Transforming growth factor beta expression in the porcine ovary: evidence that theca cells are the major secretory source during antral follicle development.

Abstract

The transforming growth factors beta (TGF beta) have been implicated as important intrafollicular regulators of follicle development in the mammalian ovary. Recent studies in this laboratory have suggested that, when cultured, both porcine theca and granulosa cells secrete TGF beta, primarily TGF beta 1 (May et al., Endocrine 2:1045-1054, 1994). In this report, evidence is presented that during follicle development in vivo, theca but not granulosa cells are the source of follicular TGF beta. Although both theca and granulosa cells secreted TGF beta when attached to culture dishes, only theca cells secreted detectable levels of TGF beta when cells were cultured in serum-free medium without attachment. Granulosa cells secreted little if any TGF beta. This difference in TGF beta secretion was not found to be due to differences in preparation of the two cell types (i.e., mechanical versus enzymatic preparation). These results suggested that theca cells may be the source of TGF beta in the follicle. To ascertain whether TGF beta is actually secreted by follicles, intact hemi-follicle linings consisting of both theca and granulosa cells were cultured in moderate-term, organ explant culture. Hemi-follicle linings secreted TGF beta at a near linear rate for at least 4 days (approximately 300 pg/follicle/day for 6-8-mm-diameter follicles). The level of TGF beta secretion was directly related to the size of the follicle (p < 0.01). Immunoneutralization studies using TGF beta subtype-specific antibodies indicated that the major form of TGF beta secreted by porcine hemi-follicle linings was TGF beta 1. To further investigate the source of TGF beta during follicle development, a combination of molecular biology procedures was employed. Using 243-bp antisense and sense cRNA probes generated from a simian TGF beta 1 cDNA, we performed in situ hybridization on sections of whole porcine ovaries containing antral follicles. Expression of TGF beta 1 mRNA was localized to both theca and granulosa cells with no expression found in the stroma, suggesting that both cell types transcribe TGF beta 1. TGF beta 1 expression was evaluated additionally by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot analysis. Oligonucleotide primers were generated from the porcine TGF beta 1 cDNA sequence for RT-PCR, and the PCR product (287-bp sequence) was amplified and used for Northern analysis. RT-PCR of total RNA isolated from theca and granulosa cells indicated that both cell types expressed TGF beta 1 mRNA. This finding was confirmed via Northern blot analysis, which further indicated the presence of two TGF beta 1 mRNAs of 2.5 and 3.5 kb, consistent with previous reports of alternate splicing of the porcine TGF beta 1 gene. These data indicate that both cell types express TGF beta 1 mRNA. To further evaluate TGF beta 1 expression, we attempted to isolate TGF beta 1 protein from freshly collected theca and granulosa cells by partial purification and immunoprecipitation. Interestingly, the growth factor could be extracted only from theca cells, not from granulosa cells, despite the presence of mRNA in both cell types. Taken together, these data suggest that whereas both theca and granulosa cells produce the TGF beta 1 gene product, only theca cells translate and secrete the actual growth factor. Thus, it is likely that the theca is the source of the growth factor during follicle development.