The transforming growth factors β (TGFβ) 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 TGFβ, primarily TGFβ1 (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 TGFβ. Although both theca and granulosa cells secreted TGFβ when attached to culture dishes, only theca cells secreted detectable levels of TGFβ when cells were cultured in serum free medium without attachment. Granulosa cells secreted little if any TGFβ. This difference in TGFβ 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 TGFβ in the follicle. To ascertain whether TGFβ 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 TGFβ at a near linear rate for at least 4 days (~300 pg/follicle/day for 6-8-mm-diameter follicles). The level of TGFβ secretion was directly related to the size of the follicle (p < 0.01). Immunoneutralization studies using TGFβ subtype-specific antibodies indicated that the major form of TGFβ secreted by porcine hemi-follicle linings was TGFβ1. To further investigate the source of TGFβ during follicle development, a combination of molecular biology procedures was employed. Using 243-bp antisense and sense cRNA probes generated from a simian TGFβ1 cDNA, we performed in situ hybridization on sections of whole porcine ovaries containing antral follicles. Expression of TGFβ1 mRNA was localized to both theca and granulosa cells with no expression found in the stroma, suggesting that both cell types transcribe TGFβ1. TGFβ1 expression was evaluated additionally by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot analysis. Oligonucleotide primers were generated from the porcine TGFβ1 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 TGFβ1 mRNA. This finding was confirmed via Northern blot analysis, which further indicated the presence of two TGFβ1 mRNAs of 2.5 and 3.5 kb, consistent with previous reports of alternate splicing of the porcine TGFβ1 gene. These data indicate that both cell types express TGFβ1 mRNA. To further evaluate TGFβ1 expression, we attempted to isolate TGFβ1 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 TGFβ1 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.
ASJC Scopus subject areas
- Reproductive Medicine