Haem oxygenase augments porcine granulosa cell apoptosis in vitro

Abstract

Haem oxygenases produce carbon monoxide, which, like nitric oxide, is a gaseous messenger molecule that is one of several important survival factors in ovarian follicles. However, little is known about the expression and possible functions of these enzymes in granulosa cells. The purpose of this study was to investigate the expression and possible role of haem oxygenases in porcine granulosa cells (PGCs). We obtained frozen sections of porcine ovaries and PGCs from ovarian follicles of various sizes by needle aspiration, and examined the expression of haem oxygenase-1 (HO-1; inducible type) and HO-2 (constitutive type) in PGCs by immunohistochemistry, RT-PCR, western blotting and flow cytometry. Both types of haem oxygenase were identified in PGCs throughout follicular development, but HO-1 was expressed primarily in granulosa cells in atretic follicles. We also investigated the effect of haem oxygenases on apoptosis of granulosa cells (flow cytometry to detect subdiploid DNA fluorescence) and on expression of Fas ligand (quantitative analysis of western blotting and flow cytometry). In tightly bound PGCs, the mean proportion of apoptotic cells treated with 1 µM haemin (a haem oxygenase substrate) was approximately 1·7-fold greater than that in untreated controls, and zinc protoporphyrin IX (ZnPP IX; a haem oxygenase inhibitor) completely inhibited the increase in apoptosis induced by haemin in 24-h culture. Conversely, in weakly associated PGCs, the proportion of apoptotic cells was not altered by haemin. The quantity of Fas ligand protein was increased in a dose-dependent manner in tightly bound PGCs treated with haemin compared with controls, and the haemin-induced increase in Fas ligand protein was inhibited by ZnPP IX. Thus we identified inducible HO-1 and constitutive HO-2 in PGCs throughout follicular development, and we conclude that products of reactions catalysed by haem oxygenases are likely to be important autocrine/paracrine factors that regulate apoptosis in PGCs.

Complement factors are secreted in human follicular fluid by granulosa cells and are possible oocyte maturation factors

“…Given the deleterious effects of C5 during pregnancy, we speculate that from an evolutionary perspective, humans with C5 and C6 present in the hFF were infertile, leading to the elimination over time of these complement factors from the oocyte milieu. Previously we demonstrated that the granulosa cells also produce the enzyme heme oxygenase −1, 13 which could provide another protective mechanism from a complement‐induced damage 14,15 . The absence of C5 and C6 from the hFF seemingly protects the oocyte from injury by the MAC.…”

The effect of heat stress on gene expression, synthesis of steroids, and apoptosis in bovine granulosa cells

“…The high temperature can stimulate the transcription of HSP70.2, HSP72, and HSP105/110 genes in ovarian follicular granulosa cells (Sirotkin 2010;Sirotkin et al 2011). Inducible HO-1 is likely to be important autocrine/ paracrine factors that regulate apoptosis in porcine GCs (Harada et al 2004). HO-1 may play a stimulatory role of endogenous carbon monoxide in the production of ovarian steroids in the rat ovary (Alexandreanu and Lawson 2003).…”

Role of vasoactive substances on endometrial and ovarian function

“…In tightly bound PGC, the mean proportion of apoptotic cells treated with 1 µmol/L hemin (a HO substrate) was approximately 1.7‐fold greater than that in untreated controls, and zinc protoporphyrin IX (ZnPP IX; a HO inhibitor) completely inhibited the increase in apoptosis induced by hemin in 24‐h culture. Conversely, in weakly associated PGC, the proportion of apoptotic cells was not altered by hemin 56 …”

“…Fas‐mediated cell‐death pathways are considered to be central to the induction of follicular atresia 59,60 . It has been shown that the quantity of Fas ligand protein increased in a dose‐dependent manner in tightly bound PGC treated with hemin compared with controls, and the hemin‐induced increase in Fas ligand protein was inhibited by ZnPP IX 56 …”