Ágnes Nemeskéri

Ontogenesis of the three parts of the fetal rat adenohypophysis

The sequential changes in the histological pattern of anterior pituitary cytodifferentiation of the rat are described. The first labeled cells were ACTH positive and were detected in the pars tuberalis on postconceptual day 13. On day 14 ACTH cells also appeared in the ventral periphery of the pars distalis. On fetal day 15 the pars tuberalis anlage was characterized by numerous well-stained ACTH cells and by some weakly labeled FSH-beta, LH-beta, TSH-beta, GH and PRL cells while the pars distalis showed only ACTH positivity. On day 16 of gestation the ACTH cells were equally distributed throughout the whole pars distalis, while LH-beta, FSH-beta, TSH-beta, PRL and GH immunoreactive cells were localized either in the ventral region of the pars distalis only or were evenly distributed throughout the pars distalis. The present immunocytochemical data suggest that in the pars distalis the hypophyseal cell differentiation follows a clear rostrocaudal, ventrodorsal direction and that the time sequence of the functional differentiation of the adenohypophysis is pars tuberalis, pars distalis and pars intermedia.

The role of PACAP in gonadotropic hormone secretion at hypothalamic and pituitary levels

The presence of pituitary adenylate cyclase-activating polypeptide (PACAP) and its mRNA in the three levels of the hypothalamo-hypophyseal-ovarian axis was previously demonstrated using immunohistochemistry, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR). In the hypothalamus, PACAP is present in neuroendocrine effector cells and in the median eminence. In the anterior pituitary and ovary, PACAP is transiently present during the proestrous stage of the estrous cycle. In the pituitary, PACAP was observed in gonadotropes. In the ovary, PACAP was demonstrated in the granulosa cells of the preovulatory ovarian follicles. The effect of PACAP on luteinizing hormone (LH) secretion was demonstrated in in vivo and in vitro models. In our work we have studied the role of PACAP in gonadotropic hormone secretion at hypothalamic and pituitary levels. At the hypothalamic level, PACAP, administered intracerebroventricularly to female rats before the critical period of the proestrus stage, can inhibit LH release and ovulation. Its inhibiting effect is mediated through corticotropin-releasing factor (CRF) and endogenous opioids. PACAP administered to neonatal female rats delayed the onset of puberty by influencing the luteinizing hormone-releasing hormone (LHRH) neuronal system. In the pituitary gland, the release of PACAP depended on the stage of the estrous cycle and on the time of day the animals were sacrificed. On the day of proestrus, the number of PACAP-releasing cells showed a diurnal change with two peaks (in the morning and in the evening). The peak was much higher in the evening at the end of the LH surge than in the morning.

Cell immunoblot assay study demonstrating the release of PACAP from individual anterior pituitary cells of rats and the effect of PACAP on LH release

The presence of pituitary adenylate cyclase activating polypeptide (PACAP) was previously demonstrated in the anterior pituitary by radioimmunoassay, immunohistochemistry, and reverse transcript-polymerase chain reaction (RT-PCR). With the use of cell immunoblot assay (CIBA), when the pituitary cells were cultured on nitrocellulose membrane, the release of PACAP by individual anterior pituitary cells was observed. The released peptide, trapped by the nitrocellulose membrane forming a blot around the cells, was demonstrated by immunocytochemistry. Double labeling revealed that a part of PACAP-immunoreactive cells can release LH as well. With the use of sandwich enzyme immunoassay (S-EIA), it was found that the concentration of PACAP in the anterior pituitaries is 10(-10) M. In cell culture in a similar concentration, PACAP stimulated the LH release from female gonadotropes, but did not influence it from male ones. The stimulated release of LH was indicated by the enhancement in the diameter of LH blots compared to the untreated control cultures. We concluded that PACAP may be released from the anterior pituitary cells in a concentration which would be able to influence LH release not only in vitro but under in vivo conditions as well. The effect of PACAP on LH release was different in female and male pituitary cultures.

Effect of PACAP on LH release studied by cell immunoblot assay depends on the gender, on the time of day and in female rats on the day of the estrous cycle

We have previously demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) can be released from cultured rat anterior pituitary cells and when added to the medium in physiological concentration it releases LH from individual gonadotropes. In the present work, we studied whether the release of PACAP and the responsiveness of LH cells to PACAP depend on the gender, on the time of day when the animals were sacrificed, and in females on the stage of the estrous cycle. Anterior pituitary cells were cultured on nitrocellulose membrane. We found that the number of PACAP releasing cells was higher in proestrous than in diestrous female or in male rats and their number was always higher in the evening than at the other times. The effect of PACAP on LH cells was stimulatory in the morning of proestrus and diestrus. In proestrous rats, PACAP did not influence LH release in the afternoon or the evening, but in diestrous rats it decreased it in the afternoon and the evening. In males, there was a decrease of LH due to PACAP treatment at 10 and 20 h; however, PACAP did not influence LH at 16 h. It was concluded that in vivo PACAP might be involved in the circadian and episodic release of LH at pituitary level.

Changes in Hypophyseal Luteinizing Hormone (LH) Content during Fetal and Early Postnatal Life, and Capacity of Fetal and Early Postnatal Pituitaries to Synthesize and Release LH in vitro

The developmental pattern of hypophyseal luteinizing hormone (LH) during fetal and early postnatal life, and the capacity of fetal and early postnatal pituitaries to synthesize, release and store LH in the absence of any hypothalamic influence were investigated. Pituitary radioimmunoassayable LH was detectable by fetal day 12; its level remained low up to fetal day 17 and thereafter continued to increase up to postnatal day 2 with sharp rises occurring between day 17 and 18 of gestation and after birth. Cultured fetal and early postnatal pituitary primordia were able to synthesize, to release and store the hormone, however, the pattern of these cellular functions varied according to the animal age when explantation was performed. Data derived from cultured pituitaries showed a gradual rise in the daily hormone content of the medium between fetal day 13 to 17. From fetal day 19 onwards, however, the daily discharge of LH decreased. These findings suggest that the fetal and early postnatal pituitary has the capacity to synthesize and release LH, furthermore after a period of independent differentiation hypothalamic signals appear to be required for pituitary hormone release.

Effect of intratesticular administration of anti-corticotropin-releasing factor antiserum (a-CRF) on testicular function in neonatal rats.

Summary. The possible physiological role of testicular corticotropin‐releasing factor (CRF) in the regulation of testicular functions was studied in neonatal rats. Two microlitres of anti‐CRF‐antiserum (dilution: 1: 10 or 1: 100) was injected intratesticularly to 5 d‐old rats with two testes and to hemicastrates. Five days after hemicastration and treatment of the remaining testis with the antiserum, serum testosterone concentration and basal testosterone secretion in vitro decreased significantly. Unilateral testicular injection of a‐CRF in rats with two testes resulted in a significant drop in serum testosterone level with no change in basal testosterone production. Data indicate that in neonatal rats testicular CRF might be a local stimulator of steroidogenesis.

The effect of neonatal vasectomy on testicular function

Summary:  The effect of unilateral or bilateral vasectomy on testicular weight and basal testosterone production in vitro was studied in neonatal intact and hemicastrated rats. Five days after right‐side vasectomy ipsilateral to vasectomy testicular weight increased, and basal testosterone production decreased. Ten days postvasectomy the changes were opposite, and affected both testes. In hemicastrated animals hemivasectomy did not interfere with compensatory hypertrophy but induced a significant decrease in basal testosterone production. The data of the present study suggest that in neonatal animals intact ductus deferens bundle(s) is also required for the full control of testicular weight and basal testosterone secretion.

PACAP and VIP in the photoneuroendocrine system. From the retina to the pituitary gland.

The concept of the photoneuroendocrine system (PNES) was first published by Scharrer in 1964. 1 According to his theory photic stimuli from the eye are conducted, not only to visual centers, but through a neuronal chain to neuroendocrine effector cells that send hormonal stimuli through the anterior pituitary to peripheral target organs. These structures form the PNES, which has four levels as follows: (1) a twoway connection between the eye and the hypothalamus; (2) retinorecipient areas; (3) neuroendocrine effector cells; and (4) pituitary gland. In the present work we have studied the distribution of PACAP and VIP immunoreactivities in all the four levels of PNES and the effect of enucleation (removal of eyes) on the intensity of PACAP and VIP immunostaining in the hypothalamic nuclei and the pituitary gland.

Prolactin-synthesizing and prolactin-releasing activity of fetal and early postnatal rat pituitaries: In vivo and in vitro studies using RIA, reverse hemolytic plaque assay and immunocytochemistry

In vivo and in vitro prolactin (PRL)-synthesizing and PRL-releasing activity of fetal (days 12-22) and early postnatal (days 1-10 after birth) rat pituitaries were studied by means of radioimmunoassay (RIA), reverse hemolytic plaque assay and immunocytochemistry. Using RIA, PRL could first be detected, both in the pituitary and in the serum, on day 17 of fetal development. From this day on, pituitary PRL gradually increased, the rise was particularly marked during the postnatal period and became depressed for the first 10 days of postnatal life. On fetal day 18, 12-15% of monodispersed pituitary cells displayed PRL immunopositivity, but only 3-5% of PRL-positive cells were plaque-forming, i.e. released PRL. By the end of gestation 19-25% and on postnatal day 10 42-45% of all pituitary cells were PRL cells and 31-35 and 15-17% of PRL-positive cells, respectively released PRL. Both pre- and postnatal PRL cells in monolayers were insensitive to TRH treatment. Pituitary primordia immunocytochemically and radioimmunologically negative for PRL (13- to 14-day-old fetal) when placed in serum-free organ culture were able to synthesize and release PRL. Fetal pituitary exhibited a highly regular increasing pattern of daily PRL release during a 7-day-culture period. Data obtained both in vivo and in vitro did not exhibit any sex differences. The present findings are consistent with all those observations suggesting an early emergence of fetal rat pituitary lactotrophs. The in vitro results support the concept that Rathkes pouch cells have substantial degree of independence from extrapituitary regulatory actions in the expression and further progression of specific functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Intratesticular injection of [d-Met2-Pro5]enkephalinamide suppresses testosterone secretion of the testis of immature rat

The possible role of enkephalin in the local control of testicular function was studied in neonatal rats. 5- and 10-day old hemicastrated rats were treated intratesticularly with an enkephalin analog [D-Met2-Pro5]enkephalinamide. In 5-day-old rats local injection of different doses (0.1-0.3 micrograms/testis) of the peptide suppressed basal testosterone secretion in vitro in a dose-dependent manner 2 h posttreatment. Intratesticular administration of naloxone prior to enkephalin treatment prevented the decrease in basal testosterone production induced by the opioid agonist. In 10-day-old animals intratesticular injection of 1.0 and 3.0 micrograms/testis of enkephalinamide reduced serum testosterone concentration and basal testosterone secretion in vitro. Systemic injection of the peptide produced no change in steroidogenesis. These results suggest that enkephalins might be among the intratesticular factors regulating Leydig cell functions.