Fibrinogen in equine pregnancy as a mediator of cell adhesion, an epigenetic and functional investigation.

Preimplantation equine embryos synthesize and secrete fibrinogen, which is a peculiar finding as fibrinogen synthesis almost exclusively occurs in the liver. This study investigated the hypothesis that conceptus-derived fibrinogen mediates cell adhesion during fixation. On day 21 of pregnancy, five integrin subunits, including ITGA5, ITGB1, ITGAV, and ITGB1, displayed significantly higher transcript abundance than on day 16 of pregnancy. Endometrial epithelial cells adhered to fibrinogen in an integrin-dependent manner in an in vitro cell adhesion assay. Bilaminar trophoblast and allantochorion expressed fibrinogen transcript, indicating that fibrinogen expression persists past fixation. Preimplantation-phase endometrium, conceptuses, and microcotyledonary tissue expressed components of the clotting cascade regulating fibrin homeostasis, leaving open the possibility that fibrinogen is converted to fibrin. Fibrinogen is likely to have functions beyond mediating cell adhesion, such trapping growth factors and triggering signaling cascades, and has remarkable parallels to the expression of fibrinogen by some tumors. The deposition of fibrinogen within tumor stroma is characteristic of breast carcinoma, and tumor-derived fibrinogen has been implicated in the metastatic potential of circulating tumor cells. DNA methylation of the fibrinogen locus in equine conceptuses was examined in comparison to liver and endometrium, and across the full gene cluster, was significantly higher for endometrium than liver and conceptus. DNA methylation of regulatory regions did not differ between liver and conceptus, and was significantly lower than in endometrium. These results, therefore, support the hypothesis of DNA methylation being a regulator of fibrinogen expression in the conceptus.

Seminal plasma does not aid in the transport of phenolsulfonphthalein across the uterotubal junction in mares.

This study tested the hypothesis that the presence of prostaglandin E2 in seminal plasma would aid in the transport of phenolsulfonphthalein (PSP) across the uterotubal junction. Five mares in estrus were inseminated during estrus with PSP dissolved in phosphate-buffered saline and during the subsequent estrus with PSP added to a standard insemination dose. Serum and urine samples were obtained at hours 0, 1, 2, and 3 following treatment and examined for the presence of PSP. Phenolsulfonphthalein could not be detected in any of the urine samples collected from mares following either treatment. None of the serum samples collected following intrauterine installation of PSP in PBS contained PSP. Phenolsulfonphthalein was detected in serum samples from 1 mare following insemination with semen containing PSP. Components in seminal plasma such as PGE2 did not facilitate the transport of PSP across the uterotubal junction as had been hypothesized.

Le plasma séminal ne facilite pas le transport de la phénolsulfonphtaléine au travers de la jonction utéro-tubaire des juments. Cette étude a testé l'hypothèse voulant que la présence de la prostaglandine E2 dans le plasma séminal facilite le transport de la phénolsulfonphtaléine (PSP) au travers de la jonction utéro-tubaire. Cinq juments en oestrus ont été inséminées avec de la PSP dissoute dans une solution saline tamponnée au phosphate et, durant l'oestrus subséquent, avec de la PSP ajoutée à une dose d'insémination standard. Des prélèvements de sérum et d'urine ont été obtenus aux heures 0, 1, 2 et 3 ainsi qu'après le traitement et examinés pour déceler la présence de la PSP. La phénolsulfonphtaléine n'a pas pu être détectée dans aucun des échantillons d'urine prélevés auprès des juments après l'un ou l'autre des traitements. Aucun des échantillons de sérum prélevés après l'installation intra-utérine de la PSP dans PBS ne contenait de PSP. La phénolsulfonphtaléine a été détectée dans des échantillons de sérum provenant d'une jument après l'insémination avec du sperme contenant de la PSP. Des composants dans le plasma séminal comme le PGE2 n'ont pas facilité le transport de la PSP au travers de la jonction utéro-tubaire conformément à l'hypothèse émise.(Traduit par Isabelle Vallières).

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.