The necessity of estrogens for male fertility was recently discovered in studies on both estrogen receptor alpha knockout and aromatase (cyp 19 gene) knockout mice. However, direct testicular effects of estrogens in male reproduction have remained unclear. Here we studied the protein expression of ERalpha and the recently described estrogen receptor beta in the human seminiferous epithelium and evaluated the role of 17beta-estradiol, the main physiological estrogen, in male germ cell survival. Interestingly, both estrogen receptors alpha and beta were found in early meiotic spermatocytes and elongating spermatids of the human testis. Furthermore, low concentrations of 17beta-estradiol (10(-9) and 10(-10) mol/L) effectively inhibited male germ cell apoptosis, which was induced in vitro by incubating segments of human seminiferous tubules without survival factors (i.e. serum and hormones). Dihydrotestosterone, which, in addition to estradiol, is an end metabolite of testosterone, was also capable of inhibiting testicular apoptosis, but at a far higher concentration (10(-7) mol/L) than estradiol. Thus, estradiol appears to be a potent germ cell survival factor in the human testis. The novel findings of the present study together with the previously reported indirect effects of estrogens on male germ cells indicate the importance of estrogens for the normal function of the testis.

The Fas-Fas ligand (FasL) system has been implicated in maintaining the immune privileged nature of the testis. The present report concerns the role of the Fas-FasL system in regulating germ cell apoptosis, another important function of this system in the human testis. Fas was localized immunohistochemically to the same types of germ cells that were identified as apoptotic, namely spermatocytes and spermatids. Strong expression of Fas was also observed in Western blot analysis of the human testis. Furthermore, an antagonistic antibody to the FasL blocked germ cell apoptosis induced in vitro by incubating segments of seminiferous tubules under serum- and hormone-free conditions (i.e., without survival factors). Thus Fas appears to mediate germ cell apoptosis. A universal caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone, also inhibited germ cell death, suggesting that Fas-associated germ cell apoptosis is mediated via the caspase pathway. The present results suggest an important role for the Fas-FasL system in the regulation of germ cell apoptosis in the human testis.

Dysregulation of male germ cell apoptosis has been associated with the pathogenesis of male infertility. Therefore, factors involved in the regulation of germ cell death are being actively investigated. Here, we studied the effects of lactate on human male germ cell death, using as a model a testis tissue culture in which physiological contacts are maintained between the germ cells and the supportive somatic Sertoli cells. Apoptosis of spermatocytes, spermatids and a few spermatogonia was induced by culturing segments of seminiferous tubules under serum-free conditions. This germ cell death was inhibited effectively and dose-dependently by lactate, indicating that it plays a crucial role in controlling cell death cascades of male germ cells. Interestingly, the anti-apoptotic role of lactate was not associated with changes in testicular adenine nucleotide (ATP, ADP and AMP) levels. In the seminiferous tubules, the final site of the death-suppressing action of lactate appeared to be downstream along the cell death pathway activated by the Fas receptor of the germ cells. In conclusion, testicular cell death was effectively regulated by lactate, which may be regarded as a potential compound for optimizing in-vitro methods involving male germ cells for assisted reproduction.

Apoptotic cell death plays an important role in limiting testicular germ cell population during spermatogenesis and its dysregulation has been shown to be associated with male infertility. The growing evidence on the role of the transcription factor nuclear factor (NF)-kappa B in controlling apoptosis prompted us to investigate NF-kappa B activity in the normal human testis and its role in testis tissue undergoing excessive apoptosis in vitro. In electrophoretic mobility shift assays, low-level constitutive NF-kappa B DNA-binding activity was found and, by immunostaining of the RelA and p50 NF-kappa B subunits, was localized to Sertoli cell nuclei. During in vitro-induced testicular apoptosis, the Sertoli cell nuclear NF-kappa B levels and whole seminiferous tubule NF-kappa B DNA-binding activity increased previous detection of germ cells undergoing apoptosis. The anti-inflammatory drug sulfasalazine effectively suppressed stress-induced NF-kappa B DNA binding and NF-kappa B-mediated I kappa B alpha gene expression. Importantly, concomitantly with inhibiting NF-kappa B, sulfasalazine blocked germ cell apoptosis. These results suggest that during testicular stress Sertoli cell NF-kappa B proteins exert proapoptotic effects on germ cells, which raises the possibility that pharmacological inhibition of NF-kappa B could be a therapeutic target in transient stress situations involving excessive germ cell death.

The necessity of estrogens for male fertility was recently discovered in studies on both estrogen receptor ␣ knockout and aromatase (cyp 19 gene) knockout mice. However, direct testicular effects of estrogens in male reproduction have remained unclear. Here we studied the protein expression of ER␣ and the recently described estrogen receptor ␤ in the human seminiferous epithelium and evaluated the role of 17␤-estradiol, the main physiological estrogen, in male germ cell survival. Interestingly, both estrogen receptors ␣ and ␤ were found in early meiotic spermatocytes and elongating spermatids of the human testis. Furthermore, low concentrations of 17␤-estradiol (10 Ϫ9 and 10 Ϫ10 mol/L) effectively inhibited male germ cell apoptosis, which was induced in vitro by incubating segments of human seminiferous tubules without survival factors (i.e. serum and hormones). Dihydrotestosterone, which, in addition to estradiol, is an end metabolite of testosterone, was also capable of inhibiting testicular apoptosis, but at a far higher concentration (10 Ϫ7 mol/L) than estradiol. Thus, estradiol appears to be a potent germ cell survival factor in the human testis. The novel findings of the present study together with the previously reported indirect effects of estrogens on male germ cells indicate the importance of estrogens for the normal function of the testis. (J Clin Endocrinol Metab 85: 2057-2067, 2000 R ECENT CONCERN over the potentially harmful effects of environmental estrogen-like chemicals on male fertility (1, 2) has aroused growing interest in understanding the physiological effects of estrogens in the male. Although estrogens have been regarded as female steroid hormones, they are now known to have profound effects on both female and male reproductive systems. In males, estrogens are synthesized mainly in the testis, where they are formed from testosterone by the enzyme P450 aromatase. In the rat, there is an age-related change in the aromatization site from Sertoli cells in the immature testis to Leydig cells in the adult testis (3, 4). In various other species, including humans, P450 aromatase has been found to be present in the Leydig cells of the adult testis (5-9). In addition, in the mouse (6), rat (7), brown bear (9), and rooster (10), aromatase has been found in meiotic and postmeiotic germ cells of the testis, predominantly in spermatids.Estrogens exert their cellular effects through estrogen receptors (ER) that exist in at least two subtypes, ER␣ (11, 12) and the recently described ER␤ (13, 14), which differ in the C-terminal ligand-binding domain and in the N-terminal trans-activation domain. These two subtypes of ER have similar high affinities for 17␤-estradiol, but some synthetic and naturally occurring ligands have different relative affinities for ER␣ and ER␤ (15). In the male reproductive tract, ER␣ has been shown to be strongly expressed in the epididymis and efferent ductules (16 -20). It has also been found in the Leydig cells of the rat testis (16), whereas the seminiferous epitheliu...