An in vitro prototype of a porcine biomimetic testis-like cell culture system: a novel tool for the study of reassembled Sertoli and Leydig cells

Abstract

At present, there is no reliable in vitro assembled prepubertal testis-like biomimetic organ culture system designed to assess the functional effects of human gonadotropins on Sertoli and Leydig cells. Spermatogenesis is regulated by endocrine, paracrine, and juxtacrine factors (testicular cross-talk), mainly orchestrated by gonadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that play a pivotal role by stimulating Leydig and Sertoli cells, respectively. The aim of our study was to set up an in vitro prepubertal porcine bioengineered construct as a new model for experimental studies on reassembled Sertoli and Leydig cells. We have evaluated Sertoli and Leydig cells obtained from 15- to 20-day-old neonatal pig testes in terms of purity and function. Subsequently, purified Sertoli and enriched Leydig cells were subjected to coincubation to obtain an in vitro prepubertal porcine testis-like culture system. We performed enzyme-linked immunosorbent assay (ELISA) for anti-Müllerian hormone (AMH), inhibin B, and testosterone secretion in the medium, and Real-Time PCR analysis of AMH, inhibin B, FSH-r, aromatase, LHr, and 3β-HSD mRNA expression levels. This in vitro testis-like system was highly responsive to the effects of human gonadotropins and testosterone. AMH mRNA expression and secretion declined, and inhibin-B increased, while FSH-receptor expression was downregulated upon FSH/LH exposure/treatment. Finally, the production of testosterone was increased selectively upon LH treatment. In summary, our proposed model could help to better determine the action of human gonadotropins on Sertoli and Leydig cells. The potential usefulness of the system for shedding light into male infertility-related issues is evident.

Keywords: Leydig cells; Sertoli cells; human gonadotropins; prepubertal biomimetic testis.

Figure 1

Testosterone secretion and SC functional competence. (a) Testosterone levels were measured in culture medium of untreated and treated SC. Functional activity of cultured SC was evaluated by measuring (b) AMH and (c) inhibin B secretion in culture medium of untreated and treated SC. Data represent the mean of three experiments ± s.d. **P < 0.001, treated SC versus untreated SC; ns: not significant, treated SC versus untreated SC. SC: Sertoli cells; s.d.: standard deviation; AMH: anti-Müllerian hormone.

Figure 2

Morphological characterization and flow cytometry analysis. (a) Morphological characterization by fluorescence microscopy: LC after immunostaining with AMH antibody and visualized by anti-goat Alexa Fluor 488 (green). (b) LC after immunostaining with 3β-HSD antibody and visualized by anti-goat Alexa Fluor 488 (green). (c) LC after immunostaining with ASMA antibody and visualized by anti-rabbit Alexa Fluor 488 (green). Nuclei are counterstained with DAPI (blue). In each figure, the magnification insert is showed in the upper right and the negative controls' insert is displayed in the bottom left. Flow cytometry analysis of cultured LC for (d) AMH, (e) 3β-HSD, and (f) ASMA. The images are representative of three separate experiments. LC: Leydig cells; AMH: anti-Müllerian hormone; ASMA: alpha-smooth muscle actin; 3β-HSD: 3β-hydroxysteroid dehydrogenase.

Figure 3

Function of cultured LC. Testosterone secretion measured in the culture medium of untreated and treated LC. Data represent the mean of three experiments ± s.d. **P < 0.001, treated LC versus untreated LC. s.d.: standard deviation; LC: Leydig cells.

Figure 4

Functional competence of in vitro prepubertal porcine system. (a) Inhibin B and (b) AMH secretion upon 72 h of FSH and LH treatment. Data represent the mean of three experiments ± s.d. **P < 0.001, treated SC + LC versus untreated SC + LC; #P < 0.05, SC + LC + FSH + LH versus SC + LC + FSH; ns: not significant, SC + LC + LH versus SC + LC. AMH: anti-Müllerian hormone; FSH: follicle-stimulating hormone; s.d.: standard deviation; LH: luteinizing hormone; SC: Sertoli cells; LC: Leydig cells.

Figure 5

Real-time PCR analysis of (a) inhibin B, (b) AMH, (c) FSH-r, (d) aromatase, (e) 3β-HSD, and (f) LHr gene expression upon 72 h of FSH and LH treatment. Data represent the mean of three experiments ± s.d. **P < 0.001, treated SC + LC versus untreated SC + LC; ns: not significant, treated SC + LC versus untreated SC + LC. PCR: polymerase chain reaction; AMH: anti-Müllerian hormone; FSH: follicle-stimulating hormone; s.d.: standard deviation; LH: luteinizing hormone; SC: Sertoli cells; LC: Leydig cells; 3β-HSD: 3β-hydroxysteroid dehydrogenase.

Figure 6

Testosterone secretion. Production of testosterone upon 72 h of LH treatment. Data represent the mean of three experiments ± s.d. **P < 0.001, SC + LC + LH versus SC + LC. s.d.: standard deviation; LH: luteinizing hormone; SC: Sertoli cells; LC: Leydig cells.