Undifferentiated spermatogonia regulate Cyp26b1 expression through NOTCH signaling and drive germ cell differentiation.

Cytochrome P450 family 26 subfamily B member 1 (CYP26B1) regulates the concentration of all-trans retinoic acid (RA) and plays a key role in germ cell differentiation by controlling local distribution of RA. The mechanisms regulating Cyp26b1 expression in postnatal Sertoli cells, the main components of the stem cell niche, are so far unknown. During gonad development, expression of Cyp26b1 is maintained by Steroidogenic Factor 1 (SF-1) and Sex-Determining Region Y Box-9 (SOX9), which ensure that RA is degraded and germ cell differentiation is blocked. Here, we show that the NOTCH target Hairy/Enhancer-of-Split Related with YRPW Motif 1 (HEY1), a transcriptional repressor, regulates germ cell differentiation via direct binding to the Cyp26b1 promoter and thus inhibits its expression in Sertoli cells. Further, using in vivo germ cell ablation, we demonstrate that undifferentiated type A spermatogonia are the cells that activate NOTCH signaling in Sertoli cells through their expression of the NOTCH ligand JAGGED-1 (JAG1) at stage VIII of the seminiferous epithelium cycle, therefore mediating germ cell differentiation by a ligand concentration-dependent process. These data therefore provide more insights into the mechanisms of germ cell differentiation after birth and potentially explain the spatiotemporal RA pulses driving the transition between undifferentiated to differentiating spermatogonia.-Parekh, P. A., Garcia, T. X., Waheeb, R., Jain, V., Gandhi, P., Meistrich, M. L., Shetty, G., Hofmann, M.-C. Undifferentiated spermatogonia regulate Cyp26b1 expression through NOTCH signaling and drive germ cell differentiation.

Donor spermatogenesis in de novo formed seminiferous tubules from transplanted testicular cells in rhesus monkey testis.

STUDY QUESTION: Can transplanted primate testicular cells form seminiferous tubules de novo, supporting complete spermatogenesis? SUMMARY ANSWER: Cryopreserved testicular cells from a prepubertal monkey can reorganize in an adult monkey recipient testis forming de novo seminiferous tubular cords supporting complete spermatogenesis. WHAT IS KNOWN ALREADY: De novo morphogenesis of testicular tissue using aggregated cells from non-primate species grafted either subcutaneously or in the testis can support spermatogenesis. STUDY DESIGN, SIZE, DURATION: Two postpubertal rhesus monkeys (Macaca mulatta) were given testicular irradiation. One monkey was given GnRH-antagonist treatment from 8 to 16 weeks after irradiation, while the other received sham injections. At 16 weeks, cryopreserved testicular cells from two different prepubertal monkeys [43 × 106 viable (Trypan-blue excluding) cells in 260 µl, and 80 × 106 viable cells in 400 µl] were transplanted via ultrasound-guided injections to one of the rete testis in each recipient, and immune suppression was given. The contralateral testis was sham transplanted. Testes were analyzed 9 months after transplantation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Spermatogenic recovery was assessed by testicular volume, weight, histology and immunofluorescence. Microsatellite genotyping of regions of testicular sections obtained by LCM determined whether the cells were derived from the host or transplanted cells. MAIN RESULTS AND THE ROLE OF CHANCE: Transplanted testis of the GnRH-antagonist-treated recipient, but not the sham-treated recipient, contained numerous irregularly shaped seminiferous tubular cords, 89% of which had differentiating germ cells, including sperm in a few of them. The percentages of donor genotype in different regions of this testis were as follows: normal tubule, 0%; inflammatory, 0%; abnormal tubule region, 67%; whole interior of abnormal tubules, >99%; adluminal region of the abnormal tubules, 92%. Thus, these abnormal tubules, including the enclosed germ cells, were derived de novo from the donor testicular cells. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: The de novo tubules were observed in only one out of the two monkeys transplanted with prepubertal donor testicular cells. WIDER IMPLICATIONS OF THE FINDINGS: These findings may represent a promising strategy for restoration of fertility in male childhood cancer survivors. The approach could be particularly useful in those exposed to therapeutic agents that are detrimental to the normal development of the tubule somatic cells affecting the ability of the endogenous tubules to support spermatogenesis, even from transplanted spermatogonial stem cells. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by research grants P01 HD075795 from Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD/NIH) to K.E.O and Cancer Center Support Grant P30 CA016672 from NCI/NIH to The University of Texas MD Anderson Cancer Center. The authors declare that they have no competing interests.

Spermatogonial behavior in rats during radiation-induced arrest and recovery after hormone suppression.

Ionizing radiation has been shown to arrest spermatogenesis despite the presence of surviving stem spermatogonia, by blocking their differentiation. This block is a result of damage to the somatic environment and is reversed when gonadotropins and testosterone are suppressed, but the mechanisms are still unknown. We examined spermatogonial differentiation and Sertoli cell factors that regulate spermatogonia after irradiation, during hormone suppression, and after hormone suppression combined with Leydig cell elimination with ethane dimethane sulfonate. These results showed that the numbers and cytoplasmic structure of Sertoli cells are unaffected by irradiation, only a few type A undifferentiated (Aund) spermatogonia and even fewer type A1 spermatogonia remained, and immunohistochemical analysis showed that Sertoli cells still produced KIT ligand (KITLG) and glial cell line-derived neurotrophic factor (GDNF). Some of these cells expressed KIT receptor, demonstrating that the failure of differentiation was not a result of the absence of the KIT system. Hormone suppression resulted in an increase in Aund spermatogonia within 3 days, a gradual increase in KIT-positive spermatogonia, and differentiation mainly to A3 spermatogonia after 2 weeks. KITL (KITLG) protein expression did not change after hormone suppression, indicating that it is not a factor in the stimulation. However, GDNF increased steadily after hormone suppression, which was unexpected since GDNF is supposed to promote stem spermatogonial self-renewal and not differentiation. We conclude that the primary cause of the block in spermatogonial development is not due to Sertoli cell factors such (KITL\GDNF) or the KIT receptor. As elimination of Leydig cells in addition to hormone suppression resulted in differentiation to the A3 stage within 1 week, Leydig cell factors were not necessary for spermatogonial differentiation.

Models and Methods for Evaluating Regeneration of Spermatogenesis After Cytotoxic Treatments.

Cytotoxic exposure, predominantly during radiation and/or chemotherapy treatment for cancer, interferes with fertility in men. While moderate doses cause temporary azoospermia allowing eventual recovery of spermatogenesis, higher doses of sterilizing agents can cause permanent sterility by killing the spermatogonial stem cells (SSCs). In this chapter, the methods involved in the following aspects of cytotoxic regeneration are described: (i) designing rodent and non-human primate models for regeneration of spermatogenesis after cytotoxic treatment by radiation and chemotherapy; (ii) analysis of SSCs with respect to the impact of the cytotoxic treatment, including analysis of spermatogonial clones, scoring the testicular section to analyze the extent of spermatogenic recovery, preparation of testicular and epididymal sperm, and collection of semen in non-human primates for sperm analysis; and (iii) preparation and delivery of a GnRH antagonist and steroids for enhancement or induction of spermatogonial differentiation, leading to the regeneration of spermatogenesis, largely applicable in the rat model.

Estrogen-regulated genes in rat testes and their relationship to recovery of spermatogenesis after irradiation.

Despite numerous observations of the effects of estrogens on spermatogenesis, identification of estrogen-regulated genes in the testis is limited. Using rats in which irradiation had completely blocked spermatogonial differentiation, we previously showed that testosterone suppression with gonadotropin-releasing hormone-antagonist acyline and the antiandrogen flutamide stimulated spermatogenic recovery and that addition of estradiol (E2) to this regimen accelerated this recovery. We report here the global changes in testicular cell gene expression induced by the E2 treatment. By minimizing the changes in other hormones and using concurrent data on regulation of the genes by these hormones, we were able to dissect the effects of estrogen on gene expression, independent of gonadotropin or testosterone changes. Expression of 20 genes, largely in somatic cells, was up- or downregulated between 2- and 5-fold by E2. The unexpected and striking enrichment of transcripts not corresponding to known genes among the E2-downregulated probes suggested that these might represent noncoding mRNAs; indeed, we have identified several as miRNAs and their potential target genes in this system. We propose that genes for which expression levels are altered in one direction by irradiation and in the opposite direction by both testosterone suppression and E2 treatment are candidates for controlling the block in differentiation. Several genes, including insulin-like 3 (Insl3), satisfied those criteria. If they are indeed involved in the inhibition of spermatogonial differentiation, they may be candidate targets for treatments to enhance recovery of spermatogenesis following gonadotoxic exposures, such as those resulting from cancer therapy.

Postpubertal spermatogonial stem cell transplantation restores functional sperm production in rhesus monkeys irradiated before and after puberty.

BACKGROUND: Cancer treatment of prepubertal patients impacts future fertility due to the abolition of spermatogonial stem cells (SSCs). In macaques, spermatogenesis could be regenerated by intratesticular transplantation of SSCs, but no studies have involved cytotoxic treatment before puberty and transplantation after puberty, which would be the most likely clinical scenario. OBJECTIVES: To evaluate donor-derived functional sperm production after SSC transplantation to adult monkeys that had received testicular irradiation during the prepubertal period. MATERIALS AND METHODS: We obtained prepubertal testis tissue by unilaterally castrating six prepubertal monkeys and 2 weeks later irradiated the remaining testes with 6.9 Gy. However, because spermatogenic recovery was observed, we irradiated them again 14 months later with 7 Gy. Three of the monkeys were treated with GnRH-antagonist (GnRH-ant) for 8 weeks. The cryopreserved testis cells from the castrated testes were then allogeneically transplanted into the intact testes of all monkeys. Tissues were harvested 10 months later for analyses. RESULTS: In three of the six monkeys, 61%, 38%, and 11% of the epididymal sperm DNA were of the donor genotype. The ability to recover donor-derived sperm production was not enhanced by the GnRH-ant pretreatment. However, the extent of filling seminiferous tubules during the transplantation procedure was correlated with the eventual production of donor spermatozoa. The donor epididymal spermatozoa from the recipient with 61% donor contribution were capable of fertilizing rhesus eggs and forming embryos. Although the transplantation was done into the rete testis, two GnRH-ant-treated monkeys, which did not produce donor-derived epididymal spermatozoa, displayed irregular tubular cords in the interstitium containing testicular spermatozoa derived from the transplanted donor cells. DISCUSSION AND CONCLUSION: The results further support that sperm production can be restored in non-human primates from tissues cryopreserved prior to prepubertal and post-pubertal gonadotoxic treatment by transplantation of these testicular cells after puberty into seminiferous tubules.

Changes in gene expression in somatic cells of rat testes resulting from hormonal modulation and radiation-induced germ cell depletion.

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 LBNF(1) 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.

Restoration of functional sperm production in irradiated pubertal rhesus monkeys by spermatogonial stem cell transplantation.

BACKGROUND: In male pre-pubertal cancer patients, radiation and chemotherapy impact future fertility by eradication of spermatogonial stem cells (SSCs). In macaques, spermatogenesis could be regenerated by intratesticular transplantation of SSCs, but only a small percentage of spermatozoa produced were of donor origin. Transient hormone suppression with a GnRH antagonist (GnRH-ant) enhanced spermatogenic recovery from transplanted SSCs. OBJECTIVES: To evaluate donor-derived and endogenous spermatogenic recovery after SSC transplantation into irradiated monkeys and to test whether hormone suppression around the time of transplantation facilitates spermatogenic recovery. MATERIALS AND METHODS: Testes of 15 adult rhesus monkeys were irradiated with 7 Gy and 4 months later transplanted, to one of the testes, with cryopreserved testicular cells containing SSCs from unrelated monkeys. Monkeys were either treated with GnRH-ant for 8 weeks before transplantation, GnRH-ant from 4 weeks before to 4 weeks after transplantation, or with no GnRH-ant. Tissues were harvested 10 months after transplantation. RESULTS: Two of the 15 monkeys, a control and a pre-transplantation GnRH-ant-treated, showed substantially higher levels of testicular spermatogenesis and epididymal sperm output in the transplanted side as compared to the untransplanted. Over 84% of epididymal spermatozoa on the transplanted side had the donor genotype and were capable of fertilizing eggs after intracytoplasmic sperm injection forming morulae of the donor paternal origin. Low levels of donor spermatozoa (~1%) were also identified in the epididymis of three additional monkeys. Transplantation also appeared to enhance endogenous spermatogenesis. DISCUSSION AND CONCLUSION: We confirmed that SSC transplantation can be used for restoration of fertility in male cancer survivors exposed to irradiation as a therapeutic agent. The success rate of this procedure, however, is low. The success of filling the tubules with the cell suspension, but not the GnRH-ant treatment, was related to the level of colonization by transplanted cells.

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.

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.

Autologous grafting of cryopreserved prepubertal rhesus testis produces sperm and offspring.

Testicular tissue cryopreservation is an experimental method to preserve the fertility of prepubertal patients before they initiate gonadotoxic therapies for cancer or other conditions. Here we provide the proof of principle that cryopreserved prepubertal testicular tissues can be autologously grafted under the back skin or scrotal skin of castrated pubertal rhesus macaques and matured to produce functional sperm. During the 8- to 12-month observation period, grafts grew and produced testosterone. Complete spermatogenesis was confirmed in all grafts at the time of recovery. Graft-derived sperm were competent to fertilize rhesus oocytes, leading to preimplantation embryo development, pregnancy, and the birth of a healthy female baby. Pending the demonstration that similar results are obtained in noncastrated recipients, testicular tissue grafting may be applied in the clinic.

p53-dependent apoptosis in the inhibition of spermatogonial differentiation in juvenile spermatogonial depletion (Utp14bjsd) mice.

In adult male mice homozygous for the juvenile spermatogonial depletion (Utp14b jsd) mutation in the Utp14b gene, type A spermatogonia proliferate, but in the presence of testosterone and at scrotal temperatures, these spermatogonia undergo apoptosis just before differentiation. In an attempt to delineate this apoptotic pathway in jsd mice and specifically address the roles of p53- and Fas ligand (FasL) /Fas receptor-mediated apoptosis, we produced jsd mice deficient in p53, Fas, or FasL. Already at the age of 5 wk, less degeneration of spermatogenesis was observed in p53-null-jsd mice than jsd single mutants, and in 8- or 12-wk-old mice, the percentage of seminiferous tubules showing differentiated germ cells [tubule differentiation index (TDI)] was 26-29% in the p53-null-jsd mice, compared with 2-4% in jsd mutants with normal p53. The TDI in jsd mice heterozygous for p53 showed an intermediate TDI of 8-13%. The increase in the differentiated tubules in double-mutant and p53 heterozygous jsd mice was mostly attributable to intermediate and type B spermatogonia; few spermatocytes were present. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining showed that most of these differentiated spermatogonia still underwent apoptosis, thereby blocking further continuation of spermatogenesis. In contrast, the percentage of tubules that were differentiated was not significantly altered in either adult Fas null-jsd mice or adult FasL defective gld-jsd double mutant mice as compared with jsd single mutants. Furthermore, caspase-9, but not caspase-8 was immunochemically localized in the adult jsd mice spermatogonia undergoing apoptosis. The results show that p53, but not FasL or Fas, is involved in the apoptosis of type A spermatogonia before/during differentiation in jsd mice that involves the intrinsic pathway of apoptosis. However, apoptosis in the later stages must be a p53-independent process.

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.

Differentiation of primate primordial germ cell-like cells following transplantation into the adult gonadal niche.

A major challenge in stem cell differentiation is the availability of bioassays to prove cell types generated in vitro are equivalent to cells in vivo. In the mouse, differentiation of primordial germ cell-like cells (PGCLCs) from pluripotent cells was validated by transplantation, leading to the generation of spermatogenesis and to the birth of offspring. Here we report the use of xenotransplantation (monkey to mouse) and homologous transplantation (monkey to monkey) to validate our in vitro protocol for differentiating male rhesus (r) macaque PGCLCs (rPGCLCs) from induced pluripotent stem cells (riPSCs). Specifically, transplantation of aggregates containing rPGCLCs into mouse and nonhuman primate testicles overcomes a major bottleneck in rPGCLC differentiation. These findings suggest that immature rPGCLCs once transplanted into an adult gonadal niche commit to differentiate towards late rPGCs that initiate epigenetic reprogramming but do not complete the conversion into ENO2-positive spermatogonia.

Testicular edema is associated with spermatogonial arrest in irradiated rats.

Irradiation of LBNF1 rat testes induces arrest of spermatogonial differentiation, which can be reversed by suppression of testosterone with GnRH antagonist treatment. The cause of the arrest is unknown. We investigated the time course and hormonal effects on radiation-induced arrest and changes in interstitial fluid volume. We postulated that the edema evident in irradiated testes caused the differentiation blockade. Rat testes were irradiated with 3.5 or 6 Gy. Interstitial fluid testosterone (IFT) increased between 2 and 6 wk after irradiation, followed by increased interstitial fluid volume at 6 wk and spermatogonial blockade at 8 wk. Additional rats irradiated with 6 Gy were given GnRH antagonist, alone or with exogenous testosterone, for 8 wk starting at 15 wk after irradiation. In rats treated with GnRH antagonist, IFT started falling within 1 wk of treatment, followed by interstitial fluid volume decreases at wk 2 and 3, with recovery of spermatogenesis starting at wk 4. Addition of exogenous testosterone largely blocked the effects of GnRH antagonist on IFT, interstitial fluid volume, and spermatogenesis. Thus the testicular edema was largely modulated by intratesticular testosterone levels. The time course of changes in the spermatogonial blockade more closely followed that of the testicular edema than of IFT, indicating that testosterone may block spermatogonial differentiation indirectly by producing edema. This conclusion was further supported by an experiment in which irradiated rats were treated with GnRH antagonist plus estrogen; the treatment further reduced IFT and interstitial fluid volume and reduced the time to initiation of recovery of spermatogonial differentiation. These results suggest that studies of the edematous process or composition of the fluid would help elucidate the mechanism of spermatogonial arrest in toxicant-treated rats.

Both testosterone and follicle-stimulating hormone independently inhibit spermatogonial differentiation in irradiated rats.

Simultaneous suppression of both testosterone and FSH with GnRH antagonists (GnRH-ant) reverses the radiation-induced block in spermatogonial differentiation in F1 hybrids of Lewis and Brown-Norway rats. Although addition of exogenous testosterone restores the block, it also raises FSH, and hence it had not been possible to conclusively determine which hormone was inhibiting spermatogonial differentiation. In the present study, we establish the relative roles of testosterone and FSH in this inhibition using three different approaches. The first approach involved the treatment of irradiated rats, in which differentiation was stimulated by GnRH-ant plus flutamide, with FSH for 2 wk; the FSH reduced the percentage of tubules that were differentiated (TDI) by about 2-fold, indicating that FSH does have an inhibitory role. The second approach involved treatment of irradiated, hypophysectomized rats with exogenous testosterone for 10 wk; testosterone also reduced the TDI, demonstrating that testosterone had a definite inhibitory effect, independent of pituitary hormones. Furthermore, in this protocol we showed that TDI in the hypophysectomized testosterone-treated group, which had higher intratesticular testosterone levels but lacked FSH, was slightly higher than the TDI in a GnRH-antagonist-testosterone-treated group of irradiated rats, which had normal physiological levels of FSH; this result supports a role for endogenous FSH in suppressing spermatogonial differentiation in the latter group. The third approach involved injection of an active anti-FSH antibody for 10 d in untreated, GnRH-ant plus flutamide-treated, or GnRH-ant plus testosterone-treated irradiated rats. This was not sufficient to increase the TDI. However, flutamide given in a similar treatment schedule did increase the TDI in GnRH-ant plus testosterone-treated rats. We conclude that both testosterone and FSH individually inhibit spermatogonial differentiation after irradiation, but testosterone is a more highly potent inhibitor than is FSH.

Spermatogonial differentiation in juvenile spermatogonial depletion (jsd) mice with androgen receptor or follicle-stimulating hormone mutations.

The jsd mice experience a single wave of spermatogenesis, followed by an arrest of spermatogenesis because of a block in spermatogonial differentiation. Previous pharmacological and surgical studies have indicated that testosterone (T) and low scrotal temperatures but not FSH block spermatogonial differentiation in jsd mice. We sought to test these observations by genetic approaches by producing male jsd mutant mice with either defective androgen receptor (AR, Tfm mutation) or a deficiency of FSH (fshb(-/-)). In adult jsd-Tfm double-mutant mice, the tubule differentiation index was 95% compared with 14% in jsd littermates, suggesting that general ablation of AR function restored spermatogonial differentiation in jsd mice. The results indicated that this enhancement of differentiation was primarily a result of elevation of temperature caused by the cryptorchid position of the testis in jsd-Tfm double-mutant mice, which resulted from the lack of AR in the gubernaculum. The low levels of T were not a factor in the release of the spermatogonial differentiation block in the jsd-Tfm mice, but we were unable to determine whether inactivation of AR in the adult jsd testis had a direct effect on the restoration of spermatogonial differentiation because the elevated temperature bypassed the T-induced block in spermatogonial differentiation. Although spermatogonia were indeed present in adult jsd-fshb double-mutant mice and were capable of differentiation after androgen deprivation, these mice had a tubule differentiation index of 0%, ruling out the possibility that endogenous FSH inhibited spermatogonial differentiation in jsd mice. The results are consistent in support of the hypothesis that inhibition of spermatogonial differentiation in jsd mice is a result of T acting through the AR only at scrotal temperatures.

Hormonal approaches to preservation and restoration of male fertility after cancer treatment.

It is important to develop methods to prevent or reverse the sterility caused by chemotherapy or radiation therapy for cancer in men. Using a rat model, we have shown that infertility after testicular exposure to moderate doses of radiation and some chemotherapeutic agents occurs as a result of the inability of spermatogonia to differentiate. There is evidence that this phenomenon also occurs in men. Spermatogenesis and fertility can be restored in rats following treatment with radiation or some chemotherapeutic agents by suppressing testosterone with gonadotropin releasing hormone (GnRH) agonists or antagonists either before or after the cytotoxic insult. However, species differences exist in the testicular response to radiation or GnRH antagonist therapy, so rescue protocols that work in rodents do not work in nonhuman primates. The applicability of this procedure to humans is still largely unknown. In rodents, suppression of testosterone with GnRH analog treatment also appears to enhance the success of spermatogonial transplantation-an option when all stem cells are killed by cytotoxic therapy.

Gonadotropin-releasing hormone antagonist (Cetrorelix) therapy fails to protect nonhuman primates (Macaca arctoides) from radiation-induced spermatogenic failure.

Treatment of men of reproductive age with radiation or alkylating agents often produces prolonged azoospermia. We previously demonstrated that suppression of testosterone (T) with gonadotropin-releasing hormone (GnRH) analogs restored spermatogenesis following atrophy induced by radiation or chemotherapy in rats. This study tested whether GnRH antagonist therapy could reverse radiation-induced testicular injury in primates with a similar protocol. Adult male stump-tailed macaques were given either 6.7 Gy radiation to the testis alone, 6.7 Gy radiation combined with GnRH-antagonist treatment starting on the day of exposure, or daily injections of the GnRH antagonist Cetrorelix for 3 months alone and were monitored for 18 months. Cetrorelix alone produced a 20-40-week fully reversible suppression of serum T, but although spermatogenic recovery was incomplete, 40%-90% of tubules contained differentiating germ cells. Following radiation alone, testis volumes were reduced to approximately 28% and sperm counts to less than 1% of pretreatment values. A biopsy at 18 months after radiation showed that only 3.0% of seminiferous tubule cross sections had germ cells. In irradiated animals that received GnRH antagonist, testis volumes were reduced to 18% of pretreatment volume, and at 18 months, only 1.9% of seminiferous tubule cross sections contained germ cells. Inhibin B values were reduced to 10% and 3% of pretreatment levels in the radiation-only and the radiation plus GnRH antagonist groups, respectively. Species differences exist in the testicular response to radiation, GnRH antagonist therapy, or both, so that rescue protocols that were successful in rodents might not work in primates.