234 REGION SPECIFIC ABUNDANCE OF INDUCIBLE NITRIC OXIDE SYNTHASE (iNOS) IN THE BOVINE OVIDUCT DURING THE ESTROUS CYCLE

Abstract

Nitric oxide synthases (NOS) are involved in a large number of biochemical processes. By generating inorganic free NO radicals, which are important modulatory agents, NOS are supposed to contribute to the regulation of mammalian reproduction. We have, therefore, studied the expression and localization of inducible NOS (iNOS) in the bovine oviduct during the estrous cycle. Simmental heifers were synchronized and slaughtered at Days 0, 3.5, 12, or 18 after standing heat. Bovine oviduct epithelial cells (BOEC) of ampulla and isthmus were collected separately for ipsi- and contralateral oviducts, as determined by the ovulatory follicle or the functional corpus luteum. In addition, a BOEC in vitro suspension culture from heifers of day 3.5 was established and stimulated with physiological doses of progesterone and estradiol-17β (10 ng/mL and 10 pg/mL, respectively). Total RNA was extracted and iNOS mRNA was absolutely quantified using real-time RT-PCR. For statistical analysis, the MIXED procedure of the SAS program package (SAS Institute, Inc., Cary, NC, USA) was used to model the experimental design (n = 4). Differences were indicated as statistically significant in the case of P < 0.05. Furthermore, immunoreactive iNOS protein was localized on serial oviductal cryosections. Transcripts of iNOS mRNA were detected in bovine oviducts throughout the estrous cycle. Since the level of 18S rRNA did not differ significantly the data were not normalized. Positive immunohistochemical staining for iNOS in the luminal epithelium gave evidence for the presence of iNOS protein in the bovine oviduct. At estrus (Day 0) and Day 18, the mRNA abundance of iNOS in epithelial cells of both ipsi- and contralateral isthmus was significantly lower than in epithelial cells of the ampulla. At Day 3.5, iNOS expression was significantly reduced in the contralateral but not the ipsilateral isthmus, whereas the abundance in the ipsilateral isthmus reached levels similar to those in the ampulla. At Day 12 the mRNA abundance was low without region specific differences. A down-regulation of iNOS could be associated with an accelerated oviductal transport due to higher contractility and increased ciliary motion in the luminal epithelium of the oviduct. This might be of special importance around ovulation time for gametes as well as for the formation of a homogeneous oviductal fluid. A modulated expression pattern of iNOS during the estrous cycle points towards a possible hormonal regulation. Preliminary in vitro results showed that in BOEC iNOS responded to progesterone, while no such up-regulation was observed after estradiol-17β stimulation. As Day 3.5 coincides with early embryonic development shortly after fertilization, the possible progesterone-dependent increase of iNOS transcripts in the ipsilateral isthmus might lead to a necessary local quiescence. Due to region-specific different transcript abundance, the presence of a local NO-system in the bovine oviduct can be postulated. Therefore, these findings point out a possible important role of iNOS in the oviduct for reproductive success. This work was supported by the DFG Ei 296/10-1 and the Ev. Studienwerk eV.