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Oviductal samples from 28 cows were used to examine the expression of NADPH oxidase (NOX) enzymes and their activation subunits in the bovine oviduct during the estrous cycle. The regional expression pattern of NOX enzymes in oviducts from ipsi- and contra-lateral site was also investigated.
Bovine oviducts collected from the slaughterhouse were classified into 4 groups: post- ovulatory (day 1-5), early-to-mid luteal (day 6-12), late luteal (day 13-18), and pre-ovulatory (day 19-21) phases. For each phase ipsi- and contra-lateral oviducts were identified according to ovulation side and further sub-divided into two separate regions, the ampulla and isthmus. Thereafter, the oviductal mucosa was recovered separately from ampulla and isthmus. For in vitro investigation of NOX enzymes mRNA expression, a primary oviductal cells culture was used. The cells were stimulated for 6h with physiological concentrations of E2 (10pg/ml), P4 (10ng/ml), AA (10µM) or PGE2 (0.25µM). Samples were collected for RNA isolation with intervals of 2 hours at time points 0h, 2h, 4h, or 6h of treatment. Cell cultures without hormonal treatment were used as control for each of the time points.
For the first time NOX and their activation subunits were found expressed in oviductal mucosal cells in ampulla and isthmus of ipsi- and contra-lateral oviduct throughout the estrus cycle. Only DUOX2 was absent in bovine oviductal mucosa confirmed by detection of its mRNA in bovine endometrial cells. NOX1, NOX2 and DUOX1 showed non-estrous cycle-dependant expression pattern, while mRNA expression of NOX4 and NOX5 were estrous cycle-dependant. NOX4 and NOX5 showed distinct expression pattern: NOX4 mRNA was up-regulated during the pre- and post-ovulatory phase, while the level of NOX5 mRNA expression was increased after ovulation and during early-to-mid luteal phase. In addition, NOX5 mRNA has a significant regional expression during post-ovulatory phase: its mRNA is up-regulated in the isthmus of ipsi-lateral oviduct compared to ampulla. Furthermore, all mRNA of the NOX subunits (p22phox, p67phox, p47phox, p40phox) were continuously detected without any significant difference between the different regions of oviduct during the estrous cycle. These subunits seemed to be expressed equally between the different phases of the estrous cycle and showed a positive correlation with expression levels of NOX1, NOX2. Furthermore, NOX4 has a strong positive correlation with its activating subunits p22phox during the post-ovulatory phase.
For the in vitro expression, all NOX enzyme components were detected in the untreated and treated primary oviductal cell cultures. E2 or P4 treatments resulted in an up-regulation of some NOX components, namely NOX2, p22phox and p47phox, while DUOX1 mRNA expression was up-regulated under P4 treatment and down-regulated by E2. One observation is that NOX5 mRNA was undetectable in untreated cells or treated cells during the whole experimental period. Generally, AA as well as PGE2 treatments showed no significant effects on the expression of NOX enzymes, although AA treatment significantly down-regulates mRNA expression of p67phox and p47phox while PGE2 induces up-regulation of p22phox and p47phox.
The obtained expression pattern of NOX components during the estrous cycle pointed to NOX enzymes as a novel source for balanced ROS levels in the oviductal environment. The enzyme activity may be regulated by endocrine and/or local factors. Presence of NOX subunits with distinct expression pattern and its responses in cell culture could be point towards precise mechanisms to regulate NOX activities. Moreover, ROS was implicated in early reproductive events occurring in oviduct and may play important new roles in physiological and pathological effects in the female reproductive tract. This leads to the hypothesis that providing controlled levels of these molecules have benefits on oocyte maturation, spermatozoa function, fertilization and early embryo development in the cow.