Inhibition of WNT/ß-catenin signalling during sex-specific gonadal differentiation is essential for normal human fetal testis development.

Sex-specific gonadal differentiation is directed by complex signalling promoting development in either male or female direction, while simultaneously inhibiting the opposite pathway. In mice, the WNT/ß-catenin pathway promotes ovarian development and the importance of actively inhibiting this pathway to ensure normal testis development has been recognised. However, the implications of alterations in the tightly regulated WNT/ß-catenin signalling during human fetal gonad development has not yet been examined in detail. Thus, the aim of this study was to examine the consequences of dysregulating the WNT/ß-catenin signalling pathway in the supporting cell lineage during sex-specific human fetal gonad development using an established and extensively validated ex vivo culture model. Inhibition of WNT/ß-catenin signalling in human fetal ovary cultures resulted in only minor effects, including reduced secretion of RSPO1 and reduced cell proliferation although this was not consistently found in all treatment groups. In contrast, promotion of WNT/ß-catenin signalling in testes severely affected development and function. This included disrupted seminiferous cord structures, reduced cell proliferation, reduced expression of SOX9/AMH, reduced secretion of Inhibin B and AMH as well as loss of the germ cell population. Additionally, Leydig cell function was markedly impaired with reduced secretion of testosterone, androstenedione and INSL3. Together, this study suggests that dysregulated WNT/ß-catenin signalling during human fetal gonad development severely impairs testicular development and function. Importantly, our study highlights the notion that sufficient inhibition of the opposite pathway during sex-specific gonadal differentiation is essential to ensure normal development and function also applies to human fetal gonads.

Identification of a window of androgen sensitivity for somatic cell function in human fetal testis cultured ex vivo.

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.

The effects of selected inhibitors on human fetal adrenal steroidogenesis differs under basal and ACTH-stimulated conditions.

BACKGROUND: Disordered fetal adrenal steroidogenesis can cause marked clinical effects including virilization of female fetuses. In postnatal life, adrenal disorders can be life-threatening due to the risk of adrenal crisis and must be carefully managed. However, testing explicit adrenal steroidogenic inhibitory effects of therapeutic drugs is challenging due to species-specific characteristics, and particularly the impact of adrenocorticotropic hormone (ACTH) stimulation on drugs targeting steroidogenesis has not previously been examined in human adrenal tissue. Therefore, this study aimed to examine the effects of selected steroidogenic inhibitors on human fetal adrenal (HFA) steroid hormone production under basal and ACTH-stimulated conditions. METHODS: This study used an established HFA ex vivo culture model to examine treatment effects in 78 adrenals from 50 human fetuses (gestational weeks 8-12). Inhibitors were selected to affect enzymes critical for different steps in classic adrenal steroidogenic pathways, including CYP17A1 (Abiraterone acetate), CYP11B1/2 (Osilodrostat), and a suggested CYP21A2 inhibitor (Efavirenz). Treatment effects were examined under basal and ACTH-stimulated conditions in tissue from the same fetus and determined by quantifying the secretion of adrenal steroids in the culture media using liquid chromatography-tandem mass spectrometry. Statistical analysis was performed on ln-transformed data using one-way ANOVA for repeated measures followed by Tukey's multiple comparisons test. RESULTS: Treatment with Abiraterone acetate and Osilodrostat resulted in potent inhibition of CYP17A1 and CYP11B1/2, respectively, while treatment with Efavirenz reduced testosterone secretion under basal conditions. ACTH-stimulation affected the inhibitory effects of all investigated drugs. Thus, treatment effects of Abiraterone acetate were more pronounced under stimulated conditions, while Efavirenz treatment caused a non-specific inhibition on steroidogenesis. ACTH-stimulation prevented the Osilodrostat-mediated CYP11B1 inhibition observed under basal conditions. CONCLUSIONS: Our results show that the effects of steroidogenic inhibitors differ under basal and ACTH-stimulated conditions in the HFA ex vivo culture model. This could suggest that in vivo effects of therapeutic drugs targeting steroidogenesis may vary in conditions where patients have suppressed or high ACTH levels, respectively. This study further demonstrates that ex vivo cultured HFAs can be used to evaluate steroidogenic inhibitors and thereby provide novel information about the local effects of existing and emerging drugs that targets steroidogenesis.

Dexamethasone affects human fetal adrenal steroidogenesis and subsequent ACTH response in an ex vivo culture model.

Introduction: Administration of dexamethasone (DEX) has been used experimentally to suppress androgenization of external genitalia in 46,XX fetuses with congenital adrenal hyperplasia. Despite this, the prenatal biological mechanism-of-action of DEX on fetal development is not known. This study aimed to examine direct effects of DEX on human fetal adrenal (HFA) steroidogenic activity including possible effects on the subsequent response to ACTH-stimulation. Methods: Human fetal adrenal (HFA) tissue from 30 fetuses (1st trimester) were cultured ex vivo with A) DEX (10 µm) for 14 days, or B) DEX (10 µm) for 10 days followed by ACTH (1 nM) for 4 days. DEX-mediated effects on HFA morphology, viability, and apoptosis (immunohistochemistry), gene expression (quantitative PCR), and steroid hormone secretion (LC-MS/MS) were investigated. Results: DEX-treatment caused decreased androstenedione (p<0.05) and increased cortisol (p<0.01) secretion suggesting that direct effects on the adrenal gland may contribute to the negative feedback on the hypothalamic-pituitary-adrenal axis in vivo. An altered response to ACTH stimulation in HFA pre-treated with DEX included increased androgen (p<0.05) and reduced cortisol production (p<0.05), supporting clinical observations of a temporary decreased ACTH-response following prenatal DEX-treatment. Additionally, the secretion of corticosterone was decreased (p<0.0001) following ACTH-stimulation in the initially DEX-treated HFAs. Discussion: The observed effects suggest that prenatal DEX-treatment can cause direct effects on HFA steroidogenesis and in the subsequent response to ACTH-stimulation. This may indicate a requirement for careful monitoring of adrenal function in prenatally DEX-treated neonates, with particular focus on their mineralocorticoid levels.

Establishment of a Novel Human Fetal Adrenal Culture Model that Supports de Novo and Manipulated Steroidogenesis.

CONTEXT: Disorders affecting adrenal steroidogenesis promote an imbalance in the normally tightly controlled secretion of mineralocorticoids, glucocorticoids, and androgens. This may lead to differences/disorders of sex development in the fetus, as seen in virilized girls with congenital adrenal hyperplasia (CAH). Despite the important endocrine function of human fetal adrenals, neither normal nor dysregulated adrenal steroidogenesis is understood in detail. OBJECTIVE: Due to significant differences in adrenal steroidogenesis between human and model species (except higher primates), we aimed to establish a human fetal adrenal model that enables examination of both de novo and manipulated adrenal steroidogenesis. DESIGN AND SETTING: Human adrenal tissue from 54 1st trimester fetuses were cultured ex vivo as intact tissue fragments for 7 or 14 days. MAIN OUTCOME MEASURES: Model validation included examination of postculture tissue morphology, viability, apoptosis, and quantification of steroid hormones secreted to the culture media measured by liquid chromatography-tandem mass spectrometry. RESULTS: The culture approach maintained cell viability, preserved cell populations of all fetal adrenal zones, and recapitulated de novo adrenal steroidogenesis based on continued secretion of steroidogenic intermediates, glucocorticoids, and androgens. Adrenocorticotropic hormone and ketoconazole treatment of ex vivo cultured human fetal adrenal tissue resulted in the stimulation of steroidogenesis and inhibition of androgen secretion, respectively, demonstrating a treatment-specific response. CONCLUSIONS: Together, these data indicate that ex vivo culture of human fetal adrenal tissue constitutes a novel approach to investigate local effects of pharmaceutical exposures or emerging therapeutic options targeting imbalanced steroidogenesis in adrenal disorders, including CAH.

Characterization of Human Adrenal Steroidogenesis During Fetal Development.

CONTEXT: The endocrine function of human fetal adrenals (HFAs) is activated already during first trimester, but adrenal steroidogenesis during fetal life is not well characterized. OBJECTIVE: This study aimed to investigate HFA steroidogenesis by analyzing adrenal glands from first and second trimesters. DESIGN AND SETTING: Male and female HFA from gestational weeks (GWs) 8 to 19 were examined, including a total of 101 samples from 83 fetuses. MAIN OUTCOME MEASURE(S): Expression level of steroidogenic genes and protein expression/localization were determined by quantitative PCR and immunohistochemistry, respectively, and intra-adrenal steroid levels were quantified by LC-MS/MS. RESULTS: Transcriptional levels of StAR, CYP11A1, CYP17A1, CYP21A2, CYP11B1/2, and SULT2A1 were significantly higher in second trimester compared to first trimester (P < 0.05), whereas expression levels of 3ß-HSD2 and ARK1C3 were unaltered between GWs 8 and 19. All investigated steroidogenic proteins were expressed in a distinct pattern throughout the investigated period, with most enzymes expressed primarily in the fetal zone, except 3ß-HSD1/2, which was expressed mainly in the definitive zone. Abundant steroidogenic enzyme expression was reflected in overall high intra-adrenal tissue concentrations of mineralocorticoids, glucocorticoids, and androgens; cortisol was the most abundant (1071 to 2723 ng/g tissue), and testosterone levels were the lowest (2 to 14 ng/g tissue). CONCLUSIONS: The expression profiles of HFA steroidogenic enzymes are distinct from first to second trimester, with no major differences between male and female samples. Intra-adrenal steroid hormone concentrations confirm that cortisol is produced throughout first and second trimesters, suggesting continued regulation of the hypothalamus-pituitary-adrenal axis during this entire period.

Involvement of the DNA mismatch repair system in cisplatin sensitivity of testicular germ cell tumours.

BACKGROUND: Testicular germ cell tumours (TGCT) are highly sensitive to cisplatin-based chemotherapy, but patients with tumours containing differentiated teratoma components are less responsive to this treatment. The cisplatin sensitivity in TGCT has previously been linked to the embryonic phenotype in the majority of tumours, although the underlying mechanism largely remains to be elucidated. The aim of this study was to investigate the role of the DNA mismatch repair (MMR) system in the cisplatin sensitivity of TGCT. METHODS: The expression pattern of key MMR proteins, including MSH2, MSH6, MLH1 and PMS2, were investigated during testis development and in the pathogenesis of TGCT, including germ cell neoplasia in situ (GCNIS). The TGCT-derived cell line NTera2 was differentiated using retinoic acid (10 µM, 6 days) after which MMR protein expression and activity, as well as cisplatin sensitivity, were investigated in both undifferentiated and differentiated cells. Finally, the expression of MSH2 was knocked down by siRNA in NTera2 cells after which the effect on cisplatin sensitivity was examined. RESULTS: MMR proteins were expressed in proliferating cells in the testes, while in malignant germ cells MMR protein expression was found to coincide with the expression of the pluripotency factor OCT4, with no or low expression in the more differentiated yolk sac tumours, choriocarcinomas and teratomas. In differentiated NTera2 cells we found a significantly (p < 0.05) lower expression of the MMR and pluripotency factors, as well as a reduced MMR activity and cisplatin sensitivity, compared to undifferentiated NTera2 cells. Also, we found that partial knockdown of MSH2 expression in undifferentiated NTera2 cells resulted in a significantly (p < 0.001) reduced cisplatin sensitivity. CONCLUSION: This study reports, for the first time, expression of the MMR system in fetal gonocytes, from which GCNIS cells are derived. Our findings in primary TGCT specimens and TGCT-derived cells suggest that a reduced sensitivity to cisplatin in differentiated TGCT components could result from a reduced expression of MMR proteins, in particular MSH2 and MLH1, which are involved in the recognition of cisplatin adducts and in activation of the DNA damage response pathway to initiate apoptosis.