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Evidence is mounting that the foetal and neonatal period of reproductive tract development is highly sensitive to hormonal disruption induced by various endocrine active compounds. Thus, we asked whether androgen withdrawal caused by prenatal (GD20, GD80) or neonatal (PD2) exposure to an anti-androgen flutamide alters Cx43 gene expression and may induce delayed effects on morphology and function of adult pig testes. Flutamide was given in five doses (50 mg/kg bw). Our histological analysis and TUNEL staining revealed varying degrees of seminiferous tubules abnormalities in all experimental pigs. Testes of pigs exposed to flutamide in utero exhibited moderate alterations of the spermatogenic process, whereas those of exposed neonatally were severely impaired. The most striking effects were spermatogenic arrest, germ cell detachment and a statistically significant increase in the frequency of germ cell apoptosis (p<0.01). Moreover, all pigs exposed to flutamide displayed Leydig cell hyperplasia. Because the network of cell-cell communication provided by gap junction channels plays an essential role in the regulation and maintenance of spermatogenesis, the physiological significance of Cx43-based gap junctions with regards to the gonadal impairment was evaluated by analysis of its expression using immunohistochemical, Western blot and qRT-PCR approaches. Significantly, lower Cx43 expression was found when flutamide was administered neonatally, which has coincided with severe disruption of spermatogenesis. Our data suggest that neonatal exposure to flutamide induces long-term effects on the spermatogenic capacity of the pig testis through alterations of Cx43-mediated intercellular communication and permanent alteration of both Sertoli and Leydig cell functions.

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This study was designed to reveal the FSHR mRNA and protein expression in the neonatal porcine ovary and to determine whether maternal administration of antiandrogen flutamide may affect FSHR expression in the ovary of newborn piglets using real-time PCR, immunohistochemistry and Western blot analysis. Pregnant sows were injected with flutamide at a dose of 50 mg/kg body weight, given five times, every second day, starting at day 20 post-coitum (p.c.) or day 80 p.c., and ovaries were obtained from neonatal pigs. The FSHR mRNA expression was significantly decreased after flutamide administration. Furthermore, higher down-regulation was observed following exposure to antiandrogen at day 20 than at day 80 p.c. Immunohistochemistry showed the positive immunostaining for FSHR in the oocytes, granulosa cells of primary follicles and the surface epithelium of the ovaries from both control and flutamide-treated pigs. However, oocytes and granulosa cells of primary follicles in the ovaries exposed in utero to flutamide were weakly immunostained when compared to those in the control ones. The presence of FSHR protein in all investigated ovaries was confirmed by Western blot analysis. Based on our findings, we suggest that FSHR may be involved in the early follicle formation in pigs, which begins during prenatal life. Furthermore, the regulation of FSHR mRNA and protein expression in neonatal porcine ovaries after maternal exposure to flutamide confirms that androgens play a crucial role in porcine folliculogenesis at the early stages.