Anandamide Levels Fluctuate in the Bovine Oviduct during the Oestrous Cycle

Mammalian oviduct acts as a reservoir for spermatozoa and provides an environment in which they may compete for the opportunity to fertilize the oocyte. Whilst in the oviduct spermatozoa undergo capacitation essential for fertilization. Sperm-oviduct interaction is essential for sperm capacitation and is a tightly regulated process influenced by the local microenvironment. Previously we reported that the endocannabinoid anandamide (AEA) regulates sperm release from epithelial oviductal cells by promoting sperm capacitation. The aims of this work were to measure the AEA content and to characterize the main AEA metabolic pathway in the bovine oviduct and determine how these change through the oestrous cycle. In this study, the levels of AEA and two other N-acylethanolamines, N-oleoylethanolamine and N-palmitoylethanolamine, were measured in bovine oviduct collected during different stages of oestrous cycle by ultra high performance liquid chromatography tandem mass spectrometry. Results indicated that intracellular oviductal epithelial levels of all three N-acylethanolamines fluctuate during oestrous cycle. Anandamide from oviductal fluid also varied during oestrous cycle, with the highest values detected during the periovulatory period. Endocannabinoid levels from ipsilateral oviduct to ovulation were higher than those detected in the contralateral one, suggesting that levels of oviductal AEA may be regulated by ovarian hormones. The expression and localization of N-acylethanolamines metabolizing enzymes in bovine oviduct were also determined by RT-PCR, Western blot, and immunohistochemistry but no change was found during the oestrous cycle. Furthermore, nanomolar levels of AEA were detected in follicular fluids, suggesting that during ovulation the mature follicle may contribute to oviductal AEA levels to create an endocannabinoid gradient conducive to the regulation of sperm function for successful fertilization.     

Bovine Steroid Hormone and SHBG Concentrations Postpartum and during the Oestrous Cycle

Changes in consecutive estimates of milk progesterone concentrations and serum steroid hormone and sex hormone-binding globulin (SHBG) concentrations in the postpartum period were examined in Finnish Ayrshire and Friesian dairy cows which were divided according to feeding into a hay group and a silage group. Milk progesterone concentrations rose above 10 nmol/1, indicating the start of ovarian luteal activity, slightly earlier in the silage group (28.4 ± 8.7 (S.D.) days, n = 19) than in the hay group (33.4 ± 10.3, n = 28) after calving. Likewise, the first normal oestrous cycles began slightly earlier in cows fed with silage. On the other hand, no differences in the beginning of ovarian luteal activity were observed between the breeds. Serum oestradiol-17β, oestrone, testosterone, 5α-dihydrotestosterone (5α-DHT), pregnenolone and progesterone concentrations were fairly unchanged during postpartum anoestrus after uterine involution and before ovarian cyclic activity. After first ovulation, considerable increases in milk and serum progesterone concentrations were observed. The increase was accompanied by elevations in serum pregnenolone and 5α-DHT concentrations. In the late luteal phase, progesterone, 5α-DHT and pregnenolone concentrations rapidly declined, leading to low hormone levels in pro-oestrus. Thereafter, serum pregenolone and 5α-DHT concentrations slightly increased during the follicular phase. On the other hand, oestradiol-17β concentrations were elevated in pro-oestrus and decreased after that, being lowest at met-oestrous. Serum testosterone concentrations appeared to be unchanged during postpartum anoestrus and over the oestrous cycle. Serum SHBG concentrations were unchanged during postpartum anoestrus and over the oestrous cycle, as well as in pregnant animals. The serum SHBG concentrations were about double those found in women with normal menstrual cycles, whereas oestradiol concentrations were much lower. At present, it cannot be explained how the biological effects of oestradiol become evident under such conditions.     

KAI1/CD82 在早孕小鼠子宫内膜组织的表达研究

目的：观察KAI1／CD82 mRNA和蛋白在小鼠妊娠D1-D8子宫内膜组织的表达。方法：以胚胎与肿瘤同源性为理论基础,胚胎植入与肿瘤侵袭转移相似为切入点,采用免疫组化和RT-PCR技术。结果：KAI1／CD82 mRNA和蛋白在早孕子宫中,KAI1／CD82mRNA的表达渐增多,蛋白表达的量和范围也渐增强。结论：KAI1／CD82mRNA和蛋白在早孕子宫组织中的动态表达,提示它在胚胎精确侵袭子宫内膜的调节中发挥作用,是滋养层细胞精确侵袭调控的分子机制之一。     

Non-identical Distribution Pattern of Epidermal Growth Factor and Platelet-derived Growth Factor in the Mouse Uterus During the Oestrous Cycle and Early Pregnancy

In the present study, we examined by immunohistochemistry the cell-specific distribution of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) in the mouse uterus during the oestrous cycle and throughout the first 7 days of pregnancy. Paraffin-embedded tissue samples were immunostained using the avidin–biotin peroxidase technique and then examined by light microscopy. Our results showed that immunostaining for EGF was detected in the stroma but not in the luminal or glandular epithelium. A high concentration of EGF was detected in the stroma around the time of embryo implantation at days 3, 4 and 5 of pregnancy. The implanted embryo at day 7 of gestation showed immunostaining for EGF between the ectoderm and endoderm layers. The cell distribution pattern for PDGF was found to be different from that observed with EGF. Luminal and glandular epithelia displayed PDGF immunostaining throughout the first 7 days of pregnancy, with the highest intensity at days 4 and 5 of gestation. In contrast, no immunostaining was observed in the luminal and glandular epithelia at post-oestrus, dioestrus and pro-oestrus stages. However, a weak reaction started to appear at oestrus. The embryo at the blastocyst stage displayed a strong immunoreaction for antibody against PDGF. In addition, the decidual boundary zone surrounding the implanted embryo at days 5, 6 and 7 of gestation also showed an immunostaining for PDGF. The present observations demonstrate clearly the presence of EGF and PDGF in the mouse uterus in high concentrations at the peri- implantation period. Thus, our results, together with what is known about the effect of EGF and PDGF in controlling the growth, differentiation and activation of a variety of cell types, suggest a possible role for these growth factors during the preparation of the endometrium for implantation in controlling the proliferation activity of stromal and/or epithelial cells.     

妊娠期家兔子宫内膜的鞘糖脂表达

 妊娠期家兎子宫内膜的神经节苷脂(Gls)的含量明显低于动情期的,而中性鞘糖脂(NGSL)的含量则以妊娠中、晚期的最高,动情期最低。鞘糖脂组成变化最显著的是妊娠早期,由动情期到早孕GM_3从28.0％增加到52.7％,CMH.CDH由未测出分别增加到29.2％和21.9％,而糖链复杂的组分GD_3,GTlb和CPH的百分含量则明显减少,到妊娠中、晚期、短糖链组分逐渐减少,而复杂糖链组分渐增。中期妊娠内膜的(GIs)以GD_3为主要组分,占45％,明显高于其它各期。NGSL在妊娠中、晚期CPH增高达70％,与动情期水平相当。结果提示,妊娠期间子宫内膜的鞘糖脂含量与组成均发生明显变化,这些变化可能与子宫功能密切相关。特别是早孕对的变化,推测与子宫内膜和胚泡的识别,粘连特性的获得有关。     

大鼠发情期和间情期下丘脑ghrelin mRNA的表达

为探索下丘脑ghrelin mRNA及GnRH mRNA在大鼠(Rattus norregicus)发情期和间情期的表达特点,通过外部观察和阴道涂片相结合的方法确定发情期和间情期,将12只未经产SD雌性大鼠分为2组,即发情期组和间情期组,每组6只。取动物下丘脑,用实时荧光RT-PCR方法检测ghrelin mRNA和GnRH mRNA的表达丰度。结果表明,间情期组大鼠下丘脑ghrelin mRNA的表达丰度显著高于发情期组(P<0.01);间情期组大鼠下丘脑GnRH mRNA的表达丰度显著低于发情期组(P<0.01)。研究发现,下丘脑ghrelin mRNA和GnRH mRNA在发情期与间情期具不同的表达模式,提示ghrelin可能在下丘脑水平上对GnRH mRNA的表达具下调作用。     

Possible Roles of mmu-miR-141 in the Endometrium of Mice in Early Pregnancy Following Embryo Implantation

Objective

Embryo implantation is directly affected by genes related to uterine receptivity. Studies have demonstrated the important roles of miRNAs in the regulation of gene expression. Our early miRNA chip analyses revealed that the mmu-miR-141 expression in endometrial tissue is lower after embryo implantation than before it. However, the possible roles of miR-141 in embryo implantation have not yet been elucidated. Here, mmu-miR-141 was designed to detect the expression and role of miR-141 in the endometria of mice in early pregnancy following embryo implantation.

Methods

Real-time PCR and in-situ hybridization were used to study mmu-miR-141 expression in mouse uterus. Cell proliferation was detected by tetrazolium dye (MTT) assay and flow cytometry. Real-time PCR and Western blot analysis were used to confirm the mRNA and protein levels of phosphatase and tensin homolog (PTEN) to determine whether it was the target gene of mmu-miR-141. Enhanced green fluorescent protein (EGFP) fluorescence reporter vector analysis was also performed. A functional study was performed by injecting mice uteri with mmu-miR-141 inhibitor or mimic vectors.

Results

mmu-miR-141 expression was lower on day 6 (D6) than day 4 (D4) and could be increased by progesterone. Reduced mmu-miR-141 could decrease the proliferation activity of stromal cells and promote apoptosis. Upregulation of mmu-miR-141 inhibited PTEN protein expression but downregulation of mmu-miR-141 increased it, while the mRNA level remained unchanged. EGFP fluorescence reporter vector analysis showed that miR-141 targets the 3′-untranslated region of the PTEN mRNA. In addition, when the physiological mmu-miR-141 level was altered on D2 by injecting with inhibitor or mimic, the embryo implantation sites were significantly decreased on D7.

Conclusions

This study demonstrated that mmu-miR-141 might influence cell proliferation and apoptosis in the endometrium by negatively regulating PTEN expression, and could also influence the number of embryo implantation sites. mmu-miR-141 plays an essential role in embryo implantation.     

Some Behavioural Changes Associated with the Guinea-pig Oestrous Cycle

This paper describes some behaviour patterns of the guinea-pig, Cavia porcellus, which vary systematically with the oestrous cycle. Oestrus is characterised by an increase in locomotion, and scent-marking, as well as by greater interest in conspecifics. All of these are behaviours known to increase at oestrus in other rodent species. The significance of these changes for the social organisation of this species is discussed.     

MicroRNA Profiles in Spontaneous Decidualized Menstrual Endometrium and Early Pregnancy Decidua with Successfully Implanted Embryos

To comparatively analyze the human microRNA (miRNA) profiles between spontaneous decidualized menstrual endometrium and early pregnancy decidua by an in-depth sequencing of miRNAs. The specific miRNAs expressed at conception might be involved in pregnancy establishment and expression of let-7f-5p and let-7g-5p was experimentally up-regulated or inhibited to assess the effect on the expression of IGF2BP-1 and IGF2R in vitro, respectively. Samples of endometria and deciduas were obtained from 25 women who suffered from tubal or male factor subfertility and from 35 early pregnant women who underwent pregnancy termination at 6–8 weeks gestation were irrespectively collected and comparatively analyzed by miRNA sequencing and differential expression of known and novel miRNAs was analyzed using bioinformatics. The 2042 miRNA expression was analyzed in the study and the differential expression of six miRNAs was validated by qRT-PCR. The expression of four miRNAs in decidua samples was down-regulated (miR-34c, miR-92a, miR-181a-5p, and miR-191), whereas the expression of miR-10a-5p and let-7f-5p was significantly up-regulated. The expression of IGF2BP-1 and IGF2R declined and increased with overexpression and inhibition of let-7f-5p and let-7g-5p, respectively. Changes in the expression of particular miRNAs might play a role in the physiology of decidualization following successful embryo implantation, ultimately resulting in continuous decidualization.     

Fluctuation of Aquaporin 1, 5, and 9 Expression in the Pig Oviduct during the Estrous Cycle and Early Pregnancy

Thirteen mammalian aquaporin (AQPs) isoforms with a unique tissue-specific pattern of expression have been identified. To date, 11 isoforms of AQP have been reported to be expressed in female and male reproductive systems. The purpose of our study was to determine the localization and quantitative changes in the expression of AQP1, 5 and 9 within the pig oviduct during different stages of the estrous cycle and early pregnancy. The results demonstrated that AQP1, 5, and 9 were clearly detected in all studied stages of the estrous cycle and pregnancy. AQP1 was localized within oviductal blood vessels. In cyclic gilts, the expression of AQP1 protein did not change significantly between days 10–12 and 14–16 but increased on days 2–4 and 18–20. AQP5 was localized in smooth muscle cells and oviductal epithelial cells. The expression of AQP5 protein did not change significantly between days 10–12 and 14–16 of the estrous cycle but increased on days 2–4 and 18–20. The anti-AQP9 antibody labeled epithelial cells of the oviduct. The expression of AQP9 did not change significantly between days 10–12 and 14–16 of the estrous cycle but increased on days 2–4 and 18–20. In pregnant gilts, expression of AQP1, 5, and 9 did not change significantly in comparison with the estrous cycle. Therefore, a functional and distinctive collaboration seems to exist among diverse AQPs in water handling during the different oviductal phases in the estrous cycle and early pregnancy.     

Plasma Progesterone in Gilts During the Prepuberal Period and the First Oestrous Cycle

The progesterone concentration of peripheral plasma in pigs has previously been studied during the oestrous cycle and in early pregnancy (Tillson & Erb 1967, Stabenfeldt et al 1969). The present study was undertaken to obtain some information on the concentration of plasma progesterone during the prepuberal period and during the first oestrous cycle in the pig.     

Inflammation-Related Molecules at the Maternal–Fetal Interface during Pregnancy and in Pathologically Altered Endometrium

The blastocyst expresses paternally derived alloantigens and induces inflammation during implantation. However, it is necessary for the onset of pregnancy. An abnormal response might result in a pathological course of pregnancy or pregnancy failure. On the other hand, a state of maternal immune tolerance is necessary to ensure the normal development of pregnancy by suppressing inflammatory processes. This article discusses recognized mechanisms and the significance of inflammatory processes for embryo implantation and pregnancy establishment. We would also like to present disorders involving excessive inflammatory response and their influence on events occurring during embryo implantation. The chain of correlation between the processes responsible for embryo implantation and the subsequent physiological course of pregnancy is complicated. Many of those interrelationships are still yet to be discovered. Undoubtedly, their recognition will give hope to infertile couples for the emergence of new treatments that will increase the chance of giving birth to a healthy child.     

The Expression Pattern of microRNAs in Granulosa Cells of Subordinate and Dominant Follicles during the Early Luteal Phase of the Bovine Estrous Cycle

This study aimed to investigate the miRNA expression patterns in granulosa cells of subordinate (SF) and dominant follicle (DF) during the early luteal phase of the bovine estrous cycle. For this, miRNA enriched total RNA isolated from granulosa cells of SF and DF obtained from heifers slaughtered at day 3 and day 7 of the estrous cycle was used for miRNAs deep sequencing. The results revealed that including 17 candidate novel miRNAs, several known miRNAs (n = 291–318) were detected in SF and DF at days 3 and 7 of the estrous cycle of which 244 miRNAs were common to all follicle groups. The let-7 families, bta-miR-10b, bta-miR-26a, bta-miR-99b and bta-miR-27b were among abundantly expressed miRNAs in both SF and DF at both days of the estrous cycle. Further analysis revealed that the expression patterns of 16 miRNAs including bta-miR-449a, bta-miR-449c and bta-miR-222 were differentially expressed between the granulosa cells of SF and DF at day 3 of the estrous cycle. However, at day 7 of the estrous cycle, 108 miRNAs including bta-miR-409a, bta-miR-383 and bta-miR-184 were differentially expressed between the two groups of granulosa cell revealing the presence of distinct miRNA expression profile changes between the two follicular stages at day 7 than day 3 of the estrous cycle. In addition, unlike the SF, marked temporal miRNA expression dynamics was observed in DF groups between day 3 and 7 of the estrous cycle. Target gene prediction and pathway analysis revealed that major signaling associated with follicular development including Wnt signaling, TGF-beta signaling, oocyte meiosis and GnRH signaling were affected by differentially expressed miRNAs. Thus, this study highlights the miRNA expression patterns of granulosa cells in subordinate and dominant follicles that could be associated with follicular recruitment, selection and dominance during the early luteal phase of the bovine estrous cycle.     

人体子宫内膜中性鞘糖脂和神经节苷脂的含量和组成在月经周期中和妊娠时的变化

从月经周期各期、早妊(6—8周)及足月妊娠子宫内膜中提取、纯化了神经节苷脂(Gls)和中性鞘糖脂(N-GSL),分别测定了其含量。对两类鞘糖脂的组成进行了HPTLC图谱分析。初步观察了不同时期子宫内膜中CMP—NeuAc:LacCer唾液酰转移酶(ST_1)和CMP-NeuAc:CM_3唾液酰转移酶(ST_2)的活性。结果表明:分泌期的GLs总含量低于增生期(P<0.02);分泌期GD_3含量较生长期增多(P<0.01);妊娠后,GM_3含量增加,而GD_3减少;相应地,ST_1活性增高,ST_2活性降低。分泌期CMH含量为增生期的4.7倍。结果提示子宫内膜鞘糖脂含量和组成的变化可能与子宫的功能有关,而且受女性激素水平的影响。