Estrous Period Research Articles

EXPRESSION OF NRDR DIFFERENT ISOFORMS IN MICE UTERUS

Uterus is an important reproductive organ for embryonic growth and development of viviparous animals. Its normal function is regulated by complex endocrine system and multiple genes. NRDR is a retinoic acid metabolizing enzyme with strong retinol oxidation and retinoaldehyde reduction activities. Its function in the reproductive system has been partially studied. In this study, the expression distribution and change of NRDR in in different physiological states and different developmental stages of mouse uterus will be clarified, so as to provide an experimental basis for further research on its function in uterus. Using immunohistochemistry and RT-qPCR, NRDR was proved mainly expressed in mouse endometrial by immunohistochemistry. The expression of two different isoforms NRDR-1 and NRDR-2 in mouse uterus increased gradually with the development of the mouse, and peaked at 15 days after birth. The expression of NRDR-1 has no significant difference during estrous and diestrus in mouse uterus. However, the expression of NRDR-2 in estrous period was significantly higher than that in diestrus; NRDR two isoforms increased during embryo recognition and implantation in mouse uterus, and their expression decreased after implantation. The experimental results of the above expression patterns indicated that NRDR played a very important role in the mouse uterus, which was related to the estrus cycle and embryo implantation, among which the isoforms NRDR-2 might play a major role. The above results provide a basis for further study of the function of NRDR in uterus. The detection of the expression of NRDR in the uterus can provide experimental basis for understanding the molecular mechanism of development and other physiological processes in uterus. Keywords: NRDR; isoforms; uterus; mice

The regulatory effect of electroacupuncture on endometrial M1-type macrophages in rats with intrauterine adhesions.

To observe the effect of electroacupuncture(EA) on endometrial fibrosis and M1-type macrophages in rats with intrauterine adhesions(IUA), so as to explore the possible mechanism of EA in the treatment of IUA. Fifteen female SD rats were randomly divided into blank group, model group and EA group, with 5 rats in each group. The IUA rat model was established by double damage method using mechanical scraping combined with lipopolysaccharide infection. Rats in the EA group were treated with acupuncture at "Guanyuan"(CV4), and EA at bilateral "Zusanli"(ST36) and "Sanyinjiao"(SP6)for 20 minutes each time, once a day, for 3 consecutive cycles of estrus. Five rats in each group were sampled during the estrous period, and the endometrial morphology, endometrial thickness and the number of blood vessels and glands were observed after HE staining. The fibrotic area of the uterus was observed after Masson staining. The positive expressions of Runt-related transcription factor(RUNX1), transforming growth factor-β1(TGF-β1), connective tissue growth factor(CTGF), α-smooth muscle actin(α-SMA), collagen type I(Col-Ⅰ), cluster of differentiation 86(CD86), interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α) in endometrial tissue were detected by immunohistochemistry. Western blot was used to detect relative protein expressions of RUNX1, TGF-β1, α-SMA, CD86, and TNF receptor 2 (TNFR2), and real-time fluorescence quantitative PCR was used to detect mRNA expressions of RUNX1, TGF-β1, α-SMA, CD86, and TNF-α in the endometrium. During the estrous phase, the endometrial layer in the model group was damaged, with reduced folds, disordered arrangement of epithelial cells, loose fibrous connective tissue, significant narrowing and adhesions in the uterine cavity, interstitial congestion, edema, and a significant infiltration of inflammatory cells with sparse glands. While uterine tissue structure of the EA group was basically intact, resembling a normal uterus, with more newly formed glands and a small amount of inflammatory cell infiltration. In comparison with the blank group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly decreased(P<0.001) in the model group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-β1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1β, and TNF-α, the protein relative expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNFR2, and the mRNA relative expression levels of RUNX1, TGF-β1, α-SMA, CD86 and TNF-α in the endometrium were significantly increased (P<0.001, P<0.01). Compared to the model group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly increased(P<0.01, P<0.05) in the EA group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-β1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1β and TNF-α in the endometrial tissue, the protein expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNFR2, and the mRNA relative expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNF-α in the endometrium were significantly decreased (P<0.001, P<0.01, P<0.05). EA can improve endometrial fibrosis in IUA rats, which may be related to its function in decreasing the level of endometrial M1-type macrophages and the secretion of related inflammatory factors.

Effect of Shoutai Pills on expression of key glycolytic proteins and apoptosis related factors at maternal fetal interface in mouse model of threatened abortion with syndrome of kidney deficiency

This study aims to explore the mechanism of Shoutai Pills in treating threatened abortion. According to the random number table method, ICR female mice were randomized into a normal group, a model group, a dydrogesterone group, and a Shoutai Pills group, with 15 mice in each group. Mice were administrated with normal saline(normal and model groups) or the suspension of Shoutai Pills or dydrogesterone by gavage at 9:00 am every day. At 16:00 every day, mice in the normal group were administrated with an equal volume of distilled water, while those in the model, Shoutai Pills, and dydrogesterone groups were administrated with hydrocortisone solution by gavage for 4 consecutive days. ICR female and male mice were caged in a ratio of 2∶1 during the pre-estrous or estrous period. From the first day of pregnancy, drug administration was continued for 5 consecutive days. On day 6, mice were administrated with mifepristone by gavage to establish the model of kidney deficiency-induced abortion. On day 6 of pregnancy, 10 female ICR mice were randomly selected from each group, and the uterus was collected for observation of the pathological changes of trophoblasts at the maternal-fetal interface by hematoxylin-eosin(HE) staining. The protein levels of key enzymes of glycolysis, hexokinase 2(HK2), enolase 1(ENO1), pyruvate kinase M2(PKM2), and lactate dehydrogenase A(LDHA), were determined by Western blot and immunofluorescence. The expression of apoptosis-related proteins including B cell lymphoma-2(Bcl-2), Bcl-2-associated protein X(Bax), and cysteinyl aspartate-specific proteinase-3(caspase-3) was determined by Western blot and real-time PCR. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling was employed to examine apoptosis. The embryo loss rate of the remaining five female mice was calculated by trypan blue staining method on day 14 of pregnancy. On day 14 of pregnancy, the embryo loss rate of the normal group was 5.00%, which was lower than that(27.78%) in the model group(P&lt;0.05). Dydrogesterone and Shoutai Pills groups showed reduced embryo loss rates(10.26% and 7.50%, respectively) compared with the model group. On day 6 of pregnancy, compared with the normal group, the model group showed down-regulated expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and up-regulated expression of Bax and caspase-3(P&lt;0.05). Compared with the model group, dydrogesterone and Shoutai Pills up-regulated the expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and down-regulated the expression of Bax and caspase-3(P&lt;0.05). Compared with that in the normal group, the apoptosis rate in the model group increased(P&lt;0.05). Compared with the model group, dydrogesterone and Shoutai Pills reduced the apoptosis rate(P&lt;0.05). In conclusion, Shoutai Pills can reduce the embryo loss rate and protect embryos by promoting aerobic glycolysis at the maternal-fetal interface and inhibiting the apoptosis of trophoblasts in mice.

Studies on Breeding Failures in Dogs: A Review

The domestic dog is a non-seasonal breeder. The female is monestrus and has a reproductive cycle that is marked by extended periods of proestrus and estrus. The estrous period is characterized by an estrogen peak that coincides with rising circulating progesterone concentration prior to ovulation. After estrus is diestrus and then anestrus, with the ovarian cycle regulated by the hypothalamic-pituitary-gonadal axis. In the male spermatogenesis is controlled by the hypothalamic-pituitary-gonadal axis with testosterone playing a vital role. The reproductive cycle of most wild canids is similar to that of domestic dogs. Diestrus is followed by an extended period of ovarian inactivity. The causes of breeding failure in dogs are numerous and require thorough investigations for accurate diagnosis. Breeding failures can be infectious or non-infectious. Among the infectious causes in the bitch, bacterial endometritis was found to be responsible for the majority of reported cases. Non-infectious causes include primary and secondary anoestrus, cystic conditions of the uterus, and degenerative diseases of the endometrium. In the male, the causes can also be infectious and non-infectious. Non-infectious causes like bilateral cryptorchidism and acquired anatomical abnormalities can also cause male infertility. Spermatocele or sperm granulomas, obstruction of the genital ducts or inguinal or scrotal hernia, and prostatitis lead to infertility. Infections can lead to orchitis/epididymitis, with alteration of the quality of the semen. Nutrition also has important implications for reproductive performance. Undernutrition can result in loss of body condition, delay the onset of puberty, and ultimately lead to infertility.

High-throughput sequencing and characterization of potentially pathogenic fungi from the vaginal mycobiome of giant panda (Ailuropoda melanoleuca) in estrus and non-estrus.

The giant panda (Ailuropoda melanoleuca) reproduction is of worldwide attention, and the vaginal microbiome is one of the most important factors affecting the reproductive rate of giant pandas. The aim of this study is to investigate the diversity of vaginal mycobiota structure, and potential pathogenic fungi in female giant pandas during estrus and non-estrus. This study combined with high-throughput sequencing and laboratory testing to compare the diversity of the vaginal mycobiota in giant pandas during estrus and non-estrus, and to investigate the presence of potentially pathogenic fungi. Potentially pathogenic fungi were studied in mice to explore their pathogenicity. The results revealed that during estrus, the vaginal secretions of giant pandas play a crucial role in fungal colonization. Moreover, the diversity of the vaginal mycobiota is reduced and specificity is enhanced. The abundance of Trichosporon and Cutaneotrichosporon in the vaginal mycobiota of giant pandas during estrus was significantly higher than that during non-estrus periods. Apiotrichum and Cutaneotrichosporon were considered the most important genera, and they primarily originate from the environment owing to marking behavior exhibited during the estrous period of giant pandas. Trichosporon is considered a resident mycobiota of the vagina and is an important pathogen that causes infection when immune system is suppressed. Potentially pathogenic fungi were further isolated and identified from the vaginal secretions of giant pandas during estrus, and seven strains of Apiotrichum (A. brassicae), one strain of Cutaneotrichosporon (C. moniliiforme), and nine strains of Trichosporon (two strains of T. asteroides, one strain of T. inkin, one strain of T. insectorum, and five strains of T. japonicum) were identified. Pathogenicity results showed that T. asteroides was the most pathogenic strain, as it is associated with extensive connective tissue replacement and inflammatory cell infiltration in both liver and kidney tissues. The results of this study improve our understanding of the diversity of the vaginal fungi present in giant pandas and will significantly contribute to improving the reproductive health of giant pandas in the future.

The state of cellular immunity link and steroid hormones in bitches under different conditions of the reproductive system

The purpose of the conducted study was to establish the correlation between indicators of the cellular immunity link and the concentration of steroid hormones in bitches under different conditions of the reproductive system. Seventeen crossbred bitches aged from 2 to 5 years were involved in the experiment. They were divided into three groups depending on the state of the reproductive system: anestrus, sexual cycle, and pyometra. The following immunophysiological indicators were determined in blood plasma: the absolute number of leukocytes, lymphocytes, and their immunoregulatory subpopulations, and the phagocytic activity of neutrophils. The content of progesterone, estradiol, and cortisol was determined in serum. According to the results of the research, it was established that the absolute number of neutrophils, as the most reactogenic cells of the innate link of cellular immunity, was 0.51 G/L higher in the group of animals with pyometra than in the group of animals during the sexual cycle, and by 0.84 G/L in bitches in conditions of anestrus. The absolute number of phagocytic active neutrophils in conditions of pyometra was 2.32 ± 0.58 G/L (Р &lt; 0.001). However, as a percentage of the absolute number of neutrophils, this indicator was 66 %. During the sexual cycle, the absolute number of neutrophils capable of phagocytosis was 2.33 ± 0.51 G/L, which is 78 % of the absolute number of all neutrophilic leukocytes. The indicator of the absolute number of lymphocytes was reliably (P &lt; 0.05) lower in the group of bitches with pyometra – 1.48 ± 0.35 G/L, which is 0.92 G/L less compared to the group of animals in the anestrous period. Analysis of the absolute number of monocytes showed that this indicator was the same in bitches during the pyometra and anestrous periods and was lower by 0,07 G/L during the sexual cycle. The absolute number of B-lymphocytes, as a humoral link of adaptive immunity, in bitches in conditions of the anestrous and estrous period was the same and amounted to 0.30 G/L. In the group of bitches with pyometra, a reliable decrease of this indicator by 33 % was established (Р &lt; 0.05). The population of natural killer (NK) lymphocytes was also the lowest in bitches with pyometra, almost twice as compared to animals during the sexual cycle. Conducted research on determining the absolute quantitative indicators of the cellular immunity link showed their dependence on the functional state of the reproductive system. It was established that the absolute number of neutrophils in pyometra was higher than in animals in anestrus. However, at the same time, their relative number capable of phagocytosis was the lowest. In conditions of pyometra, the lowest number of lymphocytes and their main subpopulations were observed.

MAMMARY GLAND DEVELOPMENT IN DOGS AND CATS IN RELATION TO THEIR SEXUAL CYCLE

"The mammary gland is a modified sweat gland specialized in milk secretion, whose development is synchronized with the sexual function, being directly influenced by the stages of the sexual cycle in both canids and felines. Knowledge of morphology and physiology is essential for a correct approach of the mammary diseases, an important pathology affecting two categories: the adult female and the newborns. There are numerous controversies in the literature regarding the terminology and duration of the dog and cat sexual cycle, leading to the existent variability of the information. However, for the bitch it is unanimously accepted that most canine breeds have two estrous periods per year, usually in spring and autumn. This period can be systematically divided into proestrus, estrus, luteal phase and anestrus. In this species, the proestrus and estrus periods are long and necessarily accompanied by a long luteal phase. These are then followed by anestrus, which is not influenced by the season, unlike the cat. The queen is a seasonally polyestrous animal, which in the absence of mating goes into estrus every 2-3 weeks throughout the breeding season. She will have repeated cycles in the breeding season, which will only be interrupted by gestation, pseudo-gestation or various ailments. The aim of this review is to describe the estrus cycle in dogs and cats, and its connection with mammary gland development, a gland with unique growth due to the fact that the final stage of development is reached in the adult female only during gestation."

Dibutyl Phthalate Adsorbed on Multiwalled Carbon Nanotubes Causes Fetal Developmental Toxicity in Balb/C Mice.

This study investigated whether using multiwalled carbon nanotubes (MWCNTs) as a carrier for dibutyl phthalate (DBP) could delay the degradation rate of DBP in mice and increase its estrogen-like interference effect. Pregnant Balb/C mice were divided into four groups and exposed to different treatments via tail-vein injection every 3 days until gestational day 20. The female and male mice were then sacrificed for toxicological study. The results showed that the combination of MWCNTs and DBP resulted in a higher fetal mortality rate than if the mice were exposed to MWCNTs or DBP alone. H&E staining showed that the estrous period of the exposed mice was delayed, the development of oocytes was blocked in the combination group, the number of spermatogenic cells decreased, and the quality of sperm decreased. Our experiment showed that the expression levels of the genes involved in sex hormone synthesis in the testis and ovaries were significantly increased after combined treatment compared with the MWCNT group (p < 0.01). The study suggests that DBP degradation is delayed when absorbed on MWCNTs, which increases its estrogen-like interference and interferes with fetal development, ultimately leading to increased fetal mortality.

DETERMINATION OF OVULATION TIME IN GAYO MARES BASED ON IMAGE OF PREOVULATORY FOLLICLE GROWTH

This study aimed to obtain the ultrasound image of preovulatory follicle growth in gayo mares to predict the ovulation time. This study used three Gayo mares and one gayo stallion for teaser aged 5-10 years and weighed 200-250 kg. Preovulatory follicle growth was observed using transrectal ultrasonography which was performed when the dominant follicle size was ≥ 3.5 cm. Observations were made for 3 consecutive estrous periods on gayo mares at relatively the same time. The absence of previous follicular appearance and the presence of hypoechoic area in the same ovary indicate that ovulation has occurred. The result revealed that the diameter of preovulatory follicle of gayo mares was 4.02±0.32 to 5.05±0.04 cm. There was no significant follicle growth in the last 48 hours before ovulation and the preovulatory follicle shape altered from spherical to pearshape. Many small follicles (2 cm in diameter) were observed in peripheral area of the ovary indicated the recruitment of follicles which form the first follicular wave. To conclude, the alteration in the size and pearshape of preovulatory follicle and the recruitment of small follicles can be used as a marker to determine the time of ovulation on gayo mares.

Obtaining follicular fluid through ovum pick up to determine steroidal concentrations of preovulatory follicles in donkeys

There is currently no evidence of reference values for steroid hormones in the follicular fluid of the species Equus asinus during the estrous cycle. The present study aimed to determine the hormonal concentrations of estradiol and progesterone in the follicular microenvironment to establish a better understanding of the role they play in the donkey and their importance in reproductive physiology of this species.. The study was carried out at the FMVZ-UNAM Equine Reproduction Clinic authorized by the Institutional Sub-committee for the Care and Use of Experimental Animals (SICUAE.MMVZ-2020/4-2). Seven Creole donkeys aged between 3 and 9 years, clinically healthy, weighing between 128 and 206 kg and with a body condition of 3 on a scale of 0 to 5, were used. In order to identify preovulatory follicles with a diameter from 27 to 38 mm (depending on the specimen), each donkey was monitored by ovarian palpation and transrectal ultrasonography. Each follicle was standardized based on its size and previous records over three estrous periods where the optimal follicular diameter was observedprior to ovulation. Subsequently, the follicular fluid was recovered using the transvaginal ultrasonography-guided follicular aspiration technique and stored at -20°C until determination of hormones by ELISA (Estradiol ELISADRG, EIA-2693 and Progesterone ELISA DRG, EIA-1561). Prior to the analysis of each sample, follicular fluid was diluted in PBS 0.1M, pH 7.2 from 1:1000 to 1:10,000. Each sample was measured in duplicate. A total of 70 follicular aspirations were obtained during 10 estrous cycles from each donkey, with an average interval of 15 to 17 days. data in Table 1 represent the mean ± standard error of the mean of 10 observations per animal. The results show for first-time concentrations of estradiol and progesterone in the donkeys, which will serve as a reference in subsequent studies.

In vitro culture of Tritrichomonas foetus in bovine cervico-vaginal fluid

Tritrichomonas foetus is the causative agent of bovine trichomonosis, a venereal disease that can lead to fetal loss. T. foetus proliferates in the vagina and cervix and invades the uterus and fetal cavities. It is not clear how T. foetus grows in the host or how its infection rarely demonstrable after 4 months, is controlled. Cervical vaginal mucus (CVM) is a protective barrier against potentially harmful microorganisms. Here, we demonstrate that bovine CVM is a medium in which this protozoan parasite can grow in vitro. T. foetus multiplied at different rates depending on the time of the estrous period from which the CVM was obtained. Growth rates were higher in CVM obtained 7–10 days after estrus.

Sire Distribution of Calves in a Beef Herd with Use of Fixed Time Artificial Insemination Followed by Immediate Bull Exposure for Natural Service in Cows and Heifers

Objective: Our objective was to determine the relative percentages of calves sired by either natural service sire or fixed time artificial insemination (FTAI) sire within the same estrous period.

Toxicity of polystyrene nanoparticles for mouse ovary and cultured human granulosa cells

The issue of global environmental contamination of microplastics has recently been receiving widespread attention. However, the effects of polystyrene nanoparticles (Nano-PS) on the female reproductive system remain unclear. We investigated the toxicity and explored the potential underlying mechanisms of Nano-PS in both mouse ovarian tissue in vivo and human ovarian granulosa cell lines in vitro. In vivo experiments: Mice were fed different concentrations of Nano-PS for 8 weeks. In vitro experiments: COV434 cells were treated with increasing concentrations of Nano-PS. In the present study, ovarian reserve was found to decrease significantly, while oxidative stress and apoptosis levels increased. Nano-PS increased the proportion of metestrum and diestrus periods, and decreased the proportion of estrous period. The implantation rates and the number of pups per litter decreased. In COV434 cells, Nano-PS reduced cell viability and mitochondrial membrane potential, increased the expression of apoptotic and oxidative stress markers and led to subsequent cell cycle arrest. Specifically, Nano-PS exert their toxic effects on mouse ovarian tissue and COV434 cells by inducing oxidative stress. A potential strategy to overcome this could be to activate the nuclear factor-E2-related factor 2 (Nrf2) signaling pathway to mitigate Nano-PS-induced oxidative stress.

Integrative Proteomics and Transcriptomics Profiles of the Oviduct Reveal the Prolificacy-Related Candidate Biomarkers of Goats (Capra hircus) in Estrous Periods

The oviduct is a dynamic reproductive organ for mammalian reproduction and is required for gamete storage, maturation, fertilization, and early embryonic development, and it directly affects fecundity. However, the molecular regulation of prolificacy occurring in estrous periods remain poorly understood. This study aims to gain a better understanding of the genes involved in regulating goat fecundity in the proteome and transcriptome levels of the oviducts. Twenty female Yunshang black goats (between 2 and 3 years old, weight 52.22 ± 0.43 kg) were divided into high- and low-fecundity groups in the follicular (FH and FL, five individuals per group) and luteal (LH and LL, five individuals per group) phases, respectively. The DIA-based high-resolution mass spectrometry (MS) method was used to quantify proteins in twenty oviducts. A total of 5409 proteins were quantified, and Weighted gene co-expression network analysis (WGCNA) determined that the tan module was highly associated with the high-fecundity trait in the luteal phase, and identified NUP107, ANXA11, COX2, AKP13, and ITF140 as hub proteins. Subsequently, 98 and 167 differentially abundant proteins (DAPs) were identified in the FH vs. FL and LH vs. LL comparison groups, respectively. Parallel reaction monitoring (PRM) was used to validate the results of the proteomics data, and the hub proteins were analyzed with Western blot (WB). In addition, biological adhesion and transporter activity processes were associated with oviductal function, and several proteins that play roles in oviductal communication with gametes or embryos were identified, including CAMSAP3, ITGAM, SYVN1, EMG1, ND5, RING1, CBS, PES1, ELP3, SEC24C, SPP1, and HSPA8. Correlation analysis of proteomics and transcriptomic revealed that the DAPs and differentially expressed genes (DEGs) are commonly involved in the metabolic processes at the follicular phase; they may prepare the oviductal microenvironment for gamete reception; and the MAP kinase activity, estrogen receptor binding, and angiotensin receptor binding terms were enriched in the luteal phase, which may be actively involved in reproductive processes. By generating the proteome data of the oviduct at two critical phases and integrating transcriptome analysis, we uncovered novel aspects of oviductal gene regulation of fecundity and provided a reference for other mammals.

THE USE OF OXYTOCIN IN COMBINATION WITH UTERINE LAVAGE AS A POST-MATING TREATMENT FOR INFERTILE MARES WITH ENDOMETRITIS

Eighteen infertile multiparous mares were used in this study. They had been barren for at least 2 years and had been bred repeatedly during those years, and were 8-20 years of age. Clinical genital, and endometrial cytology and histopathological examinations revealed that the mares suffered from acute endometritis. Each mare was subjected to an intrauterine lavage with 500-1000 ml of sterile saline solution in combination with an intravenous injection of 20 I.U. of oxytocin within 12-24 hours after mating. The mares expelled most of the flushed fluid within 20 -30 minutes of oxytocin injection. Eighteen of the treated mares, 13 (72%) conceived after mating on one estrous period. Results of this study indicates that removing intrauterine fluid containing inflammatory by-products by saline lavage and inducing uterine contractions with oxytocin after mating improved conception rates in infertile mares.

THE EFFECT OF HORMONAL TREATMENTS ON THE PREGNANCY RATES OF MARES

To determine the effect of hormonal drugs on the pregnancy rates of mares, authors analyzed this indicator in natural (n=303) and stimulated cycles (n=170). They used cloprostenol preparations to shorten the diestral period, and human chorionic gonadotropin [hCG] to induce ovulation. Natural cycles are divided into groups, depending on the duration – normal and short (19–24 and 12–18 days, respectively). Stimulated cycles for the following groups: (1) short (8–18 days); (2) normal (19–24 days) with the use of cloprostenol and hCG; (3) normal – with the use of hCG only. Short, both natural and stimulated cycles, are divided into groups – with normal (4–6 days) and short estrus (2–3 days). The analysis of natural and stimulated cycles showed that the level of pregnancy rates in stimulated cycles (73.5%–80.8%) corresponds to this indicator in normal natural cycles (79.6%). In natural short cycles, the pregnancy rate significantly decreases (26.3% ± 7.1%). When analyzing short cycles, it turned out that, both in natural and stimulated cycles, the pregnancy rate depends on the length of the estrus and decreases in cycles with a short estrus. In general, the difference in pregnancy rates was 41.0% (p ≤ 0.001). In short stimulated cycles, with an estrus length of 4–6 days, the pregnancy rate is higher (91.5% ± 4.06%) than in cycles with an estrus of 2–3 days (51.7% ± 9.3%). Thus, the shortening of the estrous period in stimulated cycles has a significant effect on the pregnancy rate.

Maternal Perinatal Exposure to Dibutyl Phthalate Promotes Ovarian Dysfunction in Adult Female Offspring via Downregulation of TGF-β2 and TGF-β3

Maternal exposure to dibutyl phthalate (DBP) may result in ovarian dysfunction in female offspring. However, the underlying mechanisms remain elusive. Pregnant Sprague-Dawley rats were intraperitoneally injected with different doses of DBP, estradiol, and corn oil from gestational day 7 until the end of lactation. The reproductive characteristics, mRNA, and protein expression of ovaries for the adult female offspring were compared. KGN cells were cultured in vitro with DBP, estrogen receptor antagonist, or ALK-5 inhibitor. Genes, proteins, estradiol, and progesterone expressed by KGN, cell proliferation, and apoptosis were measured respectively. Maternal perinatal exposure to DBP induced prolonged estrous period, increased secondary follicles, significant decreased mRNA, and protein levels of TGF-β2, TGF-β3, and TGF-βRII in ovaries of the adult female offspring, but none difference for serum levels of sex hormones, ovarian TGF-β1, and estrogen receptor. The mRNA levels of LHR, FSHR, and CYP19a in ovaries were also decreased. DBP might decrease the mRNA of TGF-β2, TGF-β3, and TGF-βR II of KGN. DBP can inhibit the mRNA of CYP19 at 24h, which might be blocked by the estrogen receptor antagonist, whose effects were attenuated at 48h. DBP combined with FSH might time-dependently regulate the gene expression of TGF-βR II, inhibitory at 24h, but stimulative at 48h, which could be blocked by the ALK5 inhibitor. However, the protein expressed by KGN was not influenced by DBP. DBP stimulated the proliferation of KGN at 24h, which could be blocked by estrogen receptor antagonist, but attenuated at 48h. The progesterone in culture medium secreted by KGN was decreased by DBP at 24h. Maternal perinatal exposure to DBP induced decreased gene expression of TGF-β signaling and functional proteins in ovaries of the adult female offspring. Molecular cross-talk between estrogen receptor and TGF-β signaling pathway may play role in the mechanism of granulosa dysfunction induced by DBP.

Influence of Dingkun Dan Combined with Estradiol Valerateon Wnt/β-catenin Signaling Pathway in Rats with Thin Endometrium with Kidney-Yang Deficiency

To explore the influence for combination of Dingkun Dan with estradiol valerateon in treating rats with thin endometrium with Kidney-Yang deficiency based on Wnt/β-catenin signaling pathway. The estrous period 40 rats were randomly divided into the normal control group, the model group, the Dingkun Dan group, the estradiol valerateon group and the combination group, with 8 rats in each group. In addition to the normal control group, the rat model of thin endometrium with Kidney-Yang deficiency was established in other groups. The control group used free diet, the model group was given distilled water, the estradiol valerateon group was treated with progynova by gavage at 0.3 mg/(kg·d) , Dingkun Dan group was treated with Dingkun Dan by gavage at 2.26 g/(kg·d), and the combined group was given Dingkun Dan at 2.26 g/(kg·d) on the basis of progynova at 0.3 mg/(kg·d). After 3 estrous cycles, the rats were killed and harvested. HE staining was used to observe histopathologic changes in endometrium. The expression of VEGF in rats endometrium were detected by immunohistochemistry. The expression of β-catenin, E-cadherin and MMP-9 protein in rat endometrium was detected by Western blot. Compared with the control group, the uterine cavity was narrowed or enlarged, the endometrium glands and blood vessels were sparse, and the endometrium was thinner significantly in the model group ( P<0.01); the levels of VEGF was decreased significantly ( P<0.01), while β-catenin, E-cadherin and MMP-9 were increased significantly ( P<0.01). Compared with the model group, more endometrial glands, rich intimal vessels, the endometrium were thickened significantly in the 3 treatment groups ( P<0.01 or P<0.05); the levels of VEGF was increased differently. The protein levels of β-catenin and E-cadherin were significantly decreased in each treatment group ( P<0.01), and MMP-9 were significantly decreased in the Dingkun Dan group and in the combination group ( P<0.01). Compared with the estradiol valerateon group, the level of β-catenin in Dingkun Dan group was higher, and MMP-9 was lower ( P<0.05 or P<0.01). The levels of β-catenin and MMP-9 in the combination group were significantly decreased ( P<0.01). Compared with the combination group, the levels of β-catenin was increased significantly, while decreased signicantly in Dingkun Dan group ( P<0.01 or P<0.05). Dingkun Dan combined with estradiol valerateon can increase the thicken of the endometrium by up-regulation of VEGF, while down-regulate of β-catenin, E-cadherin and MMP-9 in rats with Shen-Yang deficiency and thin endometrium.

Evidence and manipulation of O-GlcNAcylation in granulosa cells of bovine antral follicles†.

Glucose is a preferred energy substrate for metabolism by bovine granulosa cells (GCs). O-linked N-acetylglucosaminylation (O-GlcNAcylation), is a product of glucose metabolism that occurs as the hexosamine biosynthesis pathway (HBP) shunts O-GlcNAc sugars to serine and threonine residues of proteins. O-GlcNAcylation through the HBP is considered a nutrient sensing mechanism that regulates many cellular processes. Yet little is known of its importance in GCs. Here, O-GlcNAcylation in GCs and its effects on GC proliferation were determined. Bovine ovaries from a slaughterhouse, staged to the mid-to-late estrous period were used. Follicular fluid and GCs were aspirated from small (3–5 mm) and large (>10 mm) antral follicles. Freshly isolated GCs of small follicles exhibited greater expression of O-GlcNAcylation and O-GlcNAc transferase (OGT) than large follicles. Less glucose and more lactate was detectable in the follicular fluid of small versus large follicles. Culture of GCs revealed that inhibition of the HBP via the glutamine fructose-6-phosphate aminotransferase inhibitor, DON (50 μM), impaired O-GlcNAcylation and GC proliferation, regardless of follicle size. Direct inhibition of O-GlcNAcylation via the OGT inhibitor, OSMI-1 (50 μM), also prevented proliferation, but only in GCs of small follicles. Augmentation of O-GlcNAcylation via the O-GlcNAcase inhibitor, Thiamet-G (2.5 μM), had no effect on GC proliferation, regardless of follicle size. The results indicate GCs of bovine antral follicles undergo O-GlcNAcylation, and O-GlcNAcylation is associated with alterations of glucose and lactate in follicular fluid. Disruption of O-GlcNAcylation impairs GC proliferation. Thus, the HBP via O-GlcNAcylation constitutes a plausible nutrient-sensing pathway influencing bovine GC function and follicular growth.

Impacts of Chromium and Selenium-E on Cortisol Levels, Reproductive and productive Efficiency of Baladi Female Goats under Subtropical Conditions.

The present study was conducted during estrous cycle and pregnancy period in Baladi female goats to study the effect of chromium and selenium with vitamin E on blood serum cortisol concentration. Seventy-two of mature Baladi does with age ranged from 2 to 3 years old and the mean body weight was 25.1±1.5 kg was used under two conditions of winter and summer seasons (36 animals/season). The animals were randomly divided into three groups, the 1st group was kept as control and the 2nd group was supplemented by chromium (chromium chloridetrivalent), 0.8 mg/head/day. The 3rd group was intramuscularly injected twice a week with 2ml viteselen®, contained 0.5 mg selenium and 10.7 IU vitamin E (Se-E). Serum cortisol levels were assessed in the three experimental goats during the estrous and pregnancy periods. The results demonstrated that Goats supplementation with Cr or Se-E significantly decreased (P<0.05) serum cortisol concentrations than control during estrous cycle. In addition, Cr mostly lowered (P<0.05) cortisol than control, but Se-E thoroughly increased it during pregnancy. However, under mild season at pro-estrus phase Cr treatment increased cortisol significantly (P<0.05) than control. Also, during pregnancy period Cr decreased cortisol at Mid-Pregnancy and Late-Pregnancy periods; however, Se-E increased cortisol at the Late-Pregnancy period than the control group. Under hot season, Cr treatment decreased (P<0.05) cortisol during estrous cycle, pregnancy period. However, injection of Se-E decreased cortisol during estrous cycle and showed a contrary effect during pregnancy period. On the other side, Reproductive traits of female goats showed that, under mild season conditions Cr or Se-E supplementations did not have any effect on goat’s reproductive traits, in terms of conception rate, kidding rate, fertility rate, prolificacy and fecundity compared to control group. Under hot season conditions, Cr and Se-E improved both of fertility and conception rate, as well as, increased fecundity percentage more than control. Cr group had lowest prolificacy, however; Se-E had the highest one compared to control. Chromium group had significantly (P<0.05) higher birth weight of kids than control and Se-E either at mild or hot season. However, injection of Se-E did not affect litter birth weight under mild and hot conditions.So,It can be concluded that Cr and Se-E supplementation can enhanced the reproductive and productive traits of female Baladi goats under hot season.