Malene Lundgaard Riis1,2, Gabriele Matilionyte3, John E Nielsen1,2, Cecilie Melau1,2, David Greenald3, Kristine Juul Hare4, Lea Langhoff Thuesen4, Eva Dreisler5, Kasper Aaboe5, Pia Tutein Brenøe6, Anna-Maria Andersson1,2, Jakob Albrethsen1,2, Hanne Frederiksen1,2, Ewa Rajpert-De Meyts1,2, Anders Juul1,2,7, Rod T Mitchell3, Anne Jørgensen8,9. 1. Department of Growth and Reproduction, Copenhagen University Hospital - Rigshospitalet, Blegdamsvej 9, 2100, Copenhagen, Denmark. 2. International Research and Research Training Centre in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Blegdamsvej 9, Copenhagen, Denmark. 3. MRC Centre for Reproductive Health, The Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK. 4. Department of Obstetrics and Gynaecology, Copenhagen University Hospital - Hvidovre and Amager Hospital, Kettegård Alle 30, Hvidovre, Denmark. 5. Department of Gynaecology, Copenhagen University Hospital - Rigshospitalet, Blegdamsvej 9, DK-2100, Copenhagen, Denmark. 6. Department of Obstetrics and Gynaecology, Copenhagen University Hospital - Herlev and Gentofte Hospital, Borgmester Ib Juuls Vej 1, 2730, Herlev, Denmark. 7. Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark. 8. Department of Growth and Reproduction, Copenhagen University Hospital - Rigshospitalet, Blegdamsvej 9, 2100, Copenhagen, Denmark. aj@rh.regionh.dk. 9. International Research and Research Training Centre in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Blegdamsvej 9, Copenhagen, Denmark. aj@rh.regionh.dk.
Abstract
BACKGROUND: Reduced androgen action during early fetal development has been suggested as the origin of reproductive disorders comprised within the testicular dysgenesis syndrome (TDS). This hypothesis has been supported by studies in rats demonstrating that normal male development and adult reproductive function depend on sufficient androgen exposure during a sensitive fetal period, called the masculinization programming window (MPW). The main aim of this study was therefore to examine the effects of manipulating androgen production during different timepoints during early human fetal testis development to identify the existence and timing of a possible window of androgen sensitivity resembling the MPW in rats. METHODS: The effects of experimentally reduced androgen exposure during different periods of human fetal testis development and function were examined using an established and validated human ex vivo tissue culture model. The androgen production was reduced by treatment with ketoconazole and validated by treatment with flutamide which blocks the androgen receptor. Testicular hormone production ex vivo was measured by liquid chromatography-tandem mass spectrometry or ELISA assays, and selected protein markers were assessed by immunohistochemistry. RESULTS: Ketoconazole reduced androgen production in testes from gestational weeks (GW) 7-21, which were subsequently divided into four age groups: GW 7-10, 10-12, 12-16 and 16-21. Additionally, reduced secretion of testicular hormones INSL3, AMH and Inhibin B was observed, but only in the age groups GW 7-10 and 10-12, while a decrease in the total density of germ cells and OCT4+ gonocytes was found in the GW 7-10 age group. Flutamide treatment in specimens aged GW 7-12 did not alter androgen production, but the secretion of INSL3, AMH and Inhibin B was reduced, and a reduced number of pre-spermatogonia was observed. CONCLUSIONS: This study showed that reduced androgen action during early development affects the function and density of several cell types in the human fetal testis, with similar effects observed after ketoconazole and flutamide treatment. The effects were only observed within the GW 7-14 period-thereby indicating the presence of a window of androgen sensitivity in the human fetal testis.
BACKGROUND: Reduced androgen action during early fetal development has been suggested as the origin of reproductive disorders comprised within the testicular dysgenesis syndrome (TDS). This hypothesis has been supported by studies in rats demonstrating that normal male development and adult reproductive function depend on sufficient androgen exposure during a sensitive fetal period, called the masculinization programming window (MPW). The main aim of this study was therefore to examine the effects of manipulating androgen production during different timepoints during early human fetal testis development to identify the existence and timing of a possible window of androgen sensitivity resembling the MPW in rats. METHODS: The effects of experimentally reduced androgen exposure during different periods of human fetal testis development and function were examined using an established and validated human ex vivo tissue culture model. The androgen production was reduced by treatment with ketoconazole and validated by treatment with flutamide which blocks the androgen receptor. Testicular hormone production ex vivo was measured by liquid chromatography-tandem mass spectrometry or ELISA assays, and selected protein markers were assessed by immunohistochemistry. RESULTS: Ketoconazole reduced androgen production in testes from gestational weeks (GW) 7-21, which were subsequently divided into four age groups: GW 7-10, 10-12, 12-16 and 16-21. Additionally, reduced secretion of testicular hormones INSL3, AMH and Inhibin B was observed, but only in the age groups GW 7-10 and 10-12, while a decrease in the total density of germ cells and OCT4+ gonocytes was found in the GW 7-10 age group. Flutamide treatment in specimens aged GW 7-12 did not alter androgen production, but the secretion of INSL3, AMH and Inhibin B was reduced, and a reduced number of pre-spermatogonia was observed. CONCLUSIONS: This study showed that reduced androgen action during early development affects the function and density of several cell types in the human fetal testis, with similar effects observed after ketoconazole and flutamide treatment. The effects were only observed within the GW 7-14 period-thereby indicating the presence of a window of androgen sensitivity in the human fetal testis.
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