Sperm functionality is differentially regulated by porcine oviductal extracellular vesicles from the distinct phases of the estrous cycle.

Context Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P Conclusions poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm-oocyte interaction. Implications The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm-oocyte interaction.

Global cerebral ischemia in adult female rats interrupts estrous cyclicity and induces lasting changes in hypothalamic-pituitary-gonadal axis signaling peptides.

Persistent post-ischemic alterations to the hypothalamic-pituitary-adrenal (HPA) axis occur following global cerebral ischemia (GCI) in rodents. However, similar effects on hypothalamic-pituitary-gonadal (HPG) axis activation remain to be determined. Therefore, this study evaluated the effects of GCI in adult female rats (via four-vessel occlusion) on the regularity of the estrous cycle for 24-days post ischemia. A second objective aimed to assess persistent alterations of HPG axis activation through determination of the expression of estrogen receptor alpha (ERα), kisspeptin (Kiss1), and gonadotropin-inhibitory hormone (GnIH/RFamide-related peptide; RFRP3) in the medial preoptic area (POA), arcuate nucleus (ARC), dorsomedial nucleus (DMH) of the hypothalamus, and CA1 of the hippocampus 25 days post ischemia. Expression of glucocorticoid receptors (GR) in the paraventricular nucleus of the hypothalamus (PVN) and CA1 served as a proxy of altered HPA axis activation. Our findings demonstrated interruption of the estrous cycle in 87.5 % of ischemic rats, marked by persistent diestrus, lasting on average 11.86 days. Moreover, compared to sham-operated controls, ischemic female rats showed reduced Kiss1 expression in the hypothalamic ARC and POA, concomitant with elevated ERα in the ARC and increased GnIH in the DMH and CA1. Reduced GR expression in the CA1 was associated with increased GR-immunoreactivity in the PVN, indicative of lasting dysregulation of HPA axis activation. Together, these findings demonstrate GCI disruption of female rats' estrous cycle over multiple days, with a lasting impact on HPG axis regulators within the reproductive axis.

The Effect of Visfatin on the Functioning of the Porcine Pituitary Gland: An In Vitro Study.

Visfatin (VIS), also known as nicotinamide phosphoribosyltransferase (NAMPT), is the rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). Recently, VIS has been also recognized as an adipokine. Our previous study revealed that VIS is produced in the anterior and posterior lobes of the porcine pituitary. Moreover, the expression and secretion of VIS are dependent on the phase of the estrous cycle and/or the stage of early pregnancy. Based on this, we hypothesized that VIS may regulate porcine pituitary function. This study was conducted on anterior pituitary (AP) glands harvested from pigs during specific phases of the estrous cycle. We have shown the modulatory effect of VIS in vitro on LH and FSH secretion by porcine AP cells (determined by ELISA). VIS was also found to stimulate cell proliferation (determined by Alamar Blue) without affecting apoptosis in these cells (determined using flow cytometry technique). Moreover, it was indicated that VIS may act in porcine AP cells through the INSR, AKT/PI3K, MAPK/ERK1/2, and AMPK signaling pathways (determined by ELISA or Western Blot). This observation was further supported by the finding that simultaneous treatment of cells with VIS and inhibitors of these pathways abolished the observed VIS impact on LH and FSH secretion (determined by ELISA). In addition, our research indicated that VIS affected the mentioned processes in a manner that was dependent on the dose of VIS and/or the phase of the estrous cycle. Thus, these findings suggest that VIS may regulate the functioning of the porcine pituitary gland during the estrous cycle.

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.

Estrogen and progesterone receptors and antioxidant enzymes are expressed differently in the uterus of domestic cats during the estrous cycle.

Sex steroids and antioxidant enzymes are important in female sexual development and adequate modulation of the estrous cycle, pregnancy, and fetal development. Therefore, modifications in its signaling or expression in the genital system are associated with reproductive dysfunctions. However, the spatial-temporal expression profile of receptors for sex steroids and antioxidant enzymes in the uterus of domestic cats throughout the estrous cycle needs to be studied. Cats in proestrus/estrus (N = 6), diestrus, (N = 7), and anestrus (N = 6) were used to evaluate the uterine expression of estrogen alpha (ERα), progesterone (PR), and androgen (AR) receptors and of the antioxidant enzymes superoxide dismutase 1 (SOD1), catalase and glutathione peroxidase 1 (GPX1) by immunohistochemistry and qPCR. The uterus of cats in diestrus showed lower protein and mRNA expression of ERα and PR compared to proestrus/estrus and anestrus, mainly in the luminal and glandular epithelium and myometrium, different from catalase and SOD1, which showed higher expression in diestrus in relation to other phases of the cycle. GPX1, on the other hand, showed lower uterine gene expression in diestrus compared to proestrus/estrus and anestrus. No significant differences in AR expression were observed. In conclusion, ERα and PR sex steroid receptors and antioxidant enzymes are expressed differently in the uterus of domestic cats during the estrous cycle.

Transcriptome profile of the anterior pituitary gland in pigs during maternal recognition of pregnancy.

The activity of the pituitary gland determines the success of female reproduction. The knowledge regarding the reproductive-status-related changes in the transcriptome of the porcine pituitary is limited. This study aimed to determine and compare the transcriptome profile of the pituitary gland collected from pigs during maternal recognition of pregnancy, i.e. on days 12-13 of pregnancy and during the respective days of the estrous cycle. Analysis indicated 482 differentially expressed genes (DEGs) with an FC > 1.5 (P < 0.05) in the pituitary of pregnant vs. estrous-cyclic pigs. Among them, 68 were up-regulated and 414 were down-regulated. The evaluated DEGs were annotated into 39 gene ontology (GO) biological process terms, 13 GO cellular component terms, and 10 GO molecular function terms. Among the evaluated DEGs were selected genes coding for proteins potentially involved in the regulation of early pregnancy in pigs and used for gene interaction analysis and validation of microarray results. An analysis of the relationships among DEGs in pituitaries collected during maternal recognition of pregnancy showed that some of them are connected with, for example, TGFß signaling pathway, PRL synthesis, adipocytokines pathway and immune response during maternal recognition of pregnancy. These findings expand the knowledge regarding the molecular mechanisms appearing in the porcine pituitary during the maternal recognition period of pregnancy.

Characterization of bovine uterine fluid extracellular vesicles proteomic profiles at follicular and luteal phases of the oestrous cycle.

Extracellular vesicles (EV) have been identified in uterine fluid (UF), however the bovine UF-EV profile during different phases of the oestrous cycle has not yet been established. Therefore, we compared the UF-EV, and their protein profile at follicular and luteal phases of the oestrous cycle. UF samples were collected from healthy uteri of six live and six slaughtered cows at follicular or luteal phases. Isolation of EV was performed using tangential flow filtration followed by size exclusion chromatography. EV were characterized by nanoparticle tracking analysis (NTA), fluorescence NTA, zeta potential, and transmission electron microscopy. Mass-spectrometry was used to evaluate EV protein profile from live cows. Particle concentrations (mean ± SD) were higher (P < 0.05) at follicular than at luteal phase in both live (1.01 × 108 ± 1.66 × 107 vs 7.56 × 107 ± 1.80 × 107, respectively) and slaughtered cows (1.17 × 108 ± 2.34 × 107 vs 9.12 × 107 ± 9.77 × 106, respectively). The proportion of fluorescently labelled EV varied significantly between follicular and luteal phases across live (28.9 ± 1.9% vs 19.3 ± 2.8%, respectively) and slaughtered cows (26.5 ± 6.3% vs 27.3 ± 2 .7%, respectively). In total, 41 EV proteins were differentially expressed between the phases. Some of the proteins were involved in reproductive processes, cell adhesion and proliferation, and cellular metabolic processes. The results indicated differences in bovine UF-EV concentration and protein profile at follicular and luteal phases, which would suggest that EV modulate uterine microenvironment across the oestrous cycle. Further research is needed to understand the effect of EV changes throughout the oestrous cycle.

Hippocampus-based behavioral, structural, and molecular dynamics across the estrous cycle.

The activity of neurons in the rodent hippocampus contributes to diverse behaviors, with the activity of ventral hippocampal neurons affecting behaviors related to anxiety and emotion regulation, and the activity of dorsal hippocampal neurons affecting performance in learning- and memory-related tasks. Hippocampal cells also express receptors for ovarian hormones, estrogen and progesterone, and are therefore affected by physiological fluctuations of those hormones that occur over the rodent estrous cycle. In this review, we discuss the effects of cycling ovarian hormones on hippocampal physiology. Starting with behavior, we explore the role of the estrous cycle in regulating hippocampus-dependent behaviors. We go on to detail the cellular mechanisms through which cycling estrogen and progesterone, through changes in the structural and functional properties of hippocampal neurons, may be eliciting these changes in behavior. Then, providing a basis for these cellular changes, we outline the epigenetic, chromatin regulatory mechanisms through which ovarian hormones, by binding to their receptors, can affect the regulation of behavior- and synaptic plasticity-related genes in hippocampal neurons. We also highlight an unconventional role that chromatin dynamics may have in regulating neuronal function across the estrous cycle, including in sex hormone-driven X chromosome plasticity and hormonally-induced epigenetic priming. Finally, we discuss directions for future studies and the translational value of the rodent estrous cycle for understanding the effects of the human menstrual cycle on hippocampal physiology and brain disease risk.

Expression of phoenixin-14 and nesfatin-1 in the hypothalamo-pituitary-gonadal axis in the phases of the estrous cycle.

Phoenixin-14 (PNX-14) and nucleobindin 2 (NUCB2)/nesfatin-1 are regulatory neuropeptides expressed in the hypothalamus. These neuropeptides can be effective in hormonal regulation of the hypothalamo-pituitary-gonadal (HPG) axis and reproductive functions. In the present study, the distribution of PNX-14 and NUCB2/nesfatin-1 in the hypothalamus, pituitary, ovary, and uterus tissues during the phases of the estrous cycle in female rats was investigated. Eighteen Wistar Albino rats determined among animals showing regular estrous cycle by vaginal smear method were divided into three groups: proestrus (Group I), estrus (Group II) and diestrus (Group III). Serum gonadotropin-releasing hormone (GnRH), plasma PNX-14, and NUCB2/nesfatin-1 concentrations were the highest, moderate, and lowest in estrus, diestrus, and proestrus phases, respectively. PNX-14 immunoreactivity in the supraoptic and arcuate nuclei of the hypothalamus and NUCB2/nesfatin-1 immunoreactivity in the paraventricular nuclei were particularly evident in the estrus phase. These neuropeptide immunoreactivities were decreased in different cells of anterior pituitary during proestrus compared with those during estrus and diestrus. PNX-14 immunoreactivity in the ovary, especially during the estrus phase, was diffuse and intense in the granulosa and luteal cells and oocytes, and it was few and weak in theca cells. In addition, NUCB2/nesfatin-1 immunoreactivity was abundant and strong in granulosa and luteal cells, theca and interstitial cells, and oocytes during estrus. In the estrus phase, PNX-14 immunoreactivity was strong in the glandular epithelial cells and stromal cells of the endometrium, also NUCB2/nesfatin-1 immunoreactivity was strong in the epithelial and glandular epithelial cells. As a result, when the estrous cycle was evaluated, it was concluded that the changes in the distribution of PNX-14 and NUCB2/nesfatin-1 at all phases were related to GnRH and that these neuropeptides showed the highest immunoreactivity especially in the HPG axis and uterus tissues of estrus rats.

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.

Why the estrous cycle matters for neuroscience.

BACKGROUND: Ovarian hormone fluctuations over the rodent estrous cycle and the human menstrual cycle are known to significantly impact brain physiology and disease risk, yet this variable is largely ignored in preclinical neuroscience research, clinical studies, and psychiatric practice. METHODS: To assess the importance of the estrous cycle information for the analysis of sex differences in neuroscience research, we re-analyzed our previously published data with or without the estrous cycle information, giving a side-by-side comparison of the analyses of behavior, brain structure, gene expression, and 3D genome organization in female and male mice. We also examined and compared the variance of female and male groups across all neurobehavioral measures. RESULTS: We show that accounting for the estrous cycle significantly increases the resolution of the neuroscience studies and allows for: (a) identification of masked sex differences; (b) mechanistic insight(s) into the identified sex differences, across different neurobehavioral outcomes, from behavior to molecular phenotypes. We confirm previous findings that female data from either mixed- or staged-female groups are, on average, not more variable than that of males. However, we show that female variability is not, at all, predictive of whether the estrous cycle plays an important role in regulating the outcome of interest. CONCLUSIONS: We argue that "bringing back" the estrous cycle variable to the main stage is important in order to enhance the resolution and quality of the data, to advance the health of women and other menstruators, and to make research more gender-inclusive. We strongly encourage the neuroscience community to incorporate the estrous cycle information in their study design and data analysis, whenever possible, and we debunk some myths that tend to de-emphasize the importance and discourage the inclusion of this critically important biological variable. Highlights Ovarian hormone fluctuation impacts brain physiology and is a major psychiatric risk factor, yet this variable has been overlooked in neuroscience research and psychiatric practice. From rodent behavior to gene regulation, accounting for the estrous cycle increases the resolution of the neuroscience data, allowing identification and mechanistic insight(s) into sex differences. Female variability does not equal (and is not predictive of) the estrous cycle effect and should not be used as a proxy for the effects of ovarian hormones on the outcome of interest. Neuroscience researchers are advised to incorporate the estrous cycle information in their studies to foster more equitable, female- and gender-inclusive research. Studies of the ovarian cycle are especially important for improving women's mental health.

Automated classification of estrous stage in rodents using deep learning.

The rodent estrous cycle modulates a range of biological functions, from gene expression to behavior. The cycle is typically divided into four stages, each characterized by distinct hormone concentration profiles. Given the difficulty of repeatedly sampling plasma steroid hormones from rodents, the primary method for classifying estrous stage is by identifying vaginal epithelial cell types. However, manual classification of epithelial cell samples is time-intensive and variable, even amongst expert investigators. Here, we use a deep learning approach to achieve classification accuracy at expert level. Due to the heterogeneity and breadth of our input dataset, our deep learning approach ("EstrousNet") is highly generalizable across rodent species, stains, and subjects. The EstrousNet algorithm exploits the temporal dimension of the hormonal cycle by fitting classifications to an archetypal cycle, highlighting possible misclassifications and flagging anestrus phases (e.g., pseudopregnancy). EstrousNet allows for rapid estrous cycle staging, improving the ability of investigators to consider endocrine state in their rodent studies.

Sex- and estrous-cycle dependent dorsal hippocampal phosphoproteomic changes induced by low-dose ketamine.

Numerous emotional and cognitive processes mediated by the hippocampus present differences between sexes and can be markedly influenced by hormonal status in males and females of several species. In rodents, the dorsal hippocampus (dHPC) is known to contribute to the rapid antidepressant actions of the NMDA receptor antagonist ketamine. We and others have demonstrated a greater sensitivity to the fast-acting antidepressant ketamine in female versus male rats that is estrogen- and progesterone-dependent. However, the underlying mechanisms remain unclear. Using an acute low dose (2.5 mg/kg) of ketamine that is behaviorally effective in female but not male rats, a label-free phosphoproteomics approach was employed to identify ketamine-induced changes in signaling pathway activation and phosphoprotein abundance within the dHPC of intact adult male rats and female rats in either diestrus or proestrus. At baseline, males and females showed striking dissimilarities in the dHPC proteome and phosphoproteome related to synaptic signaling and mitochondrial function-differences also strongly influenced by cycle stage in female rats. Notably, phosphoproteins enriched in PKA signaling emerged as being both significantly sex-dependent at baseline and also the primary target of ketamine-induced protein phosphorylation selectively in female rats, regardless of cycle stage. Reduced phosphoprotein abundance within this pathway was observed in males, suggesting bi-directional effects of low-dose ketamine between sexes. These findings present biological sex and hormonal milieu as critical modulators of ketamine's rapid actions within this brain region and provide greater insight into potential translational and post-translational processes underlying sex- and hormone-dependent modulation of ketamine's therapeutic effects.

Differential Size Distribution and Estrogen Receptor Cargo of Oviductal Extracellular Vesicles at Various Stages of Estrous Cycle in Mice.

Oviductal extracellular vesicles (OEVs) play an important role in fertilization and embryo development. However, it remains largely unknown whether the size and protein cargo of OEVs change during the estrous cycle in mice. This study analyzed the changes in the size distribution and protein cargo of OEVs at four stages of the estrous cycle in mice. The distribution widths of OEVs according to the estrous cycle stage were as follows: proestrus, 20-690 nm in diameter, with two peaks at 50 and 250 nm; estrus, 22-420 nm in diameter, with two peaks at 40 and 200 nm; metestrus, 30-70 nm diameter, with a single peak at 40 nm; and diestrus, 10-26 nm diameter, with a single peak at 20 nm. The estrogen receptor (ER) level in OEVs at the proestrus stage differed significantly from that at estrus (P = 0.013) and diestrus (P = 0.005). The levels of CD9 and Hsc70 fluctuated across the four stages, although with no significant differences. Furthermore, OEVs were observed among the cilia and microvilli of epithelial cells at the proestrus, estrus, and diestrus stages, but not at the metestrus stage. The number of observed OEVs was the highest at the proestrus stage, followed by the estrus, and the diestrus stage. Endosomes were also observed at the estrus and diestrus stages. The change of the OEV size and ER cargo is associated with the estrous cycle in mice. Our findings increase the understanding of the physiological characteristics of OEVs, which may have clinical applications.

Sex, estrous cycle, and hormone regulation of CYP2D in the brain alters oxycodone metabolism and analgesia.

Opioids, and numerous centrally active drugs, are metabolized by cytochrome P450 2D (CYP2D). There are sex and estrous cycle differences in brain oxycodone analgesia. Here we investigated the mechanism examining the selective role of CYP2D in the brain on sex, estrous cycle, and hormonal regulation. Propranolol, CYP2D-specific mechanism-based inhibitor, or vehicle was delivered into cerebral ventricles 24 h before administering oxycodone (or oxymorphone, negative control) orally to male and female (in estrus and diestrus) rats. Ovariectomized and sham-operated females received no treatment, estradiol, progesterone or vehicle. Analgesia was measured using tail-flick latency, and brain drug and metabolite concentrations were measured by microdialysis. Data were analyzed by two-way or mixed ANOVA. Following propranolol (versus vehicle) inhibition and oral oxycodone, there were greater increases in brain oxycodone concentrations and analgesia, and greater decreases in brain oxymorphone/oxycodone ratios (an in vivo phenotype of CYP2D in brain) in males and females in estrus, compared to females in diestrus; with no impact on plasma drug concentrations. There was no impact of propranolol pre-treatment, sex, or cycle after oral oxymorphone (non-CYP2D substrate) on brain oxymorphone concentrations or analgesia. There was no impact of propranolol pre-treatment following ovariectomy on brain oxycodone concentrations or analgesia, which was restored in ovariectomized females following estradiol, but not progesterone, treatment. Sex, cycle, and estradiol regulation of CYP2D in brain in turn altered brain oxycodone concentration and response, which may contribute to the large inter-individual variation in response to the numerous centrally acting CYP2D substrate drugs, including opioids.

Influence of genistein and diadizine on regularity of estrous cycle in cyclic female Wistar rat: interaction with estradiol receptors and vascular endothelial growth factor.

Background: Isoflavones are estrogenic compounds that exist in soy, clover, and peanuts. They are selective estrogen receptor modulators. Aim: The study was planned to explain the interactions of isoflavones with estrogen receptors alpha (ERα), beta (ERß), and vascular endothelial growth factor (VEGF) expressions in ovarian and uterine tissues during different stages of the estrous cycle of regular cyclic female Wistar rats. Methods: Thirty-two regular cyclic females were divided equally into control group: fed casein-based diet and isoflavones group: fed casein-based diet and gavaged 50 mg/kg/day soy isoflavones extract 40%. The regularity of estrus cycles was monitored. Final body weight (FBW), weight gain (BWG), and ovarian and uterine weights were estimated. Histopathology and immunohistochemistry for ERα, Erß, and VEGF in ovarian and uterine tissues were performed. Results: All females (100%, n = 16) in control group showed regularity in estrous cycle compared to 62.5% (n = 10) in isoflavones group. Estrus and diestrus phases revealed prolongation and shortening in isoflavones rats than control, respectively. Nonsignificant variation was noted in the duration of the whole cycle of both groups. FBW and BWG significantly decreased however, ovarian and uterine weights increased significantly in all estrous phases of isoflavones group than control. Histopathology demonstrated an increase in number of follicles/ovaries besides, hyperplasia and proliferation of luminal epithelium with hydropic degeneration in the isoflavones group. Also, uterine connective tissue stroma showed edema in the isoflavones group during all estrous phases. Immunostaining percentages of ERα, Erß, and VEGF protein expression were significantly elevated in the isoflavones group during all estrous phases. Conclusion: Isoflavones induced irregularity of the estrous cycle that was encountered by increased and altered ERα, Erß, and VEGF expressions in ovarian and uterine tissues.

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.

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.

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.

Sex and Estrous Cycle Differences in Analgesia and Brain Oxycodone Levels.

Sex differences in opioid analgesia occur in rodents and humans, and could be due to differences in drug and metabolite levels. Thus, we investigated the sex and cycle differences in analgesia (nociception) from oxycodone in rats and related these to sex and cycle differences in brain and plasma oxycodone and metabolite levels. Since numerous opioids are CYP2D enzyme substrates and variation in CYP2D alters opioid drug levels and response, we also initiated studies to see if the sex and cycle differences observed might be due to differences in brain CYP2D activity. Across oxycodone doses, females in diestrus had higher analgesia (using tail flick latency) compared to males and females in estrus; we also demonstrated a direct effect of estrous cycle on analgesia within females. Consistent with the analgesia, females in diestrus had highest brain oxycodone levels (assessed using microdialysis) compared to males and females in estrus. Analgesia correlated with brain oxycodone, but not brain oxymorphone or noroxycodone levels, or plasma drug or metabolite levels. Propranolol (a CYP2D mechanism-based inhibitor), versus vehicle pre-treatments, increased brain oxycodone, and decreased brain oxymorphone/oxycodone drug level ratios (an in vivo CYP2D activity phenotype in the brain) in males and females in estrus, but not in females in diestrus. Brain oxymorphone/oxycodone inversely correlated with analgesia. Together, both sex and estrous cycle impact oxycodone analgesia and brain oxycodone levels, likely through regulation of brain CYP2D oxycodone metabolism. As CYP2D6 is expressed in human brain, perhaps similar sex and cycle influences also occur in humans.