Transgenerational effect of the endocrine disruptor vinclozolin on male spermatogenesis

The current study was designed to examine the actions of a model endocrine disruptor on embryonic testis development and male fertility. Pregnant rats (F0) that received a transient embryonic exposure to an environmental endocrine disruptor, vinclozolin, had male offspring (F1) with reduced sper-matogenic capacity. The reduced spermatogenetic capacity observed in the F1 male offspring was transmitted to the subsequent generations (F2–F4). The administration of vinclozolin, an androgen receptor antagonist, at 100 mg/kg/day from embryonic day 8–14 (E8–E14) of pregnancy to only the F0 dam resulted in a transgenera-tional phenotype in the subsequent male offspring in the F1–F4 generations. The litter size and male/female sex ratios were similar in controls and the vinclozolin generations. The average testes/body weight index of the postnatal day 60 (P60) males was not significantly different in the vinclozolin-treated generations compared to the controls. However, the testicular spermatid number, as well as the epididymal sperm number and motility, were significantly reduced in the vinclozolin generations compared to the control animals. Postnatal day 20 (P20) testis from the vinclozolin F2 generation had no morphological abnormalities, but did have an increase in spermatogenic cell apoptosis. Although the P60 testis morphology was predominantly normal, the germ cell apoptosis was significantly increased in the testes cross sections of animals from the vinclozolin generations. The increase in apoptosis was stage-specific in the testis, with tubules at stages IX–XIV having the highest increase in apoptotic germ cells. The tubules at stages I–V also had an increase in apoptotic germ cells compared to the control samples, but tubules at stages VI–VIII had no increase in apoptotic germ cells. An outcross of a vinclozolin generation male with a wild-type female demonstrated that the reduced spermatogenic cell phenotype was transmitted through the male germ line. An outcross with a vinclozolin generation female with a wild-type male had no phenotype. A similar phenotype was observed in outbred Sprague Dawley and inbred Fisher rat strains. Observations demonstrate that a transient exposure at the time of male sex determination to the antiandrogenic endocrine disruptor vinclozolin can induce an apparent epigenetic transgenerational phenotype with reduced spermatogenic capacity

Chemotactic Role of Neurotropin 3 in the Embryonic Testis That Facilitates Male Sex Determination

The first morphological event after initiation of male sex determination is seminiferous cord formation in the embryonic testis. Cord formation requires migration of pre-peritubular myoid cells from the adjacent mesonephros. The embryonic Sertoli cells are the first testicular cells to differentiate and have been shown to express neurotropin-3 (NT3), which can act on highaffinity trkC receptors expressed on migrating mesonephros cells. NT3 expression is elevated in the embryonic testis during the time of seminiferous cord formation. A trkC receptor tyrophostin inhibitor, AG879, was found to inhibit seminiferous cord formation and mesonephros cell migration. Beads containing NT3 were found to directly promote mesonephros cell migration into the gonad. Beads containing other growth factors such as epidermal growth factor (EGF) did not influence cell migration. At male sex determination the SRY gene promotes testis development and the expression of downstream sex differentiation genes such as SOX-9. Inhibition of NT3 actions caused a reduction in the expression of SOX-9. Combined observations suggest that when male sex determination is initiated, the developing Sertoli cells express NT3 as a chemotactic agent for migrating mesonephros cells, which are essential to promote embryonic testis cord formation and influence downstream male sex differentiation

Epigenetic Transgenerational Actions of Endocrine Disruptors and Male Fertility

Transgenerational effects of environmental toxins require either a chromosomal or epigenetic alteration in the germ line. Transient exposure of a gestating female rat during the period of gonadal sex determination to the endocrine disruptors vinclozolin (an antiandrogenic compound) or methoxychlor (an estrogenic compound) induced an adult phenotype in the F1 generation of decreased spermatogenic capacity (cell number and viability) and increased incidence of male infertility. These effects were transferred through the male germ line to nearly all males of all subsequent generations examined (that is, F1 to F4). The effects on reproduction correlate with altered DNA methylation patterns in the germ line. The ability of an environmental factor (for example, endocrine disruptor) to reprogram the germ line and to promote a transgenerational disease state has significant implications for evolutionary biology and disease etiology

Inhibition of platelet-derived growth factor actions in the embryonic testis influences normal cord development and morphology

Platelet-derived growth factors (PDGFs) are paracrine factors with roles in mesenchymal-epithelial interactions during normal and pathologic processes. Previously, PDGF and its receptor (PDGFR) have been shown to be present in perinatal, peripub-ertal, and adult rat testes. The role of PDGF in embryonic tes-ticular cord formation is not known. The hypothesis tested is that PDGFs and PDGFRs are expressed during cord formation and that inhibition of their action influences normal cord formation during embryonic testis development. Embryonic Day (E) 13 go-nadal organ cultures were used. Organs were cultured for 3 days and treated daily with vehicle or a PDGFR-specific tyrosine phosphorylation inhibitor (i.e., the tyrphostin AG1295 or AG1296). Vehicle-treated testes formed normal cords, whereas tyrphostin-treated testes formed ‘‘swollen cords,’ ’ a phenome

Chemotactic role of neurotropin 3 in the embryonic testis that facilitates male sex determination

The first morphological event after initiation of male sex determination is seminiferous cord formation in the embryonic testis. Cord formation requires migration of pre-peritubular myoid cells from the adjacent mesonephros. The embryonic Sertoli cells are the first testicular cells to differentiate and have been shown to express neurotropin-3 (NT3), which can act on high-affinity trkC receptors expressed on migrating mesonephros cells. NT3 expression is elevated in the embryonic testis during the time of seminiferous cord formation. A trkC receptor tyrophostin inhibitor, AG879, was found to inhibit seminiferous cord formation and mesonephros cell migration. Beads containing NT3 were found to directly promote mesonephros cell migration into the gonad. Beads containing other growth factors such as epidermal growth factor (EGF) did not influence cell migration. At male sex determination the SRY gene promotes testis development and the expression of downstream sex differentiation genes such as SOX-9. Inhibition of NT3 actions caused a reduction in the expression of SOX-9. Combined observations suggest that when male sex determination is initiated, the developing Sertoli cells express NT3 as a chemotactic agent for migrating mesonephros cells, which are essential to promote embryonic testis cord formation and influence downstream male sex differentiation

Regulation of the gonadal transcriptome during sex determination and testis morphogenesis: comparative candidate genes

Gene expression profiles during sex determination and gonadal differentiation were investigated to identify new potential regulatory factors. Embryonic day 13 (E13), E14, and E16 rat testes and ovaries were used for microarray analysis, as well as E13 testis organ cultures that undergo testis morphogenesis and develop seminiferous cords in vitro. A list of 109 genes resulted from a selective analysis for genes present in male gonadal development and with a 1.5-fold change in expression between E13 and E16. Characterization of these 109 genes potentially important for testis development revealed that cytoskeletal-associated proteins, extracellular matrix factors, and signaling factors were highly represented. Throughout the developmental period (E13-E16), sex-enriched transcripts were more prevalent in the male with 34 of the 109 genes having testis-enriched expression during sex determination. In ovaries, the total number of transcripts with a 1.5-fold change in expression between E13 and E16 was similar to the testis, but none of those genes were both ovary enriched and regulated during the developmental period. Genes conserved in sex determination were identified by comparing changing transcripts in the rat analysis herein, to transcripts altered in previously published mouse studies of gonadal sex determination. A comparison of changing mouse and rat transcripts identified 43 genes with species conservation in sex determination and testis development. Profiles of gene expression during E13-E16 rat testis and ovary development are presented and candidate genes for involvement in sex determination and testis differentiation are identified. Analysis of cellular pathways did not reveal any specific pathways involving multiple candidate genes. However, the genes and gene network identified influence numerous cellular processes with cellular differentiation, proliferation, focal contact, RNA localization, and development being predominant

Embryonic testis cord formation and mesonephric cell migration requires the phosphotidylinositol 3-kinase signaling pathway

Mesonephric cell migration and seminiferous cord formation are critical processes in embryonic testis development at the time of male sex determination. Extracellular growth factors shown to influence seminiferous cord formation such as neurotropin-3 utilize in part the phosphotidylinositol 3-kinase (PI3K) signal transduction pathway. The current study investigates the hypothesis that the PI3K pathway is critical in seminiferous cord formation and testis development. The role of the PI3K signaling pathway in testicular cord formation was examined using an Embryonic Day 13 organ culture system and a PI3K-specific inhibitor LY294002. The actions of a mitogen-activated protein (MAP) kinase-specific inhibitor PD98059 was also examined. The PI3K inhibitor blocked cord formation or reduced the number of cords in a concentration-dependent manner. The actions of LY294002 were found to have a developmental stage specificity in that cord inhibition was observed in organs from embryos with 16-17 tail somites, while organs from embryos with 19 or more tail somites had no block in cord formation and only a small reduction in cord number. In contrast, the MAP kinase inhibitor PD98059 did not block cord formation and only caused a slight reduction in cord number. Neither PI3K or MAP kinase inhibitor altered apoptotic cell number, suggesting apoptosis was not the reason for the inhibition of cord formation. Embryonic testis cell migration assays showed that the PI3K inhibitor LY294002 blocked mesonephros cell migration into the testis, while the MAP kinase inhibitor had no effect. Observations suggest the interference of cell migration is the cause for the inhibition of cord formation. Western blot analysis confirmed that LY294002 and PD98509 inhibited phosphorylation of Akt and ERK1/ERK2, respectively. Combined observations demonstrate that the PI3K signaling pathway is involved in embryonic testis cord formation and mesonephros cell migration

Effect of Transient Embryonic In Vivo Exposure to the Endocrine Disruptor Methoxychlor on Embryonic and Postnatal Testis Development

The current study was designed to examine the effects of a transient embryonic exposure to the pesticide methoxychlor, an endocrine disruptor, on in vivo rat testis development and function. Gestating female rats were transiently administered methoxychlor (MXC) from embryonic day 7 (E7; EO = plug date) through E15. Embryonic testes were collected at E16 and postnatal (PO = day of birth) testes at P4, P10, P17–20, and P60. Seminiferous cords formed in testes from MXC exposed males. However, at E16, there was a decrease in the area of cords and an increase in interstitial area in MXC exposed testes when compared with controls. At all postnatal ages collected, there did not appear to be differences in seminiferous cord/tubule area, interstitial area, or number of seminiferous cords/tubules between untreated controls and males exposed to MXC. Exposure to the endocrine disruptor also had no effect on the postnatal organ weights of a variety of different organs, nor were testosterone levels altered. Interestingly, there were reductions in the number of germ cells in testes from MXC-exposed males at P17–P20 when compared with untreated controls. Furthermore, there was a twofold increase in apoptotic cells in tubules from pubertal P17–P20–MXC exposed males when compared with untreated controls. Testes were collected from adult P60 males to determine if early embryonic and postnatal alterations in germ cell numbers or testis cellular composition had compromised spermatogenesis. In adult P60 MXC exposed testes there were no gross morphological changes in testis structure or cellular composition over that of controls. However, there was an increase in apoptotic cell number in elongating spermatids in MXC exposed testes. Four P60 males that were exposed to MXC during gestation and 4 control males were bred with unexposed females to determine their ability to produce offspring. All MXC exposed males were capable of impregnating females and had normal litter size and pup weights. Combined observations demonstrated that exposure to MXC during gestation at a critical stage of testis development (ie, sex determination) affects embryonic testis cellular composition, germ cell numbers, and germ cell survival. While alterations in these parameters does not affect the ability of males to produce offspring, there appears to be a reduced spermatogenic capacity associated with MXC treatment. Therefore, transient embryonic exposure to an endocrine disruptor (methoxychlor) during gestation can influence the germline and fertility in adult males