RESUMEN
Recent data indicate that the chromatoid body typical of rat spermatogenesis may contain RNA synthesized in early spermatids by the haploid genome. Analyses of living step-1 and step-3 spermatids by time-lapse cinephotomicrography have shown that the chromatoid body moves in relation to the nuclear envelope in two different ways. Predominantly in step 1, the chromatoid body moves along the nuclear envelope on a wide area surrounding the Golgi complex and has frequent transient contacts with the latter organelle. In step 3, the chromatoid body was shown to move perpendicular to the nuclear envelope. It was seen located very transiently at the top of prominent outpocketings of the nuclear envelope with apparent material continuities through nuclear pore complexes to intranuclear particles. The rapid movements of the chromatoid body are suggested to play a role in the transport of haploid gene products in the early spermatids, including probably nucleocytoplasmic RNA transport.
Asunto(s)
Organoides , Espermátides/ultraestructura , Espermatogénesis , Espermatozoides/ultraestructura , Animales , Masculino , Películas Cinematográficas , Movimiento , Membrana Nuclear/ultraestructura , ARN/metabolismo , Ratas , Espermátides/metabolismo , Factores de TiempoRESUMEN
The in vitro incorporation of tritiated uridine into RNA by the spermatogenic cells of the rat has been analyzed by high-resolution autoradiography. Special attention has been focused on the unique cytoplasmic organelle, the chromatoid body. After a short labeling time (2 h), this organelle remains unlabeled in the vast majority of the early spermatids although the nuclei are labeled. When the 2-h incubation with (3H)uridine is followed by a 14-h chase, the chromatoid body is seen distinctly labeled in all spermatids during early spermiogenesis from step 1 to step 8. Very few grains are seen elsewhere in the cytoplasm of these cells. When RNA synthesis in the spermatid ceases, the chromatoid body also remains unlabeled. It is likely that the chromatoid body contains RNA which is synthesized in the nuclei of the spermatids. The function of this RNA as a stable messenger RNA needed for the regulation of late spermiogenesis is discussed.
Asunto(s)
ARN/biosíntesis , Espermátides/metabolismo , Espermatogénesis , Espermatozoides/metabolismo , Animales , Núcleo Celular/metabolismo , Masculino , Organoides/metabolismo , Ratas , Espermátides/ultraestructura , Espermatocitos/metabolismo , Espermatocitos/ultraestructura , Uridina/metabolismoRESUMEN
Changes in the level of a testis-specific hsp70 gene-related transcript (hst70 RNA) and its cellular localization during the cycle of rat seminiferous epithelium have been investigated. Segments of seminiferous tubules at defined stages of the cycle were isolated in living condition by transillumination-assisted microdissection and the exact stages identified by phase-contrast microscopy of live cell squashes. The levels of the hst70 RNA were determined by Northern and slot blotting of whole cell lysates. High levels were found in stages XII-XIV and I to early VII of the cycle, and low levels were found in other stages, i.e., late VII (VIId) through VIII-XI of the cycle. The in situ hybridization revealed that the hst70 gene was activated in late pachytene primary spermatocytes during stage XII of the cycle, and that mRNA was then present in cells during differentiation through diakinesis, meiotic divisions, and early spermiogenesis (steps 1 through early 7). The activation of the gene coding for hst70 RNA shortly before meiotic divisions may indicate that the gene product is needed either during differentiation of late spermatocytes into spermatids or later during spermiogenesis, and that the mRNA may be stored in early spermatids.
Asunto(s)
Proteínas de Choque Térmico/genética , Túbulos Seminíferos/fisiología , Testículo/fisiología , Animales , Epitelio/fisiología , Regulación de la Expresión Génica , Masculino , Hibridación de Ácido Nucleico , ARN Mensajero/genética , Ratas , Espermátides/fisiología , Espermatogénesis , Espermatogonias/fisiologíaRESUMEN
A mouse protamine 1 cDNA probe was used to study P1 protamine gene expression during the cycle of the seminiferous epithelium in the rat. In situ hybridization experiments showed that transcription of the P1 protamine mRNA starts in the middle of step 7 of spermiogenesis during substage VIIc. The mRNA levels stay high in steps 7-14 spermatids but decrease during steps 15-16 and are virtually undetectable in steps 17-19 spermatids. Northern blot analyses of RNAs isolated from microdissected pools of seminiferous tubules show high P1 protamine mRNA concentrations during stages VIIc-XIV-III of the cycle and lower levels during stages IV-VIIb. Owing to a post-transcriptional shortening of the poly(A) tail by 130 bases, a decrease in the size of protamine 1 mRNA from approximately 580 to 450 nucleotides was observed in stages XIII-XIV suggesting an initiation of protamine 1 synthesis in step 13-14 spermatids. In stages II-VI (steps 16-18 spermatids), only the smaller size protamine 1 mRNA was detectable. The expression of protamine 1 mRNAs has been localized in the very last phase of the haploid gene activity. Although the in situ hybridization suggests a disappearance of protamine 1 mRNA after step 16 of spermiogenesis, Northern blot analysis shows that low levels of mRNA are present during the period of final condensation of the chromatin, reflecting the association of protamine with DNA.
Asunto(s)
Regulación de la Expresión Génica , Protaminas/genética , ARN Mensajero/análisis , Epitelio Seminífero/análisis , Testículo/análisis , Animales , Ciclo Celular , ADN/genética , Densitometría , Masculino , Hibridación de Ácido Nucleico , Protaminas/biosíntesis , Procesamiento Proteico-Postraduccional , Ratas , Ratas Endogámicas , Epitelio Seminífero/citología , Espermátides/análisis , Espermatogénesis , Transcripción GenéticaRESUMEN
Mad is a basic region helix-loop-helix leucine zipper transcription factor which can dimerize with the Max protein and antagonize transcriptional activation by the Myc-Max transcription factor heterodimer. While the expression of Myc is necessary for cell proliferation, the expression of Mad is induced upon differentiation of at least some leukemia cell lines. Here, the expression of the mad gene has been explored in developing mouse tissues. During organogenesis in mouse embryos mad mRNA was predominantly expressed in the liver and in the mantle layer of the developing brain. At later stages mad expression was detected in neuroretina, epidermis, and whisker follicles, and in adult mice mad was expressed at variable levels in most organs analyzed. Interestingly, in the skin mad was highly expressed in the differentiating epidermal keratinocytes, but not in the underlying proliferating basal keratinocyte layer. Also, in the gut mad mRNA was abundant in the intestinal villi, where cells cease proliferation and differentiate, but not in the crypts, where the intestinal epithelial cells proliferate. In the testis, mad expression was associated with the completion of meiosis and early development of haploid cells. In cell culture, Mad inhibited colony formation of a mouse keratinocyte cell line and rat embryo fibroblast transformation by Myc and Ras. The pattern of mad expression in tissues and its ability to inhibit cell growth in vitro suggests that Mad can cause the cessation of cell proliferation associated with cell differentiation in vivo.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas Represoras , Secuencia de Aminoácidos , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Encéfalo/embriología , Encéfalo/metabolismo , Diferenciación Celular , División Celular/efectos de los fármacos , Línea Celular , Proteínas de Unión al ADN/farmacología , Humanos , Hígado/embriología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/análisis , Ratas , Alineación de Secuencia , Testículo/metabolismoRESUMEN
Vascular endothelial growth factor (VEGF) is a key regulator of endothelial growth and permeability. However, VEGF may also target nonendothelial cells, as VEGF receptors and responsiveness have been detected for example in monocytes, and high concentrations of VEGF have been reported in human semen. In this work we present evidence that overexpression of VEGF in the testis and epididymis of transgenic mice under the mouse mammary tumor virus (MMTV) LTR promoter causes infertility. The testes of the transgenic mice exhibited spermatogenic arrest and increased capillary density. The ductus epididymidis was dilated, containing areas of epithelial hyperplasia. The number of subepithelial capillaries in the epididymis was also increased and these vessels were highly permeable as judged by the detection of extravasated fibrinogen products. Intriguingly, the expression of VEGF receptor-1 (VEGFR-1) was detected in certain spermatogenic cells in addition to vascular endothelium, and both VEGFR-1 and VEGFR-2 were also found in the Leydig cells of the testis. The infertility of the MMTV-VEGF male mice could thus result from VEGF acting on both endothelial and nonendothelial cells of the male genital tract. Taken together, these findings suggest that the VEGF transgene has nonendothelial target cells in the testis and that VEGF may regulate male fertility.
Asunto(s)
Factores de Crecimiento Endotelial/genética , Epidídimo/metabolismo , Infertilidad Masculina/genética , Linfocinas/genética , Testículo/metabolismo , Animales , Factores de Crecimiento Endotelial/biosíntesis , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Epidídimo/irrigación sanguínea , Epidídimo/patología , Regulación de la Expresión Génica , Vectores Genéticos , Humanos , Hiperplasia , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Linfocinas/biosíntesis , Masculino , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento/genética , Receptores Mitogénicos/genética , Receptores de Factores de Crecimiento Endotelial Vascular , Túbulos Seminíferos/patología , Espermatogénesis , Secuencias Repetidas Terminales , Testículo/irrigación sanguínea , Testículo/patología , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
beta-Nerve growth factor (NGF) is expressed in spermatogenic cells and has testosterone-downregulated low-affinity receptors on Sertoli cells suggesting a paracrine role in the regulation of spermatogenesis. An analysis of the stage-specific expression of NGF and its low affinity receptor during the cycle of the seminiferous epithelium in the rat revealed NGF mRNA and protein at all stages of the cycle. Tyrosine kinase receptor (trk) mRNA encoding an essential component of the high-affinity NGF receptor was also present at all stages. In contrast, expression of low affinity NGF receptor mRNA was only found in stages VIIcd and VIII of the cycle, the sites of onset of meiosis. The low-affinity NGF receptor protein was present in the plasma membrane of the apical Sertoli cell processes as well as in the basal plasma membrane of these cells at stages VIIcd to XI. NGF was shown to stimulate in vitro DNA synthesis of seminiferous tubule segments with preleptotene spermatocytes at the onset of meiosis while other segments remained nonresponsive. We conclude that NGF is a meiotic growth factor that acts through Sertoli cells.
Asunto(s)
Meiosis/fisiología , Factores de Crecimiento Nervioso/biosíntesis , Receptores de Superficie Celular/biosíntesis , Epitelio Seminífero/metabolismo , Animales , Diferenciación Celular/fisiología , Membrana Celular/química , Polaridad Celular , ADN/biosíntesis , Inmunohistoquímica , Masculino , Microscopía Inmunoelectrónica , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas , Receptores de Factor de Crecimiento Nervioso , Células de Sertoli/metabolismo , Espermatocitos/metabolismo , Espermatogénesis/fisiologíaRESUMEN
The molecular control of self-renewal and differentiation of stem cells has remained enigmatic. Transgenic loss-of-function and overexpression models now show that the dosage of glial cell line-derived neurotrophic factor (GDNF), produced by Sertoli cells, regulates cell fate decisions of undifferentiated spermatogonial cells that include the stem cells for spermatogenesis. Gene-targeted mice with one GDNF-null allele show depletion of stem cell reserves, whereas mice overexpressing GDNF show accumulation of undifferentiated spermatogonia. They are unable to respond properly to differentiation signals and undergo apoptosis upon retinoic acid treatment. Nonmetastatic testicular tumors are regularly formed in older GDNF-overexpressing mice. Thus, GDNF contributes to paracrine regulation of spermatogonial self-renewal and differentiation.
Asunto(s)
Proteínas de Drosophila , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/fisiología , Espermatogénesis , Espermatogonias/citología , Células Madre/citología , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular , Diferenciación Celular/efectos de los fármacos , Cobalto/metabolismo , Femenino , Expresión Génica , Marcación de Gen , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Masculino , Ratones , Ratones Transgénicos , Mitosis , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ret , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células de Sertoli/citología , Células de Sertoli/fisiología , Espermatogonias/efectos de los fármacos , Neoplasias Testiculares/patología , Testículo/anatomía & histología , Vitamina A/farmacologíaRESUMEN
In 1996-2005, ejaculates of 2048 boars were collected. All boars were intended for use in artificial insemination or natural breeding and had two descended testes. Azoospermia was present in 16 of the 1097 Yorkshire boars (1.5%) and in 2 of the 951 Landrace boars (0.2%). The two most frequent diagnoses of azoospermia were arrested spermatogenesis at the pachytene spermatocyte stage (n=8) and segmental aplasia of the Wolffian ducts (n=7). Morphometric evaluations of testicular tissues of azoospermic boars were performed using an image analyzer. The morphometric evaluations revealed decreased portions and diameter of seminiferous tubule in tissue slides from the studied azoospermic boars compared with normal boars. The use of an image analyzer for morphometric evaluations of testicular tissues proved to be a good tool to characterize findings in testicular slides of azoospermic boars.
Asunto(s)
Azoospermia/veterinaria , Enfermedades de los Porcinos/patología , Porcinos/genética , Porcinos/fisiología , Testículo/patología , Animales , MasculinoRESUMEN
Despite well-documented cardiotoxic effects, doxorubicin remains a major anticancer agent. To study the role of myocardial apoptosis following doxorubicin administration, male Wistar rats were exposed to 1.25, 2.5, and 5 mg/kg of i.p. doxorubicin and terminated on days 1-7 in groups of five. Doxorubicin caused a significant (P < 0.001) and dose-dependent induction of cardiomyocyte apoptosis at 24-48 h after the injection. Repeated injections of 2.5 mg/kg given every other day resulted in peaks of apoptosis at 24 h after each injection. However, no additive effect of repeated dosing was noted. In histological samples, alterations in the cytoskeletal apparatus with focal loss of contractile elements were seen after a single injection. Myocyte necrosis was absent. Thus, acute doxorubicin-induced cardiotoxicity involves cardiomyocyte apoptosis, a potentially preventable form of myocardial tissue loss.
Asunto(s)
Apoptosis/efectos de los fármacos , Doxorrubicina/toxicidad , Corazón/efectos de los fármacos , Miocardio/patología , Animales , Relación Dosis-Respuesta a Droga , Etiquetado Corte-Fin in Situ , Masculino , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The secretion of plasminogen activator by seminiferous tubules at defined stages of the epithelial cycle is influenced both by neighboring spermatogenic cells and by hormones. We have used cRNA probes for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators to analyze their mRNA levels in different stages of the epithelial cycle. Urokinase-type PA mRNA was most abundant in stages VII-VIII, while tPA mRNA levels showed smaller variations between the different stages. Both FSH and (Bu)2cAMP increased the steady-state level of tPA mRNA and tPA production without affecting those of uPA in stages VII-IX in vitro, whereas retinoic acid treatment selectively increased the concentration uPA mRNA and uPA production in stages II-VI. The results show that the expression of the uPA and tPA genes is differentially regulated in specific stages of the rat seminiferous epithelium.
Asunto(s)
Regulación de la Expresión Génica , Epitelio Seminífero/metabolismo , Testículo/metabolismo , Activador de Tejido Plasminógeno/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Animales , Northern Blotting , Bucladesina/farmacología , Electroforesis en Gel de Poliacrilamida , Hormona Folículo Estimulante/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Hibridación de Ácido Nucleico , Activadores Plasminogénicos/metabolismo , Sondas ARN , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas , Tretinoina/farmacologíaRESUMEN
Polyamines are believed to participate in the induction of cell growth, differentiation, and proliferation, but their role in spermatogenesis has remained obscure. Two transgenic mouse lines (K2 and K15) that overexpress the human ornithine decarboxylase (ODC) gene coding for a rate-controlling enzyme in polyamine biosynthesis and, hence, contain high levels of tissue putrescine have been used to study the stage-specific role of ODC in spermatogenesis. In K2 mice with 30-fold testicular ODC overexpression, [3H]thymidine incorporation at stages I-VI of the cycle of the seminiferous epithelium was significantly above the control level. This may reflect a specific stimulation of DNA synthesis in type A4, intermediate, and type B spermatogonia. The K15 mice that have about 70-fold ODC overexpression showed an elevation of DNA synthesis only at stage V of the cycle, suggesting a specific dependence of type B spermatogonia on putrescine. In K15 mice, [3H]thymidine incorporation of stage VIII tubule segments was decreased, suggesting that excess amounts of putrescine selectively inhibit meiotic DNA synthesis. We propose that putrescine has strictly selective local stimulatory and inhibitory actions during spermatogenic DNA synthesis, and that its excess amounts ultimately may lead to decreased fertility.
Asunto(s)
Ornitina Descarboxilasa/biosíntesis , Poliaminas/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Espermatogénesis/fisiología , Espermatogonias/metabolismo , Animales , Secuencia de Bases , Replicación del ADN , Inducción Enzimática , Citometría de Flujo , Humanos , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Ornitina Descarboxilasa/genética , Proteínas Recombinantes de Fusión/genética , Testículo/metabolismoRESUMEN
Mutations in the human DAX-1 gene lead to X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism. DAX-1 has been proposed to play a role in steroidogenesis because it is highly expressed in adrenocortical and testicular Leydig cells and because loss-of-function mutations lead to low serum levels of steroid hormones. Recent reports of DAX-1 expression in hypothalamus and pituitary, however, suggest additional functions for this protein. Here we demonstrate that DAX-1 is expressed in Sertoli cells of rat testis. This expression is regulated during spermatogenesis and peaks during the androgen-sensitive phase of the spermatogenic cycle. In addition, we show that DAX-1 expression in Sertoli cells is regulated developmentally. Maximum levels are present in the rat between postnatal days 20 and 30, during the first spermatogenic wave. Moreover, we show that activation of the cAMP-signaling pathway by the pituitary hormone FSH leads to a potent down-regulation of DAX-1 expression in cultured Sertoli cells. This down-regulation requires transcription and de novo protein synthesis. Taken together, these data indicate that DAX-1 expression in Sertoli cells may influence the development of spermatogenic cells in response to steroid and pituitary hormones.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Receptores de Ácido Retinoico/biosíntesis , Proteínas Represoras , Células de Sertoli/metabolismo , Espermatogénesis/fisiología , Testículo/crecimiento & desarrollo , Factores de Transcripción/biosíntesis , Animales , Células Cultivadas , AMP Cíclico/genética , AMP Cíclico/metabolismo , Cicloheximida/farmacología , Receptor Nuclear Huérfano DAX-1 , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Dactinomicina/farmacología , Regulación hacia Abajo , Hormona Folículo Estimulante/metabolismo , Hormona Folículo Estimulante/farmacología , Regulación del Desarrollo de la Expresión Génica , Masculino , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Ratas Wistar , Receptores de Ácido Retinoico/efectos de los fármacos , Receptores de Ácido Retinoico/genética , Células de Sertoli/efectos de los fármacos , Testículo/metabolismo , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
In situ hybridization was used to localize the mRNA expression of the high affinity activin receptor (ActR-IIB2) in the rat seminiferous epithelium. ActR-IIB2 mRNA was expressed maximally in stages IX-XI of the seminiferous epithelial cycle. The mRNA signal was detected basally in the epithelium in type A1 and A2 spermatogonia and in Sertoli cells. In the pubertal rat testis the expression was localized in Sertoli cells around primary spermatocytes and around meiotically dividing cells. The localization of ActR-IIB2 mRNA in spermatogonia lends support to the hypothesis that activin is a spermatogonial growth factor. The expression of activin receptor mRNA in pubertal rat testis suggests that activin may have a function during meiotic maturation.
Asunto(s)
ARN Mensajero/análisis , Receptores de Superficie Celular/genética , Epitelio Seminífero/química , Receptores de Activinas , Animales , Hibridación in Situ , Masculino , Ratas , Ratas Sprague-Dawley , Células de Sertoli/química , Espermatozoides/química , Testículo/químicaRESUMEN
The quantitative effects of ethane dimethane sulfonate (EDS), a Leydig cell toxin, on apoptosis in adult rat seminiferous epithelium were studied by the improved transillumination method. Nonradioactive in situ end labeling of fragmented DNA in squash preparations revealed significant increases in apoptotic cells in stages II-XI, whereas controls showed 0.5-2.3 apoptotic cells/mm tubule. Seven days post-EDS treatment, the highest numbers of apoptotic cells were scored in stages VIIab and VIIcd (74.7 +/- 23.8 and 61.3 +/- 16.0 cells/mm, respectively). The effects were suppressed by testosterone (T) supplementation, except in stages II-III and VIIcd. An opposite effect was found in stage XII, where the number of apoptotic cells decreased 1, 3, and 7 days after EDS treatment and returned to control levels in T-supplemented rats. Electrophoretic analysis of internucleosomal DNA fragmentation revealed a biphasic apoptotic process after 1 and 5-7 days due to Leydig and germ cell apoptosis, respectively. The specific germ cell apoptosis was also confirmed by electron microscopic analysis. The results suggest that T withdrawal induces apoptotic cell death in most stages of the cycle and that the effects are largely preventable. In stage XII, however, T seems to promote apoptosis in premeiotic cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Túbulos Seminíferos/citología , Testosterona/farmacología , Animales , Autorradiografía , Ciclo Celular , Hormonas/sangre , Células Intersticiales del Testículo/fisiología , Masculino , Mesilatos/farmacología , Microscopía Electrónica , Ratas , Ratas Sprague-DawleyRESUMEN
The in vitro effects of insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), truncated IGF-I, insulin, and human GH (hGH) on premitotic and premeiotic DNA synthesis of adult rat germ cells in vitro were investigated. Two-millimeter segments of seminiferous tubules from four different stages containing type A4-spermatogonia (stage I), type B spermatogonia (stage V), resting preleptotene spermatocytes (stage VIIa), and preleptotene spermatocytes in the S-phase (stage VIII-IX), respectively, were isolated by transillumination-assisted microdissection. They were cultured in serum-free medium at 34 or 37 C with and without growth factors, labeled for 4 h with tritiated thymidine, and harvested at 24, 48, and 72 h. Spontaneous progression of spermatogenesis was noted at both incubation temperatures, with a more rapid rate at 37 C. IGF-I significantly stimulated [3H]thymidine uptake in originally stage I and stage V tubule segments (type A4 and B spermatogonia, respectively) after 48 h of culture at 37 C. Improved maintenance of the DNA synthesis of stage VIII-IX tubules was found after 48 h at 37 C and 72 h at 34 C. Truncated IGF-I produced a similar response, but was more potent. IGF-II showed slight stimulation of stage V tubules after 72 h at both 34 and 37 C and maintenance of stage VIII-IX tubules after 48 h at 37 C and 72 h at 34 C. hGH was effective only at 34 C, showing slight stimulation of stage I tubule segments after 48 and 72 h of incubation. Insulin at high concentrations was effective only at 37 C and stimulated DNA synthesis in stages I, V, and VIIa after 48 h and stages V and VIIa after 72 h of incubation. It is concluded that IGFs stimulate premitotic DNA synthesis of rat germ cells in vitro and may also maintain premeiotic DNA synthesis. Whether the slight response to hGH is mediated via local production of IGF-I by the tissue cultures remains to be investigated. As IGF-I and IGF-II are locally produced in the testis, the present results suggest that these factors have a selective paracrine or autocrine role in the regulation of spermatogonial proliferation during spermatogenesis.
Asunto(s)
ADN/biosíntesis , Factor II del Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Meiosis/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , Espermatogonias/metabolismo , Animales , Hormona del Crecimiento/farmacología , Insulina/farmacología , Masculino , Meiosis/fisiología , Ratas , Ratas Sprague-Dawley , Espermatogénesis/fisiología , Espermatogonias/efectos de los fármacos , Espermatozoides/citología , Espermatozoides/metabolismo , Factores de TiempoRESUMEN
The cyclic secretion of plasminogen activator (PA) by Sertoli cells in stages VII and VIII of the rat seminiferous epithelial cycle is influenced by hormones and adjacent spermatogenic cells. To understand this interaction more in detail, we have analyzed the effects of FSH, (Bu)2cAMP, testosterone, insulin, and retinoic acid (RA) on staged seminiferous tubule segments in vitro. FSH stimulated stages VIIcd to XI of the cycle; similar results were obtained with (BU)2cAMP. RA stimulated PA secretion in stages I-VIIab, but testosterone and insulin had no effect in any stage. The secreted PA was mainly of the urokinase type, although small amounts of the tissue-type PA were found after stimulation by FSH and cAMP. These results suggest that spermatogenic cells modify the responsiveness of Sertoli cells to hormonal stimulation. Stages I-VIIab are sensitive to stimulation by RA whereas stages VIIcd-XI are preferentially stimulated by FSH and (Bu)2cAMP.
Asunto(s)
Túbulos Seminíferos/metabolismo , Espermatogénesis/efectos de los fármacos , Testículo/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Bucladesina/farmacología , Técnicas de Cultivo , Hormona Folículo Estimulante/farmacología , Insulina/farmacología , Masculino , Ratas , Testosterona/farmacología , Tretinoina/farmacologíaRESUMEN
Activin and inhibin are members of the transforming growth factor-beta (TGF beta) gene family. They are expressed in various organ systems, where they possess regulatory functions. Inhibin, activin, and TGF beta have been reported to also be expressed in the adult rat testis. We studied in vitro the action of these growth factors on premitotic and premeiotic DNA synthesis during the rat seminiferous epithelial cycle. Two-millimeter rat seminiferous tubule segments were isolated by transillumination-assisted microdissection from stages V, VIIa, VIII-IX, and I of the cycle and incubated in vitro in the presence of activin-A, inhibin-A, or TGF beta 1. During 24-, 48-, and 72-h incubation spontaneous progression of spermatogenesis was noted. The staged samples allowed us to selectively quantitate DNA synthetic activity of specific germ cell types. At the end of the culture, the tubules were pulse labeled with [3H]thymidine, and DNA synthesis was quantified by liquid scintillation counting, and the activated cells were detected by autoradiography. Activin-A stimulated preleptotene spermatocyte DNA synthesis in a dose-dependent manner. DNA synthesis of intermediate spermatogonia was also stimulated by activin-A, whereas inhibin-A inhibited DNA synthesis of these cells. TGF beta 1 had a small, but significant, stimulatory effect on DNA synthetic activity at stage VII. These results support the view that activin-A, inhibin-A, and TGF beta 1 take part in the regulation of DNA synthesis during rat spermatogenesis.
Asunto(s)
ADN/biosíntesis , Inhibinas/farmacología , Túbulos Seminíferos/metabolismo , Espermatogénesis/fisiología , Factor de Crecimiento Transformador beta/farmacología , Activinas , Animales , Técnicas de Cultivo , ADN/antagonistas & inhibidores , Masculino , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Testicular androgen receptors were measured with a recently developed exchange assay using [3H]methyltrienolone from samples of interstitial tissue, whole seminiferous tubules, and segments of tubules in different stages of the cycle of the seminiferous epithelium. The hexylene glycol method was used to isolate nuclei and pyridoxal 5'-phosphate for extraction of the androgen receptors. This method proved superior to other techniques employed by our own and other laboratories. As a consequence, higher levels of androgen receptors were detected in seminiferous tubules than previously reported. Androgen receptors in seminiferous tubules and interstitial tissue had high affinity for methyltrienolone (Kd = approximately 3 nM) and steroid binding specificity similar to that of these receptors in other tissues. The concentration of nuclear androgen receptors in whole seminiferous tubules was 670 +/- 100 fmol/mg DNA (mean +/- SE), while that of the interstitial tissue was 1070 +/- 295 fmol/mg DNA. Tubules in stages IX-XII and XIII-I of the epithelial cycle contained significantly more nuclear androgen receptors (900 +/- 170 and 805 +/- 125 fmol/mg DNA, respectively) than those in stages II-VI and VII-VIII (485 +/- 95 and 485 +/- 65 fmol/mg DNA, respectively). These results suggest that there are local differences in androgen receptor concentration along the length of the seminiferous tubule. A high concentration of nuclear androgen receptors was also present in interstitial tissue. Androgen receptors were measurable in cytosol prepared from interstitial tissue, but such measurements were obscured in cytosol from tubules, because of a high capacity binding protein for the 3H-labeled ligand. We conclude that nuclear androgen receptors can be measured in various testicular compartments, including different stages of the seminiferous tubules, using an exchange assay that maximizes recovery. The concentration of nuclear androgen receptors in the tubules varies with the cycle of the seminiferous epithelium.
Asunto(s)
Receptores Androgénicos/metabolismo , Receptores de Esteroides/metabolismo , Epitelio Seminífero/metabolismo , Testículo/metabolismo , Animales , Diferenciación Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , Estrenos/metabolismo , Masculino , Metribolona , Ratas , Ratas Endogámicas , Epitelio Seminífero/citologíaRESUMEN
Levels of rat testicular interleukin-1-like factor (tIL-1) have been shown to correlate with DNA synthetic activity during the cycle of the rat seminiferous epithelium, suggesting its role as a spermatogonial or meiotic growth factor. To explore this further, a new in vitro model system was developed. Rat seminiferous tubule segments from stages I, V, VIIa, and VIII-IX of the cycle were isolated by transillumination-assisted microdissection, cultured in chemically defined serum-free medium supplemented with human recombinant IL-1 alpha, and labeled with [3H]thymidine. During incubation, spontaneous progression of spermatogenesis was noted. Inactive stage VIIa tubule segments differentiated to stage VIII and initiated DNA synthesis, and concomitantly started to secrete IL-1-like factor. DNA synthesis of stages VIII-IX ceased through differentiation of spermatocytes to leptotene-zygotene (stages XII-XIII of the cycle). IL-1 alpha stimulated DNA synthesis significantly in spermatogonia of stage I. Meiotic DNA synthesis at stage VIIa was stimulated (48 h/34 C) and maintained at stages VIII-IX (48 h/34 C). IL-1 alpha seems to act as a regulator of spermatogenic DNA synthesis in both mitotic and meiotic phases. It has mainly stimulating and maintaining effects, but it may also be inhibitory under certain conditions.