Effects of docosahexaenoic acid individually or in combination with phthalate ester on mouse fetal testis

Abstract

Phthalates are endocrine active substances (EAS) widely used as plasticizer. Among the EAS, di-ethylhexyl phthalate (DEHP) is a very relevant endocrine disruptor (ED) for human exposure. After absorption DEHP is rapidly hydrolyzed in mono-2-ethylhexyl phthalate (MEHP), its active metabolite. Many studies confirmed the reprotoxic effects of MEHP exposure on males, such as perturbations in reproductive hormones, decreased anogenital distance and loss of germ cells, in fetus. Recent evidences indicate that phthalates interfering in signaling pathways of some nuclear receptors (NR) and deregulate lipid metabolism in human fetal testis. MEHP is ligand of peroxisome proliferator-activated receptor gamma (PPAR gamma) and decreased mRNA expression for this receptor was related to reduction in gonocyte number following in vitro exposure of human fetal testis to MEHP. In contrast to phthalates, the docasahexanoic acid (22:6 n-3), a long-chain polyunsaturated fatty acid (PUFA) abundantly found in oily fish sources, seems to have beneficial effects on the male reproductive system, especially in spermatogenesis and sperm motility. Even though PUFA supplementation has been recommended worldwide for pregnant woman, the consequences of in utero exposure to DHA on the development of the testes are still unknown. Studies in different tissues had shown that DHA may affect signaling of some NR, including PPAR³. Thus, the objective of this project is to examine the direct effects of DHA on mouse testis development, and also evaluate whether DHA acts on the adverse effects of MEHP exposure. Our analysis will be based on a powerful organ culture system, previously established by the research group with which this work will be done. Briefly, one testes of each embryo will be cultured for 3 days with 0.1% DMSO only (control), or 20µM of MEHP, or 50µM of DHA, or both (20µM of MEHP plus 50µM of DHA). Testes will be processed for light microscopy and the total number of gonocytes will be determined based on immunocytochemical tests for anti-Müllerian hormone (AMH). The kinetics of proliferation and death of these cells will be evaluated by measuring the BrdU (5-bromo-2-deoxyuridine) incorporation and by combining staining for AMH (a Sertoli-specific marker) and cleaved caspase-3, respectively. The testosterone plasma levels will also be considered. Transcriptional levels of PPAR³ and downstream genes will be evaluated by qPCR. Cell sorting analysis will be used to investigate the effects of MEHP and DHA in somatic cell populations and gonocytes. The analysis proposed here will provide new information on the physiological action of DHA on the fetal testis and the possible implications of maternal supplementation with this PUFA, as well as its interference on the effects of phthalates.