Cell death and its suppression in human ovarian tissue culture.

In women with premature ovarian failure, fertility may be preserved by ovarian tissue culture in vitro. However, techniques for tissue culture and follicle maturation have remained suboptimal. Our aim was to characterize ovarian tissue degeneration in cultures and to establish a model for cell death research in cultured ovarian tissue. Precise knowledge on the process resulting in cell death in cultured ovarian tissue will ultimately facilitate work aimed at improving long-term culture conditions. Ovarian tissue apoptosis was studied in a serum-free culture model in which nuclear DNA fragmentation was shown to occur within 24 h of the start of the culture. Activation of caspase-3 was detected in some stromal cells and a few oocytes. Since not all of the tissue exhibited signs of apoptosis and since DNA fragmentation increased over time, the tissue probably gradually dies by apoptosis. The antioxidant N-acetyl-L-cysteine (NAC; 25, 50 and 100 mmol/l) was found to inhibit this apoptosis. Thus, apoptosis appears to play a critical role in the degeneration of human ovarian cortical tissue cultures, and this cell death can be suppressed by NAC. The present tissue culture model can be used for identifying components capable of inhibiting cell death in vitro.

Lactate inhibits 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.

TNFalpha down-regulates the Fas ligand and inhibits germ cell apoptosis in the human testis.

The cytokine TNFalpha is known to be secreted by testicular germ cells. However, its effect on maturing germ cells is unknown, and its role in the regulation of spermatogenesis is unclear. Here we aimed at characterizing the effects of TNFalpha on germ cell survival in the human testis. We found that TNFalpha effectively and dose-dependently inhibited germ cell apoptosis, which was induced in vitro by incubating segments of human seminiferous tubules under serum-free culture conditions. EMSAs indicated increased activity of nuclear factor kappaB in seminiferous tubules cultured under apoptosis-inducing conditions. However, we did not observe any significant effect of TNFalpha on the activation of this transcription factor, which is often considered to be a mediator of TNFalpha-induced survival signals. As the expression of the TNF receptor protein in the human seminiferous epithelium was predominantly found in the Sertoli cells, the antiapoptotic effect of TNFalpha is probably mediated via these somatic cells. Interestingly, expression of the Fas ligand, a known inductor of testicular apoptosis, was down-regulated by TNFalpha. Thus, in the seminiferous tubules, germ cell-derived TNFalpha may regulate the level of the Fas ligand and thereby control physiological germ cell apoptosis.

TERVA: system for long-term monitoring of wellness at home.

Long-term monitoring of physiological and psychosocial variables in out-hospital conditions would be beneficial for investigating changes in wellness status of an individual or to understand interaction between physiological and behavioral processes. We aimed to design a personal wellness monitoring system (TERVA), which would allow monitoring of wellness-related variables at home for several weeks or even months. The designed TERVA system runs on a laptop computer and interfaces with different measurement devices through a serial interface. Measured variables include beat-to-beat heart rate, motor activity, blood pressure, weight, body temperature, respiration, ballistocardiography, movements, and sleep stages. In addition, self-assessments of daily well-being and activities are stored by keeping a behavioral diary. To test the system, one healthy man used the system for 10 weeks. The system was successfully applied in out-hospital conditions. The success rate of the measurements was 70-91%, depending on the variable under consideration. The pilot study indicated that the recorded data accurately reflected the health status of the subject. The TERVA system provides a method to record and investigate wellness-related data over several weeks, or even months, outside the hospital among subjects capable of using a personal computer. Several applications of the system are discussed.

Expression of transcription factor GATA-4 during human testicular development and disease.

GATA-4 is a highly conserved transcription factor that plays a critical role in regulating embryonic morphogenesis and cellular differentiation. Given the emerging role of GATA-4 in the development and function of murine gonads, we have now studied its role in human testis. We find that GATA-4 is expressed from early human fetal testicular development to adulthood. This transcription factor is evident in Sertoli cells through fetal and postnatal development. Expression of GATA-4 in Sertoli cells peaks at 19-22 weeks gestation at the time of high circulating fetal FSH. In Leydig cells, GATA-4 is expressed during fetal period and after puberty, coinciding with the periods of active androgen synthesis in the testis; this suggests a link between GATA-4 and steroidogenesis. Also, fetal germ cells and prepubertal spermatogonia express GATA-4, and it is down-regulated in these cells after puberty. As hormonal regulation of GATA-4 in human testis was not possible to study directly, we used testicular samples from patients who had endocrine abnormalities or were hormonally treated. Testicular expression of GATA-4 in hCG-treated cryptorchidism does not differ from that in controls. In androgen resistance, GATA-4 expression in Sertoli and germ cells is weak or totally absent. GATA-4 protein is abundantly present in Sertoli and Leydig cell tumors, suggesting a relationship to tumorigenesis or tumor progression in somatic cell-derived testicular neoplasms.

Estradiol acts as a germ cell survival factor in the human testis in vitro.

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.

Partial oxygen pressure and mitochondrial permeability transition affect germ cell apoptosis in the human testis.

During regular spermatogenesis, a number of testicular germ cells degenerate by an apoptotic process that is under hormonal control. Oxidative and mitochondrial changes have been proposed to play a role in apoptosis of many cell types. Previously, whether human germ cell survival is controlled by oxygen or by effectors of the mitochondrial permeability transition has not been investigated. In the present study, apoptosis was induced in human testicular germ cells by incubating segments of seminiferous tubules without survival factors (i.e. serum or hormones; 21% oxygen). Apoptosis was significantly suppressed in an inversely dose-dependent fashion at partial oxygen pressures below 10%, as detected by Southern blot analysis of DNA fragmentation, DNA labeling in situ, and electron microscopy. Cyclosporin A and its nonimmunosuppressive derivative N-methyl-Val4-cyclosporin A prevented cell death, suggesting a key role for the mitochondrial permeability transition in apoptosis. Apoptotic cells were identified as mainly spermatocytes and spermatids, the mitochondria of which underwent morphological changes during the apoptotic process. The present results imply that to improve germ cell viability in in vitro fertilization techniques, the partial oxygen pressure should be lowered.

Fas regulates germ cell apoptosis in the human testis in vitro.

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.