Uterine Fluid Extracellular Vesicles Proteome Is Altered During the Estrous Cycle.

Uterine environment is tightly and finely regulated via various signaling pathways mediated through endocrine, exocrine, autocrine, juxtacrine, and paracrine mechanisms. In utero signaling processes are paramount for normal and abnormal physiology which involves cell to cell, cells to gametes, cells to embryo, and even interkingdom communications due to presence of uterine microbiota. Extracellular vesicles (EVs) in the uterine fluid (UF) and their cargo components are known to be mediators of in utero signaling and communications. Interestingly, the changes in UF-EV proteome during the bovine estrous cycle and the effects of these differentially enriched proteins on embryo development are yet to be fully discovered. In this study, shotgun quantitative proteomics-based mass spectrometry was employed to compare UF-EV proteomes at day 0, 7, and 16 of the estrous cycle to understand the estrous cycle-dependent dynamics. Furthermore, different phase UF-EVs were supplemented in embryo cultures to evaluate their impact on embryo development. One hundred fifty-nine UF-EV proteins were differentially enriched at different time points indicating the UF-EV proteome is cycle-dependent. Overall, many identified pathways are important for normal uterine functions, early embryo development, and its nutritional needs, such as antioxidant activity, cell morphology and cycle, cellular homeostasis, cell adhesion, and carbohydrate metabolic process. Furthermore, the luteal phase UF-EVs supplementation increased in vitro blastocyst rates from 25.0 ± 5.9% to 41.0 ± 4.0% (p ≤ 0.05). Our findings highlight the importance of bovine UF-EV in uterine communications throughout the estrous cycle. Interestingly, comparison of hormone-synchronized EV proteomes to natural cycle UF-EVs indicated shift of signaling. Finally, UF-EVs can be used to improve embryo production in vitro.

Role of chemokines in regulating luteal and uterine functions in pregnant cows.

Pregnancy is intricately regulated by the interactions between various bioactive substances secreted by the conceptus, uterus, and corpus luteum (CL). Interferon-τ, synthesized and secreted by the conceptus, plays a central role in the interaction mechanism of maternal recognition in cows. Chemokines, chemotaxis mediators that are primarily secreted by immune cells, regulate various reproductive responses in various species. Although there are scattered reports on the potential roles of chemokines in the bovine CL and the uterus during the estrous cycle, there is little information on chemokines in these organs during pregnancy. Therefore, in this review, we discuss the possible physiological roles of chemokines in the CL and uterus of pregnant cows, focusing on our recent findings on chemokines and changes in their receptor expression in the CL and endometrium of cows at some stages of pregnancy.

Characterization and profiling analysis of bovine oviduct and uterine extracellular vesicles and their miRNA cargo through the estrous cycle.

Extracellular vesicles (EVs) found in various biological fluids and particularly in reproductive fluids, have gained considerable attention for their possible role in cell- to- cell communication. Among, the different bioactive molecules cargos of EVs, MicroRNAs (miRNAs) are emerging as promising diagnostic biomarkers with high clinical potential. Aiming to understand the roles of EVs in bovine reproductive tract, we intended to characterize and profile the EVs of oviduct and uterine fluids (OF-EVs, UF-EVs) and their miRNA across the estrous cycle. Nanoparticle tracking analysis and transmission electron microscopy confirmed the existence of small EV population in OF and UF at all stages, (size between 30 and 200 nm; concentration: 3.4 × 1010  EVs/ml and 6.0 × 1010  EVs/ml for OF and UF, respectively, regardless of stage). The identification of EV markers (CD9, HSP70, and ALIX proteins) was confirmed by western blot. The miRNA analysis revealed the abundance of 310 and 351 miRNAs in OF-EVs and UF-EVs, respectively. Nine miRNAs were differentially abundant in OF-EVs between stages of the cycle, eight of them displayed a progressive increase from S1 to S4 (p < .05). In UF-EVs, a total of 14 miRNAs were differentially abundant between stages. Greater differences were observed between stage 1 (S1) and stage 3 (S3), with 11 miRNAs enriched in S3 compared to S1. Functional enrichment analysis revealed the involvement of these miRNAs in relevant pathways such as cell signaling, intercellular junctions, and reproductive functions that may be implicated in oviduct and uterus modulation across the cycle, but also in their preparation for embryo/conceptus presence and development.

Transcriptome profiling of different developmental stages of corpus luteum during the estrous cycle in pigs.

To better understand the molecular basis of corpus luteum (CL) development and function RNA-Seq was utilized to identify differentially expressed genes (DEGs) in porcine CL during different physiological stages of the estrous cycle viz. early (EL), mid (ML), late (LL) and regressed (R) luteal. Stage wise comparisons obtained 717 (EL vs. ML), 568 (EL vs. LL), 527 (EL vs. R), 786 (ML vs. LL), 474 (ML vs. R) and 534 (LL vs. R) DEGs with log2(FC) ≥1 and p < 0.05. The process of angiogenesis, steroidogenesis, signal transduction, translation, cell proliferation and tissue remodelling were significantly (p < 0.05) enriched in EL, ML and LL stages, where as apoptosis was most active in regressed stage. Pathway analysis revealed that most annotated genes were associated with lipid metabolism, translation, immune and endocrine system pathways depicting intra-luteal control of diverse CL function. The network analysis identified genes AR, FOS, CDKN1A, which were likely the novel hub genes regulating CL physiology.

Sex differences in fat taste responsiveness are modulated by estradiol.

Sex as a biological variable has been the focus of increasing interest. Relatively few studies have focused, however, on differences in peripheral taste function between males and females. Nonetheless, there are reports of sex-dependent differences in chemosensitivity in the gustatory system. The involvement of endogenous changes in ovarian hormones has been suggested to account for taste discrepancies. Additionally, whether sex differences exist in taste receptor expression, activation, and subsequent signaling pathways that may contribute to different taste responsiveness is not well understood. In this study, we show the presence of both the nuclear and plasma membrane forms of estrogen receptor (ER) mRNA and protein in mouse taste cells. Furthermore, we provide evidence that estrogen increases taste cell activation during the application of fatty acids, the chemical cue for fat taste, in taste receptor cells. We found that genes important for the transduction pathway of fatty acids vary between males and females and that these differences also exist across the various taste papillae. In vivo support for the effect of estrogens in taste cells was provided by comparing the fatty acid responsiveness in male, intact female, and ovariectomized (OVX) female mice with and without hormone replacement. In general, females detected fatty acids at lower concentrations, and the presence of circulating estrogens increased this apparent fat taste sensitivity. Taken together, these data indicate that increased circulating estrogens in the taste system may play a significant role in physiology and chemosensory cellular activation and, in turn, may alter taste-driven behavior.NEW & NOTEWORTHY Using molecular, cellular, and behavioral analyses, this study shows that sex differences occur in fat taste in a mouse model. Female mice are more responsive to fatty acids, leading to an overall decrease in intake and fatty acid preference. These differences are linked to sex hormones, as estradiol enhances taste cell responsiveness to fatty acids during periods of low circulating estrogen following ovariectomy and in males. Estradiol is ineffective in altering fatty acid signaling during a high-estrogen period and in ovariectomized mice on hormone replacement. Thus, taste receptor cells are a direct target for actions of estrogen, and there are multiple receptors with differing patterns of expression in taste cells.

Effects of Radix Polygalae on Cognitive Decline and Depression in Estradiol Depletion Mouse Model of Menopause.

Postmenopausal syndrome refers to symptoms caused by the gradual decrease in female hormones after mid-40 years. As a target organ of estrogen, decrease in estrogen causes various changes in brain function such as a decrease in choline acetyltransferase and brain-derived neurotrophic factor; thus, postmenopausal women experience cognitive decline and more depressive symptoms than age-matched men. Radix Polygalae has been used for memory boosting and as a mood stabilizer and its components have shown neuroprotective, antidepressant, and stress relief properties. In a mouse model of estrogen depletion induced by 4-vinylcyclohexene diepoxide, Radix Polygalae was orally administered for 3 weeks. In these animals, cognitive and depression-related behaviors and molecular changes related to these behaviors were measured in the prefrontal cortex and hippocampus. Radix Polygalae improved working memory and contextual memory and despair-related behaviors in 4-vinylcyclohexene diepoxide-treated mice without increasing serum estradiol levels in this model. In relation to these behaviors, choline acetyltransferase and brain-derived neurotrophic factor in the prefrontal cortex and hippocampus and bcl-2-associated athanogene expression increased in the hippocampus. These results implicate the possible benefit of Radix Polygalae in use as a supplement of estrogen to prevent conditions such as postmenopausal depression and cognitive decline.

Induction of aryl hydrocarbon receptor in granulosa cells by endoplasmic reticulum stress contributes to pathology of polycystic ovary syndrome.

Recent studies have uncovered the critical role of aryl hydrocarbon receptor (AHR) in various diseases, including obesity and cancer progression, independent of its previously identified role as a receptor for endocrine-disrupting chemicals (EDCs). We previously showed that endoplasmic reticulum (ER) stress, a newly recognized local factor in the follicular microenvironment, is activated in granulosa cells from patients with polycystic ovary syndrome (PCOS) and a mouse model of the disease. By affecting diverse functions of granulosa cells, ER stress contributes to PCOS pathology. We hypothesized that expression of AHR and activation of its downstream signaling were upregulated by ER stress in granulosa cells, irrespective of the presence of EDCs, thereby promoting PCOS pathogenesis. In this study, we found that AHR, AHR nuclear translocator (ARNT), and AHR target gene cytochrome P450 1B1 (CYP1B1) were upregulated in the granulosa cells of PCOS patients and model mice. We examined CYP1B1 as a representative AHR target gene. AHR and ARNT were upregulated by ER stress in human granulosa-lutein cells (GLCs), resulting in an increase in the expression and activity of CYP1B1. Administration of the AHR antagonist CH223191 to PCOS mice restored estrous cycling and decreased the number of atretic antral follicles, concomitant with downregulation of AHR and CYP1B1 in granulosa cells. Taken together, our findings indicate that AHR activated by ER stress in the follicular microenvironment contributes to PCOS pathology, and that AHR represents a novel therapeutic target for PCOS.

Effects of supraphysiological vitamin D3 (cholecalciferol) supplement on normal adult rat ovarian functions.

This study measured the sequelae of cholecalciferol (VD3) therapy on ovarian functions in adult VD-replete rats (n = 48). The animals were distributed into the control and VD groups following estrous cycle synchronisation. The VD group received VD3 injections for 4 weeks (600 IU/Kg; 3 times/week). Vaginal cytology and cycle durations were recorded throughout the study. Serum VD (25-OH VD), Ca2+, gonadotrophins (FSH & LH) and sex steroids (E2 & progesterone) were measured following euthanasia. Follicles and corpora lutea were counted in ovarian tissue sections. VD receptor, binding protein, Ca2+-sensing receptor and retinoid X receptor-α genes and proteins were measured by quantitative RT-PCR and immunohistochemistry. Serum VD, LH, E2 and progesterone levels were significantly higher, whereas FSH declined, in the VD group than controls. VD3 therapy was also associated with markedly higher rates alongside shorter durations of estrous cycles than controls. While serum Ca2+ levels were equal between the study groups, they correlated directly with serum 25-OH VD. The numbers of small and medium size ovarian follicles were equal in both study groups, whereas large follicles and corpora lutea counts were significantly higher in the VD group. The mRNAs and proteins of targeted molecules also increased substantially in the VD group than controls. In conclusion, treating VD-sufficient female rats with supraphysiological VD3 supplements was not associated with hypercalcaemia, and could contribute to ovarian functions by regulating the hypothalamic-pituitary-ovarian hormones and ovarian VD-related molecules. However, further studies are still needed to illustrate the clinical significance of VD3 in female reproduction.

Prosaposin in the rat oviductal epithelial cells.

Prosaposin (PSAP) has two forms: a precursor and a secreted form. The secreted form has neurotrophic, myelinotrophic, and myotrophic properties. The precursor form is a precursor protein of saposins A-D. Although the distribution of PSAP in male reproductive organs is well known, its distribution in female reproductive organs, especially in the oviduct, is unclear. Immunoblots and immunohistochemistry of oviducts showed that oviductal tissues contain PSAP proteins, and a significant increase in PSAP was observed in the estrus-metestrus phase compared to the diestrus-proestrus phase in the ampulla. To identify PSAP trafficking in cells, double-immunostaining was performed with antibodies against PSAP in combination with sortilin, mannose 6 phosphate receptor (M6PR), or low-density lipoprotein receptor-related protein 1 (LRP1). PSAP and sortilin double-positive reactions were observed near the nuclei, as well as in the apical portion of microvillous epithelial cells, whereas these reactions were only observed near the nuclei of ciliated epithelial cells. PSAP and M6PR double-positive reactions were observed near the nuclei of microvillous and ciliated epithelial cells. PSAP and M6PR double-positive reactions were also observed in the apical portion of microvillous epithelial cells. PSAP and LRP1 double-positive reactions were observed in the plasma membrane and apical portion of both microvillous and ciliated epithelial cells. Immunoelectron staining revealed PSAP immunoreactive small vesicles with exocytotic features at the apical portion of microvillous epithelial cells. These findings suggest that PSAP is present in the oviductal epithelium and has a pivotal role during pregnancy in providing an optimal environment for gametes and/or sperm in the ampulla.

G Protein-Coupled Estrogen Receptor Immunoreactivity in the Rat Hypothalamus Is Widely Distributed in Neurons, Astrocytes, and Oligodendrocytes, Fluctuates during the Estrous Cycle, and Is Sexually Dimorphic.

INTRODUCTION: The membrane-associated G protein-coupled estrogen receptor 1 (GPER) mediates the regulation by estradiol of arginine-vasopressin immunoreactivity in the supraoptic and paraventricular hypothalamic nuclei of female rats and is involved in the estrogenic control of hypothalamic regulated functions, such as food intake, sexual receptivity, and lordosis behavior. OBJECTIVE: To assess GPER distribution in the rat hypothalamus. METHODS: GPER immunoreactivity was assessed in different anatomical subdivisions of five selected hypothalamic regions of young adult male and cycling female rats: the arcuate nucleus, the lateral hypothalamus, the paraventricular nucleus, the supraoptic nucleus, and the ventromedial hypothalamic nucleus. GPER immunoreactivity was colocalized with NeuN as a marker of mature neurons, GFAP as a marker of astrocytes, and CC1 as a marker of mature oligodendrocytes. RESULTS: GPER immunoreactivity was detected in hypothalamic neurons, astrocytes, and oligodendrocytes. Sex and regional differences and changes during the estrous cycle were detected in the total number of GPER-immunoreactive cells and in the proportion of neurons, astrocytes, and oligodendrocytes that were GPER-immunoreactive. CONCLUSIONS: These findings suggest that estrogenic regulation of hypothalamic function through GPER may be different in males and females and may fluctuate during the estrous cycle in females.

Estrous Cycle Modulation of Feeding and Relaxin-3/Rxfp3 mRNA Expression: Implications for Estradiol Action.

INTRODUCTION: Food intake varies during the ovarian hormone/estrous cycle in humans and rodents, an effect mediated mainly by estradiol. A potential mediator of the central anorectic effects of estradiol is the neuropeptide relaxin-3 (RLN3) synthetized in the nucleus incertus (NI) and acting via the relaxin family peptide-3 receptor (RXFP3). METHODS: We investigated the relationship between RLN3/RXFP3 signaling and feeding behavior across the female rat estrous cycle. We used in situ hybridization to investigate expression patterns of Rln3 mRNA in NI and Rxfp3 mRNA in the hypothalamic paraventricular nucleus (PVN), lateral hypothalamic area (LHA), medial preoptic area (MPA), and bed nucleus of the stria terminalis (BNST), across the estrous cycle. We identified expression of estrogen receptors (ERs) in the NI using droplet digital PCR and assessed the electrophysiological responsiveness of NI neurons to estradiol in brain slices. RESULTS: Rln3 mRNA reached the lowest levels in the NI pars compacta during proestrus. Rxfp3 mRNA levels varied across the estrous cycle in a region-specific manner, with changes observed in the perifornical LHA, magnocellular PVN, dorsal BNST, and MPA, but not in the parvocellular PVN or lateral LHA. G protein-coupled estrogen receptor 1 (Gper1) mRNA was the most abundant ER transcript in the NI. Estradiol inhibited 33% of type 1 NI neurons, including RLN3-positive cells. CONCLUSION: These findings demonstrate that the RLN3/RXFP3 system is modulated by the estrous cycle, and although further studies are required to better elucidate the cellular and molecular mechanisms of estradiol signaling, current results implicate the involvement of the RLN3/RXFP3 system in food intake fluctuations observed across the estrous cycle in female rats.

Sequential preovulatory expression of a gonadotropin-releasing hormone-inducible gene, Nr4a3, and its suppressor Anxa5 in the pituitary gland of female rats.

Functional relationship between nuclear receptor subfamily 4 group A member 3 (Nr4a3) and annexin A5 (Anxa5), which are two gonadotropin-releasing hormone (GnRH)-inducible genes, has been established while evaluating pituitary gonadotropes in relation to follicle-stimulating hormone beta (Fshb) expression. However, the physiological variations that arise due to the differential expression of these genes in the pituitary gland during rat estrous cycle remain unknown. This study aimed to evaluate the Nr4a3 and Anxa5 mRNA expression during the estrous cycle in rats in comparison with the expression of the gonadotropin subunit genes, luteinizing hormone beta (Lhb) and Fshb. Nr4a3 mRNA expression showed a single peak at 1400 h of proestrus during the 4-d estrous cycle. Anxa5 mRNA level was elevated along with increased Fshb mRNA expression after the decline of Nr4a3 mRNA until 2300 h. Lhb mRNA expression levels were not significantly changed during the estrous cycle. Notably, addition of a GnRH antagonist at 1100 h completely eradicated luteinizing hormone secretion at 1400 h and 1700 h of proestrus, and significantly reduced the Nr4a3 mRNA expression level at both the time points. These results suggest that GnRH is, at least partly, responsible for the increase in pituitary Nr4a3, and that the interaction between NR4A3 and ANXA5 is required to regulate Fshb expression during the preovulatory gonadotropin surge.

Human umbilical cord mesenchymal stem cells (hUCMSCs) promotes the recovery of ovarian function in a rat model of premature ovarian failure (POF).

AIMS: Our study was to evaluate the benefits of human umbilical cord mesenchymal stem cells (hUCMSCs) for the prevention of premature ovarian failure (POF) in a rat model. MATERIALS AND METHODS: 80 female SD rats aged between 6 and 8 weeks were randomly divided into 4 groups A, B, C and D. Rats in group A is normal control group; group B, C and D received zona pellucida glycoprotein 3 (pZP3) administration to induce POF model. Among these, group B is model control group; group C received PBS injection in ovaries and group D received hUCMSCs injection in ovaries, all injections were performed after modeling on the same day. Estrus cycle; serum hormone level of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and amount of ovarian follicles were detected 20 days after treatment. RESULTS: We successfully injected hUCMSCs in the ovary tissue of a POF rat. The estrus cycle and hormone expression of the rats in group D tends to be normal. Histological studies indicated that hUCMSCs transplantation increased the amount of ovarian follicles. CONCLUSIONS: This study shows that hUCMSCs may have a preventive effect on POF rats.

Estrogen receptor 2 mediates intraspecific aggressive behaviors of the female Cricetulus barabensis in the estrous cycle.

The social behavior mechanisms have not been thoroughly reported in the solitary female striped dwarf hamster (Cricetulus barabensis). In this study, the handling bag test and neutral arena measurements were used to detect the changes of aggression in the face of rivals of different genders of wild striped dwarf hamsters. We found that female hamsters had the highest aggressive performance in proestrus, followed by estrus, and the lowest in metestrus and the dioestrus, and the increased aggression during the proestrus or estrus period was low-intensity aggression such as intimidation, shock, boxing and counterattack, or even ritualized non-harmful behaviors to drive away opponents. When confronted with male individuals, aggression in females decreased significantly during estrus. The concentration of plasma estradiol was the highest in estrus and the lowest in metestrus and dioestrus. In contrast, estrogen receptor 2 relative expression in the hypothalamus is the lowest in proestrus and highest in metestrus and dioestrus. Besides, both estradiol levels in plasma and estrogen receptor 2 mRNA in the hypothalamus were associated with aggression. These results will broaden our understanding of the molecular mechanism of how breeding phenotype is an essential driver in changing the social behavior of female Cricetulus barabensis.

Expression and functional analysis of the Follistatin-like 3 (FSTL3) gene in the sheep ovary during the oestrous cycle.

Follistatin-like 3 (FSTL3) is a regulator of cellular apoptosis and was previously identified via RNA-Seq to be associated with follicular development in mammalian ovaries. However, the mechanism underlying the FSTL3 regulation of oestrus in sheep remained poorly understood. In this study, the oestrogen (E2) and progesterone (P4) concentrations in blood were detected, and the expression level and functional analysis of FSTL3 in the ovary were studied during the different reproductive stage in Aohan fine wool sheep (seasonal breeding breed in China). The concentrations of E2 and P4 at the anestrus were significantly lower compared to dioestrus, proestrus and oestrus stages. Higher expression levels of FSTL3 were observed in the sheep ovary, hypothalamus, and thyroid. During different reproductive stages, higher expression levels were found during the stages of dioestrus and proestrus, while lower levels were found during the oestrus and anestrus stages. Functional analysis of FSTL3 was performed in primary granulosa cells (GCs) of sheep. The concentration of E2 increased significantly after RNAi interference of FSTL3, while the P4 level decreased. FSTL3 can decrease P4 levels, which might be involved in mediating oestrous cycle in sheep.

Expression and Regulation of CD73 during the Estrous Cycle in Mouse Uterus.

Cluster of differentiation 73 (CD73, also known as ecto-5'-nucleotidase) is an enzyme that converts AMP into adenosine. CD73 is a surface enzyme bound to the outside of the plasma membrane expressed in several cells and regulates immunity and inflammation. In particular, it is known to inhibit T cell-mediated immune responses. However, the regulation of CD73 expression by hormones in the uterus is not yet clearly known. In this study, we investigated the expression of CD73 in ovariectomized mice treated with estrogen or progesterone and its regulation in the mouse uterus during the estrous cycle. The level of CD73 expression was dynamically regulated in the uterus during the estrous cycle. CD73 protein expression was high in proestrus, estrus, and diestrus, whereas it was relatively low in the metestrus stage. Immunofluorescence revealed that CD73 was predominantly expressed in the cytoplasm of the luminal and glandular epithelium and the stroma of the endometrium. The expression of CD73 in ovariectomized mice was gradually increased by progesterone treatment. However, estrogen injection did not affect its expression. Moreover, CD73 expression was increased when estrogen and progesterone were co-administered and was inhibited by the pretreatment of the progesterone receptor antagonist RU486. These findings suggest that the expression of CD73 is dynamically regulated by estrogen and progesterone in the uterine environment, and that there may be a synergistic effect of estrogen and progesterone.

Cysteine-Cysteine Motif Chemokine Receptor 5 Expression in Letrozole-Induced Polycystic Ovary Syndrome Mice.

Polycystic ovary syndrome (PCOS), which affects 5-10% of women of reproductive age, is associated with reproductive and metabolic disorders, such as chronic anovulation, infertility, insulin resistance, and type 2 diabetes. However, the mechanism of PCOS is still unknown. Therefore, this study used a letrozole-exposed mouse model in which mice were orally fed letrozole for 20 weeks to investigate the effects of letrozole on the severity of reproductive and metabolic consequences and the expression of cysteine-cysteine motif chemokine receptor 5 (CCR5) in letrozole-induced PCOS mice. The letrozole-treated mice showed a disrupted estrous cycle and were arrested in the diestrus phase. Letrozole treatment also increased plasma testosterone levels, decreased estradiol levels, and caused multicystic follicle formation. Furthermore, histological analysis of the perigonadal white adipose tissue (pgWAT) showed no significant difference in the size and number of adipocytes between the letrozole-treated mice and the control group. Further, the letrozole-treated mice demonstrated glucose intolerance and insulin resistance during oral glucose and insulin tolerance testing. Additionally, the expression of CCR5 and cysteine-cysteine motif ligand 5 (CCL5) were significantly higher in the pgWAT of the letrozole-treated mice compared with the control group. CCR5 and CCL5 were also significantly correlated with the homeostasis model assessment of insulin resistance (HOMA-IR). Finally, the mechanisms of insulin resistance in PCOS may be caused by an increase in serine phosphorylation and a decrease in Akt phosphorylation.

Decreased cued fear discrimination learning in female rats as a function of estrous phase.

Relative to males, female rats can show enhanced contextual fear generalization (demonstrating a fear response in a safe or neutral context) dependent on estrogen receptor activation. The current experiment aimed to extend this finding to cued fear conditioning. Females in low-estrogen phases of the estrous cycle showed good discrimination, similar to males, between a conditional stimulus that predicted shock (CS+) and an equally familiar one that did not (CS-), while females in the proestrus (high estrogen) phase demonstrated similar levels of fear between the CS+ and CS-. These results demonstrate that cued fear generalization is similarly influenced by endogenous estrogens.

Integrated analysis of lncRNA, miRNA and mRNA reveals novel insights into the fertility regulation of large white sows.

BACKGROUND: Improving sow fertility is extremely important as it can lead to increased reproductive efficiency and thus profitability for swine producers. There are considerable differences in fertility rates among individual animals, but the underlying molecular mechanisms remain unclear. In this study, by using different types of RNA libraries, we investigated the complete transcriptome of ovarian tissue during the luteal (L) and follicular (F) phases of the estrous cycle in Large White pigs with high (H) and low (L) fecundity, and performed a comprehensive analysis of long noncoding RNAs (lncRNAs), mRNAs and micro RNAs (miRNAs) from 16 samples by combining RNA sequencing (RNA-seq) with bioinformatics. RESULTS: In total, 24,447 lncRNAs, 27,370 mRNAs, and 216 known miRNAs were identified in ovarian tissues. The genomic features of lncRNAs, such as length distribution and number of exons, were further analyzed. We selected a threshold of P < 0.05 and |log2 (fold change)| ≥ 1 to obtain the differentially expressed lncRNAs, miRNAs and mRNAs by pairwise comparison (LH vs. LL, FH vs. FL). Bioinformatics analysis of these differentially expressed RNAs revealed multiple significantly enriched pathways (P < 0.05) that were closely involved in the reproductive process, such as ovarian steroidogenesis, lysosome, steroid biosynthesis, and the estrogen and GnRH signaling pathways. Moreover, bioinformatics screening of differentially expressed miRNAs that share common miRNA response elements (MREs) with lncRNAs and their downstream mRNA targets were performed. Finally, we constructed lncRNA-miRNA-mRNA regulation networks. The key genes in these networks were verified by Reverse Transcription Real-time Quantitative PCR (RT-qRCR), which were consistent with the results from RNA-Seq data. CONCLUSIONS: These results provide further insights into the fertility of pigs andcan contribute to further experimental investigation of the functions of these genes.

Differential and synergistic roles of 17ß-estradiol and progesterone in modulating adult female rat nucleus accumbens core medium spiny neuron electrophysiology.

Naturally occurring cyclical changes in sex steroid hormones such as 17ß-estradiol and progesterone can modulate neuron function and behavior in female mammals. One example is the estrous cycle in rats, which is composed of multiple phases. We previously reported evidence of differences between estrous cycle phases in excitatory synapse and intrinsic electrophysiological properties of rat nucleus accumbens core (AcbC) medium spiny neurons (MSNs). The AcbC is a nexus between the limbic and premotor systems and is integral for controlling motivated and reward-associated behaviors and disorders, which are sensitive to the estrous cycle and hormones. The present study expands our prior findings by testing whether circulating levels of estradiol and progesterone correlate with changes in MSN electrophysiology across estrous cycle phases. As part of this project, the excitatory synapse and intrinsic excitability properties of MSNs in late proestrus of adult female rats were assessed. Circulating levels of estradiol correlate with resting membrane potential, the time constant of the membrane, and rheobase. Circulating levels of progesterone correlate with miniature excitatory postsynaptic current (mEPSC) frequency and amplitude. Circulating levels of estradiol and progesterone together correlate with mEPSC amplitude, resting membrane potential, and input resistance. The late proestrus phase features a prominent and unique decrease in mEPSC frequency. These data indicate that circulating levels of estradiol and progesterone alone or in combination interact with specific MSN electrophysiological properties, indicating differential and synergistic roles of these hormones. Broadly, these findings illustrate the underlying endocrine actions regarding how the estrous cycle modulates MSN electrophysiology.NEW & NOTEWORTHY This research indicates that estradiol and progesterone act both differentially and synergistically to modulate neuron physiology in the nucleus accumbens core. These actions by specific hormones provide key data indicating the endocrine mechanisms underlying how the estrous cycle modulates neuron physiology in this region. Overall, these data reinforce that hormones are an important influence on neural physiology.