Oviduct and endometrial epithelium improve in vitro produced bovine embryo developmental kinetics.

In brief: Standard in vitro produced (IVP) bovine embryo culture media limit embryonic development. Culturing IVP bovine embryos in standard IVP bovine embryo culture media conditioned with oviduct and/or endometrial cells improves blastocyst formation and reduces the time to formation. Abstract: In vitro embryo production in cattle greatly impacts blastomere biochemistry, embryo rate of development and pre- and post-transfer survival. In vivo, the bovine embryo migrates through the oviduct isthmus before entering the uterus on approximately day 4 of development where it remains unattached within the uterine lumen until day 20 of gestation. During this time, the embryo is sequentially exposed to oviduct followed by endometrial secretions that support embryonic development. Considering this, we tested the effect of culturing in vitro produced (IVP) bovine embryos sequentially in oviduct epithelial- (OEp; days 1-3) followed by endometrial epithelial- (EEp) or EEp and fibroblast cell (EEp/F; days 4-8)-conditioned media on embryonic development using a time-lapse monitoring system. Compared to control, culturing IVP embryos in EEp- or EEp/F-conditioned media without prior culture in OEp-conditioned media increased blastocyst formation (P < 0.05) and reduced the time to blastocyst formation (P < 0.05). Culturing IVP bovine embryos in OEp-conditioned media followed by EEp- or EEp/F-conditioned media, however, had the greatest impact on embryo developmental kinetics and increased morula and blastocyst formation (P < 0.05) and reduced time to formation (P < 0.05). Day 8 blastocyst cell numbers, diameter and quality were not significantly different, although, blastocyst quality scores were less (indicative of better quality) for all cell-conditioned media compared to control. In conclusion, IVP bovine embryo development may be improved using a sequential embryo culture system involving bovine oviduct followed by endometrial cell-conditioned media.

Progesterone-stimulated endometrial cell conditioned media increases in vitro produced bovine embryo blastocyst formation.

The early bovine embryo is supported by histotroph molecules secreted by endometrial epithelial (EPI) and stroma fibroblast (SF) cells in response to luteal progesterone (P4). We hypothesized that specific histotroph molecule transcript abundance depends on cell type and P4 concentration and that endometrial cell conditioned media (CM) could improve in vitro produced (IVP) embryo development in culture. Primary bovine EPI and SF cells from seven uteri were incubated for 12 h with RPMI medium containing 0 (Control), 1, 15, or 50 ng of P4. RPMI was also incubated without cells (N-CM) and CM from EPI or SF cultures (EPI- or SF-CM) or a combination of the two (1:1; EPI/SF-CM) was used to culture IVP embryos from days 4-8 of development (n = 117). There was an effect of cell type (SLC1A1, SLC5A6, SLC7A1, FGF-2, FGF-7, CTGF, PRSS23 and NID2) and/or P4 concentration (FGF-7 and NID2) on endometrial cell histotroph molecule mRNA (P < 0.05). Compared to N-CM, blastocyst development on day 7 was greater in the EPI or SF-CM (P ≤ 0.05) and tended to be greater in the EPI/SF-CM (P = 0.07). On day 8, blastocyst development was greater only in the EPI-CM (P < 0.05). Further, culturing embryos with endometrial cell CM reduced day 8 blastocyst transcript abundance of cell adhesion molecule LGALS1 (P < 0.01). In conclusion, endometrial cell CM or histotroph molecules may be used to improve IVP embryo development in cattle.

Ruminant conceptus-maternal interactions: interferon-tau and beyond.

Embryonic or fetal loss in cattle is associated with problems that occur during oocyte maturation, early embryonic development, conceptus elongation, maternal recognition of pregnancy (MRP), and/or placental attachment and implantation. Many of these problems manifest as inadequate or asynchronous communication between the developing conceptus and endometrium, resulting in pregnancy failure. This review will provide an overview of how various conceptus-endometrial paracrine signaling systems control the fate of early pregnancy in cattle and other ruminants. We begin by summarizing the actions of interferon-tau, the classic MRP signal in ruminates, and then explore how other secretory factors derived from either the conceptus or endometrium influence establishment and maintenance of pregnancy. Insight into how the endometrium responds to male vs. female conceptuses or conceptuses produced by in vitro methods will also be described. Specific focus will be placed on describing how "omic" technologies and other cutting-edge techniques have assisted with identifying novel conceptus and/or endometrial factors and their functions. Recent findings indicate that the endometrial transcriptome and histotroph are altered by conceptus sex, quality, and origin, suggesting that the endometrium is a sensor of conceptus biochemistry. Although the endometrium has a certain level of flexibility in terms of conceptus-maternal interactions, this interplay is not sufficient to retain some pregnancies. However, new information inspires us to learn more and will help develop technologies that mitigate early embryonic loss and reproductive failure in ruminants and other animals.

Early pregnancy losses are common in cattle. This review describes how critical the interplay between the developing conceptus (embryo and extraembryonic membranes) and endometrium is to maintaining pregnancies in cattle and other ruminants. The discovery of interferon-tau more than 40 yr ago initiated a new field of reproductive biology focused on describing how the conceptus and endometrium communicate with one another through the secretion of paracrine factors, extracellular vesicles, and other molecules. The use of "omic" and gene editing technologies has assisted with identifying novel functions for many conceptus and endometrial secreted factors. This review provides examples of how conceptus sex, quality, and in vitro vs. in vivo development influences endometrial function. The endometrium appears to have some flexibility in its response to conceptuses, and this insight could be used to our advantage as we work towards developing schemes to rescue conceptuses that are in danger of experiencing pregnancy loss.

Galectin-1 induces gene and protein expression related to maternal-conceptus immune tolerance in bovine endometrium†.

Conceptus secretory factors include galectins, a family of carbohydrate binding proteins that elicit cell adhesion and immune suppression by interacting with intracellular and extracellular glycans. In rodents, galectin-1 (LGALS1) promotes maternal-fetal immune tolerance in the decidua through expansion of tolerogenic cluster of differentiation 11c (CD11c) positive dendritic cells, increased anti-inflammatory interleukin (IL)-10, and activation of forkhead box P3 (FOXP3) positive regulatory T cells (Treg). This study characterized galectin expression in early ruminant conceptuses and endometrium. We also tested the effect of recombinant bovine LGALS1 (rbLGALS1) and progesterone (P4) on endometrial expression of genes and protein related to maternal-conceptus immune tolerance in cattle. Elongating bovine and ovine conceptuses expressed several galectins, particularly, LGALS1, LGALS3, and LGALS8. Within bovine endometrium, expression of LGALS3, LGALS7, and LGALS9 was greater on Day 16 of pregnancy compared to the estrous cycle. Within ovine endometrium, LGALS7 was greater during pregnancy compared to the estrous cycle and endometrium of pregnant sheep tended to have greater LGALS9 and LGALS15. Expression of endometrial LGALS4 was less during pregnancy in sheep. Treating bovine endometrium with rbLGALS1 increased endometrial expression of CD11c, IL-10, and FOXP3, within 24 h. Specifically, within caruncular endometrium, both rbLGALS1 and P4 increased FOXP3, suggesting that both ligands may promote Treg expansion. Using IHC, FOXP3+ cells with a leukocyte phenotype were localized to the bovine uterine stratum compactum near the uterine surface and increased in response to rbLGALS1. We hypothesize that galectins have important functions during establishment of pregnancy in ruminants and bovine conceptus LGALS1 and luteal P4 confer mechanisms of maternal-conceptus immune tolerance in cattle.

Conceptus-induced, interferon tau-dependent gene expression in bovine endometrial epithelial and stromal cells†.

Bovine endometrium consists of epithelial and stromal cells that respond to conceptus interferon tau (IFNT), the maternal recognition of pregnancy (MRP) signal, by increasing expression of IFN-stimulated genes (ISGs). Endometrial epithelial and stromal-cell-specific ISGs are largely unknown but hypothesized to have essential functions during pregnancy establishment. Bovine endometrial epithelial cells were cultured in inserts above stromal fibroblast (SF) cells for 6 h in medium alone or with IFNT. The epithelial and SF transcriptomic response was analyzed separately using RNA sequencing and compared to a list of 369 DEGs recently identified in intact bovine endometrium in response to elongating bovine conceptuses and IFNT. Bovine endometrial epithelial and SF shared 223 and 70 DEGs in common with the list of 369 endometrial DEGs. Well-known ISGs identified in the epithelial and SF were ISG15, MX1, MX2, and OAS2. DEGs identified in the epithelial but not SF included a number of IRF molecules (IRF1, IRF2, IRF3, and IRF8), mitochondria SLC transporters (SLC25A19, SLC25A28, and SLC25A30), and a ghrelin receptor. Expression of ZC3HAV1, an anti-retroviral gene, increased specifically within the SF. Gene ontology analysis identified the type I IFN signaling pathway and activation of nuclear factor kappa B transcription factors as biological processes associated with the epithelial cell DEGs. This study has identified biologically relevant IFNT-stimulated genes within specific endometrial cell types. The findings provide critical information regarding the effects of conceptus IFNT on specific endometrial compartments during early developmental processes in cattle.

Bovine endometrium responds differentially to age-matched short and long conceptuses†.

This study combined in vitro production of bovine blastocysts, multiple embryo transfer techniques, and a conceptus-endometrial explant co-culture system to test the hypothesis that bovine endometrium exposed to long vs. short day 15 conceptuses would exhibit a different transcriptome profile reflective of potential for successful pregnancy establishment. Bovine endometrial explants collected at the late luteal stage of the estrous cycle were cultured in RPMI medium for 6 h with nothing (control), 100 ng/mL recombinant ovine interferon tau (IFNT), a long day 15 conceptus, or a short day 15 conceptus. Transcriptional profiling of the endometrial explants found that exposure of endometrium to IFNT, long conceptuses, or short conceptuses altered (P < 0.05) expression of 491, 498, and 230 transcripts, respectively, compared to the control. Further analysis revealed three categories of differentially expressed genes (DEG): (i) commonly responsive to exposure to IFNT and conceptuses, irrespective of size (n = 223); (ii) commonly responsive to IFNT and long conceptuses only (n = 168); and genes induced by the presence of a conceptus but independent of IFNT (n = 108). Of those 108 genes, 101 were exclusively induced by long conceptuses and functional analysis revealed that regulation of molecular function, magnesium-ion transmembrane transport, and clathrin coat assembly were the principal gene ontologies associated with these DEG. In conclusion, bovine endometrium responds differently to age-matched conceptuses of varying size in both an IFNT-dependent and -independent manner, which may be reflective of the likelihood of successful pregnancy establishment.

Do differences in the endometrial transcriptome between uterine horns ipsilateral and contralateral to the corpus luteum influence conceptus growth to day 14 in cattle?

Embryo transfer to the uterine horn contralateral to the ovary containing the corpus luteum (CL) negatively impacts pregnancy establishment in cattle. Our aim was to compare the transcriptome and ability of the ipsilateral and contralateral uterine horns to support preimplantation conceptus survival and growth to day 14. In experiment 1, endometrial samples from both horns were collected from synchronized heifers slaughtered on day 5, 7, 13, or 16 post-estrus (n = 5 per time) and subjected to RNA sequencing. In experiment 2, 10 day 7 in vitro produced blastocysts were transferred into the uterine horn ipsilateral (n = 9) or contralateral to the CL (n = 8) or into both horns (i.e., bilateral, n = 9) of synchronized recipient heifers. Reproductive tracts were recovered at slaughter on day 14, and the number and dimensions of recovered conceptuses were recorded for each horn. A total of 217, 54, 14, and 18 differentially expressed genes (>2-fold change, FDR P < 0.05) were detected between ipsilateral and contralateral horns on days 5, 7, 13, and 16, respectively, with signaling pathways regulating pluripotency of stem cells, ErbB signaling pathway, and mTOR signaling pathway amongst the top canonical pathways. Site of embryo transfer did not affect recovery rate (48.0%, 168/350) or length of conceptuses (mean ± SE 2.85 ± 0.27 mm). Although differences in gene expression exist between the endometrium of uterine horns ipsilateral and contralateral to the CL in cattle, they do not impact conceptus survival or length between day 7 and 14.

Interferon tau-dependent and independent effects of the bovine conceptus on the endometrial transcriptome†.

This study investigated bovine conceptus-induced modifications to the endometrial transcriptome related to effects of interferon tau (IFNT), conceptus origin (in vivo vs. in vitro), and conceptus sex. In vitro (IVF) or in vivo (superovulation and artificial insemination, AI) produced blastocysts were transferred into recipient heifers on day 7 of the estrous cycle. On day 15, IVF- or AI-derived conceptuses were obtained by uterine flushing and individually placed on endometrial explants in media for 6 h. Explants were also cultured with media alone as a control or media containing 100 ng/mL IFNT. Total explant RNA was analyzed by RNA-Seq. Incubation of endometrium with IFNT or IVF- or AI-derived conceptuses changed (P ≤ 0.001) expression of 491, 498, and 576 transcripts, respectively, compared to the control. Further, 369 differentially expressed genes (DEGs) were common between explants exposed to IFNT or a conceptus. A total of 240 DEGs were uniquely altered by conceptuses (IVF- and AI-derived) but not IFNT. Of these transcripts, 46 were shared between the IVF and AI groups, while 61 and 133 were specific to IVF and AI conceptuses, respectively. Five genes [melanophilin (MLPH), prominin-2 (PROM2), myeloid associated differentiation marker (MYADM), vomeronasal 1 receptor 4 like (VN1R4L) and 5-hydroxytryptamine receptor 1A (HTR1A)] were more abundant in endometrium exposed to female compared to male conceptuses (P < 0.001). A single gene [ADP-ribosylation factor like GTPase 4C (ARL4C)] was more abundant in response to male conceptuses (P < 0.001) than female conceptuses. These data support the hypothesis that conceptus regulation of gene expression in the endometrium is complex and involves factors other than IFNT that may have a biological role in pregnancy establishment.

Embryonic maternal interaction in cattle and its relationship with fertility.

Embryo mortality is a major contributor to poor reproductive efficiency and profitability in cattle production systems. While conception is achieved (i.e., the oocyte is fertilized) in the vast majority of cases if insemination is carried out correctly, a significant proportion of the resulting embryos fail to develop to term. Appropriate communication between the developing conceptus and the maternal endometrium is essential for the establishment and maintenance of pregnancy in all mammals. Up to the blastocyst stage, around Days 7-9, contact worth the female reproductive system is not required. However, the process of conceptus elongation after hatching and prior to implantation is entirely maternally driven and is essential to ensure that sufficient quantities of interferon-tau (IFNT) are secreted by the developing conceptus to abrogate the mechanisms that bring about luteolysis. While the importance of conceptus-derived IFNT in maternal recognition of pregnancy and prevention of luteolysis in cattle is unequivocal, many questions, such as the threshold level of IFNT required for pregnancy maintenance, remain unanswered. Furthermore, the precise role of IFNT-independent mechanisms in pregnancy establishment remains to be elucidated. Irrespective of this, failure of the conceptus to elongate undoubtedly results in embryonic loss and is thus believed to contribute greatly to reproductive failure in cattle. This review will address some of these answered questions and try to shed some light on those gaps in knowledge that could potentially contribute to improved embryo survival and reproductive efficiency.

Embryo development in cattle and interactions with the reproductive tract.

Embryo mortality is a major contributor to poor reproductive efficiency and profitability in cattle production systems. Coordinated interaction between the developing embryo or conceptus and the maternal reproductive tract is essential for pregnancy establishment in mammals. Up to the blastocyst stage, the embryo can grow in the absence of contact with the oviduct or uterus; however, conceptus elongation after hatching and before implantation, a characteristic of ruminant early development, is entirely maternally driven and is essential to ensure that sufficient quantities of interferon-τ (IFNT) are secreted by the developing conceptus to abrogate the mechanisms that bring about luteolysis. Surprisingly, many questions, such as the threshold level of IFNT required for pregnancy maintenance, remain unanswered. Failure of the conceptus to elongate undoubtedly results in embryonic loss and is thus believed to contribute greatly to reproductive failure in cattle.

Rapid conceptus elongation in the pig: An interleukin 1 beta 2 and estrogen-regulated phenomenon.

Establishment and maintenance of pregnancy in the pig involves activating many physiological, cellular, and molecular signaling pathways between the developing conceptus and hormonally regulated maternal endometrium. Rapid elongation of the pig trophoblast allows for the establishment of sufficient placental surface area for the transport of nutrients to the fetus throughout pregnancy. Estrogens secreted by the conceptus during elongation act on uterine epithelia to induce secretion of uterine factors required for conceptus development and for preventing endocrine secretion of prostaglandin F2α, which would cause luteolysis. Thus, trophoblast expansion within the uterine lumen during early gestation is an essential process for implantation and maintenance of pregnancy in species with an epitheliochorial form of placentation. In the pig, rapid conceptus elongation involves the unique expression of interleukin-1 beta 2 (IL1B2), which establishes pro-inflammatory effects that may be tempered by the spatiotemporal secretion of estrogen from the conceptuses. The present review provides current information on pig conceptus remodeling and signaling via estrogen and IL1B2 pathways, as well as endometrial responses to those conceptus factors leading to establishment of pregnancy.

Interleukins, interferons, and establishment of pregnancy in pigs.

Early pregnancy in mammals requires complex and highly orchestrated cellular and molecular interactions between specialized cells within the endometrium and the conceptus. Proinflammatory cytokines are small signaling proteins released by leukocytes that augment innate and adaptive immune responses. They are also released by the mammalian trophectoderm as the conceptus apposes the uterine surface for implantation. On approximately day 12 of development in pigs, the conceptus undergoes a rapid morphological transformation referred to as elongation while simultaneously releasing estrogens and a novel conceptus form of interleukin-1 beta (IL1ß). Following elongation, pig conceptuses express interferon gamma (IFNγ) and, in lesser amounts, interferon delta (IFNδ). Significant IFN signaling takes place within the endometrium between day 14 and 18 of pregnancy as the conceptus intimately associates with the uterine epithelium. Based on studies carried out in pigs and other mammals, the combined spacio-temporal activities of conceptus estrogens, IL1ß, and IFN set in motion a series of coordinated events that promote establishment of pregnancy. This is achieved through enhancement of conceptus development, uterine receptivity, maternal-fetal hemotropic exchange, and endometrial leukocyte function. These events require activation of specific signaling pathways within the uterine luminal epithelium, glandular epithelium, and stroma. Here, we review proinflammatory cytokine expression by pig conceptuses and the hypothesized actions of these molecules during establishment of pregnancy.

Activation of the transcription factor nuclear factor-kappa B in uterine luminal epithelial cells by interleukin 1 Beta 2: a novel interleukin 1 expressed by the elongating pig conceptus.

Conceptus mortality is greatest in mammals during the peri-implantation period, a time when conceptuses appose and attach to the uterine surface epithelium while releasing proinflammatory molecules. Interleukin 1 beta (IL1B), a master proinflammatory cytokine, is released by the primate, rodent, and pig blastocyst during the peri-implantation period and is believed to be essential for establishment of pregnancy. The gene encoding IL1B has duplicated in the pig, resulting in a novel gene. Preliminary observations indicate that the novel IL1B is specifically expressed by pig conceptuses during the peri-implantation period. To verify this, IL1B was cloned from mRNA isolated from Day 12 pig conceptuses and compared with IL1B cloned from mRNA isolated from pig peripheral blood leukocytes (PBLs). The pig conceptuses, but not the PBLs, expressed a novel IL1B, referred to here as interleukin 1 beta 2 (IL1B2). Porcine endometrium was treated with recombinant porcine interleukin 1 beta 1 (IL1B1), the prototypical cytokine, and IL1B2 proteins. Immunohistochemistry and real-time RT-PCR were used to measure activation of nuclear factor-kappa B (NFKB) and NFKB-regulated transcripts, respectively, within the endometrium. Both IL1B1 and IL1B2 activated NFKB in the uterine luminal epithelium within 4 h. The NFKB activation and related gene expression, however, were lower in endometrium treated with IL1B2, suggesting that the conceptus-derived cytokine may have reduced activity within the uterus. In conclusion, the peri-implantation pig conceptus expresses a novel IL1B that can activate NFKB within the uterine surface epithelium, likely creating a proinflammatory microenvironment during establishment of pregnancy in the pig.

Cytokines from the pig conceptus: roles in conceptus development in pigs.

Establishment of pregnancy in pigs involves maintaining progesterone secretion from the corpora lutea in addition to regulating a sensitive interplay between the maternal immune system and attachment of the rapidly expanding trophoblast for nutrient absorption. The peri-implantation period of rapid trophoblastic elongation followed by attachment to the maternal uterine endometrium is critical for establishing a sufficient placental-uterine interface for subsequent nutrient transport for fetal survival to term, but is also marked by the required conceptus release of factors involved with stimulating uterine secretion of histotroph and modulation of the maternal immune system. Many endometrial genes activated by the conceptus secretory factors stimulate a tightly controlled proinflammatory response within the uterus. A number of the cytokines released by the elongating conceptuses stimulate inducible transcription factors such as nuclear factor kappa B (NFKB) potentially regulating the maternal uterine proinflammatory and immune response. This review will establish the current knowledge for the role of conceptus cytokine production and release in early development and establishment of pregnancy in the pig.

Uterine progesterone receptor expression, conceptus development, and ovarian function in pigs treated with RU 486 during early pregnancy.

Establishment of pregnancy in the pig depends on down-regulation of progesterone receptor (PGR) in the uterine luminal and glandular epithelium during the first week after breeding. The present study evaluated the regulation of endometrial PGR by progesterone and the possible role of endometrial tumor necrosis factor (ligand) superfamily member 11 (TNFSF11) and nuclear factor-kappa B (NFKB) activation in PGR expression. Mature, cycling gilts were inseminated (Day 0) and assigned to either untreated control (n = 9) or one of two treatments that employed RU 486 to block progesterone action either before (treatment 1 [T1]) or after (treatment 2 [T2]) the initiation of PGR down-regulation. The T1 gilts were treated with RU 486 (400 mg/day) on Days 3-5 of pregnancy (n = 9), and T2 gilts were treated with RU 486 on Days 6 and 7 of pregnancy (n = 9). Uteri and ovaries were collected on Day 8 or 12 of gestation. The diameter of the conceptuses in T1 gilts was approximately half that in controls by Day 8, and normal conceptuses were not collected from any T1 gilts on Day 12. Endometrial PGR mRNA was more abundant in T1 and T2 gilts compared with control gilts. The PGR-B protein decreased from Day 8 to Day 12 in the luminal epithelium and, to some extent, in superficial glandular epithelium in control and T2 gilts. In T1 gilts, the PGR-B protein remained elevated (i.e., failed to undergo down-regulation) on Day 12. Blocking PGR action early in the cycle (i.e., on or before Day 5), therefore, prevented normal conceptus development, caused elevated PGR mRNA, and prevented the decrease in PGR protein that typically occurs in pigs. We could not confirm a role for NFKB activation in PGR down-regulation, because pigs with extreme differences in PGR and TNFSF11 expression (T1 and controls) had similar NFKB activation on Day 8. Activated NFKB within the luminal epithelium and glandular epithelium (both superficial and deep) was observed in T2 and control pigs on Day 12 when elongating conceptuses (presumably releasing interleukin 1 beta to activate NFKB) were recovered. Gilts treated with RU 486 had greater ovarian follicular growth and greater plasma estradiol concentrations. We conclude that the mechanisms controlling PGR down-regulation are progesterone-dependent and occur between Day 3 and Day 6 of pregnancy. NFKB activation did not appear to have a role in PGR down-regulation within the period that we studied. Blocking progesterone action after Day 6 did not reverse the process of PGR down-regulation, nor did it appear to affect the development of conceptuses collected on Day 12.