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L. E. REICHERT JR.
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M. V. L'HEUREUX
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SUMMARY

Reduced triphosphopyridine nucleotide (TPNH) incubated with parathyroid gland extract (Injection Parathyroid, Lilly & Co.) for 6hr at pH 7·4 and at room temperature exhibits a reduction in u.v. absorption at 340 mμ. No correlation could be shown to exist between this decrease in absorbing capacity of TPNH at 340 mμ and the calcium-mobilizing activity of the extract.

Dialysis of the active extract against distilled water or treatment with ethanol-diethyl ether or with formaldehyde resulted in a marked reduction or virtual elimination of biological activity, and in a marked reduction in the ability of the treated preparation to affect the absorbing capacity of TPNH at 340 mμ. Inactivation of the calcium-mobilizing principle of the extract by pepsin resulted in a preparation which retained its ability to affect the u.v. absorption of TPNH. Dialysis of the pepsin-inactivated extract against distilled water or treatment with ethanol-diethyl ether decreased this spectrophotometric effect.

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S. G. HILLIER
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AGNES M. J. VAN DEN BOOGAARD
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L. E. REICHERT JR
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E. V. VAN HALL
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Locally produced androgens and oestrogens are thought to be important factors in the hormonal regulation of follicular development. In the present study the relationship between follicular maturity and granulosa cell aromatase activity has been examined in vitro. Granulosa cells harvested from the largest antral follicles in adult rat ovaries produced negligible amounts of immunoreactive oestradiol when incubated for 3 h in vitro irrespective of the day of the oestrous cycle upon which they were obtained. However, the addition of aromatizable C19 steroid substrate (testosterone, androstenedione or 19-hydroxyandrostenedione) to the incubation medium resulted in time- and concentration-dependent increases in oestradiol production which were related to the level of follicular maturity attained in vivo. By measuring oestradiol production using testosterone (10−7 mol/l) as substrate, the aromatase activity of granulosa cells obtained on the first day of vaginal dioestrus was shown to be only a fraction (less than 5%) of that observed for cells obtained on the morning of pro-oestrus. Cells obtained on the second day of dioestrus displayed an intermediate level of activity which remained approximately five times lower than that of granulosa cells at pro-oestrus. These observations, therefore, establish the induction or activation of granulosa cell aromatase activity as a correlate of normal preovulatory follicular development. However, intrafollicular androgen/oestrogen ratios may also be influenced by quantitative and/or qualitative alterations in the C19 steroidal substrate available for the aromatase reaction. Thus, the naturally occurring non-aromatizable 5α-reduced androgen metabolites, 5α-dihydrotestosterone and 5α-androstanedione, proved to be potent competitive inhibitors of the granulosa cell aromatase reaction in vitro. In this respect each of these biologically active androgens was more effective than 1-enetestololactone, an established C19 steroidal aromatase inhibitor. Since C19 steroid 5α-reductase is known to be an ovarian enzyme, it is suggested that by affecting the androgenic/oestrogenic composition of the hormonal milieu, local alterations in the activity of this enzyme may be an additional determinant of preovulatory follicular development and function.

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S. G. Hillier
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E. J. Wickings
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P. T. K. Saunders
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A. F. Dixson
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S. Shimasaki
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I. A. Swanston
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L. E. Reichert Jr
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A. S. McNeilly
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ABSTRACT

In-vitro data from experiments on rats implicate granulosa cells as primary sites of hormone-dependent ovarian inhibin biosynthesis, but no equivalent data exist for primates. We have used the common marmoset (Callithrix jacchus) to investigate inhibin biosynthesis in primate granulosa cells in vitro and to determine its relationship to preovulatory follicular development. To relate the production of immunoactive inhibin to follicular maturity, we studied primary granulosa cell cultures from follicles at progressive stages of preovulatory development. Granulosa cells from 'large' (≥2·0 mm diameter) follicles expressed high rates of inhibin production and steroidogenesis (progesterone), and were positively regulated by human (h)LH in vitro. Less mature granulosa cells from 'medium' (1·1–1·9 mm) and 'small' (≤ 1·0 mm) follicles expressed proportionately lower rates of inhibin production and steroidogenesis, but each parameter was stimulated in a dose- and time-dependent manner by hFSH in vitro. The stimulatory action of hFSH on immunoactive inhibin was augmented by the presence of testosterone or oestradiol; testosterone (but not oestradiol) also augmented the steroidogenic response to hFSH. Marmoset luteal tissue also produced inhibin in vitro and expressed an ∼1·5 kb inhibin α-subunit mRNA, confirming the corpus luteum as a source of ovarian inhibin in primates.

These results provide direct experimental evidence that primate granulosa cells produce inhibin. They suggest that production of inhibin by immature granulosa cells is initially induced by FSH and subject to modulation by follicular steroids. During advanced preovulatory development, granulosa cell inhibin production becomes directly responsive to LH, thereby indicating a role for LH in the control of peri- and postovulatory inhibin secretion by the primate ovary.

Journal of Endocrinology (1989) 123, 65–73

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