Embryo gene expression in pig pregnancy

Pregnancy is a complex process in which significant changes occur continually in both the corpora lutea and in the endometrium of the females and varies depending on the embryonic, pre-implantation or foetal stages. In the embryonic stages, the majority of genes expressed in the pig embryo correspond to the loss of cellular pluripotency. In contrast, the implantation consists of three phases: elongation of the conceptus, adhesion and union of the embryo to the endometrial epithelium. During these phases, many factors are expressed, including growth factors, molecules that facilitate adhesion and cytokines. All these changes are ultimately regulated by different lipid and hormonal substances, specifically by progesterone, oestradiol and prostaglandins, which regulate the expression of many proteins necessary for the development of the embryo, endometrial remodelling and embryo–maternal communication. This paper is a review of primary gene regulatory mechanisms in pigs during different stages of implantation.     

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。     

哺乳动物子宫内膜腺的发育与调控

哺乳动物的子宫内膜腺可合成和分泌或转运一些蛋白及相关物质 ,这些物质在胎儿存活、发育、妊娠识别信号的启动及胚胎着床等方面起重要的作用。子宫内膜腺的发育包括内膜上皮出芽形成内膜腺上皮、内膜腺上皮管穿过固有层及内膜腺上皮卷曲和分支等几个连续的过程。哺乳动物子宫内膜腺的形态发生受许多因素的调控 ,但确切的调控机理目前尚不完全清楚     

Concentration effect of prostaglandin E2 on the growth factor expression and cell proliferation in bovine endometrial explants and their kinetic characteristics

Bovine endometrium undergoes various physiological and histological changes that are necessary for blastocyst implantation during oestrous cycle. From pro‐oestrus to late‐oestrus, endometrium thickens gradually for implantation preparation and exhibits remarkable capacity for self‐repair after uterine lining shedding while implantation does not occur. The prostaglandin E₂ (PGE ₂) secretion pattern is synchronized with endometrial growth during oestrous cycles in bovine endometrium; however, limited information is available regarding the association between PGE ₂ secretion and endometrial growth. In this study, the concentration (10^?9 to 10^?5 M) and time effect (2–36 hr) of PGE ₂ treatment on a series of growth factors are essential for endometrial growth including connective tissue growth factor (CTGF ), fibroblast growth factor‐2 (FGF ‐2), interleukin‐8 (IL ‐8), transforming growth factor‐β1 (TGF ‐β1), matrix metalloproteinase‐2 (MMP ‐2), and vascular endothelial growth factor A (VEGFA ) mRNA and protein expression, and proliferation of epithelial and fibroblast cells was investigated in bovine endometrial explants in vitro. The results indicated that PGE ₂ at concentration about 10^?7 to 10^?5 M could up‐regulate CTGF , FGF ‐2, IL ‐8, MMP ‐2, TGF ‐β1, VEGFA mRNA and protein expression, and could induce the proliferation of epithelial and fibroblast cells and reduce the proapoptotic factor (caspase‐3) expression in bovine endometrial explants in vitro. These results collectively improved the possibility of PGE ₂ functions in endometrial growth during oestrous cycles.     

The potential function of endometrial‐secreted factors for endometrium remodeling during the estrous cycle

Uterine has a pivotal role in implantation and conceptus development. To prepare a conducive uterine condition for possibly new gestation during the estrous cycle, uterine endometrium undergoes dramatic remodeling. In addition, angiogenesis is an indispensable biological process of endometrium remodeling. Furthermore, essential protein expressions related to important biological processes of endometrium remodeling, which are vascular endothelial growth factor (VEGF), myoglobin (MYG), collagen type IV (COL4), fucosyltransferase IV (FUT4), and cysteine‐rich protein 2 (CRP2), were detected in the endometrial tissue reported in many previous studies and recently discovered in histotroph substrates during the estrous cycle. Those proteins, which are liable for provoking new vessel development, cell proliferation, cell adhesion, and cell migration, were expressed higher in the histotroph during the luteal phase than follicular phase. Histotroph proteins considerably contribute to endometrium remodeling during the estrous cycle. To that end, the following review will discuss and highlight the relevant information and evidence of the uterine fluid proteins as endometrial‐secreted factors that adequately indicate the potential role of the uterine secretions to be involved in the endometrial remodeling process.     

Production of calcium maintenance factor Stanniocalcin-1 (STC1) by the equine endometrium during the early pregnant period

A factor responsible for progression to pregnancy establishment in the mare has not been definitively characterized. To identify factors possibly involved in the establishment of equine pregnancy, the endometrium was collected from day 13 (day 0=day of ovulation) cyclic and day 13, 19 and 25 pregnant animals. From initial subtractive hybridization studies, a calcium regulating factor, Stanniocalcin-1 (STC1) mRNA, was found as a candidate molecule expressed uniquely in the pregnant endometrium. Endometrial expression of STC1 mRNA was noted on day 19 and was markedly increased in the day 25 gravid endometrium. STC1 protein was found in the extracts of day 25 gravid endometrium and immunochemically localized in the uterine glands. In addition, STC1 protein was detected in uterine flushing media collected from day 25 pregnant mares. High concentrations of estradiol-17 β (E(2)) were detected in day 25 conceptuses. E(2) levels were much higher in the gravid endometrium than in other regions, whereas progesterone levels did not differ among the samples from different endometrial regions. Expression of STC1 mRNA, however, was not significantly upregulated in cultured endometrial explants treated with various concentrations of E(2) (0.01-100 ng/ml) with or without 10 ng/ml progesterone. These results indicate that an increase in STC1 expression appears to coincide with capsule disappearance in the conceptus, and suggest that STC1 from the uterine glands likely plays a role in conceptus development during the pregnancy establishment period in the mare.     

Two concepts on the immunological aspect of blastocyst implantation

The process of blastocyst implantation is a series of interactions between the blastocyst and maternal tissues. The purpose of this process is (1) to provide nourishment to the embryo for developmental growth in appropriate physiological and endocrinological environment until a placenta is established, and (2) to protect the (semi-)allogeneic embryo from any attacks from the maternal immune system. To facilitate successful implantation, therefore, these two aspects of the embryonic demand must be satisfied in the embryo-maternal interface throughout the entire process of implantation. The first concept I present in this paper is that blastocyst implantation essential factors (BIEFs) have dual functions: one, for structural and functional modification of the endometrium to accommodate the developing embryo and provide nourishment and suitable environment for its development, and the other, for modulation, directly or indirectly, of the maternal immune system to prevent attacks by the maternal immune system. The second concept is that BIEFs convert the endometrium (or uterus) from an immunologically non-privileged site to a privileged site. This endometrial (uterine) conversion is the immunological aspect of the blastocyst implantation process. When the endometrium has become receptive for blastocyst implantation, it signifies that the immunological conversion of the endometrium by BIEFs has been sufficiently attained to let the embryo start contacting maternal tissues. During the early stages of placentation, as the trophoblast cells differentiate and make their way to the maternal blood vessels to establish the placenta, BIEFs continuously provide nourishment and immunological protection to the developing embryo. The immunological protection of the embryo/fetus from potential attacks by the maternal immune system appears to reach a peak at the time of establishment of the placenta. Thus, clarification of the roles of BIEFs in both the physiological/endocrinological aspect as well as the immunological aspect is essential for understanding the biological process of implantation.     

猪胚胎附植部分因子研究进展

产仔数性状是养猪业中一个重要的经济性状,而胚胎附植对猪产仔数的高低有重要的影响。胚胎附植的调控涉及到多个生物学过程,在其中发挥作用的物质（附植因子）很多。作者针对孕酮、孕酮受体、雌二醇、雌激素受体α（ESR1）和促红细胞生成素产生肝细胞受体配体（Eph-ephrin）系统这几种附植因子的相关研究进行了归纳总结,重点阐述了这些因子在母胎对话过程中的时空表达、功能、突变、调控等方面的研究进展。其中,孕酮及其受体在猪胚胎附植活动中全程发挥着重要作用,黄体分泌的孕酮与母猪子宫内膜腔上皮、腺上皮、基质和肌细胞中的孕酮受体结合发挥作用,使这些组织细胞分泌多种附植因子,这些附植因子进一步参与胚胎附植过程。雌二醇是雌激素中含量最多、活性最强的一种激素,主要由卵巢颗粒细胞分泌,雌激素受体α是子宫内雌二醇的主要受体,二者结合可使母猪发情;此外,附植中处于游离状态的胚泡也分泌雌二醇,其是一个让母猪子宫内膜得以识别的信号,允许胚泡附植。Eph-ephrin系统作用广泛,其在人、小鼠和猪的胚胎附植过程中发挥着重要作用。系统中的EphA1、ephrin A1、EphA4等基因在猪的胚胎附植期表达量显著高于空怀猪,它们在子宫内膜附植点的表达量显著高于附植点间的部位,且ephrin A1的抑制表达会降低子宫内膜上皮细胞的迁移和黏附能力。这些附植因子都在猪胚胎附植活动中发挥着重要作用,对它们的深入研究将有利于明确猪胚胎附植调控机理,进一步揭示猪高繁机理。     

The effect of progesterone on oxytocin-stimulated intracellular Ca2+ mobilisation and prostaglandin secretion in porcine endometrium

We have studied in the porcine endometrium the expression of oxytocin receptor (OTR) mRNA and the effect of progesterone (P4) on oxytocin/oxytocin receptor (OT/OTR) function concerning intracellular Ca2+ mobilisation ([Ca2+]i), prostaglandin F2alpha (PGF2alpha) and E2 (PGE2; PG) secretion. Tissue was taken from cyclic and early pregnant pigs (days 14-16). A higher expression of OTR mRNA (P < 0.05) was observed in the endometrium of cyclic than pregnant pigs. The stimulatory (P < 0.05) effect of OT (10(-7) M) on [Ca2+]i mobilisation was noticed within 15-60 s and 30-60 s in endometrial stromal cells of cyclic and pregnant pigs, respectively. In the presence of P4 (10(-5) M) basal and OT-stimulated [Ca2+]i concentrations decreased in stromal cells during luteolysis and pregnancy. In stromal cells P4 delayed mobilisation of [Ca2+]i in response to OT by 15 s during luteolysis and had no effect during pregnancy. In cyclic and pregnant epithelial cells OT stimulated mobilisation of [Ca2+]i in 45 s and 60 s, respectively. Oxytocin increased (P < 0.05) PGF2alpha secretion during luteolysis and pregnancy and PGE2 during luteolysis from endometrial slices. Progesterone did not inhibit this stimulatory effect. During luteolysis OT increased (P < 0.05) PGF2alpha in epithelial and stromal cells and PGE2 secretion in epithelial cells. In the presence of P4 this effect of OT was reduced only in stromal cyclic cells (6 h culture). The presence of P4 decreased the effect of OT on [Ca2+]i mobilisation only in stromal cells. We found that, in most conditions, P4 did not inhibit the OT-stimulated secretion of PG in the porcine endometrium.     

Tumour necrosis factor in the canine endometrium: an immunohistochemical study

Tumour necrosis factor (TNF), a pleiotropic cytokine that regulates cell growth and differentiation as well as the synthesis of other cytokines, has been identified in the uterus of several species describing a cyclic pattern, eventually under ovarian steroid regulation. Information is yet limited on the presence of TNF protein in the canine endometrium during the oestrous cycle and early pregnancy. This study depicts the temporal immunolocalization of TNF in the bitch endometrium along the oestrous cycle and changes associated with the early steps of embryo invasion. TNF immunolabelling was found in both the stromal fibroblasts and epithelial components of the canine endometrium in all stages studied. Stromal immunostaining was more intense than that of the epithelia, in all the stages of the oestrous cycle. In addition, a tendency for a decrease in the surface epithelium intensity score was found in early dioestrus. A positive glandular content was only observed in anoestrus and proestrus stages. In early pregnancy (days 13-16), TNF immunolabelling was detected at the embryo-maternal surface, in the syncytium cords and the trophoblast, as well in the endometrial stroma and the basal endometrial glands, but not in the lacunar epithelium. The overall TNF immunoreactivity was higher in early pregnancy samples in comparison with those of the early dioestrus and dioestrus stages, suggesting it plays a role during implantation.     

secretion of the major progesterone-induced proteins of the sheep uterus by caruncular and intercaruncular endometrium of the pregnant ewe from days 20–140 of gestation

An experiment was performed to evaluate the types and quantity of proteins secreted by intercaruncular endometrium at days 20, 60, 100 and 140 of gestation and caruncular endometrium from days 100 and 140 of gestation. Tissues were obtained from ewes made unilaterally pregnant. Based on SDS polyacrylamide gel electrophoresis (SDS-PAGE), the major proteins present in uterine fluid at days 60–140 of gestation were the uterine milk proteins (UTM-proteins), a pair of structurally-related, progesterone-induced polypeptides with molecular weights of 55,000 and 57,000. These proteins were also present in uterine fluid at day 20, but the major protein at this time migrated coincident with albumin. Cultured explants of endometrium at all days of pregnancy produced UTM-proteins as their major radiolabelled product for both caruncular and intercaruncular endometrium. The amount of protein secretion in vitro was greater (P<.04) for intercaruncular endometrium than for caruncular endometrium but was not significantly affected by stage of gestation or local presence of the conceptus. Immunohistochemical analysis revealed that UTM-proteins were present in glandular and luminal epithelium of intercaruncular endometrium and in both epithelial and stromal elements of caruncular endometrium. It was concluded that the UTM-proteins are produced earlier than previously described (i.e., day 20). In addition, caruncles contribute to the uterine secretory protein milieu through the secretion of proteins that are similar to that produced by the glandular intercaruncular epithelium.     

Analyses of circRNA profiling during the development from pre-receptive to receptive phases in the goat endometrium

Background: Recent studies have revealed that noncoding RNAs play important regulatory roles in the formation of endometrial receptivity. Circular RNAs(circRNAs) are a universally expressed noncoding RNA species that have been recently proposed to act as miRNA sponges that directly regulate expression of target genes or parental genes.Results: We used Illumina Solexa technology to analyze the expression profiles of circRNAs in the endometrium from three goats at gestational day 5(pre-receptive endometrium, PE) and three goats at gestational day 15(receptive endometrium, RE). Overall, 21,813 circRNAs were identified, of which 5,925 circRNAs were specific to the RE and 9,078 were specific to the PE, which suggested high stage-specificity. Further analysis found 334 differentially expressed circRNAs in the RE compared with PE(P 0.05). The analysis of the circRNA-miRNA interaction network further supported the idea that circRNAs act as miRNA sponges to regulate gene expression.Moreover, some circRNAs were regulated by estrogen(E2)/progesterone(P4) in endometrial epithelium cell lines(EECs) and endometrial stromal cell line(ESCs), and each circRNA molecule exhibited unique regulation characteristics with respect to E2 and P4.Conclusions: These data provide an endometrium circRNA expression atlas corresponding to the biology of the goat receptive endometrium during embryo implantation.     

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.     

Comparative analysis between endometrial proteomes of pregnant and non-pregnant ewes during the peri-implantation period

Background:Early pregnancy failure has a profound impact on both human reproductive health and animal production.2/3 pregnancy failures occur during the peri-implantation period;however,the underlying mechanism(s)remains unclear.Well-organized modification of the endometrium to a receptive state is critical to establish pregnancy.Aberrant endometrial modification during implantation is thought to be largely responsible for early pregnancy loss.Result:In this study,using well-managed recipient ewes that received embryo transfer as model,we compared the endometrial proteome between pregnant and non-pregnant ewes during implantation period.After embryo transfer,recipients were assigned as pregnant or non-pregnant ewes according to the presence or absence of an elongated conceptus at Day 17 of pregnancy.By comparing the endometrial proteomic profiles between pregnant and non-pregnant ewes,we identified 94 and 257 differentially expressed proteins(DEPs) in the endometrial caruncular and intercaruncular areas,respectively.Functional analysis showed that the DEPs were mainly associated with immune response,nutrient transport and utilization,as well as proteasome-mediated proteolysis.Conclusion:These analysis imply that dysfunction of these biological processes or pathways of DEP in the endometrium is highly associated with early pregnancy loss.In addition,many proteins that are essential for the establishment of pregnancy showed dysregulation in the endometrium of non-pregnant ewes.These proteins,as potential candidates,may contribute to early pregnancy loss.     

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.