Evening Of Proestrus Research Articles

Musashi as a Regulator of the Follicle-Stimulating Hormone in the Gonadotropes

The cyclic expression of gonadotropin releasing-hormone receptors (GnRHR), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) by pituitary gonadotropes is critical in the female reproductive process. We have shown that the translational regulator Musashi (MSI) binds to Gnrhr mRNA and inhibits its translation, and the gonadotrope-specific deletion of Msi1 and Msi2 (Gon-Msi-null) leads to increased pituitary GnRHR protein levels. An in silico analysis of gonadotropin mRNAs revealed 5 different MSI binding elements in the 3’UTR of Fshb mRNA. We hypothesize that, in addition to Gnrhr, MSI may also bind and repress Fshb mRNA translation in the gonadotropes. To test if MSI does target the Fshb transcript in the pituitary, we performed RNA immunoprecipitation (IP) on pooled control female mouse pituitaries using a MSI1 antibody and measured Fshb mRNA by qRT-PCR. To study the in vivo effects of MSI on Fshb, we harvested the pituitaries of the Gon-Msi-null (MUT) female mice and their littermate controls (CTL) during the estrous cycle. We collected serum and protein for EIAs to measure the levels of FSH and LH, and RNA for Fshb qRT-PCR. We harvested proestrous ovaries and fixed them for embedding, sectioning, and H&E staining. Our RNA IP experiments show a 7-fold enrichment for Fshb with the MSI1 antibody. The Gon-Msi-null females have significantly higher pituitary FSH protein content than controls on estrous morning (MUT: 4.8±1.3 vs. CTL: 1.8±2.6 ng/ml/μg protein, p<0.0001, n=9-10/group). These mice also have increased serum FSH levels (MUT: 56.9±6.4 vs. CTL: 44.5±9.6 ng/ml, p=0.0147, n=9-10/group). No changes at the Fshb mRNA level were detected. Analysis of Gon-Msi-null ovaries revealed a 50% decrease in the number of follicles, with significant decreases in the average numbers of maturing follicles (p<0.0175) and corpora lutea (p<0.0215). Interestingly, the LH levels in these mice were also altered. The Gon-Msi-null females show a decrease in the pituitary LH protein content in the evening of proestrus (MUT: 11.8±1.4 vs. CTL: 15.1±2.0 ng/ml/μg protein, p=0.0333, n=7/group), in addition to a delayed and blunted LH surge (MUT: 2.6±1.9 vs. CTL: 7.3±3.5 ng/ml, p=0.0089, n=7-11/group). Taken together, our data indicate that Fshb is a Musashi target in the gonadotropes. By deleting MSI from the pituitary gonadotropes, we observe an increase in FSH protein content and serum levels. These Gon-Msi-null female mice have significantly fewer maturing follicles and corpora lutea, which might suggest lower levels of estrogens and progesterone. This, together with the increased GnRHR pituitary protein content, affects LH secretion, leading to a blunted LH surge in the Gon-Msi-null females. Our studies thus reveal a novel translational regulatory mechanism to govern levels of critical reproductive hormones in the pituitary.

Circadian Function in Multiple Cell Types Is Necessary for Proper Timing of the Preovulatory LH Surge.

The timing of the preovulatory surge of luteinizing hormone (LH), which occurs on the evening of proestrus in female mice, is determined by the circadian system. The identity of cells that control the phase of the LH surge is unclear: evidence supports a role of arginine vasopressin (AVP) cells of the suprachiasmatic nucleus (SCN), but it is not known whether vasopressinergic neurons are necessary or sufficient to account for circadian control of ovulation. Among other cell types, evidence also suggests important roles of circadian function of kisspeptin cells of the anteroventral periventricular nucleus (AvPV) and gonadotropin-releasing hormone (GnRH) neurons of the preoptic area (POA), whose discharge is immediately responsible for the discharge of LH from the anterior pituitary. The present studies used an ovariectomized, estradiol-treated preparation to determine critical cell types whose clock function is critical to the timing of LH secretion. As expected, the LH surge occurred at or shortly after ZT12 in control mice. In further confirmation of circadian control, the surge was advanced by 2 h in tau mutant animals. The timing of the surge was altered to varying degrees by conditional deletion of Bmal1 in AVPCre, KissCreBAC, and GnRHCreBAC mice. Excision of the mutant Cnsk1e (tau) allele in AVP neurons resulted in a reversion of the surge to the ZT12. Conditional deletion of Bmal1 in Kiss1 or GnRH neurons had no noticeable effect on locomotor rhythms, but targeting of AVP neurons produced variable effects on circadian period that did not always correspond to changes in the phase of LH secretion. The results indicate that circadian function in multiple cell types is necessary for proper timing of the LH surge.

GnRH Pulse Generator Activity Across the Estrous Cycle of Female Mice.

A subpopulation of kisspeptin neurons located in the arcuate nucleus (ARN) operate as the GnRH pulse generator. The activity of this population of neurons can be monitored in real-time for long periods using kisspeptin neuron-selective GCaMP6 fiber photometry. Using this approach, we find that ARN kisspeptin neurons exhibit brief (∼50 seconds) periods of synchronized activity that precede pulses of LH in intact female mice. The dynamics and frequency of these synchronization episodes (SEs) are stable at approximately one event every 40 minutes throughout metestrus, diestrus, and proestrus, but slow considerably on estrus to occur approximately once every 10 hours. Evaluation of ARN kisspeptin neuron activity across the light-dark transition, including the time of onset of the proestrus LH surge, revealed no changes in SE frequency. Longer 24-hour recordings across proestrus into estrus demonstrated that an abrupt decrease in SEs occurred ∼4 to 5 hours after the onset of the LH surge to reach the low frequency of SEs observed on estrus. The frequency of SEs was stable across the 24-hour period from metestrus to diestrus. Administration of progesterone to diestrus mice resulted in the abrupt slowing of SEs. These observations show that the GnRH pulse generator exhibits an unvarying pattern of activity from metestrus through to the late evening of proestrus, at which time it slows dramatically, likely in response to postovulation progesterone secretion. The GnRH pulse generator maintains a constant frequency of activity across the time of the LH surge, demonstrating that it is not involved directly in surge generation.

Neuronal SIRT1 Regulates Metabolic and Reproductive Function and the Response to Caloric Restriction.

Sirt1 is an NAD-dependent, class III deacetylase that functions as a cellular energy sensor. In addition to its well-characterized effects in peripheral tissues, emerging evidence suggests that neuronal Sirt1 activity plays a role in the central regulation of energy balance and glucose metabolism. In this study, we generated mice expressing an enzymatically inactive form (N-MUT) or wild-type (WT) SIRT1 (N-OX) in mature neurons. N-OX male and female mice had impaired glucose tolerance, and N-MUT female, but not male, mice had improved glucose tolerance compared with that of WT littermates. Furthermore, glucose tolerance was improved in all mice with caloric restriction (CR) but was greater in the N-OX mice, who had better glucose tolerance than their littermates. At the reproductive level, N-OX females had impaired estrous cycles, with increased cycle length and more time in estrus. LH and progesterone surges were absent on the evening of proestrus in the N-OX mice, suggesting a defect in spontaneous ovulation, which was confirmed by the ovarian histology revealing fewer corpora lutea. Despite this defect, the mice were still fertile when mated to WT mice on the day of proestrus, indicating that the mice could respond to normal pheromonal or environmental cues. When subjected to CR, the N-OX mice went into diestrus arrest earlier than their littermates. Together, these results suggested that the overexpression of SIRT1 rendered the mice more sensitive to the metabolic improvements and suppression of reproductive cycles by CR, which was independent of circadian rhythms.

Implantation and pregnancy outcome of Sprague–Dawley rats fed with low and high salt diet

Abstract Objective: To investigate the influence of low salt diet and high salt diet on implantation and pregnancy in rats. Design: This is an experimental animal study. Materials and methods : Female rats were acclimatized and divided into three groups consisting of control/normal salt diet (0.3% salt), low salt diet (0.14% salt) and high salt diet (8% salt). Rats were fed for six weeks and the cycles of the rats were observed in the 6th week for the introduction of male rats to female rats on the evening of proestrus for mating. Implantation studies were carried out on days 6 and 8 of pregnancy while fetal parameters were ascertained on day 19 of pregnancy and at term. Levels of progesterone, estradiol, prostaglandin E 2 , nitric oxide (NO) and cyclic guanosine monophosphate (cGMP), were measured on days 6, 8 and 19 of pregnancy. Results: The results showed decreased implantation sites on days 6 and 8 in high salt fed rats, decreased birth weight in low salt fed rats and increased placental to birth weight ratio in the high salt fed rats when compared with control. A significant decrease in PGE 2 level was found on day 8 of pregnancy in the high salt fed rats, while progesterone:estradiol ratio was decreased on days 6 and 8 of pregnancy in both low salt and high salt fed rats. Also, NO and cGMP levels were decreased on day 19 of pregnancy in the low salt fed rats when compared with control ( p Conclusion: The results showed that a shift in salt homeostasis either to a high or a low salt level causes hormonal imbalance and impaired reproductive outcome during pregnancy.

Maternal Vitamin D Deficiency Programs Reproductive Dysfunction in Female Mice Offspring Through Adverse Effects on the Neuroendocrine Axis.

Vitamin D (VitD) deficiency affects more than 1 billion people worldwide with a higher prevalence in reproductive-aged women and children. The physiological effects of maternal VitD deficiency on the reproductive health of the offspring has not been studied. To determine whether maternal VitD deficiency affects reproductive physiology in female offspring, we monitored the reproductive physiology of C57BL/6J female offspring exposed to diet-induced maternal VitD deficiency at three specific developmental stages: 1) in utero, 2) preweaning, or 3) in utero and preweaning. We hypothesized that exposure to maternal VitD deficiency disrupts reproductive function in exposed female offspring. To test this hypothesis, we assessed vaginal opening and cytology and ovary and pituitary function as well as gonadotropin and gonadal steroid levels in female offspring. The in utero, preweaning, and in utero and preweaning VitD deficiency did not affect puberty. However, all female mice exposed to maternal VitD deficiency developed prolonged and irregular estrous cycles characterized by oligoovulation and extended periods of diestrus. Despite similar gonadal steroid levels and GnRH neuron density, females exposed to maternal VitD deficiency released less LH on the evening of proestrus. When compared with control female offspring, there was no significant difference in the ability of females exposed to maternal VitD deficiency to respond robustly to exogenous GnRH peptide or controlled ovarian hyperstimulation. These findings suggest that maternal VitD deficiency programs reproductive dysfunction in adult female offspring through adverse effects on hypothalamic function.

Differential DNA methylation pattern in the A and B promoters of the progesterone receptor is associated with differential mRNA expression in the female rat hypothalamus during proestrus

In rodents, the display of reproductive behavior occurs during the proestrus–estrus transition of the estrus cycle. This behavior is regulated by estradiol and progesterone mainly via their intracellular receptors. Two isoforms of the progesterone receptor have been described (A and B), and they have different promoters for their regulation. It has been demonstrated that the mRNA for both isoforms changes during the proestrus–estrus transition. It has been recently established that DNA methylation can be transient and cyclical in gene promoters, however, these changes have only been reported in vitro but not in physiological models. The aim of this study was to analyze the pattern of DNA methylation in the PR (A and B) promoter regions during the proestrus–estrus transition in the rat hypothalamus and its correlation with the regulation of mRNA expression. The results demonstrated a differential mRNA expression of the progesterone receptor (A and B) isoforms. The expression of total PR did not change significantly during the proestrus day, while the expression of isoform B increased significantly at 17:00h, followed by a significant decrease at 21:00h of the proestrus day. Interestingly, we also found that the isoform A promoter was mainly unmethylated at all studied time points. In contrast, the isoform B promoter showed a transient methylation increase during the evening of proestrus. The overall results indicate that there is a switch of progesterone receptor isoforms expression during the evening of proestrus that is related to the differential gene methylation patterns of their promoter regions, mainly for the isoform B promoter.

The effect of Vitamin C on amodiaquine‐induced oxidative stress in the ovary of Sprague‐Dawley rats

AQ‐induced oxidative stress leads to increased atresia which in turn leads to reduced follicle numbers, and reduced oocyte numbers at ovulation. This research study the effect of the co‐administration of amodiaquine hydrochloride (AQ.HCl) with Vitamin C on oestrous cycle, ovulation, oxidative stress markers and the histomorphology of the ovary of mature Sprague‐Dawley rats.Two study experimental groups were observed, Group 1: the effect of the co‐administration of AQ.HCl with Vitamin C on pattern and length of the oestrous cycle, histomorphology of the ovary and antioxidant activities of superoxide dismutase (SOD) and catalase (CAT). Group 2: the effect of the co‐administration of a single dose of AQ.HCl with Vitamin C administered in the evening of proestrus on ovulation and serum concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin (PRL).Co‐administration of AQ.HCl with Vitamin C resulted in an irregularity in cycle pattern and also increased (p &lt; 0.05) the length of the oestrous cycle by prolonging the diestrus phase compared to the control. Histological sections showed increased number of atretic follicles compared to the control. A statistical significant decrease (p &lt; 0.05) was observed in the activities of CAT and SOD. Serum levels of FSH, LH and PRL were comparable to control values however; the number of ova shed in the oviduct was significantly reduced (p &lt; 0.05). The result of this study showed that Vitamin C could not confer protection against the deleterious effect of AQ.HCl on the ovary of Sprague‐Dawley rats.

Short term hypothyroidism affects ovarian function in the cycling rat

BackgroundRats made hypothyroid with propilthyouracil start showing abnormal cycling on the second cycle after the start of the treatment, with a high proportion of spontaneous pseudopregnancies and reduced fertility.MethodsTo investigate some of the mechanisms involved in these reproductive abnormalities, hypothyroidism was induced in virgin rats by propilthyouracil (0.1 g/L in the drinking water) and we determined circulating hormones by radioimmunoassay and whole ovary expression of ovarian hormone receptors, growth factors and steroidogenic enzymes using semi-quantitative RT-PCR.The study was performed on days 6 to 9 of treatment, corresponding to diestrus I (at 20.00-22.00 h), diestrus II (at 20.00-22.00 h), proestrus and estrus (both at 8.00-10.00 h and 20.00-22.00 h) of the second estrous cycle after beginning propilthyouracil treatment. Another group of rats was mated on day 8 and the treatment continued through the entire pregnancy to evaluate reproductive performance.ResultsHypothyroidism increased circulating prolactin and estradiol on estrus 5 to 7-fold and 1.2 to 1.4-fold respectively. Growth hormone and insulin-like growth factor 1 diminished 60 and 20% respectively on proestrus morning. Hypothyroidism doubled the ovarian mRNA contents of estrogen receptor-beta on proestrus and estrus evenings, cyp19A1 aromatase mRNA on estrus evening and of growth hormone receptor on proestrus evening. Hypothyroidism did not influence ovulation rate or the number of corpora lutea at term, but a diminished number of implantation sites and pups per litter were observed (Hypothyroid: 11.7 +/- 0.8 vs. Control: 13.9 +/- 0.7).ConclusionsShort term hypothyroidism alters normal hormone profile in the cycling rat increasing the expression of estrogen receptor-beta and cyp19A1 aromatase on estrus, which in turn may stimulate estradiol and prolactin secretion, favouring corpus luteum survival and the subsequent instauration of pseudopregnancy.

Female sexual behavior, estrous cycle and gene expression in sexually dimorphic brain regions after pre- and postnatal exposure to endocrine active UV filters

The developing female brain represents a potential target for estrogenic environmental chemicals because it depends on estrogen but is exposed to low endogenous estrogen levels, thus facilitating competition by exogenous estrogen receptor (ER) agonists. We investigated effects of two estrogenic UV filters, 4-methylbenzylidene camphor (4-MBC) and 3-benzylidene camphor (3-BC). 4-MBC has been detected in human milk, indicating potential exposure of fetus and infant. The two chemicals were administered in chow to rats of the parent generation before mating, during pregnancy and lactation, and to their offspring until adulthood. Female sexual behavior was recorded on videotape in adult female offspring on proestrus evening at the beginning of the dark phase. 4-MBC (7 and 24 mg/kg bw/day) and 3-BC (2.4 and 7 mg/kg bw/day) reduced proceptive behavior (jump and ear wiggling) and receptive behavior (lordosis quotient), and increased rejection behavior towards the male. Estrous cycles were not affected by 4-MBC but disturbed by 3-BC. mRNAs encoding for genes involved in female sexual behavior, ERα, ERβ, progesterone receptor (PR) and steroid receptor coactivator-1 (SRC-1), were measured by real-time RT-PCR in ventromedial hypothalamic nucleus (VMH) and medial preoptic area of adult male and female offspring (studied in diestrus) after pre- and postnatal exposure to 3-BC (0.24, 0.7, 2.4 and 7 mg/kg bw/day). Gene expression was affected in a sex- and region-specific manner. PR mRNA in female VMH was reduced to male levels at dose levels of 2.4 and 7 mg/kg bw/day 3-BC. Our data demonstrate that female sexual behavior represents a sensitive target of endocrine disrupters and point to an involvement of PR in VMH.

PACAP is transiently expressed in anterior pituitary gland of rats: In situ hybridization and cell immunoblot assay studies

In this work the expression of PACAP (pituitary adenylate cyclase activating polypeptide) in rat anterior pituitary was demonstrated for the first time using in situ hybridization. The number of cells showing PACAP signal in intact male rats was negligible similarly to that of diestrous rats. In proestrous rats sacrificed at 10 h there was a moderate increase in the expression and after a decrease at 16 h and 18 h, there was a transient peak at 20 h and then the number of labeled cells was declined again (22 h). In the cell immunoblot assay study it was observed that the number of PACAP blot forming (PACAP releasing) cells in an anterior pituitary cell culture changed according to a similar pattern as the number of PACAP expressing cells. The number of blots was also the highest when the animals were sacrificed in the evening of proestrus at 20 h. The results obtained by in situ hybridization and cell immunoblot assay well correlate with each other. The above-mentioned results support our hypothesis that the enhanced expression and secretion of PACAP in the pituitary gland may be involved in ceasing the LH surge.

Global gene expression profiles of ovarian surface epithelial cells in vivo

Epithelial ovarian cancer, the leading cause of death from gynecological malignancy in Western countries, is thought to arise from the ovarian surface epithelium (OSE). It has been postulated that the constant rounds of proliferation and repair following ovulation contributes to neoplastic transformation. However, there is little information on the genes and pathways which are involved in the normal functions of the ovarian epithelium, in particular genes that are hormone responsive and those central to functions such as proliferation and apoptosis during ovulation. We used laser microdissection and cDNA microarrays to profile gene expression specifically in mouse ovarian epithelial cells, first compared with other ovarian cells, and secondly between ovarian epithelium collected at different physiological stages. We identified over 1000 transcripts that were consistently more highly expressed in the ovarian epithelium compared with remaining ovarian cell types, including genes involved in cell growth, transcription, and cell adhesion. At the various physiological stages examined, the highest number of regulated genes was found during the estrous cycle, specifically on the evening of proestrus, coincident with the ovulatory surge of hormones and just prior to ovulation. The expression of several selected genes, identified by the microarray analysis, including Villin 2, Keratin 8, Arginine-rich mutated in epithelial tumors, and Tumor-associated calcium signal transducer 1, was validated by independent methods. The identification of genes expressed and regulated in the OSE, and characterization of the pathways involved, will contribute to a more detailed understanding of the ovarian epithelium transcriptome and ultimately lead to a better understanding of the aberrations leading to malignant transformation in the ovarian epithelium.

Genital sensory stimulation shifts estradiol intraoviductal signaling from nongenomic to genomic pathways, independently from prolactin surges.

Estradiol (E2) accelerates oviductal egg transport through nongenomic pathways involving oviductal protein phosphorylation in non-mated rats, and through genomic pathways in mated rats. Here we investigated the ability of cervico-vaginal stimulation (CVS) to switch the mode of action of E2 in the absence of other male-associated components. Pro-estrous rats were subjected to CVS with a glass rod and 12 hours later were injected subcutaneously with E2 and intrabursally with the RNA synthesis inhibitor Actinomycin D or the protein phosphorylation inhibitor H-89. The number of eggs in the oviduct, assessed 24 h later, showed that Actinomycin D, but not H-89 blocked the E2-induced egg transport acceleration. This clearly indicates that CVS alone, without other mating-associated signals, is able to shift E2 signaling from nongenomic to genomic pathways. Since mating and CVS activate a neuroendocrine reflex that causes iterative prolactin (PRL) surges, the involvement of PRL pathway in this phenomenon was evaluated. Prolactin receptor mRNA and protein expression in the rat oviduct was demonstrated by RT-PCR and Western blot, but their levels were not different on day 2 of the cycle (C2) or pregnancy (P2). Activated ST AT 5a/b (phosphorylated) was detected by Western blot on P2 in the ovary, but not in the oviduct, showing that mating does not stimulate this PRL signalling pathway in the oviduct. Other rats subjected to CVS in the evening of pro-estrus were treated with bromoergocriptine to suppress PRL surges. In these rats, H-89 did not block the E2-induced acceleration of egg transport suggesting that PRL surges are not essential to shift E2 signaling pathways in the oviduct. We conclude that CVS is one of the components of mating that shifts E2 signaling in the oviduct from nongenomic to genomic pathways, and this effect is independent of PRL surges elicited by mating.

Expression of progesterone receptors A and B in the mouse ovary during the estrous cycle.

Progesterone plays a central role in the regulation of ovarian function. The progesterone receptor (PR) has been shown to be essential for ovulation because mice lacking PR fail to ovulate and are infertile. PR is expressed as two isoforms, PRA and PRB, which have been shown to have different functional activities. In this study, we investigated the cellular distribution of PRA and PRB in the ovaries and oviducts of cycling mice using immunohistochemistry with isoform-specific monoclonal antibodies. In the ovary, on the evening of proestrus before ovulation, both the granulosa and theca cells of the preovulatory follicles expressed both PR isoforms. PRA and PRB staining was also observed in the theca cells of preantral and antral follicles, whereas only PRB was observed in the granulosa cells of primary, preantral, and antral follicles and in the corpus luteum. In the oviduct, PRA was the predominant isoform observed, expressed in both the epithelial and stromal cells, whereas PRB was only detected in the epithelial cells. The differences in PRA and PRB localization in the ovary and oviduct may reflect diverse functions for PRA and PRB in reproductive tissues and may have important implications in understanding the mechanisms of progesterone action.

Calcitonin Expression in Rat Anterior Pituitary Gland Is Regulated by Ovarian Steroid Hormones

Gonadotroph-derived calcitonin-like peptide (pit-CT) is a potent inhibitor of lactotroph function. We investigated the effect of ovarian hormones on pit-CT mRNA expression in the anterior pituitary (AP) gland of cycling female rats. Levels of mRNAs for pit-CT, CT receptor, prolactin (PRL), and beta-LH during 4-d estrous cycle were determined. In a second study, the effects of estrogens and progesterone on pit-CT and PRL mRNA levels were investigated. In a third group, the effect of estrogen or progesterone depletion on pit-CT mRNA expression was studied. In a fourth group, the effect of passive pit-CT immunization on PRL and LH mRNA expression was examined. Pit-CT mRNA levels varied during estrous cycle. They were highest in diestrus, but lowest in the evening of proestrus. CT-receptor mRNA levels displayed smaller fluctuations. Estrogen repletion caused a decline in pit-CT mRNA expression in ovariectomized rats, but progesterone produced a marked increase. ICI 182,780 prevented the decline of pit-CT mRNA levels during late proestrus-estrus, but RU 486 attenuated pit-CT mRNA levels. Passive CT immunization in diestrus altered PRL and LH mRNA expression, and advanced the estrus cycle. These results suggest that pit-CT mRNA expression is regulated by ovarian hormones, and depletion of pit-CT advances their estrous cycle.

Estrous cycle-regulated expression of CYP1B1 mRNA in the rat ovary

CYP1B1, a member of the cytochrome P450 superfamily, is expressed constitutively in the steroidogenic tissues of mammals and is inducible by peptide hormones, cAMP and aromatic hydrocarbon receptor (AHR) ligands. The mechanism of induction of this cytochrome P450 is similar to that for CYP1A1, i.e. through the aromatic hydrocarbon receptor (AHR) signaling pathway. We have recently reported that CYP1B1, but not CYP1A1, is expressed in rat granulosa cells (GC) in the absence of any external stimulus. The induction of CYP1B1 mRNA in rat GC by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in vitro was followed by an increase in AHR and estrogen receptor (ER-β) RNA levels. Estrous cycle-dependent expression of AHR, AHR-nuclear translocator (ARNT) and ER-mRNAs in the rat ovary was reported. We suggest that CYP1B1 may play a major role in the regulation of rat ovarian function/cycle but until now this has been unexplored experimentally. The present study was therefore aimed at examining the expression of CYP1A1, CYP1B1 and ER-mRNA in rat ovarian tissues throughout the estrous cycle to establish any correlation in the expressions of these mRNAs in rat ovary. Total RNA was extracted from the ovary and liver of cycling adult rats and the mRNAs were analyzed using relative RT-PCR with gene-specific primers for the target mRNA and for RPL 19 or S16 primers as an internal control. The results indicated that in the ovary, CYP1B1 mRNA increased significantly on the evening of proestrus and dramatically decreased on the morning of estrus, while ER-mRNA remained unaltered throughout the estrous cycle. CYP1A1 mRNA in the ovary and both CYP1A1 and CYP1B1 mRNAs in the liver were undetectable. That the sudden decrease of ovarian CYP1B1 mRNA on the morning of estrus was not an effect of the LH surge was verified in vitro using our short-term GC culture model. GC prepared from rats super-stimulated with equine chorionic gonadotropin (eCG) were cultured for 6 h with or without LH and TCDD. It was observed that both CYP1A1 and CYP1B1 mRNAs were induced by TCDD with no apparent effect of LH. It is suggested that the high level of CYP1B1 mRNA expression on the evening of proestrus in rat ovary might be involved in metabolism of estrogens to catecholestrogen (a known effect of CYP1B1), and that expression is unaffected in GC by LH.

The preovulatory rise of ovarian ornithine decarboxylase is required for progesterone secretion by the corpus luteum

Ovarian progesterone secretion during the diestrus stage of the estrous cycle is produced by luteal cells derived from granulosa and thecal cells after the differentiation process that follows ovulation. Our results show that blockade of the preovulatory rise of ovarian ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, by treatment with the specific inhibitor α-difluoromethylornithine (DFMO) leads to a significant decrease in the ovarian progesterone content and a dramatic fall in the plasma levels of this hormone during the following diestrus. The same inhibition was produced in spite of the fact that both luteinizing and follicle stimulating hormones were given concomitantly with DFMO. On the other hand, the acute rise in the plasma progesterone levels observed after administration of human chorionic gonadotropin to mice at different periods of the estrous cycle was not affected by DFMO administration. Our results indicate that although elevated levels of ODC are not required for acute ovarian steroidogenesis, the preovulatory peak of ovarian ODC activity observed in the evening of proestrus may be critical for the establishment of a constitutive steroidogenic pathway and progesterone secretion by the corpus luteum during the diestrus stage of the murine estrous cycle.

Excitotoxic Lesions of the Medial Amygdala Differentially Disrupt Prolactin Secretory Responses in Cycling and Mated Female Rats

AbstractThis study examined the role of the posterodorsal medial amygdala (MePD) in the control of prolactin secretion in gonadally intact female rats 20 min after mating, during the oestrous cycle, and during early pregnancy/pseudopregnancy (P/PSP). Cycling females received bilateral infusions of an excitotoxic dose of N‐methyl‐d‐aspartate (NMDA) or vehicle into the MePD. Two to 4 weeks later, they were surgically implanted with intra‐atrial catheters for repeated blood sampling and were mated on the evening of proestrus until receiving 5, 10, 15 or 20 intromissions or 15 mounts‐without‐intromission (MO) from males. The percentages of rats becoming P/PSP increased as a function of numbers of intromissions received. All groups receiving intromissions showed similar approximately four‐fold increases in plasma prolactin concentrations 20 min after mating, while MO rats showed no increase at this time. There was no effect of NMDA lesion on this acute secretory response. Among rats that continued cycling, bilateral MePD lesion completely abolished the diurnal preovulatory prolactin surge, while incomplete and sham lesions did not. In rats that subsequently became P/PSP, bilateral lesion of the MePD resulted in dampening of prolactin concentrations at all sampling times 6–7 days after mating, while incomplete or sham lesions did not alter prolactin secretion at these times. These results demonstrate that the MePD is selectively important for the secretion of prolactin on the afternoon of proestrus and that this structure may also modulate prolactin release in P/PSP rats. Activity within MePD neurones does not appear to be required for the acute prolactin response to vaginocervical stimulation which occurs within minutes after mating.

Excitotoxic lesions of the medial amygdala differentially disrupt prolactin secretory responses in cycling and mated female rats.

This study examined the role of the posterodorsal medial amygdala (MePD) in the control of prolactin secretion in gonadally intact female rats 20 min after mating, during the oestrous cycle, and during early pregnancy/pseudopregnancy (P/PSP). Cycling females received bilateral infusions of an excitotoxic dose of N-methyl-D-aspartate (NMDA) or vehicle into the MePD. Two to 4 weeks later, they were surgically implanted with intra-atrial catheters for repeated blood sampling and were mated on the evening of proestrus until receiving 5, 10, 15 or 20 intromissions or 15 mounts-without-intromission (MO) from males. The percentages of rats becoming P/PSP increased as a function of numbers of intromissions received. All groups receiving intromissions showed similar approximately four-fold increases in plasma prolactin concentrations 20 min after mating, while MO rats showed no increase at this time. There was no effect of NMDA lesion on this acute secretory response. Among rats that continued cycling, bilateral MePD lesion completely abolished the diurnal preovulatory prolactin surge, while incomplete and sham lesions did not. In rats that subsequently became P/PSP, bilateral lesion of the MePD resulted in dampening of prolactin concentrations at all sampling times 6-7 days after mating, while incomplete or sham lesions did not alter prolactin secretion at these times. These results demonstrate that the MePD is selectively important for the secretion of prolactin on the afternoon of proestrus and that this structure may also modulate prolactin release in P/PSP rats. Activity within MePD neurones does not appear to be required for the acute prolactin response to vaginocervical stimulation which occurs within minutes after mating.

Impact of uremia on female reproductive cyclicity, ovulation, and luteinizing hormone in the rat

Impaired reproductive function accompanies chronic renal insufficiency (uremia) in both the human and experimental animal. Clinical hypogonadism occurs in both genders. The present studies were designed to investigate possible anti-ovulatory effects of uremia in the female rat, a species that produces multiple ova during the normal estrous cycle. Renal insufficiency (uremia) was induced by 5/6 nephrectomy. Two control groups comprised sham-operated animals fed ad libitum (sham) or pair-wise with the uremic animals (pair-fed). Estrous cycles were determined by cytology of vaginal lavage. We examined concomitant changes in the preovulatory luteinizing hormone (LH) surge by radioimmunoassay (RIA), immunoradiometric assay (IRMA), and bioassay. Repetitive LH measurements were made from blood samples taken by intra-atrial catheter throughout the afternoon and evening of proestrus. The following morning (estrus), ovaries were collected, and ova were enumerated per oviduct. Experimentally uremic animals manifested a threefold elevation of plasma creatinine and urea nitrogen and concomitantly a more than 50% impoverishment of ova production. Analyses of a large group of animals (N = 83) by RIA revealed uremia-associated attenuation of the preovulatory LH surge. Further measurements of the preovulatory LH surge by independent IRMA and LH bioassay (N = 26) corroborated this attenuation. Additional experiments indicated that these hormonal changes, but not changes in ovulation, might further reflect modulation of LH release by the anesthesia used in the preparative nephrectomy and catheterization surgeries. When normalized to body weight, the ovaries of uremic rats were found to weigh more than those of either the sham or pair-fed animals. The present experiments take advantage of an experimental uremic model to document a consistent decrease in the number of ova released during estrus in the uremic animal. Possible disruption of hypothalamic-pituitary-ovarian regulation is further highlighted by attenuation in the preovulatory LH surge. These results provide a basis for further studies of neuroendocrine pathophysiology in a rodent model of uremia-associated ovulatory disruption.