Abstract
The aims of this study were to detect spontaneously occurring apoptosis in cultured porcine ovarian cells, to examine the role of growth hormone (GH), tyrosine kinase (TK), protein kinase G (PKG) and cyclin-dependent kinase (CDK) in the control of this process, and to determine whether the effect of GH on apoptosis is mediated by TK-, PKG- and cdc2-dependent intracellular mechanisms. We studied the action of pGH (10 ng/ml), blockers of TK (genistein, lavendustin, both 100 ng/ml), PKG (Rp-Br-PET- cGMPS, 50 nM; KT5823, 100 ng/ml) and CDK (olomoucine, 1 µg/ml), as well as combinations of GH with these blockers, on the onset of apoptosis in cultured granulosa cells isolated from antral (3-6 mm) porcine follicles. The functional characteristics of an early apoptotic event, DNA fragmentation, were determined using terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labelling (TUNEL), whilst morphological signs of advanced apoptosis such as pyknosis, chromatin marginalization, shrinkage and fragmentation of nucleus, were detected using routine light microscopy. After culture, some ovarian granulosa cells exhibited DNA fragmentation, which in some cases was associated with morphological apoptosis-related changes (pyknosis, shrinkage and fragmentation of the nucleus). GH significantly reduced the proportion of TUNEL-positive cells. Neither TK nor CDK blockers when given alone, significantly affected the percentage of TUNEL-positive cells although both PKG blockers significantly increased this index. Furthermore, TK and PKG blockers given together with GH, prevented or reversed the inhibitory effect of GH on apoptosis, whilst the CDK blocker olomoucine promoted it. These observations demonstrate apoptosis in porcine ovaries and suggest the involvement of GH, TK, PKG and CDK in the control of this process. They also suggest that the effect of GH on ovarian apoptosis is mediated or regulated by multiple signalling pathways including TK-, PKG- and CDK-dependent intracellular mechanisms. (Asian- Aust. J. Anim. Sci. 2002. Vol 15, No. 7 : 1045-1050)
Highlights
Apoptosis can occur in tissues and organs spontaneously or under the influence of several inducers
We studied the action of pGH, blockers of tyrosine kinase (TK), protein kinase G (PKG) (Rp-BrPET-cGMPS or KT5823) and CDK, as well as combinations of blockers and growth hormone (GH), on apoptosis in cultured porcine granulosa cells
The signs of apoptosis in ovarian cells observed in the present study are similar to those described previously in rodents (Chun et al, 1996; Eisenhauer et al, 1995), cows (Sirotkin and Makarevich, 1999) and pigs (Guthrie et al, 1998, 2000; Sirotkin et al, 2000)
Summary
Apoptosis can occur in tissues and organs spontaneously or under the influence of several inducers. Occurring or hormone-induced apoptosis plays an important role in the control of ovarian cyclicity, remodelling, follicular development, selection and atresia. Information concerning the hormonal regulation of apoptosis in the ovary is poor. The pattern of hormonal effects seems to be dependent on the stage of the follicular cycle. The anti-apoptotic actions of gonadotropins, cAMP analogues (Hirshfield, 1991) and growth hormone (GH; Eisenhauer et al, 1995; Danilovich et al, 2000) were observed in preovulatory but not in preantral (Danilovich et al, 2000) or early antral (Chun et al, 1996) rodent follicles, whilst in rodent luteal cells GH promoted apoptosis (Kiya et al, 1999).
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