Gunapala Shetty

Enhancement of A Spermatogonial Proliferation and Differentiation in Irradiated Rats by Gonadotropin-Releasing Hormone Antagonist Administration

The initial changes in the numbers, proliferation, and differentiation of A spermatogonia in irradiated rats after the administration of a GnRH antagonist, which is known to induce differentiation in this system, were investigated. LBNF1 rats were given 6 Gy ofγ -irradiation; some were treated with the GnRH antagonist Cetrorelix beginning 15 weeks after irradiation. Although the spermatogonia in the irradiated rats without hormone treatment continue to proliferate (labeling and mitotic indexes of 24% and 18%, respectively), they underwent apoptosis (apoptotic indexes of 21% by the terminal transferase-mediated end labeling assay and 9% by nuclear morphology), resulting in a constant number of A spermatogonia. Whole mount analysis of clones of A spermatogonia revealed that larger clones were more likely to undergo apoptosis than mitosis. Hormone administration decreased the intratesticular testosterone concentration to 6% of the level in irradiated rats within 1 week. Concomitantly, there was a decrease in...

Hormonal suppression for fertility preservation in males and females

Methods to restore fertility of men and women sterilized by medical treatments and environmental toxicant exposures are under investigation. Rendering spermatogenesis and ovarian follicular development kinetically quiescent by suppression of gonadotropins has been proposed to protect them from damage by cytotoxic therapy. Although the method fails to protect the fertility of male mice and monkeys, gonadotropin and testosterone suppression in rats before or after cytotoxic therapy do enhance the recovery of spermatogenesis. However, the mechanism involves not the induction of quiescence but rather the reversal, by suppression of testosterone, of a block in differentiation of surviving spermatogonia caused by damage to the somatic environment. In men, only one of eight clinical trials was successful in protecting or restoring spermatogenesis after cytotoxic therapy. In women, protection of primordial follicles in several species from damage by cytotoxic agents using GnRH analogs has been claimed; however, only two studies in mice appear convincing. The protection cannot involve the induction of quiescence in the already dormant primordial follicle but may involve direct effects of GnRH analogs or indirect effects of gonadotropin suppression on the whole ovary. Although numerous studies in female patients undergoing chemotherapy indicate that GnRH analogs might be protective of ovarian function, none of the studies showing protection were prospective randomized clinical trials and thus they are inconclusive. Considering interspecies differences and similarities in the gonadal sensitivity to cytotoxic agents and hormones, mechanistic studies are needed to identify the specific beneficial effects of hormonal suppression in select animal models that may be applicable to humans.

Germline stem cells: toward the regeneration of spermatogenesis

Improved therapies for cancer and other conditions have resulted in a growing population of long-term survivors. Infertility is an unfortunate side effect of some cancer therapies that impacts the quality of life of survivors who are in their reproductive or prereproductive years. Some of these patients have the opportunity to preserve their fertility using standard technologies that include sperm, egg, or embryo banking, followed by IVF and/or ET. However, these options are not available to all patients, especially the prepubertal patients who are not yet producing mature gametes. For these patients, there are several stem cell technologies in the research pipeline that may give rise to new fertility options and allow infertile patients to have their own biological children. We will review the role of stem cells in normal spermatogenesis as well as experimental stem cell-based techniques that may have potential to generate or regenerate spermatogenesis and sperm. We will present these technologies in the context of the fertility preservation paradigm, but we anticipate that they will have broad implications for the assisted reproduction field.

Androgen-responsive microRNAs in mouse Sertoli cells.

Although decades of research have established that androgen is essential for spermatogenesis, androgens mechanism of action remains elusive. This is in part because only a few androgen-responsive genes have been definitively identified in the testis. Here, we propose that microRNAs – small, non-coding RNAs – are one class of androgen-regulated trans-acting factors in the testis. Specifically, by using androgen suppression and androgen replacement in mice, we show that androgen regulates the expression of several microRNAs in Sertoli cells. Our results reveal that several of these microRNAs are preferentially expressed in the testis and regulate genes that are highly expressed in Sertoli cells. Because androgen receptor-mediated signaling is essential for the pre- and post-meiotic germ cell development, we propose that androgen controls these events by regulating Sertoli/germ cell-specific gene expression in a microRNA-dependent manner.

The Missing Niche for Spermatogonial Stem Cells: Do Blood Vessels Point the Way?

Although spermatogonial stem cell niches have been defined in lower organisms, their definitive localization in mammalian seminiferous tubules has been elusive. In a recent Science paper, Yoshida et al. (2007) elegantly demonstrated a vascular and interstitial tissue-associated niche for undifferentiated spermatogonia in the mouse.

Fetal Cyclophosphamide Exposure Induces Testicular Cancer and Reduced Spermatogenesis and Ovarian Follicle Numbers in Mice

Exposure to radiation during fetal development induces testicular germ cell tumors (TGCT) and reduces spermatogenesis in mice. However, whether DNA damaging chemotherapeutic agents elicit these effects in mice remains unclear. Among such agents, cyclophosphamide (CP) is currently used to treat breast cancer in pregnant women, and the effects of fetal exposure to this drug manifested in the offspring must be better understood to offer such patients suitable counseling. The present study was designed to determine whether fetal exposure to CP induces testicular cancer and/or gonadal toxicity in 129 and in 129.MOLF congenic (L1) mice. Exposure to CP on embryonic days 10.5 and 11.5 dramatically increased TGCT incidence to 28% in offspring of 129 mice (control value, 2%) and to 80% in the male offspring of L1 (control value 33%). These increases are similar to those observed in both lines of mice by radiation. In utero exposure to CP also significantly reduced testis weights at 4 weeks of age to ∼70% of control and induced atrophic seminiferous tubules in ∼30% of the testes. When the in utero CP-exposed 129 mice reached adulthood, there were significant reductions in testicular and epididymal sperm counts to 62% and 70%, respectively, of controls. In female offspring, CP caused the loss of 77% of primordial follicles and increased follicle growth activation. The results indicate that i) DNA damage is a common mechanism leading to induction of testicular cancer, ii) increased induction of testis cancer by external agents is proportional to the spontaneous incidence due to inherent genetic susceptibility, and iii) children exposed to radiation or DNA damaging chemotherapeutic agents in utero may have increased risks of developing testis cancer and having reduced spermatogenic potential or diminished reproductive lifespan.

Donor Sertoli cells transplanted into irradiated rat testes stimulate partial recovery of endogenous spermatogenesis

Irradiation of rat testes leads to the failure to support differentiation of the surviving spermatogonia due to damage of the somatic environment. To determine the involvement of Sertoli cells in this somatic damage, we transplanted seminiferous tubule cells from normal immature GFP-transgenic rats into the testes of irradiated rats. The donor Sertoli cells colonized and developed in the host testes. In many seminiferous tubules, the donor Sertoli cells formed abnormal spherical structures in the lumen, but in some tubules they formed a normal-appearing epithelium, but with only isolated spermatogonia, on the basement membrane. When the donor cells were injected into the interstitial region of the testis, they formed tubule-like structures containing Sertoli cells and occasional isolated spermatogonia, both of donor origin. Surprisingly, in host tubules adjacent to these newly formed donor-cell tubules or adjacent to the endogenous tubules with abnormal donor Sertoli-cell structures, endogenous spermatogonia differentiated to the spermatocyte or even to spermatid stages. Around these newly donor cell-formed tubules and the host tubules with abnormal donor Sertoli-cell structures, many cells including macrophages, which perhaps represented chronic inflammation, accumulated in the interstitium. We conclude that the donor Sertoli cells that colonized the seminiferous tubules did not directly support recovery of spermatogenesis. Instead, the colonizing Sertoli cells acted indirectly on the interstitium to stimulate localized differentiation of endogenous spermatogonia.

Dickkopf-Like1 Regulates Postpubertal Spermatocyte Apoptosis and Testosterone Production

Dickkopf-like1 (Dkkl1) encodes a glycoprotein secreted by postmeiotic male germ cells. We report here that adult Dkkl1-deficient males have elevated sperm counts caused by a decrease in postpubertal spermatocyte apoptosis and display, upon aging, increased local production of testosterone. Molecular analyses identified the Fas death ligand (FasL) as a target for Dkkl1 pro-apoptotic activity in adult mice. Accordingly, adult FasL-deficient gld mice display an increased sperm count and decreased spermatocyte apoptosis phenotype similar to the one observed in Dkkl1-deficient mice. We also show that the elevated testosterone level observed in aging Dkkl1-deficient males is secondary to increased expression in Leydig cells of CYP11A and CYP17, two genes implicated in steroidogenesis. Furthermore, treatment of Leydig cells with Dkkl1 decreases DNA binding and transcriptional activity of steroidogenic factor 1, a pivotal regulator of gene expression in testis. Thus, this study establishes Dkkl1 as a negative regulator of adult testis homeostasis and identifies a novel, Dkkl1/FasL-dependent, regulation that specifically controls the number of postpubertal spermatocytes.

Changes in Gene Expression in Somatic Cells of Rat Testes Resulting from Hormonal Modulation and Radiation-Induced Germ Cell Depletion

Abstract Although gonadotropins and androgen are required for normal spermatogenesis and both testosterone and follicle-stimulating hormone (FSH) are responsible for the inhibition of spermatogonial differentiation that occurs in irradiated rats, it has been difficult to identify the specific genes involved. To study specific hormonally regulated changes in somatic cell gene expression in the testis that may be involved in these processes, without the complication of changing populations of germ cells, we used irradiated LBNF1 rats, the testes of which contain almost exclusively somatic cells except for a few type A spermatogonia. Three different groups of these rats were treated with various combinations of gonadotropin-releasing hormone antagonist, an androgen receptor antagonist (flutamide), testosterone, and FSH, and we compared the gene expression levels 2 wk later to those of irradiated-only rats by microarray analysis. By dividing the gene expression patterns into three major patterns and 11 subpatterns, we successfully distinguished, in a single study, the genes that were specifically regulated by testosterone, by luteinizing hormone (LH), and by FSH from the large number of genes that were not hormonally regulated in the testis. We found that hormones produced more dramatic upregulation than downregulation of gene expression: Testosterone had the strongest upregulatory effect, LH had a modest but appreciable upregulatory effect, and FSH had a minor upregulatory effect. We also separately identified the somatic cell genes that were chronically upregulated by irradiation. Thus, the present study identified gene expression changes that may be responsible for hormonal action on somatic cells to support normal spermatogenesis and the hormone-mediated block in spermatogonial development after irradiation.

Hormone suppression with GnRH antagonist promotes spermatogenic recovery from transplanted spermatogonial stem cells in irradiated cynomolgus monkeys

Hormone suppression given before or after cytotoxic treatment stimulates the recovery of spermatogenesis from endogenous and transplanted spermatogonial stem cells (SSC) and restores fertility in rodents. To test whether the combination of hormone suppression and transplantation could enhance the recovery of spermatogenesis in primates, we irradiated (7 Gy) the testes of 12 adult cynomolgus monkeys and treated six of them with gonadotropin‐releasing hormone antagonist (GnRH‐ant) for 8 weeks. At the end of this treatment, we transfected cryopreserved testicular cells with green fluorescent protein‐lentivirus and autologously transplanted them back into one of the testes. The only significant effect of GnRH‐ant treatment on endogenous spermatogenesis was an increase in the percentage of tubules containing differentiated germ cells (tubule differentiation index; TDI) in the sham‐transplanted testes of GnRH‐ant–treated monkeys compared with radiation‐only monkeys at 24 weeks after irradiation. Although transplantation alone after irradiation did not significantly increase the TDI, detection of lentiviral DNA in the spermatozoa of one radiation‐only monkey indicated that some transplanted cells colonized the testis. However, the combination of transplantation and GnRH‐ant clearly stimulated spermatogenic recovery as evidenced by several observations in the GnRH‐ant–treated monkeys receiving transplantation: (i) significant increases (~20%) in the volume and weight of the testes compared with the contralateral sham‐transplanted testes and/or to the transplanted testes of the radiation‐only monkeys; (ii) increases in TDI compared to the transplanted testes of radiation‐only monkeys at 24 weeks (9.6% vs. 2.9%; p = 0.05) and 44 weeks (16.5% vs. 6.1%, p = 0.055); (iii) detection of lentiviral sequences in the spermatozoa or testes of five of the GnRH‐ant–treated monkeys and (iv) significantly higher sperm counts than in the radiation‐only monkeys. Thus hormone suppression enhances spermatogenic recovery from transplanted SSC in primates and may be a useful tool in conjunction with spermatogonial transplantation to restore fertility in men after cancer treatment.