Sex differences in avoidance behavior and cued threat memory dynamics in mice: Interactions between estrous cycle and genetic background.

Anxiety disorders are the most prevalent mental illnesses worldwide, exhibit high heritability, and affect twice as many women as men. To evaluate potential interactions between genetic background and cycling ovarian hormones on sex differences in susceptibility to negative valence behaviors relevant to anxiety disorders, we assayed avoidance behavior and cued threat memory dynamics in gonadally-intact adult male and female mice across four common inbred mouse strains: C57Bl/6J, 129S1/SVlmJ, DBA/2J, and BALB/cJ. Independent of sex, C57Bl/6J mice exhibited low avoidance but high threat memory, 129S1/SvlmJ mice high avoidance and high threat memory, DBA/2J mice low avoidance and low threat memory, and BALB/cJ mice high avoidance but low threat memory. Within-strain comparisons revealed reduced avoidance behavior in the high hormone phase of the estrous cycle (proestrus) compared to all other estrous phases in all strains except DBA/2J, which did not exhibit cycle-dependent behavioral fluctuations. Robust and opposing sex differences in threat conditioning and extinction training were found in the C57Bl/6J and 129S1/SvlmJ lines, whereas no sex differences were observed in the DBA/2J or BALB/cJ lines. C57Bl/6J males exhibited enhanced acute threat memory, whereas 129S1/SvlmJ females exhibited enhanced sustained threat memory, compared to their sex-matched littermates. These effects were not mediated by estrous cycle stage or sex differences in active versus passive defensive behavioral responses. Our data demonstrate that core features of behavioral endophenotypes relevant to anxiety disorders, such as avoidance and threat memory, are genetically driven yet dissociable and can be influenced further by cycling ovarian hormones.

A nonsense mutation in mouse Adamtsl2 causes uterine hypoplasia and an irregular estrous cycle.

The spontaneous mutation stubby (stb) in mice causes chondrodysplasia and male infertility due to impotence through autosomal recessive inheritance. In this study, we conducted linkage analysis to localize the stb locus within a 1.6 Mb region on mouse chromosome 2 and identified a nonsense mutation in Adamtsl2 of stb/stb mice. Histological analysis revealed disturbed endochondral ossification with a reduced hypertrophic chondrocyte layer and stiff skin with a thickened dermal layer. These phenotypes are similar to those observed in humans and mice with ADAMTSL2/Adamtsl2 mutations. Moreover, stb/stb female mice exhibited severe uterine hypoplasia at 5 weeks of age and irregular estrous cycles at 10 weeks of age. In normal mice, Adamtsl2 was more highly expressed in the ovary and pituitary gland than in the uterus, and this expression was decreased in stb/stb mice. These findings suggest that Adamtsl2 may function in these organs rather than in the uterus. Thus, we analyzed Gh expression in the pituitary gland and plasma estradiol and IGF1 levels, which are required for the development of the female reproductive tract. There was no significant difference in Gh expression and estradiol levels, whereas IGF1 levels in stb/stb mice were significantly reduced to 54-59% of those in +/+ mice. We conclude that Adamtsl2 is required for the development of the uterus and regulation of the estrous cycle in female mice, and decreased IGF1 may be related to these abnormalities.

Dynamic transcriptome analysis of Maiwa yak corpus luteum during the estrous cycle.

Maiwa yak is a special breed of animal living on the Qinghai-Tibet Plateau, which has great economic value, but its fertility rate is low. The corpus luteum (CL) is a temporary tissue that plays a crucial role in maintaining the physiological cycle. However, little is known about the transcriptome profile in Maiwa yak CL. In the present study, the transcriptome of Maiwa yak CL at early (EYCL), middle (MYCL) and late-stages (LYCL) was studied employing high-throughput sequencing. A total of 25,922 transcripts were identified, including 22,277 known as well as 3,645 novel ones. Furthermore, 690 and 212 differentially expressed (DE) mRNAs were detected in the EYCL vs. MYCL and MYCL vs. LYCL groups, respectively. KEGG pathway enrichment analysis of DEGs illustrated that the most enriched pathway was PI3K-Akt pathway. Furthermore, twenty-six DEGs were totally found to be associated with different biological processes of CL development. One of these genes, PGRMC1, displayed a dynamical expression trend during the lifespan of yak CL. The knockdown of PGRMC1 in luteinized yak granulosa cells resulted in defective steroidogenesis. In conclusion, this study analyzed the transcriptome profiles in yak CL of different stages, and provided a novel database for analyzing the gene network in yak CL.HIGHLIGHTSThe manuscript analyzed the transcriptome profiles in yak CL during the estrous cycle.Twenty-six DEGs were found to be associated with the development or function of CL.One of the DEGs, PGRMC1, was found to be responsible for steroidogenesis in luteinized yak granulosa cells.

Cnih3 Deletion Dysregulates Dorsal Hippocampal Transcription across the Estrous Cycle.

In females, the hippocampus, a critical brain region for coordination of learning, memory, and behavior, displays altered physiology and behavioral output across the estrous or menstrual cycle. However, the molecular effectors and cell types underlying these observed cyclic changes have only been partially characterized to date. Recently, profiling of mice null for the AMPA receptor trafficking gene Cnih3 have demonstrated estrous-dependent phenotypes in dorsal hippocampal synaptic plasticity, composition, and learning/memory. We therefore profiled dorsal hippocampal transcriptomes from female mice in each estrous cycle stage, and contrasted it with that of males, across wild-type (WT) and Cnih3 mutants. In wild types, we identified only subtle differences in gene expression between the sexes, while comparing estrous stages to one another revealed up to >1000 differentially expressed genes (DEGs). These estrous-responsive genes are especially enriched in gene markers of oligodendrocytes and the dentate gyrus, and in functional gene sets relating to estrogen response, potassium channels, and synaptic gene splicing. Surprisingly, Cnih3 knock-outs (KOs) showed far broader transcriptomic differences between estrous cycle stages and males. Moreover, Cnih3 knock-out drove subtle but extensive expression changes accentuating sex differential expression at diestrus and estrus. Altogether, our profiling highlights cell types and molecular systems potentially impacted by estrous-specific gene expression patterns in the adult dorsal hippocampus, enabling mechanistic hypothesis generation for future studies of sex-differential neuropsychiatric function and dysfunction. Moreover, these findings suggest an unrecognized role of Cnih3 in buffering against transcriptional effects of estrous, providing a candidate molecular mechanism to explain estrous-dependent phenotypes observed with Cnih3 loss.

Expression of IRS2 in the female reproductive system during the estrous cycle in mice.

Insulin receptor substrate 2 (IRS2) participates in reproduction; however, the location and expression of IRS2 in the reproductive system of female mice is not clear. We used real-time quantitative polymerase chain reaction (RT-PCR), western blot and immunohistochemical staining to investigate the expression of IRS2 in the ovary, oviduct and uterus of female mice during the estrous cycle. We found that IRS2 was expressed in all reproductive organs of mouse and that the expression level changed with the estrous phases. The expression of IRS2 in reproductive organs was greatest during estrus.

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.

Molecular Mechanism of Equine Endometrosis: The NF-κB-Dependent Pathway Underlies the Ovarian Steroid Receptors' Dysfunction.

Endometrosis is a frequently occurring disease decreasing mares' fertility. Thus, it is an important disease of the endometrium associated with epithelial and stromal cell alterations, endometrium gland degeneration and periglandular fibrosis. Multiple degenerative changes are found in uterine mucosa, the endometrium. However, their pathogenesis is not well known. It is thought that nuclear factor-κB (NF-κB), a cell metabolism regulator, and its activation pathways take part in it. The transcription of the profibrotic pathway genes of the NF-κB in fibrotic endometria differed between the follicular (FLP) and mid-luteal (MLP) phases of the estrous cycle, as well as with fibrosis progression. This study aimed to investigate the transcription of genes of estrogen (ESR1, ESR2) and progesterone receptors (PGR) in equine endometria to find relationships between the endocrine environment, NF-κB-pathway, and fibrosis. Endometrial samples (n = 100), collected in FLP or MLP, were classified histologically, and examined using quantitative PCR. The phase of the cycle was determined through the evaluation of ovarian structures and hormone levels (estradiol, progesterone) in serum. The transcription of ESR1, ESR2, and PGR decreased with the severity of endometrial fibrosis and degeneration of the endometrium. Moreover, differences in the transcription of ESR1, ESR2, and PGR were noted between FLP and MLP in the specific categories and histopathological type of equine endometrosis. In FLP and MLP, specific moderate and strong correlations between ESR1, ESR2, PGR and genes of the NF-κB pathway were evidenced. The transcription of endometrial steroid receptors can be subjected to dysregulation with the degree of equine endometrosis, especially in both destructive types of endometrosis, and mediated by the canonical NF-κB pathway depending on the estrous cycle phase.

A functional cellular framework for sex and estrous cycle-dependent gene expression and behavior.

Sex hormones exert a profound influence on gendered behaviors. How individual sex hormone-responsive neuronal populations regulate diverse sex-typical behaviors is unclear. We performed orthogonal, genetically targeted sequencing of four estrogen receptor 1-expressing (Esr1+) populations and identified 1,415 genes expressed differentially between sexes or estrous states. Unique subsets of these genes were distributed across all 137 transcriptomically defined Esr1+ cell types, including estrous stage-specific ones, that comprise the four populations. We used differentially expressed genes labeling single Esr1+ cell types as entry points to functionally characterize two such cell types, BNSTprTac1/Esr1 and VMHvlCckar/Esr1. We observed that these two cell types, but not the other Esr1+ cell types in these populations, are essential for sex recognition in males and mating in females, respectively. Furthermore, VMHvlCckar/Esr1 cell type projections are distinct from those of other VMHvlEsr1 cell types. Together, projection and functional specialization of dimorphic cell types enables sex hormone-responsive populations to regulate diverse social behaviors.

Estrous activity in lactating cows with divergent genetic merit for fertility traits.

This observational study aimed to determine the effect of genetic merit for fertility traits on estrous expression and estrous cycle duration in grazing dairy cows, as measured by an activity monitoring device. A secondary aim was to describe changes in expression of estrus that occur during successive estrous cycles postpartum. Neck-mounted, activity-monitoring devices (Heatime, SCR Engineers Ltd.) were fitted to nulliparous Holstein-Friesian heifers with positive (POS FertBV) or negative genetic merit for fertility traits (NEG FertBV) to capture activity data during their first and second lactations (POS FertBV: n = 242, n = 188; NEG FertBV: n = 159, n = 87 in lactation 1 and 2, respectively). An estrous event was identified when the activity change index exceeded 26 activity units (AU) for 4 h. A total of 1,254 and 892 estrous events were identified in lactation 1 and 2, respectively. Estrous duration was defined as the interval between when the threshold was first exceeded and when activity dropped below the threshold, with no new event starting within 24 h of the end of the previous event. This definition of estrus included cows in which activity crossed the threshold multiple times in a day and were classified as a single estrous event. A second measure, high activity duration, was defined as the total hours that activity exceeded the threshold. To characterize estrous activity, peak activity (above baseline) and total activity (area under the curve of activity above baseline) were measured. Compared with NEG FertBV cows, POS FertBV cows had more active, longer estrous events. In lactation 1, the POS FertBV group had a mean estrous duration and a high activity duration of 12.5 and 12.4 h compared with 11.4 and 11.3 h for the NEG FertBV group [standard error of the difference (SED) = 0.5 and 0.4 h, respectively]. This significant difference also occurred in lactation 2, with a mean estrous duration of 13.1 versus 11.8 h (SED = 0.5 h) and a high activity duration of 13.0 versus 11.8 h (SED = 0.4 h) in the POS and NEG FertBV groups, respectively. Total activity and peak activity were greater in the POS compared with the NEG FertBV group in lactation 1 (peak activity: 65.5 vs. 55.8 AU, SED = 2.4 AU; total activity: 588 vs. 494 AU, SED = 25 AU) and lactation 2 (peak activity: 72.5 vs. 61.2 AU, SED = 2.9 AU; total activity: 648 vs. 541 AU, SED = 30 AU). Estrous cycle duration did not differ between the POS and NEG FertBV groups (lactation 1: 20.4 vs. 20.6 d, SED = 0.25; lactation 2: 20.8 vs. 21.0 d, SED = 0.28). Less estrous activity of the cow was associated with the first postpartum estrus. In contrast, the number of previous estrous events did not consistently affect the duration of the subsequent estrous cycle. The outcomes of this study provide evidence that positive genetic merit for fertility traits is associated with more overt estrous expression. Selection for these traits may improve estrous expression and thus estrous detection in commercial herds.

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.

MC3R links nutritional state to childhood growth and the timing of puberty.

The state of somatic energy stores in metazoans is communicated to the brain, which regulates key aspects of behaviour, growth, nutrient partitioning and development1. The central melanocortin system acts through melanocortin 4 receptor (MC4R) to control appetite, food intake and energy expenditure2. Here we present evidence that MC3R regulates the timing of sexual maturation, the rate of linear growth and the accrual of lean mass, which are all energy-sensitive processes. We found that humans who carry loss-of-function mutations in MC3R, including a rare homozygote individual, have a later onset of puberty. Consistent with previous findings in mice, they also had reduced linear growth, lean mass and circulating levels of IGF1. Mice lacking Mc3r had delayed sexual maturation and an insensitivity of reproductive cycle length to nutritional perturbation. The expression of Mc3r is enriched in hypothalamic neurons that control reproduction and growth, and expression increases during postnatal development in a manner that is consistent with a role in the regulation of sexual maturation. These findings suggest a bifurcating model of nutrient sensing by the central melanocortin pathway with signalling through MC4R controlling the acquisition and retention of calories, whereas signalling through MC3R primarily regulates the disposition of calories into growth, lean mass and the timing of sexual maturation.

Deletion of Gremlin-2 alters estrous cyclicity and disrupts female fertility in mice†.

Members of the differential screening-selected gene aberrative in neuroblastoma (DAN) protein family are developmentally conserved extracellular binding proteins that antagonize bone morphogenetic protein (BMP) signaling. This protein family includes the Gremlin proteins, GREM1 and GREM2, which have key functions during embryogenesis and adult physiology. While BMPs play essential roles in ovarian follicle development, the role of the DAN family in female reproductive physiology is less understood. We generated mice null for Grem2 to determine its role in female reproduction in addition to screening patients with primary ovarian insufficiency (POI) for variants in GREM2. Grem2-/- mice are viable, but female Grem2-/- mice have diminished fecundity and irregular estrous cycles. This is accompanied by significantly reduced production of ovarian anti-Müllerian hormone (AMH) from small growing follicles, leading to a significant decrease in serum AMH. Surprisingly, as AMH is a well-established marker of the ovarian reserve, morphometric analysis of ovarian follicles showed maintenance of primordial follicles in Grem2-/- mice like wild-type (WT) littermates. While Grem2 mRNA transcripts were not detected in the pituitary, Grem2 is expressed in hypothalami of WT female mice, suggesting the potential for dysfunction in multiple tissues composing the hypothalamic-pituitary-ovarian axis that contribute to the subfertility phenotype. Additionally, screening 106 women with POI identified one individual with a heterozygous variant in GREM2 that lies within the predicted BMP-GREM2 interface. In total, these data suggest that Grem2 is necessary for female fecundity by playing a novel role in regulating the HPO axis and contributing to female reproductive disease.

Effects of FSH treatment and withdrawal during proestrus on uterine proliferation and steroid hormone receptor expression in beef heifers.

To determine the effects of Follicle Stimulating Hormone (FSH) treatment and subsequent withdrawal on uterine proliferation and estrogen receptor (ESR), Brahman crossbred heifers (n = 12) were twice daily injected with FSH (4, 3, and 2 mg/injection) on Days 17-19 of the estrous cycle (FSH 3 days) and (4 and 3 mg/injection) on Days 17-18 (FSH 2 days) and withdrawal with saline on Day 19 and (4 mg/injection) on Day 17 (FSH 1 day) and withdrawal with saline on Days 18-19. Uterine tissue was subjectively collected on Day 20 and microscopically classified to four regions: endometrial stroma (ES), surface endometrial gland (EG), deep endometrial gland (DG), and myometrium (Myo). The cell proliferation marker, Ki-67, was quantified as labeling index (LI) in uterine regions, and tissues were immunostained to detect ESR2 followed by image analysis. The LI of ES, EG, and DG was greater (P = 0.0018, P = 0.0005, and P = 0.0103; respectively) in heifers received FSH for 3 days. The expression of ESR2 protein on ES and EG was greatest (P < 0.0001 and P = 0.0036, respectively) in FSH 3 days-treated group. Thus, FSH administration during proestrus stimulates uterine cell proliferation, and ESR2 expressions are affected by FSH during proestrus and differentially distributed in the uterine regions.

Evidence of haptoglobin in the porcine female genital tract during oestrous cycle and its effect on in vitro embryo production.

Recent evidence supports involvement of the acute phase protein haptoglobin in numerous events during mammalian reproduction. The present study represents an in-depth investigation of haptoglobin expression and secretion in the porcine oviduct and uterus, and assesses its effect on porcine in vitro embryo production. A systematic study was made of sows in different oestrous stages: late follicular, early luteal and late luteal stages. Relative haptoglobin mRNA abundance was quantified by RT-qPCR. In addition, expression of the protein was analysed by immunohistochemistry and the results were complemented by Western-blot and proteomic analyses of the oviductal and uterine fluids. In vitro porcine fertilization and embryo culture were carried out in the presence of haptoglobin. The results indicate that haptoglobin mRNA expression in the porcine oviduct and uterus is most abundant during the late luteal stage of the oestrous cycle. By means of Western blot and proteomic analyses haptoglobin presence was demonstrated in the oviduct epithelium and in the oviductal and uterine fluids in different stages of the oestrous cycle. The addition of haptoglobin during gamete co-incubation had no effect on sperm penetration, monospermy or efficiency rates; however, compared with the control group, blastocyst development was significantly improved when haptoglobin was present (haptoglobin: 64.50% vs. control: 37.83%; p < 0.05). In conclusion, the presence of haptoglobin in the oviduct and uterus of sows at different stages of the oestrous cycle suggests that it plays an important role in the reproduction process. The addition of haptoglobin during in vitro embryo production improved the blastocyst rates.

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.

Spatiotemporal transcriptional dynamics of the cycling mouse oviduct.

Female fertility in mammals requires iterative remodeling of the entire adult female reproductive tract across the menstrual/estrous cycle. However, while transcriptome dynamics across the estrous cycle have been reported in human and bovine models, no global analysis of gene expression across the estrous cycle has yet been reported for the mouse. Here, we examined the cellular composition and global transcriptional dynamics of the mouse oviduct along the anteroposterior axis and across the estrous cycle. We observed robust patterns of differential gene expression along the anteroposterior axis, but we found surprisingly few changes in gene expression across the estrous cycle. Notable gene expression differences along the anteroposterior axis included a surprising enrichment for genes related to embryonic development, such as Hox and Wnt genes. The relatively stable transcriptional dynamics across the estrous cycle differ markedly from other mammals, leading us to speculate that this is an evolutionarily derived state that may reflect the extremely rapid five-day mouse estrous cycle. This dataset fills a critical gap by providing an important genomic resource for a highly tractable genetic model of mammalian female reproduction.

Characterization of Long Non-Coding RNA Profiles in Porcine Granulosa Cells of Healthy and Atretic Antral Follicles: Implications for a Potential Role in Apoptosis.

Long non-coding RNAs (lncRNAs) play important roles in multiple biological processes including ovarian follicular development. Here we aimed to gain novel information regarding lncRNAs transcriptome profiles in porcine granulosa cells of advanced atretic antral (AA) and healthy antral (HA) follicles using RNA-seq. A total of 11,321 lncRNAs including 10,813 novel and 508 annotated lncRNAs were identified, of which 173 lncRNAs were differentially expressed (DE-lncRNAs); ten of these were confirmed by qRT-PCR. Gene Ontology indicated that DE-lncRNAs associated with developmental processes were highly enriched. Pathway analysis demonstrated predicted cis- and trans-targets of DE-lncRNAs. Potential mRNA targets of up-regulated DE-lncRNAs were mainly enriched in apoptosis related pathways, while targeted genes of downregulated DE-lncRNAs were primarily enriched in metabolism and ovarian steroidogenesis pathways. Linear regression analyses showed that expression of upregulated DE-lncRNAs was significantly associated with apoptosis related genes. NOVEL_00001850 is the most-downregulated DE-lncRNA (FDR = 0.04, FC = -6.53), of which miRNA binding sites were predicted. KEGG analysis of its downregulated target genes revealed that ovarian steroidogenesis was the second most highlighted pathway. qRT-PCR and linear regression analysis confirmed the expression and correlation of its potential targeted gene, CYP19A1, a key gene involved in estradiol synthesis. Our results indicate that lncRNAs may participate in granulosa cells apoptosis and thus antral follicular atresia.

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.

Steroidal Regulation of Oviductal microRNAs Is Associated with microRNA-Processing in Beef Cows.

Information on molecular mechanisms through which sex-steroids regulate oviductal function to support early embryo development is lacking. Here, we hypothesized that the periovulatory endocrine milieu affects the miRNA processing machinery and miRNA expression in bovine oviductal tissues. Growth of the preovulatory follicle was controlled to obtain cows that ovulated a small follicle (SF) and subsequently bore a small corpus luteum (CL; SF-SCL) or a large follicle (LF) and large CL (LF-LCL). These groups differed in the periovulatory plasmatic sex-steroid's concentrations. Ampulla and isthmus samples were collected on day four of the estrous cycle. Abundance of DROSHA, DICER1, and AGO4 transcripts was greater in the ampulla than the isthmus. In the ampulla, transcription of these genes was greater for the SF-SCL group, while the opposite was observed in the isthmus. The expression of the 88 most abundant miRNAs and 14 miRNAs in the ampulla and 34 miRNAs in isthmus were differentially expressed between LF-LCL and SF-SCL groups. Integration of transcriptomic and miRNA data and molecular pathways enrichment showed that important pathways were inhibited in the SF-SCL group due to miRNA control. In conclusion, the endocrine milieu affects the miRNA expression in the bovine oviduct in a region-specific manner.

Pluripotent Stem (VSELs) and Progenitor (EnSCs) Cells Exist in Adult Mouse Uterus and Show Cyclic Changes Across Estrus Cycle.

We have earlier reported pluripotent, very small embryonic-like stem cells (VSELs) and slightly bigger endometrial stem cells (EnSCs) in adult mouse uterus and their regulation by gonadotropin and steroid hormones. VSELs can differentiate into cells of all three lineages in vitro; however, they neither expand readily in vitro nor compliment a developing embryo. In the present study, a robust protocol is described to enrich uterine stem/progenitor cells along with their characterization and variation across estrus cycle. After enzymatic digestion of adult mouse uterus, single-cell suspension obtained was spun at 1000 rpm (250 g) to pellet majority of cells. Stem cells remain buoyant at this speed and were pelleted by spinning supernatant at 3000 rpm (1000 g). Spherical, darkly stained VSELs (2-6 µm) with high nucleo-cytoplasmic ratio and EnSCs (> 6 µm) expressed OCT-4, NANOG, SSEA-1, SCA-1, and c-KIT. OCT-4-positive cells co-expressed SSEA-1, ERα, ERß, PR, and FSHR. Transcripts specific for pluripotent state (Oct-4, Oct-4a, Sox-2, Nanog), primordial germ cells (Stella, Fragilis), and receptors for pituitary and steroid hormones (ERα, ERß, PR, FSHR 1 and 3) were studied by RT-PCR in 3000 rpm pellet. Cell pellet collected at 3000 rpm showed 10-fold enrichment of VSELs (2-6 µm, viable cells with surface phenotype of LIN-CD45-SCA-1+) by flow cytometry and upregulation of pluripotent transcripts by qRT-PCR compared with 1000 rpm pellet. VSELs were maximal during estrus and metestrus phases of estrus cycle. To conclude, VSELs/EnSCs can be enriched from adult uterus using the strategy described here, vary in numbers across estrus cycle, and are vulnerable to endocrine disruption as they express steroid receptors.