Leydig cells are present after 30 days of organotypic culture but are partially mature.

(A) Representative images of 3β-HSD expression by Leydig cells (LC) during mouse postnatal development (6.5 dpp, 22.5 dpp and 36.5 dpp) and in in vitro cultured tissues after 16 days of culture (D16) or 30 days (D30). Testicular tissue sections were counterstained with Hoechst (blue). Dotted lines delineate seminiferous tubules. Scale: 15 μm. (B) Number of 3β-HSD+ LC per cm2 of testicular tissue during mouse postnatal development (6.5 dpp, 22.5 dpp and 36.5 dpp) and in in vitro cultured tissues (D16 and D30). (C) Percentage of LC in apoptosis (3β-HSD and cleaved caspase 3 positive) or in proliferation (3β-HSD and Ki67 positive) in in vivo and in vitro matured tissues. (D) Percentage of 3β-HSD positive LC expressing AR in in vivo and in vitro matured tissues. (E) Relative mRNA levels of differentiation factors for LC (Igf1, Dhh), ILC (Srd5a1) and ALC markers (Sult1e1, Insl3) (normalized to Gapdh and Actb or to Hsd3b1). (F) Intratesticular concentration of INSL3 (pg/mg of tissue) or in the culture medium (pg/mL).

Data are presented as means ± SEM with n = 4 biological replicates for each group. A value of *P < 0.05 was considered statistically significant. n.d.: not determined (under the detection limit)

FT: Fresh Tissue; CSF: Controlled Slow Freezing.

The expression of several actors of steroidogenesis is downregulated in 30-day organotypic cultures.

(A) Relative mRNA levels of Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3 and Hsd17b2 (normalized to Gapdh and Actb or to Hsd3b1) and relative protein levels of 3β-HSD and CYP17A1 (normalized to ACTB) during mouse postnatal development (6.5 dpp, 22.5 dpp and 36.5 dpp) and in in vitro cultured FT or CSF tissues (D16 and D30). (B) Representative images of CYP17A1 expression during mouse postnatal development (22.5 dpp and 36.5 dpp) and in in vitro cultured tissues (D16 and D30). Testicular tissue sections were counterstained with Hoechst (blue). Dotted lines delineate seminiferous tubules. Scale: 15 μm.

Data are presented as means ± SEM with n = 4 biological replicates for each group. A value of *P < 0.05 was considered statistically significant.

An increased production of progesterone and estradiol and a decreased production of androstenedione are observed after 16 and 30 days of culture of prepubertal mouse testicular tissues.

Intratesticular concentrations of (A) progesterone, (B) androstenedione, (C) testosterone and (D) estradiol during mouse postnatal development (6.5 dpp, 22.5 dpp and 36.5 dpp) and in in vitro cultured fresh (FT) or frozen/thawed (CSF) tissues (D16 and D30). Steroid concentrations were normalized to protein levels. (E) Concentrations of progesterone, androstenedione, testosterone and estradiol in the culture medium of FT tissues. (F) Intratesticular concentrations of total, free and esterified cholesterol normalized to tissue mass.

Data are presented as means ± SEM with n = 4 biological replicates for each group. A value of *P < 0.05 was considered statistically significant.

Androgen signaling is altered in 30-day organotypic cultures.

(A) Relative mRNA levels of Ar (normalized to Gapdh and Actb) and relative protein levels of AR (normalized to ACTB) during mouse postnatal development (6.5 dpp, 22.5 dpp and 36.5 dpp) and in in vitro cultured FT or CSF tissues (D16 and D30). (B) Representative images of AR expression at 22.5 dpp and 36.5 dpp and at corresponding in vitro time points (D16 and D30). Testicular tissue sections were counterstained with Hoechst (blue). Solid arrowheads: peritubular myoid cells. Open arrowheads: Sertoli cells. Arrows: Leydig cells. Dotted lines delineate seminiferous tubules (ST). Scale: 15 μm. (C) Relative mRNA levels of Rhox5, Septin12, Eppin and Abp (normalized to Gapdh and Actb).

Data are presented as means ± SEM with n = 4 biological replicates for each group. A value of *P < 0.05 was considered statistically significant.

The expression of aromatase and estrogen signaling are impaired after 30 days of organotypic culture.

(A) Relative mRNA levels of Cyp19a1 (normalized to Gapdh and Actb) and relative protein levels of CYP19A1 (normalized to ACTB) during mouse postnatal development (6.5 dpp, 22.5 dpp and 36.5 dpp) and in in vitro cultured FT or CSF tissues (D16 and D30). The bands on the Western Blot correspond to different isoforms of CYP19. (B) Representative images of CYP19A1 expression at 6.5 dpp, 22.5 dpp and 36.5 dpp and at corresponding in vitro time points (D16 and D30). Testicular tissue sections were counterstained with Hematoxylin. Scale: 15 μm. Arrow: Leydig cells. Arrowheads: elongated spermatids. (C) Aromatase activity (normalized to tissue weight). (D) Relative mRNA levels of Esr1, Esr2 and Gper1 (normalized to Gapdh and Actb). (E) Relative mRNA levels of Faah (normalized to Gapdh and Actb) and relative protein levels of FAAH (normalized to ACTB). The second band at 80 kDa is an isoform of FAAH (Q8BRM1, UniProtKB).

Data are presented as means ± SEM with n = 4 biological replicates for each group. A value of *P < 0.05 was considered statistically significant.

Leydig cells are functional as they respond to stimulation by 1 nM hCG.

(A) Levels of androgens (androstenedione and testosterone) in testicular tissues and in culture medium with or without hCG supplementation. (B) Relative mRNA levels of genes involved in steroidogenesis and androgen/estrogen signaling pathways (normalized to Gapdh and Actb or to Hsd3b1) with or without hCG supplementation. (C) Proportion of seminiferous tubules containing the most advanced type of germ cells after in vitro culture with or without hCG. P < 0.05: * vs 36.5 dpp and # vs D30 FT. (D) Sertoli cell number in seminiferous tubules after in vitro culture with or without hCG.

Data are presented as means ± SEM with n = 4 biological replicates for each group. A value of *P < 0.05 was considered statistically significant. FT: Fresh Tissue; CSF: Controlled Slow Freezing.

Altered steroidogenesis and androgen/estrogen signaling pathways in in vitro matured testicular tissues of prepubertal mice

A similar density of Leydig cells (LC) is found after 30 days of organotypic culture (D30) and at 36.5 days postpartum, the corresponding in vivo time point. However, LC are partially mature in vitro with a decrease in Sult1e1 and Insl3 mRNA levels (adult LC markers). The mRNA levels of Cyp11a1, Cyp17a1 and Hsd17b3 encoding steroidogenic enzymes and the protein levels of CYP17A1 are decreased in vitro. Increased amounts of cholesterol, progesterone and estradiol and decreased androstenedione intratesticular levels are observed at D30. Furthermore, despite testosterone levels similar to in vivo, the expression of the androgen receptor (AR) and of the androgen binding protein (Abp), androgen signaling is altered at D30, with decreased transcript levels of androgen target genes (Rhox5, Septin12). Moreover, with severely decreased expression and activity of aromatase and decreased estrogen receptor expression, estrogen signaling is impaired at D30, leading to decreased transcript and protein levels of the estrogen target gene Faah.