Time Of Vaginal Opening Research Articles

Effects of constant infusion with insulin-like growth factor-I (IGF-I) to immature female rats on body weight gain, tissue growth, and sexual function : Evidence that such treatment does not affect sexual maturation or fertility.

Plasma levels for insulin-like growth factor-I (IGF-I) steadily increase in female rats between 20 and 40 d of life, and this increase is intimately related to the wellknown growth spurt occurring at this age. Since specific actions of IGF-I related to sexual function have been described at the ovarian and hypothalamic levels, an endocrine role of rising circulating IGF-I levels during sexual maturation cannot be excluded. Therefore, the impact of adult-type plasma IGF-I levels during the juvenile age, on body weight (BW) gain, growth of several organs, sexual development, and fertility has been evaluated. Female Sprague-Dawley rats were infused with rhIGF-I (2 and 4 μg/g BW/d, using Alzet minipumps), between 20 and 41 d of life. When infusing 2 μg/g BW/d, plasma levels for IGF-I were increased 1.5- to 2-fold over controls at all ages studied. They were further increased with the higher dosage, but only after 35 d of age. Plasma levels for insulin-like growth factor binding protein (IGFBP)-1 to-3 were clearly increased. BW gain was significantly increased, but only with the higher dosage. Tail length was never modified. In contrast, a growth acceleration for spleen, kidneys, adrenals, and ovaries was observed with both dosages. The ovarian weight of treated animals represented approx 140% of control animals with the 4 μg/g BW/d dosage. Histology of the enlarged ovaries did not reveal any abnormalities. No meaningful modification of the timing of vaginal opening was observed, and fertility was not compromised by previous rhIGF-I infusion during the 20-41 d age period. In summary, early exposure to increased (adult-like) plasma IGF-I levels did not modify BW gain or tail length, but affected the development of spleen, kidneys, adrenals, and ovaries. Exposure to supraphysiological plasma IGF-I levels (>1200 ng/mL), accelerated BW gain and increased the weight of all organs studied. No signs of precocious sexual maturation were seen and fertility was normal. In conclusion, prematurely increased plasma IGF-I levels affected somatotropic parameters, but not the onset of sexual function.

Opioids regulate cell proliferation in the developing rat uterus: effects during the period of sexual maturation.

The present studies demonstrate, for the first time, that the rate of DNA synthesis in rat uterus of 21-32 days of age is inhibited by opioid peptides [D-Met2, Pro5]enkephalinamide. At around the time of vaginal opening (approximately 33 days) the opioids failed to act. High-affinity nuclear [3H]naloxone binding sites with linear Scatchard plots were detected in the uteri during the opioid-sensitive periods of DNA synthesis. Characteristics of these binding sites and the opioid sensitivity of uterine DNA synthesis are dependent on the age of the animals, the level of circulatory oestradiol and/or the maturity of the nuclear oestrogen receptor system.

Evaluation of the developmental neuroendocrine and reproductive toxicology of aluminium

Two experiments evaluating functional endpoints pertaining to the developmental neuroendocrine effects of aluminium in the rat are reported. A total of 31 timed mated dams were fed by daily gastric gavage 0, 5, 25, 50, 250, 500 or 1000 mg/kg body weight/day aluminium as a solution of aluminium lactate in distilled water from days 5 to 15 of gestation. The 390 offspring were evaluated for morphological and physiological parameters of reproductive functioning, including birth weight, anogenital distance (AGD), timing of vaginal opening, regularity of oestrous cycles, duration of pseudopregnancy (PSP), number of superovulated oocytes, and gonadal weight. No consistent or reproducible findings suggestive of toxic effect were found in the parameters of birth weight, AGD, timing of vaginal opening, duration of PSP, number of superovulated oocytes, and adult gonadal weight. A temporary increase in the proportion of aberrant oestrous cycles was detected during the first four cycles after vaginal opening, in the 250 mg/body weight/day group, with none by the fifth consecutive oestrous cycle. These results suggest that, apart from a transient disturbance of oestrous cycle regularity, aluminium does not have a developmental reproductive toxic effect.

Temporal Energy Restriction and the Timing of Vaginal Opening in Female Rats.

The effect of a temporary dietary restriction (10 g/day) duirng gestation or lactation on the subsequent maturation of female offspring was examined. Mother rats were divided into 6 groups according to the period of restriction: namely, Group A (between days 0 and 7 of gestation), Group B (between days 8-14 of gestation), Group C (between days 15-21 of gestation), Group D (between days 1-7 of lactation), Groupt E (between days 8-14 of lactation), Group F (no restriction). In Group E, the mother's body weight decreased by 17% compared to that on day 0 of gestation. Female pups in this group showed retardation in growth rate concurrent with a delay in vaginal opening. No significant effects were found in any other groups. These results suggest that adequate nutrition during midlactation is critical for optimal growth of female pups and subsequent opening of the vagina.

The Effect of Prenatal Exposure to the Phytoestrogen Genistein on Sexual Differentiation in Rats

Exposure to naturally occurring estrogens during critical periods of development can alter morphologic and physiologic markers of sexual differentiation. The current experiment characterizes the effects of in utero treatment with genistein, an isoflavonoid phytoestrogen, on birth weight, anogenital distance (AGD) at birth. GnRH stimulated luteinizing hormone (LH) secretion, volume of the sexually dimorphic nucleus in the preoptic area of the hypothalamus (SDN-POA), puberty onset, and vaginal cyclicity. Pregnant Charles River CD rats were injected sc daily on gestation day 16-20 with either 25,000 micrograms genistein (G25), 5,000 micrograms genistein (G5), 5 micrograms diethylstillbestrol (DES), 50 micrograms estradiol benzoate (E), or corn oil alone for controls. Birth weights and anogenital distance was taken and exposed progeny were subsequently used in two experiments. In Experiment 1 intra-atrial catheters were placed in adult castrated rats, GnRH was given iv, serial blood samples were drawn and sera were assayed for LH by radioimmunoassay (RIA). Brains obtained by subsequent decapitation were saved for histology. In Experiment 2, females were monitored for timing of vaginal opening as a marker of puberty onset, and vaginal smears were taken to monitor cyclicity. G25-treated females and DES- and E-treated animals of both sexes had decreased weights at birth compared with controls. G5- and E-treated animals of both sexes and DES males had smaller AGD than controls. No significant differences in pituitary responsiveness to GnRH were found among treatment groups. There was a nonsignificant decrease in SDN-POA volume in G5-treated females while DES- and E-treated females had increased SDN-POA volume compared with controls. G5-treated females had delayed puberty onset, and DES-treated females had atypical vaginal cycles in comparison with controls. The results confirm that prenatal exposure to estrogens in the environment can influence sexual differentiation. Our previous experiments have demonstrated that castrate female rats exposed as neonates to genistein have decreased pituitary responsiveness to GnRH challenge and enlarged SDN-POA volume in comparison with controls. Prenatal genistein at these dosages did not significantly alter these markers. However, genistein did mimic other estrogens' effects on AGD and birth weight and had a unique influence on puberty onset. Not only are genistein's effects different from other estrogens, but dosage and timing of exposure during development appear to be important factors in genistein's ability to modify these end points.

Glutamate: a major excitatory transmitter in neuroendocrine regulation.

Excitatory amino acids (EAAs), such as glutamate and aspartate, are found in large concentrations in presynaptic boutons of a variety of important hypothalamic nuclei, including the arcuate nucleus, supraoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, organum vasculosa of the lamina terminalis (OVLT) and preoptic area (POA). Likewise, the different ionotropic/metabotropic EAA receptor subtypes are found in the same regions of the hypothalamus although there are differences in their individual patterns of localization. Furthermore, there is evidence supporting the presence of ionotropic N-methyl-D-aspartate (NMDA) receptors and non-NMDA (kainate and AMPA) receptors in the anterior lobe, intermediate lobe and posterior lobe of the pituitary. The majority of work to date has focused on the role of EAAs in the control of LH secretion. Administration of glutamate, NMDA, kainate or AMPA leads to rapid LH release mediated through the stimulation of hypothalamic GnRH release. The major site of NMDA action appears to be the OVLT/preoptic area--where GnRH cell bodies reside, whereas AMPA and kainate have been suggested to act primarily at the arcuate nucleus/median eminence--the site of GnRH nerve terminals. There is evidence that some of the effects of glutamate on GnRH release may involve activation of the novel neurotransmitter nitric oxide and possibly catecholamines. The physiological importance of EAAs in the control of LH surge expression is evidenced by the findings that the steroid-induced LH surge in ovariectomized animals and the preovulatory LH surge in cycling animals and in PMSG-primed animals is blocked by treatment with specific NMDA receptor antagonists, or non-NMDA receptor antagonists. EAAs also appear to be important in regulating the normal pulsatile pattern of LH release as evidenced by the finding that both the NMDA antagonist, AP5, and the AMPA/kainate antagonist, DNQX, lower mean LH levels, LH pulse amplitude and LH pulse frequency in the adult ovariectomized rat. A role for NMDA receptors in the achievement of puberty has been suggested since activation of NMDA receptors has been shown to advance the time of vaginal opening in the immature female rat, while kainate and DNQX were without effect. Steroids have been reported not to affect NMDA receptor binding in the hypothalamus; however, steroids appear to up-regulate AMPA receptor GluR1 subunit levels and non-NMDA receptor binding in the hypothalamus. Steroids also increase the release rates of glutamate and aspartate in the POA during the steroid-induced LH surge in the ovariectomized adult rat.(ABSTRACT TRUNCATED AT 400 WORDS)

Pubertal Development of Estrogen Receptors in the Rat Brain

The regional distribution of estrogen receptors (ER) was studied in the brain of the female rat over the peripubertal period. Increases were observed in nuclear ER occupation as well as cytosolic progestin receptor induction in the period leading up to the time of vaginal opening, consistent with an increased availability of estrogen to the brain. ER binding capacity, however, was remarkably similar in pre- and postpubertal rats. Using a microdissection-based nuclear ER assay, small increases in the total ER content of the hypothalamic ventromedial and arcuate nuclei were observed in the female at around the time of the onset of reproductive cyclicity. In vivo autoradiographic studies confirmed the existence of a small increase in estrogen binding in these two brain regions at around the time of vaginal opening, as well as transient increases in estrogen retention within the preoptic area and bed nucleus of the stria terminalis. These changes in estrogen binding in the brain may play a role in pubertal alterations in the feedback sensitivity of the brain to circulating estrogen.

Excitatory Amino Acids: Function and Significance in Reproduction and Neuroendocrine Regulation

Excitatory amino acid neurotransmission is an essential component of the neuroendocrine transmission line that regulates anterior pituitary luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. Excitatory amino acids (EAAs), such as glutamate and aspartate, are found in largo concentrations in presynaptic boutons of a variety of important hypothalamic nuclei, including the arcuate nucleus, the suprachiasmatic nucleus, the supraoptic nucleus, the paraventricular nucleus, and the preoptic area. EAA receptors can be divided into two broad groups, namely, ionotropic and metabotropic receptors. Ionotropic receptors are subdivided into NMDA ( N-methyl-D-aspartate), kainate, and AMPA (DL-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Their main mode of action is by the modulation of Na +, K +, and Ca 2+ ion channels. Metabotropic receptors, on the other hand, act by a G-protein-stimulated release of intracellular Ca 2+ or modulation of adenylate cyclase activity. The different EAA receptor subtypes are found in a variety of areas of the hypothalamus and the brain. In a variety of species, the administration of glutamate, NMDA, or kainate leads to LH release mediated through the stimulation of hypothalamic gonadotropin hormone-releasing hormone (GnRH) release. The major site of NMDA action appears to be the preoptic area—where GnRH cell bodies reside. AMPA and kainate appear to act primarily at the arcuate nucleus/median eminence, the site of GnRH nerve terminals. NMDA may also act upon noradrenergic neurons in the locus coeruleus to influence hypothalamic GnRH release. The steroid-induced LH surge in ovariectomized animals and the preovulatory surge of LH in cycling animals and in pregnant mare's serum gonadotropic-primed animals are blocked by the NMDA antagonist MK801 and the AMPA/kainate antagonist DNQX. MK801 also suppressed FSH surges in most instances, whereas DNQX had no effect on FSH surges. In the ovariectomized female rat, both the NMDA antagonist AP5 and the AMPA/kainate antagonist DNQX, lowered mean LH levels, LH pulse amplitude, and LH pulse frequency. Activation of NMDA receptors advanced the time of vaginal opening in the immature female rat, while kainate and DNQX were without effect. Gonadal steroid removal (castration) did not alter NMDA receptor levels or affinity in the hypothalamus of female or male rats. Likewise, steroid replacement to castrate rats did not affect hypothalamic NMDA receptor levels or NMDA R 1 mRNA levels. Similarly, NMDA and kainate receptor levels in the hypothalamus did not change during the time of puberty in the female rat. In contrast, AMPA receptor (GluR 1) immunoreactive levels in the magnocellular preoptic area (mPOA), the arcuate nucleus (ARC), and the suprachiasmatic nucleus (SCN) were found to be markedly elevated during the time of the LH surge in estradiol-progesterone-treated castrate rats compared to those of the vehicle-only-treated castrate rat. The release rates of glutamate and aspartate in the POA were found to be significantly elevated during the steroid-induced LH surge in the ovariectomized adult rat. Hence, the induction of the LH surge by steroids may involve enhanced EAA neurotransmission mediated by steroid-induced elevations in the release of glutamate and aspartate in the POA and AMPA receptors in the POA, the ARC, and the SCN. Evidence also exists supporting a role of EAAs in the release of other pituitary hormones, such as prolactin, ACTH, growth hormone, oxytocin, and vasopressin, as well as circadian rhythm induction and regulation. The above evidence points to a pivotal role of EAAs acting at the level of the hypothalamus in the neuroendocrine regulation of a variety of hormonal systems with the largest amount of data available on the control of gonadotropin secretion.

A phytoestrogen diet induces the premature anovulatory syndrome in lactationally exposed female rats.

The effects of a phytoestrogen diet on sexual differentiation were examined in lactationally exposed rat pups. Rat dams were provided a semipurified diet containing the isoflavonoid coumestrol at a concentration (0.01%) previously found to be uterotrophic. Coumestrol treatment did not significantly alter the time of vaginal opening, although vaginal opening did occur at a lighter body weight. By 132 days of age, 83% of coumestrol-treated females exhibited the cornified smears of a persistent estrous state. By contrast, 91% of control animals were cycling regularly at 132 days of age. Estradiol stimulation failed to elicit an LH elevation in the coumestrol-treated animals, suggesting the possibility of neuroendocrine impairments. These findings indicate that the female offspring of mothers fed a low-level phytoestrogen diet during lactation manifest early and nearly universal disruption of cyclicity of the persistent-estrus type.

Role of Excitatory Amino Acid Neurotransmission during Puberty in the Female Rat

The purpose of the present study was to determine the contribution to and role of N-methyl- d-aspartate (NMDA) and non-NMDA receptor-mediated neurotransmission in the initiation of puberty in the female rat. The ability of NMDA and the non-NMDA receptor agonist kainate to facilitate puberty was compared, the effect of a non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) on the timing of puberty was investigated, and the question of whether NMDA or kainate receptor binding in the hypothalamus changes during sexual maturation was examined. NMDA treatment effectively and significantly advanced puberty by an average of 2.5 days compared to that in vehicle controls ( P < 0.001). The body weight of NMDA-treated rats at the time of vaginal opening was significantly lower than that of vehicle controls, reflecting that puberty had occurred at an earlier age. In contrast to NMDA, kainate treatment had no effect on the timing of puberty in female rats. Body weights of kainate-treated rats at the time of vaginal opening were also similar to those of vehicle controls. Treatment with the non-NMDA receptor antagonist DNQX (15 nmol at 1400 and 1600 h) through third ventricle administration from Days 29 to 36 had no effect on the timing of puberty in female rats, further suggesting that non-NMDA receptor activation may not play a critical role in puberty. Finally, it was demonstrated that neither NMDA nor kainate receptor binding in the hypothalamus changes during sexual maturation in the female rat. The results of these studies suggest that NMDA receptors are more important than kainate receptors in the process of puberty in the female rat.

Effects of methoxychlor treatment of pregnant mice on female offspring of the treated and subsequent pregnancies

This study was designed to assess whether exposure to the estrogenic pesticide methoxychlor (MXC) during pregnancy would affect reproductive parameters not only in female offspring exposed prenatally, but also in those of a subsequent litter. Mice were exposed via oral gavage to 7.5, 5.0, or 2.5 mg technical grade MXC (50%) or 0.025 mg estradiol-17β (E-17β) from days 6 to 15 of pregnancy. Following delivery, female offspring (F 1a) were cross-fostered and sacrificed at 8 weeks of age. Mothers exposed during their first pregnancy were allowed to mate again and their second set of offspring (F 1b) were similarly evaluated to detect any latent effects from the initial exposure. Mice exposed to 7.5 mg MXC were unable to carry their litters to term. Results revealed a significant increase in the length of gestation of mice exposed to both E-17β and 5.0 mg MXC. A larger percentage of atretic follicles appeared in the ovaries of F 1a females exposed prenatally to 5.0 mg MXC when compared to controls. Females from the F 1b litter displayed a significant advance in time of vaginal opening, an apparent residual effect of MXC from a mother exposed during a previous pregnancy.

Effects of neonatal exposure to the antiprogestin mifepristone, RU 486, on the sexual development of the rat

RU 38486 (RU 486, mifepristone) is a potent progesterone receptor antagonist that has been used in humans in the pharmacologic induction of abortion. The effects of exposure to RU 486 during the neonatal period of the rat has not been previously reported. We examined the consequences of such exposure in the context of sexual development. Long-Evans rat pups were subcutaneously injected with either 100 μg RU 486, 300 μg RU 486, 500 μg progesterone (P), or 50 μg testosterone propionate (TP) in 0.05 ml sesame oil, or oil vehicle alone within 8 hours of birth, and 24 and 48 hours later. Treatment with either dose of RU 486 significantly advanced the onset of vaginal opening in females and attenuated defeminization of the lordosis response measured in males castrated as adults. As expected, TP-treated subjects were masculinized and defeminized, with females displaying fused vaginas and neither males nor females demonstrating lordosis behavior. Treatment with P caused no significant alterations in either the timing of vaginal opening or sexual behavior. These results indicate that RU 486 has clear developmental effects in the rat. Since this may well be a result of progesterone receptor blockade, further research is needed to clarify the processes involved.

Central administration of neuropeptide Y induces precocious puberty in female rats.

To examine the role of neuropeptide Y (NPY) in the regulation of puberty, the effects of various doses of NPY or saline injected into the 3rd ventricle of peripubertal female rats were determined. A single injection of NPY at a dose range of 10-20 micrograms into the 3rd ventricle of 30-day-old female rats advanced the time of vaginal opening (VO) and first ovulation by 4 days, as compared to saline-treated controls. Ova number and ovarian, adrenal and uterine weights at first estrous were similar in all animals regardless of treatment. The NPY-treated precocious animals showed lower body weight than did the VO-matched controls, but showed similar body weight as that of age-matched controls. Determination of plasma luteinizing hormone (LH) levels in the peripubertal female rats revealed that plasma LH was increased transiently immediately after NPY administration. Also, NPY-treated rats which had precocious puberty showed elevated pituitary and plasma LH levels on the day of VO. These results suggest that NPY can activate the hypothalamic-pituitary-gonadal axis to precociously initiate puberty.

Sexual maturation in female rats: Hereditary, developmental and environmental aspects

Two physiological components of sexual maturation, vaginal opening and first estrus, apparently evolve similarly in Wistar and Sprague-Dawley rats. However, a bimodal distribution in the frequency of the days of vaginal opening is observed within a given strain, which is less related to heredity than to the timing and type of experiment. In addition, when the modulators of sexual maturation are reviewed, it can be observed that sensitivity to external stimuli can vary even within a strain. For a defined set of breeding conditions, one group of rats can be more susceptible to changes in the lighting regimen and not be affected by controlled stressors, while another group responds more to stress and less to light. The reason for susceptibility to one rather than another environmental factor under similar breeding conditions is not understood. In that context, it is difficult to evaluate the role of heredity when we cannot understand the full impact of the environment, not to mention maternal influence in fetal and early life. Using two lines of psychogenetically selected rats, it was possible to show that they had differences in sexual maturation, which strongly suggested a genetic predisposition. Nevertheless, the question arises as to whether the genetic locus directly affects organs implicated in sexual maturation or whether it acts on some unknown factor which only secondarily modifies sexual maturation. In summary, there is more need to understand the role of the environment, including that of the mother early in fetal and neonatal life. It is suggested that the mechanisms underlying organ growth are set for a given species, while developmental and environmental factors fix the timing of vaginal opening and first ovulation. In the rat, there appear to be two times which are preferred for vaginal opening, given the laboratory conditions that have been used in the last 20 or so years: an early period, at 31-35 days, and a late period, at 36-40 days. An explanation for this dichotomy would be that a combination of parameters (not necessarily always the same) is needed for vaginal opening. These parameters oscillate during sexual maturation with different frequencies, which can achieve resonance to lead to vaginal opening and ovulation only during given periods.

Guanethidine-Mediated Destruction of Ovarian Sympathetic Nerves Disrupts Ovarian Development and Function in Rats*

Immunosympathectomy produced by treatment of newborn rats with antibodies to nerve growth factor (NGF) delays ovarian development and disrupts estrous cyclicity. While these alterations have been ascribed to loss of sympathetic neurons innervating the ovary, the treatment also causes partial loss of ovarian sensory innervation. The present experiments were undertaken to determine if selective interference with ovarian noradrenergic/sympathetic action would result in alterations of ovarian development similar to those caused by NGF antibodies (NGF Ab). We have used two approaches to disrupt catecholamine action on ovarian cells: 1) inhibition of beta-adrenoreceptors by local delivery of receptor blockers to the ovaries of juvenile rats; and 2) elimination of the sympathetic innervation by long term postnatal treatment with guanethidine (GD), an adrenergic neuron blocking agent. When GD is administered chronically it produces an autoimmune-mediated destruction of peripheral sympathetic nerves, without affecting cholinergic or sensory neurons. Of the receptor blockers tested, FM-24, a nonreversible antagonist, resulted in a sustained 70% decrease in available receptors throughout the 10-day period studied. In spite of this, the timing of puberty, assessed by the age at vaginal opening and first ovulation, was not delayed, suggesting that activation of the remaining receptors by an intact innervation suffices to maintain a normal noradrenergic influence. GD treatment initiated at the end of the first week of postnatal life and maintained for three weeks slowed the juvenile-peripubertal rate of body growth, delayed the time of vaginal opening and first ovulation, and disrupted subsequent estrous cyclicity, but did not affect the animals' fertility. The ovaries of GD-treated rats exhibited a striking loss of sympathetic (norepinephrine and neuropeptide Y) nerves but a normal sensory innervation (represented by fibers containing calcitonin gene-related peptide). The concentration of beta-adrenoreceptors in granulosa cells was reduced, suggesting follicular immaturity. Direct assessment of this inference by morphometric analysis of the ovaries revealed that follicular development was retarded. The progesterone and estrogen response of juvenile ovaries to gonadotropins in vitro were also reduced. At this time, circulating LH levels were slightly decreased, but neither LHRH content in the median eminence nor the LHRH response to prostaglandin E2 in vitro were affected.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of a Luteinizing Hormone-Releasing Hormone Antagonist in Late-Juvenile Female Rats: Blockade of Follicle Growth and Delay of First Ovulation Following Suppression of Gonadotropin Concentrations

Subcutaneous injections of an antagonist against luteinizing hormone-releasing hormone (LHRH-A, Org, 30276) were administered to late-juvenile female rats. The effects on timing of vaginal opening and first ovulation on serum gonadotropin concentrations and on follicle growth were studied. The dose of 100 micrograms LHRH-A/100 g body wt, given on Days 28, 31, and 34, did not influence timing of first ovulation. After administration of 500 micrograms LHRH-A/100 g body wt, ovulation was retarded by 4.7 days if injections were given on Days 28 and 31; by 6.7 days if given on Days 28, 31, and 34; and by 11.5 days if given on Days 28, 31, 34, and 37. Serum LH and FSH concentrations 3 days after the first, second, and third injections of 500 micrograms LHRH-A were significantly (p less than 0.01) lower than in saline-treated controls. Ovarian follicle counts showed decreased numbers of (antral) Class 2, 3, and 4 follicles 3 days after injection of 500 micrograms LHRH-A/100 g body wt on Day 28; a significantly higher number of Class 1 follicles and a further decrease in Class 2, 3, and 4 follicles 3 days after the second LHRH-A injection; and total absence of Class 3, 4, and 5 follicles 3 days after the third LHRH-A injection. Six days after the third LHRH-A injection, Class 3 and 4 follicles reappeared in the ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)

N‐Methyl‐DL‐Aspartate Receptor Antagonism by D‐2‐Amino‐5‐Phosphonovaleric Acid Delays Onset of Puberty in the Female Rat

Abstract To investigate the possible role of N-methyl-DL-aspartate (NMDA) receptor activation in the initiation of puberty, we examined the effects of the selective competitive antagonist 2-amino-5-phosphonovaleric acid (AP5) on the timing of vaginal opening. Paired and weight-matched litter mates of immature female rats were implanted with osmotic minipumps for the intracerebroventricular infusion of DL- or D-AP5 or artificial cerebrospinal fluid from 27 to 30 days of age for 14 days. Each animal was weighed and examined daily for vaginal opening as the indicator of first oestrous. Infusion of 20 or 40 mM DL-AP5 beginning on Day 30 failed to delay vaginal opening. Administration 50mM of the single enantiomer D-AP5 beginning on Day 27 significantly delayed the age of vaginal opening to 40.6+/-1.1 (mean +/- SEM) days compared to the cerebrospinal fluid-infused controls (36.5 +/- 0.6 days). Blockade of NMDA receptors in the D-AP5-treated animals was confirmed on Day 32 by the suppression of luteinizing hormone response to intravenous NMDA (20 mg/kg) while the response to exogenous luteinizing hormone-releasing hormone (50 ng/kg) remained intact. AP5-treated animals had a slower rate of growth (3.1 +/- 0.2 g/day) compared to controls (4.2 +/- 0.2 g/day). However, a similar degree of growth retardation produced by a 75% restricted diet in untreated juvenile animals did not delay vaginal opening. This suggests that the slower growth rate in the D-AP5-treated animals could not account for the delayed onset of puberty. In conclusion, these data suggest that blockade of central NMDA receptors inhibits excitatory mechanisms which may be important in the control of pubertal onset in the female rat.

Genetic Influences on the Timing of Puberty in Mice1

Genetic influences on the timing of three pubertal events--vaginal opening, first vaginal cornification, and onset of cyclicity--were studied in C57BL/6J, DBA/2J, and C3H/HeJ mice and in two F1 hybrid strains (B6D2F1 and B6C3HF1). Marked genotypic differences were found. Among inbred strains, differences in the onset of vaginal opening and first vaginal cornification (C3H less than DBA less than C57) did not parallel those for the onset of cyclicity (C3H much greater than DBA = C57). Compared to parental strains, F1 hybrid strains were intermediate for times of vaginal opening and first vaginal cornification, consistent with the model in which the genetic effects on the timing of these events are additive. By contrast, onset of cyclicity occurred significantly earlier in the F1 hybrids than in their parent strains, indicating heterosis for one or more genes specifying this event. Body weights also differed among the genotypes from weaning onward, but these differences were only partially correlated with the differences in the timing of the pubertal events. Thus, genetic influences other than those affecting body weight contribute to the differential timing of pubertal events in these mouse strains. These results reveal marked genetic variation in the timing of puberty, and indicate that the set of genes specifying the timing of vaginal opening and first vaginal cornification differs from those specifying the onset of cyclicity.

Intracellular CA 2+ and excitatory amino acids in hippocampal neurons : Robert K.S. Wong, Department of Neurology, Columbia University, NY

Excitatory amino acid neurotransmission is an essential component of the neuroendocrine transmission line that regulates anterior pituitary luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. Excitatory amino acids (EAAs), such as glutamate and aspartate, are found in largo concentrations in presynaptic boutons of a variety of important hypothalamic nuclei, including the arcuate nucleus, the suprachiasmatic nucleus, the supraoptic nucleus, the paraventricular nucleus, and the preoptic area. EAA receptors can be divided into two broad groups, namely, ionotropic and metabotropic receptors. Ionotropic receptors are subdivided into NMDA (N-methyl-D-aspartate), kainate, and AMPA (DL-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Their main mode of action is by the modulation of Na+, K+, and Ca2+ ion channels. Metabotropic receptors, on the other hand, act by a G-protein-stimulated release of intracellular Ca2+ or modulation of adenylate cyclase activity. The different EAA receptor subtypes are found in a variety of areas of the hypothalamus and the brain. In a variety of species, the administration of glutamate, NMDA, or kainate leads to LH release mediated through the stimulation of hypothalamic gonadotropin hormone-releasing hormone (GnRH) release. The major site of NMDA action appears to be the preoptic area—where GnRH cell bodies reside. AMPA and kainate appear to act primarily at the arcuate nucleus/median eminence, the site of GnRH nerve terminals. NMDA may also act upon noradrenergic neurons in the locus coeruleus to influence hypothalamic GnRH release. The steroid-induced LH surge in ovariectomized animals and the preovulatory surge of LH in cycling animals and in pregnant mare's serum gonadotropic-primed animals are blocked by the NMDA antagonist MK801 and the AMPA/kainate antagonist DNQX. MK801 also suppressed FSH surges in most instances, whereas DNQX had no effect on FSH surges. In the ovariectomized female rat, both the NMDA antagonist AP5 and the AMPA/kainate antagonist DNQX, lowered mean LH levels, LH pulse amplitude, and LH pulse frequency. Activation of NMDA receptors advanced the time of vaginal opening in the immature female rat, while kainate and DNQX were without effect. Gonadal steroid removal (castration) did not alter NMDA receptor levels or affinity in the hypothalamus of female or male rats. Likewise, steroid replacement to castrate rats did not affect hypothalamic NMDA receptor levels or NMDA R1 mRNA levels. Similarly, NMDA and kainate receptor levels in the hypothalamus did not change during the time of puberty in the female rat. In contrast, AMPA receptor (GluR1) immunoreactive levels in the magnocellular preoptic area (mPOA), the arcuate nucleus (ARC), and the suprachiasmatic nucleus (SCN) were found to be markedly elevated during the time of the LH surge in estradiol-progesterone-treated castrate rats compared to those of the vehicle-only-treated castrate rat. The release rates of glutamate and aspartate in the POA were found to be significantly elevated during the steroid-induced LH surge in the ovariectomized adult rat. Hence, the induction of the LH surge by steroids may involve enhanced EAA neurotransmission mediated by steroid-induced elevations in the release of glutamate and aspartate in the POA and AMPA receptors in the POA, the ARC, and the SCN. Evidence also exists supporting a role of EAAs in the release of other pituitary hormones, such as prolactin, ACTH, growth hormone, oxytocin, and vasopressin, as well as circadian rhythm induction and regulation. The above evidence points to a pivotal role of EAAs acting at the level of the hypothalamus in the neuroendocrine regulation of a variety of hormonal systems with the largest amount of data available on the control of gonadotropin secretion.

Progesterone and Estradiol Plasma Levels in Neonatally Irradiated Cycling Rats

Female rats which were exposed to a single low dose of gamma irradiation (6R or 15R) at the age of 8 days produce smaller litters when mature than untreated controls. The possibility that the impaired fertility resulted from altered ovarian activity as reflected by changes in plasma levels of progesterone or estardiol was investigated. Plasma levels of both steroids were determined throughout the day of proestrus. Progesterone level was also determined in 6R animals on the day of weaning. The maturity of such irradiated rats was assessed by observing the time of vaginal opening. The results indicated that the preovulatory peak of progesterone was delayed in the 6R rats whereas in the 15R group its levels were significantly lower. On the other hand no differences in estradiol plasma levels were noticed between the groups. The higher level of progesterone in the 6R animals was not evident on the day of weaning and was even in both groups, but vaginal opening in the irradiated rats was significantly delayed. The elevated level of progesterone might be responsible, among other endocrine changes, for the lower fertility of neonatally irradiated mature female rats.