Induction of apoptosis in rat thecal/interstitial cells by transforming growth factor alpha plus transforming growth factor beta in vitro.
ABSTRACT In each estrous cycle dominant follicles are selected from a growing pool to develop to the preovulatory stage and to ovulate. Those follicles that do not ovulate must be eliminated in order to maintain the constant mass and homeostasis of the ovary. Granulosa cells are lost by apoptosis at the onset of follicular atresia, whereas apoptotic thecal cells are identified at later stages of atresia. Since transforming growth factor (TGF) alpha and TGF beta 1 have been implicated in the regulation of thecal cell physiology we have localized these growth factors by immunohistochemistry in sections of ovaries from 25-day-old rats, an age at which the ovary exhibits a wave of atresia of preantral follicles. Thecal cells contained TGF alpha and TGF beta 1 throughout the entire process of follicular atresia. To determine if these growth factors could influence thecal cell death, thecal/interstitial cells were isolated from 25-day-old rats, and maintained in culture with growth factors. Subconfluent cultures treated with TGF alpha or TGF beta 1 alone remained healthy whereas in the presence of both TGF alpha and TGF beta 1 there was light microscopical evidence of rounding up of cells and detachment from the monolayer. Chromatin condensation and internucleosomal fragmentation, characteristic of apoptosis, were observed by nucleic acid staining and fluorescence microscopy of thecal/interstitial cells treated with TGF alpha plus TGF beta 1. Further evidence that these cells were undergoing apoptosis came from DNA analysis and the demonstration of DNA laddering. This response of thecal/interstitial cells to TGF alpha plus TGF beta 1 was density dependent; confluent cultures were protected from the induction of apoptosis under these conditions. We conclude that thecal cells are eliminated from atretic follicles by the active and strictly regulated process of involving the combined actions of TGF alpha and TGF beta 1.
- SourceAvailable from: Laurel D Quirke[show abstract] [hide abstract]
ABSTRACT: Recently, several members of the transforming growth factor-beta (TGF-beta) superfamily have been shown to be essential for regulating the growth and differentiation of ovarian follicles and thus fertility. Ovaries of neonatal and adult sheep were examined for expression of the TGF-betas 1-3 and their receptors (RI and RII) by in situ hybridization using ovine cDNAs. The effects of TGF-beta 1 and 2 on proliferation and differentiation of ovine granulosa cells in vitro were also studied. The expression patterns of TGF-beta 1 and 2 were similar in that both mRNAs were first observed in thecal cells of type 3 (small pre-antral) follicles. Expression of both mRNAs continued to be observed in the theca of larger follicles and was also present in cells within the stroma and associated with the vascular system of the ovary. There was no evidence for expression in granulosa cells or oocytes. Expression of TGF-beta 3 mRNA was limited to cells associated with the vascular system within the ovary. TGFbetaRI mRNA was observed in oocytes from the type 1 (primordial) to type 5 (antral) stages of follicular growth and granulosa and thecal cells expressed this mRNA at the type 3 (small pre-antral) and subsequent stages of development. The TGFbetaRI signal was also observed in the ovarian stroma and vascular cells. In ovarian follicles, mRNA encoding TGFbetaRII was restricted to thecal cells of type 3 (small pre-antral) and larger follicles. In addition, expression was also observed in some cells of the surface epithelium and in some stromal cells. In granulosa cells cultured for 6 days, both TGF-beta 1 and 2 decreased, in a dose dependent manner, both the amount of DNA and concentration of progesterone. In summary, mRNA encoding both TGF-beta 1 and 2 were synthesized by ovarian theca, stroma and cells of the vascular system whereas TGF-beta 3 mRNA was synthesized by vascular cells. Luteinizing granulosa cells also responded to both TGF-beta 1 and beta 2 in vitro. These findings in sheep are consistent with TGF-beta potentially being an important autocrine regulator of thecal cell function and possibly a paracrine regulator of ovarian cell function at various development stages.Reproductive Biology and Endocrinology 12/2004; 2:78. · 2.14 Impact Factor
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ABSTRACT: Theca-interstitial (T-I) cells play a fundamental role in the control of ovarian function. Steroidogenic activity and growth of the T-I cells are regulated by many paracrine and endocrine factors. However, little is known about the mechanisms controlling T-I death. In an in vitro model of apoptosis, purified rat T-I cells were cultured for 24 h with serum and subsequently for up to an additional 24 h with serum or in serum-free medium with or without insulin, insulin-like growth factors (IGF-I and IGF-II) and LH or 8-bromo-cyclic AMP (8Br-cAMP). Apoptosis was identified by histological assessment of nuclear morphology and by detection of internucleosomal cleavage and quantified by determination of [alpha32P]-dideoxy-ATP 3'-end labeling of low molecular weight DNA. Serum withdrawal resulted in nuclear condensation and fragmentation into apoptotic bodies of T-I cells and led to pronounced DNA cleavage. Insulin (10 nM) protected T-I cells from apoptosis, reducing DNA fragmentation by 39 +/- 8% compared to serum-free controls. IGF-I (10 nM) and IGF-II (10 nM) had comparable antiapoptotic effects, decreasing DNA fragmentation by 55 +/- 9% and 37 +/- 14%, respectively. In contrast, LH (100 ng/ml) and 8Br-cAMP (1 mM) augmented the pro-apoptotic effect of serum withdrawal, increasing DNA fragmentation by 85 +/- 55% and 72 +/- 42%, respectively. The antiapoptotic effects of insulin and IGFs and the pro-apoptotic effect of LH, acting via the cAMP system, may be important in the maintenance of T-I homeostasis. Moreover, excessive levels of insulin and free IGFs may lead to T-I cell hyperplasia characteristic of conditions such as polycystic ovary syndrome.Molecular Human Reproduction 06/2005; 11(5):319-24. · 4.54 Impact Factor
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ABSTRACT: Transforming growth factor-beta (TGF-beta) isoforms are important paracrine and autocrine signaling molecules for the regulation of ovarian follicle growth and physiology. Effective communication between the epithelial granulosa cells, the mesenchymal theca cells, and the oocyte is vital for ovarian function and reproductive success. The expression, localization, and regulation of TGF-beta isoforms in the developing bovine follicle was examined using both immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (RT-PCR) procedures. TGF-beta1 protein was found to be present in the granulosa cells of early pre-antral, early antral, and 1-2 mm follicles. Interestingly, there was no visible staining of granulosa cells of 3-5 or 5-10 mm follicles. There was also no TGF-beta1 staining of theca cells. TGF-beta2 and TGF-beta3 staining were present in the granulosa and theca cells of all follicle stages examined. The levels of TGF-beta mRNA expression in granulosa and theca cells from antral follicles was measured using quantitative RT-PCR. For each isoform mRNA expression levels did not change in different sized antral follicles. TGF-beta3 mRNA levels were much higher than those of TGF-beta1 and TGF-beta2 in both granulosa and theca. Expression levels were higher in theca than in granulosa for TGF-beta2 and TGF-beta3. FSH was found to decrease TGF-beta1 mRNA expression in granulosa cells, but had no effect on TGF-beta2 and TGF-beta3. Bovine ovarian follicles were found to have a unique pattern of TGF-beta isoform expression and regulation when compared to other species (i.e., rodent, pig, quail, and human). The similarities and differences between the various species is discussed to help elucidate common functions of TGF-beta in the ovary. In summary, observations demonstrate that as antral follicles develop, TGF-beta3 is the most abundant TGF-beta isoform and TGF-beta1 protein levels decline in large follicles. Granulosa cell TGF-beta1 expression was decreased by FSH and this correlated with reduced levels in large antral follicles. TGF-betas involved in antral follicular growth and development appear to act as paracrine/autocrine signaling molecules having a species-specific pattern of expression.Molecular Reproduction and Development 12/2003; 66(3):237-46. · 2.81 Impact Factor