Temporal and hormonal regulation of inhibin protein and subunit mRNA expression by post-natal and immature rat ovaries

Abstract

The contribution of specific follicle populations to dimeric inhibin production and inhibin subunit mRNA expression by the rat ovary has been investigated in two model systems, granulosa cells isolated from 25-day-old diethylstilboestrol (DES)-treated rats and post-natal rat ovaries, dispersed in culture or whole ovaries, using specific two-site immunoassays and 'real time' PCR. Media from FSH-stimulated granulosa cell cultures fractionated by gel filtration and RP-high performance liquid chromatography revealed two predominant peaks of subunit activity which were attributed to subunit and 31 k dimeric inhibin-A. The corresponding inhibin-B levels were low. FSH stimulation did not alter the ratio of inhibin-A: subunit produced by granulosa cells. All three inhibin subunit mRNAs were expressed by granulosa cells, with eight-fold more subunit mRNA relative to either of the subunits. Administration of DES to immature rats prior to the isolation of granulosa cells from the ovary led to A and B mRNA expression being down-regulated in the absence of any significant change in subunit expression by the granulosa cells. Inhibin-A, -B and -subunit were produced by basal and stimulated cultures of ovarian cells prepared from 4-, 8-and 12-day-old rats, indicating that primary, preantral and antral follicles contribute to total inhibin production. Consistent with these results, follicles within these ovaries expressed all three inhibin subunit mRNAs, with maximal expression observed in the ovaries of 8-day-old rats. The appearance of antral follicles in the ovary at day 12 led to a decline in the mRNA levels of each of the subunits but was most evident for the subunits. There was a profound influence of secondary preantral follicles on dimeric inhibin-A production, with FSH stimulation increasing inhibin-A relative to subunit levels in cultures of ovarian cells prepared from 8-day-old rats. Thus, preantral follicles exposed to FSH contribute significantly to A subunit production by the ovary. In contrast, primary and preantral follicles did not produce inhibin-B in response to FSH stimulation. Transforming growth factor-(TGF-) enhanced, in a time-dependent manner, the production of the inhibin forms by ovarian cells in culture, although inhibin-B production was not responsive until day 8. The simultaneous treatment of ovarian cell cultures with FSH and TGF-elicited the greatest increases in production of all the inhibin forms.In summary, ovaries of 4-, 8-and 12-day-old rats expressed inhibin subunit mRNAs and produced dimeric inhibin-A and -B and free subunit. Preantral follicles (day-8 ovarian cell cultures) were particularly sensitive to stimulation by FSH and TGF-and had a substantial capacity for inhibin production. The production of oestrogen by follicles may be instrumental in regulating inhibin production given that subunit mRNA expression was down-regulated by DES. The mechanisms by which inhibin-A and inhibin-B are individually regulated are likely to be similar during the post-natal period, when folliculoge...

Reciprocal regulation of activin A and inhibin B by interleukin-1 (IL-1) and follicle-stimulating hormone (FSH) in rat Sertoli cells in vitro

“…The data in the present study clearly indicate that FSH inhibits formation of both of the A -subunit-containing dimers (activin A and inhibin A) in the Sertoli cell. This is quite different from the situation in the ovary, where FSH and cAMP stimulate inhibin A formation (Tuuri et al 1996, Drummond et al 2000. It is equally interesting that, even though IL-1 stimulates A -subunit expression by the Sertoli cell, this does not result in an increase in inhibin A.…”

“…For every sample, a no-RT control also was performed, to verify the absence of contaminating genomic DNA in the PCR. mRNA expression was quantified by the Roche LightCycler (Roche, Mannheim, Germany), as previously described (Drummond et al 2000). For PCR, 2 µl of each cDNA preparation were diluted to a final concentration of 1:50 and added to individual capillary tubes with dNTP, Mg 2+ , SYBR Green and relevant primers.…”

Rodent adrenocortical cells display high affinity binding sites and proteins for inhibin A, and express components required for autocrine signalling by activins and bone morphogenetic proteins

“…For each RT-PCR reaction, a 1 or 2 µg aliquot of total RNA was reverse transcribed with 50 U of expand reverse transcriptase in a volume of 20 µl using 250 ng oligo-dT primers. Primers and conditions for inhibin subunit, glyceraldehyde-3-phosphate dehydrogenase and activin receptor type I, IB, II and IIB amplifications were as previously described (Drummond et al 2000(Drummond et al , 2002. Primers and product sizes for other reactions are shown in Table 1.…”

Reduced leukocyte–endothelial cell interactions in the inflamed microcirculation of macrophage migration inhibitory factor–deficient mice

“…Total RNA (1 g) was reverse transcribed using Superscript II reverse transcriptase (Gibco BRL) and oligo(dT) [12][13][14][15][16][17][18] . PCR amplification was performed on a LightCycler (Roche Diagnostics, Castle Hill, New South Wales, Australia) using SYBR Green I, as previously described (33). Murine MIF and ␤-actin PCR products were used as assay standards.…”