RESUMEN
SPATS1 (spermatogenesis-associated, serine-rich 1) is an evolutionarily conserved, testis-specific protein that is differentially expressed during rat male meiotic prophase. Some reports have suggested a link between SPATS1 underexpression/mutation and human pathologies such as male infertility and testicular cancer. Given the absence of functional studies, we generated a Spats1 loss-of-function mouse model using CRISPR/Cas9 technology. The phenotypic analysis showed no overt phenotype in Spats1-/- mice, with both males and females being fertile. Flow cytometry and histological analyses did not show differences in the testicular content and histology between WT and knockout mice. Moreover, no significant differences in sperm concentration, motility, and morphology, were observed between WT and KO mice. These results were obtained both for young adults and for aged animals. Besides, although an involvement of SPATS1 in the Wnt signaling pathway has been suggested, we did not detect changes in the expression levels of typical Wnt pathway-target genes in mutant individuals. Thus, albeit Spats1 alteration might be a risk factor for male testicular health, we hereby show that this gene is not individually essential for male fertility and spermatogenesis in mouse.
Asunto(s)
Fertilidad/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Espermatogénesis/fisiología , Secuencia de Aminoácidos , Animales , Femenino , Infertilidad Masculina/metabolismo , Masculino , Meiosis/fisiología , Ratones , Ratones Noqueados , Neoplasias de Células Germinales y Embrionarias/metabolismo , Serina/metabolismo , Recuento de Espermatozoides/métodos , Motilidad Espermática/fisiología , Espermatozoides/metabolismo , Neoplasias Testiculares/metabolismo , Testículo/metabolismoRESUMEN
BACKGROUND: Spermatogenesis is a complex differentiation process that involves the successive and simultaneous execution of three different gene expression programs: mitotic proliferation of spermatogonia, meiosis, and spermiogenesis. Testicular cell heterogeneity has hindered its molecular analyses. Moreover, the characterization of short, poorly represented cell stages such as initial meiotic prophase ones (leptotene and zygotene) has remained elusive, despite their crucial importance for understanding the fundamentals of meiosis. RESULTS: We have developed a flow cytometry-based approach for obtaining highly pure stage-specific spermatogenic cell populations, including early meiotic prophase. Here we combined this methodology with next generation sequencing, which enabled the analysis of meiotic and postmeiotic gene expression signatures in mouse with unprecedented reliability. Interestingly, we found that a considerable number of genes involved in early as well as late meiotic processes are already on at early meiotic prophase, with a high proportion of them being expressed only for the short time lapse of lepto-zygotene stages. Besides, we observed a massive change in gene expression patterns during medium meiotic prophase (pachytene) when mostly genes related to spermiogenesis and sperm function are already turned on. This indicates that the transcriptional switch from meiosis to post-meiosis takes place very early, during meiotic prophase, thus disclosing a higher incidence of post-transcriptional regulation in spermatogenesis than previously reported. Moreover, we found that a good proportion of the differential gene expression in spermiogenesis corresponds to up-regulation of genes whose expression starts earlier, at pachytene stage; this includes transition protein-and protamine-coding genes, which have long been claimed to switch on during spermiogenesis. In addition, our results afford new insights concerning X chromosome meiotic inactivation and reactivation. CONCLUSIONS: This work provides for the first time an overview of the time course for the massive onset and turning off of the meiotic and spermiogenic genetic programs. Importantly, our data represent a highly reliable information set about gene expression in pure testicular cell populations including early meiotic prophase, for further data mining towards the elucidation of the molecular bases of male reproduction in mammals.
Asunto(s)
Fase Paquiteno/genética , Espermatogénesis/genética , Transcriptoma , Animales , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Profase Meiótica I/genética , Ratones , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN , Espermatogonias/citología , Cromosoma X/genéticaRESUMEN
MTCH2 has been described in liver as a protein involved in the intrinsic apoptotic pathway, although new evidence also associates this protein with cellular metabolism. In this work, the expression of MTCH2 in testis (an organ in which high levels of apoptosis normally take place as part of the spermatogenic process) is analyzed in rat, both at the mRNA and at the protein levels. Our results showed that MTCH2 was highly expressed in testis compared with other tissues and was differentially expressed according to developmental stage and testicular cell type. Protein expression was initially detected during the first spermatogenic wave at the time of meiosis onset and its levels increased in adulthood, with the highest expression levels being detected in meiotic prophase I. Specific differences in MTCH2 expression levels at the various stages of the adult seminiferous epithelium were also observed. Co-staining with TUNEL revealed a differential MTCH2 staining pattern in TUNEL-positive cells, mainly in dying primary spermatocytes, i.e., meiotic prophase I cells. Furthermore, upon mild hyperthermia (treatment shown to increase apoptosis in testis), MTCH2 levels rose concomitantly with a massive appearance of TUNEL-positive cells within the seminiferous tubules; these cells exhibited a differential MTCH2 distribution. Thus, MTCH2 is related to testicular apoptosis, especially during meiotic prophase.
Asunto(s)
Apoptosis/fisiología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Túbulos Seminíferos/metabolismo , Espermatocitos/metabolismo , Testículo/metabolismo , Animales , Etiquetado Corte-Fin in Situ/métodos , Masculino , Meiosis/fisiología , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Espermatogénesis/fisiologíaRESUMEN
Spats1 encodes the first reported testis-specific protein containing a long serine stretch. Besides, it bears a probable bipartite nuclear localization signal. Here, we describe the expression pattern of Spats1 in rat along embryonic and postnatal testis development by immunoblots and confocal immunohistochemistry. Spats1 is first expressed in the embryo at 17.5 days post-coitum, coinciding with the time when gonocytes acquire a quiescent state. At this time expression is detected in peritubular myoid cells and gonocytes. Spats1 attains maximum levels during meiosis of the first spermatogenic wave, mainly in pachytene spermatocytes, while a lower signal is also observed in spermatogonia, Sertoli cells and myoid cells. Protein levels dramatically decay afterwards, with minimum expression in adult individuals, where no signal was detected in elongating spermatids or spermatozoa. Spats1 is mostly cytoplasmic, although in pachytene spermatocytes it mapped to nuclei as well. Alkaline phosphatase treatment showed that this protein would be highly phosphorylated. Moreover, we show that the protein is highly conserved along metazoan evolution. Our results suggest a role in the initiation of the first spermatogenic wave, and in the establishment or progression of the first male meiotic division.