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MARGARET RYLE

Summary.

Ovaries from 15-day-old mice were cultured with or without fsh and lh for 3 or 6 days. Paired control ovaries were fixed at the time of dissection. The number of follicles in each ovary having at least three layers of granulosa cells was determined, and changes in the follicle population during culture were examined. The diameter of each follicle with at least four layers was measured. fsh stimulated follicle growth throughout the experiment but lh stimulated it only between Day 3 and Day 6. lh significantly increased the proportion of follicles with diameters exceeding 119 μ on Day 6; fsh tended to increase this proportion but its effect was not significant. lh reduced the incidence of pycnotic nuclei in peripheral follicles but fsh had no such effect. It is suggested that lh stimulates follicle growth by stimulating production of an oestrogen which must accumulate to a critical level before becoming effectively mitogenic.

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MARGARET RYLE

Summary.

A series of six factorial experiments is described in which ovaries of 15-day-old mice were cultured in vitro for 2 to 4 days in the presence of various concentrations of highly purified fsh and lh. The uptake of [14C]thymidine by the tissue was measured in all experiments. fsh consistently and significantly increased the uptake where the mice used were above a certain size. In only two experiments did lh significantly increase thymidine uptake; this effect appeared to depend on certain precise conditions of culture. The uptake of [3H]lysine was also measured in two of the six experiments; it was significantly increased by fsh in one of them. lh had no significant effect on lysine uptake. There were no significant interactions between fsh and lh. It is concluded that the uptake of labelled thymidine in response to fsh should have useful experimental applications. The possibility that the exclusive role of fsh is to initiate DNA synthesis in follicle cells is briefly discussed.

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MARGARET RYLE

Both FSH and LH stimulate ovarian follicular growth during prolonged incubation in vitro, the response to FSH being detectable after 1 day whereas that to LH first appears after 3 days (Ryle, 1971a, b). Both hormones also enhance the synthesis of cyclic adenosine 3':5' monophosphate (cyclic AMP) by ovarian tissue during brief periods of incubation (Fontaine, Fontaine-Bertrand, Delerue-Labelle & Salmon, 1971; Koch, Zor, Pomerantz, Chobsiang & Lindner, 1973; Mason, Schaffer & Toomey, 1973). Most studies on the effects of cyclic AMP on endocrine target tissues have involved only short exposures and rapid responses. Both rabbit and rat Graafian follicles, however, undergo luteinization following treatment with either LH or cyclic AMP (Miller & Keyes, 1972; Ellsworth & Armstrong, 1973) and adrenal cortical cells of the fetal rat exposed to cyclic AMP for several days eventually undergo changes similar to those induced by ACTH (Milner, 1972). It was therefore of interest

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MARGARET RYLE

Summary.

Ovaries of infantile mice were cultured in vitro with no hormone, lh or fsh for 2 or 5 days, [3H] thymidine being present in the medium for the last 24 hr of culture. Each ovary was serially sectioned and autoradiographs were prepared. The effects of the treatments on follicle numbers and on the extent to which they were labelled were examined in groups of follicles of different sizes. Growth was stimulated by fsh in follicles of all sizes both on Day 2 and Day 5, but lh did not stimulate growth in primordial follicles and had relatively little effect on one- and two-cell layer follicles. It greatly accelerated growth in some with three- and four-cell layers on Day 5, but not on Day 2. It also appeared to stimulate mitosis in the theca interna whereas fsh appeared to diminish it. A few follicles grew rapidly on Day 5 in the absence of any hormone. Spindle formation in oocytes was associated with rapid follicle growth but not with hormone treatment. Possible explanations of the delayed effect of lh are discussed.

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MARGARET RYLE

Summary.

Ovaries from 16- to 17-day-old mice were cultured for 4 days in the presence of various concentrations of highly purified fsh and lh. Serial sections were prepared and parameters of all follicles with four or more layers of granulosa cells were measured. fsh significantly increased the total number of such follicles and in particular the number of small ones. It did not significantly increase the number of large ones although it markedly augmented the number of follicles with small antra. lh enlarged the mean follicle diameter by multiplying the number of large follicles while having no effect on the number of small ones, but it did not significantly increase the number of follicles with antra.

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MARGARET RYLE

Summary.

Mouse ovaries were incubated with fsh then removed to fresh medium lacking hormone. At various times after removal, [14C]thymidine was added to the dishes. After 24-hr further incubation, the tissue was prepared for counting. Parallel cultures, never exposed to fsh, received thymidine and were counted simultaneously. The difference in counts between paired fsh-treated and untreated cultures indicated the residual fsh effect. The differences were significant up to 9 hr after removal from fsh, but not thereafter. The results suggest that a constant supply of fsh is needed for the continued growth of early follicles.

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JOYCE KENT and MARGARET RYLE

Summary.

The activities of alkaline phosphatase, NAD diaphorase and NADP diaphorase increased in infantile mouse ovaries in response to injected gonadotrophins. The distribution and activity of these enzymes were studied in detail in the ovaries of normal mice from 1 to 41 days after birth and in mice injected at various ages with FSH, LH and HCG. Granulosa cells contained NAD and NADP diaphorases. Thecal cells contained NADP diaphorase and alkaline phosphatase with NAD diaphorase first appearing in the thecae of larger follicles 11 days after birth. All three enzymes occurred in interstitial tissue, in the interfollicular stroma and in groups of gonadotrophin-responsive cells in the medulla. These medullary cells and the interstitial tissue were stimulated by exogenous LH and HCG but not by FSH. Granulosa, theca and interfollicular tissue were stimulated at some stage by each of the three injected hormones. The normal pattern of development is discussed in relation to the changing serum levels of endogenous gonadotrophin found in similar mice. It is concluded that the enzyme changes were closely and reciprocally related to endogenous hormone concentrations.

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Margaret Ryle and Jane Sage

Summary. A procedure is described for testing potential inhibitors of hCG activity in vivo, based on a hormone-responsive alkaline phosphatase in the ovaries of young mice. Standard injections of an antiserum inhibited the response to a subsequent injection of 2 i.u. hCG after an interval of up to 24 h.

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Janice Boggis and Margaret Ryle

When intact ovaries of infantile mice are cultured in the presence of gonadotrophins, steroids or dibutyryl cyclic adenosine 3′:5′ monophosphate follicular growth is stimulated. If [3H]thymidine is supplied in the medium, its accumulation in the tissue is enhanced as a result of this growth (Ryle, 1969, 1972, 1974; Kent, 1973). During other experiments it was noted that lithium lactate also increased the uptake of [3H]thymidine by cultured ovaries, and the following studies were designed to extend this observation.

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Margaret Ryle and Jayne Carrier

Summary. Replicate cultures of 50000 mouse Leydig cells were used to obtain linear log dose–response curves to LH. This hormone augmented the activity of an endogenous alkaline phosphatase, which was subsequently allowed to act on p-nitrophenyl phosphate, the coloured end-point being measured with a spectrophotometer. The linear portion of the curve extended from 0·2 to 3·2 mi.u. LH/ml.