Amino acids in oviduct and uterine fluid and blood plasma during the estrous cycle in the bovine

Up to 40% of cattle embryos die within 3 weeks of fertilization while they are nutritionally dependent on the maternal environment provided by the oviduct and uterine fluids for their development and survival. Despite this dependence there is limited information on the composition of these fluids in cattle. Amino acids are essential for the normal growth and development of the early embryo, acting as precursors of proteins and nucleic acids and as energy sources, osmolytes and signaling molecules. The objective of this study was to measure and compare the amino acid concentrations of oviduct and uterine fluid and blood plasma on different days of the estrous cycle. Oviduct fluid was collected in situ from anaesthetised heifers on Days 0, 2, 3, 4 and 6 and uterine fluid on Days 6, 8 and 14 of the estrous cycle and the concentrations of 19 amino acids determined. Glycine was the most abundant amino acid in both oviduct and uterine fluid. However, the concentrations of many amino acids differed between oviduct and uterus and many were present at higher concentrations in oviduct and uterine fluid than in blood plasma. Oviduct fluid concentrations of amino acids were not affected by day of cycle in contrast to uterine fluid for which there was a day of cycle effect on most of the amino acids. These results provide novel information on the amino acid concentrations in the maternal environment of the early cattle embryo and could form the basis for devising improved media for the production of embryos in vitro.     

LEFTY2 expression and localization in rat oviduct during early pregnancy

In mammals, fertilization and preimplantation embryo development occurs in the oviduct. Cross-talk between the developing embryos and the maternal reproductive tract has been described in such a way as to show that the embryos modulate the physiology and gene expression of the oviduct. Different studies have indicated that transforming growth factor beta (TGF-β) can modulate the oviductal microenvironment and act as an autocrine/paracrine factor on embryo development. LEFTY2, a novel member of the TGF-β superfamily is involved in the negative regulation of other cytokines in this family such as nodal, activin, BMPs, TGF-β1 and Vg1. In previous studies, we have reported that LEFTY2 is differentially expressed in the rat oviduct during pregnancy. In this study, we describe the temporal pattern of LEFTY2 in pregnant and non-pregnant rat oviduct by western blotting, which showed higher levels of LEFTY2 on day 4 of pregnancy, a time at which the embryos are ending their journey along the oviduct. The cellular location of LEFTY2 was assessed by immunohistochemistry, which showed immunolabelling in the cytoplasm and at the apical surface of the oviductal epithelial cells. The oviductal fluid also presented a 26 kDa band, which corresponds to the biologically active form of this protein, at the preimplantation period of pregnancy, indicating LEFTY2 secretion to the lumen. As LEFTY2 is expressed at a high level just before the embryos pass to the uterus, its biological effect might be relevant and significant for the preimplantation stage of embryo development in the oviduct. The fact that embryos do not express LEFTY2 at this stage of development supports this hypothesis.     

Autocrine embryotropins revisited: how do embryos communicate with each other in vitro when cultured in groups?

In the absence of the maternal genital tract, preimplantation embryos can develop in vitro in culture medium where all communication with the oviduct or uterus is absent. In several mammalian species, it has been observed that embryos cultured in groups thrive better than those cultured singly. Here we argue that group‐cultured embryos are able to promote their own development in vitro by the production of autocrine embryotropins that putatively serve as a communication tool. The concept of effective communication implies an origin, a signalling agent, and finally a recipient that is able to decode the message. We illustrate this concept by demonstrating that preimplantation embryos are able to secrete autocrine factors in several ways, including active secretion, passive outflow, or as messengers bound to a molecular vehicle or transported within extracellular vesicles. Likewise, we broaden the traditional view that inter‐embryo communication is dictated mainly by growth factors, by discussing a wide range of other biochemical messengers including proteins, lipids, neurotransmitters, saccharides, and microRNAs, all of which can be exchanged among embryos cultured in a group. Finally, we describe how different classes of messenger molecules are decoded by the embryo and influence embryo development by triggering different pathways. When autocrine embryotropins such as insulin‐like growth factor‐I (IGF‐I) or platelet activating factor (PAF) bind to their appropriate receptor, the phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K) pathway will be activated which is important for embryo survival. On the other hand, the mitogen‐activated protein kinase (MAPK) pathway is activated when compounds such as hyaluronic acid and serotonin bind to their respective receptors, thereby acting as growth factors. By activating the peroxisome‐proliferator‐activated receptor family (PPAR) pathway, lipophilic autocrine factors such as prostaglandins or fatty acids have both survival and anti‐apoptotic functions. In conclusion, considering different types of messenger molecules simultaneously will be crucial to understanding more comprehensively how embryos communicate with each other in group‐culture systems. This approach will assist in the development of novel media for single‐embryo culture.     

Expression and localization of nodal in bovine oviduct and uterus during different functional stages of oestrus cycle and pregnancy

Members of TGF-β superfamily play a major role in the endometrial changes involved in the establishment and maintenance of pregnancy. Their deregulated expression and action could lead to absolute or partial failure of embryo implantation. Nonetheless, the precise function and mechanism of many of these cytokines remain unclear. Nodal, a transforming growth factor beta (TGF-β) superfamily member, was characterized in the human and rodent uterus and implicated in the tissue remodeling events during menstruation and embryo implantation. In order to study its possible role in the cattle reproductive process, we have analyzed Nodal expression pattern and localization in the oviduct and uterine horn during the oestrus cycle and early pregnancy (day 20). Nodal was detected both in oviduct and uterus during either the oestrus cycle or pregnancy; however, it shows a differential expression profile in the uterine horn at dioestrus and pregnancy, decreasing 1.5 and 1.4 folds in comparison with oestrus. Nodal immunostaining intensity was observed in stromal and in epithelial cells of the surface and the glandular epithelium. The staining pattern correlates with the RT-qPCR expression profile. This work is the first to evidence the presence of Nodal in the bovine reproductive tract; our data suggest that Nodal is a novel cytokine that would be involved in the remodelling occurring in the endometrium of cattle during the oestrus cycle and in the embryo implantation. The identification of new molecules that participate in endometrium cycling and/or pregnancy may be useful for predicting the ability of the uterine tissue to establish and maintain pregnancy or for detecting the infertility processes. These results highlight Nodal as a possible novel marker of the fertility process, nevertheless further studies should be done to determine its role in the reproductive system.     

In vivo analysis of progesterone receptor action in the uterus during embryo implantation

In order for a successful pregnancy to occur, the embryo must attach to the luminal epithelial cells and invade into the stroma. Then, the surrounding stromal cells need to undergo decidualization in order to establish the vasculature necessary for survival of the embryo. These events in early pregnancy are tightly regulated by the steroid hormones, estrogen (E2) and progesterone (P4), through their cognate receptors, the estrogen receptor (ER) and the progesterone receptor (PR), respectively. Using a mouse model in which the PR has been ablated, it was demonstrated that the PR is necessary for embryo implantation and decidualization. Therefore, understanding the mechanism of PR action in the adult uterus is necessary in order to understand the events of early pregnancy. Insights from both mouse models and human samples have been integral in elucidating uterine PR action. These studies have shown that not only PR target genes, but also mediators of PR action are important for correct PR action in early pregnancy. Many of the genes involved in PR action in early pregnancy have also been shown to have roles in uterine diseases such as endometriosis and endometrial cancer. Therefore, the integration of mouse and human studies on PR action in the uterus will be important for the future understanding of uterine diseases and in the development of treatment for these diseases.     

Possible expansion of "Window of Implantation" in pseudopregnant mice: time of implantation of embryos at different stages of development transferred into the same recipient

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and maternal environment. It is believed that the growth of transferred embryos of different ages is synchronized during preimplantation development and that such embryos are implanted in the uterus at the same time. To define the time of synchronization for developing embryos of different ages, embryos at two different stages of development were transferred separately into the oviducts of the same recipient. We then examined the subsequent development of the embryos at various time intervals after transfer. Pronucleus (PN) stage eggs were transferred separately to the right or left oviduct of recipients on Day 0, while eight-cell embryos (8C) were transferred to the other oviduct. For 8C, 5%, 63%, and 74% of transferred embryos were implanted in the uterus at 42, 66, and 90 h posttransfer, respectively. In contrast, none of the transferred PN was implanted until 90 h posttransfer. At 90 h posttransfer, 59% of the PN had successfully implanted. Histological examination revealed that developmental stage of the embryos in both groups synchronized around 162 h posttransfer, even though the implantation was accelerated in 8C compared with PN. Our results indicate that embryos of advanced stage transferred to the oviduct implant in the uterus in advance of younger embryos and that the uterine development is synchronized at the neural plate, presomite stage. Our results strongly suggest that uterine receptivity for implantation is expandable in pseudopregnant mice.     

In vivo analysis of progesterone receptor action in the uterus during embryo implantation

In order for a successful pregnancy to occur, the embryo must attach to the luminal epithelial cells and invade into the stroma. Then, the surrounding stromal cells need to undergo decidualization in order to establish the vasculature necessary for survival of the embryo. These events in early pregnancy are tightly regulated by the steroid hormones, estrogen (E2) and progesterone (P4), through their cognate receptors, the estrogen receptor (ER) and the progesterone receptor (PR), respectively. Using a mouse model in which the PR has been ablated, it was demonstrated that the PR is necessary for embryo implantation and decidualization. Therefore, understanding the mechanism of PR action in the adult uterus is necessary in order to understand the events of early pregnancy. Insights from both mouse models and human samples have been integral in elucidating uterine PR action. These studies have shown that not only PR target genes, but also mediators of PR action are important for correct PR action in early pregnancy. Many of the genes involved in PR action in early pregnancy have also been shown to have roles in uterine diseases such as endometriosis and endometrial cancer. Therefore, the integration of mouse and human studies on PR action in the uterus will be important for the future understanding of uterine diseases and in the development of treatment for these diseases.     

GABA consumption during early pregnancy impairs endometrial receptivity and embryo development in mice

γ‐Aminobutyrate (GABA) is commonly used as a food supplement and a health care product by young females, due to its positive roles in relieving stress, alleviating anxiety, and improving sleep. However, its recommended daily dose in different products varies widely. Besides, it is unknown whether, and how, GABA consumption during early pregnancy influences pregnancy establishment. In this study, we found that when pregnant mice were treated with a high (12.5 mg/g) dose of GABA (orally) during preimplantation, there was a reduction in the number of implantation sites on day 5 of pregnancy. Also, among these unimplanted embryos, most exhibited morphological degeneration and developmental retardation, and only a few of them developed into blastocysts but could not implant into the uterus. Moreover, the expression of uterine receptivity–related factors—LIF, E‐cadherin, and HOXA10—were all downregulated, while the number of uterine glands was reduced in the high GABA dose group. Finally, in vitro results demonstrated that GABA (ranging from 10 to 50 μg/μL) markedly inhibited preimplantation embryo development in a dose‐response manner. However, this inhibitory effect was not observed when the embryos were pretreated with 40 μΜ 2‐hydroxysaclofen, a GABA_B antagonist, indicating that GABA exerts its inhibitory effects via its B‐type receptor. Our results suggest that exposure to certain GABA concentrations, during early pregnancy, can impair preimplantation embryo development via its B‐type receptor, and endometrial receptivity, which greatly disturbs early embryo implantation in mice. These findings could raise concerns about GABA consumption during the early stages of pregnancy.     

Blastocysts don't go it alone. Extrinsic signals fine-tune the intrinsic developmental program of trophoblast cells

The preimplantation embryo floats freely within the oviduct and is capable of developing into a blastocyst independently of the maternal reproductive tract. While establishment of the trophoblast lineage is dependent on expression of developmental regulatory genes, further differentiation leading to blastocyst implantation in the uterus requires external cues emanating from the microenvironment. Recent studies suggest that trophoblast differentiation requires intracellular signaling initiated by uterine-derived growth factors and integrin-binding components of the extracellular matrix. The progression of trophoblast development from the early blastocyst stage through the onset of implantation appears to be largely independent of new gene expression. Instead, extrinsic signals direct the sequential trafficking of cell surface receptors to orchestrate the developmental program that initiates blastocyst implantation. The dependence on external cues could coordinate embryonic activities with the developing uterine endometrium. Biochemical events that regulate trophoblast adhesion to fibronectin are presented to illustrate a developmental strategy employed by the peri-implantation blastocyst.     

Advances in understanding the physiological mechanism of maternal immune tolerance to the embryo

The results of immunological studies, especially research conducted in the last decade, have shown that free (not bound to protein) progesterone molecules (fP₄) can block the ability of dendritic cells, monocytes and macrophages to present antigens to Th cells and thereby reduce maternal immune activity and increase the maternal tolerance to the semiallogenic embryo. Endocrine studies have shown that fP₄ in the female reproductive organs are transferred at high concentrations into the arterial blood that supplies the oviduct and uterus due to the special adaptations of the blood circulation and lymph flow. Here, the authors present the results of numerous studies documenting their thesis that an important element of maternal tolerance of the semiallogenic embryo in the uterus is conditioned by the close interaction of two processes that occur in the reproductive organs: (1) the local decrease of maternal immune system activity, in which the ability of dendritic cells, macrophages and monocytes to present embryonic antigens to Th cells is blocked by fP₄; and (2) the proper function of the system governing the local retrograde and destination transfer of hormones, which increases the concentration of fP₄ that are able to immediately bind to their receptors in dendritic cells and in the monocytes and macrophages present in the blood supplying the oviduct and uterus. The authors believe that the local interaction of the immune and endocrine systems in the female reproductive tract reduces local maternal immunoreactivity and thus fulfils a critical physiological role; this mechanism protects the embryo but does not change the general immunological resistance of the mother.     

Thrombosis during pregnancy: Risks,prevention, and treatment for mother and fetus—harvesting the power of omic technology,biomarkers and in vitro or in vivo models to facilitate the treatment of thrombosis

Maternal thromboembolism and a spectrum of placenta‐mediated complications including the pre‐eclampsia syndromes, fetal growth restriction, fetal loss, and abruption manifest a shared etiopathogenesis and predisposing risk factors. Furthermore, these maternal and fetal complications are often linked to subsequent maternal health consequences that comprise the metabolic syndrome, namely, thromboembolism, chronic hypertension, and type II diabetes. Traditionally, several lines of evidence have linked vasoconstriction, excessive thrombosis and inflammation, and impaired trophoblast invasion at the uteroplacental interface as hallmark features of the placental complications. “Omic” technologies and biomarker development have been largely based upon advances in vascular biology, improved understanding of the molecular basis and biochemical pathways responsible for the clinically relevant diseases, and increasingly robust large cohort and/or registry based studies. Advances in understanding of innate and adaptive immunity appear to play an important role in several pregnancy complications. Strategies aimed at improving prediction of these pregnancy complications are often incorporating hemodynamic blood flow data using non‐invasive imaging technologies of the utero‐placental and maternal circulations early in pregnancy. Some evidence suggests that a multiple marker approach will yield the best performing prediction tools, which may then in turn offer the possibility of early intervention to prevent or ameliorate these pregnancy complications. Prediction of maternal cardiovascular and non‐cardiovascular consequences following pregnancy represents an important area of future research, which may have significant public health consequences not only for cardiovascular disease, but also for a variety of other disorders, such as autoimmune and neurodegenerative diseases. Birth Defects Research (Part C) 105:209–225, 2015. © 2015 Wiley Periodicals, Inc.     

From Peptide Masses to Pregnancy Maintenance: A Comprehensive Proteomic Analysis of The Early Equine Embryo Secretome,Blastocoel Fluid,and Capsule

Early pregnancy in the mare is a poorly understood, high risk period during which the embryo communicates its presence to the maternal endometrium. Remarkably, the maternal recognition of pregnancy signal is unknown in the horse. This study aimed to profile the proteins secreted by equine blastocysts into their immediate environment, along with proteins contained in the blastocoel and within the acellular embryo capsule. Embryos were recovered on day 8 after ovulation and cultured for 48 hours. Secretomes of day 9 and day 10 embryos were analyzed by LC‐MS/MS and supported by analysis of blastocoel fluid and embryo capsule. Analyses revealed 72 (24 h) and 97 (48 h) unique protein IDs in the embryo secretome, 732 protein IDs in blastocoel fluid, and 11 proteins IDs in the embryo capsule. Novel findings of interest include secretion of a pregnancy specific proteinase (PAG) by the equine embryo at day 10, along with detection of a prostaglandin receptor inhibiting protein (PTGFRN) and a progesterone potentiating factor (FKBP4) in blastocoel fluid. This is the first comprehensive proteomic analysis of the equine embryo secretome, and provides new insights into the unique physiology of early pregnancy in this species.     

Effects of cervical dilation and intrauterine infusions on the timing of oviductal transport of equine embryos

Equine embryos spend 5 to 6 d in the oviduct before entering the uterus as expanded blastocysts, and cannot be consistently collected nonsurgically until Day 7. Technologies such as cryopreservation and embryo splitting, which are most successful with embryos at the morula or early blastocyst stage, have not been used in mares because equine morulae and early blastocysts are located in the oviduct and cannot be recovered nonsurgically. These experiments test the hypothesis that transport of equine embryos through the oviduct can be hastened by cervical dilation or by acute, sterile endometritis induced by intrauterine oyster glycogen treatment. Cervical dilation with or without intrauterine infusion of 0.5 ml PBS on Day 4 did not appear to hasten the transport of embryos into the uterus since Day 5 uterine embryo recovery rates were not higher (P > 0.1) for mares with cervical dilation or cervical dilation plus PBS infusion vs mares receiving no treatments (0 of 5 and 0 of 5 vs 0 of 10, respectively). Intrauterine infusions of 40 ml of 1% oyster glycogen or 40 ml of PBS on Day 3 did not appear to hasten the transport of embryos into the uterus since Day 5 uterine embryo recovery rates were not higher (P > 0.1) for oyster glycogen- or PBS-treated vs untreated mares (2 of 12 and 3 of 11 vs 0 of 10, respectively). Cervical and uterine treatments on Day 3 or Day 4 and uterine lavages on Day 5 decreased (P < 0.05) Days 11 to Day 15 pregnancy rates compared with that of untreated mares. Day 11 to Day 15 pregnancy rates were 1 of 5 for mares with Day 4 cervical dilation and Day 5 uterine lavage, 1 of 5 for mares with Day 4 PBS infusion and Day 5 uterine lavage, 2 of 12 for mares with Day 3 oyster glycogen infusion and Day 5 uterine lavage, and 3 of 11 for mares with Day 3 PBS infusion and Day 5 uterine lavage vs 7 of 10 for mares that received no treatment or lavage. Cervical and uterine manipulations on Day 3 or 4 and uterine lavage on Day 5 appeared to decrease pregnancy rates by Days 11 to 15. The results of these experiments do not support the hypothesis that cervical dilation or uterine infusion hasten oviductal transport, since neither cervical manipulation nor transcervical infusion of oyster glycogen or PBS into the uterus significantly hastened the rate of embryo transport into the uterus.     

PGF2α levels in Day 8 blood plasma are increased by the presence of one or more embryos in the uterus

In cattle, the detection of very early endometrial responses is considered to be hampered by the presence of only a single embryo. Therefore, we have previously developed a model of multiple embryo transfer to circumvent this hindrance. In this work, we analysed embryo–maternal interactions in the bovine uterus on day 8 of development while comparing the presence of multiple v. single embryos using embryo transfer and artificial insemination, respectively. Concentration of proteins (β-actin, NFkB, clusterin and immunoproteosome 20S β5i subunit–i20S), by western blot, and hexoses (glucose and fructose) were measured in paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus and were compared with UF obtained after artificial insemination. Prostaglandin (PG) F₂α and PGE₂ concentrations were also analysed in blood plasma. The four proteins analysed and hexoses were unaffected by the presence of one or more embryos in the uterus. However, blood PGF₂α showed similar, significant increases with one or more embryos over cyclic animals; such changes were not observed in blood PGE₂. Although multiple embryo transfer may appear to be non-physiological, we showed that the uterus, at the very early embryonic stages, does exhibit physiological reactions. Multiple embryo transfer can, therefore, be used for studies of very early embryo–maternal interactions in vivo in monotocous species.     

Smoking and reproduction: The oviduct as a target of cigarette smoke

The oviduct is an exquisitely designed organ that functions in picking-up ovulated oocytes, transporting gametes in opposite directions to the site of fertilization, providing a suitable environment for fertilization and early development, and transporting preimplantation embryos to the uterus. A variety of biological processes can be studied in oviducts making them an excellent model for toxicological studies. This review considers the role of the oviduct in oocyte pick-up and embryo transport and the evidence that chemicals in both mainstream and sidestream cigarette smoke impair these oviductal functions. Epidemiological data have repeatedly shown that women who smoke are at increased risk for a variety of reproductive problems, including ectopic pregnancy, delay to conception, and infertility. In vivo and in vitro studies indicate the oviduct is targeted by smoke components in a manner that could explain some of the epidemiological data. Comparisons between the toxicity of smoke from different types of cigarettes, including harm reduction cigarettes, are discussed, and the chemicals in smoke that impair oviductal functioning are reviewed.     

胚胎体外共培养：影响因素及作用机理

综述了近年来关于哺乳动物早期胚胎与体细胞共培养的研究进展。重点讨论了早期胚胎与不同类型体细胞共培养,血清、发情周期和体细胞传代次数对胚胎共培养效果的影响,以及胚胎体外共培养的作用机理。体细胞共培养体系可以改善早期胚胎体外培养的条件,促进胚胎发育,提高着床率和妊娠率,在发育生殖研究领域有着广泛的应用前景。然而,对其影响因素和作用机理尚欠系统深入研究,许多问题还亟待解决。