RESUMO
Why both testosterone (T) suppression and cryptorchidism reverse the block in spermatogonial differentiation in adult mice homozygous for the juvenile spermatogonial depletion (jsd) mutation has been a conundrum. To resolve this conundrum, we analyzed interrelations between T suppression, testicular temperature, and spermatogonial differentiation and used in vitro techniques to separate the effects of the two treatments on the spermatogonial differentiation block in jsd mice. Temporal analysis revealed that surgical cryptorchidism rapidly stimulated spermatogonial differentiation whereas androgen ablation treatment produced a delayed and gradual differentiation. The androgen suppression caused scrotal shrinkage, significantly increasing the intrascrotal temperature. When serum T or intratesticular T (ITT) levels were modulated separately in GnRH antagonist-treated mice by exogenous delivery of T or LH, respectively, the inhibition of spermatogonial differentiation correlated with the serum T and not with ITT levels. Thus, the block must be caused by peripheral androgen action. When testicular explants from jsd mice were cultured in vitro at 32.5 C, spermatogonial differentiation was not observed, but at 37 C significant differentiation was evident. In contrast, addition of T to the culture medium did not block the stimulation of spermatogonial differentiation at 37 C, and androgen ablation with aminoglutethimide and hydroxyflutamide did not stimulate differentiation at 32.5 C, suggesting that T had no direct effect on spermatogonial differentiation in jsd mice. These data show that elevation of temperature directly overcomes the spermatogonial differentiation block in adult jsd mice and that T suppression acts indirectly in vivo by causing scrotal regression and thereby elevating the testicular temperature.
Assuntos
Androgênios/farmacologia , Temperatura Corporal/efeitos dos fármacos , Ribonucleoproteínas Nucleolares Pequenas/genética , Espermatogênese/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testosterona/farmacologia , Animais , Temperatura Corporal/fisiologia , Criptorquidismo , Homozigoto , Hormônio Luteinizante/farmacologia , Masculino , Camundongos , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Escroto/efeitos dos fármacos , Escroto/fisiologia , Espermatogênese/fisiologia , Espermatogônias/efeitos dos fármacos , Espermatogônias/fisiologia , Testículo/fisiologiaRESUMO
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.
Assuntos
Estradiol/uso terapêutico , Estrogênios/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatogênese/efeitos da radiação , Testículo/efeitos dos fármacos , Testículo/metabolismo , Antagonistas de Androgênios/uso terapêutico , Animais , Cruzamentos Genéticos , Quimioterapia Combinada , Flutamida/uso terapêutico , Raios gama , Regulação da Expressão Gênica/efeitos da radiação , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Antagonistas de Hormônios/uso terapêutico , Insulina/genética , Insulina/metabolismo , Masculino , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oligopeptídeos/uso terapêutico , Proteínas/genética , Proteínas/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Testículo/patologia , Testículo/efeitos da radiação , Testosterona/antagonistas & inibidoresRESUMO
Irradiation interrupts spermatogenesis and causes prolonged sterility in male mammals. Hormonal suppression treatment with gonadotropin-releasing hormone (GnRH) analogues has restored spermatogenesis in irradiated rats, but similar attempts were unsuccessful in irradiated mice, monkeys, and humans. In this study, we tested a stronger hormonal suppression regimen (the GnRH antagonist, acyline, and plus flutamide) for efficacy both in restoring endogenous spermatogenesis and in enhancing colonization of transplanted stem spermatogonia in mouse testes irradiated with a total doses between 10.5 and 13.5 Gy. A 4-week hormonal suppression treatment, given immediately after irradiation, increased endogenous spermatogenic recovery 1.5-fold, and 11-week hormonal suppression produced twofold increases compared with sham-treated irradiated controls. Furthermore, 10-week hormonal suppression restored fertility from endogenous surviving spermatogonial stem cells in 90% of 10.5-Gy irradiated mice, whereas only 10% were fertile without hormonal suppression. Four- and 11-week hormonal suppression also enhanced spermatogenic development from transplanted stem spermatogonia in irradiated recipient mice, by 3.1- and 4.8-fold, respectively, compared with those not given hormonal treatment. Moreover, the 10-week hormonal suppression regimen, but not a sham treatment, restored fertility of some 13.5-Gy irradiated recipient mice from donor-derived spermatogonial stem cells. This is the first report of hormonal suppression inducing recovery of endogenous spermatogenesis and fertility in a mouse model treated with anticancer agents. The combination of spermatogonial transplantation with hormonal suppression should be investigated as a treatment to restore fertility in young men after cytotoxic cancer therapy.
Assuntos
Antagonistas de Androgênios/uso terapêutico , Hormônio Liberador de Gonadotropina/uso terapêutico , Infertilidade Masculina/terapia , Espermatogônias/transplante , Testículo/efeitos da radiação , Animais , Infertilidade Masculina/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espermatogênese , Testosterona/sangueRESUMO
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.
Assuntos
Hormônio Foliculoestimulante/metabolismo , Regulação da Expressão Gênica , Hormônio Luteinizante/metabolismo , Testículo/metabolismo , Testosterona/farmacologia , Animais , Flutamida/farmacologia , Raios gama , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Células Germinativas/efeitos dos fármacos , Células Germinativas/metabolismo , Células Germinativas/efeitos da radiação , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Oligopeptídeos/farmacologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testículo/efeitos dos fármacos , Testículo/efeitos da radiação , Testosterona/sangueRESUMO
Testosterone acting through the androgen receptor (AR) maintains the arrest of spermatogonial differentiation in juvenile spermatogonial depletion (jsd mutation in the Utp14b gene) mutant adult male mice. It is not known which of the somatic cell types expressing AR mediates this inhibition. To determine whether Sertoli cells are responsible, we selectively eliminated AR in Sertoli cells in jsd mice containing a floxed-Ar gene and an anti-Müllerian hormone-Cre transgene. In these Sertoli AR-knockout (SCARKO)-jsd mice, spermatogonial differentiation did not recover. However, the normal organization of Sertoli cell nuclei was drastically disrupted in SCARKO-jsd mice compared with SCARKO or jsd mice. In addition, the extent of ectoplasmic specializations was reduced; tight junctions were not found; vinculin, an anchoring protein found in ectoplasmic specializations, became uniformly distributed in the cytoplasm; and the adult Sertoli cells showed excess heterochromatin subjacent to their nuclear envelope. Despite the abnormalities in Sertoli cells in SCARKO-jsd mice, global suppression of testosterone action and levels was still effective in restoring the differentiated germ cells, and this was accompanied by an improved arrangement of Sertoli cell nuclei. We conclude that Sertoli cells are not targets for the testosterone-mediated inhibition of spermatogonial differentiation in jsd mice, and that both AR in Sertoli cells and the presence of differentiated germ cells contribute to maintaining the organization of Sertoli cells within the seminiferous tubules.
Assuntos
Receptores Androgênicos/metabolismo , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Espermatogênese/fisiologia , Testosterona/metabolismo , Animais , Diferenciação Celular/fisiologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Receptores Androgênicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermatogônias/citologia , Espermatogônias/metabolismoRESUMO
p27 mediates Cdk2 inhibition and is also found in cyclin D1-Cdk4 complexes. The present data support a role for p27 in the assembly of D-type cyclin-Cdk complexes and indicate that both cyclin D1-Cdk4-p27 assembly and kinase activation are regulated by p27 phosphorylation. Prior work showed that p27 can be phosphorylated by protein kinase B/Akt (PKB/Akt) at T157 and T198. Here we show that PKB activation and the appearance of p27pT157 and p27pT198 precede p27-cyclin D1-Cdk4 assembly in early G(1). PI3K/PKB inhibition rapidly reduced p27pT157 and p27pT198 and dissociated cellular p27-cyclin D1-Cdk4. Mutant p27 allele products lacking phosphorylation at T157 and T198 bound poorly to cellular cyclin D1 and Cdk4. Cellular p27pT157 and p27pT198 coprecipitated with Cdk4 but were not detected in Cdk2 complexes. The addition of p27 to recombinant cyclin D1 and Cdk4 led to cyclin D1-Cdk4-p27 complex formation in vitro. p27 phosphorylation by PKB increased p27-cyclin D1-Cdk4 assembly in vitro but yielded inactive Cdk4. In contrast, Src pretreatment of p27 did not affect p27-cyclin D1-Cdk4 complex formation. However, Src treatment led to tyrosine phosphorylation of p27 and catalytic activation of assembled cyclin D1-Cdk4-p27 complexes. Thus, while PKB-dependent p27 phosphorylation appears to increase cyclin D1-Cdk4-p27 assembly or stabilize these complexes in vitro, cyclin D1-Cdk4-p27 activation requires the tyrosine phosphorylation of p27. Constitutive activation of PKB and Abl or Src family kinases in cancers would drive p27 phosphorylation, increase cyclin D1-Cdk4 assembly and activation, and reduce the cyclin E-Cdk2 inhibitory function of p27. Combined therapy with both Src and PI3K/PKB inhibitors may reverse this process.
Assuntos
Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Substituição de Aminoácidos/efeitos dos fármacos , Catálise/efeitos dos fármacos , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Humanos , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Treonina/metabolismoRESUMO
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.
Assuntos
Ribonucleoproteínas Nucleolares Pequenas/genética , Espermatogônias/citologia , Proteína Supressora de Tumor p53/genética , Animais , Apoptose , Criptorquidismo/patologia , Proteína Ligante Fas/genética , Células Germinativas/citologia , Células Germinativas/fisiologia , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Knockout , Ribonucleoproteínas Nucleolares Pequenas/deficiência , Espermatogônias/fisiologia , Testículo/citologia , Testículo/fisiologia , Receptor fas/genéticaRESUMO
Nutrients and bioenergetics are prerequisites for proliferation and survival of mammalian cells. We present evidence that the cyclin-dependent kinase inhibitor p27(Kip1), is phosphorylated at Thr 198 downstream of the Peutz-Jeghers syndrome protein-AMP-activated protein kinase (LKB1-AMPK) energy-sensing pathway, thereby increasing p27 stability and directly linking sensing of nutrient concentration and bioenergetics to cell-cycle progression. Ectopic expression of wild-type and phosphomimetic Thr 198 to Asp 198 (T198D), but not unstable Thr 198 to Ala 198 (p27(T198A)) is sufficient to induce autophagy. Under stress conditions that activate the LKB1-AMPK pathway with subsequent induction of autophagy, p27 knockdown results in apoptosis. Thus LKB1-AMPK pathway-dependent phosphorylation of p27 at Thr 198 stabilizes p27 and permits cells to survive growth factor withdrawal and metabolic stress through autophagy. This may contribute to tumour-cell survival under conditions of growth factor deprivation, disrupted nutrient and energy metabolism, or during stress of chemotherapy.