In our brief report “Evaluation of ex-vivo adrenocorticotropic hormone-responsiveness of human fetal testis” (doi to be provided) we evaluated the Adrenocorticotropic Hormone (ACTH) responsiveness of ex-vivo cultured human fetal testis (HFT) tissue. Human fetal adrenal (HFA) tissue was included as positive control. In this repository, we provide the Supplementary Methods and Supplementary Data.The following Supplementary files are provided:Schroder_2023_Supplementary_Table1.pdf contains Supplementary Table 1, presenting median (full range) steroid hormone concentrations (nmol/L) in culture media of ex vivo cultured HFA and HFT tissue fragments (n=6). Culture media was collected after an initial 24-hour culture period and after two subsequent 48-hour periods of culture with or without exposure to 10 nM ACTH. Ratios between the ACTH-exposed and control condition are provided (ACTH/Media).Schroder_2023_Supplementary_Table2.pdf contains Supplementary Table 2, presenting Median (full range) normalized steroid hormone levels relative to baseline steroid hormone levels (24 hours of initial culture) quantified in conditioned culture media of HFA tissue fragments and HFT tissue fragments cultured with or without exposure to 10 nM ACTH.Schroder_2023_Supplementary_Figure1.pdf contains Supplementary Figure 1, presenting Hematoxylin and Eosin staining of ex-vivo cultured Fetal Testis (A) and Fetal Adrenal (B) fragments fixed (4% normal buffered formalin) either prior to culture (preculture control [PCC]), or after 5 days of culture with four days of 10 nM ACTH treatment or basal control.Schroder_2023_Supplementary_Figure2.pdf contains Supplementary Figure 2, presenting normalized steroid hormone levels in culture media collected after a second 48-hour period of ex-vivo culture of HFA tissue fragments and HFT tissue fragments with or without exposure to 10 nM ACTH. Steroid hormone levels were normalized for steroid hormone levels in culture media collected after a preceding 24-hour basal culture period without ACTH exposure. Data of individual biological replicates (n=6) are presented with unique symbols.Schroder_2023_Supplementary_Methods contains Supplementary Methods on the Liquid Chromatography tandem mass spectrometry (LC-MS/MS) method to quantify steroid hormones in culture media and on the quantitative polymerase chain reaction (qPCR) to confirm male sex.Abbreviations: 11dF, 11-deoxycortisol; 17OHP, 17-hydroxyprogesterone; A4, Androstenedione; ACTH; Adrenocorticotropic hormone; F, Cortisol; HFA, human fetal adrenal; HFT, human fetal testis; T, Testosterone; A4, Androstenedione.

Date: 2023-11-01

Date Submitted: 2023-10-23