RESUMEN
In mammals, the transition from mitosis to meiosis facilitates the successful production of gametes. However, the regulatory mechanisms that control meiotic initiation remain unclear, particularly in the context of complex histone modifications. Herein, we show that KDM2A, acting as a lysine demethylase targeting H3K36me3 in male germ cells, plays an essential role in modulating meiotic entry and progression. Conditional deletion of Kdm2a in mouse pre-meiotic germ cells results in complete male sterility, with spermatogenesis ultimately arrested at the zygotene stage of meiosis. KDM2A deficiency disrupts H3K36me2/3 deposition in c-KIT+ germ cells, characterized by a reduction in H3K36me2 but a dramatic increase in H3K36me3. Furthermore, KDM2A recruits the transcription factor E2F1 and its co-factor HCFC1 to the promoters of key genes required for meiosis entry and progression, such as Stra8, Meiosin, Spo11, and Sycp1. Collectively, our study unveils an essential role for KDM2A in mediating H3K36me2/3 deposition and controlling the programmed gene expression necessary for the transition from mitosis to meiosis during spermatogenesis.
RESUMEN
Spermatogenesis depends on the crosstalk of Sertoli cells (SCs) and germ cells. However, the gene regulatory network establishing the communications between SCs and germ cells remains unclear. Here, we report that heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) in SCs is essential for the establishment of crosstalk between SCs and germ cells. Conditional knockout of hnRNPH1 in mouse SCs leads to compromised blood-testis barrier function, delayed meiotic progression, increased germ cell apoptosis, sloughing of germ cells and, eventually, infertility of mice. Mechanistically, we discovered that hnRNPH1 could interact with the splicing regulator PTBP1 in SCs to regulate the pre-mRNA alternative splicing of the target genes functionally related to cell adhesion. Interestingly, we also found hnRNPH1 could cooperate with the androgen receptor, one of the SC-specific transcription factors, to modulate the transcription level of a group of genes associated with the cell-cell junction and EGFR pathway by directly binding to the gene promoters. Collectively, our findings reveal a crucial role for hnRNPH1 in SCs during spermatogenesis and uncover a potential molecular regulatory network involving hnRNPH1 in establishing Sertoli-germ cell crosstalk.
Asunto(s)
Células de Sertoli , Espermatogénesis , Animales , Masculino , Ratones , Fertilidad/fisiología , Células Germinativas/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Ratones Noqueados , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Células de Sertoli/metabolismo , Espermatogénesis/genética , Testículo/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive. We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored. Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly. BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice. In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion. Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8's affinity for substrate proteins. Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.
Asunto(s)
Proteínas del Citoesqueleto , Infertilidad Masculina , Teratozoospermia , Tiazoles , Animales , Humanos , Masculino , Ratones , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Dineínas/metabolismo , Proteínas del Choque Térmico HSC70/genética , Proteínas del Choque Térmico HSC70/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Semen/metabolismo , Cabeza del Espermatozoide/fisiología , Espermatogénesis/genética , Espermatozoides/metabolismo , Teratozoospermia/metabolismo , Teratozoospermia/patologíaRESUMEN
Transcription factor-like 5 (TCFL5) is a testis-specific protein that contains the basic helix-loop-helix domain, but the in vivo functions of TCFL5 remain unknown. Herein, we generated CRISPR/Cas9-mediated knockout mice to dissect the function of TCFL5 in mouse testes. Surprisingly, we found that it was difficult to generate homozygous mice with the Tcfl5 deletion as the heterozygous males (Tcfl5+/-) were infertile. However, we did observe markedly abnormal phenotypes of spermatids and spermatozoa in the testes and epididymides of Tcfl5+/- mice. Mechanistically, we demonstrated that TCFL5 transcriptionally and post-transcriptionally regulated a set of genes participating in male germ cell development via TCFL5 ChIP-DNA and eCLIP-RNA high-throughput sequencing. We also identified a known RNA-binding protein, FXR1, as an interacting partner of TCFL5 that may coordinate the transition and localization of TCFL5 in the nucleus. Collectively, we herein report for the first time that Tcfl5 is haploinsufficient in vivo and acts as a dual-function protein that mediates DNA and RNA to regulate spermatogenesis. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Espermatogénesis , Testículo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , ADN/metabolismo , Fertilidad/genética , Masculino , Ratones , Ratones Noqueados , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Espermatogénesis/genética , Espermatozoides/metabolismo , Testículo/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The mouse WD repeat and FYVE domain containing 1 (Wdfy1) gene is located in chromosome 1qC4 and spans over 73.7 kilobases. It encodes a protein of 410-amino acid protein that shares 97.8% amino acid sequence identity with the human WDFY1 protein. However, the expression pattern of WDFY1 in reproductive organs and its function in male fertility remain unknown. In this study, we generated transgenic mice expressing FLAG-Wdfy1-mCherry cDNA driven by the Wdfy1 promoter to clarify the expression of WDFY1. The results showed that WDFY1 is highly expressed in mouse testes and located in the cytoplasm of late pachytene spermatocytes to elongated spermatids. Interestingly, the global Wdfy1 knockout (KO) male mice displayed normal growth, development, and fertility. Further histological analysis of Wdfy1 knockout mouse testes revealed that all spermatogenic cells are present in Wdfy1 KO seminiferous tubules. Together, our data demonstrate that WDFY1 is dispensable for mouse spermatogenesis and male fertility.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Fertilidad/genética , Regulación de la Expresión Génica , Espermatogénesis/genética , Testículo/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Western Blotting , Femenino , Perfilación de la Expresión Génica/métodos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espermátides/citología , Espermátides/metabolismo , Testículo/citología , Repeticiones WD40/genéticaRESUMEN
During male meiosis, the constitutively unsynapsed XY chromosomes undergo meiotic sex chromosome inactivation (MSCI), and the DNA damage response (DDR) pathway is critical for MSCI establishment. Our previous study showed that UHRF1 (ubiquitin-like, with PHD and ring finger domains 1) deletion led to meiotic arrest and male infertility; however, the underlying mechanisms of UHRF1 in the regulation of meiosis remain unclear. Here, we report that UHRF1 is required for MSCI and cooperates with the DDR pathway in male meiosis. UHRF1-deficient spermatocytes display aberrant pairing and synapsis of homologous chromosomes during the pachytene stage. In addition, UHRF1 deficiency leads to aberrant recruitment of ATR and FANCD2 on the sex chromosomes and disrupts the diffusion of ATR to the XY chromatin. Furthermore, we show that UHRF1 acts as a cofactor of BRCA1 to facilitate the recruitment of DDR factors onto sex chromosomes for MSCI establishment. Accordingly, deletion of UHRF1 leads to the failure of meiotic silencing on sex chromosomes, resulting in meiotic arrest. In addition to our previous findings, the present study reveals that UHRF1 participates in MSCI, ensuring the progression of male meiosis. This suggests a multifunctional role of UHRF1 in the male germline.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Emparejamiento Cromosómico , Cromosomas Sexuales , Ubiquitina-Proteína Ligasas , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Daño del ADN , Masculino , Meiosis/genética , Ratones , Cromosomas Sexuales/genética , Espermatocitos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Emerging evidence shows that m6A, one of the most abundant RNA modifications in mammals, is involved in the entire process of spermatogenesis, including mitosis, meiosis, and spermiogenesis. "Writers" catalyze m6A formation on stage-specific transcripts during male germline development, while "erasers" remove m6A modification to maintain a balance between methylation and demethylation. The different functions of RNA-m6A transcripts depend on their recognition by "readers". m6A modification mediates RNA metabolism, including mRNA splicing, translation, and degradation, as well as the maturity and biosynthesis of non-coding RNAs. Sperm RNA profiles are easily affected by environmental exposure and can even be inherited for several generations, similar to epigenetic inheritance. Here, we review and summarize the critical role of m6A in different developmental stages of male germ cells, to understand of the mechanisms and epigenetic regulation of m6A modifications. In addition, we also outline and discuss the important role of non-coding RNAs in spermatogenesis and RNA modifications in epigenetic inheritance.
Asunto(s)
Epigénesis Genética , ARN/metabolismo , Espermatozoides/metabolismo , Enzimas AlkB/metabolismo , Animales , Humanos , Masculino , Metiltransferasas/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , EspermatogénesisRESUMEN
BACKGROUND: The mitosis-to-meiosis switch during spermatogenesis requires dynamic changes in gene expression. However, the regulation of meiotic transcriptional and post-transcriptional machinery during this transition remains elusive. RESULTS: We report that methyltransferase-like protein 16 (METTL16), an N6-methyladenosine (m6A) writer, is required for mitosis-to-meiosis transition during spermatogenesis. Germline conditional knockout of Mettl16 in male mice impairs spermatogonial differentiation and meiosis initiation. Mechanistically, METTL16 interacts with splicing factors to regulate the alternative splicing of meiosis-related genes such as Stag3. Ribosome profiling reveals that the translation efficiency of many meiotic genes is dysregulated in METTL16-deficient testes. m6A-sequencing shows that ablation of METTL16 causes upregulation of the m6A-enriched transcripts and downregulation of the m6A-depleted transcripts, similar to Meioc and/or Ythdc2 mutants. Further in vivo and in vitro experiments demonstrate that the methyltransferase activity site (PP185-186AA) of METTL16 is necessary for spermatogenesis. CONCLUSIONS: Our findings support a molecular model wherein the m6A writer METTL16-mediated alternative splicing and translation efficiency regulation are required to control the mitosis-to-meiosis germ cell fate decision in mice, with implications for understanding meiosis-related male fertility disorders.
Asunto(s)
Adenosina , Empalme Alternativo , Meiosis , Metiltransferasas , Espermatogénesis , Animales , Espermatogénesis/genética , Masculino , Metiltransferasas/metabolismo , Metiltransferasas/genética , Ratones , Adenosina/análogos & derivados , Adenosina/metabolismo , Biosíntesis de Proteínas , Ratones Noqueados , Mitosis , Testículo/metabolismo , Espermatogonias/metabolismoRESUMEN
Genetic and epigenetic changes in sperm caused by male aging may be essential factors affecting semen parameters, but the effects and specific molecular mechanisms of aging on male reproduction have not been fully clarified. In this study, to explore the effect of aging on male fertility and seek the potential molecular etiology, we performed high-throughput RNA-sequencing in isolated spermatogenic cells, including pachytene spermatocytes (marked by the completion of chromosome synapsis) and round spermatids (produced by the separation of sister chromatids) from the elderly and the young men. Functional enrichment analysis of differentially expressed genes (DEGs) in round spermatids between the elderly and young showed that they were significantly enriched in gamete generation, spindle assembly, and cilium movement involved in cell motility. In addition, the expression levels of DEGs in round spermatids (post-meiotic cells) were found to be more susceptible to age. Furthermore, ten genes (AURKA, CCNB1, CDC20, CCNB2, KIF2C, KIAA0101, NR5A1, PLK1, PTTG1, RAD51AP1) were identified to be the hub genes involved in the regulation of sperm quality in the elderly through Protein-Protein Interaction (PPI) network construction and measuring semantic among GO terms and gene products. Our data provide aging-related molecular alterations in meiotic and post-meiotic spermatogenic cells, and the information gained from this study may explain the abnormal aging-related male fertility decline.
Asunto(s)
Semen , Espermátides , Masculino , Humanos , Anciano , Espermátides/metabolismo , Espermatozoides/metabolismo , Perfilación de la Expresión Génica , Fertilidad/genética , Espermatogénesis/genéticaRESUMEN
The continuous regeneration of spermatogonial stem cells (SSCs) underpins spermatogenesis and lifelong male fertility, but the developmental origins of the SSC pool remain unclear. Here, we document that hnRNPU is essential for establishing the SSC pool. In male mice, conditional loss of hnRNPU in prospermatogonia (ProSG) arrests spermatogenesis and results in sterility. hnRNPU-deficient ProSG fails to differentiate and migrate to the basement membrane to establish SSC pool in infancy. Moreover, hnRNPU deletion leads to the accumulation of ProSG and disrupts the process of T1-ProSG to T2-ProSG transition. Single-cell transcriptional analyses reveal that germ cells are in a mitotically quiescent state and lose their unique identity upon hnRNPU depletion. We further show that hnRNPU could bind to Vrk1, Slx4, and Dazl transcripts that have been identified to suffer aberrant alternative splicing in hnRNPU-deficient testes. These observations offer important insights into SSC pool establishment and may have translational implications for male fertility.
Asunto(s)
Espermatogénesis , Espermatogonias , Animales , Masculino , Ratones , Células Madre Germinales Adultas/metabolismo , Empalme Alternativo/genética , Diferenciación Celular , Espermatogénesis/genética , Espermatogonias/metabolismo , Espermatogonias/citología , Células Madre/metabolismo , Células Madre/citología , Testículo/metabolismo , Testículo/citología , Ribonucleoproteína Heterogénea-Nuclear Grupo U/metabolismoRESUMEN
Pachytene piRNA biogenesis is a hallmark of the germline, distinct from another wave of pre-pachytene piRNA biogenesis with regard to the lack of a secondary amplification process known as the Ping-pong cycle. However, the underlying molecular mechanism and the venue for the suppression of the Ping-pong cycle remain elusive. Here, we showed that a testis-specific protein, ADAD2, interacts with a TDRD family member protein RNF17 and is associated with P-bodies. Importantly, ADAD2 directs RNF17 to repress Ping-pong activity in pachytene piRNA biogenesis. The P-body localization of RNF17 requires the intrinsically disordered domain of ADAD2. Deletion of Adad2 or Rnf17 causes the mislocalization of each other and subsequent Ping-pong activity derepression, secondary piRNAs overproduced, and disruption of P-body integrity at the meiotic stage, thereby leading to spermatogenesis arrested at the round spermatid stage. Collectively, by identifying the ADAD2-dependent mechanism, our study reveals a novel function of P-bodies in suppressing Ping-pong activity in pachytene piRNA biogenesis.
Asunto(s)
ARN de Interacción con Piwi , Cuerpos de Procesamiento , Masculino , Profase Meiótica I , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Espermatogénesis/genéticaRESUMEN
Coordinated regulation of alternative pre-mRNA splicing is essential for germ cell development. However, the underlying molecular mechanism that controls alternative mRNA expression during germ cell development remains elusive. Herein, we show that hnRNPH1 is highly expressed in the reproductive system and recruits the PTBP2 and SRSF3 to modulate the alternative splicing in germ cells. Conditional knockout Hnrnph1 in spermatogenic cells causes many abnormal splicing events, thus affecting the genes related to meiosis and communication between germ cells and Sertoli cells. This is characterized by asynapsis of chromosomes and impairment of germ-Sertoli communications, which ultimately leads to male sterility. Markedly, Hnrnph1 germline-specific mutant female mice are also infertile, and Hnrnph1-deficient oocytes exhibit a similar defective synapsis and cell-cell junction as seen in Hnrnph1-deficient male germ cells. Collectively, our data support a molecular model wherein hnRNPH1 governs a network of alternative splicing events in germ cells via recruitment of PTBP2 and SRSF3.