Regulation of immunoreactive inhibin A and B secretion in cultured human granulosa-luteal cells by gonadotropins, activin A and insulin-like growth factor type-1 receptor

Abstract

Inhibins are gonadal glycoproteins with endocrine effects on pituitary FSH secretion and para/autocrine effects on ovarian and testicular function. The purpose of this study was to investigate the endocrine and para/autocrine regulation of inhibin A and inhibin B secretion in human ovarian granulosa-luteal cells. The cells were obtained from women undergoing in vitro fertilization, and the primary cultures were treated with FSH, LH, human chorionic gonadotropin (hCG), activin A, 8-bromo cyclic AMP (8-BrcAMP), staurosporine (a protein kinase C inhibitor) and an antagonist of IGF action (type-1 IGF receptor antibody IR3). The secretion of inhibins was measured by ELISA assays capable of reliably distinguishing between inhibin A and B.FSH, LH, hCG and 8-BrcAMP increased inhibin A secretion on average up to 180% (P<0·01), 192% (P<0·05), 210% (P<0·01) and 243% (P<0·01) respectively of the control level, while their stimulatory effect on inhibin B secretion was less pronounced (up to 167%, P<0·01; 139%, P<0·05; 127%, P>0·05; 133%, P>0·05 of the controls respectively). IR3 decreased inhibin A and B secretion down to 70% (P<0·01) and 50% (P<0·01) respectively of the control. Staurosporine decreased inhibin B secretion down to 49% (P<0·01) of the control; its effect on inhibin A secretion was not significant. Activin A increased inhibin B secretion up to fourfold of the control (P<0·05) while its effect on inhibin A secretion was insignificant.We conclude that gonadotropins via the protein kinase A signal transduction pathway are the main positive regulators of inhibin A and B secretion in human granulosa-luteal cells. The protein kinase C signal transduction pathway seems to be important especially for inhibin B secretion. Locally produced IGFs are probably important inducers of the production of both forms of inhibin in human ovaries while activins seem to upregulate inhibin B secretion.

Modulatory effects of gonadotrophins and insulin-like growth factor on the secretion of inhibin A and progesterone by granulosa cells from chicken preovulatory (F1-F3) follicles

“…Secretion of dimeric inhibins has recently been investigated in rat (Lanuza et al, 1999) and bovine (Boudjemaa et al, 2000;Glister et al, 2001) granulosa cells, human granulosa-lutein cells (Vanttinen et al, 2000) and mouse preantral follicles (Smitz and Cortvrindt, 1999). Increased expression of inhibin/activin α-and β A -subunit mRNA in mammals has been demonstrated in response to FSH (Su and Hsueh, 1994) or cAMP analogues (Tuuri et al, 1996).…”

Biphasic regulation of activin A secretion by gonadotropins in cultured human ovarian granulosa-luteal cells leads to decreasing activin:inhibin ratios during continuing gonadotropin stimulation

“…Human granulosa cells were obtained from women undergoing an in vitro fertilization (IVF) program in Helsinki University Hospital as described previously (Vänttinen et al 2000, Liu et al 2001. Before follicular aspiration, the patients were treated with a gonadotropin-releasing hormone analog to suppress endogenous gonadotropin secretion.…”

“…The granulosa-luteal cells were purified and cultured as described previously (Vänttinen et al 2000). In brief, the cells were dispersed and separated from red blood cells, washed and plated with a medium containing DMEMHam's F12 (1:1) supplemented with 10% fetal calf serum (Gibco or Bioclear UK Ltd, Calne, Wilts, UK), 2 mM -glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin sulfate (Gibco) at a density of 2-5 10 5 cells/ well on 35-mm six-well dishes.…”

“…In preovulatory and early luteal granulosa cells, the -and A-subunits are highly expressed, whereas the expression of the B-subunit is absent, which suggests that activin A and inhibin A are the most important activin/inhibin forms during these stages. Gonadotropins up-regulateand A-subunit expression (Erämaa et al 1994) and activin A and inhibin A peptide secretion in human granulosa-luteal cells (Muttukrishna et al 1997, Vänttinen et al 2000.…”

Human granulosa-lutein cell in vitro production of progesterone, inhibin A, inhibin B, and activin A are dependent on follicular size and not the presence of the oocyte

“…Although numerous studies have been performed to investigate GC function by detecting different hormones and cytokines during each stage of follicular development, most experiments were based on cells that were isolated from pooled follicular fluid samples (13)(14)(15)(16)(17)(18), which cannot be correlated to specific cell populations harvested from individual follicles for which fertilization rates can be established.…”