Interleukin-1 receptor antagonist expression in the equine endometrium during the peri-implantation period

To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0 = day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was found in the extracts of day 25 gravid endometrium and was immunochemically localized to the glandular epithelium/luminal cavity of the pregnant uterus. High concentrations of estradiol-17β (E₂) were detected in day 25 conceptuses. Concentrations of E₂ were higher in the gravid endometrial portion than in other endometrial regions. On the other hand, progesterone concentrations did not differ among endometrial samples analyzed. Furthermore, the expression of IL-1RN mRNA was up-regulated in endometrium culture samples treated with 10 ng/mL E₂ and 10 ng/mL progesterone. In the analysis of related gene expression, increased amounts of IL-1α and IL-6 mRNA were also found in the day 25 gravid endometrium; however, these expressions in endometrial culture samples were not up-regulated by the steroid treatment. These results indicate that expression of IL-1RN in the endometrium is likely regulated by E₂ and progesterone and suggest that IL-1RN regulates the degree of IL-1 signal transduction and thereby plays an important role in the establishment of equine pregnancy.     

Endometrial gland surface density and hyperaemia of the endometrium during early pregnancy in the mare

REASON FOR PERFORMING STUDY: The morphology and surface density of endometrial glands responsible for the nutrition of the placenta requires further study employing computer assisted morphometric analysis. OBJECTIVES: To analyse changes in the morphology and surface density of endometrial glands situated adjacent to, and remote from, the conceptus during the first 30 days of gestation. METHODS: Endometrial biopsies were recovered, under videoendoscopic control, from both nonpregnant and pregnant mares 12 days after ovulation, as well as from pregnant mares on Days 16, 20, 25 and 30 of gestation. One biopsy was taken at the base of the nongravid uterine horn and the other from beneath the conceptus at the base of the gravid horn. The samples were evaluated by computer assisted morphometric analysis to determine the surface area per unit volume (Sv) of endometrial glands in the stratum spongiosum. RESULTS: No differences in morphology or density of the glands were discernable between pregnant and nonpregnant mares on Day 12 day after ovulation. On Day 16, however, the glands beneath the conceptus appeared to be at a significantly lower density than the clearly more tortuous glands at the base of the nongravid horn. Between Days 20 and 30 of gestation the endometrium in direct contact with the conceptus was visibly more reddened and hyperaemic than elsewhere in the uterus. However, the Sv of glands beneath the conceptus had now increased again so they appeared similar to those in the nongravid horn, due mainly to a reduction in the level of stromal oedema in the former. CONCLUSIONS AND POTENTIAL RELEVANCE: The embryo-maternal interactions that are vital during early pregnancy to suppress the cyclical luteolytic cascade and support nidation of the conceptus, were characterised here with local endometrial hyperaemia, histotroph production and a decrease in endometrial gland Sv at the commencement of the stationary phase. Both the components secreted by the endometrial glands and the factors liberated by the conceptus during early pregnancy need further investigation.     

Effect of Conceptus on Transforming Growth Factor (TGF) β1 mRNA Expression and Protein Concentration in the Porcine Endometrium—In Vivo and In Vitro Studies

Transforming growth factor (TGF) β and its receptors are expressed at the conceptus-maternal interface during early pregnancy in the pig. The present studies were conducted to examine: (1) the effect of conceptus products on TGFβ1 mRNA expression and protein concentration in the porcine endometrium using in vivo and in vitro models, and (2) the effect of TGFβ1 on proliferation of porcine trophoblast cells in vitro. During in vivo experiments, gilts with one surgically detached uterine horn were slaughtered on days 11 or 14 of the estrous cycle and pregnancy. For in vitro studies, endometrial explants and luminal epithelial (LE) cells co-cultured with stromal (ST) cells were treated with conceptus-exposed medium (CEM). Moreover, porcine trophoblast cells were treated with TGFβ1, and the number of viable cells was measured. On day 11, the presence of conceptuses had no effect on TGFβ1 mRNA expression, but decreased the TGFβ1 protein concentration in the connected uterine horn compared with the detached uterine horn. In contrast to day 11, on day 14 after estrus, TGFβ1 mRNA expression and protein content in the endometrium collected from the gravid uterine horn were greater when compared with the contralateral uterine horn. The treatment of endometrial slices with CEM resulted in greater TGFβ1 mRNA expression and protein secretion. LE cells responded to CEM with an increased TGFβ1 mRNA level. Moreover, TGFβ1 stimulated the proliferation of day 14 trophoblast cells. In summary, porcine conceptuses may regulate TGFβ1 synthesis in the endometrium at the time of implantation. TGFβ1, in turn, may promote conceptus development by increasing the proliferation of trophoblast cells.     

αv β3 Integrin may participate in conceptus attachment by regulating morphologic changes in the endometrium during peri-implantation in ovine

The objective of this study was to determine expression and potential functions of α(v) and β(3) integrin subunits in ovine endometrium during the peri-implantation period (days 8-17 after fertilization). The morphologic changes in the endometrium were observed histochemically following haematoxylin/eosin (HE) staining, whereas the expressions of α(v) and β(3) integrin subunits were analysed by RT-PCR, immunohistochemistry and Western blot. The filamentous conceptus attached to the luminal epithelium (LE) on day 17 of pregnancy, with no differences in endometrial morphology between days 8-12 of pregnancy. However, endometrial glands in the endometrial stroma (S) underwent extensive hyperplasia from day 14 to day 17, increased reductus of the LE with an obvious proliferation of caruncles, and an increased number and diameter of blood vessels (V) in the endometrium. The relative expression levels of α(v) and β(3) integrin subunits mRNA gradually increased until day 16, but sharply declined on day 17. Western blot analysis revealed that the expression pattern of α(v) and β(3) integrin subunit proteins paralleled that of the corresponding mRNA. In addition, immunohistochemical localization of α(v) and β(3) integrin subunits confirmed their presence in the glandular epithelium (GE), LE and endometrial stroma. Immunostaining on LE and stroma varied with the increasing days of pregnancy, with the strongest immunostaining on days 16 and 17. In conclusion, expression of α(V) and β(3) integrin subunits was closely related to the early progression of pregnancy and conceptus attachment; therefore, we inferred that α(v) β(3) integrin may participate in conceptus attachment by the regulation of endometrial morphology during peri-implantation in ovine.     

Equine Endometrial Gland Density and Endometrial Thickness Vary among Sampling Sites in Thoroughbred Mares

The secretions of the equine endometrial glands are essential for the survival, growth, and development of the conceptus in early pregnancy, and endometrial gland density is directly related to successful pregnancy outcome. Endometrial biopsy is routinely used to assess the reproductive potential of broodmares. Some previous studies have shown that equine endometrial glands are uniformly distributed throughout the uterus; however, other work has shown variation of the endometrial architecture between biopsy sites, suggesting that a single biopsy is not representative of the entire endometrium. The aims of this study were to assess and compare the endometrial gland density and thickness at four sampling sites in the uterus (the central segment of each uterine horn, the uterine horn-body junction, and the caudal portion of the uterine body). Endometrial samples from five nulliparous Thoroughbred mares in diestrus were obtained at necropsy and used for subsequent histomorphometric analysis. The caudal uterine body had a significantly lower endometrial gland density and endometrial thickness than the other sites. This may result in nutrient deprivation and reduced survival of embryos or fetuses in this region of the uterus. The endometrial gland density and endometrial thickness did not significantly differ between the other regions sampled, indicating that they are similarly suitable for embryonic implantation and fetal development. Our results suggest that the endometrial structure of the caudal uterine body of the mare is not representative of the endometrial morphology at other sites. Thus, the caudal uterine body is not a suitable site for routine endometrial biopsy.     

Uteroferrin (ACP5) in the Ovine Uterus:Ⅰ. Regulation by Pregnancy and Progesterone

Uteroferrin,also known as type 5 tartrate resistant acid phosphatase ( ACP5 ) or TRAP,is an iron-containing glycoprotein secreted by uterine gland epithelium (GE) in response to progesterone and transported across the placental areoalae into the fetal circulation and allantoic fluid to deliver iron and to stimulate hematopoeisis in pigs.This study determined if ACP5 was expressed in the ovine uterus in response to pregnancy,progesterone,interferon tau,placental lactogen,and placental growth hormone.ACP5 protein was present in uterine GE of cycfic and early pregnant ewes,particularly between days 18 and 120 of pregnancy.ACP5 mRNA was expressed in uterine GE of cyclic and pregnant ewes in the same temporal and cell-specific manner.ACP5 was present in secretions from uterine glands,i.e.,uterine milk,and aUantoic fluid from days 40 to 80 of pregnancy,and in uterine flushings from cyclic and early pregnant ewes.Progesterone induced expression of ACP5 mRNA and intrauterine infusion of recombinant ovine interferon tau further stimulated ACP5 expression in uterine GE of ewes,but intrauterine injections of ovine placental lactogen and ovine growth hormone had no effect on ACP5 expression in uterine GE.These results indicate that ACP5 is:1 ) expressed only in GE in response to progesterone ;2 ) secreted into the uterine lumen and transported into the conceptus via plaeental areolae during pregnancy;and 3) present in secretions from uterine GE and in allantoic fluid.The roles of ACP5 in the ovine uterus may include transport of iron across the placenta and stimulation of hematopoiesis.     

Endometrial and conceptus expression of HoxA10, transforming growth factor β1, leukemia inhibitory factor,and prostaglandin H synthase-2 in early pregnant pigs with gonadotropin-induced estrus

This study was conducted to evaluate the effect of estrus induction with gonadotropins on endometrial and conceptus expression of HoxA10, transforming growth factor (TGF) β1, leukemia inhibitory factor (LIF), and prostaglandin H synthase-2 (PGHS-2) during early pregnancy in pigs. Twenty-four prepubertal gilts received 750 IU of pregnant mare serum gonadotropin (PMSG) and 500 IU of human chorionic gonadotropin (hCG) 72 h later. Gilts in the control group (n = 23) were observed daily for estrus behavior. Endometrial tissue samples, conceptuses, blood serum, and uterine luminal flushings (ULFs) were collected on days 10, 11, 12, and 15 after insemination. There was no effect of estrus induction on estradiol content in ULFs, or on ovulation and fertilization rates in studied gilts. However, the content of progesterone in the blood serum was greater in naturally ovulated gilts in comparison to gonadotropin-treated animals on day 12 of pregnancy (P < 0.05). HoxA10 expression was up-regulated in the endometrium of pregnant gilts, with natural ovulation on days 12 (P < 0.05) and 15 (P < 0.001) in comparison to days 10 and 11. When compared to control gilts, administration of PMSG/hCG resulted in decreased expression of endometrial HoxA10, TGFβ, LIF, and PGHS-2 on day 12 of pregnancy (P < 0.05). Conceptus expression of studied factors was not affected by gonadotropin treatment. Overall, these results suggest improper endometrial preparation for implantation in prepubertal gilts induced to ovulate with PMSG/hCG.     

Uterine secretions of the cow contain proteins that are immunochemically related to the major progesterone-induced proteins of the sheep uterus

Proteins that cross-react with antiserum to the major progesterone-induced proteins found in the pregnant sheep uterus, the uterine milk proteins (UTM-proteins), were detected as radiolabelled secretory products of endometrium from pregnant cows. Cross-reactive proteins included a form at 57,000 molecular weight as well as other lower-molecular-weight variants found in lower amounts. Similar proteins were also detected in uterine fluid from a cow at day 270 of gestation. Using immunohistochemical procedures, proteins that cross-reacted with antiserum to sheep UTM-proteins could be localized to the epithelial cells of endometrial glands in the cow. Results indicate that UTM-protein-like molecules are secreted by the endometrium of the cow during pregnancy.     

The expression of interferon-stimulated gene 15 in equine endometrium

Establishment of pregnancy is critically dependent upon a precisely orchestrated embryo-maternal interaction leading to a receptive uterine environment. The up-regulation of the interferon-stimulated protein 15 kDa (ISG15) during pregnancy has been described in various species and has been hypothesized to be part of the molecular repertoire that makes the uterus receptive to conceptus development. In the current study, the expression of ISG15 and enzymes involved in ISG15ylation was examined at the mRNA and protein level in equine endometrium at Day 14 of the luteal phase and at Day 14 and 50 of pregnancy. ISG15 mRNA showed a 2.63-fold higher expression at Day 14 of pregnancy when compared to Day 14 of the cycle, while mRNA abundance at Day 50 of pregnancy was unchanged compared to Day 14 of the cycle. Upon Western blot analysis using anti-ISG15 antibody, several higher molecular weight bands could be observed, representing proteins conjugated to ISG15. No free ISG15 could be detected. The pattern of ISG15 reactive proteins differed from those observed in non-uterine samples. Upon immunohistochemistry, ISG15 reactive proteins located primarily to luminal and glandular epithelial cells, while stromal cells showed weaker staining. In conclusion, the expression of ISG15-conjugated proteins in equine endometrium did not differ between cyclic and pregnant 14 days after ovulation and Day 50 of pregnancy. It is hypothesized that the unique subset of ISG15ylated proteins expressed in endometrial tissue contributes to normal cellular function and that, unlike other species, the modification of ISG15-conjugated proteins is not an active contributor to conceptus-maternal interaction in the mare.     

绵羊妊娠识别、建立和维持的信号系统

妊娠的建立和维持归因于胚体的信号和黄体对孕酮的产生,在反刍动物,胚体滋养层分泌的激素抑制子宫内膜产生前列腺素PGF2α。在发情周期的绵羊,孕酮下调子宫内膜腔上皮和子宫腺上皮孕酮受体基因的表达,并伴随着上皮雌激素受体和催产素受体的增加。在催产素的作用下,子宫开始分泌溶黄体作用的前列腺素F2α。在妊娠绵羊,孕体滋养层产生的干扰素可作用于子宫内膜而直接抑制雌激素受体基因和催产素受体基因的转录。孕酮、干扰素、胎盘催乳素和生殖激素共同组成了一个"作用网络"来调节子宫的功能分化和子宫腺的形态发生,从而维持绵羊妊娠。本文综述了绵羊妊娠识别、建立和维持的信号系统。     

Retinol and estradiol regulation of retinol binding protein and prostaglandin production by porcine uterine epithelial cells in vitro

Secretion into the uterine lumen follows a precise pattern during early pregnancy. Near the end of the second week of pregnancy and coincident with elongation of conceptuses, retinol, retinol binding protein (RBP), estradiol (E2), and prostaglandins E (PGE) and F (PGF) increase in the uterine lumen, and RBP mRNA increases in the endometrium. In the present studies the potential for E2 (0.1 microM) and retinol (10 microM) to regulate RBP and PG production by cultured luminal (LEC) and glandular (GEC) epithelial cells collected from postpubertal females and LEC from prepubertal gilts was examined. Endometrial tissue was collected surgically from cyclic and pregnant females (n = 8) on d 10 and 13 postestrus (first day of estrus = d 0) and from 120- and 150-d-old prepubertal gilts that were treated with progesterone (P4) (2.2 mg x kg(-1) x d(-1), n = 6) or corn oil (n = 6) for 14 d prior to tissue collection. The LEC from postpubertal females responded to retinol with increased (P < 0.05) RBP, PGE, and PGF in culture medium and increased (P < 0.07) RBP mRNA but E2 decreased (P < 0.05) RBP and RBP mRNA and had no effect on prostaglandins. No E2 or retinol effects on secretions of GEC occurred in vitro, but a day x pregnancy status interaction (P < 0.06) affected PGE output by the GEC. Secretion of PGE was greater when GEC were collected on d 10 of pregnancy than from d-10 cyclic or d-13 pregnant or cyclic females. Both E2 and retinol stimulated (P < 0.05) secretion of RBP by LEC isolated from prepubertal gilts, but their effects were not additive. In vivo treatment of prepubertal gilts with P4 increased (P < 0.05) RBP and decreased (P < 0.05) PG production by LEC in vitro. Therefore responses to E2 and retinol differ between pre- and post-pubertal females, and retinol may function in the regulation of endometrial RBP and PG secretion.