反刍动物双核滋养层巨细胞及其妊娠特异蛋白家族的研究进展

双核滋养层巨细胞是反刍动物胎盘特有的滋养层巨细胞群。作为母胎界面的第1层细胞,其从胚胎附着启动直接参与子宫上皮的变形和母胎交换过程。研究发现,牛、绵羊和山羊的该细胞群能特异表达胎盘催乳素等妊娠特异蛋白家族,以参与胚胎附着、胎盘形成、血管发生、免疫调节和母胎交换等生理过程,在妊娠维持中起着不同的重要作用,但具体生物学功能和调节机制仍有待深入研究。本文综述了反刍动物双核滋养层巨细胞的特征和其妊娠特异蛋白家族表达及生物学功能的研究进展。     

Expression of ADAMTS1 mRNA in bovine endometrium and placenta during gestation

A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) is a secreted protease. Through the regulation of extracellular matrix remodeling or developmental processes or both, ADAMTS1 is involved in several biological functions, including ovulation and embryo receptivity. However, the expression and possible role of ADAMTS1 in bovine endometrium is unknown. In this study, we analyzed ADAMTS1 mRNA expression in bovine endometrium during the estrous cycle, peri-implantation period, and at different stages of gestation by using quantitative real-time RT-PCR (qPCR) and in situ hybridization. The qPCR results indicated that the expression of ADAMTS1 mRNA was not affected by the day of the estrous cycle and was similar to cyclic levels on day 35 of gestation; however, the expression was more abundant in cotyledonary tissues of the placenta during late gestation. The in situ hybridization study showed that ADAMTS1 mRNA was detected mainly in uterine luminal epithelia and stromal cells during the estrous cycle and peri-implantation period. A disintegrin and metalloproteinase with thrombospondin motifs 1 mRNA was also expressed in the peri-implantation conceptus as well as in trophoblast cells, which include binucleate cells, and increased during late gestation. Furthermore, treatment of stromal cell with progesterone (300 nM) stimulated the expression of ADAMTS1 mRNA. This study indicates that ADAMTS1 participates in bovine endometrial remodeling, which is required for implantation and placental development in coordination with ovarian steroids.     

Spatiotemporal heterogeneity of PPARγ expression in porcine uteroplacenta for regulating of placental angiogenesis through VEGF-mediated signalling

Non-infectious prenatal mortality severely affects the porcine industry, with pathological placentation as a likely key reason. Previous studies have demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) deficiency causes defects in the uteroplacental vasculature and induces embryonic losses in mice. However, its role in porcine placental angiogenesis remains unclear. In the present study, PPARγ expression was investigated in porcine uteroplacental tissues at gestational day (GD) 25, GD40 and GD70 via quantitative polymerase chain reaction (qPCR), Western blot and immunohistochemistry (IHC). Moreover, the roles of PPARγ in porcine placental angiogenesis were investigated using a cell model of porcine umbilical vein endothelial cells (PUVECs) to conduct proliferation, migration and tube formation assays in vitro and a mouse xenograft model to assess capillary formation in vivo. The results showed that PPARγ was mainly located in the glandular epithelium, trophoblast, amniotic chorion epithelium and vascular endothelium, as indicated by the higher expression levels at GD25 and GD40 than at GD70 in endometrium and by higher expression levels at GD40 and GD70 than at GD25 in placenta. Moreover, PPARγ expression was significantly downregulated in placenta with dead foetus. In PUVECs, knocking out PPARγ significantly inhibited proliferation, migration and tube formation in vitro and inhibited capillary formation in mouse xenografts in vivo by blocking S-phase, promoting apoptosis and downregulating the angiogenic factors of VEGF and its receptors. Overall, the spatiotemporal heterogeneity of PPARγ expression in porcine uteroplacental tissue suggests its vital role in endometrial remodelling and placental angiogenesis, and PPARγ regulates placental angiogenesis through VEGF-mediated signalling.     

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.     

Biology of the prolactin family in bovine placenta. I. Bovine placental lactogen: Expression, structure and proposed roles

Bovine placenta produces an array of proteins that are structurally and functionally similar to pituitary prolactin. Bovine placental lactogen (bPL) is a glycoprotein hormone that has lactogenic and somatogenic properties. Purified bPL contains several kinds of isoforms that are created by alternative splicing and/or multiple glycosylation patterns. bPL can activate the prolactin (PRL) receptor‐mediated signaling pathway as well as PRL does. The bPL mRNA is transcribed in trophoblast binucleate cells, and synthesized bPL protein is stored in membrane‐bound secretory granules. The message encoding bPL is first detectable in trophoblast binucleate cells at approximately day 20 of gestation at, or shortly after, the appearance of binucleate cells in the trophoblast. Most binucleate cells are detected as expressed bPL in the placenta. Bovine PL may be the determinant in trophoblast differentiation. Although the biological activities of bPL have long been studied, the precise role of bPL is still largely unclear. This article reviews and discusses the biological roles of bPL, focusing on luteal function, fetal growth and pregnancy‐associated maternal adaptation, mammogenesis and lactogenesis, and placental angiogenesis. The precise biological function of bPL needs to be further evaluated.     

Comparative analysis of proteomic profiles between endometrial caruncular and intercaruncular areas in ewes during the peri-implantation period

The endometrium of sheep consists of plenty of raised aglandular areas called caruncular (C), and intensely glandular intercaruncular areas (IC). In order to better understand the endometrium involved mechanisms of implantation, we used LC-MS/MS technique to profile the proteome of ovine endometrial C areas and IC areas separately during the peri-implantation period, and then compared the proteomic profiles between these two areas. We successfully detected 1740 and 1813 proteins in C areas and IC areas respectively. By comparing the proteome of these two areas, we found 170 differentially expressed proteins (DEPs) (P < 0.05), functional bioinformatics analysis showed these DEPs were mainly involved in growth and remodeling of endometrial tissue, cell adhesion and protein transport, and so on. Our study, for the first time, provided a proteomic reference for elucidating the differences between C and IC areas, as an integrated function unit respectively, during the peri-implantation period. The results could help us to better understand the implantation in the ewes. In addition, we established a relatively detailed protein database of ovine endometrium, which provide a unique reference for further studies.     

Comparative analysis of proteomic profiles between endometrial caruncular and intercaruncular areas in ewes during the peri-implantation period

The endometrium of sheep consists of plenty of raised intercaruncular areas （IC）. In order to better understand aglandular areas called caruncular （C）, and intensely glandular the endometrium involved mechanisms of implantation, we used LC-MS/MS technique to profile the proteome of ovine endometrial C areas and IC areas separately during the peri-implantation period, and then compared the proteomic profiles between these two areas. We successfully detected 1740 and 1813 proteins in C areas and IC areas respectively. By comparing the proteome of these two areas, we found 170 differentially expressed proteins （DEPs） （P 〈 0.05）, functional bioinformatics analysis showed these DEPs were mainly involved in growth and remodeling of endometrial tissue, cell adhesion and protein transport, and so on Our study, for the first time, provided a proteomic reference for elucidating the differences between C and IC areas, as an integrated function unit respectively, during the peri-implantation period. The results could help us to better understand the implantation in the ewes. In addition, we established a relatively detailed protein database of ovine endometrium, which provide a unique reference for further studies.     

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.     

Ruminant placental lactogens: structure and biology.

Ruminant placental lactogens (PL) are members of the somatotropin, prolactin gene family that are synthesized by trophectodermal binucleate cells. The structure and biology of PL has been studied in the cow, sheep, and goat. Ruminant PL have greater structural identity to prolactin than somatotropin, although they bind to both lactogenic and somatogenic receptors. The molecular weights of ovine and caprine PL are approximately 23,000, whereas bovine PL is larger (31,000 to 34,000) due to glycosylation. Placental lactogen is secreted into both the fetal and maternal circulations. The concentration of PL in the fetus decreases with advancing gestation, whereas PL concentration peaks in the maternal circulation during the last third of pregnancy then reaches a plateau. Furthermore, the maternal concentration of PL is 100- to 1,000-fold higher in sheep and goats than in cows. The precise factors that modulate secretion of PL are unknown, although placental mass and nutrition seem to play a role. Ruminant PL have both lactogenic and somatogenic biological activities and may also have unique activities mediated through a specific receptor. There is circumstantial evidence to suggest that PL plays a role in stimulating mammogenesis. Placental lactogen secreted into the fetal compartment may also help regulate fetal growth. Direct experimental data indicate that PL can regulate maternal intermediary metabolism. Thus, it may act as a partitioning agent to regulate nutrient supply for fetal growth. The precise biological function of PL in ruminants, therefore, still needs to be defined.     

The dynamic expression of extracellular matrix in the bovine endometrium at implantation

Remodeling of uterine endometrial extracellular matrix (ECM) is pivotal to successful implantation and placentation, and has been well described in the rodents and humans. However, bovine endometrial ECM remodeling is still vaguely defined, especially at the time of implantation. Therefore, this study investigated the distribution of four ECMs namely, types I and IV collagen, laminin and fibronectin, from days 0 to 30 of gestation in bovine endometrium by immunofluorescence microscopy. A change in the distribution pattern of ECMs was evident by day 14 of gestation as features at this stage were clearly different from those of day 14 of the estrous cycle. The immunoreactivity of type I collagen, fibronectin and laminin decreased from day 14 of gestation and was obscured by day 24 of gestation. The type I collagen fibers formed were of thinner consistency than those of the estrous cycle and showed a coarser meshwork within the epithelium sites during the implantation period. In addition, the type IV collagen and laminin immunoreactivities of epithelial basement membrane also remarkably declined at exactly the same time. By day 30 of gestation, the four ECMs had regenerated with the formation of the placentome. In conclusion, this study reveals that remodeling of ECM is essential for the successful establishment of pregnancy in the bovine.     

Quantitative unbiased estimates of endometrial gland surface area and volume in cycling cows and heifers

Entire reproductive tracts were removed from seven normal healthy heifers and qualitative unbiased estimates made of endometrial gland volume density and glandular surface density. After examining approximately 55 microscopic fields of endometrium from each tract, a mean glandular surface density value of 10.2 mm(2)/mm(3) (CE 3.1%) was obtained. The stereological method was then employed in optimising the design of the main study. The endometrial height was measured for 17 healthy cycling heifers and 19 similar cows. Subsequently, unbiased estimates were made of intercaruncular endometrial gland volume per unit surface for all cattle were investigated; differences between heifers and cows generally, and the possible effect of the follicular and luteal phases of the oestrous cycle were compared. The mean surface area of glands per unit area of endometrium at the intercaruncular site in heifers and cows was approximately 18 mm(2)/mm(2) in the follicular phase and 26 mm(2)/mm(2) in the luteal phase, figures similar to the gland area found in women. The intercaruncular gland volume increased significantly, by about 30% during the luteal phase of the bovine oestrous cycle in heifers, from 0.01 to 0.13 per mm(3). The differences in endometrial anatomy between site of sampling and either follicular or luteal phases of the oestrous cycle were always more significant in heifers than cows. The endometrial thickness in cows was always greater than for heifers, irrespective of the site of sampling. It was concluded that the intercaruncular endometrium of cattle was far more active physiologically than recognised previously.     

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.     

Immunohistochemical Localization of Vascular Endothelial Growth Factor (VEGF) and its Two Receptors (Flt-I and KDR) in the Endometrium and Placenta of the Mare During the Oestrous Cycle and Pregnancy

Polyclonal antisera for vascular endothelial growth factor (VEGF) and its two main receptor molecules, VEGF-I (Flt) and VEGF-II (KDR), were used in a conventional immunocytochemical staining method to localize these angiogenic ligand molecules in the endometrium and placenta of the mare during the oestrous cycle and pregnancy. The anti-VEGF and anti-Flt sera both labelled the lumenal and glandular epithelia of the endometrium throughout the oestrous cycle and both the invasive trophoblast cells of the endometrial cups and the non-invasive trophoblast of the allantochorion in pregnancy. The anti-KDR serum likewise stained the maternal and foetal epithelial layers during the oestrous cycle and pregnancy and it also labelled fibroblast-like cells in the endometrial and allantoic stromas and the endothelium of foetal and maternal capillaries. The results demonstrated that constant supplies of the principal vasculogenic and angiogenic factor, VEGF, and its two major receptors, Flt and KDR, are available on both the maternal and foetal sides of the placental barrier throughout gestation in the mare. They are presumed to facilitate the continuing development of the extensive foetal and maternal capillary networks that are such prominent features within the microplacentomes of the diffuse, epitheliochorial equine placenta.     

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.     

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.