Early Proestrus Research Articles

Sex-dependent effects of multiple acute concurrent stresses on memory: a role for hippocampal estrogens.

Memory disruption commonly follows chronic stress, whereas acute stressors are generally benign. However, acute traumas such as mass shootings or natural disasters—lasting minutes to hours and consisting of simultaneous physical, social, and emotional stresses—are increasingly recognized as significant risk factors for memory problems and PTSD. Our prior work has revealed that these complex stresses (concurrent multiple acute stresses: MAS) disrupt hippocampus-dependent memory in male rodents. In females, the impacts of MAS are estrous cycle-dependent: MAS impairs memory during early proestrus (high estrogens phase), whereas the memory of female mice stressed during estrus (low estrogens phase) is protected. Female memory impairments limited to high estrogens phases suggest that higher levels of estrogens are necessary for MAS to disrupt memory, supported by evidence that males have higher hippocampal estradiol than estrous females. To test the role of estrogens in stress-induced memory deficits, we blocked estrogen production using aromatase inhibitors. A week of blockade protected male and female mice from MAS-induced memory disturbances, suggesting that high levels of estrogens are required for stress-provoked memory impairments in both males and females. To directly quantify 17β-estradiol in murine hippocampus we employed both ELISA and mass spectrometry and identified significant confounders in both procedures. Taken together, the cross-cycle and aromatase studies in males and females support the role for high hippocampal estrogens in mediating the effect of complex acute stress on memory. Future studies focus on the receptors involved, the longevity of these effects, and their relation to PTSD-like behaviors in experimental models.

Salivary cell-free HSD17B1 and HSPA1A transcripts as potential biomarkers for estrus identification in buffaloes (Bubalus bubalis)

Estrus detection is a major problem in buffaloes because of the poor expression of estrus signs leading to low reproductive efficiency. Salivary transcripts analysis is a promising tool to identify biomarkers; therefore, the present study was carried out to evaluate their potential as estrus biomarkers. The levels of HSD17B1, INHBA, HSPA1A, TES transcripts were compared in saliva during estrous cycle stages [early proestrus (day −2, EP), late proestrus (day-1, LP), estrus (E), metestrus (ME) and diestrus (DE)] of cyclic heifers (n = 8) and pluriparous (n = 8) buffaloes by employing quantitative real-time polymerase chain reaction (qRT-PCR). The levels of HSD17B1 (EP/DE 1.46–2.43 fold, LP/DE 2.49–3.06 fold; E/DE 7.21–11.9-fold p < 0.01; ME/D 1.0–1.16 fold) and HSPA1A (EP/DE 0.93–2.39 fold, LP/DE 2.68–3.23 fold; E/DE 8.52–15.18 fold p < 0.01; ME/D 0.86–1.01 fold) were significantly altered during the estrus than other estrous cycle stages in both cyclic heifers and pluriparous buffaloes. Receiver operating characteristic curve analysis revealed the ability of salivary HSD17B1 (AUC 0.96; p < 0.001) and HSPA1A (AUC 0.99; p < 0.01) to differentiate E from other stages of the estrous cycle. Significantly higher levels of HSD17B1 and HSPA1A transcripts in saliva during the estrus phase suggest their biomarkers potential for estrus detection in buffaloes.

Results of ultrasound-guided transvaginal collection of oocytes (TVA-OPU) from mares to be fertilized by intracytoplasmic sperm injection (ICSI).

The aim of this study was to present the results of transvaginal ultrasound-guided collection of oocytes (TVA-OPU) from 21 mares to be fertilized in vitro by the ICSI method. One week before the planned OPU, the mares underwent a gynecological examination with particular emphasis on the functional status of the ovaries. The recommendation for the procedure was the presence of at least 12 ovarian follicles with a diameter of at least 4 mm on both ovaries. The selected mares were given PGF2α in a luteolytic dose 3 days before the planned OPU, so that the mares were in the early proestrus stage during the procedure and did not have a corpus luteum on the ovary. If PGF2α administration was believed to increase the risk of estrus on the day of the OPU procedure, the hormone was not administered. Immediately prior to OPU, the mares were premedicated with detomidine and butorphanol. During the procedure, symptoms of anxiety or pain were controlled using the bolus agents listed above, plus flunixin as needed. A mixture of hyascin butylbromide and sodium metamizole was also used in order to eliminate an excessive tension of the rectum. After each treatment, the mares were administered procaine penicillin, benzathine penicillin, and streptomycin. All mares underwent the OPU treatment without complications. During 24 OPU procedures (3 mares underwent the procedure twice), a total of 239 oocytes (an average of 10 oocytes from 1 mare) were obtained, 137 of which were qualified for the ICSI procedure (57% of the total). After fertilization, embryonic division occurred in 95 (70% of the oocytes qualified for ICSI). Ultimately, 23 embryos were grown and preserved in liquid nitrogen (which resulted in an average of 1 embryo per 1 OPU treatment in mares) and accounted for 17% of oocytes qualified for the treatment. According to the authors' best knowledge, it was the first project of this type in our country.

Evaluation of changes in blood flow of the uterine artery by Doppler ultrasonography during the estrous cycle in lactating Bos indicus cows

The objective of this study was to evaluate the changes in uterine blood flow (UBF) based on the resistance index (RI) and the pulsatility index (PI) by Doppler ultrasonography throughout the estrous cycle in lactating Sahiwal cows (n=9). Cows were randomly selected during their spontaneous estrus. UBF was examined on alternate days in all cows during the estrous cycle. The results revealed that the mean RI values were higher (P<0.05) on day −1 (estrus) than on day 0 (ovulation). The mean RI values followed a consistent pattern on days 2, 4, 6, and 8 (metestrus to early diestrus) and were lower (P<0.05) than on days 10, 14 16, and 18 (diestrus to early proestrus). The RI and corpus luteum (CL) size negatively correlated during its static phase (r=−0.99; P<0.05). The mean RI was greater (P<0.05) in high producers than in low producing cows. The mean PI value was higher (P<0.05) on day 10 than on day −1 and day 0. Similarly, the mean PI values were lower (P <0.05) on days 2 and 4 than on day 10. The PI value remained significantly lower (P<0.05) on day 2 than on day 8 of the estrous cycle. There was a positive correlation between RI and P4 (r=0.70; P<0.05) and PI and P4 (r=0.56; P<0.05) during the estrous cycle. It is concluded that the RI of uterine arteries, as a measure of blood flow, is considerably lower, while the PI is substantially elevated during diestrus compared to estrus and ovulation in Sahiwal cows.

Antinociception and sedation following intracerebroventricular administration of Δ 9-tetrahydrocannabinol in female vs. male rats

Systemically administered cannabinoids produce greater antinociceptive and sedative effects in female compared to male rats. Sex differences in the brain endocannabinoid system have also been reported. The aim of this study was to determine whether sex differences in antinociceptive and motoric effects of a cannabinoid can be attributed to supraspinal mechanisms. Vehicle or Δ 9-tetrahydrocannabinol (THC, 100 μg) was administered i.c.v., and behavioral effects were compared between gonadally intact male and female rats, and among females in different estrous stages (early proestrus, late proestrus, estrus and diestrus). Antinociception on the tail withdrawal and paw pressure tests after i.c.v. THC was slightly but not significantly greater in females (pooled across estrous stages) compared to males. THC suppressed locomotor activity similarly in all groups, with the exception that only males showed hyperlocomotion at 4 h post-injection. When females in the four estrous stages were compared, females in late proestrus showed significantly greater THC-induced antinociception than females in estrus (and males). These results suggest that supraspinal mechanisms may contribute to greater systemic THC effects in females compared to males, and to estrous stage-dependent differences in THC effects among females.

Initial body condition score affects hormone and metabolite response to nutritional restriction and repletion in yearling postpubertal beef heifers1

Twenty Simmental x Angus, half-sibling, postpubertal heifers (initial BW of 443 +/- 9 kg) were allotted randomly into 2 treatment groups to evaluate if initial BCS affects response of the hypothalamic-pituitary-ovarian axis to metabolic signals elicited by energy restriction and repletion. During a preliminary feeding period, diets were formulated so that each heifer in the designated treatment would reach a BCS of 5 (moderate condition; MOD) or a BCS of 7 (heavy condition; FAT). Once each heifer had reached desired BCS, diets were formulated to supply 30% of NE(m) requirements until each heifer became anestrous (serum concentrations of progesterone < 1 ng/mL; restriction period). Blood collections took place on d 1 of each period, on d 43 of energy restriction and d 44 of energy repletion, and when heifers were confirmed to recommence estrous cycles. When heifers were cycling, their estrous cycles were synchronized to ensure hormone sampling occurred during late diestrus or early proestrus. Energy restriction resulted in decreased concentrations of LH (FAT, P = 0.02; MOD, P < 0.001), IGF-1 (FAT, P < 0.001; MOD, P = 0.003), and insulin (P < 0.001); in contrast, concentrations of GH (P < 0.001) and plasma urea nitrogen (P < 0.001) increased. During repletion, LH concentration increased (P = 0.03) in MOD condition heifers but was still less (P = 0.002) than d 1 of restriction, whereas LH concentration tended to increase in FAT heifers (P = 0.06) until it was similar (P = 0.40) to d 1 of restriction. Repletion also increased concentrations of IGF-1 (P < 0.001), insulin (P < 0.001), and glucose (P < 0.001), whereas concentrations of GH (P < 0.001), NEFA (P < 0.001), and plasma urea nitrogen (P < 0.001) decreased. For both treatments, concentrations of GH after repletion were similar (FAT, P = 0.88; MOD, P = 0.10) to those on d 1 of restriction. After repletion, FAT condition heifers had decreased concentrations of IGF-1 (P < 0.001), insulin (P < 0.05), and glucose (P < 0.001), but greater concentrations of acetate (P < 0.01) and butyrate (P < 0.05), than MOD heifers. Anestrus or resumption of estrous cycles seems to be activated gradually in response to dietary manipulation, unrelated to certain metabolite changes.

The sympathetic neural apparatus of the rat uterus during the sexual cycle

A microspectral fluorescence-histochemical method was used to study the sympathetic neural apparatus of the rat uterus at different phases of the sexual cycle. Most nerve fibers, as perivascular plexuses and terminals emanating from them, were located in the myometrium. It is suggested that the uterus contains reserve ("silent" nerve fibers, which explains the variability in their appearance in this organ. Microspectrofluorimetric studies identified catecholamines and serotonin in varicose swellings and intervaricosity segments of nerve fibers; levels at test points showed a strong linear correlation at all stages of the sexual cycle. The greatest bioamine levels in most structural elements of the sympathetic neural apparatus of the uterus were seen in late diestrus. Early and late estrus and proestrus were characterized by low monoamine levels in adrenergic fibers. A number of significant differences were seen between the level of saturation with bioamines and the spatial distribution density of nerve fibers in the body and cervix of the uterus.

Participation of vasoactive intestinal polypeptide in ovarian steroids production during the rat estrous cycle and in the development of estradiol valerate-induced polycystic ovary

Vasoactive intestinal polypeptide (VIP) stimulates estradiol and progesterone release from ovarian granulosa cells in vitro. Very little information is available as to the role VIP plays in the control of steroid secretion during reproductive cyclicity and in ovarian pathologies involving altered steroid secretion. In this study, we determined the involvement of VIP in regulating ovarian androgen and estradiol release during estrous cyclicity and estradiol valerate (EV)-induced polycystic ovarian development in rats. Our findings show that androgen and estradiol release from ovaries obtained during different stages of rat estrous cycle mimic cyclic changes in steroid release observed in vivo with maximal release occurring during late proestrus. VIP increased androgen release from ovaries of all cycle stages except late proestrus and estradiol release from all cycle stages. Increases in VIP-induced androgen and estradiol release were maximal at early proestrus. Inclusion of saturating concentrations of androstenedione increased magnitude of VIP-induced estradiol release at diestrus and estrus but not proestrus. Magnitude of VIP-induced androgen and estradiol release tended to be greater in the ovaries from EV-treated rats with polycystic ovary compared with estrous controls. At the tissue level, ovarian VIP concentration was cycle stage dependent with highest level seen in diestrus. Maximum concentration of VIP was found in EV-treated rats. Changes in VIP were inversely related to changes in ovarian nerve growth factor, a neuropeptide involved in ovarian androgen secretion. These results strongly suggest that intraovarian VIP participates in the control of estradiol secretion during the rat estrous cycle and possibly in the maintenance of increased ovarian estradiol secretory activity of EV-treated rats.

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.

BMP7/ActRIIB regulates estrogen-dependent apoptosis: new biomarkers for environmental estrogens.

A ligand-receptor pair, bone morphogenetic protein-7 (BMP7) and activin receptor IIB (actRIIB), was identified from a pool of DNA fragments recovered from MCF7 cells treated with 17beta-estradiol (E2) by chromatin immunoprecipitation with antiestrogen receptor-alphaantibody. The E2 responsiveness of both genes was confirmed in MCF cells and in the mouse uterus. Repeated treatment with E2 resulted in decreased expression of both actRIIB and BMP7 mRNA in the uteri of ovariectomized mice. A single oral administration of bisphenol A (BPA), an environmental estrogen, inhibited actRIIB and BMP7 expression and apoptosis in the luminal epithelium of the mouse uterus at diestrus (or early proestrus). This decrease, due to BPA administration, was restored by an estrogen receptor (ER) antagonist suggesting that it is mediated through ERs. These results suggest that E2 and BPA suppress estrogen-dependent apoptosis of epithelial cells of the endometrium through down-regulation of actRIIB and BMP7. Thus, we propose that BMP7 and actRIIB, a ligand-receptor pair, are involved in regulation of the apoptotic signaling pathway and might therefore be new biomarkers of the effects of environmental estrogens on the female reproductive tract.

Treatment of rats with 17beta-estradiol or relaxin rapidly inhibits uterine estrogen receptor beta1 and beta2 messenger ribonucleic acid levels.

Estrogen regulates the growth and differentiation of the uterus via binding to estrogen receptors (ERs), members of the nuclear receptor family of transcription factors. Two forms of ER exist: ERalpha and ERbeta. The former is a well-characterized mediator of estrogen-induced transcription, but the function of the latter is unclear. Recent in vitro studies suggest that both splicing forms of ERbeta expressed in rat tissues, beta1 and beta2, may function as inhibitors of ERalpha transcriptional activity. To gain insight into the role of ERbeta in estrogen action, we examined the effects of estrogen and relaxin, a ligand-independent activator of ERs, on the expression of ERbeta1 and ERbeta2 mRNA in the uterus in vivo. Eighteen-day-old female rats were ovariectomized and, after recovery, treated with 17beta-estradiol, relaxin, or vehicle. Quantitative reverse transcription-polymerase chain reaction analyses of uterine RNA from estrogen-treated animals revealed marked decreases in the steady-state levels of the mRNAs for both ERbeta1 and ERbeta2 at 3, 6, and 24 h after treatment. Relaxin induced a similar effect. Neither hormone had any significant effect on ERalpha mRNA levels. To determine if endogenous estrogen exerts this effect, we examined the expression of ERbetas in the uterus during the estrous cycle. Levels of both isoforms were highest at diestrus (low estrogen), were significantly lower at early proestrus (rising estrogen), reached a nadir during late proestrus (peak estrogen), and rebounded at estrus (declining estrogen). These data suggest that down-regulation of ERbeta expression may be required for estrogen to exert its full trophic effects on the uterus.

Central administration of an antisense oligodeoxynucleotide against type I pituitary adenylate cyclase-activating polypeptide receptor suppresses synthetic activities of LHRH-LH axis during the pubertal process

In the present study, we determined the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP receptor type I (PAC 1) genes during juvenile development and the pubertal process. Female rats were assigned — based on uterine weights, the presence and abundance of uterine fluid, and their vaginal patency — to one of the following: anestrus (AE), early proestrus (EP), late proestrus (LP) or first estrus (E). The hypothalami from 22-, 24- and 26-day-old animals and from those in the peripubertal phases of AE, EP, LP and E were collected, and the content of PACAP and PAC 1 mRNA was assessed. These levels were found to decrease in EP and LP. To determine the effect of PACAP on prepubertal luteinizing hormone-releasing hormone (LHRH) and LH synthesis through PAC 1, a PAC 1 antisense oligodeoxynucleotide (ODN) was i.c.v.-administered, and mRNA levels of LHRH, LH β, and LHRH receptor (LHRH-R) were determined. Prepubertal increases in LHRH, LH β, and LHRH-R mRNA levels were markedly suppressed, and the onset of puberty was delayed by the i.c.v. injection of the antisense PAC 1 ODN. These data suggest that PACAP may play a role in the regulation of hypothalamic LHRH neurons, through which it regulates synthetic machinery of pituitary LH, during the pubertal process.

Distribution of NADPH-diaphorase and nitric oxide synthase (NOS) in different regions of porcine oviduct during the estrous cycle.

Nitric oxide synthase (NOS) is responsible for the biological production of nitric oxide (NO) in several organs, including those of the reproductive tract. We investigated potential changes in NADPH-diaphorase (NADPH-d) activity (marker for NOS activity) and the presence and distribution of NOS in the porcine oviduct. Tissues were obtained from gilts (n=16) on different days of the estrous cycle. One fallopian tube was used for histo- and immunohistochemistry and the other for Western blotting analysis. NADPH-d activity was much higher in the epithelium of the mucosa than in the myosalpinx. The highest activity of NADPH-d was always found in the epithelium of the isthmus. The intensity of the reaction (arbitrary units +/- SEM) in isthmus epithelium increased from the postovulatory period until early proestrus (96.2 +/- 11.2) and then gradually decreased. The lowest intensity of NADPH-d reaction in the epithelium of the isthmus was seen at estrus (58.4 +/- 7.7). The most intense NADPH-d activity in myosalpinx of all parts of the oviduct was observed at the postovulatory stage of the estrous cycle (isthmus 38.3 +/- 2.5; ampulla 35.6 +/- 4.2; infundibulum 24.7 +/- 0.8) and then decreased during the remaining stages of the estrous cycle (p< 0.001). The presence of endothelial NOS (eNOS) was detected in epithelial cells of mucosa and in endothelium of vascular tissues and myosalpinx during all studied days of the estrous cycle. The positive reaction for inducible NOS (iNOS) was restricted only to the endothelium of lymph vessels and some blood vessels. Because our Western blotting analysis revealed that porcine oviduct contains eNOS but not iNOS, we suggest that eNOS is the main isoform of NOS expressed in the porcine oviduct. We concluded that the different activity of NADPH-d in the various regions of the oviduct, accompanied by changes in its activity during the course of the estrous cycle, could indicate an important role of NO in regulation of tubal function.

Differential expression of growth hormone messenger ribonucleic acid by somatotropes and gonadotropes in male and cycling female rats.

Past studies have reported the appearance of cells sharing phenotypic characteristics of gonadotropes and GH cells. During diestrus and early proestrus, a subset of somatotropes (40-60%) expressed both GH antigens and gonadotropin (LH-beta, LHbeta, or FSH-beta) messenger RNAs (mRNAs) or GnRH receptors. More recently, we reported that subsets of gonadotropes identified by LHbeta or FSHbeta antigens expressed GH- releasing hormone (GHRH) binding sites. The present studies were designed to learn if these putative multipotential cells also expressed GH mRNA. Biotinylated sense and antisense oligonucleotide probes were developed and cytochemical in situ hybridization tests were optimized for the detection of GH mRNA with GH, LHbeta, and FSHbeta antigens. RNase protection assays were developed with a complementary RNA probe that detected a 380-bp region at the 5' end of the GH mRNA. Both the in situ hybridization and RNase protection assays detected changes in expression of GH mRNA during the estrous cycle with the lowest expression occurring during metestrus and peak expression occurring on the morning of proestrus. Cell counts confirmed the results of the RNase protection assays showing that increases in mRNA levels seen from metestrus to proestrus reflected increased percentages of GH mRNA-bearing cells. In addition, densitometric analyses demonstrated that the higher GH mRNA levels assayed from diestrus to proestrus reflected increased area and density of label per cell. Both types of assays showed sex differences in expression of GH mRNA; male rat cell populations had higher values than female rats in metestrus, diestrus, or estrus. However, percentages of GH cells in male rats were equal to those from proestrous female rats and levels of GH mRNA were lower in male rats than proestrous females. Dual labeling experiments showed that, in male rats and diestrous, proestrous, or estrous females, GH mRNA was expressed in over 70% of GH cells. Expression of GH mRNA was also found in 50-57% of cells with LHbeta or FSHbeta antigens in the same groups. The lowest expression was seen in the metestrous groups (30-40% of GH cells or gonadotropes expressed GH mRNA). Expression of GH mRNA was first increased from metestrus to diestrous largely in GH cells, and slightly in cells with LHbeta antigens. Further increases were seen in GH and LH cells by the morning of proestrus. In contrast, FSH gonadotropes did not show an increased expression of GH mRNA until the morning of proestrus (reaching the same peak reached by LH cells). These data confirm the working hypothesis that a multihormonal cell type develops during diestrus to support both the somatotrope and gonadotrope populations. Collectively, our studies suggest that this multihormonal cell may function to help support the regulatory functions of the gonadotrope during the periovulatory period. In addition, the appearance of significant levels of expression of GH mRNA by male rat gonadotropes suggests that this multihormonal cell may play a role in regulation of the male reproductive system as well.

Ovarian follicle apoptosis in bovine growth hormone transgenic mice.

Growth hormone directly or via insulin like-growth factor-I has been shown to inhibit preovulatory follicle apoptosis, which is the underlying mechanism of follicular atresia. We studied the levels of apoptosis in the ovaries of transgenic mice expressing bovine growth hormone. Female bovine growth hormone transgenic mice (n = 10) and nontransgenic litter mates (n = 8) were killed at early proestrus. Ovaries were collected, sectioned, and processed using a nonradioactive in situ method for apoptosis detection. Follicles were classified and counted on the basis of size and level of apoptosis. Our results demonstrate that the percentage of ovarian follicles containing apoptotic cells was lower in transgenic versus normal mice (30% vs. 46%; P < 0.05). The percentage of follicles undergoing heavy apoptosis was lower (P < 0.05) in transgenic versus control animals in preovulatory and early antral follicles, but it was not different in preantral follicles. The percentage of healthy preovulatory follicles was also higher in transgenic versus normal mice (7.4% vs. 4.3%; P < 0.05). These results indicate that growth hormone overexpression in transgenic mice significantly decreases follicle apoptosis, and thus atresia in the mouse ovary, therefore leading to increased propensity for ovulation in these animals.

Localization of bradykinin B2 receptors in the endometrium and myometrium of rat uterus and the effects of estrogen and progesterone.

In the uterus, bradykinin is a potent inducer of smooth muscle contraction, which is mediated by the bradykinin B2 receptor subtype. However, little is known about the distribution or regulation of this receptor in this tissue. The aim of this study was to localize the B2 receptor in the uterus and determine whether the levels of this receptor were altered during the estrous cycle and modulated by estrogen and/or progesterone in ovariectomized rats. At diestrus, uterine B2 receptors were localized to both the circular and longitudinal smooth muscle layers of the myometrium, the endometrial stroma, the glandular epithelium, and the layer subjacent to the luminal epithelium. B2 receptor levels in both myometrium and endometrium were lowest during early proestrus, when estrogen levels are low, whereas myometrial B2 receptor protein and messenger RNA levels were highest during late proestrous, when estrogen levels peak. Similar findings were observed for the estrogen-supplemented group after ovariectomy, with progesterone appearing to inhibit the estrogen-induced rise in bradykinin B2 receptor density in estrogen/progesterone-treated animals. Using in vitro receptor autoradiography employing the specific B2 receptor antagonist analog, HPP-HOE140, immunostaining with specific antipeptide antibodies generated against the B2 receptor, and in situ hybridization using a specific bradykinin B2 receptor riboprobe, our findings show a discrete distribution of the bradykinin B2 receptor throughout the different layers of the uterus and suggest that bradykinin B2 receptor levels in the rat uterus are regulated by estrogen, and possibly progesterone, in both myometrium and endometrium.

Evaluation of LH secretory dynamics during the rat proestrous LH surge.

The preovulatory luteinizing hormone (LH) surge results from the integration of complex interactions among gonadal steroids and hypothalamic and pituitary hormones. To evaluate changes in LH secretory dynamics that occur during the rat LH surge, we have 1) obtained frequently sampled serum LH concentration time series, 2) used both waveform-dependent and waveform-independent convolution analyses, and 3) independently assessed proestrous LH half-life and basal non-gonadotropin-releasing hormone (GnRH)-dependent LH secretion during the LH surge. Waveform-independent pulse analysis revealed a 24-fold increase in the maximal pulsatile LH secretory rate attained during late proestrus compared with early proestrus. A 15-fold increase was quantified for the mean LH secretory rate. In complementary analyses, we applied a measured LH half-life of 17 +/- 2.7 min and a median basal LH secretion rate of 0.0046 microgram. l-1. min-1 for convolution analysis, revealing a 16-fold increase in the mass of LH released/burst and more than sixfold rise in the amplitude of the secretory peaks. Evaluation of the approximate entropy of the LH surge profiles was performed, showing an increase in the orderliness of the LH release process during the surge. We conclude that both quantitative (mass/burst) and qualitative (approximate entropy) features of LH release are regulated during the proestrous LH surge.

Temporal and spatial expression of tissue inhibitors of metalloproteinases during the natural ovulatory cycle of the mouse.

The role of tissue inhibitors of metalloproteinases (TIMPs) in ovarian function has primarily been documented by studies that utilize hormone-primed animals. In this study, our objectives were to elucidate the spatiotemporal expression of individual TIMP genes during the natural ovulatory cycle, and to correlate these with specific biological events. Two models of spontaneous ovulation used were the murine estrous cycle and the first ovulation postpartum. Ovaries were collected from mice at diestrus, estrus, and metestrus, or at early and late proestrus, and from pregnant females on Days 17 and 18 of gestation (D17, D18) and within 24 or 48 h postpartum (PP1, PP2). We observed that TIMP-1 mRNA was elevated at early proestrus and D18 and was maximal at late proestrus and PP1. The TIMP-3 pattern was distinct from that of TIMP-1, maximal expression occurring at early proestrus and D17 and D18. In both models, TIMP-2 mRNA remained constant and at very low levels throughout ovulation. In situ hybridization localized TIMP-1 mRNA to the corpus luteum at D18 and PP1, and to oocytes at specific stages of follicular development. Expression of TIMP-1 in granulosa and thecal cells was not observed at any stage. Demonstrating a distinct distribution, TIMP-3 mRNA was localized to oocytes, thecal and granulosa cells of small and large follicles, and corpora lutea only at D17. These data suggest specific hormonal regulation of individual TIMP gene expression in the ovary associated with distinct physiological functions. We propose that in the natural ovulatory process, TIMP-1 is probably a factor that regulates corpus luteum regression while TIMP-3 is important in maintaining the structural integrity of the corpus luteum.

Plasma selenium and glutathione peroxidase activity fluctuate during the rat estrous cycle

The effect of the estrous cycle on selenium status was studied in chronically-catheterized Sprague-Dawley rats. Plasma and red blood cell selenium concentrations and glutathione peroxidase (GPx) activities were measured in five chronically-catheterized rats throughout two consecutive 4-d estrus cycles. Fifteen blood samples were serially drawn from each animal during each 4-d cycle. Plasma selenium and GPx activity and RBC selenium concentrations fluctuated significantly throughout the estrus cycle. Plasma selenium concentration reached a peak during proestrus and was significantly greater (P<0.05) than that observed during the other days of the estrous cycle. Plasma GPx activity peaked during early proestrus and late metestrus. RBC selenium peaked during estrus. These results suggest that hormonal fluctuations and the stage of the reproductive cycle should be considered when assessing female selenium status and when establishing dietary selenium recommendations.

Prolactin receptor messenger ribonucleic acid expression in the ovary during the rat estrous cycle.

We have investigated the relative amounts and sites of synthesis during the rat estrous cycle of the two ovarian mRNAs encoding the long and short PRL receptors (PRL-R). Quantitative analysis has revealed that the mRNA encoding the short PRL-R is consistently present throughout the cycle in lower quantities than the long receptor mRNA. Both receptor mRNAs are at maximal levels during proestrus, decline to their lowest level of expression during estrus, then gradually rise in metestrus and diestrus. By in situ hybridization, both receptor mRNAs are present during early proestrus in corpora lutea, in the granulosa cell layers of large Graafian follicles, and in the interstitial cells closely associated with these follicles. The short PRL-R mRNA was detected at significant levels in the granulosa-derived cumulus oophorus and in the thecal cell region at this time, whereas the long PRL-R mRNA was only weakly expressed in these cell types. In contrast, the long PRL-R mRNA was present at higher levels, compared to the short receptor mRNA, in the granulosa cells of preantral follicles in the interior of the ovary. On late proestrus, the long PRL-R mRNA was found predominantly in the mural granulosa cells of large Graafian follicles and in corpora lutea, but by estrous morning this mRNA appeared to be mostly restricted to the corpora lutea. This distribution was maintained through estrous evening and metestrous morning. On diestrus, both mRNAs were present in some corpora lutea and in the granulosa cell layer in a subset of the larger Graafian follicles, but were detected at even higher levels in the interstitial cells surrounding these follicles; again, the long receptor mRNA appeared to be only weakly expressed in the thecal cell region of these follicles. These results indicate that the levels and locations of PRL-R mRNA expression in the ovary, and therefore, the potential responsiveness of the ovary to PRL, change throughout the reproductive cycle. Furthermore, the presence of both receptor mRNAs in several different ovarian cell types suggests that both of these receptor forms play important roles in PRL physiology in the ovary.