper-aggressive individual, the potential queen (PQ), will develop her ovaries, lose her hyper-aggression, and become the next colony queen. Because of the non-aggressive nature of the queen, and because the PQ loses her aggression by the time she starts laying eggs, we hypothesized that regulation of worker reproduction in R marginata is mediated by pheromones rather than by physical aggression. Based on the immediate loss of aggression by the PQ upon return of the queen, we developed a bioassay to test whether the queen's Dufour's gland is, at least, one of the sources of the queen pheromone. Macerates of the queen's Dufour's gland, but not that of the worker's Dufour's gland, mimic the queen in making the PQ decrease her aggression. We also correctly distinguished queens and workers of R. marginata nests by a discriminant function analysis based on the chemical composition of their respective Dufour's glands.

Effect of anti-luteinizing hormone serum on the ovulation of rats

IN the cyclic female albino rat, a release of pituitary luteinizing hormone (LH) occurs on the afternoon of proestrus1-5. This apparently induces ovulation, for ova are seen in the Fallopian tube 12 h later. Similarly, it is well known that in immature rats primed with pregnant mare serum gonadotrophin (PMS), ovulation can be induced by the administration of human chorionic gonadotrophin (HCG) or LH, the ova being seen in the Fallopian tube 12 h later. No information is available, however, about the mode of action of LH, released or administered, in bringing about ovulation. We have approached this problem by blocking the action of the ovulating hormone (LH) at various times after administration. © 1970 Nature Publishing Group.

Detoxication of benzoate by glycine conjugation in the silkworm, Bombyx mori L

The role of hippuric acid formation as a mechanism for detoxication of benzoic acid in the silkworm has been investigated. Benzoate is inhibitory to the growth of the silkworm and excreted as hippuric acid, which is not toxic. Hippuric acid is not a normal constituent of excreta. Synthesis of hippuric acid has been shown to occur in the intestines of the silkworm. Hippuricase activity is present in the fat body and silk-gland tissue.

Stimulation by adenosine 3′,5′-cyclic monophosphate of protein synthesis by adenohypophyseal polyribosomes

Addition of dibutyryl 3′,5′-cyclic AMP to slices of bovine pituitary stimulated incorporation of [3H]leucine into protein, whether or not actinomycin D was present; therefore the influence of 3′,5′-cyclic AMP on protein synthesis by bovine pituitary polysomes was studied. If the cyclic nucleotide was added to the complete protein-synthesizing system (including pH 5.0 enzyme), stimulation of [3H]leucine incorporation occurred only with pH 5.0 enzyme from rat liver; there was no stimulation when homologous enzyme, i.e., from bovine pituitary, was used. Addition of 3′,5′-cyclic AMP to the polysomes, before addition of pH 5.0 enzyme, resulted in stimulation of protein synthesis with either source of enzyme, but stimulation was facilitated to a greater degree, over the range 0.5-2 mM 3′,5′-cyclic AMP, when rat liver was the source. The stimulation of protein synthesis was prevented by the addition of cycloheximide. With rat liver pH 5.0 enzyme the product of hydrolysis of 3′,5′-cyclic AMP was mainly 5′-AMP whereas with pituitary pH 5.0 enzyme there was also dephosphorylation and deamination resulting in production of hypoxanthine and other bases. However, using either source of pH 5.0 enzyme and the complete protein-synthesizing system (i.e., including an ATP-regenerating mechanism) most of the 3H from hydrolysis of [3H]3′,5′-cyclic AMP was incorporated into ATP. The data are seen as compatible with a stimulation by 3′,5′-cyclic AMP of translation by pituitary polysomes; the significance of the importance of the source of pH 5.0 enzyme used in the system is obscure.

Mechanism of glycine uptake by the silk glands of the silkworm Bombyx mori L.

The transport of glycine in vitro into the silk glands of the silkworm has been studied. Glycine accumulates inside the tissue to a concentration higher than that present outside, indicating an active transport mechanism. The kinetics of uptake show a biphasic curve and two apparent Km values for accumulation, 0.33 mM and 5.00 mM. The effect of inhibitors on the energy metabolism of glycine transport is inconclusive. Exchange studies indicate the existence of two pools inside the gland, one that is easily removed by exchange and osmotic shock, and the other which is not. The results obtained conform with the carrier model of Britten and McClure concerning the amino-acid pool in E. coli.

A possible role for a low molecular weight peptide in regulation of testosterone production by rat Leydig cells

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of Percoll purified Leydig cell proteins from 20- and 120-day-old rats revealed a significant decrease in a low molecular weight peptide in the adult rats. Administration of human chorionic gonadotropin to immature rats resulted in a decrease in the low molecular weight peptide along with increase in testosterone production. Modulation of the peptide by human chorionic gonadotropin could be confirmed by Western blotting. The presence of a similar peptide could be detected by Western blotting in testes of immature mouse, hamster, guinea pig but not in adrenal, placenta and corpus luteum. Administration of testosterone propionate which is known to inhibit the pituitary luteinizing hormone levels in adult rats resulted in an increase in the low molecular weight peptide, as checked by Western blotting. It is suggested that this peptide may have a role in regulation of acquisition of responsiveness to luteinizing hormone by immature rat Leydig cells.

Chronic suppression of testicular function by constant infusion of gonadotropin-releasing hormone agonist and testosterone supplementation in the bonnet monkey (Macaca radiata)

Objective: To study the efficacy of long-term buserelin acetate infusion to desensitize pituitary and block testicular function in adult male monkeys (Macaca radiata). Animals: Proven fertile male monkeys exhibiting normal testicular function. Protocol: Each of the control (n = 5) and experimental monkeys (n = 10) received a fresh miniosmotic pump every 21 days, whereas pumps in controls delivered vehicle of experimentals released 50-mu-g buserelin acetate every 24 hours. On day 170 (renewed every 60 days) a silastic capsule containing crystalline testosterone (T) was implanted in the experimental monkeys. At the end of 3 years, treatment was stopped, and recovery of testicular function and fertility monitored. Results: (1) Treatment resulted in marked reduction of nocturnal but not basal serum T; (2) the pituitary remained desensitized to buserelin acetate throughout the 3-year period; (3) animals were largely azoospermic with occasional oligospermia exhibited by two monkeys; and (4) withdrawal of treatment restored testicular function, with 70% of animals regaining fertility. Conclusion: Long-term infertility (but restorable) can be induced in male monkeys by constant infusion of buserelin acetate and T.