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.

Sleep- and sleep deprivation-related changes of vertex auditory evoked potentials during the estrus cycle in female rats.

The estrus cycle in female rodents has been shown to affect a variety of physiological functions. However, little is known about its presumably thorough effect on auditory processing during the sleep-wake cycle and sleep deprivation. Vertex auditory evoked potentials (vAEPs) were evoked by single click tone stimulation and recorded during different stages of the estrus cycle and sleep deprivation performed in metestrus and proestrus in female rats. vAEPs showed a strong sleep-dependency, with the largest amplitudes present during slow wave sleep while the smallest ones during wakefulness. Higher amplitudes and longer latencies were seen in the light phase during all vigilance stages. The largest amplitudes were found during proestrus (light phase) while the shortest latencies were seen during estrus (dark phase) compared to the 2nd day diestrus baseline. High-amplitude responses without latency changes were also seen during metestrus with increased homeostatic sleep drive. More intense and faster processing of auditory information during proestrus and estrus suggesting a more effective perception of relevant environmental cues presumably in preparation for sexual receptivity. A 4-h sleep deprivation resulted in more pronounced sleep recovery in metestrus compared to proestrus without difference in delta power replacement suggesting a better tolerance of sleep deprivation in proestrus. Sleep deprivation decreased neuronal excitability and responsiveness in a similar manner both during metestrus and proestrus, suggesting that the negative consequences of sleep deprivation on auditory processing may have a limited correlation with the estrus cycle stage.

Uterine injury during diestrus leads to placental and embryonic defects in future pregnancies in mice†.

Uterine injury from procedures such as Cesarean sections (C-sections) often have severe consequences on subsequent pregnancy outcomes, leading to disorders such as placenta previa, placenta accreta, and infertility. With rates of C-section at ~30% of deliveries in the USA and projected to continue to climb, a deeper understanding of the mechanisms by which these pregnancy disorders arise and opportunities for intervention are needed. Here we describe a rodent model of uterine injury on subsequent in utero outcomes. We observed three distinct phenotypes: increased rates of resorption and death, embryo spacing defects, and placenta accreta-like features of reduced decidua and expansion of invasive trophoblasts. We show that the appearance of embryo spacing defects depends entirely on the phase of estrous cycle at the time of injury. Using RNA-seq, we identified perturbations in the expression of components of the COX/prostaglandin pathway after recovery from injury, a pathway that has previously been demonstrated to play an important role in embryo spacing. Therefore, we demonstrate that uterine damage in this mouse model causes morphological and molecular changes that ultimately lead to placental and embryonic developmental defects.

Characterization and analysis of transcriptomes of multiple tissues from estrus and diestrus in pigs.

A comprehensive understanding of the complex regulatory mechanisms governing estrus and ovulation across multiple tissues in mammals is imperative to improve the reproductive performance of livestock and mitigate ovulation-related disorders in humans. To comprehensively elucidate the regulatory landscape, we analyzed the transcriptome of protein-coding genes and long intergenic non-coding RNAs (lincRNAs) in 58 samples (including the hypothalamus, pituitary, ovary, vagina, and vulva) derived from European Large White gilts and Chinese Mi gilts during estrus and diestrus. We constructed an intricate regulatory network encompassing 358 hub genes across the five examined tissues. Furthermore, our investigation identified 85 differentially expressed lincRNAs that are predicted to target 230 genes associated with critical functions including behavior, receptors, and apoptosis. Importantly, we found that vital components of estrus and ovulation events involve "Apoptosis" pathway in the hypothalamus, "Autophagy" in the ovary, as well as "Hypoxia" and "Angiogenesis" in the vagina and vulva. We have identified several differentially expressed transcription factors (TFs), such as SPI1 and HES2, which regulate these pathways. SPI1 may suppress transcription in the autophagy pathway, promoting apoptosis and inhibiting the proliferation of ovarian granulosa cells. Our study provides the most comprehensive transcriptional profiling information related to estrus and ovulation events.

Aged and induced-premature ovarian failure mouse models affect diestrus profile and ovarian features.

Sex hormones exert a wide influence on several systems of the human body, especially in women, who undergo intense changes in the trans and postmenopausal periods. Different experimental models are used to mimic these conditions; however, the impact on hormonal profile may be different. This study aimed to analyze and compare vaginal cytology of different post-estropausal mice models, along with their microscopical ovarian features. Forty-six C57BL/6J female mice with the ages of 4, 6 and 18 months at the beginning of the experiment, weighing about 25-28 grams, constituted five groups: NC-(negative control) animals with no treatment, OVX-SHAM-sham ovariectomized, OVX-ovariectomized, VCD-medicated with 160 mg/kg/day of 4-vinylcyclohexene diepoxide via IP for 20 consecutive days, and Aged-senescent mice under physiological estropause. Euthanasia was performed at different periods for the removal of the ovaries, and after diestrus was confirmed by vaginal cytology for 10 consecutive days. For daily vaginal cytology, morphological and histomorphometric microscopic analyzes were performed. Aged mice presented significant increased neutrophils when compared to VCD group, as well as increased cornified epithelial cells when compared to OVX mice, and also increased nucleated epithelial cells when compared to VCD and OVX. NC and OVX-SHAM ovaries presented innumerous follicles at different stages of development, while VCD showed marked follicular atresia, depleted of primordial or developing follicles and a predominance of interstitial cells. The ovaries of aged mice were predominantly constituted by corpus luteum degenerated into corpus albicans, with rare antral follicles. All analyzed models led to different permanent diestrus profiles caused by each model, as indicated by ovarian features. This should be carefully considered when choosing a post-estropausal experimental model, in order to better correlate this challenging phase of female's life with physiological/pathological conditions.

CFTR Modulates Hypothalamic Neuron Excitability to Maintain Female Cycle.

Cystic fibrosis transmembrane conductance regulator (CFTR), known as an epithelial Cl- channel, is increasingly noted to be expressed in the nervous system, although whether and how it plays a role in neuronal excitability is unclear. Given the association of CFTR with fertility, we tested here possible involvement of CFTR in regulating hypothalamic neuron excitability. Patch-clamp and Ca2+ imaging showed that pharmacological inhibition of CFTR evoked electrical pulses and Ca2+ spikes in primary rat hypothalamic neurons, which was dependent on extracellular Cl-. Hypothalamic neurons in brain-slice preparations from adult female mice with CFTR mutation (DF508) exhibited significantly reduced electrical pulses as compared to the wild-type controls. Removal of extracellular Cl- eliminated hypothalamic electrical pulses in the wild-type brain slices, which was reversible by subsequent addition of Cl-. In adult female mice, Ca2+ indicator (GCaMP6s)-based fiber-photometry showed that hypothalamic Ca2+ activities in vivo were enhanced at the proestrus/estrus phase as compared to the diestrus phase of the female cycle. Such estrus-associated hypothalamic activities were largely diminished in DF508 female mice, together with delayed puberty and disturbed female cycles. Therefore, these findings suggest a critical role of CFTR in modulating hypothalamic neuron excitability, which may account for the disturbed female cycles and reduced female fertility associated with CFTR mutations.

The effect of orexin on the hypoxic ventilatory response of female rats is greatest in the active phase during diestrus.

We recently showed that in male rats, orexin contributes to the hypoxic ventilatory response (HVR), with a stronger effect in the active phase. The effect of orexin on the HVR in females has not been investigated. As estrogen can inhibit orexin neurons, here we hypothesized that orexin neurons are activated by hypoxia and facilitate the HVR only in diestrus, when estrogen is low. We exposed female rats (n = 10) to near-isocapnic hypoxia ([Formula: see text] from 0.21 to 0.09) over ∼5 min, after vehicle and again after suvorexant (a dual OxR antagonist; 20 mg/kg ip), with ventilation measured using whole body plethysmography. Each rat was tested in proestrus or estrus (p/estrus), and again in diestrus, during both inactive and active phases. We also performed immunohistochemistry (IHC) to determine the proportion of orexin neurons activated by acute hypoxia during diestrus (n = 6) or proestrus/estrus (n = 6) in the active phase. In the inactive phase, the HVR was unaffected by OxR blockade, irrespective of estrus stage. In the active phase, the effect of OxR blockade depended on stage: the slope of the HVR was significantly reduced by OxR blockade only during diestrus. IHC revealed that hypoxia activated more orexin neurons during diestrus compared with p/estrus. We conclude that in females, orexin neurons are activated by hypoxia and contribute to the HVR only in diestrus when estrogen levels are low. Stage of the estrus cycle should be considered when examining the physiological function of orexin neurons in females.NEW & NOTEWORTHY We previously showed that orexin facilitates the hypoxic ventilatory response (HVR) of adult male rats during the active phase. Others have shown that estrogen inhibits orexin neurons. Here we show that orexin neurons are activated by hypoxia and facilitate the HVR of adult female rats during the active phase, but only in diestrus. These data suggest that orexin neurons facilitate the HVR in females when they are free from the inhibitory effects of estrogen.

The Correlation of Endogenous Progesterone Concentration in Diestrus on Early Pregnancy Rate in Thoroughbred Mares.

The aim of this project was to test the hypothesis that progesterone concentration 5 days after ovulation did not differ between pregnant and nonpregnant Thoroughbred mares on stud farms located in the Waikato region of New Zealand. A prospective cohort study was performed involving five stud farms in the Waikato region of New Zealand during the 2018 breeding season. A total of 275 mares were enrolled in the study. Mares were served by 34 individual stallions. Blood samples were taken from each mare 5 days after ovulation (D0) and measured for progesterone concentration. Early pregnancy was confirmed at D14 by transrectal palpation and ultrasonography of the mares reproductive tract. Progesterone concentration at Day 5 post-ovulation was higher in mares determined to pregnant at Day 14 of gestation than in mares determined to be non-pregnant at Day 14 (6.4 ± 3.0 ng/ml vs. 5.5 ± 3.3 ng/ml respectively; P = .02). A negative association between increasing mare age and pregnancy rate was found but mare age had no effect on progesterone concentration at D5. In this study we found that although higher serum progesterone concentration at Day 5 post ovulation was associated with a higher pregnancy rate at Day 14, no predictive or definitive minimum required progesterone concentration could be identified. Additional studies are required to determine if a synthetic progestogens can serve to supplant natural progesterone to increase pregnancy rate in naturally bred mares.

Effects of Different Energy Diets on FSHR mRNA Expression and DNA Methylation in Promoter Region of Duolang Sheep During Diestrus.

The aim of this study was to study the relationship between the methylation level of the promoter region of follicle-stimulating hormone receptor (FSHR) gene and the mRNA expression of Duolang sheep fed different energy diets. In this experiment, three groups of diets with different energy levels (dietary nutrient level reference (NY/T816 - 2004)) were designed to obtain medium energy level diets with metabolic energy of 10.88 MJ/d. The treatments with high and low energy levels increased and decreased by 15%, 12.51, and 9.25 MJ/d, respectively, on the basis of medium energy level. Total RNA and genomic DNA were extracted from the ovaries of Duolang sheep, and qPCR was performed. Bisulfite genomic sequencing PCR and sequence alignment were used to detect the relationship between the relative expression level of FSHR and methylation. The results showed that the expression of FSHR in high-energy group and medium-energy group was significantly higher than that in low-energy group (p < 0.01), and there was no significant difference between high-energy group and medium-energy group (p > 0.05). The methylation rate of the target fragment in the promoter region of FSHR gene was 41.67% in the high-energy group, 75.00% in the medium-energy group, and 83.33% in the low-energy group. This study revealed that different dietary energy levels had certain effects on DNA methylation level of FSHR gene and then affected gene expression, and the expression level was negatively correlated with methylation level.

Use of serum progesterone and prostaglandin F2α metabolite levels to predict onset of parturition in the bitch.

The prediction of time to onset of parturition in a preparturient bitch is of great clinical value, particularly for bitches at high risk of dystocia and those lacking relevant clinical data from the time of breeding. In a previous study, four cut-offs for plasma progesterone levels, measured by radioimmunoassay, were shown to be useful for predicting the likelihood of a bitch entering stage one of parturition within defined time intervals. The first aim of the current study was to evaluate these cut-offs in a clinical setting, using serum progesterone samples drawn from preparturient bitches 12-hourly instead of 6-hourly and assessed using chemiluminescence immunoassay. Furthermore, the use of 13,14-dihydro-15-keto-prostaglandin F2α, (PGFM), a metabolite of prostaglandin F2α , in predicting the time to onset of parturition was evaluated. Forty bitches carrying two or more foetuses were admitted to a specialist veterinary reproduction hospital 53 d after the onset of cytological dioestrus when that date was known, or 57 d after the last mating. Vaginal speculum examinations were performed every 6 hr until cervical dilatation was visualized (time of cervical dilatation; TCD). Serum samples were collected at 08h00 and 18h00 daily until TCD. All bitches underwent elective caesarean section at TCD. Results of this study show that approximately 5% and 10% of preparturient bitches will reach TCD within 12 hr despite a serum progesterone level of at least 15.8 nmol/L and 8.7 nmol/L respectively. In addition, there is a 95% probability that a preparturient bitch will reach TCD within 48 hr if her serum progesterone level is below 8.7 nmol/L, and a 91% probability of her reaching TCD within 24 hr if her serum progesterone level is below 3.18 nmol/L. Approximately 90% of bitches that demonstrate a 20% increase in PGFM over a 12-hr period are likely to be within 36 hr of TCD. These results provide useful benchmarks for the management of canine parturition.

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

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

Activity of the glycosidases ß-galactosidase, α-l-fucosidase, ß-N-acetyl-hexosaminidase, and sialidase in uterine tissues from female dogs in diestrus with and without pyometra.

This study aimed to compare the activity of selected glycosidases (ß-galactosidase, α-l-fucosidase, ß-N-acetyl-hexosaminidase, and sialidase) in homogenates of uterine tissues obtained from female dogs with and without pyometra. In addition, it examined the availability of substrates for these glycosidases in the homogenates. The study was carried out on female dogs undergoing ovariohysterectomy for pyometra (n = 10) and clinically healthy dogs (n = 10) undergoing elective spaying. The activity of ß-galactosidase, α-l-fucosidase, and ß-N-acetyl-hexosaminidase was analyzed using a spectrofluorometer and that of sialidase using a colorimetric method. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis with Alcian Blue (AB) and Periodic Acid-Schiff (PAS) staining was performed to determine the presence of substrates for these glycosidases in the homogenates of uterine tissues. The results revealed that the activity of all the examined glycosidases was significantly higher (P < 0.05) in the uterine tissues isolated from dogs with pyometra in comparison to healthy dogs. The electrophoretic patterns of the selected samples showed several proteins, which contained different sugar moieties stained by AB and PAS and the profiles differed significantly between the pyometra group and the healthy group. Densitometric analysis of AB staining showed patterns between 233 and 148, 86 and 55, and 43 and 20 kDa, which differed markedly in sugar content between the examined groups of animals. Similarly, PAS staining analysis revealed patterns of different molecular weights, between 233 and 117 and between 55 and 32 kDa, which also differed in sugar content. These findings suggest that canine pyometra is accompanied by the increase in the activity of selected glycosidases in the uterus. This could potentially modify the glycan structures of uterine glycoproteins and in result their biological functions. Further studies are needed to elucidate the potential role of the increased activity of glycosidases in the pathogenesis of this disease.

Luteal expression of factors involved in the metabolism and sensitivity to oestrogens in the dog during pregnancy and in non-pregnant cycle.

The canine corpus luteum (CL) is the main source of reproductive steroids during dioestrus in the dog and remains active even in the absence of pregnancy (non-pregnant dioestrus, physiological pseudopregnancy). Whereas the biological effects of 17ß-oestradiol (E2) in the canine CL remain unclear, the transcriptional availability of oestrogen receptors, ESR1 and ESR2, as well as other modulators of local availability of E2, for example, HSD17B7 (converts oestrone into oestradiol), SULT1E1 (inactivates E2 binding capacity to its own receptors through sulphonation) and STS (reverts E2 sulphonation), were previously detected in the CL of non-pregnant bitches. The aim of the present work was to evaluate the mRNA amounts of these factors involved in luteal sensitivity and metabolism of E2 in the canine CL during the course of non-pregnant dioestrus (days 10, 20, 30, 40, 50 and 60 post-ovulation, n = 5/group) and at different stages of pregnancy (n = 4-6/group): pre-implantation (days 8-12), post-implantation (days 18-25), mid-gestation (days 35-40) and prepartum luteolysis. During pregnancy, the availability of ESR1, HSD17B7, SULT1E1 and STS decreased from mid-pregnancy to prepartum luteolysis. The main findings during non-pregnant dioestrus were as follows: increased ESR2:ESR1 ratio on days 40 and 50 after ovulation, decreasing during luteal regression (day 60); increased STS at day 30 when SULT1E1 levels decreased; increased availability of SULT1E1 transcripts during luteal regression; and decreased amounts of HSD17B7 mRNA in early dioestrus, increasing towards later stages. These results suggest that E2 signalling and biologically active local concentrations could diverge in response to time and pregnancy status of the bitch.

Changes in fluid composition and expression of ion channels in rat cervix during different phases of the estrus cycle.

We investigated changes in the composition of cervical fluid at different phases of the female rat reproductive cycle. Fluid was collected from the cervix of rats by direct cervical flushing and analyzed for changes in Na+ and Cl- content and osmolarity. Following sacrifice, the cervix was harvested and expressions of mRNA and protein for ENaCs, CFTR and AQPs were measured using qPCR and immunohistochemistry, respectively. Cervical fluid Na+ and Cl- content was high during estrus, but osmolarity was high during metestrus and diestrus. Expressions of CFTR, AQP-1 and AQP-2 in the cervix were high during estrus, but low during diestrus. Expression of ENaC (α, ß, γ), AQP-5 and AQP-7 was high during metestrus and diestrus and low during estrus. Changes in expression of ion channels in the cervix could explain changes in cervical fluid composition during the estrus cycle phases that could affect female fertility.

Morphological features of the rat uterus.

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

Enhanced responsiveness to hypoxic panicogenic challenge in female rats in late diestrus is suppressed by short-term, low-dose fluoxetine: Involvement of the dorsal raphe nucleus and the dorsal periaqueductal gray.

BACKGROUND: Acute hypoxia, which is panicogenic in humans, also evokes panic-like behavior in male rats. Panic disorder is more common in women and susceptibility increases during the premenstrual phase of the cycle. AIMS: We here investigated for the first time the impact of hypoxia on the expression of panic-like escape behavior by female rats and its relationship with the estrous cycle. We also evaluated functional activation of the midbrain panic circuitry in response to this panicogenic stimulus and whether short-term, low-dose fluoxetine treatment inhibits the hyper-responsiveness of females in late diestrus. METHODS: Male and female Sprague Dawley rats were exposed to 7% O2. Females in late diestrus were also tested after short-term treatment with fluoxetine (1.75 or 10 mg/kg, i.p.). Brains were harvested and processed for c-Fos and tryptophan hydroxylase immunoreactivity in the periaqueductal gray matter (PAG) and dorsal raphe nucleus (DR). RESULTS: Acute hypoxia evoked escape in both sexes. Overall, females were more responsive than males and this is clearer in late diestrus phase. In both sexes, hypoxia induced functional activation (c-Fos expression) in non-serotonergic cells in the lateral wings of the DR and dorsomedial PAG, which was greater in late diestrus than proestrus (lowest behavioral response to hypoxia). Increased responding in late diestrus (behavioral and cellular levels) was prevented by 1.75, but not 10 mg/kg fluoxetine. DISCUSSION: The response of female rats to acute hypoxia models panic behavior in women. Low-dose fluoxetine administered in the premenstrual phase deserves further attention for management of panic disorders in women.

Modulation of pulsatile GnRH dynamics across the ovarian cycle via changes in the network excitability and basal activity of the arcuate kisspeptin network.

Pulsatile GnRH release is essential for normal reproductive function. Kisspeptin secreting neurons found in the arcuate nucleus, known as KNDy neurons for co-expressing neurokinin B, and dynorphin, drive pulsatile GnRH release. Furthermore, gonadal steroids regulate GnRH pulsatile dynamics across the ovarian cycle by altering KNDy neurons' signalling properties. However, the precise mechanism of regulation remains mostly unknown. To better understand these mechanisms, we start by perturbing the KNDy system at different stages of the estrous cycle using optogenetics. We find that optogenetic stimulation of KNDy neurons stimulates pulsatile GnRH/LH secretion in estrous mice but inhibits it in diestrous mice. These in vivo results in combination with mathematical modelling suggest that the transition between estrus and diestrus is underpinned by well-orchestrated changes in neuropeptide signalling and in the excitability of the KNDy population controlled via glutamate signalling. Guided by model predictions, we show that blocking glutamate signalling in diestrous animals inhibits LH pulses, and that optic stimulation of the KNDy population mitigates this inhibition. In estrous mice, disruption of glutamate signalling inhibits pulses generated via sustained low-frequency optic stimulation of the KNDy population, supporting the idea that the level of network excitability is critical for pulse generation. Our results reconcile previous puzzling findings regarding the estradiol-dependent effect that several neuromodulators have on the GnRH pulse generator dynamics. Therefore, we anticipate our model to be a cornerstone for a more quantitative understanding of the pathways via which gonadal steroids regulate GnRH pulse generator dynamics. Finally, our results could inform useful repurposing of drugs targeting the glutamate system in reproductive therapy.

Influence of Reproductive Status on Equine Serum Proteome: Preliminary Results.

The reproductive cycle and early pregnancy represent dynamic physiological states in mammals, but mechanisms involved in early pregnancy in the domestic horse remain poorly understood. Proteins in uterine secretions have been studied, but the proteome of peripheral serum during various reproductive states has not been investigated. This study characterized and compared the serum proteome in the domestic horse during various reproductive states. Serum was collected from three mares during: (1) estrus (day [d] -1; d 0 = ovulation), (2) diestrus (d 12.5, non-mated), (3) early pregnancy (d 12.5, pregnant), and (4) nonpregnant (d 12.5, unsuccessfully mated) states. Serum proteins in each sample were analyzed by Nano LC-MS/MS, and 308 proteins were identified. Differentially-expressed proteins (DEP; > 1.5-fold or < - 0.5-fold) were identified by comparison of protein relative abundance between reproductive states: (1) diestrus compared to estrus (DEP = 71), (2) pregnant compared to diestrus (DEP = 72), and (3) non-pregnant compared to pregnant (DEP = 81). DEPs were analyzed for biological function using PANTHER (pantherdb.org). Several pregnancy-specific proteins previously identified in equine pregnant histotroph, including Apolipoprotein A-I, Complement C3, and Histone H4, were detectable in the serum. The ability to detect these biomarkers in serum provides a more readily available option for investigating and understanding early equine pregnancy.

Acute LPS exposure increases synaptosomal metabolism during estrus but not diestrus.

The hormones estrogen and progesterone alter physiological functions, including the estrus cycle and relevant neurological and synaptic activity. Here, we determined the extent to which estrus cycle stage interacts with an inflammatory stimulus, lipopolysaccharide (LPS), to alter synaptic mitochondrial respiration in female rats. LPS elevated synaptic mitochondrial respiration of rats in estrus, but not diestrus. Likewise, estrogen concentration correlated with multiple respiratory metrics in LPS treated females in estrus. These data suggest estrogen likely modulates synaptic mitochondrial respiration in a high progesterone environment.

In vivo measurement of pH and CO2 levels in the uterus of sows through the estrous cycle and after insemination.

The pH-CO2-HCO3- system is a ubiquitous biological regulator with important functional implications for reproduction. Knowledge of the physiological values of its components is relevant for reproductive biology and the optimization of Assisted Reproductive Technologies (ARTs). However, in situ measurements of these parameters in the uterus are scarce or null. This study describes a non-invasive method for in situ time-lapse recording of pH and CO2 within the uterus of non-anesthetized sows. Animals were at three different reproductive conditions, estrous with no insemination and two hours after insemination, and diestrous. From pH and CO2 data, HCO3- concentration was estimated. The non-invasive approach to the porcine uterus with novel optical probes allowed the obtaining of in situ physiological values of pH, CO2, and HCO3-. Variable oscillatory patterns of pH, CO2 and HCO3- were found independently of the estrous condition. Insemination did not immediately change the levels of uterine pH, CO2 (%) and HCO3- concentration, but all the values were affected by the estrous cycle decreasing significantly at diestrous condition. This study contributes to a better understanding of the in vivo regulation of the pH-CO2-HCO3- system in the uterus and may help to optimize the protocols of sperm treatment for in vitro fertilization.