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.

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.

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.

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.

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.

Dynamics of lipid droplets in the endometrium and fatty acids and oxylipins in the uterine lumen, blood, and milk of lactating cows during diestrus.

Our objective was to investigate the lipid content of uterus, blood plasma, and milk at early, mid, and late diestrus. Lactating cows (n = 30) had the estrous cycle and ovulation synchronized by administration of exogenous hormones. Cows were blocked by parity and assigned randomly to receive transcervical uterine flushing and biopsy on d 5 (early diestrus), 10 (mid diestrus) or 15 (late diestrus) of the estrous cycle. Flushing and endometrial biopsy were performed in the uterine horn ipsilateral to the corpus luteum. The recovered flushing was used for analyses of lipid composition by liquid chromatography-tandem mass spectrometry and the biopsy was used for investigation of lipid droplet abundance in endometrial cryosections using a neutral lipid fluorescent dye. In addition, blood and milk samples were collected from all cows on d 5, 10, and 15. All blood samples were used to measure the concentration of progesterone in plasma, and all milk samples were used to determine milk composition. Subsamples of blood plasma and milk were also used to evaluate the composition of fatty acids and oxylipins using the same methodology used for uterine flushing samples. The abundance of lipid droplets in the endometrium increased 1.9-fold from d 5 to 10, and 2-fold from d 10 to 15. Concentration of long-chain fatty acids and oxylipins in uterine flushing were, on average, 2.2 and 2.5 times greater in samples collected on d 15 compared with those collected on d 5 and 10. These differences were not observed in blood and milk, suggesting that accumulation of fatty acids and oxylipins in the uterus is regulated locally. In addition to concentration, the profile of individual fatty acids and oxylipins in uterine lumen changed substantially during diestrus. The main categories with increased abundance at late diestrus were mono- and polyunsaturated fatty acids, and oxylipins derived from arachidonic acid, dihomo-γ-linolenic acid, and docosahexaenoic acid. In conclusion, fatty acids and oxylipins accumulate in the uterine lumen during diestrus and might work as a mechanism to supply these lipids to the developing conceptus at late diestrus, when the onset of elongation occurs and substantial synthesis of biomass and cell signaling by lipid mediators are required.

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

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

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.

Characterization of the endometrial transcriptome in early diestrus influencing pregnancy status in dairy cattle after transfer of in vitro-produced embryos.

Modifications of the endometrial transcriptome at day 7 of the estrus cycle are crucial to maintain gestation after transfer of in vitro-produced (IVP) embryos, although these changes are still largely unknown. The aim of this study was to identify genes, and their related biological mechanisms, important for pregnancy establishment based on the endometrial transcriptome of recipient lactating dairy cows that become pregnant in the subsequent estrus cycle, upon transfer of IVP embryos. Endometrial biopsies were taken from Holstein Friesian cows on day 6-8 of the estrus cycle followed by embryo transfer in the following cycle. Animals were classified retrospectively as pregnant (PR, n = 8) or nonpregnant (non-PR, n = 11) cows, according to pregnancy status at 26-47 days. Extracted mRNAs from endometrial samples were sequenced with an Illumina platform to determine differentially expressed genes (DEG) between the endometrial transcriptome from PR and non-PR cows. There were 111 DEG (false discovery rate < 0.05), which were mainly related to extracellular matrix interaction, histotroph metabolic composition, prostaglandin synthesis, transforming growth factor-ß signaling as well as inflammation and leukocyte activation. Comparison of these DEG with DEG identified in two public external data sets confirmed the more fertile endometrial molecular profile of PR cows. In conclusion, this study provides insights into the key early endometrial mechanisms for pregnancy establishment, after IVP embryo transfer in dairy cows.

Analysis of the uterine flush fluid proteome of healthy mares and mares with endometritis or fibrotic endometrial degeneration.

The objective of this study was to evaluate the differences in the uterine flush fluid proteome between healthy mares and mares with endometritis or fibrotic endometrial degeneration (FED). Uterine flush fluid samples were collected from healthy mares (n=8; oestrus n=5 and dioestrus n=3) and mares with endometritis (n=23; oestrus n=14 and dioestrus n=9) or FED (n=7; oestrus n=6 and dioestrus n=1). Proteomic analysis was performed using label-free liquid chromatography-tandem mass spectrometry. Of 216 proteins identified during oestrus, 127 were common to all three groups, one protein was exclusively detected in healthy mares, 47 proteins were exclusively detected in mares with endometritis and four proteins were exclusively detected in mares with FED. Of 188 proteins identified during dioestrus, 113 proteins were common between healthy mares and mares with endometritis, eight proteins were exclusively detected in healthy mares and 67 proteins were exclusively detected in mares with endometritis. Quantitative analysis revealed a subset of proteins differing in abundance between the three groups during oestrus and between healthy mares and mares with endometritis during dioestrus. These results provide a springboard for evaluation of specific proteins as biomarkers of uterine health and disease and for investigation of their roles in the establishment and maintenance of pregnancy.

Role of the superior ovarian nerve in the regulation of follicular development and steroidogenesis in the morning of diestrus 1.

PURPOSE: Little is known about the role of the superior ovarian nerve (SON) in follicular development during the estrus cycle. The aim of the present study was to analyze the role of neural signals arriving through the SON at the ovaries in the regulation of follicular development and ovarian steroid secretion in diestrus 1 of cyclic rats. METHODS: Cyclic rats were subjected to left, right, or bilateral SON sectioning or to unilateral or bilateral laparotomy at diestrus 1 at 11:00 h. Animals were sacrificed 24 h after surgery. RESULTS: Compared to laparotomized animals, unilateral SON sectioning decreased the number of preovulatory follicles, while bilateral SON sectioning resulted in a decreased number of atretic preantral follicles. An important observation was the presence of invaginations in the follicular wall of large antral and preovulatory follicles in animals with denervation. Furthermore, left SON sectioning increased progesterone levels but decreased testosterone levels, which are effects that were not observed in animals that were subjected to right denervation. CONCLUSIONS: At 11:00 h of diestrus 1, the SON was found to stimulate follicle development, possibly via neural signals, such as noradrenaline and/or vasoactive intestinal peptide, and this stimulation induced the formation of follicle-stimulating hormone receptors. The role of the SON in the regulation of ovarian steroid secretion is asymmetric: the left SON inhibits the regulation of progesterone and stimulates testosterone secretion, and the right nerve does not participate in these processes.

Qualitative analysis and functional classification of the uterine proteome of mares in oestrus and dioestrus.

Quantitative analysis of the uterine flush fluid proteome of mares in oestrus and dioestrus has been previously reported. The objectives of this study were to: a) evaluate qualitative differences in the uterine flush fluid proteome between mares in oestrus and mares in dioestrus and b) perform a functional classification of proteins either unique to each stage or common between the two stages. Uterine flush fluid samples were collected from 8 light breed mares in either oestrus (n = 5) or dioestrus (n = 3). Proteomic analysis of the samples was conducted using liquid chromatography-tandem mass spectrometry. Proteins exclusively detected in oestrus or dioestrus and those common to both stages were identified using the Scaffold software (version 4.4.8, Proteome Software Inc., Portland, OR). The identified proteins were classified into gene ontology (GO) categories (cellular component [CC], molecular function [MF] and biological process [BP]) using the PANTHER (www.pantherdb.org) classification system version 14.0. Of 172 proteins identified, 51 and 28 were exclusively detected in mares in oestrus and dioestrus, respectively, and 93 proteins were common to both stages. The most represented terms in various GO categories were similar among the three subsets of proteins. The most represented CC terms were extracellular region and cell, the most represented MF terms were catalytic activity and binding, and the most represented BP terms were metabolic process and cellular process. In conclusion, proteomic analysis of the uterine flush fluid enabled the identification of subsets of proteins unique to oestrus or dioestrus, or common to both stages. The results of this study can serve as a baseline for future research focused on finding stage-specific protein markers or evaluating differences in the uterine flush fluid proteome between normal mares and those with uterine disease.

Endometrial expression of key genes related to fertility in repeat breeder and non-repeat breeder cows.

The aim of the present study was to compare the endometrial gene expression of epidermal growth factor receptor (EGFR), nodal growth differentiation factor (NODAL), prostaglandin-endoperoxide synthase 2 (PTGS2), oestrogen receptor 1 (ESR1) and progesterone receptor (PGR) in repeat breeder cows (RBC) and non-RBC during diestrus. Endometrial samples were collected by cytobrush technique and stored in RNA stabilizing solution at -20°C until RT-qPCR analysis. Differences in endometrial mRNA expression of selected genes were assessed by ANOVA and simple (r) and the partial correlations (rp) among selected genes were performed. Results demonstrated that mRNA expression of EGFR and NODAL were higher in RBC than in non-RBC (3 and 25-fold change, p < .01 and p < .01, respectively), while the mRNA expression of PTGS2 was lower (1.56-fold change, p < .01). Although there were no differences detected in the mRNA expression of ESR1 and PGR, there was a positive correlation between the expression of ESR1 and EGFR (0.84, p < .05) and a negative correlation between PGR and PTGS2 (-0.49, p < .05). In conclusion, the difference on the endometrial mRNA expression of the genes included in the study between RBC and non-RBC indicates a deregulation of important mechanisms that are vital to establish a successful pregnancy. Thus, the present study provides useful insight as a base for future studies to elucidate the causes of RBC.

Comparing Gene Expression in the Parabrachial and Amygdala of Diestrus and Proestrus Female Rats after Orofacial Varicella Zoster Injection.

The orofacial pain pathway projects to the parabrachial and amygdala, and sex steroids have been shown to affect neuronal activity in these regions. GABA positive cells in the amygdala are influenced by sex steroid metabolites to affect pain, and sex steroids have been shown to alter the expression of genes in the parabrachial, changing neuronal excitability. Mechanisms by which sex steroids affect amygdala and parabrachial signaling are unclear. The expression of genes in the parabrachial and amygdala in diestrus (low estradiol) and proestrus (high estradiol) female rats were evaluated in this study. First, varicella zoster virus was injected into the whisker pad of female rats to induce a pain response. Second, gene expression was quantitated using RNA-seq one week after injection. Genes that had the greatest change in expression and known to function in pain signaling were selected for the quantitation of protein content. Protein expression of four genes in the parabrachial and seven genes in the amygdala were quantitated by ELISA. In the parabrachial, neurexin 3 (Nrnx3) was elevated at proestrus. Nrnx3 has a role in AMPA receptor and GABA signaling. Neuronatin (Nnat) and protein phosphatase, Mg2+/Mn2+ dependent 1E (Ppm1e) were elevated in the parabrachial of diestrus animals both genes having a role in pain signaling. Epoxide hydroxylase (Ephx2) was elevated in the parabrachial at proestrus and the vitamin D receptor (Vdr) was elevated in the amygdala. Ephx2 antagonists and vitamin D have been used to treat neuropathic pain. In conclusion, sex steroids regulate genes in the parabrachial and amygdala that might result in the greater pain response observed during diestrus.

Quality of Preovulatory (GV) Oocytes in Mice after Injection of eCG at Various Stages of the Estrous Cycle.

We studied the influence of the estrous cycle on the morphology of preovulatory (germinal vesicle, GV) oocytes in mice and their capacity to meiotic maturation in vitro. After standard injections of eCG gonadotropin (PMSG, Follimag) to females at different stages of the estrous cycle, the maximum levels of GV oocytes (26±1/mouse) were isolated from the ovaries of animals injected with the hormone during estrus. The capacity of isolated GV oocytes to meiotic maturation in vitro decreased in the following order: estrus (75.5±2.3%), metestrus (67.9±3.4%), proestrus (57.8±4.4%), and diestrus (50.6±5.6%); the differences between estrus and diestrus/proestrus were significant (p<0.05). After eCG injections during estrus, GV oocytes differed from other oocytes by lesser total diameter, lesser diameter of cytoplasm, lesser thickness of zona pellucida, and moderately dilated perivitelline space. These signs reflected higher competence of the "estrous" GV oocytes for meiotic maturation in vitro. Hormone stimulation of females with eCG, with consideration for the stage of the estrous cycle, seems to be an effective method for improving the quality of GV oocytes isolated from mouse ovaries.

First proteomic analysis of diestrus and anestrus canine oocytes at the germinal vesicle reveals candidate proteins involved in oocyte meiotic competence.

In domestic dogs, oocyte maturation rates are low and the percentage of oocytes that remain in the stage of germinal vesicle (GV) regardless of culture conditions is high. The present study was conducted to characterize the proteome of canine oocyte at the germinal vesicle stage using label-free mass spectrometry. Ovaries were collected from 415 adult domestic dogs and oocytes were divided anestrus and diestrus group. Protein lysates were subjected to quantitative proteomic analysis to identify differentially expressed proteins in different status reproductive. All runs for each sample were performed on an Easy nLC1000 nano-LC chromatograph system directly connected to a quadrupole-type Orbitrap mass spectrometer. For identification of peptides and proteins, raw data of the spectra were loaded into MaxQuant software version 1.5.2.8. Proteomic data were analysed according to gene ontology and a protein-protein interaction network. 312 proteins were identified and grouped according to their biological processes, molecular functions and cellular component. Forty-six differentially expressed proteins among diestrus and control group were associated with at least one GO term in the biological process database. Several proteins involved in the cell cycle, fertilization, regulation of transcription and signalling pathways that are essential for the full development of oocytes and fertilization were expressed. This study identified proteins that were absent, and more or less expressed in different status reproductive. These differentially expressed proteins revealed a framework of molecular reorganization within a GV that renders its competency. This knowledge will enable the identification of target competence biomarkers and thus the establishment of more adequate means of cultivation to improve the M-I and II indexes in this species and also to better understand the physiology of the domestic dog, promoting the development of new reproduction biotechniques.

Expression of Bcl-2 Family Proteins in the Ovarian Follicular Apparatus in the Acute Period after Experimental Hyperthermia.

The expression of apoptosis regulators (proapoptotic protein Bad and anti-apoptotic protein Bcl-2) was analyzed and Bcl-2/Bad ratio in the follicular apparatus of the rat ovary was determined on day 3 after hyperthermia (rectal temperature 43.5°C). Hyperthermia in the catabolic phase leads to different degrees of activation of the molecular "switches" of apoptosis in cells of ovarian follicular epithelium. This was seen from increased intensity of immunohistochemical staining for Bad protein against the background of more pronounced expression of Bcl-2 protein. On day 3 after exposure to hyperthermia, Bcl-2/Bad ratio increased, which reflects antiapoptotic protection of cells and conditions for blockade of mitochondrial pathway of apoptosis in the follicular apparatus of the ovaries during the acute period after hyperthermia.

Hexokinase 2 drives glycogen accumulation in equine endometrium at day 12 of diestrus and pregnancy.

BACKGROUND: Secretion of histotroph during the prolonged pre-implantation phase in mares is crucial to pregnancy maintenance, manifested as increased embryonic loss in mares with age-related endometrial degeneration. Glycogen content of uterine histotroph is higher during the progesterone-dominated phase of the estrous cycle in mares, but regulatory mechanisms are not well understood. METHODS: mRNA expression of glycogen-metabolizing enzymes (HK1, HK2, GSK3B, GYS1, PEPCK, PKM, PYGM) in endometrial samples were compared among mares in anestrus, estrus, and at Day 12 of diestrus and pregnancy. In addition, hexokinase 2 (HK2) activity was assessed using a colorimetric assay. RESULTS: HK2 was the key regulator of glycogen accumulation during diestrus and pregnancy; hexokinase transcript abundance and enzyme activity were significantly higher during diestrus and pregnancy than estrus and anestrus. In addition, despite similar relative transcript abundance, hexokinase activity was significantly greater in the pregnant versus diestrous endometrium. Therefore, we inferred there was regulation of hexokinase activity through phosphorylation, in addition to its regulation at the transcriptional level during early pregnancy. Based on immunohistochemistry, HK2 was localized primarily in luminal and glandular epithelial cells, with weaker staining in stromal cells. CONCLUSION: Among glycogen metabolizing enzymes identified, expression of HK2 was significantly greater during the progesterone-dominated phase of the cycle.

Peri-ovulatory endocrine regulation of the prostanoid pathways in the bovine uterus at early dioestrus.

We hypothesised that different endocrine profiles associated with pre-ovulatory follicle (POF) size would impact on uterine prostanoid pathways and thereby modulate the histotroph composition. Beef cows (n=15 per group) were hormonally manipulated to have small (SF-SCL group) or large (LF-LCL group) pre-ovulatory follicles (POF) and corpora lutea (CL). Seven days after induction of ovulation, animals were slaughtered and uterine tissues and flushings were collected for quantification of prostanoids. The POF and CL size and the circulating progesterone concentrations at Day 7 were greater (P<0.05) in the LF-LCL cows than in the SF-SCL group, as expected. The abundance of 5 out of 19 genes involved in prostanoid regulation was different between groups. Transcript abundance of prostaglandin F2α, E2 and I2 synthases was upregulated (P<0.05) and phospholipase A2 was downregulated (P<0.05) in endometrium of the LF-LCL group. No difference (P>0.1) in prostanoid concentrations in the endometrium or in uterine flushings was detected between groups. However, prostaglandin F2α and E2 concentrations in the uterine flushings were positively correlated with the abundance of transcripts for prostaglandin endoperoxide synthase 2 (0.779 and 0.865, respectively; P<0.002). We conclude that endometrial gene expression related to prostanoid synthesis is modulated by the peri-ovulatory endocrine profile associated with POF size, but at early dioestrus differences in transcript abundance were not reflected in changes in prostanoid concentrations in the uterine tissue and fluid.