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J. H. DORRINGTON and R. KILPATRICK

SUMMARY

Luteinizing hormone (LH) stimulated synthesis of both progesterone and 20α-hydroxypregn-4-en-3-one by rabbit ovarian tissue in vitro. Progesterone synthesis was stimulated by nicotinamide adenine dinucleotide phosphate (NADP), but 20α-hydroxypregn-4-en-3-one production was only slightly affected by NADP compared with the effect of LH. When NADP was added with glucose-6-phosphate (G-6-P) or 6-phospho-gluconate (6-P-G), no apparent stimulation of progestational steroid synthesis occurred.

Specific activity measurements suggested that stimulation of synthesis was masked by increased conversion of progestational steroids to other products.

When NADP and submaximal concentrations of LH were used together, potentiation rather than addition of effects on progesterone synthesis was found, and addition of effects with supramaximal concentrations of LH. No potentiation was found when NADP was replaced by NADP and G-6-P or 6-P-G, or by NADPH2. NADP, unlike LH, caused striking stimulation of progesterone synthesis by separated corpora lutea. It is suggested that the present results provide further support for the view that the actions of LH and NADP are related.

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J. H. DORRINGTON and R. KILPATRICK

SUMMARY

Ovine luteinizing hormone (LH) increased the output of progestational steroids (20α-hydroxypregn-4-en-3-one and progesterone) in rabbit ovarian venous blood. Similar increases were found with ovine follicle-stimulating hormone (FSH) and growth hormone, but much larger amounts were necessary. Ovine prolactin was without effect. The increased output was due to increased synthesis and not only to release of stored steroids.

Synthesis of these progestational steroids was stimulated by LH incubated with rabbit ovarian tissue. The stimulation produced by FSH was probably due to contamination by LH since the log dose-response lines for LH and FSH were parallel, and FSH was approximately 100 times less active than LH. Ovine prolactin had no stimulatory activity in concentrations up to 20 μg./ml.

The stimulatory action of LH was unrelated to the presence of corpora lutea. Separated corpora lutea showed only a slight response to LH, whereas the response of interstitial tissue was similar to that found with undissected ovaries. Hence LH caused progestational steroid synthesis by stimulating the ovarian interstitial tissue.

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H. C. K. LALJEE, R. N. SMITH and K. J. DORRINGTON

SUMMARY

A mouse assay for thyrocalcitonin using either intravenous or intraperitoneal injections is described. Responses to porcine thyroid extracts were similar to those in rats. Extracts of human thyroid glands obtained at autopsy or surgical operation caused hypocalcaemia in the mouse. The rat was less sensitive to human thyrocalcitonin. Studies on autopsy tissue showed that human thyrocalcitonin could only be detected if thyroids were removed within 12 hr. after death. Crude extracts of human thyroids were assayed in mice by intraperitoneal injection and the thyrocalcitonin content was determined in tissue obtained from patients with thyrotoxicosis or non-toxic goitre.

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A Foghi, K J Teerds, H van der Donk and J Dorrington

Abstract

In each estrous cycle dominant follicles are selected from a growing pool to develop to the preovulatory stage and to ovulate. Those follicles that do not ovulate must be eliminated in order to maintain the constant mass and homeostasis of the ovary. Granulosa cells are lost by apoptosis at the onset of follicular atresia, whereas apoptotic thecal cells are identified at later stages of atresia. Since transforming growth factor (TGF) α and TGFβ1 have been implicated in the regulation of thecal cell physiology we have localized these growth factors by immunohistochemistry in sections of ovaries from 25-day-old rats, an age at which the ovary exhibits a wave of atresia of preantral follicles. Thecal cells contained TGFα and TGFβ1 throughout the entire process of follicular atresia. To determine if these growth factors could influence thecal cell death, thecal/interstitial cells were isolated from 25-day-old rats, and maintained in culture with growth factors. Subconfluent cultures treated with TGFα or TGFβ1 alone remained healthy whereas in the presence of both TGFα and TGFβ1 there was light microscopical evidence of rounding up of cells and detachment from the monolayer. Chromatin condensation and internucleosomal fragmentation, characteristic of apoptosis, were observed by nucleic acid staining and fluorescence microscopy of thecal/interstitial cells treated with TGFα plus TGFβ1. Further evidence that these cells were undergoing apoptosis came from DNA analysis and the demonstration of DNA laddering. This response of thecal/interstitial cells to TGFα plus TGFβ1 was density dependent; confluent cultures were protected from the induction of apoptosis under these conditions. We conclude that thecal cells are eliminated from atretic follicles by the active and strictly regulated process of apoptosis involving the combined actions of TGFα and TGFβ1.

Journal of Endocrinology (1997) 153, 169–178

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A Foghi, KJ Teerds, H van der Donk, NC Moore and J Dorrington

Follicular atresia is characterized by the initial rapid loss of granulosa cells by apoptosis, followed by the loss of thecal cells at a slower rate. We have previously shown that treatment of subconfluent cultures of thecal/interstitial cells (T/I) with transforming growth factor (TGF) alpha plus TGF beta caused chromatin condensation and internucleosomal fragmentation characteristic of apoptosis, whereas in the presence of either TGF alpha or TGF beta alone the cells remained healthy. In this study we have examined the effect of TGF alpha and TGF beta alone and in combination on the levels of mRNA encoding bcl-2 and interleukin-1 beta-converting enzyme (ICE) in T/I cells using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. Bcl-2, a cell survival gene, has been implicated in regulating the balance between cell proliferation and cell death in physiological processes. ICE, the homolog of the C. elegans cell death gene, ced-3, is also involved in apoptotic signal transduction. The levels of mRNA encoding specific PCR products for bcl-2 (430 bp) and ICE (453 bp) were amplified from T/I cell cDNA. Untreated T/I cells and TGF alpha- or TGF beta-treated cells contained comparable levels of bcl-2 mRNA. Treatment of T/I cells with TGF alpha plus TGF beta significantly decreased the levels of bcl-2 mRNA expression. TGF alpha plus TGF beta caused a significant decrease in bcl-2 mRNA levels within 3 h of treatment of T/I cells, followed by a progressive decline to 10% of control levels after 24 h of treatment. In contrast, in control T/I cells, the levels of ICE mRNA were low. TGF alpha plus TGF beta caused a progressive increase in ICE mRNA, reaching levels 2- and 3-fold higher than control cells after 5 and 7 h respectively. DNA analysis showed that DNA fragmentation, indicative of apoptosis, occurred after 10 h of treatment with TGF alpha plus TGF beta. These studies demonstrated that treatment of T/I cells with TGF alpha plus TGF beta influenced gene expression of bcl-2 and ICE prior to the time at which DNA fragmentation was observed. We propose that the gene products of bcl-2 and ICE are involved in the apoptotic signal transduction pathway induced by TGF alpha plus TGF beta in T/I cells.

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K A Slot, M de Boer-Brouwer, M Houweling, A B Vaandrager, J H Dorrington and K J Teerds

Gonadotrophins including LH have been suggested to play an important role in the etiology of epithelial ovarian cancers. The goal of the present study was to obtain more insight in the mechanism of gonadotrophin action on ovarian surface epithelium (OSE) cells. As the Fas system is known to be a major player in the regulation of the process of apoptosis in the ovary, we investigated whether LH interfered with Fas-induced apoptosis in the human OSE cancer cell lines HEY and Caov-3. Activation of Fas receptor by an agonistic anti-Fas receptor antibody induced apoptosis, as was evaluated by caspase-3 activation, poly(ADP-ribose) polymerase fragmentation, phosphatidylserine externalization and morphological changes characteristic of apoptosis. Co-treatment with LH reduced the number of apoptotic cells following activation of Fas in a transient manner, while LH by itself did not affect apoptosis or cell proliferation. The anti-apoptotic effect of LH could be mimicked by the membrane-permeable cAMP analog 8-(4-chlorophenylthio) cAMP (8-CPT-cAMP), and blocked by H89, a specific inhibitor of protein kinase A (PKA). In conclusion, these findings suggest that LH protects HEY cells against Fas-induced apoptosis through a signaling cascade involving PKA. Although it is plausible that in vivo LH might also enhance OSE tumor growth through inhibition of apoptosis, further research is necessary to confirm this hypothesis.