雌牛生殖道内游离氨基酸种类及含量分析

根据黄体的大小和形态，判断屠宰母牛的发情时期，分别收集发情第3天(D3)和第7天(D7)的输卵管液(OF)和子宫液(UF)，通过HPLC分析游离氨基酸种类及含量。总共检测到23种氨基酸，包括除Cys外的19种必需和非必需氨基酸，以及另外四种(βAla,Tau,Orm和Cit)非蛋白质氨基酸。OFD3、UFD3、OFD7和UFD7的游离氨基酸总量分别为31．6、46．9、26．3和17．2mmol/L，其中，Gly含量最高，分别为14．7、14．4、11．1和4．4，其次为Glu、Gln和A1a，其他氨基酸的含量均接近或低于lmmol/L。结果表明：氨基酸总量及个别氨基酸含量在不同体液及不同发育时期均存在一定程度的差异.     

The proliferative phase underpins endometrial development: Altered cytokine profiles in uterine lavage fluid of women with idiopathic infertility

Endometrial gland development occurs during the proliferative phase of a woman’s menstrual cycle, laying the foundation for the subsequent receptive, secretory phase when pregnancy is established. Idiopathic infertility has been rarely investigated with respect to the proliferative phase endometrium. We investigated whether gland development and/or altered secretion of cytokines during the proliferative phase is associated with infertility. Area of the glandular epithelium (GE) was measured in proliferative phase endometrial tissue collected from fertile (n = 18) and infertile (n = 14) women. Cytokines were measured in proliferative phase uterine lavage of fertile (n = 15) and infertile (n = 15) women. Immunohistochemistry determined cellular localisation of transforming growth factor alpha (TGFα) and interferon gamma (IFNγ) in proliferative phase endometrial tissue. For statistical analysis the cohort was divided into women <35 years and ⩾35 years. There were no significant differences in GE area of infertile and fertile women. C-C motif chemokine 11 (P = 0.048), TGFα (P = 0.049), IFNγ (P = 0.033) and interleukin-1 alpha (P = 0.047) were significantly elevated in uterine lavage from infertile women <35 years compared to fertile but not in women ⩾35 years. TGFα and IFNγ localised predominantly to GE in both the fertile and infertile endometrium. The potential impact of this altered proliferative phase environment on subsequent receptivity is discussed.     

Comparison of the cytobrush, cottonswab, and low-volume uterine flush techniques to evaluate endometrial cytology for diagnosing endometritis in chronically infertile mares

Endometritis is the most important cause of infertility in barren mares. The quick method of endometrial cytology (EC) has a relatively high reliability in diagnosing endometrial inflammation in the mare. For reliable cytological results, a collection technique that yields many well-preserved cells representative of a large uterine surface area without causing harm to the reproductive tract is required. The aim of the study was to compare three usually employed techniques for collection of endometrial and inflammatory cells (guarded cotton swab, uterine lavage, and cytobrush) in chronically infertile mares. Twenty Standardbred mares were used. In each mare, samples for EC were collected, first by a cotton swab (DGS), then by a cytobrush (CB), and finally by low volume flush (LVF). The slides were stained using the Diff Quick stain. The following parameters were assessed for each tested technique: background content of the slides; quality of the cells harvested; total cellularity; neutrophils; ratio PMN/uterine epithelial cells; inflammatory cells; vaginal epithelium cells. Categorical variables were compared using contingency tables and Pearson Chi-square tests, whereas continuous variables were compared using one-way analysis of variance (ANOVA); P < 0.05 was considered significant. Samplings by DGS and CB resulted easy and quick to perform via a single operator in all cases. LVF was performed easily, but required the presence of 2-3 players and took more time. The background content of the slides prepared by DGS appeared proteinaceous, slides prepared by LVF appeared contaminated by red blood cells or debris, whereas slides prepared by CB appeared clear. All smears showed a good total cellularity. The CB yielded significantly more cells (P < 0.0001) than DGS and LVF. The DGS produced significant more cells than LVF (P < 0.0001). The DGS produced significantly more (P = 0.003) intact cells than CB and LVF. Distorted cells were significantly (P = 0.001) more frequent in smears by LVF. The CB harvested significantly (P = 0.009) more fragmented cells. CB and LVF produced significantly (P < 0.0001; P = 0.02) more PMNs/HPF than DGS. In smears collected by LVF the proportion of PMNs/uterine epithelial cells was significantly (P = 0.0062; P = 0.0023) higher than in smears by CB and DGS. CB collected a significantly higher (P = 0.0011) proportion of PMNs than DGS. Acute endometritis was diagnosed in 50% (10/20) of the mares by DGS cytological samples, 25% (5/20) by CB, and 75% (15/20) by LVF. Inflammatory cells other than PMN (lymphocytes, macrophages, eosinophils) were collected exclusively by CB method. Epithelial cells from the vagina were only detected in LVF slides. The agreement of the diagnosis of endometritis between the three techniques of collection and between the different criteria adopted to evaluate smears obtained with the same technique was poor (k ≤ 0.3). In conclusion, results show that cytobrush and flush specimens were superior in all parameters to cotton swab smears. Even though the cytobrush technique requires specialized equipment, sample collection by this method was easier, more consistent, and quicker than the lavage method, indicating that the brush would be the preferred collection method for use on field in the mare. More studies are needed to establish criteria for interpretation of inflammation in the mare on cytobrush samples.     

Differential label‐free quantitative proteomic analysis of avian eggshell matrix and uterine fluid proteins associated with eggshell mechanical property

Eggshell strength is a crucial economic trait for table egg production. During the process of eggshell formation, uncalcified eggs are bathed in uterine fluid that plays regulatory roles in eggshell calcification. In this study, a label‐free MS‐based protein quantification technology was used to detect differences in protein abundance between eggshell matrix from strong and weak eggs (shell matrix protein from strong eggshells and shell matrix protein from weak eggshells) and between the corresponding uterine fluids bathing strong and weak eggs (uterine fluid bathing strong eggs and uterine fluid bathing weak eggs) in a chicken population. Here, we reported the first global proteomic analysis of uterine fluid. A total of 577 and 466 proteins were identified in uterine fluid and eggshell matrix, respectively. Of 447 identified proteins in uterine fluid bathing strong eggs, up to 357 (80%) proteins were in common with proteins in uterine fluid bathing weak eggs. Similarly, up to 83% (328/396) of the proteins in shell matrix protein from strong eggshells were in common with the proteins in shell matrix protein from weak eggshells. The large amount of common proteins indicated that the difference in protein abundance should play essential roles in influencing eggshell strength. Ultimately, 15 proteins mainly relating to eggshell matrix specific proteins, calcium binding and transportation, protein folding and sorting, bone development or diseases, and thyroid hormone activity were considered to have closer association with the formation of strong eggshell.     

Delayed follicular development and ovulation following inhibition of FSH with equine follicular fluid in the mare

In two experiments, PGF(2alpha) was given to all mares on Day 10 (ovulation = Day 0). In experiment 1, mares received either whole follicular fluid or saline i.v. every 12 hours on Days 10 to 14. Experiment 2 was similar to experiment 1, except the follicular fluid was extracted with charcoal to remove steroids. Analysis of the FSH data for Days 10 to 21 indicated an effect of treatment (P<0.08) with whole follicular fluid, but not with charcoal-extracted follicular fluid. However, there was an effect of day (P<0.05) and an interaction (P<0.01) of treatment with day for both experiments. The interaction of treatment with day seemed primarily due to a marked post-treatment increase in FSH concentrations between Days 15 and 17 for mares treated with either whole follicular fluid or charcoal-extracted follicular fluid. Analysis of the diameter of the largest follicle for Days 10 to 18 indicated a main effect of treatment (P<0.05) and day (P<0.05) and an interaction (P<0.05) of treatment with day for both experiments. The interaction of treatment with day was attributable to the inhibition of follicular growth by Day 14 for mares treated with whole follicular fluid and by Day 15 for mares treated with charcoal-extracted follicular fluid. The length of the interovulatory interval was longer (P<0.05) in the treated group than in controls for both experiments. Results indicated that equine follicular fluid contained a proteinaceous substance that suppressed circulating concentration of FSH. The inhibited follicular growth and the delay in ovulation were attributed to the reduced concentrations of circulating FSH.     

Treatment with metformin and sorafenib alleviates endometrial hyperplasia in polycystic ovary syndrome by promoting apoptosis via synergically regulating autophagy

This study aimed to investigate the molecular mechanisms underlying the roles of metformin (MET) and Sorafenib (SOR) in the treatment of endometrial hyperplasia (EH) in polycystic ovary syndrome (PCOS). Effects of MET and SOR on the area of endometrium and myometrium were detected. Western blot analysis and immunohistochemistry assays were carried out to detect the levels of mammalian target of rapamycin complex 1 (mTORC1), mTORC2, LC3-II, P62, and Caspase-3 in rats and cultured cells. Furthermore, cell counting kit-8 assay and flow cytometry analysis was carried out to determine the apoptotic profiles of treated cells. MET and SOR could apparently decrease the areas of endometrium and myometrium in PCOS. MET notably enhanced the expression of LC3-II and Caspase-3 in PCOS while substantially reducing the level of mTORC1 and P62. Similarly, SOR also enhanced the expression of LC3-II and Caspase-3 in PCOS while substantially reducing the level of mTORC2 and P62. Treatment with MET and SOR significantly inhibited the proliferation of HCC-94 and HEC-1-A cells while promoting their apoptosis by upregulating the expression of Caspase-3. In cells treated with MET, the expression of mTORC1 and LC3-II was upregulated while the expression of P62 was downregulated. Similarly, in cells treated with SOR, the expression of mTORC2 and LC3-II was also upregulated while the expression of P62 was also downregulated. Furthermore, MET showed no effect on mTORC2 expression, while SOR showed no effect on mTORC1 expression. In this study, we suggested that MET and SOR alleviated the risk of EH in PCOS via the mTORC1/autophagy/apoptosis axis and mTORC2/autophagy/apoptosis axis, respectively.     

Inflammatory cytokine concentrations in uterine flush and serum samples from dairy cows with clinical or subclinical endometritis

The objective of this study was to compare the concentrations of inflammatory cytokines in uterine flush and serum from healthy postpartum dairy cows and cows with clinical or subclinical endometritis. Clinical endometritis was diagnosed by observation of vaginal discharges (>50% pus) and subclinical endometritis was diagnosed by evaluation of uterine cytology (neutrophils >18%) at 4 weeks postpartum. Uterine flush was obtained from 48 cows at 4, 6, and 8 weeks postpartum for evaluation of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, and IL-10 concentrations. Serum samples were obtained from 34 cows just after calving and at 1, 2, 4, 6, and 8 weeks postpartum for evaluation of TNF-α, IL-1β, and IL-6 concentrations. Concentrations of TNF-α, IL-6, and IL-10 were greater (P < 0.05) in cows with clinical endometritis than in cows with subclinical endometritis and healthy controls, whereas concentrations of IL-8 in both cows with clinical and subclinical endometritis were greater (P < 0.005) than in controls. Overall, IL-6 and IL-10 concentrations decreased during the postpartum period. IL-1β concentrations in cows with clinical endometritis decreased (P < 0.0005) during the postpartum, whereas concentrations in cows with subclinical endometritis and controls did not change significantly with time; at 4 weeks postpartum, concentrations were greater (P < 0.0001) in cows with clinical endometritis. There were no significant effects of group, sampling time, or interaction on serum cytokine concentrations. In conclusion, cows with endometritis have greater inflammatory cytokine concentrations in uterine flush than healthy cows, but no differences were observed in serum.     

Follicular fluid vascular endothelial growth factor is associated with type of infertility and interferon alpha correlates with endometrial thickness in natural cycle in vitro fertilization

The aim of this study was to analyse the presence of vascular endothelial growth factor (VEGF) and interferon alpha (IFN-α) in the follicular fluid (FF) and their possible influence, as pro-angiogenic or anti-angiogenic factors, on in vitro fertilization outcome. The concentrations of VEGF and IFN-α were correlated with oocyte and embryo quality, concentrations of hormones in the serum, perifollicular blood flow and endometrial thickness. VEGF was detected in all FF samples (median 706.6?pg/ml, range 182.9–6638?pg/ml). IFN-α was detected in 60% of the samples (median 6.5?pg/ml, range 0–79.4?pg/ml), while in 40% of the samples its levels were below the test detection limit. VEGF and IFN-α concentrations did not correlate with the cause of infertility, concentrations of FSH, LH, E2 and prolactin, oocyte or embryo quality. Significantly higher concentrations of VEGF have been found in women with primary compared with secondary infertility (p?=?0.011, Mann Whitney test). The concentrations of VEGF and IFN-α did not correlate with the resistance index (RI) on days of hCG administration, follicular aspiration and embryo transfer. However, the concentrations of IFN-α correlated with endometrial thickness on the day of embryo transfer (Spearman correlation coefficient ρ?=?0.4107; P?<?0.05) but not on days of hCG administration and follicular aspiration. The mechanism of VEGF association with the previous ability of having a child needs to be clarified in future studies. The results of this study indicate a possible role of IFN-α in pathways of endometrial remodelling.