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Equine endometrial gene expression changes during and after maternal recognition of pregnancy
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The mechanism for maternal recognition of pregnancy (MRP) in horses is unknown. To maintain a pregnancy, a mobile conceptus must be recognized by the uterus before d 14 postovulation (PO). This recognition prevents endometrial secretion of PGF2α on d14 through 16, which would otherwise initiate luteolysis. The objective of this study was to evaluate gene expression in the endometrium of pregnant and nonpregnant mares during and after MRP to identify possible genes involved during this time. Twelve normally cycling mares were used in a crossover design and randomly assigned to a specific collection day. Endometrial samples were collected from a pregnant and nonpregnant (nonmated) mare on cycle d 12, 14, 16, and 18 (n = 3/d) PO. Microarray analysis comparing the endometrial gene expression in pregnant and nonpregnant mares revealed no differences at d 12. Ten genes were identified to have consistently higher or lower expression levels in the endometrium from pregnant versus nonpregnant mares on d 14, 16, and 18 (P < 0.001). The expression of these 10 genes was further analyzed with real-time PCR. d 14, 16, and 18 gene expression patterns were consistent with the microarray analysis, but on d 12, 4 of the 10 were identified as differentially expressed. Endometrial samples were then collected on d 13 PO (n = 3) and processed for western blot and immunohistochemical analysis of 2 proteins due to their reproductive significance. SPLA2 and DKK1 antibody specificity were confirmed via western blot analysis but were not different in samples from pregnant and nonpregnant mares (P = 0.114 and P = 0.514, respectively) and cellular localization was examined by immunohistochemical analysis. This is the first study to describe gene expression and cellular localization in the endometrium at the time of MRP for these genes and suggests that the uterus does not prepare to support a pregnancy until d 14. The function of these genes may be critical in the process of MRP.
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... The specific factors secreted by the conceptus may also prepare the uterus for a receptive environment to support maintenance of pregnancy. Attempts to unravel the signaling molecule(s) have been focused on examining the embryo and the receptive endometrium as well as the uterine environment (uterine fluid) by using different transcriptomics and proteomics approaches [6][7][8][9][10][11][12][13][14] . However, the molecule or variety of molecules responsible for MRP remained still a mystery in the horse. ...
... two times per day) to avoid intrauterine fluid accumulation. The days of sampling (10,11,12, and 13 after ovulation), 100 ml of phosphate-buffered saline (PBS) (Corning cellgro; Virginia, USA) were introduced in the uterus of each mare using an equine uterine flushing catheter (8 mm inner and 10 mm outer diameter; JorgVet, Jorgensen Labs; Colorado, USA) in the C groups or an endotracheal tube for small ruminants (13.0 mm inner and 17.3 mm outer diameter; DEE Veterinary Products) for the P groups. Then, a careful transrectal massage of the uterus was performed to assure an equal passage of the fluid throughout the entire uterus. ...
In contrast to other domestic mammals, the embryo-derived signal(s) leading to maternal recognition of pregnancy (MRP) are still unknow in the mare. We hypothesize that these embryonic signals could be packed into uterine extracellular vesicles (uEVs), acting as multi-signal messengers between the conceptus and the maternal tract, and contributing to MRP. To unveil these signals, the RNA and protein cargos of uEVs isolated from uterine lavages collected from pregnant mares (P; day 10, 11, 12 and 13 after ovulation) and cyclic control mares (C; day 10 and 13 after ovulation) were analyzed. Our results showed a fine-tuned regulation of the uEV cargo (RNAs and proteins), by the day of pregnancy, the estrous cycle, and even the size of the embryo. A particular RNA pattern was identified with specific increase on P12 related to immune system and hormonal response. Besides, a set of proteins as well as RNAs was highly enriched in EVs on P12 and P13. Differential abundance of miRNAs was also identified in P13-derived uEVs. Their target genes were linked to down- or upregulated genes in the embryo and the endometrium, exposing their potential origin. Our study identified for first time specific molecules packed in uEVs, which were previously associated to MRP in the mare, and thus bringing added value to the current knowledge. Further integrative and functional analyses will help to confirm the role of these molecules in uEVs during MRP in the mare.
... With the advent of new techniques for comprehensive transcriptome analysis, several studies have been performed to analyze endometrial gene expression changes in response to the presence of a conceptus during the period of MRP in the mare, mainly focusing on days 8, 12, 13, 13.5 or 16 [11][12][13][14][15][16] . Two studies analyzed endometrial miRNA expression during MRP in addition to mRNAs 13,16 , and Smits et al. 17 performed also a comparison to gene expression in the embryo and protein expression in the uterine fluid. ...
... On the basis of the experiences in our previous studies 11 and the results of others 12,14 , a robust approach for identification of DGE was used based on a higher number of biological replicates. Particularly, the studies of Klohonatz et al. 12,14 indicated that a low number of replicates can lead to controversial results since they did not find DEGs on day 12 of P in their first study 28 , but later they even identified DEGs on days 9 and 11 of P 12 . Unfortunately, these and other authors did not provide information about sizes of obtained conceptuses. ...
During the period of maternal recognition of pregnancy (MRP) in the mare, the embryo needs to signal its presence to the endometrium to prevent regression of the corpus luteum and prepare for establishment of pregnancy. This is achieved by mechanical stimuli and release of various signaling molecules by the equine embryo while migrating through the uterus. We hypothesized that embryo’s signals induce changes in the endometrial gene expression in a highly cell type-specific manner. A spatiotemporal transcriptomics approach was applied combining laser capture microdissection and low-input-RNA sequencing of luminal and glandular epithelium (LE, GE), and stroma of biopsy samples collected from days 10–13 of pregnancy and the estrous cycle. Two comparisons were performed, samples derived from pregnancies with conceptuses ≥ 8 mm in diameter (comparison 1) and conceptuses ≤ 8 mm (comparison 2) versus samples from cyclic controls. The majority of gene expression changes was identified in LE and much lower numbers of differentially expressed genes (DEGs) in GE and stroma. While 1253 DEGs were found for LE in comparison 1, only 248 were found in comparison 2. Data mining mainly focused on DEGs in LE and revealed regulation of genes related to prostaglandin transport, metabolism, and signaling, as well as transcription factor families that could be involved in MRP. In comparison to other mammalian species, differences in regulation of genes involved in epithelial barrier formation and conceptus attachment and implantation reflected the unique features of equine reproduction at the time of MRP at the molecular level.
... On the endometrium side, transcriptomic gene expression studies on whole endometrial biopsies using microarrays or RNA-sequencing (RNA-seq) have been performed. [16][17][18][19][20][21][22] Moreover, two spatial transcriptomics studies have focused on specific alterations of the different endometrial compartments (luminal epithelium [LE], glandular epithelium [GE], and stroma [ST]) by combining laser capture microdissection (LCM) and low input RNA-seq. 23,24 On the embryo side, only a few studies at the mRNA level have been carried out. ...
During initial maternal recognition of pregnancy (MRP), the equine embryo displays a series of unique events characterized by rapid blastocyst expansion, secretion of a diverse array of molecules, and transuterine migration to interact with the uterine surface. Up to date, the intricate transcriptome and proteome changes of the embryo underlying these events have not been critically studied in horses. Thus, the objective of this study was to perform an integrative transcriptomic (including mRNA, miRNAs, and other small non‐coding RNAs) and proteomic analysis of embryos collected from days 10 to 13 of gestation. The results revealed dynamic transcriptome profiles with a total of 1311 differentially expressed genes, including 18 microRNAs (miRNAs). Two main profiles for mRNAs and miRNAs were identified, one with higher expression in embryos ≤ 5 mm and the second with higher expression in embryos ≥ 7 mm. At the protein level, similar results were obtained, with 259 differentially abundant proteins between small and large embryos. Overall, the findings demonstrated fine‐tuned transcriptomic and proteomic regulations in the developing embryo associated with embryo growth. The identification of specific regulation of mRNAs, proteins, and miRNAs on days 12 and 13 of gestation suggested these molecules as pivotal for embryo development and as involved in MRP, and in establishment of pregnancy in general. In addition, the results revealed new insights into prostaglandin synthesis by the equine embryo, miRNAs and genes potentially involved in modulation of the maternal immune response, regulation of endometrial receptivity and of late implantation in the mare.
... In this study, conceptus ESR1 was correlated with OXTR on D10. The conceptus may engage paracrine mechanisms to regulate steroid receptors, as pregnant mares have a decreased endometrial expression of ESR1 on D13.5, 14 and 15 compared to nonpregnant mares [60][61][62]. Similar to the conceptus ESR1 pattern of expression, conceptus PGR expression was higher on D12 and D21; however, this was reversed on D10, where ESR1 had lower expression and PGR higher expression, which then decreased by D14 [4]. ...
Leucyl and cystinyl aminopeptidase (LNPEP/oxytocinase) is an enzyme that metabolizes oxytocin in serum and tissues. The presence of oxytocin/neurophysin I (OXT), oxytocin and LNPEP and their relationship to other genes is unknown in the equine conceptus. Our objective was to characterize gene expression of LNPEP and OXT on D8, 10, 12, 14, 15, 16 and 21 conceptuses in relationship to other genes. Immunohistochemistry, western blot and liquid chromatography with tandem mass spectrometry (LC-MS/MS) were used for identification of oxytocin and LNPEP in D15, 16 and 18 conceptuses. LNPEP was increased at D15 compared to D10, was immunolocalized in the equine trophectoderm and endoderm, and protein was confirmed by LC-MS/MS. Maximal abundance of OXT was at D21, and lowest on D12 and D14, but no protein was identified. OXTR abundance was highest on D14 and D21. LNPEP was correlated with PTGFR and PTGES on D12 and D14–D15, and high expression of PTGES, PTGS2 was found on D14, D15 and D21; PTGFR was found on D8 and D12–21. LNPEP may have a role in prostaglandin regulation and conceptus fixation by decreasing the availability of oxytocin. Further investigation on the role embryonic LNPEP during pregnancy is warranted.
... The lower gene expression of ESR1 in the pregnancy group than in the nonbred group agree with previous reports that in pregnant mares downregulation of ESR1 occurs on Days 10 [9] and 12 [54]. The ESR1 plays a major role in the uterotrophic effect of estrogen [11], and a decrease of ESR1 have been described as one of the regulators of the luteal response to pregnancy in mares [5]. ...
Prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) are involved in equine embryo mobility throughout the uterus on Days 11–15 (ovulation = Day 0). On a day (Day 12) of maximal embryo mobility in pregnant mares (n = 13) and before luteolysis in nonbred mares (n = 10), gene expressions were compared between the uterine horns that did and did not contain the mobile embryo and between pregnant and nonbred mares. A cytobrush was used to collect an endometrial sample from the middle of each uterine horn. In nonbred mares, there was no difference for any of the considered gene expressions between the uterine horn ipsilateral and contralateral to the CL or for side (left vs right). For endometrial estrogen receptors, ESR1 was lower (P < 0.03) and ESR2 was greater (P < 0.04) for pregnant than nonbred mares. The mRNA abundance for PGE2 synthase (PTGES) was greater (P < 0.05) in the horn with (1.40 ± 0.10) than without (0.89 ± 0.10) the embryo and was greater (P < 0.05) in the horn with the embryo than in the combined horns of nonbred mares (1.06 ± 0.10). The hypothesis that the embryo locally upregulates PGE2 and PGF2α synthesis in the endometrium adjacent to the embryo in the pregnant group but not in the uterine horns of the nonbred group, was partially supported; only PGE2 synthase (PTGES) was locally upregulated in the endometrium adjacent to the mobile embryo.
... Therefore, it seems reasonable to suspect that some sort of physical, rather than biochemical, interaction between the capsule and the endometrium is involved in initiating the so-called "maternal recognition of pregnancy signal" in the mare which suppresses the release of PGF2α from the endometrium during days 14-16 after ovulation that would normally occur to induce luteolysis and a return to estrus in the cycling mare (Stabenfeldt et al. 1974;Allen 1981). However, the identity and mode of action of the equine "maternal recognition of pregnancy signal" remains unproven and is a matter of considerable conjecture in the equine reproduction research community at the present time (Klohonatz et al. 2015;Klein 2016;Smits et al. 2018). ...
This chapter focuses on the early stages of placental development in horses and their relatives in the genus Equus and highlights unique features of equid reproductive biology. The equine placenta is classified as a noninvasive, epitheliochorial type. However, equids have evolved a minor component of invasive trophoblast, the chorionic girdle and endometrial cups, which links the equine placenta with the highly invasive hemochorial placentae of rodents and, particularly, with the primate placenta. Two types of fetus-to-mother signaling in equine pregnancy are mediated by the invasive equine trophoblast cells. First, endocrinological signaling mediated by equine chorionic gonadotrophin (eCG) drives maternal progesterone production to support the equine conceptus between days 40 and 100 of gestation. Only in primates and equids does the placenta produce a gonadotrophin, but the evolutionary paths taken by these two groups of mammals to produce this placental signal were very different. Second, florid expression of paternal major histocompatibility complex (MHC) class I molecules by invading chorionic girdle cells stimulates strong maternal anti-fetal antibody responses that may play a role in the development of immunological tolerance that protects the conceptus from destruction by the maternal immune system. In humans, invasive extravillous trophoblasts also express MHC class I molecules, but the loci involved, and their likely function, are different from those of the horse. Comparison of the cellular and molecular events in these disparate species provides outstanding examples of convergent evolution and co-option in mammalian pregnancy and highlights how studies of the equine placenta have produced new insights into reproductive strategies.
Embryo-maternal crosstalk is essential to establish pregnancy, with the equine embryo moving throughout the uterus on days 9–15 (ovulation = day 0) as part of this interaction. We hypothesized that the presence of a mobile embryo induces local changes in the gene expression of the endometrium. On Day 12, the endometrial transcripts were compared among three groups: uterine horn with an embryo (P+, n = 7), without an embryo (P−, n = 7) in pregnant mares, and both uterine horns of nonbred mares (NB, n = 6). We identified 1,101 differentially expressed genes (DEGs) between P+ vs. NB and 1,229 DEGs between P− vs. NB. The genes upregulated in both P+ and P− relative to NB were involved in growth factor pathway and fatty acid activation, while downregulated genes were associated with oxytocin signaling pathway and estrogen receptor signaling. Comparing the transcriptome of P+ to that of P−, we found 59 DEGs, of which 30 genes had a higher expression in P+. These genes are associated with regulating vascular growth factors and the immune system, all known to be essential in early pregnancy. Overall, this study suggests that the mobile embryo influences the endometrial gene expression locally.
Our objectives were to examine changes in endometrial and luteal gene expression during estrus, diestrus, pregnancy and treatments to induce luteolysis and putatively induce luteostasis. Groups were: Diestrus (DIEST), Estrus (ESTR), Pregnant (PREG), Oxytocin (OXY), Carbetocin (CARB), and Meclofenamic acid (MFA). Blood was obtained from day (D)12 to D15 for measurement of oxytocinase, also referred to as leucyl-cysteinyl aminopeptidase (LNPEP) and progesterone. Luteal biopsies were obtained on D12 and D15 and an endometrial biopsy on D15. Real-time RT-PCR was performed for the following genes: PGR, ESR1, OXTR, OXT, LNPEP, PTGS2, PTGFR, PLA2G2C, PTGES, SLC2A4, and SLC2A1. Regarding serum LNPEP, PREG and OXY (p-value<0.001) had higher concentrations than DIEST mares. Endometrial PTGES expression was higher (p-value <0.04) in DIEST, PREG and OXY than other groups. Endometrium from ESTR had increased expression of OXT (p-value < 0.02) compared to MFA and OXY mares. Carbetocin treatment: decreased serum progesterone and LNPEP; increased endometrial PLA2G2C; decreased endometrial PTGES; and decreased luteal aromatase and PTGES. Treatment with MFA: decreased endometrial PLA2G2C, increased endometrial PTGES; and resulted in less OXTR and OXT luteal abundance on D12 compared to D15. Endometrial and luteal expression of LNPEP is affected by physiologic stage and treatment and is involved in luteal function and pregnancy recognition pathways through effects on oxytocin and prostaglandin synthesis in the horse.
Our understanding of the temporal changes in endometrial and luteal gene transcripts related to the actions of oxytocin and prostaglandin during early equine pregnancy is incomplete. Additionally, the role of oxytocinase, also known as Leucyl-cystinyl aminopeptidase (LNPEP), during early pregnancy in mares has not been previously investigated. Luteal and endometrial biopsies were obtained on Day (D)8, D10, D12 and D15 post-ovulation in pregnant (PREG) and diestrus (DIEST) mares for real-time qPCR. Differences in endometrial gene expression occurred over time in: SLC2A4, SLC2A1, PTGES, OXTR and LNPEP. PTGFR and PLA2G2C had lower relative abundance in PREG D15 endometrium compared to D10. OXT and OXTR were increased on D10 and 15 PREG, respectively. Regarding luteal mRNA relative abundance, ESR1, PTGS2, PTGFR, and PTGES had higher relative abundance in D12 of DIEST and PREG. Luteal expression of OXTR and OXT had higher relative abundance in D15 compared to D8, and LNPEP had higher relative abundance in D10 and 12. Endometrial and luteal PTGES had an increased mRNA abundance in both D12 DIEST and PREG mares, which may lead to additional luteoprotective prostaglandin E2 (PGE2) secretion. Furthermore, luteal SLC2A1 had higher relative abundance in pregnancy, and likely supports the high metabolic activity of luteal tissue by increasing glucose uptake. Oxytocinase is present in endometrial and luteal tissue and its role in oxytocin induced prostaglandin secretion is uncertain.
The current study used RNA sequencing to determine transcriptional profiles of equine endometrium collected 14, 22, and 28 days after ovulation from pregnant mares. In addition, the transcriptomes of endometrial samples obtained 20 days after ovulation from pregnant mares, and from non-pregnant mares which displayed and failed to display extended luteal function following the administration of oxytocin, were determined and compared in order to delineate genes whose expressions depend on the presence of the conceptus as opposed to elevated progesterone alone. A mere fifty-five transcripts were differentially expressed between samples collected from mares at Day 22 and Day 28 of pregnancy. This likely reflects the longer-term exposure to a relatively constant, progesterone-dominated environment with little change in factors secreted by the conceptus that would affect endometrial gene expression. The complement system was amongst the canonical pathways significantly enriched in transcripts differentially expressed between Day 14 and Day 22/28 of pregnancy. The expression of complement components 7 and 8 was confirmed using in situ hybridization. The expression of SERPING1, an inhibitor of the complement system, was confirmed by immunohistochemistry. In line with the resumed capacity of the endometrium to produce prostaglandin, prostaglandin G/H synthase 1 was expressed at higher levels at Days 22 and 28 than at Day 14 of pregnancy. Our data suggest that this up-regulation is enhanced by the presence of the conceptus; samples obtained from mares at Day 20 of pregnancy had significantly higher levels of prostaglandin G/H synthase 1 transcript than mares with extended luteal function.
Despite enormous efforts, biochemical and molecular mechanisms associated with equine reproduction, particularly processes of pregnancy establishment, have not been well characterized. Previously, PCR-selected suppression subtraction hybridization analysis was executed to identify unique molecules functioning in the equine endometrium during periods of pregnancy establishment, and granzyme B (GZMB) cDNA was found in the pregnant endometrial cDNA library. Because GZMB is produced from natural killer (NK) cells, endometrial expression of GZMB and immune-related transcripts were characterized in this study. The level of GZMB mRNA is higher in the pregnant endometrium than in non-pregnant ones. This expression was also confirmed through Western blot and immunohistochemical analyses. IL-2 mRNA declined as pregnancy progressed, while IL-15, IFNG and TGFB1 transcripts increased on day 19 and/or 25. Analyses of IL-4 and IL-12 mRNAs demonstrated the increase in these transcripts as pregnancy progressed. Increase in CCR5 and CCR4 mRNAs indicated that both Th1 and Th2 cells coexisted in the day 25 pregnant endometrium. Taken together, the expression of immune-related transcripts suggests that immunological responses are present even before the trophectoderm actually attaches to the uterine epithelial cells.
Establishment and maintenance of pregnancy in equids is only partially understood. To provide new insights into early events of this process, we performed a systematic analysis of transcriptome changes in the endometrium at Days 8 and 12 of pregnancy. Endometrial biopsy samples from pregnant and nonpregnant stages were taken from the same mares. Composition of the collected biopsy samples was analyzed using quantitative stereological techniques to determine proportions of surface and glandular epithelium and blood vessels. Microarray analysis did not reveal detectable changes in gene expression at Day 8, whereas at Day 12 of pregnancy 374 differentially expressed genes were identified, 332 with higher and 42 with lower transcript levels in pregnant endometrium. Expression of selected genes was validated by quantitative real-time RT-PCR. Gene set enrichment analysis, functional annotation clustering, and cocitation analysis were performed to characterize the genes differentially expressed in Day 12 pregnant endometrium. Many known estrogen-induced genes and genes involved in regulation of estrogen signaling were found, but also genes known to be regulated by progesterone and prostaglandin E2. Additionally, differential expression of a number of genes related to angiogenesis and vascular remodeling suggests an important role of this process. Furthermore, genes that probably have conserved functions across species, such as CRYAB, ERRFI1, FGF9, IGFBP2, NR2F2, STC1, and TNFSF10, were identified. This study revealed the potential target genes and pathways of conceptus-derived estrogens, progesterone, and prostaglandin E2 in the equine endometrium probably involved in the early events of establishment and maintenance of pregnancy in the mare.
Progesterone (P) down-regulation of uterine estradiol (E) receptor (ER) appears to be a general mechanism by which P modulates E action in the uterus. Our present studies focus on the regulation of ER by P during the changeover from E to P dominance during artificial menstrual cycles in the rhesus monkey. Because of differential cell-type response and the cellular zonation of the primate uterus, we used immunohistochemical analysis in addition to biochemical assays to study the regulation of ER by P. Ki-67 immunoreactivity was used as an index of endometrial proliferation. We performed our analyses on Days 13 (peak of E), 14 (declining E and rising P), 17 (basal E and rising P), and 21 (basal E and peak P). ER immunoreactivity was present throughout the endometrium in luminal and glandular epithelia and stromal fibroblasts on Day 13. As E was withdrawn and P rose on Day 14 there were few distinct changes in ER staining in stromal and epithelial cells. On Day 17, immunoreactive staining showed a distinct reduction for stromal cells in all zones. Although luminal epithelial cells showed a decrease in immunoreactivity on Day 17, zones II, III, and IV retained positive staining for ER in glandular epithelia. ER staining in stromal cells on Day 21 was similar to the pattern observed on Day 17, whereas epithelial cells in zones I, II, and III showed a reduction in staining. Glandular epithelia in zone IV maintained strong positive staining for ER on Day 21.(ABSTRACT TRUNCATED AT 250 WORDS)
The expression of calbindin-D9K (CaBP9K) and calbindin-D28K (CaBP28K) genes in the reproductive system is well established for rodent and avian species, but not for domestic livestock. This investigation expanded the study of these proteins to include the bovine uterus and examined the levels of CaBP9K and CaBP9K mRNA in the nonpregnant bovine uterus during the estrous cycle. Immunohistochemical studies revealed that CaBP9K was present in all uterine glandular and luminal epithelial cells. In contrast, the closely related calcium binding protein CaBP28K was present in only one to two glandular cells in the samples examined. Neither protein was localized in the myometrium or in the stromal cells of the endometrium. RIA and dot blot hybridization were used to quantify the amount of CaBP9K and CaBP9K mRNA. The levels of both the protein and its mRNA were threefold higher during the luteal phase than during the follicular phase. RIA was also used to determine bovine uterine levels of 17 beta-estradiol and progesterone. Progesterone levels were higher during the luteal phase than during the follicular phase, while 17 beta-estradiol levels were higher during the follicular phase. This investigation represents the first characterization of CaBP9K gene expression in the bovine uterus. It demonstrated that the expression of CaBP9K and CaBP9K mRNA was greatest during the progesterone-dominated luteal phase of the bovine estrous cycle. These results indicated that CaBP9K may be involved in uterine glandular function during the luteal phase.
Adrenomedullin (AM) is a 52-amino acid peptide with a pluripotential activity. AM is expressed in many tissues throughout the body, and plays a critical role in several diseases such as cancer, diabetes, cardiovascular and renal disorders, among others. While AM is a protective agent against cardiovascular disorders, it behaves as a stimulating factor in other pathologies such as cancer and diabetes. Therefore, AM is a new and promising target for the development of molecules which, through their ability to regulate AM levels, could be used in the treatment of these pathologies.
Previous reports documenting progesterone receptors (PR) and oestrogen receptors (ER) in the endometrium of early pregnant mares included specimens only up to Day 20 post ovulation. This study aimed to localise PR and ERα on equine feto-maternal tissues between Days 20 and 68 to encompass the period around fixation of the conceptus, development of the endometrial cups and attachment and initial interdigitation of the allantochorion. During early pregnancy mares had the same pattern of PR in the endometrium as that reported for other mammals; namely, a loss of PR from the endometrial epithelia but continued localisation in stromal cells. The spatial arrangement of ERα over the same time period showed cytoplasmic staining of endometrial epithelia and in the nuclei of occasional stromal cells. In the fetal tissues, no cells had PR although ERα was evident in some tissue compartments. No major change in localisation of either receptor was noted throughout the time period examined despite important changes occurring at the placental interface. Nevertheless, these steroid receptor molecules probably play important roles in the production of histotroph and growth factors by the endometrium which go on to stimulate differentiation and growth of the feto-maternal tissues.
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A critical period of early gestation in the mare involves the immobilization (fixation) of the encapsulated conceptus at around days 16–17. We compared the major proteins in the normal equine embryonic capsule and endometrial secretions around the period of fixation with those from pregnancies in the process of termination induced by administration of an analogue of prostaglandin F 2α (PGF 2α ). Uterocalin and β 2 ‐microglobulin (β 2 M) associated with the embryonic capsule were proteolytically converted to smaller forms during the fixation period. These conversions were similar in conceptuses from control and treated mares. A 17 kDa cationic protein identified as a secretory phospholipase A2 (sPLA2) type IIA was detected bound to normal capsules but increased substantially in response to PGF 2α . Two forms of uteroglobin were distinguished by partial amino acid sequences of ∼6 kDa bands in flush fluids from normal pregnant uteri. After administration of PGF 2α one immunoreactive form of uteroglobin was preferentially increased. These studies demonstrate that failure of pregnancy in this model is associated with an increase in secretory phospholipase in the capsule and a change in the forms of uteroglobin in the uterine secretions.
WNT signaling pathway plays important roles in reproductive events. Aims were to (1) determine presence of WNT genes and their antagonists in equine endometrium; and (2) to evaluate their expression profiles during early pregnancy. Endometrial biopsies were obtained from mares on day of ovulation (d0, n=4) and on days of 14 (P14, n=4), 18 (P18, n=4), 22 (P22, n=4) of early pregnancy. Biopsies were also collected from cyclic mares during late diestrus (LD, on day of 13.5-14, n=4) and after luteolysis in estrus phase (AL, on day of 17.5-18, n=4) of the cycle. PCR was used to detect expression of genes studied and then relative expression levels were quantified using real-time PCR analysis. A mixed model was fitted on the normalized data and least significant difference test (α=0.05) was employed. Eleven WNT genes (WNT2, WNT2B, WNT4, WNT5A, WNT5B, WNT7A, WNT8A, WNT9B, WNT10B, WNT11 and WNT16) and their antagonists (SFRP1, SFRP2, SFRP5, DKK1, DKK2 and WIF-1) were detected in equine endometrium. Compared to d0, WNT2, WNT5B, WNT7A and SFRP1 expressions were downregulated by the pregnancy while DKK1 was upregulated. WNT5A, WNT11 and WIF-1 were upregulated on P14 and P18, but WNT2B increased only on P14. When LD and P14 were compared, level of WNT8A decreased on P14 while increase in WNT4 level on P14 was slightly significant (P<0.06). Levels of WNT7A and SFRP1 decreased while DKK1 and WIF-1 increased by the pregnancy on P18 compared to AL. Moreover, WNT2B, WNT5A, WNT9B, WNT10B, WNT11, WNT16 DKK1 and WIF-1 were upregulated on LD compared to AL whereas WNT4, WNT7A, SFRP1 were downregulated. In conclusion, the results demonstrate that WNT genes and their antagonists appear to be regulated during early pregnancy in equine endometrium possibly due to embryonic factors and/or maternal progesterone.
Establishment and maintenance of pregnancy are critically dependent on embryo-maternal communication during the preimplantation period. The horse is one of the few domestic species in which the conceptus-derived pregnancy recognition signal has not been identified. To gain new insights into the factors released by the equine conceptus, transcriptional profiling analyses of conceptuses retrieved 8, 10, 12, and 14 days after ovulation were performed using a whole-genome microarray. Selected array data were confirmed using quantitative PCR, and the expression of proteins of interest was confirmed using immunohistochemistry and Western blotting. Gene ontology classification of differentially regulated transcripts underlines the ongoing embryo-maternal dialogue. Transcript showing higher expression levels as conceptus' development proceeds mainly localizes to the extracellular environment, thereby having the potential to act upon the uterine environment. Genes involved in the positive regulation of the immune system are enriched among transcripts displaying decreased expression, reflecting the need of the semiallograft conceptus to be protected from the immune system. A subset of differentially expressed genes, such as BRCA1 and FGF2, has previously been described to be expressed by early stages of embryonic development, whereas other transcripts are apparently unique to equine conceptuses, as their expression has not been reported in other species. These transcripts include fibrinogen subunits, the expressions of which were confirmed at the mRNA and protein level. Furthermore, results indicate the counteraction of trophoblast invasion, and that the conceptus appears to regulate changes in sialic acid content of its capsule, an event suggested to be essential for successful establishment of pregnancy.
The maternal recognition of pregnancy (MRP) signal in the mare has not been determined, although oestrogens have been proposed as a potential candidate.
To determine effects of intrauterine administration of oestrogen and various oils on cyclic luteolysis in the mare. Hypothesis: Intrauterine oestradiol or fatty acids may suppress luteolysis in the cycling mare when administered during late dioestrus.
A single 1 ml dose of slow-release oestradiol (10 mg/ml) in fractionated coconut oil was infused into the uterine lumen of cycling mares on Days 6, 8, 10, 12 or 14 post ovulation (n=12 in each group). Four further groups, each of 12 mares, received an intrauterine infusion of either 1 ml of fractionated coconut oil, peanut oil, mineral oil or a slow-release preparation of oestradiol (10 mg/ml) in mineral oil on Day 10 post ovulation. Serial blood samples were assayed for progesterone concentrations to monitor luteal function.
Intrauterine administration of oestradiol in fractionated coconut oil showed peak efficacy at Day 10 when luteolysis was delayed in 11/12 (92%) mares. The ability of the treatment to delay luteolysis was not significantly different when administered on Days 8 (9/12; 75%), 12 (10/12; 83%) or 14 (6/12; 50%) of dioestrus, but declined significantly when given on Day 6 (3/12; 25%). Oestradiol was not needed to initiate luteostasis since fractionated coconut oil alone or peanut oil administered at Day 10 induced the same high rate of luteal persistence (11/12; 92% for both oils). In contrast, mineral oil did not prolong luteal lifespan, either when administered alone (2/12; 17%) or combined with oestradiol (3/12; 25%).
These results do not unequivocally rule out a possible involvement of embryonic oestrogens in MRP in the mare but suggest it is unlikely. The results demonstrate that plant oils can postpone luteolysis, suggesting they may modulate synthesis or release of prostaglandins from the mare's endometrium.
Administration of fractionated coconut or peanut oil on Day 10 post ovulation provides an effective and practical method of prolonging luteal function ('pseudopregnancy') thereby suppressing unwanted oestrous behaviour. Further studies to elucidate the mechanism by which this is achieved may increase understanding of both luteostasis and MRP signal in the mare.