Afternoon Of Proestrus Research Articles

Impact of proestrous milieu on expression of orexin receptors and prepro-orexin in rat hypothalamus and hypophysis: actions of Cetrorelix and Nembutal

Orexins and their receptors OX1 and OX2 regulate energy balance and the sleep-wake cycle. We studied the expression of prepro-orexin (PPO), OX1, and OX2 in brain and pituitary under the influence of the hormonal status in adult rats. Primarily, PPO, OX1, and OX2 expression was determined in Sprague-Dawley female cycling rats during proestrus and in males. Animals were killed at 2-h intervals. Anterior (AH) and mediobasal (MBH) hypothalamus, anterior pituitary (P), and frontoparietal cortex (CC) were homogenized in TRIzol, and mRNAs were obtained for screening of PPO, OX1, OX2 expression by semiquantitative RT-PCR. Main findings were confirmed and extended to all days of the cycle by quantitative real-time RT-PCR. Hormones and food consumption were determined. Finally, OX1, OX2, and PPO were measured by real-time RT-PCR in tissues collected at 1900 of proestrus after treatments at 1400 with ovulation-blocking agents Cetrorelix or pentobarbital. OX1 and OX2 expression increased at least threefold in AH, MBH, and P, but not in CC, between 1700 and 2300 of proestrus, without variations in estrus, diestrus, or in males. PPO in AH and MBH showed a fourfold or higher increase only during proestrus afternoon. Cetrorelix or pentobarbital prevented increases of OX1 and OX2 only in the pituitary and blunted gonadotropin surges, but left OX1, OX2, and PPO brain expression unchanged. Reproduction, energy balance, and sleep-wake cycle are integrated. Here, we demonstrate that, in the physiological neuroendocrine condition leading to ovulation, information to the orexinergic system acts in hypothalamus and pituitary by different mechanisms.

Vasopressin Regulation of the Proestrous Luteinizing Hormone Surge in Wild-Type and Clock Mutant Mice1

In the female mouse, ovulation and estrous cyclicity are under both hormonal and circadian control. We have shown that mice with a mutation in the core circadian gene Clock have abnormal estrous cycles and do not have a luteinizing hormone (LH) surge on the afternoon of proestrus due to a defect at the hypothalamic level. In the present study, we tested the hypotheses that vasopressin (AVP) can act as a circadian signal to regulate the proestrous release of LH, and that this signal is deficient in the Clock mutant. We found that Avp expression in the suprachiasmatic nucleus (SCN) and AVP 1a receptor (Avpr1a) expression in the hypothalamus is reduced in Clock mutant mice compared to wild-type mice. Intracerebroventricular (i.c.v.) injection of AVP on the afternoon of proestrus is sufficient to induce LH secretion, which reaches surge levels in 50% of Clock mutant mice. The effect of AVP on the Clock mutant LH surge is mediated by AVPR1A, as co-infusion of AVP and an AVPR1A-specific antagonist prevents AVP induction of LH release, although infusion of an AVPR1A antagonist into wild-type mice failed to prevent a proestrous LH surge. These results suggest that reduced hypothalamic AVP signaling plays a role in the absence of the proestrous LH surge in Clock mutant mice. The results also support the hypothesis that AVP produced by the SCN may be a circadian signal that regulates LH release.

Expression of KiSS-1 in Rat Ovary: Putative Local Regulator of Ovulation?

Kisspeptins, the products of KiSS-1 gene, and their receptor, GPR54, have recently emerged as essential gatekeepers of reproduction, mainly through regulation of GnRH secretion at the hypothalamus. However, the profound hypogonadotropism linked to GPR54 inactivation is likely to mask additional functions of this system at other levels of the gonadal axis, in which expression of KiSS-1 and GPR54 has been preliminarily reported. We describe herein the expression of KiSS-1 gene and kisspeptin immunoreactivity (IR) in rat ovary and evaluate its developmental and hormonal regulation. KiSS-1 and GPR54 mRNAs were persistently detected in adult ovary along estrous cycle. Yet, contrary to GPR54, ovarian KiSS-1 levels fluctuated in a cyclic-dependent manner, with a robust increase in the afternoon of proestrus, i.e. preceding ovulation. In addition, kisspeptin-IR was observed in rat ovary, with strong signals in theca layers of growing follicles, corpora lutea, and interstitial gland, compartments in which modest GPR54-IR was also detected. Interestingly, the rise in ovarian KiSS-1 mRNA at proestrus was prevented by blockade of preovulatory gonadotropin surge and restored by replacement with human chorionic gonadotropin as superagonist of LH. In addition, immature ovaries showed low to negligible levels of KiSS-1 mRNA, which were significantly enhanced by gonadotropin priming. In summary, we present novel evidence for the developmental and hormonally regulated expression of the KiSS-1 gene, and the presence of kisspeptin-IR, in rat ovary. The ability of the LH surge to timely induce ovarian expression of KiSS-1 at the preovulatory period strongly suggests a previously unsuspected role of locally produced kisspeptin in the control of ovulation.

Effects of Ghrelin upon Gonadotropin-Releasing Hormone and Gonadotropin Secretion in Adult Female Rats: In vivo and in vitro Studies

A reproductive facet of ghrelin, a stomach-derived orexigenic peptide involved in energy homeostasis, has been recently suggested, and predominantly inhibitory effects of ghrelin upon luteinizing hormone (LH) secretion have been demonstrated in rat models. Yet, the modulatory actions of ghrelin on the gonadotropic axis remain scarcely evaluated. We report herein a detailed analysis of the effects of ghrelin upon LH and follicle-stimulating hormone (FSH) secretion in the female rat, using a combination of in vivo and in vitro approaches. Intracerebroventricular administration of ghrelin (3 nmol/rat) evoked a significant inhibition of LH secretion in cyclic female rats throughout the estrous cycle (proestrus afternoon, estrus, metestrus), as well as in ovariectomized females. In good agreement, gonadotropin-releasing hormone (GnRH) secretion by hypothalamic fragments from ovariectomized females was significantly inhibited by ghrelin. In contrast, ghrelin dose-dependently stimulated basal LH and FSH secretion by pituitary tissue in vitro; a phenomenon that was proven dependent on the phase of estrous cycle, as it was neither detected at estrus nor observed after ovariectomy. Conversely, GnRH-stimulated LH secretion in vitro was persistently inhibited by ghrelin regardless of the stage of the cycle, whereas stimulated FSH secretion was only inhibited by ghrelin at estrus. In addition, cyclic fluctuations in mRNA levels of growth hormone secretagogue receptor (GHS-R)1a, i.e. the functional ghrelin receptor, were observed in the pituitary, with low values at estrus and metestrus. GHS-R1a mRNA levels, however, remained unchanged after ovariectomy. In summary, our data illustrate a complex mode of action of ghrelin upon the gonadotropic axis, with predominant inhibitory effects at central (hypothalamic) levels and upon GnRH-induced gonadotropin secretion, but direct stimulatory actions on basal LH and FSH secretion. Overall, our results further document the reproductive role of ghrelin, which might be relevant for the integrated control of energy balance and reproduction.

Involvement of Central Metastin in the Regulation of Preovulatory Luteinizing Hormone Surge and Estrous Cyclicity in Female Rats

Ovulation is caused by a sequence of neuroendocrine events: GnRH and LH surges that are induced by positive feedback action of estrogen secreted by the mature ovarian follicles. The central mechanism of positive feedback action of estrogen on GnRH/LH secretion, however, is not fully understood yet. The present study examined whether metastin, the product of metastasis suppressor gene KiSS-1, is a central neuropeptide regulating GnRH/LH surge and then estrous cyclicity in the female rat. Metastin had a profound stimulation on LH secretion by acting on the preoptic area (POA), where most GnRH neurons projecting to the median eminence are located, because injection of metastin into the third ventricle or POA increased plasma LH concentrations in estrogen-primed ovariectomized rats. Metastin neurons were immunohistochemically found in the arcuate nucleus (ARC) to be colocalized with estrogen receptors with some fibers in the preoptic area (POA) in close apposition with GnRH neuronal cell bodies or fibers. Quantitative RT-PCR has revealed that KiSS-1 and GPR54 mRNAs were expressed in the ARC and POA, respectively. The blockade of local metastin action in the POA with a specific monoclonal antibody to rat metastin completely abolished proestrous LH surge and inhibited estrous cyclicity. Metastin-immunoreactive cell bodies in the ARC showed a marked increase and c-Fos expression in the early proestrus afternoon compared with the day of diestrus. Thus, metastin released in the POA is involved in inducing the preovulatory LH surge and regulating estrous cyclicity.

Pituitary Adenylate Cyclase Activating Polypeptide Messenger RNA in the Paraventricular Nucleus and Anterior Pituitary During the Rat Estrous Cycle1

The neuropeptide pituitary adenylate cyclase activating polypeptide (ADCYAP 1, or PACAP) has been demonstrated to enhance gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion and regulate gonadotropin subunit gene expression in cultures of anterior pituitary cells. In the present study, we used in situ hybridization and real-time polymerase chain reaction to examine the expression of Pacap mRNA within the paraventricular nucleus (PVN) and anterior pituitary throughout the estrous cycle of the rat. Levels of luteinizing hormone in serum and pituitary gonadotropin subunit mRNAs were evaluated and displayed cyclic fluctuations similar to those reported previously. Pacap mRNA expression in the PVN and pituitary varied significantly during the estrous cycle, with the greatest changes occurring on the day of proestrus. Pacap mRNA levels in the PVN declined significantly on the morning of diestrus. During proestrus, PVN Pacap mRNA levels significantly increased 3 h before the gonadotropin surge and then declined. Pituitary expression of Pacap mRNA also varied on the afternoon of proestrus with a moderate decline at the time of the gonadotropin surge and a significant increase later in the evening. Expression of the mRNA species encoding the 288 amino acid form of follistatin increased significantly following the rise in pituitary Pacap mRNA, at the termination of the secondary surge in follicle-stimulating hormone beta (Fshb) gene expression. These results suggest that PACAP is involved in events before and following the gonadotropin surge, perhaps through increased gonadotroph sensitivity to GnRH and suppression of Fshb subunit expression through increased follistatin, as previously observed in vitro.

Differential Gonadotropin-Releasing Hormone (GnRH) and GnRH Receptor Messenger Ribonucleic Acid Expression Patterns in Different Tissues of the Female Rat across the Estrous Cycle

GnRH, the main regulator of reproduction, is produced in a variety of tissues outside of the hypothalamus, its main site of synthesis and release. We aimed to determine whether GnRH produced in the female rat pituitary and ovaries is involved in the processes leading to ovulation. We studied the expression patterns of GnRH and GnRH receptor (GnRH-R) in the same animals throughout the estrous cycle using real-time PCR. Hypothalamic levels of GnRH mRNA were highest at 1700 h on proestrus, preceding the preovulatory LH surge. No significant changes in the level of hypothalamic GnRH-R mRNA were detected, although fluctuations during the day of proestrus are evident. High pituitary GnRH mRNA was detected during the day of estrus, in the morning of diestrus 1, and at noon on proestrus. Pituitary GnRH-R displayed a similar pattern of expression, except on estrus, when its mRNA levels declined. Ovarian GnRH mRNA levels increased in the morning of diestrus 1 and early afternoon of proestrus. Here, too, GnRH-R displayed a somewhat similar pattern of expression to that of its ligand. To the best of our knowledge, this is the first demonstration of a GnRH expression pattern in the pituitary and ovary of any species. The different timings of the GnRH peaks in the three tissues imply differential tissue-specific regulation. We believe that the GnRH produced in the anterior pituitary and ovary could play a physiological role in the preparation of these organs for the midcycle gonadotropin surge and ovulation, respectively, possibly via local GnRH-gonadotropin axes.

Lactation reduces prolactin levels in reproductively experienced female rats

Long-term alterations in prolactin (PRL) secretion following reproductive experience have been demonstrated in both women and female rats. In the rat, these changes include decreased PRL secretion in response to a dopamine antagonist challenge following ovariectomy, decreased post-coital diurnal and nocturnal prolactin surges in multigravid versus primigravid females, as well as decreased suckling-induced prolactin release in multiparous versus primiparous females. To date, there have been no studies examining PRL secretion following reproductive experience in cycling female rats. Studies in women, however, have demonstrated a reduction in basal PRL secretion during the menstrual cycle. The purpose of the present work was to determine whether similar changes occur in the rat during the estrous cycle and to what extent lactation is involved in these effects. In addition to examining PRL, potential parity-induced changes in estradiol secretion were also studied. The findings revealed a significant decrease in PRL levels during the afternoon of proestrus, which was only observed in primiparous females that had lactated. Significant differences in estradiol secretion were not detected following reproductive experience. Thus, a reduction in the PRL surge on the afternoon of proestrus is a consequence of reproductive experience that requires both pregnancy and lactation.

Interaction Between Norepinephrine, Oxytocin, and Nitric Oxide in the Stimulation of Gonadotropin‐Releasing Hormone Release From Proestrous Rat Basal Hypothalamus Explants

In the proestrous female rat, norepinephrine, oxytocin and nitric oxide (NO) all participate in the regulation of the preovulatory gonadotropin-releasing hormone (GnRH) surge. Recent studies from our laboratory have demonstrated that oxytocin induces dose-dependent release of GnRH from proestrous basal hypothalamus explants. The present studies were undertaken to determine whether norepinephrine could also stimulate GnRH release from similar explants, to identify the receptors responsible for this effect and to investigate interactions between norepinephrine, oxytocin and NO. Norepinephrine significantly stimulated GnRH release from proestrous basal hypothalamus explants, and coadministration of the alpha(1)-adrenergic antagonist prazosin blocked this effect. Combined administration of oxytocin and norepinephrine stimulated significantly more GnRH release than either drug alone, and this stimulation was blocked by inhibition of NO synthase, or by an oxytocin receptor antagonist. NO production was measured from the same samples using a modified Griess reaction. Oxytocin, but not norepinephrine, significantly increased NO production, as did norepinephrine and oxytocin in combination. Oxytocin receptor antagonist administration attenuated the stimulation of NO production by norepinephrine/oxytocin. These results demonstrate for the first time that oxytocin and norepinephrine dramatically stimulate GnRH release from basal hypothalamus explants harvested on the afternoon of proestrus, and indicate that this involves oxytocin receptor and NO-dependent mechanisms.

A secondary surge of prolactin on the estrus afternoon

It has been described that throughout the estrous cycle of the rat, plasma prolactin (PRL) is basal except on proestrus afternoon when a preovulatory surge occurs. However, there have been controversies about PRL levels on the estrus day. Thus, the aim of this study was to evaluate the existence of a secondary surge of PRL on estrus afternoon and correlate it with plasma estradiol levels. The jugular vein of cycling rats was cannulated at 14:00 h on proestrus and a blood sample was withdrawn at 17:00 h for plasma LH measurement and determination of the preovulatory LH surge occurrence. In order to exclude the regular cycling rats that do not present the gonadotropins preovulatory surge and do not ovulate, only rats showing the LH surge on proestrus were considered in this study. Blood samples were collected hourly during estrus from midnight to 9:00 h (group 1) and from 10:00 to 18:00 h (group 2). In group 1, PRL showed a descending profile from midnight to 9:00 h, whereas the estradiol concentrations were constant. In group 2, a secondary surge of PRL was observed in 20 of 25 (80%) rats and plasma estradiol remained constant, but was higher in animals with the PRL surge. Thus the present data suggest the occurrence of a secondary surge of PRL in the afternoon of estrus that seems to be related to plasma estradiol levels of estrus day, which might exert only a permissive role in this surge generation.

Estrogen Induces Neuropeptide Y (NPY) Y1 receptor gene expression and responsiveness to NPY in gonadotrope-enriched pituitary cell cultures.

We showed previously that neuropeptide Y1 receptor (Y1R) expression is increased in the hypothalamus on proestrus afternoon and that this up-regulation of Y1R mRNA may permit neuropeptide Y (NPY) to facilitate release of the preovulatory GnRH surge. Because NPY also modulates LH release directly, we examined steroid regulation of Y1R expression in the female rat anterior pituitary. Treatment of female rats with estrogen in vivo decreased the levels of Y1R mRNA in the whole pituitary gland. In lactotrope/somatotrope-enriched pituitary cells separated by unit gravity sedimentation, 17beta-estradiol (E(2)) treatment likewise suppressed Y1R expression. In contrast, E(2) elevated Y1R mRNA in gonadotrope-enriched cell populations, indicating that estrogen regulates Y1R mRNA expression differently in gonadotropes vs. other pituitary cell types. After exposure to E(2), NPY augmented GnRH-induced LH release from gonadotrope-enriched cells in a manner requiring Y1R activation. Without steroid exposure, this augmentation disappeared, and with progesterone alone, NPY reduced GnRH-induced LH release. In addition, NPY inhibited prolactin secretion from primary pituitary cells in a steroid-free environment, but not in the presence of estrogen. These findings demonstrate that E(2) can directly up-regulate gonadotrope responsiveness to NPY and suggest that this action is mediated at least in part by E(2)'s ability to stimulate Y1R gene expression in gonadotropes. Our observations are consistent with the idea that this regulatory mechanism represents a component of E(2)'s positive feedback actions in pituitary gonadotropes. The biological importance of E(2)'s opposite effects on Y1R expression in other pituitary cell types remains to be determined.

Immunization against endogenous inhibin increases normal oocyte/embryo production in adult mice.

The objectives of the present study were to induce super-ovulation in adult mice by immunoneutralization of endogenous inhibin and to investigate embryo development in vivo and in vitro. Adult female mice of the ddY strain at 3 mo of age were superovulated with a single ip injection of inhibin antiserum (inhibin-AS; 50, 100, or 200 micro L) at 12:00 h on metestrus of the 4-d estrous cycle. The control group was treated with 100 micro L normal goat serum (control serum). Five mice were sacrificed every 6 h and blood samples were collected for hormonal assay. Individual female mice were bred in the afternoon of proestrus with individual fertile males. Embryos were collected at 08.00 h on d 2 post-coitus and cultured in KSOM solution. Treatment with inhibin-AS significantly increased the concentrations of plasma FSH. Furthermore, there was a significant elevation in plasma concentrations of estradiol-17beta and progesterone in the inhibin-immunized groups in comparison with controls. The superovulated oocytes that were fertilized normally in vivo were able to develop normally to blastocysts in vitro. The number of oocytes and blastocysts developed in animals treated with inhibin-AS was significantly higher than that for mice treated with control serum. Moreover, the rate of fertilization and the rate of blastocyst development were similar in inhibin-AS treated and control groups. These results indicate that immunoneutralization of endogenous inhibin induces superovulation in adult mice without additional treatment with human chorionic gonadotropin (hCG). In addition, the superovulated oocytes obtained by administration of inhibin-AS have normal embryonic developmental competence.

The Luteinizing Hormone Surge Is Preceded by an Estrogen-Induced Increase of Hypothalamic Progesterone in Ovariectomized and Adrenalectomized Rats

As circulating estrogen levels rise on the afternoon of proestrus, they stimulate the hypothalamo-pituitary axis. This estrogen positive feedback is pivotal to stimulate the luteinizing hormone (LH) surge required for ovulation and luteinization of ovarian follicles. In addition to estrogen, pre-LH surge progesterone is critical for an LH surge as was demonstrated by blocking progesterone synthesis. In ovariectomized (OVX) rats treated with trilostane, a blocker of the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) that catalyzes the conversion of pregnenolone to progesterone, estrogen did not induce an LH surge. Further, estrogen induced an LH surge in OVX and adrenalectomized (ADX) rats, indicating that the source of progesterone was neither the ovary nor adrenal gland. This estrogen-only LH surge was inhibited by pretreatment with trilostane, indicating that although the adrenal gland and ovary were not necessary for positive feedback, progesterone synthesis was critical for estrogen-induced positive feedback in an OVX/ADX rat. This suggested that the LH surge is dependent on the pre-LH surge synthesis of progesterone. Estrogen-induced progesterone receptors in the hypothalamus are vital for the LH surge, so a potential location for progesterone synthesis is the hypothalamus. OVX/ADX female rats were treated with 17β-estradiol (50 µg) and progesterone levels were assayed by RIA. Progesterone levels were elevated in hypothalamic tissue following estrogen treatment. No increases in tissue progesterone levels were found in parietal cortex, cerebellum, medulla, pituitary or plasma. Additionally, male rats that do not have an estrogen positive feedback-induced LH surge were examined. Castrated/ADX male rats had no increase in hypothalamic progesterone levels after estrogen treatment. Together, these data strongly suggest that estrogen enhances neuroprogesterone synthesis in the hypothalamus that is involved in the positive feedback regulating the LH surge.

Presence of κ-opioid tone at the onset of the ovulatory luteinizing hormone surge in the proestrous rat

A decrease in endogenous opioid peptide inhibitory tone on the afternoon of proestrus is one event underlying generation of the ovulatory luteinizing hormone (LH) surge. Whether this disinhibition involves a complete loss of opioid suppression at the time of the LH surge is controversial. The objective of the present study was to determine whether a total loss specifically of the κ-opioid inhibitory component suppressing LH secretion occurs on proestrus at the onset of the LH surge. Proestrous rats were infused intraventricularly with either artificial cerebrospinal fluid (aCSF) or aCSF containing nor-binaltorphimine (nor-BNI), a selective κ-opioid receptor antagonist, from 15:30 or 16:30 h (the approximate onset time of the spontaneous LH surge) to 18:50 h. The LH surge in rats treated with nor-BNI beginning at 15:30 h started 0.5 h earlier than the spontaneous surge in aCSF controls, and had significantly higher plasma LH levels from 16:30 to 17:30 h. Nor-BNI administration begun at 16:30 h also produced an LH surge with more elevated plasma LH levels at 17:30 and 18:00 h than in aCSF-treated controls. These results demonstrate that significant amounts of κ-opioid tone are still present during the hours when the LH surge is initiated. Thus, a complete loss of κ-opioid inhibition is not required for the onset of the LH surge on proestrus.

Functional expression of the dopamine-activated K(+) current in lactotrophs during the estrous cycle in female rats: correlation with prolactin secretory responses.

It is well established that hypothalamic dopamine (DA) is the major physiologic regulator of prolactin (PRL) secretion, exerting a tonic inhibition throughout most of the estrous cycle. A dramatic drop in the amount of DA perfusing the anterior pituitary occurs in the afternoon of proestrus and is critical for the production of the surge of PRL that occurs at that time. In my laboratory, we have identified and characterized a DA-activated K(+) channel (K(DA)) in lactotrophs derived from proestrous rats that underlies DA-induced membrane hyperpolarization of lactotrophs. We have also demonstrated that this hyperpolarization plays a critical role in both the inhibition of PRL release from proestrous cells and the PRL secretory rebound that occurs following DA withdrawal. We now report that the ability of DA to activate the K(DA) channel and elicit hyperpolarization in primary lactotrophs changes dramatically during the estrous cycle. Lactotrophs isolated from cycling female rats were studied using whole-cell voltage clamp. DA (1 microM) elicited a robust membrane K(+) current in the majority of proestrous lactotrophs (86%; 24.0 +/- 2.9 pA). By contrast, DA activated a considerably smaller membrane current (3.3 pA) in very few lactotrophs isolated from rats on either diestrus or estrus (8 and 0%, respectively). Using a perifusion system to examine temporal patterns of PRL release, we found that following application and withdrawal of DA, proestrous cells produced a robust secretory rebound, but diestrous and estrous cells did not. However, DA inhibited PRL release to the same extent regardless the stage of the cycle from which the cells were derived. These data are consistent with the presence of multiple DA effectors in lactotrophs and demonstrate that their relative importance shifts dramatically with changes in the endocrine status of the animal. We propose that the DA-activated K(+) channel (K(DA)) is a critical effector governing the unique secretory profile of PRL observed in proestrous animals.

Proestrous Surge of Gonadotropin-Releasing Hormone Secretion Inhibits Apoptosis of Anterior Pituitary Cells in Cycling Female Rats

Estrogen affects apoptotic cell death in estrogen-responsive tissues. The purpose of the present study was to examine dynamic changes in apoptotic cell death in the anterior pituitary gland during the estrous cycle and to investigate neuroendocrine regulation of these changes in cycling female rats. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) for in situ detection of DNA strand breaks revealed that the number of TUNEL-positive anterior pituitary cells was changing during the estrous cycle, with a maximum in the morning of proestrus and a minimum in the morning of estrus. A similar pattern was observed with Bax immunostaining; however, no difference was observed in Bcl-2 immunostaining. Most of Bax-immunoreactive cells were identified as a subpopulation of gonadotropes. Pentobarbital administered in the afternoon of proestrus attenuated the decrease in TUNEL-positive or Bax-immunoreactive cells in the morning of estrus, although estradiol treatment failed to affect it. This action of pentobarbital was reduced by simultaneous treatment with an ovulation-inducing dose of gonadotropin-releasing hormone (GnRH), but not with progesterone, an ovarian steroid also released after GnRH treatment. These results suggest that anterior pituitary cells, mostly gonadotropes, undergo a cyclic change in apoptotic cell death during the estrous cycle and that the inhibition of apoptosis on estrus is due, at least in part, to the proestrous surge of GnRH secretion.

Inhibition of in vivo and in vitro fertilization in rodents by gonadotropin-releasing hormone antagonists.

We have examined the effect of two GnRH antagonists, Ac-D-Nal(1)-Cl-D-Phe(2)-3-Pyr-D-Ala(3)-Arg(5)-D-Glu(AA)(6)-GnRH (Nal-Glu) and Ac(3,4)-dehydro-Pro(1),-p-fluoro-D-Phe(2),D-Trp(3,6)-GnRH (4pF), on in vivo and in vitro fertilization in rodents. Female rats were treated in the afternoon of proestrus with 2 micro l of Nal-Glu or 4pF (0.5 and 5 mM) injected directly into one oviductal horn (experimental); saline was injected into the contralateral horn (control). Females were then mated and the oviducts were perfused for egg and sperm recovery. The results indicate that both antagonists inhibited in vivo fertilization. Thus, the percentage of fertilized eggs in control oviducts ranged from 92% +/- 5% to 100% +/- 0%, whereas in treated oviducts, fertilization ranged from 25% +/- 6% to 73% +/- 5%. GnRH antagonists did not interfere with the process of ovulation, sperm migration to the site of fertilization, or early embryo development. In additional experiments with mice, GnRH antagonists inhibited in vitro fertilization. One fertilization event that was specifically inhibited by GnRH antagonists was the process of sperm binding to the zona pellucida. This step was precisely monitored using the hemizona assay. GnRH antagonists did not affect sperm movement or acrosomal status. These observations indicated that local treatment with GnRH antagonists inhibit in vivo fertilization and give additional support to the idea that endogenous GnRH may play an important role during fertilization by increasing the efficiency of sperm-zona binding.

Hindbrain glucoprivic inhibition of the proestrus lutenizing hormone surge in the female rat is attenuated by exogenous lactate administration

AbstractCentral glucose availability is a critical monitored variable in the regulation of reproductive neuroendocrine function. While current studies implicate the periventricular caudal brainstem as a source of signaling of decreased availability of catabolic intermediates and/or glycolytic end products, the mechanisms by which local metabolic ‘sensors’ signal diminished glucose utilization are not clear. The present experiments investigated whether fourth ventricular infusion of the aerobic catabolic substrate, lactate, attenuates glucoprivic suppression of luteinizing hormone (LH). Cyclic female rats were injected at 13.00h on proestrus with the glucose antimetabolite, 2‐deoxy‐D‐glucose (2DG; 50 vs. 150ug), or vehicle into the rostral fourth ventricle. Additional proestrus animals were treated by continuous fourth ventricular infusion of 10.0 µM lactate/hr or vehicle, initiated prior to ip injection of 400mg 2DG/kg bw or saline. Serial blood samples were obtained hourly during proestrus afternoon for radioimmunoassay of plasma LH. The results demonstrate a reduction in the LH surge following icv 2DG administration, while showing that fourth ventricular infusion of lactate reversed inhibition of hormone release by systemic antimetabolite administration. These findings support the view that hindbrain signaling of glucose metabolic dysfunction initiates neural mechanisms that inhibit the proestrus LH surge, and that lactate utilization may be monitored as an indicator of local cellular energy stasis.

GABA release in the medial preoptic area of cyclic female rats

GABA is a potent regulator of gonadotropin-releasing hormone neurons in the hypothalamus. To determine the profile of GABA release in the medial preoptic area where the gonadotropin surge generator resides, an in vivo microdialysis study was performed in cyclic female rats. The microdialysis samples were collected and sequential blood samples (150 μl each) were also obtained, at 1-h intervals. During estrus and diestrus 1, GABA release in the medial preoptic area was relatively low. A small increase in the GABA release began in the afternoon of diestrus 1 and attained its peak in the morning of diestrus 2, but declined in the afternoon of that day. The GABA release markedly increased from late in the night of diestrus 2 through the morning of proestrus, when it attained its peak, and thereafter it declined sharply until the critical period of proestrus. A distinct preovulatory luteinizing hormone surge was observed in the afternoon of proestrus in all proestrous rats. From these results we suggest that the preovulatory elevation of the GABA release from the night through to the morning of proestrus, followed by a sharp decline, is closely associated with the onset of the preovulatory luteinizing hormone surge in cyclic female rats. The present study is the first to report the 4-day profile of GABA release in the medial preoptic area during the estrous cycle.

Dynamics of messenger RNAs encoding inhibin/activin subunits and follistatin in the ovary during the rat estrous cycle.

Quantitative changes in ovarian inhibin/activin subunit and follistatin mRNAs during the rat estrous cycle were examined by ribonuclease protection assay using digoxygenin-labeled RNA probes. Levels of ovarian inhibin alpha subunit mRNA remained low throughout estrus, metestrus, and diestrus; abruptly increased on the morning of proestrus; then rapidly decreased when the primary gonadotropin surge occurred. A similar changing pattern was observed in inhibin/activin beta(A) subunit mRNA. On the other hand, inhibin/activin beta(B) subunit mRNA showed a different changing pattern. Levels of beta(B) subunit mRNA remained constant during metestrus and diestrus, abruptly decreased on the afternoon of proestrus, then quickly recovered from the nadir by 1100 h on estrus. Throughout the rat estrous cycle, especially during the periovulatory period, alpha subunit mRNA levels were considerably higher than beta(A) and beta(B) subunit mRNA levels. In addition, changes in plasma concentrations of inhibin A and inhibin B were very similar to that in ovarian beta(A) and beta(B) subunit mRNA levels, respectively, with several-hour delays. These results suggest that levels of beta subunit mRNAs restrict secretion of dimeric inhibins. Levels of follistatin mRNA remained low from the midnight of metestrus to the midnight of diestrus, then increased until initiation of the primary gonadotropin surge. Thereafter, follistatin mRNA decreased, reached the nadir at 0200 h on estrus, then increased abruptly at 1100 h on estrus. Afterward, follistatin mRNA levels remained high until the morning of metestrus. The changing pattern of ovarian follistatin mRNA was similar to, and preceded, the changes in plasma concentrations of progesterone, suggesting that ovarian follistatin may modulate progesterone secretion during the rat estrous cycle.