Submission Number: ROD-4-17-21

Abstract Number: 527

PORCINE ENDOMETRIAL GENE EXPRESSION OF KALLIKREIN AND KININOGEN DURING THE ESTROUS CYCLE AND EARLY PREGNANCY.

BR Zhang, Jerry R Malayer*, MJ Allen*, AM Hettinger*, D Goad and Rodney D Geisert*

Oklahoma State University, Stillwater, OK 74078 ¹

Abstract:
Previously, we had demonstrated the presence of kallikrein (KAL) mRNA, using RTPCR, and KAL enzymatic activity in the porcine endometrium during the estrous cycle and early pregnancy. Activation of KAL maybe involved with early conceptus attachment to the uterine surface, changes in capillary flow through cleavage of LMWkininogen (KIN) to release bradykinin and proteolysis of insulinlike growth factor binding proteins. Objective of the present study is to determine endometrial KAL and KIN gene expression during the estrous cycle and early pregnancy of the pig. Cyclic gilts were hysterectomized on D0, 5, 12, 15 and 18; while uteri were collected from pregnant gilts on D12, 15 and 18 (4 gilts/d). Total endometrial RNA was obtained following TRIzol extraction. Endometrial RNA was transcribed into cDNA, and primers specific to KAL and KIN were used to generate PCR products from the cDNA templates. PCR products were resolved on agarose, extracted from the gel, and inserted into plasmid vectors. The cDNA inserts were purified and sequenced to validate identity of the PCR products. Plasmids containing KAL and KIN fragments were linearized and transcribed to form cRNA riboprobes using ³²PUTP isotope labeling. Total RNA (35 g) from the endometrial samples was separated by electrophoresis through a denaturing agarose gel and transferred to a HybondN nylon membrane. cRNA probes were hybridized at 42C overnight. After washing, the membranes were subjected to autoradiography with Kodak Diagnostic film at -80C. Quantitative densitometric analysis of northern blots was performed using a phosphorimaging system (BioRad) and analytical software (MultiAnalyst, BioRad). Endometrial gene expression was significantly different across the days of the estrous cycle for KAL (p<0.001) and KIN (p<0.05). Gene expression for both KAL and KIN were greatest on D5 of the estrous cycle followed by a decline on D12. Endometrial KIN gene expression was not effected by pregnancy. A significant status x day (p<0.005) effect was detected for KAL. Changes in kallikrein and kininogen gene expression during the estrous cycle suggest a possible role of the kallikreinkininogenkinin system in establishment of pregnancy in the pig. Supported by USDA grant 983520376224.

Keywords: Uterus Kallikrein Kininogen Porcine

This abstract is being presented at: 8:00 AM in session:
Implantation and Early Development