Claudia Klein

Transcriptional Profiling of Equine Endometrium During the Time of Maternal Recognition of Pregnancy

Establishment and maintenance of pregnancy are critically dependent on embryo-maternal communication during the preimplantation period. To gain new insights into this complex process in the horse, transcriptional profiling of Day 13.5 pregnant and cyclic endometrial tissue samples was carried out using custom-designed microarrays. Selected array data were validated using quantitative RT-PCR, and proteins of interest were localized using immunohistochemistry. One hundred and six transcripts were up-regulated, whereas 47 transcripts showed lower expression levels in pregnant mares, that is, were down-regulated in pregnant mares. Half of the genes with known or inferred function are classically regulated by estrogens. Elevated transcript levels were found for genes involved in cell-cell signaling, heat shock response, and secretory proteins, among others. Solute carrier family 36 (proton/amino acid symporter), member 2, SLC36A2, was one of the most highly up-regulated genes, potentially reflecting the nutritional needs of the rapidly developing embryo. Among the genes showing lower expression in pregnant mares, estrogen receptor 1 was of particular interest because of its potential involvement in the initiation of luteolysis in cyclic mares. We hypothesize that either conceptus estrogens or luteinizing hormone of uterine origin is involved in the observed down-regulation of estrogen receptor 1. Several of the genes identified in the current study are known to play a role in early pregnancy in species other than the horse. Thus, products of these commonly expressed genes likely contain universal activities for controlling endometrial receptivity to the conceptus, whereas other factors play unique roles within specific species in ensuring ongoing corpus luteum function. This is the first systematic study of endometrial transcriptome changes in response to the presence of an embryo during maternal recognition of pregnancy and an important step toward deciphering the embryo-maternal dialogue in equids.

Expression stability of putative reference genes in equine endometrial, testicular, and conceptus tissues

BackgroundQuantitative RT-PCR data are commonly normalized using a reference gene. A reference gene is a transcript which expression does not differ in the tissue of interest independent of the experimental condition. The objective of this study was to evaluate the stability of mRNA expression levels of putative reference genes in three different types of equine tissue, endometrial, testicular, and conceptus tissue.FindingsThe expression stability of four (uterine tissue) and six (testicular and conceptus tissue) was assessed using descriptive data analysis and the software programs Normfinder and geNorm. In uterine samples, 18S showed the largest degree of variation in expression while GAPDH, B2M, and ACTB were stably expressed. B2M and GAPDH were identified as the most stably expressed genes in testicular samples, while 18S showed some extent of regulation between samples. Conceptus tissue overall was characterized by very low variability of the transcripts analyzed with GAPDH, YWHZ, and 18S being the most stably expressed genes.ConclusionsIn equine endometrium, GAPDH, B2M, and ACTB transcript levels are equally stable, while 18S is less stably expressed. In testes and associated structures, B2M and GAPDH are the transcripts showing the least amount of variation, while in conceptus tissue GAPDH, YWHZ, and 18S were identified as the most suitable reference genes. Overall, transcripts analyzed in conceptus tissue were characterized by less variation than transcripts analyzed in uterine and testicular tissue.

Transcriptional Profiling of Equine Conceptuses Reveals New Aspects of Embryo-Maternal Communication in the Horse

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.

Maternal recognition of pregnancy in the horse: a mystery still to be solved

Maternal recognition of pregnancy in the horse is the sum of events leading to maintenance of pregnancy; in a narrow sense, maternal recognition of pregnancy refers to the physiological process by which the lifespan of the corpus luteum is prolonged. The horse is one of the few domestic species in which the conceptus-derived pregnancy recognition signal has not been identified. The presence of the conceptus reduces pulsatile prostaglandin F(2α) secretion by the endometrium during early gestation in the mare, partly attributed to the reduced expression of cyclooxygenase-2. Cyclooxygenase-2 has therefore been suggested as one of the regulators of endometrial prostaglandin F(2α) release modified by the antiluteolytic factor secreted by the conceptus. In addition, altered oxytocin responsiveness has been implicated in the adjustment of prostaglandin release in pregnant mares. While conceptus mobility has proven to be essential for establishment of pregnancy, conceptus-derived oestrogens and prostaglandins, principally prostaglandin E(2), have not been confirmed as the critical antiluteolytic factor. Various ways to induce prolonged luteal function in the non-pregnant mare will be highlighted in the current review, specifically, how they may pertain to the process of maternal recognition of pregnancy. Furthermore, recently published microarray experiments comparing the transcriptome of pregnant and non-pregnant endometria and different stages of conceptus development will be reviewed. Findings include the prevention of conceptus adhesion, the provision of nutrients to the conceptus and the avoidance of immunological rejection, among others.

Conceptus-mediated endometrial vascular changes during early pregnancy in mares – An anatomic, histomorphometric and vascular endothelial growth factor receptor system immunolocalization and gene expression study.

This work examined how the conceptus modulates endometrial tissue remodeling and vascular development prior to implantation in mares. A macroscopic uterine examination was completed at day 21 of pregnancy. In situ morphology revealed that the endometrium involved in encroachment is restricted to the dorsal endometrium immediately overlying the yolk sac. The amount of stromal area occupied by blood vessels and the number of endometrial glands were increased during early pregnancy. Endometrial histomorphometry as well as the endometrial mRNA abundance and immunolocalization of VEGF, VEGFR1, VEGFR2, and Ki-67 was completed at days 14 and 21 of pregnancy, at day 10 of the estrous cycle, and during estrus. No obvious differences in VEGF and VEGFR1 protein localization were detected between pregnant and cycling mares but differential staining pattern for VEGFR2 and Ki-67 was observed. VEGFR2 localized to luminal and glandular epithelium of pregnant mares, while luminal epithelium was negative in cycling mares. Ki-67 staining was weak during the luteal phase but exhibited prominent luminal epithelium staining during estrus. In pregnant mares, all endometrial layers were Ki-67 positive. Quantitative RT-PCR revealed a greater abundance of VEGF mRNA during pregnancy. VEGFR2 transcript abundance was greatest in pregnant mares on day 21. This study supports the concept that the conceptus plays an active role in directing vasculogenesis within the uterus and thereby establishing hemotrophic nutrition that supports pregnancy after implantation.

Induction of ovulation in seasonally anestrous mares under ambient lights using recombinant equine FSH (reFSH)

Traditionally, mares are put under artificial lights to advance the first ovulation of the year. The aim of the present study was to determine the efficacy of recombinant equine FSH (reFSH) in stimulating follicular development and advancing the first ovulation of the year in seasonally anestrous mares compared with anestrous mares given a placebo. Both groups of mares were housed under ambient light conditions. Sixty deep anestrous mares of light horse breeds (follicular diameters ≤ 20 mm in diameter and progesterone <1 ng/mL) were maintained under a natural photoperiod at three different sites: University of California, Davis, Colorado State University, and University of Kentucky Gluck Centre. Twenty mares at each site were randomly allocated to receive either 0.65 mg of reFSH (group A: treatment; n = 10) or a placebo (group B: control; n = 10) twice daily by im beginning on January 31. Treatment continued until one or more preovulatory follicles developed or up to a maximum of 15 days. Randomized treatments were blinded. Follicular development was closely monitored by transrectal ultrasonography. When the largest follicle reached ≥ 35 mm in diameter, reFSH treatment was discontinued and an injection of 2500 international units of hCG was administered iv 36 hours later to induce ovulation. Jugular blood samples were collected daily from all mares at University of California, Davis, and processed for LH, FSH, progesterone, estradiol-17β, and immunoreactive-inhibin by RIA. All 30 mares receiving reFSH (group A) developed follicles ≥ 35 mm within 7.4 ± 1.6 days of treatment. Twenty-three of the 30 reFSH-treated mares (group A) ovulated within 72 hours after hCG administration. In contrast, mares in group B (placebo, control) did not exhibit significant follicular development and none ovulated within the 15-day observation period. Mares in group A had significantly higher plasma levels of FSH, estradiol-17β, and immunoreactive-inhibin during treatment but did not exhibit a preovulatory LH surge. Mares administered reFSH returned to anestrus and spontaneously ovulated at a similar calendar date as control mares. These data indicate that reFSH was effective in stimulating the development of ovarian follicles and advancing the first ovulation of the year in seasonally anestrous mares under ambient lights but was not successful in inducing continued cyclicity.