Gregory W. Burns

Extracellular vesicles in luminal fluid of the ovine uterus.

Microvesicles and exosomes are nanoparticles released from cells and can contain small RNAs, mRNA and proteins that affect cells at distant sites. In sheep, endogenous beta retroviruses (enJSRVs) are expressed in the endometrial epithelia of the uterus and can be transferred to the conceptus trophectoderm. One potential mechanism of enJSRVs transfer from the uterus to the conceptus is via exosomes/microvesicles. Therefore, studies were conducted to evaluate exosomes in the uterine luminal fluid (ULF) of sheep. Exosomes/microvesicles (hereafter referred to as extracellular vesicles) were isolated from the ULF of day 14 cyclic and pregnant ewes using ExoQuick-TC. Transmission electron microscopy and nanoparticle tracking analysis found the isolates contained vesicles that ranged from 50 to 200 nm in diameter. The isolated extracellular vesicles were positive for two common markers of exosomes (CD63 and HSP70) by Western blot analysis. Proteins in the extracellular vesicles were determined by mass spectrometry and Western blot analysis. Extracellular vesicle RNA was analyzed for small RNAs by sequencing and enJSRVs RNA by RT-PCR. The ULF extracellular vesicles contained a large number of small RNAs and miRNAs including 81 conserved mature miRNAs. Cyclic and pregnant ULF extracellular vesicles contained enJSRVs env and gag RNAs that could be delivered to heterologous cells in vitro. These studies support the hypothesis that ULF extracellular vesicles can deliver enJSRVs RNA to the conceptus, which is important as enJSRVs regulate conceptus trophectoderm development. Importantly, these studies support the idea that extracellular vesicles containing select miRNAs, RNAs and proteins are present in the ULF and likely have a biological role in conceptus-endometrial interactions important for the establishment and maintenance of pregnancy.

Oviduct-Embryo Interactions in Cattle: Two-Way Traffic or a One-Way Street?

ABSTRACT This study examined the effect of the presence of single or multiple embryos on the transcriptome of the bovine oviduct. In experiment 1, cyclic (nonbred, n = 6) and pregnant (artificially inseminated, n = 11) heifers were slaughtered on Day 3 after estrus, and the ampulla and isthmic regions of the oviduct ipsilateral to the corpus luteum were separately flushed. Oviductal epithelial cells from the isthmus region, in which all oocytes/embryos were located, were snap-frozen for microarray analysis. In experiment 2, heifers were divided into cyclic (nonbred, n = 6) or pregnant (multiple embryo transfer, n = 10) groups. In vitro-produced presumptive zygotes were transferred endoscopically to the ipsilateral oviduct on Day 1.5 postestrus (n = 50 zygotes/heifer). Heifers were slaughtered on Day 3, and oviductal isthmus epithelial cells were recovered for RNA sequencing. Microarray analysis in experiment 1 failed to detect any difference in the transcriptome of the oviductal isthmus induced by the presence of a single embryo. In experiment 2, following multiple embryo transfer, RNA sequencing revealed 278 differentially expressed genes, of which 123 were up-regulated and 155 were down-regulated in pregnant heifers. Most of the down-regulated genes were related to immune function. In conclusion, the presence of multiple embryos in the oviduct resulted in the detection of differentially expressed genes in the oviductal isthmus; failure to detect changes in the oviduct transcriptome in the presence of a single embryo may be due to the effect being local and undetectable under the conditions of this study.

Extracellular Vesicles Originate from the Conceptus and Uterus During Early Pregnancy in Sheep

ABSTRACT Cells release diverse types of membrane-bound vesicles of endosomal and plasma membrane origin, termed exosomes and microvesicles, respectively. Extracellular vesicles (EVs) represent an important mode of intercellular communication by transferring select RNAs, proteins, and lipids between cells. The present studies tested the hypothesis that the elongating ovine conceptus and uterus produces EVs that mediate conceptus-maternal interactions during early pregnancy. In Study 1, EVs were purified from uterine luminal fluid of Day 14 cyclic sheep. The EVs were fluorescently labeled with PKH67 dye and infused into the uterine lumen of pregnant sheep for 6 days using an osmotic pump. On Day 14, labeled EVs were observed in the conceptus trophectoderm and uterine epithelia, but not in the uterine stroma or myometrium. In Study 2, Day 14 conceptuses were cultured ex vivo for 24 h and found to release EVs into the culture medium. Proteomics analysis of the Day 14 conceptus-derived EVs identified 231 proteins that were enriched for extracellular space and several protein classes, including proteases, protease inhibitors, chaperones and chaperonins. RNA sequencing of Day 14 conceptus-derived EVs detected expression of 512 mRNAs. The top-expressed genes were overrepresented in ribosomal functions and components. Isolated EVs from conceptuses were fluorescently labeled with PKH67 and infused into the uterine lumen of cyclic sheep for 6 days using an osmotic pump. On Day 14, labeled EVs were observed in the uterine epithelia, but not in the uterine stroma or myometrium. Labeled EVs were not observed in the ovary or in other maternal tissues. These studies support the ideas that EVs emanate from both the conceptus trophectoderm and uterine epithelia, and are involved in intercellular communication between those cells during the establishment of pregnancy in sheep.

Peroxisome Proliferator Activator Receptor Gamma (PPARG) Regulates Conceptus Elongation in Sheep

ABSTRACT The ovine blastocyst hatches from the zona pellucida by Day 8 and develops into an ovoid or tubular conceptus (embryo and associated extraembryonic membranes) that grows and elongates into a filamentous form between Days 12 and 16. The trophectoderm of the elongating conceptus synthesizes and secretes interferon tau (IFNT) as well as prostaglandins (PGs) via prostaglandin synthase two (PTGS2). Intrauterine infusion of a PTGS2 inhibitor prevents conceptus elongation in sheep. Although many PGs are secreted, PGI2 and PGJ2 can activate nuclear peroxisome proliferator activator receptors (PPARs) that heterodimerize with retinoic X receptors (RXRs) to regulate gene expression and cellular function. Expression of PPARD, PPARG, RXRA, RXRB, and RXRG is detected in the elongating ovine conceptus, and nuclear PPARD and PPARG are present in the trophectoderm. Consequently, PPARD and PPARG are hypothesized to have essential roles in conceptus elongation in ruminants. In utero loss-of-function studies of PPARD and PPARG in the ovine conceptus trophectoderm were conducted using morpholino antisense oligonucleotides (MAOs) that inhibit mRNA translation. Elongating, filamentous-type conceptuses were recovered from ewes infused with a control morpholino or PPARD MAO. In contrast, PPARG MAO resulted in severely growth-retarded conceptuses or conceptus fragments with apoptotic trophectoderm. In order to identify PPARG-regulated genes, PPARG chromatin immunoprecipitation sequencing and RNA sequencing were conducted using Day 14 ovine conceptuses. These analyses revealed candidate PPARG-regulated genes involved in biological pathways, including lipid and glucose uptake, transport, and metabolism. Collectively, results support the hypothesis that PTGS2-derived PGs and PPARG are essential regulators of conceptus elongation, with specific roles in trophectoderm survival and proliferation.

Spatial differences in gene expression in the bovine oviduct

The aim of this study was to compare the transcriptome of the oviductal isthmus of pregnant heifers with that of cyclic heifers as well as to investigate spatial differences between the transcriptome of the isthmus and ampulla of the oviduct in pregnant heifers. After synchronizing crossbred beef heifers, those in standing oestrus (=Day 0) were randomly assigned to cyclic (non-bred, n=6) or pregnant (artificially inseminated, n=11) groups. They were slaughtered on Day 3 and both oviducts from each animal were isolated and cut in half to separate ampulla and isthmus. Each portion was flushed to confirm the presence of an oocyte/embryo and was then opened longitudinally and scraped to obtain epithelial cells which were snap-frozen. Oocytes and embryos were located in the isthmus of the oviduct ipsilateral to the corpus luteum Microarray analysis of oviductal cells revealed that proximity to the corpus luteum did not affect the transcriptome of the isthmus, irrespective of pregnancy status. However, 2287 genes were differentially expressed (P<0.01) between the ampulla and isthmus of the oviduct ipsilateral to the corpus luteum in pregnant animals. Gene ontology revealed that the main biological processes overrepresented in the isthmus were synthesis of nitrogen, lipids, nucleotides, steroids and cholesterol as well as vesicle-mediated transport, cell cycle, apoptosis, endocytosis and exocytosis, whereas cell motion, motility and migration, DNA repair, calcium ion homeostasis, carbohydrate biosynthesis, and regulation of cilium movement and beat frequency were overrepresented in the ampulla. In conclusion, large differences in gene expression were observed between the isthmus and ampulla of pregnant animals at Day 3 after oestrus.

Biological Roles of Hydroxysteroid (11-Beta) Dehydrogenase 1 (HSD11B1), HSD11B2, and Glucocorticoid Receptor (NR3C1) in Sheep Conceptus Elongation

ABSTRACT In sheep, the elongating conceptus synthesizes and secretes interferon tau (IFNT) as well as prostaglandins (PGs) and cortisol. The enzymes, hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1) and HSD11B2 interconvert cortisone and cortisol. In sheep, HSD11B1 is expressed and active in the conceptus trophectoderm as well as in the endometrial luminal epithelia; in contrast, HSD11B2 expression is most abundant in conceptus trophectoderm. Cortisol is a biologically active glucocorticoid and ligand for the glucocorticoid receptor (NR3C1 or GR) and mineralocorticoid receptor (NR3C2 or MR). Expression of MR is not detectable in either the ovine endometrium or conceptus during early pregnancy. In tissues that do not express MR, HSD11B2 protects cells from the growth-inhibiting and/or proapoptotic effects of cortisol, particularly during embryonic development. In study one, an in utero loss-of-function analysis of HSD11B1 and HSD11B2 was conducted in the conceptus trophectoderm using morpholino antisense oligonucleotides (MAOs) that inhibit mRNA translation. Elongating, filamentous conceptuses were recovered on Day 14 from ewes infused with control morpholino or HSD11B2 MAO. In contrast, HSD11B1 MAO resulted in severely growth-retarded conceptuses or conceptus fragments with apoptotic trophectoderm. In study two, clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing was used to determine the role of GR in conceptus elongation and development. Elongating, filamentous-type conceptuses (12–14 cm in length) were recovered from ewes gestating control embryos (n = 7/7) and gestating GR-edited embryos (n = 6/7). These results support the idea that the effects of HSD11B1-derived cortisol on conceptus elongation are indirectly mediated by the endometrium and are not directly mediated through GR in the trophectoderm.

Identification of Beef Heifers with Superior Uterine Capacity for Pregnancy.

ABSTRACT Infertility and subfertility represent major problems in domestic animals and humans, and the majority of embryonic loss occurs during the first month of gestation that involves pregnancy recognition and conceptus implantation. The critical genes and physiological pathways in the endometrium that mediate pregnancy establishment and success are not well understood. In study one, predominantly Angus heifers were classified based on fertility using serial embryo transfer to select animals with intrinsic differences in pregnancy loss. In each of the four rounds, a single in vitro-produced, high-quality embryo was transferred into heifers on Day 7 postestrus and pregnancy was determined on Days 28 and 42 by ultrasound and then terminated. Heifers were classified based on pregnancy success as high fertile (HF), subfertile (SF), or infertile (IF). In study two, fertility-classified heifers were resynchronized and bred with semen from a single high-fertility bull. Blood samples were collected every other day from Days 0 to 36 postmating. Pregnancy rate was determined on Day 28 by ultrasound and was higher in HF (70.4%) than in heifers with low fertility (36.8%; SF and IF). Progesterone concentrations in serum during the first 20 days postestrus were not different in nonpregnant heifers and also not different in pregnant heifers among fertility groups. In study three, a single in vivo-produced embryo was transferred into fertility-classified heifers on Day 7 postestrus. The uteri were flushed on Day 14 to recover embryos, and endometrial biopsies were obtained from the ipsilateral uterine horn. Embryo recovery rate and conceptus length and area were not different among the heifer groups. RNA was sequenced from the Day 14 endometrial biopsies of pregnant HF, SF, and IF heifers (n = 5 per group) and analyzed by edgeR-robust analysis. There were 26 differentially expressed genes (DEGs) in the HF compared to SF endometrium, 12 DEGs for SF compared to IF endometrium, and three DEGs between the HF and IF endometrium. Several of the DEG-encoded proteins are involved in immune responses and are expressed in B cells. Results indicate that preimplantation conceptus survival and growth to Day 14 is not compromised in SF and IF heifers. Thus, the observed difference in capacity for pregnancy success in these fertility-classified heifers is manifest between Days 14 and 28 when pregnancy recognition signaling and conceptus elongation and implantation must occur for the establishment of pregnancy.

Analysis of the Uterine Epithelial and Conceptus Transcriptome and Luminal Fluid Proteome During the Peri-Implantation Period of Pregnancy in Sheep

ABSTRACT Studies support the idea that uterine epithelia and their secretions have important biological roles in conceptus survival, elongation, and implantation in sheep. The present study evaluated the transcriptome of the uterine luminal epithelium (LE) and glandular epithelium (GE) and the conceptus and proteome of uterine luminal fluid (ULF) during the peri-implantation period of pregnancy. Transcriptome (RNA-sequencing) analysis was conducted in LE and GE isolated from uteri of Day 10, 12, 14, 16, and 20 pregnant sheep by laser capture microdissection. In the LE, the total number of expressed genes increased between Days 10 and 20, whereas expressed genes in the GE increased from Days 10 to 14 and then decreased to Day 20. Most of the expressed genes in LE and GE from Days 10 to 14 are involved in cell survival and growth, whereas genes involved in cell organization and protein synthesis were most abundant on Days 16 and 20. Total expressed genes in the conceptus was greatest on Day 12, decreased to Day 16, and then increased to Day 20. Genes abundantly expressed in the elongating conceptus included IFNT, PTGS2, MGST1, FADS1, and FADS2, whereas SERPINA1, CSH1, and PLET1 were most abundant in the Day 20 conceptus. Proteins, identified by mass spectrometry, increased in the ULF from Days 10 to 16 and are involved in cellular reorganization or are proteases or chaperone proteins. These results support the idea that conceptus elongation and implantation is regulated by both extrinsic and intrinsic factors. This study provides critical information that serves as a foundation to discover new regulatory pathways governing uterine receptivity, conceptus elongation, trophectoderm differentiation, conceptus-endometrial interactions, and pregnancy establishment in ruminants.

Uterine glands impact uterine receptivity, luminal fluid homeostasis and blastocyst implantation.

Uterine glands are essential for pregnancy in mice and likely humans, because they secrete or transport bioactive substances that regulate uterine receptivity for blastocyst implantation. In mice, the uterus becomes receptive to blastocyst implantation on day 4, but is refractory by day 5. Here, blastocysts could be recovered from progesterone-induced uterine gland (PUGKO) but not wildtype (WT) mice on day 5 post-mating. Anti-adhesive Muc1 protein and microvilli were present on the luminal epithelium of PUGKO but not WT uteri. A number of known uterine receptivity genes and gland-specific genes were altered in the PUGKO uterus. Next, the uterus and uterine luminal fluid (ULF) were obtained from WT and PUGKO mice on day 3, 4 and 5. Transcriptome analysis revealed that 580 genes were decreased in the PUGKO uterus, however ULF secrotome analysis revealed that many proteins and several amino acids were increased in the PUGKO ULF. Of note, many proteins encoded by many gland-specific genes were not identified in the ULF of WT mice. These results support the ideas that uterine glands secrete factors that regulate ULF homeostasis and interact with other cell types in the uterus to influence uterine receptivity and blastocyst implantation for the establishment of pregnancy.

Progesterone effects on extracellular vesicles in the sheep uterus

Abstract Progesterone (P4) acts via the endometrium to promote conceptus growth and implantation for pregnancy establishment. Many cells release extracellular vesicles (EVs) that are membrane-bound vesicles of endosomal and plasma membrane origin. In sheep, endometrial-derived EVs were found to traffic to the conceptus trophectoderm. Thus, EVs are hypothesized to be an important mode of intercellular communication by transferring select RNAs, proteins, and lipids between the endometrium and conceptus. Electronmicroscopy analysis found that the endometrial luminal and glandular epithelia were the primary source of EVs in the uterus of cyclic sheep. Size exclusion chromatography and nanoparticle tracking analysis (NTA) found that total EV number in the uterine lumen increased from day 10 to 14 in cyclic sheep. Next, ewes were ovariectomized and hormone replaced to determine effects of P4 on the endometrium and EVs in the uterine lumen. Transcriptome analyses found that P4 regulated 1611 genes and nine miRNAs in the endometrium. Total EV number in the uterine lumen was increased by P4 treatment. Small RNA sequencing of EVs detected expression of 768 miRNAs and determined that P4 regulated seven of thosemiRNAs. These studies provide fundamental new information on how P4 influences endometrial function to regulate conceptus growth for pregnancy establishment in sheep. Summary Sentence Progesterone regulates extracellular vesicles and miRNAs in the ovine uterus.