Parallel expression patterns of NR4A nuclear receptor family genes in the pituitary gland of proestrus rats.

The NR4A nuclear receptor family (NR4As), encompassing NR4A1, NR4A2, and NR4A3, exerts pivotal roles in cellular processes through intricate expression patterns and interactions. Despite the influence of some NR4As on anterior pituitary functions regulated by the hypothalamus, their physiological expression patterns remain unclear. In our prior work, we demonstrated the specific upregulation of NR4A3 in the rat anterior pituitary gland during the proestrus afternoon, coinciding with a gonadotropin surge. In this study, we investigated changes in pituitary Nr4a gene expression throughout the estrous cycle in rats and a gonadotropin surge-induced model. Nr4a1 and Nr4a2 gene expression significantly increased during proestrus, aligning with previous observations for Nr4a3. Furthermore, prolactin gene expression increased sequentially with rising Nr4a gene expression, while thyroid-stimulating hormone beta gene expression remained stable. Immunohistochemistry revealed a widespread and differential distribution of NR4A proteins in the anterior pituitary, with NR4A1 and NR4A3 being particularly abundant in thyrotrophs, and NR4A2 in gonadotrophs. In estrogen-treated ovariectomized rats, elevated luteinizing hormone secretion corresponded to markedly upregulated expression of Nr4a1, Nr4a2, and Nr4a3. In gonadotroph and somatomammotroph cell lines, gonadotropin- and thyrotropin-releasing hormones transiently and dose-dependently increased the expression of Nr4a genes. These findings suggest that hypothalamic hormone secretion during proestrus may induce the parallel expression of pituitary Nr4a genes, potentially influencing the pituitary gene expression program related to endocrine functions before and after ovulation.

ACTH impairs the migratory and secretory profile of mononuclear cells during proestrus in cattle.

The aim was to evaluate the effect of ACTH on the mechanisms involved in peripheral blood mononuclear cells (PBMCs) infiltration into the ovary during dairy cattle proestrus. Regarding this, proper expression pattern of adhesion molecules must take place both in PBMCs and in endothelial cells. Argentinian Holstein cows (n = 12) were treated with 100 IU of ACTH every 12 h for 4 days before ovulation when ovariectomy was performed (day 18). Blood samples were taken on day 15 (0 h) and immediately before (72 h) and after (74 h) the last ACTH administration. In PBMCs, flow cytometry was performed to analyze CD44, CD11b and CD62-L expression along with gene expression of chemokines' receptors. Interleukin (IL)-4 and tumor necrosis factor-α (TNF-α) production was analyzed by flow cytometry after exposing PBMCs to autologous follicular fluid. In ovarian blood vessels, expression of the vascular endothelium cell adhesion-1 (VCAM-1) and the platelet endothelial cell adhesion molecule-1 was evaluated by immunohistochemistry. In T-lymphocytes, the expression of CD44 and CD11b was lower at 72 h in ACTH-treated cows (P < 0.05). In monocytes, the expression of CD11b and CD62-L was lower at 72 h in ACTH-treated cows (P < 0.05). Also, the percentage of IL-4+ cells was higher in ACTH-treated cows, meanwhile, the percentage TNF-α+ cells was lower in ACTH-treated cows (P < 0.05). Finally, in the vessels associated with the preovulatory follicle VCAM-1 immunoexpression was lower in ACTH-treated cows (P < 0.05). Here, we present novel insights into the effect of stress during the preovulatory period on the inflammatory pathway necessary for ovulation.

Thyrotropin-releasing hormone stimulates NR4A3 expression in the pituitary thyrotrophs of proestrus rats.

The secretion of several hypothalamic peptide hormones is activated during the preovulatory period. Hypothalamic thyrotropin-releasing hormone (TRH) is one such hormone with reproductive and/or metabolic significance. However, it remains unclear whether thyroid-stimulating hormone (TSH)-producing thyrotrophs are produced during the preovulatory period. We previously found a transient increase in the expression of the nuclear receptor NR4A3, a well-known immediate early gene, in the proestrus afternoon in the anterior pituitary glands of rats. To investigate the relationship between TRH secretion and pituitary NR4A3 expression during proestrus, we used proestrus and thyroidectomized rats to identify NR4A3-expressing cells and examined the regulation of Nr4a3 gene expression via the hypothalamus-pituitary-thyroid (HPT) axis. The percentage of NR4A3-expressing cells increased in thyrotrophs at 14:00 h of proestrus. Incubation of rat primary pituitary cells with TRH transiently stimulated Nr4a3 expression. Thyroidectomy to attenuate the negative feedback effects led to increased serum TSH levels and Nr4a3 gene expression in the anterior pituitary, whereas thyroxine (T4) administration conversely suppressed Nr4a3 expression. Additionally, the administration of T4 or TRH antibodies significantly suppressed the increase in Nr4a3 expression at 14:00 h of proestrus. These results demonstrate that pituitary NR4A3 expression is regulated by the HPT axis, and that TRH stimulates thyrotrophs and induces NR4A3 expression during the proestrus afternoon. This suggests the potential involvement of NR4A3 in the regulation of the HPT axis during pre- and post-ovulatory periods.

The injections of mitochondrial fusion promoter M1 during proestrus disrupt the progesterone secretion and the estrous cycle in the mouse.

After ovulation, the mitochondrial enzyme CYP11A1 cleavage the cholesterol into pregnenolone for progesterone synthesis, suggesting that mitochondrial dynamics play a vital role in the female reproductive system. The changes in the mitochondria dynamics throughout the ovarian cycle have been reported in literature, but the correlation to its role in the ovarian cycle remains unclear. In this study, mitochondrial fusion promotor, M1, was used to study the impact of mitochondria dynamics in the female reproductive system. Our results showed that M1 treatment in mice can lead to the disruptions of estrous cycles in vagina smears. The decrease in serum LH was recorded in the animal. And the inhibitions of progesterone secretion and ovulations were observed in ovarian culture. Although no significant changes in mitochondrial networks were observed in the ovaries, significant up-regulation of mitochondrial respiratory complexes was revealed in M1 treatments through transcriptomic analysis. In contrast to the estrogen and steroid biosynthesis up-regulated in M1, the molecules of extracellular matrix, remodeling enzymes, and adhesion signalings were decreased. Collectively, our study provides novel targets to regulate the ovarian cycles through the mitochondria. However, more studies are still necessary to provide the functional connections between mitochondria and the female reproductive systems.

Metagenomic analysis of bacterial communities during estrous cycle in Bos indicus.

The present study, using 16 s rRNA sequencing of the V3-V4 hypervariable region, was aimed to check diversity of vaginal microbiota throughout different stages of the estrous cycle in Bos indicus, with attention to changes in progesterone hormone and microorganism diversity. Metagenomic research was conducted on vaginal swabs obtained from nine healthy Indian Gir cows at estrus (day 0), metestrus (day 04), diestrus (day 12), and proestrus (day 16) phases of the estrous cycle. The findings revealed that the diestrus phase has a different bacterial diversity than the other three estrous cycle phases, implying that progesterone affects bacterial diversity. Proteobacteria and Firmicutes were the most abundant phyla at the phylum level, accounting for 94% of bacterial diversity. Actinobacteriota, Patescibacteria, Cyanobacteria, and Bacteroidota were among the less prevalent phyla observed in all samples. After statistical analysis, Bacillaceae, Alcaligenes, Enterobacteriaceae, and Morganellaceae families were more significant. The Enterobacteriaceae family was found to be lower in the diestrus phase than in the other three phases; in contrast, all statistically significant genera were high at the diestrus phase. The luteal stage had higher levels of Micrococcus, Stenotrophomonas, UGC-010, Massilia, and Methylobacillus than the follicular stage, and statistical analysis revealed substantial difference between the luteal and follicular stages. Lactobacillus genus was present in both the estrus and diestrus phases. This study represents an important step toward the understanding of microbial diversity within different stages of the estrous cycle of Indian cows.

Association between length of proestrus, follicular size, estrus behavior, and pregnancy rate in beef heifers subjected to fixed-time artificial insemination.

This study evaluated the relationship between proestrus length and follicular size, estrous behavior, and pregnancy rate in Bos taurus beef heifers subjected to fixed-time artificial insemination (FTAI). A total of 911 heifers received a synchronization treatment protocol for FTAI (J-Synch) consisting of an intravaginal progesterone device for 6 d, estradiol benzoate at the time of device insertion cloprostenol sodium and eCG at device removal and GnRH at the time of FTAI. The presence or absence of a corpus luteum (CL) was determined by ultrasonography at device insertion and all heifers were tail painted at device removal for estrus detection at the time of FTAI. For the establishment of different periods of proestrus length (i.e., interval from device removal to FTAI), GnRH was administered i.m. at 48 h (n = 308), 60 h (n = 290) or 72 h (n = 313) after device removal. The diameter of the largest follicle at the time of GnRH administration was determined by ultrasonography, expression of estrous was determined by percentage of tail paint removal, and FTAI was performed at the time of GnRH administration in all heifers. The diameter of the largest follicle was greater when GnRH/FTAI was performed at 72 or 60 h (12.9 ± 0.2 mm and 12.8 ± 0.1 mm, respectively) than at 48 h (12.2 ± 0.1 mm, P < 0.05). The proportion of heifers in estrus tended to be greater when GnRH/FTAI was performed at 72 h (77.0%, 137/178) than at 48 h (68.2%, 122/179; P = 0.06), and intermediate at 60 h (71.4%, 120/168). Pregnancy rate tended to be greater in heifers with the longest (72 h: 70.0%, 219/313) than the shortest (48 h: 63.6%, 196/308; P < 0.1) proestrus length, while 60 h proestrus length was intermediate (63.1%, 183/290; P= NS). Pregnancy rate was affected by the presence of a CL at device insertion (71.3%, 352/494 in heifers with a CL, vs. 59.0%, 246/417 for those without a CL; P < 0.01). For those heifers bearing a CL, pregnancy rate was greater in heifers with a 72 h proestrus length (77.0%, 134/174) than with 48 or 60 h proestrus length (67.7%, 107/158 and 68.5%, 111/162; respectively; P < 0.05). In heifers without a CL, proestrus length did not affect pregnancy rate. In summary, extending proestrus length by delaying the interval from device removal to GnRH/FTAI from 48 to 72 h, was associated with a greater diameter of the preovulatory follicle, greater proportion of heifers expressing estrus at the time of FTAI, and greater pregnancy rate in cycling beef heifers.

The effects of artificially induced proestrus on heroin intake: A critical role for estradiol.

Heroin intake decreases markedly during proestrus in normally cycling female rats; however, it is not known whether estradiol, progesterone, or both hormones are responsible for these decreases in heroin intake. The purpose of the present study was to examine the roles of estradiol and progesterone in heroin intake by artificially inducing a proestrus state in ovariectomized rats. To this end, ovariectomized female rats were implanted with intravenous catheters and trained to self-administer heroin (0.0075 mg/kg/infusion) on a fixed ratio (FR1) schedule of reinforcement. After 1 week of training, rats were tested at weekly intervals with estradiol (0.005 mg, sc) or vehicle 22 hr before a test session and progesterone (0.125 mg, sc) or vehicle 0.5 hr before a test session to artificially mimic the naturally occurring hormone concentrations characteristic of late proestrus. Administration of estradiol 22 hr prior to testing and progesterone 0.5 hr prior to testing significantly reduced heroin intake relative to the previous training day and vehicle control. It is interesting that this same effect was observed when only estradiol, but not progesterone, was administered. These data suggest that estradiol but not progesterone is responsible for the proestrus-induced decreases in heroin intake previously reported in normally cycling female rats. These findings differ from those reported previously with stimulants and suggest that estrogen-based pharmacotherapies may be of value to women with opioid use disorder. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Morphological features of the rat uterus.

The rat uterus is an important female reproductive organ that has essential for the organism's reproduction. That is why it is necessary to understand all the rat uterus' morphological features as a perfect biomodel for studying the molecular peculiarities of the female reproductive system and pathologies development in experimental studies. AIM: The aim of research was to perform the comprehensive morphological analysis of the uterine in intact female rats. MATERIALS AND METHODS: The uterine of reproductive-aged intact female rats were used in this research. The cytological study of vaginal smears, histological (H and E), and immunohistochemical (estrogen, progesterone, and Ki-67 receptors) analysis of uterus tissues were used for light microscopic examination. RESULTS: The rat's vaginal smears' cytological features showed a specific qualitative cellular composition (variation of leukocytes, nucleated and anucleated cornified epithelial cells) in different estrous cycle phases (proestrus, estrus, metestrus, and diestrus). Uterine histology showed the structural regularities of parenchymal and stromal components with clear differentiation on the endometrium, myometrium and perimetrium. It was presented uterus sensitivity to the influence of the sex hormones (positive to estrogen and progesterone receptors) and the variable cellular proliferation activity (Ki-67 expression) in the organ wall. CONCLUSIONS: Our research demonstrated that the rats« uterus has a unique structural organization, sex hormones sensitivity, and variable proliferation in the parenchymal and stromal components. The rat estrous cycle should be considered while studying the morphological features of the uterus. The rat's uterus may serve as an acceptable object for modeling various pathological processes with the following results' extrapolation.

Estrous Cycle Plasticity in the Central Clock Output to Kisspeptin Neurons: Implications for the Preovulatory Surge.

Coordination of ovulation and behavior is critical to reproductive success in many species. During the female estrous cycle, the preovulatory gonadotropin surge occurs when ovarian follicles reach maturity and, in rodents, it begins just before the daily onset of activity, ensuring that ovulation coincides with sex behavior. Timing of the surge relies on projections from the suprachiasmatic nucleus (SCN), the locus of the central circadian clock, to hypothalamic circuits that regulate gonadotropin secretion. The cellular mechanisms through which the SCN controls these circuits and gates the preovulatory surge to the appropriate estrous cycle stage, however, are poorly understood. We investigated in mice the functional impact of SCN arginine-vasopressin (AVP) neuron projections to kisspeptin (Kiss1) neurons in the rostral periventricular area of the third ventricle (RP3VKiss1), responsible for generating the preovulatory surge. Conditional anterograde tracing revealed that SCNAVP neurons innervate approximately half of the RP3VKiss1 neurons. Optogenetic activation of SCNAVP projections in brain slices caused an AVP-mediated stimulation of RP3VKiss1 action potential firing in proestrus, the cycle stage when the surge is generated. This effect was less prominent in diestrus, the preceding cycle stage, and absent in estrus, following ovulation. Remarkably, in estrus, activation of SCNAVP projections resulted in GABA-mediated inhibition of RP3VKiss1 neuron firing, an effect rarely encountered in other cycle stages. Together, these data reveal functional plasticity in SCNAVP neuron output that drives opposing effects on RP3VKiss1 neuron activity across the ovulatory cycle. This might contribute to gating activation of the preovulatory surge to the appropriate estrous cycle stage.

Modulation of heroin intake by ovarian hormones in gonadectomized and intact female rats.

RATIONALE: Heroin intake decreases during the proestrus phase of the estrous cycle in female rats. Circulating concentrations of both estradiol and progesterone peak during proestrus, and it is not known which of these hormones, or their combination, are responsible for these effects. OBJECTIVES: The purpose of this study was to determine the effects of estradiol, progesterone, and their combination on heroin self-administration in female rats. METHODS: In Experiment 1, the estrous cycle of intact female rats was tracked daily. If a rat was in proestrus, either the estrogen receptor antagonist, raloxifene, the progesterone receptor antagonist, mifepristone, or their combination was administered 30 min prior to a heroin self-administration session. In Experiment 2, separate groups of ovariectomized female rats were treated chronically with exogenous estradiol, progesterone, estradiol + progesterone, or vehicle, and heroin intake was examined over a 100-fold dose range. RESULTS: In Experiment 1, raloxifene, but not mifepristone, significantly blocked proestrus-associated decreases in heroin intake. In Experiment 2, estrogentreated rats self-administered less heroin than any other group and significantly less heroin than rats treated with progesterone. CONCLUSIONS: These data suggest that (1) estradiol but not progesterone is responsible for proestrus-associated decreases in heroin intake and (2) estradiol decreases heroin intake relative to progesterone. These data differ from those reported previously with stimulants and suggest that estrogen-based pharmacotherapies may be of value to women with opioid use disorder.

Improvement in identification of pro-estrous mice by using a novel method of detecting vaginal mucous cells.

Accurate identification of the murine estrous cycle using vaginal exfoliative cytology is the initial and crucial step for controlled reproduction of this species. However, it is generally difficult to discriminate each stage of the cycle, and thus to select pro-estrous mice for mating. To increase the accuracy of identification of the pro-estrous stage, we re-evaluated the vaginal fold histology and modified the method of exfoliative cytology. Tissue fixation using methanol in Carnoy's solution but not paraformaldehyde, combined with Alcian blue staining but not the conventional Giemsa staining, resulted in better manifestation of mucosal cell layers in the vaginal epithelium just above the keratinized layer. This mucous layer in the fold histology was found to form specifically in the pro-estrous and late di-estrous stages, and the mucous cells exfoliated in smear samples only in the pro-estrous stage. This novel method was found, by a blinded test, to increase the rate of accurate identification of the pro-estrous stage compared to the conventional method (80% vs 50%). Consistent with this finding, the mating experiment with "pro-estrous" females selected by the novel method revealed a significantly higher success rate than that with the conventional method (78.0% vs 47.5%). Thus, our study demonstrates vaginal exfoliative mucous cells as a better potential marker to detect the "receptive" state of female mice that leads to an improved success rate of mating.

Changes in the localization of ovarian visfatin protein and its possible role during estrous cycle of mice.

Visfatin is a crucial adipokine, which also regulates ovarian functions in many animals. Mice estrous cycle is characterized by a dynamic complex physiological process in the reproductive system. Expression of various factors changes during the estrous cycle in the ovary. To the best of our knowledge, no previous study has been conducted on the expression of visfatin in mice ovaries during the estrous cycle. Therefore, we investigated the localization and expression of visfatin protein in the ovary of mice during the estrous cycle. Western blot analysis showed the elevated expression of visfatin in proestrus and lowest in diestrus. Immunohistochemical localization of visfatin showed intense staining in the corpus luteum of proestrus and diestrus ovaries. Thecal cells, granulosa cells, and oocytes also showed the presence of visfatin. Expression of ovarian visfatin was correlated to BCL2 and active caspase3 expression and exhibited a significant positive correlation. Furthermore, in vivo inhibition of visfatin by FK866 in the proestrus ovary down-regulated active caspase3 and PCNA expression, and up-regulated the BCL2 expression. These results suggest the role of visfatin in the proliferation and apoptosis of the follicles and specific localization of visfatin in the corpus luteum also indicate its role in corpus luteum function, which may be in progesterone biosynthesis and regression of old corpus luteum. However, further study is required to support these findings. In conclusion, visfatin may also be regulating follicular growth during the estrous cycle by regulating proliferation and apoptosis.

Comparing Gene Expression in the Parabrachial and Amygdala of Diestrus and Proestrus Female Rats after Orofacial Varicella Zoster Injection.

The orofacial pain pathway projects to the parabrachial and amygdala, and sex steroids have been shown to affect neuronal activity in these regions. GABA positive cells in the amygdala are influenced by sex steroid metabolites to affect pain, and sex steroids have been shown to alter the expression of genes in the parabrachial, changing neuronal excitability. Mechanisms by which sex steroids affect amygdala and parabrachial signaling are unclear. The expression of genes in the parabrachial and amygdala in diestrus (low estradiol) and proestrus (high estradiol) female rats were evaluated in this study. First, varicella zoster virus was injected into the whisker pad of female rats to induce a pain response. Second, gene expression was quantitated using RNA-seq one week after injection. Genes that had the greatest change in expression and known to function in pain signaling were selected for the quantitation of protein content. Protein expression of four genes in the parabrachial and seven genes in the amygdala were quantitated by ELISA. In the parabrachial, neurexin 3 (Nrnx3) was elevated at proestrus. Nrnx3 has a role in AMPA receptor and GABA signaling. Neuronatin (Nnat) and protein phosphatase, Mg2+/Mn2+ dependent 1E (Ppm1e) were elevated in the parabrachial of diestrus animals both genes having a role in pain signaling. Epoxide hydroxylase (Ephx2) was elevated in the parabrachial at proestrus and the vitamin D receptor (Vdr) was elevated in the amygdala. Ephx2 antagonists and vitamin D have been used to treat neuropathic pain. In conclusion, sex steroids regulate genes in the parabrachial and amygdala that might result in the greater pain response observed during diestrus.

Interactions of circulating estradiol and progesterone on changes in endometrial area and pituitary responsiveness to GnRH†.

Changes in circulating progesterone (P4) and estradiol (E2) during proestrus produce dynamic changes in endometrial function and pituitary release of gonadotropins. Independent and combined effects of P4 and E2 on endometrium and pituitary were evaluated. In a preliminary study, an exogenous hormone model of proestrus was created by removal of corpus luteum and follicles ≥5 mm followed by gradual removal of intravaginal P4 implants during 18 h and treatment with increasing doses of estradiol benzoate during 48 h to mimic proestrus using high E2 (n = 9) or low E2 (n = 9). Decreased P4, increased E2, and increased endometrial area (EA) simulated proestrus in high-E2 cows and this was used subsequently. The main experiment used a 2 × 2 factorial design with: high E2 and low P4 (n = 11); high E2 and high P4 (n = 11); low E2 and high P4 (n = 11); low E2 and low P4 (n = 10). At 48 h, gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) and follicle stimulating hormone (FSH) release was determined. Variables were analyzed using PROCMIXED of Statistical Analysis System. The EA increased dramatically during 48 h only in high-E2 and low-P4 cows. For FSH, high-E2 cows had greater area under the curve (AUC) and FSH peak after GnRH than low E2, with mild negative effects of high P4. For LH, concentration at peak and AUC were 2-fold greater in high E2 compared to low-E2 groups, with low P4 also 2-fold greater than high-P4 groups. Thus, maximal changes in uterus and pituitary during proestrus depend on both low P4 and high E2, but different physiologic responses are regulated differently by E2 and P4. Changes in endometrium depend on low P4 and high E2, whereas GnRH-induced FSH secretion primarily depends on high E2, and GnRH-induced LH secretion is independently increased by high E2 or reduced by high P4.

"Rapid actions of the neurosteroid allopregnanolone on ovarian and hypothalamic steroidogenesis: Central and peripheral modulation".

The present study aimed to determine whether an i.c.v. administration of allopregnanolone (ALLO) rapidly modifies the hypothalamic and ovarian 3ß-hydroxysteroid dehydrogenase (3ß-HSD) enzymatic activity and gene expression in in vivo and ex vivo systems in pro-oestrus (PE) and dioestrus I (DI) rats. Animals were injected with vehicle, ALLO, bicuculline or bicuculline plus ALLO and were then killed. In the in vivo experiment, the hypothalamus, ovaries and serum were extracted and analysed. In the ex vivo experiment, the superior mesenteric ganglion - ovarian nerve plexus - ovary system was extracted and incubated during 120 minutes at 37 ºC. The serum and ovarian compartment fluids were used to determine progesterone by radioimmunoanalysis. In the in vivo experiments, ALLO caused a decrease in hypothalamic and ovarian 3ß-HSD enzymatic activity during PE. During DI, ALLO increased hypothalamic and ovarian 3ß-HSD activity and gene expression. The ovarian 3ß-HSD activity increased in both stages in the ex vivo system; gene expression increased only during DI. ALLO induced an increase in serum progesterone only in D1 and in the ovarian incubation liquids in both stages. All findings were reversed by an injection of bicuculline before ALLO. Ovarian steroidogenic changes could be attributed to signals coming from ganglion neurones, which are affected by the acute central neurosteroid stimulation. The i.c.v. administration of ALLO via the GABAergic system altered 3ß-HSD activity and gene expression, modulating the neuroendocrine axis. The present study reveals the action that ALLO exerts on the GABAA receptor in both the central and peripheral nervous system and its relationship with hormonal variations. ALLO is involved in the "fine tuning" of neurosecretory functions as a potent modulator of reproductive processes in female rats.

Influence of age and gender on sex steroid receptors in rat masticatory muscles.

The temporomandibular muscle dysfunction is characterized by myofascial pain and is more prevalent in women of reproductive age. Sex steroid hormones are hypothetically involved in the dysfunction, but few are the studies of steroid receptors in masticatory and mastication-related muscles. Our aim was to determine estrogen and testosterone receptor expression in rat masticatory and mastication-related muscles within the context of age and gender. Twelve rats were equally divided into four groups: (a) 10-month-old females; (b) 10-month-old males; (c) 24-month-old females; and (d) 24-month-old males. Euthanasia of the females was performed in the proestrous phase (vaginal smears) and the masticatory and accessory muscles were removed for immunohistochemical analysis. Statistical analysis was performed with ANOVA and the Tukey test. Estrogen receptor expression was similarly low in all muscles and groups. Testosterone receptor expression in the Masseter muscle of the 24-month-old male rats was higher than that in the other groups and significantly superior to its expression in the Posterior Digastric muscle. In short, testosterone receptor expression was highest in old male rats. If we generalize to humans, this fact could indicate age- and sex-related hormonal influence on temporomandibular muscle dysfunction. Further studies, however, are necessary to strengthen this hypothesis.

Stimulation of nicotinic receptors in the suprachiasmatic nucleus results in a higher number of growing follicles and ova shed.

NEW FINDINGS: What is the central question of this study? What is the role of the nicotinic system of the suprachiasmatic nucleus (SCN) in the regulation of follicular growth and ovulation? What is the main finding and its importance? The stimulation of the nicotinic system of the pro-oestrus rat SCN results in an increase in the number of ova shed, in the number of growing ovarian follicles and in the secretion of oestradiol. ABSTRACT: The timing of the preovulatory luteinizing hormone surge that leads to ovulation depends to a large extent on a functional circadian clock that is localized in the suprachiasmatic nucleus (SCN). The activities of the SCN are regulated by several neurotransmitter systems, including the muscarinic system. Given that acetylcholine binds to muscarinic (mAChRs) and nicotinic (nAChRs) receptors, in the present study, we analysed the effects of unilaterally stimulating nAChRs in the left or right SCN. Stimulation treatment was administered in rats in pro-oestrus at 09.00 or 19.00 h by injecting 0.3 µl of a nicotine solution (200 µm). The effects of the stimulation were assessed by evaluating the number of ova shed, the number of ovarian follicles, and the levels of oestradiol and progesterone in serum 24 h after treatment. We observed that regardless of the time (4 h after lights on, 09.00 h, or immediately after lights off, 19.00 h) or the side of the SCN treated, the unilateral microinjection of nicotine resulted in a higher number of ova shed and higher number of growing follicles in the ovaries as well as higher oestradiol serum levels. When the nicotine microinjection treatment failed to reach the SCN, the oestradiol levels in serum were similar to those of animals treated with vehicle solution. Based on the current results, we suggest that during pro-oestrus, the nicotinic neuronal information in the SCN modulates follicular growth and ovulation in a stimulatory manner.

Effects of non-contingent cocaine on 3 alpha-androstanediol. II. Disruption of lordosis of proestrous rats.

Drug use influences sexual behavior, performance, and can be associated with increased sexual risk-taking. Our prior results using an animal model indicate that progestogens contribute to hormonally-mediated changes in sexual behavior of female rodents during acute cocaine exposure. Androgens, such as testosterone, and its metabolite 3ɑ-androstanediol (3α-diol), and estradiol, are known to influence male sexual behavior, but can also alter the expression of sexual behavior of female rodents. As such, we investigated the influence of endogenous androgen and estradiol fluctuations on cocaine-mediated changes in motor behavior and sexual receptivity of rats during diestrous or proestrous phases of the estrous cycle. Female rats were administered saline or cocaine (5, 10, or 20mg/kg, i.p.). Motor behavior was observed in the first 30min following drug administration, and then sexual responding was assessed for 15min. Cocaine decreased aggressive behavior in response to attempted mounts by a male among non-receptive (diestrous) rats and inhibited sexual behavior among sexually receptive (proestrous) rats. Cocaine dose-dependently altered concentrations of testosterone metabolites (estradiol and 3α-diol), but not testosterone, which correlated to motor and sexual behaviors of diestrous and proestrous rats, respectively. These data suggest that actions of 3α-diol may be involved in female sexual and motor behavior in response to cocaine, in a cycle-dependent manner.

Estrogen and progesterone dependent expression of visfatin/NAMPT regulates proliferation and apoptosis in mice uterus during estrous cycle.

Visfatin is an adipokine which has an endocrine effect on reproductive functions and regulates ovarian steroidogenesis. There is scant information about the expression, regulation, and functions of visfatin in the mammalian uterus. The present study examined expression and localization of visfatin in the mouse uterus at various stages of the natural estrous cycle, effects of estrogen and progesterone on localization and expression of visfatin in the ovariectomised mouse uterus and effect of visfatin inhibition by a specific inhibitor, FK866 on proliferation and apoptosis in the uterus. Western blot analysis of visfatin showed high expression in proestrus and metestrus while it declined in estrus and diestrus. Immulocalization study also showed strong immunostaining in the cells of endometrium, myometrium, luminal and glandular epithelium during proestrus and metestrus that estrus and diestrus. The uterine visfatin expression closely related to the increased estrogen levels in proestrus and suppressed when progesterone rose to a high level in diestrus. The treatment with estrogen to ovariectomised mice up-regulates visfatin, PCNA, and active caspase3 whereas progesterone up-regulates PCNA and down-regulates visfatin and active caspase3 expression in mouse uterus. The co-treatment with estrogen and progesterone up-regulates visfatin and down-regulates PCNA and active caspase3. In vitro study showed endogenous visfatin inhibition by FK866 increased expression of PCNA and BCL2 increased catalase activity while FK866 treatment decreased expression of active caspase3 and BAX with decreased SOD and GPx activity. BrdU labeling showed that inhibition of visfatin modulates the uterine proliferation. This study showed that expression of visfatin protein is steroid dependent in mouse uterus which is involved in the regulation of proliferation and apoptosis via modulating antioxidant system in the uterus of mice during the reproductive cycle.

Role of GPER in the anterior pituitary gland focusing on lactotroph function.

Ovarian steroids control a variety of physiological functions. They exert actions through classical nuclear steroid receptors, but rapid non-genomic actions through specific membrane steroid receptors have been also described. In this study, we demonstrate that the G-protein-coupled estrogen receptor (GPER) is expressed in the rat pituitary gland and, at a high level, in the lactotroph population. Our results revealed that ~40% of the anterior pituitary cells are GPER positive and ~35% of the lactotrophs are GPER positive. By immunocytochemical and immuno-electron-microscopy studies, we demonstrated that GPER is localized in the plasmatic membrane but is also associated to the endoplasmic reticulum in rat lactotrophs. Moreover, we found that local Gper expression is regulated negatively by 17ß-estradiol (E2) and progesterone (P4) and fluctuates during the estrus cycle, being minimal in proestrus. Interestingly, lack of ovarian steroids after an ovariectomy (OVX) significantly increased pituitary GPER expression specifically in the three morphologically different subtypes of lactotrophs. We found a rapid estradiol stimulatory effect on PRL secretion mediated by GPER, both in vitro and ex vivo, using a GPER agonist G1, and this effect was prevented by the GPER antagonist G36, demonstrating a novel role for this receptor. Then, the increased pituitary GPER expression after OVX could lead to alterations in the pituitary function as all three lactotroph subtypes are target of GPER ligand and could be involved in the PRL secretion mediated by GPER. Therefore, it should be taken into consideration in the response of the gland to an eventual hormone replacement therapy.