RESUMEN
The primary goal of this study was to determine the 5'region of the Insl3 gene that specifically targets the expression of human insulin to Leydig cells, and to explore whether the testicular proinsulin is efficiently processed to insulin that is able to rescue the diabetes in different mouse models of diabetes. We show here that the sequence between nucleotides -690 and +4 of mouse Insl3 promoter is sufficient to direct the Leydig cell-specific expression of the human insulin transgene (Insl3-hIns). We also found that the 3'untranslated region (3'UTR) of Insl3 was effective in enhancing transgene expression of the insulin in vivo. Expression analysis revealed that the temporal expression pattern of the hIns transgene in Leydig cells of transgenic testes is roughly the same as that of the endogenous Insl3. Despite the Leydig cells translate human proinsulin and secrete a significant level of free C-peptide into the serum, the Leydig cell-derived insulin is not able to overcome the diabetes in different mouse models of diabetes, suggesting a lack of glucose sensing mechanisms in the Leydig cells. A consequence of overexpression of the human proinsulin in Leydig cells was the decrease of fertility of transgenic males at older ages. Germ cells in transgenic males were able to initiate and complete spermatogenesis. However, there was a progressive and age-dependent degeneration of the germ cells that lead to male infertility with increasing age.
Asunto(s)
Insulina/metabolismo , Células Intersticiales del Testículo/metabolismo , Proinsulina/metabolismo , Regiones Promotoras Genéticas , Proteínas/genética , Espermatozoides/metabolismo , Regiones no Traducidas 3' , Factores de Edad , Animales , Glucemia/metabolismo , Péptido C/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Insulina/genética , Células Intersticiales del Testículo/patología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Proinsulina/genética , Espermatozoides/patología , Factores de Tiempo , Regulación hacia ArribaAsunto(s)
Gonadotropina Coriónica/sangre , Síndrome de Down/diagnóstico , Trofoblastos/metabolismo , Biomarcadores/sangre , Gonadotropina Coriónica/metabolismo , Gonadotropina Coriónica Humana de Subunidad beta/sangre , Femenino , Humanos , Embarazo , Primer Trimestre del Embarazo , Proteína Plasmática A Asociada al Embarazo/análisisRESUMEN
Triple knockout mice were used to investigate the interactions of five genes that were expressed in meiotic and haploid spermatogenic cells in mice, transition protein 2 (Tnp2), proacrosin (Acr), histone H1.1 (H1.1), histone H1t (H1t), and sperm mitochondria-associated cysteine-rich protein (Smcp). TNP2 functions in the replacement of histones and the initial condensation of the spermatid nucleus. The linker histone subtypes H1.1 and H1t are expressed at high levels in meiotic and early haploid cells. ACR, a protease that is stored as a proenzyme in the acrosome, is activated during the acrosome reaction and functions in binding of sperm to the zona pellucida. SMCP is a structural protein in the outer membranes of sperm mitochondria that functions in motility. Previous work demonstrates that homozygous knockout mice lacking each of these proteins individually exhibit no defect in fertility on mixed genetic backgrounds. In contrast, the present study demonstrates that five triple knockout lines, Acr/H1.1/Smcp, Acr/Tnp2/Smcp, Tnp2/H1.1/Smcp, Acr/H1t/Smcp, Tnp2/H1t/Smcp, exhibit drastic reductions in fertility on mixed genetic backgrounds. Analysis of fertility parameters reveal that the decreased fertility is due to line-dependent defects in sperm motility in vitro correlated with reduced migration in the female reproductive tract, and decreased fertilization due to defects in adhesion of sperm to the zona pellucida, the membrane surrounding the egg. It was also found that triple knockout males, that are hemizygous for one locus and homozygous for two other loci, are as subfertile as homozygous triple knockout males, a phenomenon known as haploinsufficiency. These findings demonstrate that male fertility involves synergistic interactions of genes that function in sperm motility and sperm-egg adhesion during fertilization.
Asunto(s)
Fertilización/genética , Infertilidad Masculina/genética , Motilidad Espermática/genética , Espermatozoides/metabolismo , Reacción Acrosómica/genética , Reacción Acrosómica/fisiología , Animales , Epidídimo/citología , Fertilización/fisiología , Infertilidad Masculina/metabolismo , Masculino , Ratones , Ratones Noqueados , Motilidad Espermática/fisiologíaRESUMEN
To analyze the biological role of factor XII (FXII, Hageman Factor) in vivo, we generated mice deficient for FXII using a gene targeting approach on two distinct genetic backgrounds, i.e. mixed C57Bl/6J X 129X1/SvJ and inbred 129X1/SvJ. Homozygous FXII knockout (FXII(-)/(-)) mice showed no FXII plasma activity and had a markedly prolonged activated partial thromboplastin time (aPTT). In contrast, coagulation factors XI, VIII, IX, X,VII, V, II and fibrinogen did not differ between FXII(-/-) mice and their wild-type littermates. Heterozygous matings segregated according to the Mendelian inheritance indicating that FXII deficiency does not increase fetal loss. Furthermore, matings of FXII(-/-) males and FXII(-/-) females resulted in normal litter sizes demonstrating that total FXII deficiency in FXII(-/-) females does not affect pregnancy outcome. Also, gross and histological anatomy of FXII(-/-) mice was indistinguishable from that of their wild-type littermates on both genetic backgrounds. Thus it appears that deficiency of murine FXII does not cause thrombophilia or impaired fibrinolysis in vivo. These results indicate that FXII deficiency does not affect hemostasis in vivo and we anticipate that the FXII(-/-) mice will be helpful to elucidate the biological role(s) of FXII in health and disease.
Asunto(s)
Factor XII/genética , Factor XII/fisiología , Ratones Noqueados , Animales , Activación Enzimática , Femenino , Fibrinólisis , Hemostasis , Humanos , Patrón de Herencia , Masculino , Ratones , Modelos Animales , Tiempo de Tromboplastina Parcial , Embarazo , Resultado del Embarazo , TrombofiliaRESUMEN
In humans, male and female partners contribute more or less equally to the infertility problem. In approximately 20% of infertile couples, the concurrence of male and female factors is suggested to be responsible for infertility. Neither of these factors are known nor is there a model system to prove this assumption. We present such a model system in the mouse, in which the lack of acrosin in the male and modifications of the zona pellucida (ZP) in the female result in a significant reduction of the fertilization rate in vitro. We generated mice carrying a deletion in the proline-rich region (PRR) of the proacrosin gene, resulting in the absence of proacrosin in the homozygous PRR(-/-) male mouse. Under normal conditions, sperm from the proacrosin-deficient mice are still capable of ZP penetration and fertilization. In this study, modifications of the ZP of oocytes after superovulation were achieved by treatment with dimethylsulphoxide or aroclor-1254 or by in-vitro ageing. It is known that under these conditions, a time-dependent hardening of the ZP occurs. The rates of fertilization in vitro of treated and aged oocytes using sperm from PRR(-/-) mice were found to be significantly reduced when compared with those reached with wild-type sperm. The relevance of the acrosin status and ZP condition for fertilization success were further substantiated by the finding that the fertilization rate with PRR(-/-) sperm is affected by the thickness of the ZP. Our results demonstrate that the lack of acrosin in sperm in combination with modifications to the ZP can affect fertility and can be an experimental model for the study of unexplained infertility in human couples in which both male- and female-derived factors are suggested to be the underlying causes.
Asunto(s)
Acrosina/genética , Acrosina/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Infertilidad/fisiopatología , Zona Pelúcida/fisiología , Acrosina/deficiencia , Animales , Dimetilsulfóxido/efectos adversos , Modelos Animales de Enfermedad , Precursores Enzimáticos/deficiencia , Femenino , Fertilización In Vitro , Expresión Génica , Infertilidad/inducido químicamente , Infertilidad/genética , Masculino , Ratones , Ratones Endogámicos , Ratones Mutantes , Oocitos/química , Oocitos/fisiología , Prolina/genética , Estructura Terciaria de Proteína , Factores de Tiempo , Zona Pelúcida/efectos de los fármacosRESUMEN
The sperm mitochondria-associated cysteine-rich protein (SMCP) is a cysteine- and proline-rich structural protein that is closely associated with the keratinous capsules of sperm mitochondria in the mitochondrial sheath surrounding the outer dense fibers and axoneme. To investigate the function of SMCP, we generated mice with a targeted disruption of the gene Smcp by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J x 129/Sv) are fully fertile, while they are infertile on the 129/Sv background, although spermatogenesis and mating are normal. Homozygous Smcp(-/-) female mice are fertile on both genetic backgrounds. Electron microscopical examination demonstrated normal structures of sperm head, mitochondria, and tail. In vivo experiments with sperm of Smcp(-/-) 129/Sv mice revealed that the migration of spermatozoa from the uterus into the oviduct is reduced. This result is supported by the observation that sperm motility as determined by the computer-assisted semen analysis system (CASA) is significantly affected as compared to wild-type spermatozoa. In vitro fertilization assays showed that Smcp-deficient spermatozoa are able to bind to the oocyte but that the number of fertilized eggs is reduced by more than threefold relative to the wild-type control. However, removal of the zona pellucida resulted in an unaffected sperm-egg fusion which was monitored by the presence of pronuclei and generation of blastocyts. These results indicate that the infertility of the male Smcp(-/-) mice on the 129/Sv background is due to reduced motility of the spermatozoa and decreased capability of the spermatozoa to penetrate oocytes.