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M. Heistermann, E. Pröve, H.-J. Wolters and G. Mika

Summary. Oestrone, oestradiol-17β and progesterone were measured by radioimmunoassay in daily urine samples after pairing and during subsequent pregnancy in a pied bare-face tamarin. On the basis of excretion profiles an ovarian cycle length of about 3 weeks and a gestation length of about 160 days are suggested. Oestrone was the predominant urinary oestrogen excreted by the non-pregnant and pregnant pied bareface tamarin, the oestrone/oestradiol ratio being greater than 100:1. The results suggest that steroid monitoring can provide useful information about reproductive physiology in this species of tamarin.

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K Augustowska, E EL Gregoraszczuk, A Grochowalski, T Milewicz, M Mika, J Krzysiek and R Chrzaszcz

Explants of human placental tissue harvested immediately after expulsion were used to determine differences between accumulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated dibenzo-p-dioxin (PCDD)-polychlorinated dibenzo-p-furans (PCDF) environmental mixture, and their influence on placental steroidogenesis. Explants were cultured in vitro for 5 days in media supplemented each day with either TCDD or a mixture of PCDD-PCDF. Media were collected every day for steroid content analysis by radioimmunoassay. At 24 h after the last treatment, the tissue was frozen for further analysis of the content of TCDD or other congeners present in the mixture. Determinations of TCDD and all 17 PCDDs and PCDFs were performed using gas chromatography equipped with DB-5 MS and DB-17 capillary columns. In the control tissue, the amounts of both TCDD and mixture components were close to the limit of detection of the method. In the treated tissue, the TCDD accumulation was 94% of the total exposure to TCDD. The most toxic congeners 2,3,7,8-TCDD, 2,3,7,8-tetrachlorodibenzofuran, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 1,2,3,7,8-pentachlorodibenzo-p-furans (PeCDF) and 2,3,4,7,8-PeCDF showed the highest accumulation, which covered >50% of the total toxic equivalents present in this mixture. During the first 3 days of exposure to TCDD there was no effect on the conversion of dehydroepiandrosterone to oestradiol, whereas on days 4 and 5 of exposure, a twofold decrease in oestradiol secretion was observed. However, a small but significant increase in oestradiol secretion was noted at all times of exposure to the PCDD-PCDF mixture. All observed changes in oestradiol secretion were not accompanied by changes in progesterone secretion after exposure to TCDD or the PCDD-PCDF mixture. In conclusion, a high accumulation of TCDD in the placental tissue resulted in a decrease in oestradiol secretion and in vivo this could result in a decrease in blood flow through the placenta. From the mixture, PeCDD and PeCDF in the higher amount accumulated in the placental tissue caused an increase in oestrogen secretion and as a consequence could activate oxytocin secretion from the pituitary and early pregnancy outcome.

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HE Paczoska-Eliasiewicz, A Gertler, M Proszkowiec, J Proudman, A Hrabia, A Sechman, M Mika, T Jacek, S Cassy, N Raver and J Rzasa

Thirty-four-week-old laying hens received injections of recombinant chicken leptin to assess the role of leptin in avian ovarian function. In the first experiment, the hens (n=60) were divided into three groups: (i). fed ad libitum; (ii). fasted; and (iii). fasted + leptin. Hens were fasted for 5 days and those treated with leptin received 250 microg leptin kg-1 body weight twice a day, i.p. In the second experiment, the hens (n=72) were divided into four groups: (i). fed ad libitum; (ii). fasted; (iii). fasted + leptin given only during fasting (5 days); or (iv). fasted and leptin given during both fasting and 5 days of re-feeding (10 days). LH was measured in blood plasma, and progesterone and oestradiol were measured in blood plasma and the ovary by radioimmunoassay. Apoptosis was examined in the walls of the three largest yellow hierarchical follicles (F3-F1; F3 25-35 mm) by the TdT-mediated dUTP nick-end labelling method. Results showed that the injections of leptin during fasting: (i). delayed cessation of egg laying; (ii). attenuated regression of yellow hierarchical follicles; (iii). altered ovarian steroidogenesis; and (iv). abolished the fasting-induced apoptosis in the wall of F3-F1 follicles during the first 2 days of fasting and partially attenuated apoptosis after 5 days of fasting. Prolongation of leptin injections into the re-feeding period considerably delayed the restoration of the ovary. Expression of leptin receptor in laying hens was determined by RT-PCR. The highest expression of leptin receptor was observed in the hypothalamus. Lower receptor mRNA expression was found in the hypophysis, whereas the lowest expression was observed in the ovary. Within the ovary, a relatively high expression of leptin receptor was found in the stroma with cortical follicles <1 mm, the wall of white (1-8 mm) and small yellow follicles (>8-12 mm), and the granulosa layer of F3 follicles. The expression of leptin receptor in the granulosa layer of F2 and F1 follicles was barely detectable. This was in contrast to a much higher expression of leptin receptor maintained in the theca layer of F3-F1 follicles. The present results indicate that in chickens leptin might be involved in the adaptation to starvation due to attenuation of follicular apoptosis. The presence of leptin receptors in the ovary indicates the possibility of a peripheral effect of the hormone.

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Samu Myllymaa, Arja Pasternack, David G Mottershead, Matti Poutanen, Minna M Pulkki, Lauri J Pelliniemi, Olli Ritvos and Mika P E Laitinen

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are among the key regulators transmitting the signaling between the oocyte and the surrounding granulosa cells. Previously, it has been shown that a recombinant BMP type II receptor ectodomain–Fc fusion protein (BMPR2ecd–Fc) is able to inhibit the actions of GDF9 and BMP15 in vitro. Here, we have produced bioactive BMPR2ecd–Fc, which was injected i.p. into neonatal mice. Early folliculogenesis was first studied by injecting mice five times with various doses of BMPR2ecd–Fc during the postnatal days 4–12. Folliculogenesis was affected dose dependently, as evidenced by a decreased mitogenesis of granulosa cells of the growing follicles. Furthermore, we also noticed a decrease in the number of secondary and tertiary follicles as well as an increase in the oocyte size. Electron microscopic analysis revealed that the ultrastructure of the granulosa cells of the primary follicles was not affected by the BMPR2ecd–Fc treatment. A second study was conducted to investigate whether a longer treatment with 12 injections during postnatal days 4–28 would inhibit folliculogenesis. Similar effects were observed in the two studies on the early follicular developmental stages. However, in the long-term study, later stages of folliculogenesis were not blocked but rather increased numbers of antral follicles, preovulatory follicles, and corpora lutea were found. We conclude that BMPR2ecd–Fc is a potent modulator of ovarian folliculogenesis in vivo, and thus, is a valuable tool for studying the physiology and downstream effects of oocyte-derived growth factors in vivo.

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Inger B Carlsson, Mika P E Laitinen, Jennifer E Scott, Henna Louhio, Louiza Velentzis, Timo Tuuri, Johanna Aaltonen, Olli Ritvos, Robert M L Winston and Outi Hovatta

The receptor tyrosine c-Kit and its cognate ligand, c-Kit ligand (KL, stem cell factor, SCF), are involved in ovarian follicular development in several animal species. We studied the expression of KL and c-Kit using in situ hybridization and immunohistochemistry in donated human ovarian cortical tissue. The KL transcripts were expressed in granulosa cells of primary follicles, whereas the expression of c-Kit was confined to the oocyte and granulosa cells in primary and secondary follicles. We employed an ovarian organ culture using firstly serum-containing and then serum-free medium to study the effects of KL and an anti-c-Kit antibody, ACK2, on the development and survival of ovarian follicles in vitro. Culture of ovarian cortical slices for 7 days resulted in a 37% increase in the number of primary follicles and a 6% increase in secondary follicles. The proportion of viable follicles decreased in all cultures. The addition of KL (1, 10 and 100 ng/ml) into the culture media did not affect the developmental stages of the follicles or the proportion of atretic follicles. Inclusion of ACK2 (800 ng/ml) in the culture medium significantly increased the proportion of atretic follicles on days 7 (49 vs 28% in control cultures) and 14 (62 vs 38%) of culture. In conclusion, c-Kit and KL are expressed in human ovaries during follicular development. Blocking the c-Kit receptor induces follicular atresia. The KL/c-Kit signaling system is likely to control the survival of human ovarian follicles during early follicular development.