NEUROENDOCRINE REGULATION OF PROLACTIN SECRETION IN THE FEMALE RHESUS MONKEY IN RELATION TO GONADAL STATUS -INVOLVEMENT OF EXCITATORY AMINO ACIDS

Abstract

The secretion of Prolactin (PRL) from the anterior pituitary is under the dual control of two hypothalamic factors; the stimulatory, prolactin releasing factors (PRF) and the inhibitory, prolactin inhibitory factors (PIF). The synthesis and secretion of these hormones is influenced by a variety of neuromodulators, neuropeptides and neurotransmitters, including glutamate and aspartate. N-methyl-D-aspartate (NMDA) and N-methyl D, L-aspartate (NMA) are excitatory amino acid analogues of aspartate which bind to a specific receptors in brain known as NMDA receptor. In both rats and primates, the activation of these receptors has been shown to stimulate the release of PRL, probably through discharge of hypophysiotropic factors. The present study attempts to examine the role of NMDA receptors in the central regulation of PRL secretion that may have potential involvement in ovarian function and its alteration by glutamate in various phases of menstrual cycle of rhesus monkey (Macaca mulatta).In addition, a possible modulation of PRL secretion by steroids has been assessed in this primate species. The involvement of NMDA receptors in the regulation of PRL secretion in ovariectomized animal has also been investigated. Immature, immature ovariectomized, and adult intact female monkeys were used in this investigation. NMA (15mglkg BW) or normal saline was infused through, a teflon cannula implanted in the saphenous vain. Blood samples were collected 20-60 min before and 30-60 min after the injection of the drug. NMA was dissolved in normal saline immediately before use and passed through a 0.22 ~m filter at the time of injection. All bleedings were carried under ketamine hydorchloride anesthesia (initial dose Smg/kg BW, im followed by 2.S mg/kg at 30 min. intervals). The plasma level of PRL, Estradiol (E2) and Progesterone (P) were determined by using specific assay systems. In the first set of experiments, the hypothalamic-lactotrope activity under basal conditions as well as the sensitivity ofNMDA receptors to NMA stimulation in immature female rhesus monkey were studied before and after induction of ovulation. Immature (n=5) monkeys were chronically treated with FSH (201 U daily for 12 days, im) to initiate follicular development followed by a bolus injection of human chorionic gonadotrpin (hCG; 1250 IU im) to achieve ovulation. Ovarian conditions during FSH/hCG treatment were examined by ultrosonography, and peripheral estradiol and progesterone were monitored. During the course of FSHlhCG-induced ovarian cycle, all animals were challenged with, NMA at different days of the cycle. The mean basal plasma concentration of PRL during different days of the cycle showed marked differences (F=8.39, P<O.OO I). A single iv injection of NMA produced differential effect on PRL secretion during different days of cycle depending upon the level of PRL secretion under basal condition whereas NMA had no demonstrable effect on PRL secretion before the gonadotropins treatment. Maximum increase (213%) in plasma PRL concentration in response to NMA was observed on the 21 day of the induced cycle. Similarly, NMA had no effect on E2 and P secretion before gonadotropian treatment while on day 12 maximum increase over the basal level (P<0.0006) was induced by a single iv injection of NMA on E2 secretion. NMA has no effect on progesterone secretion over this period except during menstruation (P<0.04, 5.55±0.61nmollL at 0 min and 8.77±1.20 at 10 min). Ovaries of treated monkey exhibited a marked response to gonadotropin treatment by way of enlargement in size, follicular development and formation of corpous lutem as assessed by ultrasonography. In another set of experiments, role of FSH and hCG were studied on fo llicular development and on the induction of normal ovarian cycle in immature female rhesus monkeys. The protocol used was similar as described earlier. Laproscopy was preformed to evaluate the ovarian conditions during different days of the induced cycle. Selected animals were ovariectomized at various time points, before and following ovulation and ovaries were thoroughly studied for follicle/luteal parameters and the tissues were processed for histological observations and for evaluation by light microscopy. Results indicated that before gonadotropin treatment the ovaries showed abundant primordial, primary and preantral follicles. Fluids filled antral follicles were also observed. Ovaries of treated monkey showed a marked response to the gonadotropin treatment by way of enlargement in size, follicular development and corpous luteum formation. Ovarian size was significantly different before and after treatment (P<0.05, P<O.OI , P<O.OOI). Large antral, atretic follicles were observed on day 11 , and on day 15 ovulation points on the surface of large follicles were also observed. Abundant luteal tissues were observed in ovaries of animals on day 22 of treatment. Corpora lutea of various sizes (category A were <6 mm, category B were >6 mm) were also noticed. The ovaries of the monkey examined on day 39 showed regressed corpora lutea. In the third set of experiments the role of E2, P and E2 plus P in influencing the secretion of PRL at the level of hypothalamus was studied by measuring the NMA-induced PRL secretion in ovariectomized immature female monkeys. Treatment of immature monkey with estradiol valerate (500 Jlg/animallweek) or progesterone (50 Jlg/animal/week) resulted in a significant (P<0.03, P<0.005, respectively) increase in PRL concentrations. A single iv injection of NMA (15mg/kg BW) resulted in a rapid and large increase in PRL circulating levels reaching at peak within 15 mm of injection (3171.02±472.70mIU/L at 0 min and 4395.28±242.70 mIU/L at 15 min), whereas a similar dose regimen of NMA failed to induced a significant rise in peripheral concentrations of PRL in ovariectomized animals (P>0.33). Combination of these steroids also induced a significant (P<O.02) increase in PRL secretion after 15 min of NMA injection but these steroids failed to cause further increase in basal PRL level because of the high level of PRL already present. Steroid replacement in ovariectomized monkeys reestablished the PRL responsiveness to NMA stimulation. In the final set of experiment, the hypothalamic lactotropes activity under basal condition as well as the sensitivity ofNMDA receptors to NMA stimulation in adult normal cycling female rhesus monkey (n=4) were studied. The adult animals were studied during follicular, luteal and menstrual phase of the cycle. The animals were challenged with NMA during these days. Mean basal plasma PRL profile was also studied during these days of the cycle. A single iv injection of NMA (15mg/kg BW) produced differential effect of PRL secretion during different stages of the cycle (F=4.51, P<0.02). NMA injection resulted in rapid and large increase in PRL circulating levels reaching a peak within 10 min of injection during these days. But the greatest response was observed during the luteal phase (355.87±87mIU/L at 0 min and 1079.59±189.49 at 10 min) of the cycle. Basal plasma PRL concentrations were also significantly different in the luteal phase as compared to the follicular and menstrual phase of the cycle (F= 11.22, P<O. 001). In conclusion the present study suggests that glutamatergic component of the control system that governs PRL secretion by utilizing NMDA receptor may play an important role in the regulation of changes in the secretion of PRL in the adult and immature female rhesus monkeys. 1. Present finding are noteworthy in demonstrating the enhancing role of FSH on ovarian morphology and the FSH priming is the primary stimulus for early follicle growth in primates. Moreover, it appears that exogenous FSH administration stimulates aspects of early antral follicle development. 2. Pharmacological doses of FSH could induce a normal ovarian cycle in immature rhesus monkeys with profound gonadotropins deficiency. This study confirms the enhancing role of FSH in ovarian steroidogensis. More importantly, the results highlight the predominant role of gonadotropins in inducing ovulation when negligible amount of gonadotropins are present. Our results demonstrate that FSH is of greater importance in the induction of normal ovarian cycle. In conclusion, it is suggested that sexual precocity observed in these animals is due to an increase in estrogen and progesterone secretions by the ovaries. These steroids in turn may affect the hypothalamo-pituitary axis, thus bringing about an alteration of normal process leading to puberty. 3. NMDA driven PRL-release is dramatically influenced by the steroid milieu, in the immature non-human primates. Hypothalamic NMA induced PRL release may be regulated by either estrogen or progesterone but combination of these steroids failed to cause any further increase in PRL levels. This inhibition in further release of PRL may be attributed to the high levels of PRL itself. 4. Prolactin response to NMA in adult female rhesus monkeys during the luteal phase of the menstrual cycle is different from that observed in follicular and menstrual phases and that the steroids may overtly influence the NMDA dependent drive to prolactin release. Results of the present study indicate that NMA involvement in central regulation of PRL secretion may occurs through activation of PRL stimulating system depending upon the physiological state or steroids milieu. It is possible therefore, that the NMA induced release of PRF and PRL is enhanced in the presence of ovarian feedback.