Serum progesterone and oxytocinase, and endometrial and luteal gene expression in pregnant, nonpregnant, oxytocin, carbetocin and meclofenamic acid treated mares.

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.

Endometrial and luteal gene expression of putative gene regulators of the equine maternal recognition of pregnancy.

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.

Interferon epsilon is constitutively expressed in equine endometrium and up-regulated during the luteal phase.

Interferon epsilon (IFNE) is type I interferon which stands out through its unusual expression profile and differing regulation compared to classic type I interferons such as interferon alpha and interferon beta. Unlike other type I interferons, the expression of IFNE is not stimulated through exposure to viral agents. Expression of IFNE is most abundant in mouse and human endometrium where it is constitutively expressed in luminal and glandular epithelial cells and expression levels are up-regulated with estrogen exposure. The aim of the current study was to determine whether a cycle or pregnancy dependent expression pattern of IFNE is existent in equine endometrium and to localize IFNE expression within the endometrium. Additionally, endometrial explant culture and culture of mixed epithelial/stromal cells populations was used to determine the effects estrogen and seminal plasma on IFNE transcript abundance. Samples collected during diestrus and pregnancy expressed significantly higher levels of IFNE than samples obtained from anestrous or estrous mares (P < 0.001). Exposure of mixed endometrial epithelial/stromal cell populations and endometrial explants to 10% seminal plasma and estradiol 17-beta did not affect IFNE expression levels (P > 0.05). Upon in situ hybridization, staining was exclusively present in luminal and glandular epithelial cells, with stromal displaying absent staining intensity. Both diestrous and pregnant samples were characterized by markedly stronger staining of glandular epithelial cells than anestrous and estrous samples. The progesterone-dependent increase in IFNE abundance during the estrous cycle likely implies that IFNE is part of the innate immune system in endometrium that gives protection against uterine infections during progesterone-dominated phase of the estrous cycle.

Hexokinase 2 drives glycogen accumulation in equine endometrium at day 12 of diestrus and pregnancy.

BACKGROUND: Secretion of histotroph during the prolonged pre-implantation phase in mares is crucial to pregnancy maintenance, manifested as increased embryonic loss in mares with age-related endometrial degeneration. Glycogen content of uterine histotroph is higher during the progesterone-dominated phase of the estrous cycle in mares, but regulatory mechanisms are not well understood. METHODS: mRNA expression of glycogen-metabolizing enzymes (HK1, HK2, GSK3B, GYS1, PEPCK, PKM, PYGM) in endometrial samples were compared among mares in anestrus, estrus, and at Day 12 of diestrus and pregnancy. In addition, hexokinase 2 (HK2) activity was assessed using a colorimetric assay. RESULTS: HK2 was the key regulator of glycogen accumulation during diestrus and pregnancy; hexokinase transcript abundance and enzyme activity were significantly higher during diestrus and pregnancy than estrus and anestrus. In addition, despite similar relative transcript abundance, hexokinase activity was significantly greater in the pregnant versus diestrous endometrium. Therefore, we inferred there was regulation of hexokinase activity through phosphorylation, in addition to its regulation at the transcriptional level during early pregnancy. Based on immunohistochemistry, HK2 was localized primarily in luminal and glandular epithelial cells, with weaker staining in stromal cells. CONCLUSION: Among glycogen metabolizing enzymes identified, expression of HK2 was significantly greater during the progesterone-dominated phase of the cycle.