Abstract
The significant role that estrogens play in spermatogenesis has opened up an exciting area of research in male reproductive biology. The realization that estrogens are essential for proper maintenance of spermatogenesis, as well as growing evidence pointing to the deleterious effects of estrogen-like chemicals on male reproductive health, has made it imperative to dissect the role estrogens play in the male. Using a model estrogen, diethylstilbestrol (DES), to induce spermatogenic cell apoptosis in vivo in the male rat, we provide a new insight into an estrogen-dependent regulation of the Fas-FasL system specifically in spermatogenic cells. We show a distinct increase in Fas-FasL expression in spermatogenic cells upon exposure to diethylstilbestrol. This increase is confined to the spermatid population, which correlates with increased apoptosis seen in the haploid cells. Testosterone supplementation is able to prevent DES-induced Fas-FasL up-regulation and apoptosis in the spermatogenic cells. DES-induced germ cell apoptosis does not occur in Fas-deficient lpr mice. One other important finding is that spermatogenic cells are type II cells, as the increase in Fas-FasL expression in the spermatogenic cells is followed by the cleavage of caspase-8 to its active form, following which Bax translocates to the mitochondria and precipitates the release of cytochrome c that is accompanied by a drop in mitochondrial potential. Subsequent to this, activation of caspase-9 occurs that in turn activates caspase-3 leading to the cleavage of poly(ADP-ribose) polymerase. Taken together, the data indicate that estrogen-like chemicals can precipitate apoptotic death in spermatogenic cells by increasing the expression of spermatogenic cell Fas-FasL, thus initiating apoptosis in the same lineage of cells through the activation of the apoptotic pathway chosen by type II cells.
Highlights
The study of the role of estrogens in spermatogenesis has attracted significant interest because of the increasing awareness that estrogen is essential for spermatogenesis [1, 2], spermatogenic cells express estrogen receptors (ER)1 [1,2,3], and Regular apoptosis of spermatogenic cells is required to maintain proper testicular homeostasis, increased cell death can result in defective spermatogenesis leading to infertility [11]
One other important finding is that spermatogenic cells are type II cells, as the increase in Fas-FasL expression in the spermatogenic cells is followed by the cleavage of caspase-8 to its active form, following which Bax translocates to the mitochondria and precipitates the release of cytochrome c that is accompanied by a drop in mitochondrial potential
The DES-induced spermatogenic cell apoptosis model was ideal to study the mechanism of estrogen-induced spermatogenic cell death, as DES can mimic estrogen action and has been widely used as a model estrogen to study the effects on the neonatal male rat reproductive tract [8, 9]
Summary
Materials—The Apoptosis Detection System was procured from Promega (Madison, WI). Bicinchoninic protein assay reagents A & B were purchased from Pierce. RNase treatment was given at 37 °C for 30 min and PI (50 g/ml) was added to the cells, which were analyzed in a flow cytometer as described above. Detection of DNA fragmentation by TUNEL staining was carried out as described previously [19, 23] using a TUNEL assay kit according to instructions from the manufacturer. Deparaffinized tissue sections were treated with equilibration buffer (200 mM potassium cacodylate, 25 mM Tris-HCl, pH 8.0, 0.2 mM DTT, 0.25 mg/ml BSA, 2.5 mM cobalt chloride) for 10 min at room temperature followed by incubation with TdT buffer containing nucleotide mix (50 M dUTP-biotin, 100 M dATP, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 7.6) for 1 h at 37 °C. For colocalization of Fas-FasL, isolated spermatogenic cells fixed in 2% paraformaldehyde were blocked for nonspecific sites with 10% normal
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