RESUMEN
DMRT1 is an evolutionarily conserved transcriptional factor expressed only in the postnatal testis, where it is produced in Sertoli cells and germ cells. While deletion of Dmrt1 in mice demonstrated it is required for postnatal testis development and fertility, much is still unknown about its temporal- and cell-specific functions. This study characterized a novel mouse model of DMRT1-deficient germ cells that was generated by breeding Dmrt1-null (Dmrt1(-/-)) mice with Wt1-Dmrt1 transgenic (Dmrt1(+/-;tg)) mice, which express a rat Dmrt1 cDNA in gonadal supporting cells by directing it from the Wilms tumor 1 locus in a yeast artificial chromosome transgene. Like Dmrt1(-/-) mice, male Dmrt1(-/-) transgenic mice (Dmrt1(-/-;tg)) were infertile, while female mice were fertile. Immunohistochemistry and Western blot analysis showed transgenic DMRT1 expressed in supporting cells of the newborn gonads of both sex and in Sertoli cells of the testis afterbirth. Sertoli cells were evaluated by electron microscopy, revealing that maturation of Dmrt1(-/-;tg) Sertoli cells was incomplete. Morphological analysis of testes from 42-day-old mice showed that, compared to Dmrt1(-/-) mice, Dmrt1(-/-;tg) mice have improved seminiferous tubule structure, with lumens present in many. Immunohistochemistry of the polarity markers ESPIN and NECTIN-2 showed that DMRT1 in Sertoli cells is required for NECTIN-2 expression and influences organization of ectoplasmic specializations. Further functional analyses of the transgene on a Dmrt1(-/-) background showed that it did not rescue the decrease in Dmrt1(-/-) testis size, but when expressed on a wild-type background, exogenous DMRT1 prevented the normal age-related decline in testis size and enhanced sperm progressive motility. The studies suggest that DMRT1 in Sertoli cells regulates tubule morphology, spermatogenesis, and sperm function via its effects on Sertoli cell maturation and polarity. Furthermore, expression and function of transgenic DMRT1 in Sertoli cells establishes a novel mouse model of DMRT1-deficient germ cells generated by breeding Dmrt1-null mice with Wt1-Dmrt1 transgenic mice (rescue; Dmrt1(-/-;tg)).
Asunto(s)
Modelos Animales , Células de Sertoli/metabolismo , Testículo/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transgenes/genética , Animales , Moléculas de Adhesión Celular/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas de Microfilamentos/metabolismo , Microscopía Electrónica de Transmisión , Nectinas , Células de Sertoli/patología , Células de Sertoli/ultraestructura , Motilidad Espermática/fisiología , Testículo/patología , Testículo/ultraestructura , Factores de Transcripción/metabolismoRESUMEN
Immunohistochemistry was used to examine GCNA1, a germ cell-specific protein, together with DMRT1 (Doublesex and Mab-3-related transcription factor-1), a transcription factor implicated in Sertoli cell and germ cell function, in order to resolve DMRT1's cellular profile during pre- and postnatal gonad development in the mouse. In the indifferent gonad (10.5-11.5 days postcoitus [dpc]), DMRT1 localized to somatic cells and GCNA1(+) germ cells and was indistinguishable in males and females. By 12.5 dpc, a clear sexual preference for DMRT1 in male somatic cells was observed, with male DMRT1 localized to testicular cords and more abundant in Sertoli cells than in germ cells and female DMRT1 diffusely labeled and markedly lower in somatic cells than in germ cells. A male somatic preference continued throughout development, with DMRT1 evident in Sertoli cells at all ages examined and absent in ovarian somatic cells from 13.5 dpc onward. In contrast, expression in primordial germ cells was not sexually distinct, and both sexes showed DMRT1 increasing through 13.5 dpc and absent by 15.5 dpc. Notably, sexual differences in germ cell DMRT1 were detected after birth, when it was detected only in spermatogonia of the testis. Colocalization of DMRT1 with proliferation markers KI67 and proliferating cell nuclear antigen (PCNA) and stem cell markers OCT4 (also known as POU5F1) and NGN3 indicated that, in postnatal testes, DMRT1 was present in both stem and proliferating spermatogonia. Together, the findings implicate opposite functions for DMRT1 in somatic and germ cells of the testis. In Sertoli cells, DMRT1 expression correlated with differentiation, whereas in germ cells, it suggested a role in expansion and maintenance of undifferentiated spermatogonia.
