S. R. Ojeda

Dopamine and norepinephrine stimulate somatostatin release by median eminence fragments in vitro

Abstract The effect of catecholamines on somatostatin release by median eminence (ME) fragments was evaluated using an in vitro incubation system. Adult male rats were used as tissue donors. Somatostatin release was readily detected during short-term incubations (10 and 30 minutes). Dopamine (DA) significantly stimulated somatostatin release during a 30 minute incubation period at the two doses tested (0.6 and 6 μM). Under similar conditions, norepinephrine (NE) stimulated somatostatin release only at the 6 μM dose. Using a shorter incubation period (10 min) and a 6 μM dose, only DA stimulated somatostatin release. The effects of DA and NE were specifically blocked by the in vitro addition of pimozide or phentolamine, respectively, suggesting that dopaminergic and noradrenergic receptors may be present in the somatostatinergic terminals of the ME. The results indicate that both DA and NE may be involved in the regulation of somatostatin secretion.

The Onset of Female Puberty: Studies in the Rat

Publisher Summary nThis chapter discusses the onset of female puberty from the studies in rat. The developmental regulation of the neuroendocrine reproductive axis at the level of its three basic components: the hypothalamus, the anterior pituitary gland, and the ovaries were investigated in both in vivo and in vitro. The developmental process that leads to puberty in the female rat is composed of an extraordinary, complex series of interrelated events. The CNS plays a pivotal role in the process by controlling both anterior pituitary function through the secretion of hypothalamic factors and the ovary via pituitary hormones and direct neural inputs. Based on the results, it was noticed that the first activational period occurs during infantile development and is expressed as an enhancement in FSH secretion, with sporadic elevation in LH levels. The second activational period signals the end of juvenile development and represents the first neuroendocrine manifestation of the onset of puberty. This activational period is also determined by a centrally driven, gonadal-independent mechanism and is expressed as a diurnal change in pulsatile LH release. The third and final activational period occurs more abruptly, and is predominantly determined by an increased output of ovarian steroids, especially E2. This period corresponds to the preovulatory discharge of LHRH which directly promotes the first surge of gonadotropins. The ovaries grow under the influence of the gonadotropins, which is modulated by milk LHRH during the neonatal–infantile development, and facilitated afterward by the growth hormone and prolactin. An additional facilitatory control mechanism is provided by adrenergic and peptidergic nerves, and by EPI of adrenal medullary origin.

A rapid microprocedure for isolating RNA from multiple samples of human and rat brain

In order to establish a routine procedure for isolating undegraded RNA from small amounts of rat and human brain tissue, several techniques were investigated. Initial studies demonstrated that undegraded RNA could not be reproducibly isolated from milligram amounts of brain tissue homogenized in an aqueous medium. Several isolation techniques utilizing tissue homogenization in the denaturing agent guanidinium chloride were compared. This method of homogenization, followed by sedimentation of RNA through cesium chloride, resulted in good yields of undegraded translationally active RNA. A maximum of 6 RNA samples could be processed simultaneously. In contrast, when homogenization in guanidinium chloride was followed by repeated guanidinium chloride-ethanol precipitations many samples could be processed simultaneously. The resulting RNA yields were low. The introduction of several modifications in the guanidinium chloride-ethanol precipitation technique resulted in a high yield of undegraded translationally active RNA. DNA was removed by two guanidinium-ethanol precipitations. Residual protein was digested with proteinase K. RNA was precipitated after extraction with phenol-chloroform-isoamyl alcohol. This refined procedure allows the recovery, in high yields, of translationally active undegraded RNA which is both DNA and protein free. Thirty-six samples can be processed in one day.

The role of prostaglandins in the control of gonadotropin and prolactin secretion

Abstract Experimental evidence has recently accumulated indicating that administration of some prostaglandins (PGs), particularly those of the E and F series can evoke release of gonadotropic hormones (LH and FSH) and prolactin (PRL) by the anterior pituitary gland. Rather than acting on the pituitary directly, PGs are thought to exert their effects on the hypothalamus. In the case of LH and FSH they stimulate the release of lutenizing hormone-releasing hormone (LHRH). In the case of PRL, PGEs may act by inhibiting the release of PIF (prolactin-inhibiting factor) and/or by enhancing the release of PRF (prolactin-releasing factor). Pharmacologic inhibition of PG synthesis depresses the PRL release induced by estrogen without reducing pituitary PRL release from TRH. Blockade of PG synthesis also decreases LH release in a variety of circumstances, but does not inhibit the pituitary response to LHRH, thus supporting the concept that PGs may play a physiological role in the neural control of the release of this hormone. At the hypothalamus, PGE2 appears to stimulate LHRH release by acting directly on LHRH-secreting elements located in the medial basal hypothalamic-median eminence region (MBH-ME) and the preoptic-anterior hypothalamic (POA-AHA) areas. Radioimmunoassayable PGE and bicassayable PGF content of the ME of the rat is much greater than that of the MBH. Similarly, in vitro release of PGEs by the ME is several times greater than that of the MBH. In vitro incubation of MEs with norepinephrine (NE) results in PGE ands TI-release by the tissue. Inhibition of PG synthesis with indomethacin prevents not only the elevation in PGE levels induced by NE but also the increase in LHRH release induced by both dopamine (DA) and NE suggesting that PGs are physiologically involved in the process of neurotransmitter induced release of LHRH. Moreover, release of PGEs from the ME is reduced by incubation of the tissue with α-adrenergic or dopaminergic receptor blockers and the in vitro response of the ME LHRH terminals to PGEs is enhanced by in vivo pretreatment with ovarian steroids. In conclusion, these recent observations coupled to the aforementioned findings provide strong evidence in support of a physiological role for PGs in the hypothalamic control of gonadotropin release.

The first proestrus in the female rat: circulating steroid levels preceding and accompanying the preovulatory LH surge.

Summary On the day of the first proestrus in the female rat, serum LH levels were very low throughout the morning and increased markedly between 1200 and 1600 hr to reach maximum values at this time. By 1800 hr the LH titers, though still elevated, were already declining. Serum estradiol (E2) levels were maximally increased at 0800 hr (76 pg/ml) and decreased somewhat between this time and 1600 hr to finally return to prepubertal values by 1800 hr. Although morning progesterone (P) levels were higher than those of prepubertal rats, the greatest increase in serum P occurred in the afternoon of proestrus with a time course that slightly lagged behind that of LH, so that maximal P values (56 ng/ml) were observed by 1800 hr. At 0800 hr on proestrus serum testosterone (T) was higher than in prepubertal rats, declined thereafter to a nadir at 1200 hr and increased again briefly at the time of the LH surge. Serum dihydrotestosterone (DHT) did not vary throughout the proestrus day and did not differ from levels in prepubertal animals. Ovarian weight increased dramatically between 0800 and 1800 hr, with some increment already being observed at a time in which serum LH was still at basal levels. The occurrence of elevated E2 levels, in the absence of any apparent elevation in mean basal radioimmunoassayable LH levels prior to the afternoon LH surge, supports the view that the onset of puberty in the female rat is a consequence of the triggering of E2 positive feedback on LH release.

Effect of experimental alterations in serum levels of 5α-androstane-3β, 17β-diol on the timing of puberty in the female rat

It has been suggested that in the rat, 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) is physiologically involved in restraining the onset of female puberty. To test this hypothesis several experiments were performed. In normal rats, serum levels of 3 alpha-diol decline slightly during the initial phases of puberty and then sharply several hours before the afternoon preovulatory LH surge on the day of first proestrus. Inhibition of 5 alpha-reductase activity with a highly specific inhibitor, 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstane-3-one (4-MA) strikingly depressed both ovarian content and serum levels of 3 alpha-diol, but failed to advance vaginal opening or first ovulation. Administration of different doses of 3 alpha-diol to juvenile rats via Silastic capsules produced a dose-related increase in serum 3 alpha-diol levels. Titers attained ranged from values similar to those of untreated juvenile rats to levels more than 10-fold higher. None of the concentrations, however, inhibited the LH surge and ovulation induced by pregnant mare serum gonadotropin (PMSG). When a similar treatment was administered to normally maturing rats, only the high dose of 3 alpha-diol delayed the age of vaginal opening and of first ovulation. Serum 3 alpha-diol levels attained with this dose were markedly higher than those of untreated juvenile rats (1,086 +/- 267 vs. 124 +/- 14 pg/ml, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the central effect of prolactin in inducing precocious puberty in the female rat

Implantation of prolactin (PRL) into the median eminence (PRL-ME implants) of 23 day old female rats markedly advanced the onset of puberty, as measured by the age at vaginal opening and at first ovulation. Precocious puberty was preceded by steroidogenic activation of the ovary, as reflected by increases in uterine weight and an enhanced in vitro steroidal responsiveness of the ovary to hCG. The stimulatory effect of PRL-ME implants could not be attributed to alterations in the release of LH, FSH, GH or TSH from the anterior pituitary. Likewise, the PRL effect was neither exerted through the adrenal gland nor involved activation of a direct neural, vagal-mediated influence on the ovary. Furthermore, the effect of PRL-ME implants was not due to a decrease in pituitary secretion of opioid substances, which appear to restrain chronically gonadotropin release during female prepubertal development. These latter experiments also showed that administration of the opioid agonist, morphine, can delay the onset of puberty in the female rat. Although local exposure of the medial basal hypothalamus to high PRL levels is extremely effective in accelerating puberty, the mechanisms by which this effect is exerted remains to be elucidated.

Cyclic GMP and cyclic AMP levels in median eminence, hypothalamus and pituitary gland of the rat after decapitation or microwave irradiation.

Cyclic AMP and cyclic GMP content of the median eminence (ME), medial basal hypothalamus (MBH), posterior pituitary (PP) and anterior pituitary (AP) of inact male rats was measured by radioimmunoassay following decapitation or exposure to microwave irradiation (MW). Levels of both nucleotides in MW irradiated animals were higher in all three nervous tissues studied than in the AP. Following decapitation cyclic AMP levels increased strikingly in the MBH and ME, slightly in the AP, but remained unchanged in the PP. Cyclic GMP levels increased more markedly in the ME than in the MBH, slightly in the PP and did not change in the AP. The distinct synthesizing capability of the cyclic AMP and cyclic GMP generating systems in the ME, a region rich in neurohormones known to control anterior pituitary function, raises the possibility that these nucleotides are involved in the process of neurohormone secretion by the ME.

Physiological peripubertal activation of the ovary is not reproduced by pregnant mare serum gonadotropin (PMSG) administration.

Abstract During the days preceding the first ovulation the ovary of the rat exhibits a remarkable increase in estradiol (E2) and progesterone (P) release in response to gonadotropins. No such increase is observed in the case of androgens (A, testosterone + dihydrotestosterone). The present experiments were undertaken to examine the possibility of reproducing these developmental events by stimulating the ovary with a gonadotropin that has substantial FSH-like activity. In vivo administration of pregnant mare serum gonadotropin (PMSG) to juvenile 29-day-old rats greatly increased the in vitro E2 and A response to human chorionic gonadotropin (hCG) measured 2 days later in the morning. The magnitude of the A response was significantly larger than that of ovaries from juvenile animals or rats in first proestrus. The E2 response was much greater than that of juvenile ovaries but similar to that of ovaries from late proestrous rats. In contrast, the P response to hCG was not enhanced by PMSG. In fact the response was similar to that of juvenile ovaries and markedly less than that of first proestrous rats. This decreased P response was not due to a greater conversion of P to its less active metabolite 20α-hydroxy-4-pregnen-3-one (20α-OH-P). The results suggest that PMSG enhances the E2 and A response of immature ovaries to hCG at the expense of that of P. Treatment of immature rats with PMSG may represent a useful model to study E2 release from preovulatory ovaries, but it cannot be used to reproduce in its entirety the developmental changes in steroidal response to gonadotropins associated with normal puberty.

Physiological and Biochemical Dissection of Mechanisms Underlying Puberty

The last few years have witnessed the emergence of a more comprehensive understanding of the mechanisms underlying the onset of mammalian puberty. At present, several groups utilizing different animal models are actively engaged in studying various aspects of the pubertal process, more noticeably the luteinizing hormone-releasing hormone (LHRH)-gonadotropin component of the reproductive axis.