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G. C. B. Randall and B. K. Tsang

Summary. Fetal and maternal plasma progesterone and unconjugated oestrone and oestradiol-17β concentrations were measured in intact pig fetuses and those in which the pituitary had been destroyed. Progesterone concentrations were significantly higher (P < 0·05) and oestrogen concentrations significantly lower (P < 0·01) in hypophysectomized fetuses than in intact fetuses. When fetuses in one uterine horn only were hypophysectomized, oestrogen concentrations in the uterine vein draining this horn were lower than those from the contralateral vein. The results indicate that both fetal and maternal oestrogen concentrations are influenced by the fetal pituitary. When dexamethasone was infused (at 27μg/h for 96 h) into 5 chronically-catheterized hypophysectomized fetuses no changes in peripheral fetal progesterone or oestrone were observed.

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B. K. Tsang, M. Li and J. A. Carnegie

Summary. The secretion of progesterone and 20α-hydroxypregn-4-en-3-one (20α-dihydroprogesterone) by granulosa cells from 30-day-old rats pretreated with PMSG (4 i.u.; i.p.) was significantly increased in a time- and concentration-dependent manner by FSH or cytochalasin B. Whereas FSH markedly stimulated progestagen secretion during 3 h of incubation, a significant enhancement of the steroidogenic response was not noted until 12 h of exposure to the inhibitor in vitro. Although cytochalasin B also enhanced the submaximal stimulation of progestagen production by FSH (15 ng/ml), it was ineffective in the presence of maximal stimulatory concentration of the gonadotrophin (150 ng/nl). With increasing concentrations of cytochalasin B, the ability of FSH to further stimulate progestagen secretion was progressively reduced. Granulosa cells cultured in medium alone contained a prominent cytoplasmic array of microfilaments which was markedly reduced by FSH or cytochalasin B. FSH and, to a greater extent, cytochalasin B elicited concentration-dependent reductions in the mean area occupied by the cells on the culture surface, the contour index (a size-independent representation of cell profile irregularity) and cell perimeter, indicating that the cells underwent less spreading and were more spherical and regular in outline in the presence of either agent. The FSH-induced reductions in the three shape-related parameters were augmented by cytochalasin B although the influence of the FSH on the mean area and perimeter was progressively reduced in the presence of higher concentrations of cytochalasin B. These findings are consistent with the concept that microfilaments influence cell shape and steroidogenesis in granulosa cells in vitro and that FSH alters microfilament distribution and shape of cultured granulosa cells in eliciting its steroidogenic influence.

Keywords: microfilaments; steroidogenesis; FSH; granulosa cells

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B. K. Tsang, L. Ainsworth, B. R. Downey and G. J. Marcus

Summary. Dispersed granulosa and theca interna cells were recovered from follicles of prepubertal gilts at 36, 72 and 108 h after treatment with 750 i.u. PMSG, followed 72 h later with 500 i.u. hCG to stimulate follicular growth and ovulation. In the absence of aromatizable substrate, theca interna cells produced substantially more oestrogen than did granulosa cells. Oestrogen production was increased markedly in the presence of androstenedione and testosterone in granulosa cells but only to a limited extent in theca interna cells. The ability of both cellular compartments to produce oestrogen increased up to 72 h with androstenedione being the preferred substrate. Oestrogen production by the two cell types incubated together was greater than the sum produced when incubated alone. Theca interna cells were the principal source of androgen, predominantly androstenedione. Thecal androgen production increased with follicular development and was enhanced by addition of pregnenolone or by LH 36 and 72 h after PMSG treatment. The ability of granulosa and thecal cells to produce progesterone increased with follicular development and addition of pregnenolone. After exposure of developing follicles to hCG in vivo, both cell types lost their ability to produce oestrogen. Thecal cells continued to produce androgen and progesterone but no longer responded to LH in vitro. These studies indicate that several functional changes in the steroidogenic abilities of the granulosa and theca interna compartments occur during follicular maturation.

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B. K. Tsang, D. F. Mattice, M. Li and E. K. Asem

Summary. The gonadotrophic regulation of progesterone production by rat granulosa cells was examined in a chemically-defined medium containing FSH, dibutyryl cyclic AMP ((Bu)2cAMP) and the calcium ionophore, A23187. FSH and A23187 alone significantly enhanced the production of pregnenolone, progesterone and its metabolite, 20α-hydroxypregn-4-en-3-one (20α-OH-P) from endogenous substrate(s). Stimulation of progesterone production by A23187 was accompanied by an increase in 3β-hydroxysteroid dehydrogenase (3β-HSD) but not 20α-hydroxysteroid dehydrogenase (20α-HSD) activity, as attested by enhancement of the metabolism of exogenous pregnenolone to progesterone but not of progesterone to 20α-OH-P. In contrast, although (Bu)2cAMP increased pregnenolone and progesterone production and the metabolism of exogenous progesterone to 20α-OH-P, it failed to stimulate the conversion of exogenous pregnenolone to progesterone. The increase in progesterone production and in the conversion of exogenous pregnenolone to progesterone by FSH and A23187 was concentration- and time-dependent. Whereas maximal stimulation of de-novo progesterone synthesis by FSH was evident by 6 h (earliest time examined), a significant increase in the conversion of exogenous pregnenolone to progesterone in the presence of FSH or the ionophore was not noted until 12 h of incubation. Although a small but significant increase in progesterone production was also noted as early as 6 h of incubation in the presence of the calcium ionophore, this was markedly smaller than that elicited by FSH.

We conclude that the calcium ionophore A23187 and (Bu)2cAMP have similar as well as distinct effects on progesterone production in rat granulosa cells in vitro. We suggest that, while cAMP may be involved in the more rapid control by gonadotrophin of the production of the steroid via increased synthesis of pregnenolone and/or its metabolism to 20α-OH-P, calcium may be important in the synthesis and metabolism of pregnenolone for the maintenance of steroidogenic capacity of the granulosa cells.

Keywords: calcium; cAMP; 3β-hydroxysteroid dehydrogenase; 20α-hydroxysteroid dehydrogenase; steroidogenesis; granulosa cell; gonadotrophin action; rat

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Q Qiu, M Yang, B K Tsang and A Gruslin

Epidermal growth factor (EGF) is present in the maternal-fetal environment and has an important role in placental development. Matrix metalloproteinase-9 (MMP-9) expression/activation is a pre-requisite in extravillous trophoblast invasion. Whereas EGF up-regulates MMP-9 activity in a variety of cell types, there is no direct evidence for the stimulation of MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) secretion by EGF in extravillous trophoblasts. In addition, the signalling pathways involved in this regulation are not clear. In the present study, we have examined the possible involvement of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways in the regulation of the MMP-9/TIMP-1 system by EGF in vitro. We used a well-established invasive extravillous trophoblast cell line (HTR8/Svneo) and measured gene and protein expression by semi-quantitative RT-PCR and western analysis respectively. MMP activity was determined by zymography. We showed for the first time that EGF activated both PI3K/Akt and MAPK/extracellular-signal regulated kinase (ERK) signalling in HTR8/SVneo, and increased both MMP-9 and TIMP-1 mRNAs and protein concentrations. Interfering with either signalling pathway via PI3K inhibitor LY294002 or MEK inhibitor U0126 in EGF-stimulated HTR8/SVneo cells blocked the induction of MMP-9 and TIMP-1. LY294002 inhibited Akt phosphorylation, but had no effect on ERK phosphorylation; U0126 suppressed ERK phosphorylation without interfering with the phosphorylation of Akt. In addition, expression of constitutively active Akt (Myr-Akt1, Myr-Akt2, Myr-Akt3) was not sufficient to induce proMMP-9 and TIMP-1 secretion. Our results suggest that the activation of both PI3K and MAPK pathways in extravillous trophoblasts is necessary for the up-regulation of MMP-9 and TIMP-1 expression by EGF.