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.

Ovarian PERK/NRF2/CX43/StAR/progesterone pathway activation mediates female reproductive dysfunction induced by cold exposure.

Ambient air temperature is a key factor affecting human health. Female reproductive disorders are representative health risk events under low temperature. However, the mechanism involving in cold-induced female reproductive disorders remains largely unknown. Female mice were intermittently exposed to cold conditions (4 °C) to address the health risk of low temperature on female reproductive system. Primary granulosa cells (GCs) were prepared and cultured under low temperature (35 °C) or exposed to ß3-adrenoreceptor agonist, isoproterenol, to mimic the condition of cold exposure. Western-blot, RT-PCR, co-IP, ELISA, pharmacological inhibition or siRNA-mediated knockdown of target gene were performed to investigate the possible role of hormones, gap conjunction proteins, and ER stress sensor protein in regulating female reproductive disorders under cold exposure. Cold exposure induced estrous cycle disorder and follicular dysplasia in female mice, accompanying with abnormal upregulation of progesterone and its synthetic rate-limiting enzyme, StAR, in the ovarian granulosa cells. Under the same conditions, an increase in connexin 43 (CX43) expressions in the GCs was also observed, which contributed to elevated progesterone levels in the ovary. Moreover, ER stress sensor protein, PERK, was activated in the ovarian GCs after cold exposure, leading to the upregulation of downstream NRF2-dependent CX43 transcription and aberrant increase in progesterone synthesis. Most importantly, blocking PERK expression in vivo significantly inhibited NRF2/CX43/StAR/progesterone pathway activation in the ovary and efficiently rescued the prolongation of estrous cycle and the increase in follicular atresia of the female mice induced by cold stress. We have elucidated the mechanism of ovarian PERK/NRF2/CX43/StAR/progesterone pathway activation in mediating female reproductive disorder under cold exposure. Targeting PERK might be helpful for maintaining female reproductive health under cold conditions.

[Individual hormonal profiles of blood progesterone and estradiol-17ß during the course of a reproductive cycle in mares]. / Individueller Konzentrationsverlauf von Progesteron und Östradiol-17ß im Blut bei Stuten während des Zyklus.

OBJECT AND AIM: This study presents the individual course of estradiol-17ß and progesterone concentrations in blood during the reproductive cycle in mares in order to point out physiological differences between individual animals and to aid in the interpretation of hormone values. MATERIAL AND METHODS: Concentrations of estradiol-17ß and progesterone were determined in seven mares over the course of their cycle. One mare was excluded from the study due to a physiologically deviating cycle. In addition, the mares' ovaries were examined via ultrasound on a daily basis in order to match the hormone values to morphological changes of the ovaries. RESULTS: In some cases, the mares showed considerable individual differences in their hormone concentrations, which also differed from the published comparative values in the literature. For example, two mares showed progesterone levels above basal levels at the time of ovulation. The postovulatory progesterone concentrations of the mares are characterized by marked fluctuations, which makes it difficult to provide reference values in the different sections of the corpus luteum phase. The length of the plateau phases averaged 12.3±1.5 days. The mare with double ovulation showed the highest progesterone concentrations. CONCLUSION: The measurement of plasma progesterone levels in mares should be interpreted only in the context of other test results. The very wide variation in estradiol-17ß concentrations makes it questionable whether the determination of this hormone value is of diagnostic value. CLINICAL RELEVANCE: When interpreting steroid hormone values in the ingravid cycle of a mare, the individual concentration courses must be taken into consideration, as they may deviate significantly from the published reference values.

Consistent and effective method to define the mouse estrous cycle stage by a deep learning-based model.

The mouse estrous cycle is divided into four stages: proestrus (P), estrus (E), metestrus (M), and diestrus (D). The estrous cycle affects reproductive hormone levels in a wide variety of tissues. Therefore, to obtain reliable results from female mice, it is important to know the estrous cycle stage during sampling. The stage can be analyzed from a vaginal smear under a microscope. However, it is time-consuming, and the results vary between evaluators. Here, we present an accurate and reproducible method for staging the mouse estrous cycle in digital whole-slide images (WSIs) of vaginal smears. We developed a model using a deep convolutional neural network (CNN) in a cloud-based platform, Aiforia Create. The CNN was trained by supervised pixel-level multiclass semantic segmentation of image features from 171 hematoxylin-stained samples. The model was validated by comparing the results obtained by CNN with those of four independent researchers. The validation data included three separate studies comprising altogether 148 slides. The total agreement attested by the Fleiss kappa value between the validators and the CNN was excellent (0.75), and when D, E, and P were analyzed separately, the kappa values were 0.89, 0.79, and 0.74, respectively. The M stage is short and not well defined by the researchers. Thus, identification of the M stage by the CNN was challenging due to the lack of proper ground truth, and the kappa value was 0.26. We conclude that our model is reliable and effective for classifying the estrous cycle stages in female mice.

Transcriptome analysis during 4-vinylcyclohexene diepoxide exposure-induced premature ovarian insufficiency in mice.

The occupational chemical 4-Vinylcyclohexene diepoxide (VCD) is a reproductively toxic environmental pollutant that causes follicular failure, leading to premature ovarian insufficiency (POI), which significantly impacts a woman's physical health and fertility. Investigating VCD's pathogenic mechanisms can offer insights for the prevention of ovarian impairment and the treatment of POI. This study established a mouse model of POI through intraperitoneal injection of VCD into female C57BL/6 mice for 15 days. The results were then compared with those of the control group, including a comparison of phenotypic characteristics and transcriptome differences, at two time points: day 15 and day 30. Through a comprehensive analysis of differentially expressed genes (DEGs), key genes were identified and validated some using RT-PCR. The results revealed significant impacts on sex hormone levels, follicle number, and the estrous cycle in VCD-induced POI mice on both day 15 and day 30. The DEGs and enrichment results obtained on day 15 were not as significant as those obtained on day 30. The results of this study provide a preliminary indication that steroid hormone synthesis, DNA damage repair, and impaired oocyte mitosis are pivotal in VCD-mediated ovarian dysfunction. This dysfunction may have been caused by VCD damage to the primordial follicular pool, impairing follicular development and aggravating ovarian damage over time, making it gradually difficult for the ovaries to perform their normal functions.

Taurine dynamics in serum during the oestrous cycle in buffaloes.

Estrus identification is one of the common issues in buffaloes because of their short estrus duration and silent estrus problem. Hence, specific biomarkers facilitating in identifying the estrus stage would be helpful to buffalo farmers and researchers. In our previous studies, taurine, a non-protein amino acid that helps in the secretion of reproductive hormones such as GnRH, was found to be associated with postpartum anestrus in buffaloes. Therefore, the present study was conducted to explore the level of taurine in serum during different stages of the oestrous cycle in healthy cyclic buffaloes. Blood samples were collected from healthy cyclic buffaloes (n = 4), and taurine was estimated at the estrus (0th day), proestrus (-2nd day), metestrus (3rd day) and diestrus (+10th day) stages using TLC method. The days of the oestrous cycle were determined by ultrasonography and observation of behavioural signs by trained professionals. The results revealed that taurine was consistently present in the serum. However, the highest concentration of taurine was observed at the proestrus (0.20 ± 0.03 mg/mL) stage, which was greater (p < .05) than metestrus (0.10 ± 0.05 mg/mL) and diestrus (0.13 ± 0.03 mg/mL) stages, but comparable with the estrus stage. These results were also validated in the simulated population datasets of population size 6 to 10,000. Further, ROC curve analysis for the large simulated population indicated the efficiency of taurine to distinguish proestrus from metestrus and diestrus stages at a lower cutoff value of <0.1643 mg/mL with 60% sensitivity and specificity. Therefore, the present study concludes that serum taurine concentration could help in detecting proestrus stage of buffalo estrous cycle.

Dehydroepiandrosterone with a high-fat diet treatment at inducing polycystic ovary syndrome in rat model.

OBJECTIVE: This study aimed to evaluate the effects of dehydroepiandrosterone (DHEA) and DHEA combined with a high-fat diet (HFD) treatment of reproductive and endocrine metabolism in rats and then identify an ideal model of polycystic ovary syndrome (PCOS). METHODS: Three-week-old female Sprague-Dawley rats were injected subcutaneously with DHEA or oil, fed with or without a HFD, for 21 days, during which body weight, feed intake, and estrous cycle monitoring were carried out. Fasting blood glucose was measured, and serum fasting insulin, testosterone, dihydrotestosterone (DHT), estradiol, progesterone, luteinizing hormone (LH), anti-Müllerian hormone (AMH), and follicle-stimulating hormone (FSH) were estimated by ELISA. Serum total cholesterol (TC), total triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured by colorimetric assay. Whereas, histologic changes in rat ovaries were evaluated by H&E staining. Ovarian steroid hormone synthases and their protein levels (StAR, 3ß-HSD2, 17ß-HSD1, CYP11A1, CYP17A1, and CYP19A1) were examined by Western blotting. RESULTS: Both DHEA and DHEA + HFD-treated rats lost a regular estrous cycle; had polycystic ovarian changes, significantly higher serum fasting insulin and testosterone levels; and increased ovarian StAR, 3ß-HSD2, and CYP11A1 protein levels. Additionally, rats in the DHEA + HFD-treated group were obese; had elevated fasting blood glucose, TG, DHT, AMH levels and LH:FSH ratios; increased ovarian 17ß-HSD1 protein levels. CONCLUSION: DHEA combined with HFD treatment is more effective at inducing PCOS than DHEA alone. The reproductive and endocrine metabolic aspects of this method are more consistent with the clinical characteristics of PCOS patients.

Sleep and local field potential effect of the D2 receptor agonist bromocriptine during the estrus cycle and postpartum period in female rats.

BACKGROUND: Pituitary lactotrophs are under tonic dopaminergic inhibitory control and bromocriptine treatment blocks prolactin secretion. METHODS: Sleep and local field potential were addressed for 72 h after bromocriptine treatments applied during the different stages of the estrus cycle and for 24 h in the early- and middle postpartum period characterized by spontaneously different dynamics of prolactin release in female rats. RESULTS: Sleep changes showed strong dependency on the estrus cycle phase of the drug application. Strongest increase of wakefulness and reduction of slow wave sleep- and rapid eye movements sleep appeared during diestrus-proestrus and middle postpartum treatments. Stronger sleep-wake effects appeared in the dark phase in case of the estrus cycle treatments, but in the light phase in postpartum treatments. Slow wave sleep and REM sleep loss in case of estrus cycle treatments was not compensated at all and sleep loss seen in the first day post-injection was gained further later. In opposition, slow wave sleep loss in the light phase after bromocriptine injections showed compensation in the postpartum period treatments. Bromocriptine treatments resulted in a depression of local field potential delta power during slow wave sleep while an enhancement in beta and gamma power during wakefulness regardless of the treatment timing. CONCLUSIONS: These results can be explained by the interplay of dopamine D2 receptor agonism, lack of prolactin release and the spontaneous homeostatic sleep drive being altered in the different stages of the estrus cycle and the postpartum period.

Characterizing the relationship between gonadotropin releasing hormone (GnRH), kisspeptin, and RFamide related peptide 3 (RFRP-3) neurons in the equine hypothalamus across the estrous cycle and in the anovulatory seasons.

To understand better the role that kisspeptin plays in regulating seasonal and estrous cycle changes in the mare, this study investigated the number, location and interactions between GnRH, kisspeptin and RFRP-3 neurons in the equine hypothalamus. Hypothalami were collected from mares during the non-breeding season, vernal transition and various stages of the breeding season. Fluorescent immunohistochemistry was used to label the neuropeptides of interest. GnRH cells were observed primarily in the arcuate nucleus (ARC), while very few labeled cells were identified in the pre-optic area (POA). Kisspeptin cells were identified primarily in the ARC, with a small number of cells observed dorsal to the ARC, surrounding the third ventricle (3V). The mean number of kisspeptin cells varied between animals and typically showed no pattern associated with season or stage of estrous cycle, but a seasonal difference was identified in the ARC population. Small numbers of RFRP-3 cells were observed in the ARC, ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH). The mean number of RFRP-3 cells appeared higher in pre-ovulatory animals compared to all other stages. The percentage of GnRH cell bodies with kisspeptin appositions did not change with season or stage of estrous cycle. The percentage of kisspeptin cells receiving inputs from RFRP-3 fibers did not vary with season or stage of estrous cycle. These interactions suggest the possibility of the presence of an ultra-short loop feedback system between these three peptides. The changes in RFRP-3 neurons suggest the possibility of a role in the regulation of reproduction in the horse, but it is unlikely to be as a gonadotropin inhibitory factor.

Histological, immunohistochemical and serological investigations of the ovary during follicular phase of estrous cycle in Saidi sheep.

BACKGROUND: Saidi sheep are the most abundant ruminant livestock species in Upper Egypt, especially in the Assiut governorate. Sheep are one of the most abundant animals raised for food in Egypt. They can convert low-quality roughages into meat and milk in addition to producing fiber and hides therefore; great opportunity exists to enhance their reproduction. Saidi breed is poorly known in terms of reproduction. So this work was done to give more information on some hormonal, oxidative, and blood metabolites parameters in addition to histological, histochemical and immunohistochemical investigations of the ovary during follicular phase of estrous cycle. The present study was conducted on 25 healthy Saidi ewes for serum analysis and 10 healthy ewes for histological assessment aged 2 to 5 years and weighted (38.5 ± 2.03 kg). RESULTS: The follicular phase of estrous cycle in Saidi sheep was characterized by the presence of ovarian follicles in different stages of development and atresia in addition to regressed corpus luteum. Interestingly, apoptosis and tissue oxidative markers play a crucial role in follicular and corpus luteum regression. The most prominent features of the follicular phase were the presence of mature antral (Graafian) and preovulatory follicles as well as increased level of some blood metabolites and oxidative markers. Here we give a new schematic sequence of ovarian follicles in Saidi sheep and describing the features of different types. We also clarified that these histological pictures of the ovary was influenced by hormonal, oxidative and blood metabolites factors that characterizes the follicular phase of estrous cycle in Saidi sheep. CONCLUSION: This work helps to understanding the reproduction in Saidi sheep which assist in improving the reproductive outcome of this breed of sheep. These findings are increasingly important for implementation of a genetic improvement program and utilizing the advanced reproductive techniques as estrous synchronization, artificial insemination and embryo transfer.

Changes in fecal microbiota during estrous cycle in healthy thoroughbred mares.

Gut microbiota plays a crucial role in various physiological processes, including the regulation of the reproductive system and steroid sex hormones. Throughout the normal estrous cycle of healthy mares, the levels of estradiol-17ß (E2) and progesterone (P4) in the blood exhibit periodic changes. To investigate the relationship between cyclic changes in steroid sex hormones and the gut microbiome of mares, we analyzed the fecal microbiota composition in healthy mares during the typical estrous cycle. Blood and fecal samples from five healthy mares were collected, E2 and P4 levels in serum were analyzed using radioimmunoassay (RIA), and the gut microbiome was analyzed by 16S rRNA sequencing. The overall richness and composition of the gut microbiota remained relatively stable during the normal estrous cycle in mares. The Linear Discriminant Analysis Effect Size analysis of the microbial composition during the follicular and luteal phases identified the Rhodococcus genus as differentially abundant. These findings indicate that the mare's gut microbiota's significant composition remains consistent throughout the estrous cycle. At the same time, specific low-abundance pathogenic bacteria exhibit changes that align with sexual hormonal fluctuations.

Repeated exposure to physiologically effective doses of contraceptive hormones ethinyl estradiol or levonorgestrel do not alter the reinforcing effects of a brief visual stimulus in ovary-intact rats.

Estradiol and progesterone potentiate and attenuate reward processes, respectively. Despite these well-characterized effects, there is minimal research on the effects of synthetic estrogens (e.g., ethinyl estradiol, or EE) and progestins (e.g., levonorgestrel, or LEVO) contained in clinically-utilized hormonal contraceptives. The present study characterized the separate effects of repeated exposure to EE or LEVO on responding maintained by a reinforcing visual stimulus. Forty ovary-intact female Sprague-Dawley rats received either sesame oil vehicle (n = 16), 0.18 µg/day EE (n = 16), or 0.6 µg/day LEVO (n = 8) subcutaneous injections 30-min before daily one-hour sessions. Rats' responding was maintained by a 30-sec visual stimulus on a Variable Ratio-3 schedule of reinforcement. The day after rats' last session, we determined rats estrous cycle phase via vaginal cytology before sacrifice and subsequently weighing each rat's uterus to further verify the contraceptive hormone manipulation. The visual stimulus functioned as a reinforcer, but neither EE nor LEVO enhanced visual stimulus maintained responding. Estrous cytology was consistent with normal cycling in vehicle rats and halting of normal cycling in EE and LEVO rats. EE increased uterine weights consistent with typical uterotrophic effects observed with estrogens, further confirming the physiological impacts of our EE and LEVO doses. In conclusion, a physiologically effective dose of neither EE nor LEVO did not alter the reinforcing efficacy of a visual stimulus reinforcer. Future research should characterize the effects of hormonal contraceptives on responding maintained by other reinforcer types to determine the generality of the present findings.

Modulatory effects of estrous cycle on ingestive behaviors and energy balance in young adult C57BL/6J mice maintained on a phytoestrogen-free diet.

The estrous cycle is known to modify food, fluid, and electrolyte intake behaviors and energy homeostasis in various species, in part through fluctuations in estrogen levels. Simultaneously, commonly commercially available rodent dietary formulations greatly vary in soy protein content, and thereby the delivery of biologically active phytoestrogens. To explore the interactions among the estrous cycle, sodium, fluid, and caloric seeking behaviors, and energy homeostasis, young adult C57BL/6J female mice were maintained on a soy protein-free 2920x diet and provided water, or a choice between water and 0.15 mol/L NaCl drink solution. Comprehensive metabolic phenotyping was performed using a multiplexed Promethion (Sable Systems International) system, and estrous stages were determined via daily vaginal cytology. When provided food and water, estrous cycling had no major modulatory effects on intake behaviors or energy balance. When provided a saline solution drink choice, significant modulatory effects of the transition from diestrus to proestrus were observed upon fluid intake patterning, locomotion, and total energy expenditure. Access to saline increased total daily sodium consumption and aspects of energy expenditure, but these effects were not modified by the estrous stage. Collectively, these results indicate that when supplied a phytoestrogen-free diet, the estrous cycle has minor modulatory effects on ingestive behaviors and energy balance in C57BL/6J mice that are sensitive to sodium supply.NEW & NOTEWORTHY When provided a phytoestrogen-free diet, the estrous cycle had very little effect on food and water intake, physical activity, or energy expenditure in C57BL/6J mice. In contrast, when provided an NaCl drink in addition to food and water, the estrous cycle was associated with changes in intake behaviors and energy expenditure. These findings highlight the complex interactions among estrous cycling, dietary formulation, and nutrient presentation upon ingestive behaviors and energy homeostasis in mice.

Postweaning Social Isolation Alters Puberty Onset by Suppressing Electrical Activity of Arcuate Kisspeptin Neurons.

INTRODUCTION: Postweaning social isolation (PWSI) in rodents is an advanced psychosocial stress model in early life. Some psychosocial stress, such as restrain and isolation, disrupts reproductive physiology in young and adult periods. Mechanisms of early-life stress effects on central regulation of reproduction need to be elucidated. We have investigated the effects of PWSI on function of arcuate kisspeptin (ARCKISS1) neurons by using electrophysiological techniques combining with monitoring of puberty onset and estrous cycle in male and female Kiss1-Cre mice. METHODS: Female mice were monitored for puberty onset with vaginal opening examination during social isolation. After isolation, the estrous cycle of female mice was monitored with vaginal cytology. Anxiety-like behavior of mice was determined by an elevated plus maze test. Effects of PWSI on electrophysiology of ARCKISS1 neurons were investigated by the patch clamp method after intracranial injection of AAV-GFP virus into arcuate nucleus of Kiss1-Cre mice after the isolation period. RESULTS: We found that both male and female isolated mice showed anxiety-like behavior. PWSI caused delay in vaginal opening and extension in estrous cycle length. Spontaneous-firing rates of ARCKISS1 neurons were significantly lower in the isolated male and female mice. The peak amplitude of inhibitory postsynaptic currents to ARCKISS1 neurons was higher in the isolated mice, while frequency of excitatory postsynaptic currents was higher in group-housed mice. CONCLUSION: These findings demonstrate that PWSI alters pre- and postpubertal reproductive physiology through metabolic and electrophysiological pathways.

Long-term Recordings of Arcuate Nucleus Kisspeptin Neurons Across the Mouse Estrous Cycle.

The arcuate nucleus kisspeptin (ARNKISS) neurons represent the GnRH pulse generator that likely drives pulsatile gonadotropin secretion in all mammals. Using an improved GCaMP fiber photometry system enabling long-term continuous recordings, we aimed to establish a definitive profile of ARNKISS neuronal activity across the murine estrous cycle. As noted previously, a substantial reduction in the frequency of ARNKISS neuron synchronization events (SEs) occurs on late proestrus and extends into estrus. The SE amplitude remains constant throughout the cycle. During metestrus, we unexpectedly detected many multipeak SEs where many SEs occurred rapidly, within 160 seconds of each other. By applying a machine learning-based, k-means clustering analysis, we were further able to detect substantial within-stage variability in the patterns of pulse generator activity. Estrous cycle-dependent changes in SE activity occurred around the time of lights on and off. We also find that a mild stressor such as vaginal lavage reduces ARNKISS neuron SE frequency for up to 3 hours. These observations provide a comprehensive account of ARNKISS neuron activity across the estrous cycle, highlight a new pattern of multipeak SE activity, and introduce a new k-means clustering approach for analyzing ARNKISS neuron population behavior.

REPRODUCTIVE CYCLE STAGE ASSESSMENT IN THE TWO-TOED SLOTH (CHOLOEPUS HOFFMANNI), PRELIMINARY RESULTS.

The number of two-toed sloths (Choloepus hoffmanni) has significantly decreased in the last years. Deepening the knowledge of this tropical mammal's reproductive physiology is essential to improve captive breeding within conservation programs for this species. However, several aspects of its reproductive biology remain unexplored and have not been described sufficiently. The aim of this work was to describe the estrous cycle and reproductive physiology of two adult female C. hoffmanni by vaginal cytology, appearance of the external genitalia, and behavior. Vaginal cytology assay showed that the average duration of the estrous cycle was 15.1 ± 4.53 d. Positive correlations (P < 0.05) were found between the peak presence of superficial cells (estrous phase) and four parameters: aggressive behavior, pursuing behavior, vulvar swelling, and vaginal discharge. This pilot study, conducted on just two animals, forms a basis for a study design that may be employed for a more comprehensive assessment of the two-toed sloth reproductive physiology and behavior.

Microbial, proteomic, and metabolomic profiling of the estrous cycle in wild house mice.

Symbiotic microbial communities affect the host immune system and produce molecules contributing to the odor of an individual. In many mammalian species, saliva and vaginal fluids are important sources of chemical signals that originate from bacterial metabolism and may act as honest signals of health and reproductive status. In this study, we aimed to define oral and vaginal microbiomes and their dynamics throughout the estrous cycle in wild house mice. In addition, we analyzed a subset of vaginal proteomes and metabolomes to detect potential interactions with microbiomes. 16S rRNA sequencing revealed that both saliva and vagina are dominated by Firmicutes and Proteobacteria but differ at the genus level. The oral microbiome is more stable during the estrous cycle and most abundant bacteria belong to the genera Gemella and Streptococcus, while the vaginal microbiome shows higher bacterial diversity and dynamics during the reproductive cycle and is characterized by the dominance of Muribacter and Rodentibacter. These two genera cover around 50% of the bacterial community during estrus. Proteomic profiling of vaginal fluids revealed specific protein patterns associated with different estrous phases. Highly expressed proteins in estrus involve the keratinization process thus providing estrus markers (e.g., Hrnr) while some proteins are downregulated such as immune-related proteins that limit bacterial growth (Camp, Clu, Elane, Lyz2, and Ngp). The vaginal metabolome contains volatile compounds potentially involved in chemical communication, for example, ketones, aldehydes, and esters of carboxylic acids. Data integration of all three OMICs data sets revealed high correlations, thus providing evidence that microbiomes, host proteomes, and metabolomes may interact.IMPORTANCEOur data revealed dynamic changes in vaginal, but not salivary, microbiome composition during the reproductive cycle of wild mice. With multiple OMICs platforms, we provide evidence that changes in microbiota in the vaginal environment are accompanied by changes in the proteomic and metabolomics profiles of the host. This study describes the natural microbiota of wild mice and may contribute to a better understanding of microbiome-host immune system interactions during the hormonal and cellular changes in the female reproductive tract. Moreover, analysis of volatiles in the vaginal fluid shows particular substances that can be involved in chemical communication and reproductive behavior.

Effects of maternal antral follicle count in Bos taurus indicus cattle on the genetic merit of male offspring and antral follicle count of female offspring.

This study evaluated the effects of antral follicle count (AFC) in female cattle on offspring characteristics. Recently calved multiparous Bos indicus cows (Nelore; n = 222) were evaluated using ultrasonography on random days of their estrous cycle to determine the AFC and were classified into "low" (≤15 follicles), "intermediate" (≥16 and ≤ 29 follicles), and "high" (≥30 follicles) AFC groups. Weight and scrotal circumference (SC) of male offspring from these cows (n = 127) were determined from 20 to 27 months, and the data were added to a genetic evaluation program (economic total genetic merit, MGTe and TOP value) that uses the kinship matrix to evaluate the genetic relationship between animals. The AFC of female offspring from these cows (n = 95) was evaluated to analyze the relationship between the AFC of mothers and daughters. The effects of maternal AFC on the genetic merit of male and female offspring were analyzed using GLIMMIX and GLM, respectively. Correlations were assessed using the Pearson's coefficient. Male offspring of cows with high AFC had superior MGTe (P = 0.005) and TOP values (P = 0.01) than those from cows with low AFC. Additionally, the AFC of mothers was positively correlated with MGTe (R = 0.33; P < 0.0001) and negatively correlated with TOP values (R = -0.32; P < 0.0001). The SC (P = 0.01), but not body weight of the offspring (P = 0.46) was affected by maternal AFC. The daughters' AFC were correlated (R = 0.29; P = 0.004) with mothers' AFC and were influenced by maternal (P = 0.05) but not paternal (P = 0.77) effect. In conclusion, cows with high AFC produced males with greater MGTe, superior TOP values and higher SC. Maternal AFC did not influence the weight of male offspring but was correlated with the AFC of daughters.

Track-by-Day: A standardized approach to estrous cycle monitoring in biobehavioral research.

Despite known sex differences in brain function, female subjects are underrepresented in preclinical neuroscience research. This is driven in part by concerns about variability arising from estrous cycle-related hormone fluctuations, especially in fear- and anxiety-related research where there are conflicting reports as to whether and how the cycle influences behavior. The inconsistency may arise from a lack of common standards for tracking and reporting the cycle as opposed to inherent unpredictability in the cycle itself. The rat estrous cycle is conventionally tracked by assigning vaginal cytology smears to one of four qualitatively-defined stages. Although the cytology stages are of unequal length, the stage names are often, but not always, used to refer to the four cycle days. Subjective staging criteria and inconsistent use of terminology are not necessarily a problem in research on the cycle itself, but can lead to irreproducibility in neuroscience studies that treat the stages as independent grouping factors. We propose the explicit use of cycle days as independent variables, which we term Track-by-Day to differentiate it from traditional stage-based tracking, and that days be indexed to the only cytology feature that is a direct and rapid consequence of a hormonal event: a cornified cell layer formed in response to the pre-ovulatory 17ß-estradiol peak. Here we demonstrate that cycle length is robustly regular with this method, and that the method outperforms traditional staging in detecting estrous cycle effects on Pavlovian fear conditioning and on a separate proxy for hormonal changes, uterine histology.

Rat brown adipose tissue thermogenic markers are modulated by estrous cycle phases and short-term fasting.

Brown adipose tissue (BAT) converts chemical energy into heat to maintain body temperature. Although fatty acids (FAs) represent a primary substrate for uncoupling protein 1 (UCP1)-dependent thermogenesis, BAT also utilizes glucose for the same purpose. Considering that estrous cycle effects on BAT are not greatly explored, we examined those of 6-h fasting on interscapular BAT (iBAT) thermogenic markers in proestrus and diestrus. We found that the percentage of multilocular adipocytes was lower in proestrus than in diestrus, although it was increased after fasting in both analyzed estrous cycle stages. Furthermore, the percentage of paucilocular adipocytes was increased by fasting, unlike the percentage of unilocular cells, which decreased in both analyzed stages of the estrous cycle. The UCP1 amount was lower in proestrus irrespectively of the examined dietary regimens. Regarding FA transporters, it was shown that iBAT CD36 content was increased in fasted rats in diestrus. In contrast to GLUT1, the level of GLUT4 was interactively modulated by selected estrous cycle phases and fasting. There was no change in insulin receptor and ERK1/2 activation, while AKT activation was interactively modulated by fasting and estrous cycle stages. Our study showed that iBAT exhibits morphological and functional changes in proestrus and diestrus. Moreover, iBAT undergoes additional dynamic functional and morphological changes during short-term fasting to modulate nutrient utilization and adjust energy expenditure.