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1.
Cell Rep ; 43(7): 114430, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38963760

RESUMO

Cancer cells undergo major epigenetic alterations and transcriptomic changes, including ectopic expression of tissue- and cell-type-specific genes. Here, we show that the germline-specific RNA helicase DDX4 forms germ-granule-like cytoplasmic ribonucleoprotein granules in various human tumors, but not in cultured cancer cells. These cancerous DDX4 complexes contain RNA-binding proteins and splicing regulators, including many known germ granule components. The deletion of DDX4 in cancer cells induces transcriptomic changes and affects the alternative splicing landscape of a number of genes involved in cancer growth and invasiveness, leading to compromised capability of DDX4-null cancer cells to form xenograft tumors in immunocompromised mice. Importantly, the occurrence of DDX4 granules is associated with poor survival in patients with head and neck squamous cell carcinoma and higher histological grade of prostate cancer. Taken together, these results show that the germ-granule-resembling cancerous DDX4 granules control gene expression and promote malignant and invasive properties of cancer cells.

2.
Andrology ; 11(4): 710-723, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36624638

RESUMO

BACKGROUND: Germ granules are large cytoplasmic ribonucleoprotein complexes that emerge in the germline to participate in RNA regulation. The two most prominent germ granules are the intermitochondrial cement (IMC) in meiotic spermatocytes and the chromatoid body (CB) in haploid round spermatids, both functionally linked to the PIWI-interacting RNA (piRNA) pathway. AIMS: In this study, we clarified the IMC function by identifying proteins that form complexes with a well-known IMC protein PIWIL2/MILI in the mouse testis. RESULTS: The PIWIL2 interactome included several proteins with known functions in piRNA biogenesis. We further characterized the expression and localization of two of the identified proteins, Exonuclease 3'-5' domain-containing proteins EXD1 and EXD2, and confirmed their localization to the IMC. We showed that EXD2 interacts with PIWIL2, and that the mutation of Exd2 exonuclease domain in mice induces misregulation of piRNA levels originating from specific pachytene piRNA clusters, but does not disrupt male fertility. CONCLUSION: Altogether, this study highlights the central role of the IMC as a platform for piRNA biogenesis, and suggests that EXD1 and EXD2 function in the IMC-mediated RNA regulation in postnatal male germ cells.


Assuntos
RNA de Interação com Piwi , Espermatócitos , Camundongos , Masculino , Animais , Espermatogênese/fisiologia , Grânulos de Ribonucleoproteínas de Células Germinativas , Exonucleases/metabolismo , Proteínas/metabolismo , RNA/metabolismo , RNA Interferente Pequeno/genética , Testículo/metabolismo
3.
Reproduction ; 165(1): 49-63, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36194437

RESUMO

In brief: Proper regulation of heterochromatin is critical for spermatogenesis. This study reveals the dynamic localization patterns of distinct chromatin regulators during spermatogenesis and disrupted sex chromatin status in spermatocytes in the absence of DICER. Abstract: Heterochromatin is dynamically formed and organized in differentiating male germ cells, and its proper regulation is a prerequisite for normal spermatogenesis. While heterochromatin is generally transcriptionally silent, we have previously shown that major satellite repeat (MSR) DNA in the pericentric heterochromatin (PCH) is transcribed during spermatogenesis. We have also shown that DICER associates with PCH and is involved in the regulation of MSR-derived transcripts. To shed light on the heterochromatin regulation in the male germline, we studied the expression, localization and heterochromatin association of selected testis-enriched chromatin regulators in the mouse testis. Our results show that HELLS, WDHD1 and BAZ1A are dynamically expressed during spermatogenesis. They display limited overlap in expression, suggesting involvement in distinct heterochromatin-associated processes at different steps of differentiation. We also show that HELLS and BAZ1A interact with DICER and MSR chromatin. Interestingly, deletion of Dicer1 affects the sex chromosome heterochromatin status in late pachytene spermatocytes, as demonstrated by mislocalization of Polycomb protein family member SCML1 to the sex body. These data substantiate the importance of dynamic heterochromatin regulation during spermatogenesis and emphasize the key role of DICER in the maintenance of chromatin status in meiotic male germ cells.


Assuntos
Cromatina , Proteínas Cromossômicas não Histona , DNA Helicases , Heterocromatina , Animais , Masculino , Camundongos , Cromatina/metabolismo , DNA Helicases/genética , Heterocromatina/metabolismo , Espermatócitos/metabolismo , Espermatogênese/fisiologia , Testículo/metabolismo , Proteínas Cromossômicas não Histona/genética
4.
Semin Reprod Med ; 41(6): 213-225, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-38346711

RESUMO

Male reproductive functions are strictly regulated in order to maintain sperm production and fertility. All processes are controlled by precise regulation of gene expression, which creates specific gene expression programs for different developmental stages and cell types, and forms the functional basis for the reproductive system. Small non-coding RNAs (sncRNAs) are involved in gene regulation by targeting mRNAs for translational repression and degradation through complementary base pairing to recognize their targets. This review article summarizes the current knowledge on the function of different classes of sncRNAs, in particular microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), during male germ cell differentiation, with the focus on sncRNAs expressed in the germline. Although transcriptionally inactive, mature spermatozoa contain a complex population of sncRNAs, and we also discuss the recently identified role of sperm sncRNAs in the intergenerational transmission of epigenetic information on father's environmental and lifestyle exposures to offspring. Finally, we summarize the current information on the utility of sncRNAs as potential biomarkers of infertility that may aid in the diagnosis and prediction of outcomes of medically assisted reproduction.


Assuntos
MicroRNAs , Pequeno RNA não Traduzido , Humanos , Masculino , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Sêmen/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Espermatozoides/metabolismo , Reprodução/genética
5.
Endocrinology ; 164(2)2022 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-36461763

RESUMO

Spermatogenesis is a complex differentiation process that takes place in the seminiferous tubules. A specific organization of spermatogenic cells within the seminiferous epithelium enables a synchronous progress of germ cells at certain steps of differentiation on the spermatogenic pathway. This can be observed in testis cross-sections where seminiferous tubules can be classified into distinct stages of constant cellular composition (12 stages in the mouse). For a detailed analysis of spermatogenesis, these stages have to be individually observed from testis cross-sections. However, the recognition of stages requires special training and expertise. Furthermore, the manual scoring is laborious considering the high number of tubule cross-sections that have to be analyzed. To facilitate the analysis of spermatogenesis, we have developed a convolutional deep neural network-based approach named "STAGETOOL." STAGETOOL analyses histological images of 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI)-stained mouse testis cross-sections at ×400 magnification, and very accurately classifies tubule cross-sections into 5 stage classes and cells into 9 categories. STAGETOOL classification accuracy for stage classes of seminiferous tubules of a whole-testis cross-section is 99.1%. For cellular level analysis the F1 score for 9 seminiferous epithelial cell types ranges from 0.80 to 0.98. Furthermore, we show that STAGETOOL can be applied for the analysis of knockout mouse models with spermatogenic defects, as well as for automated profiling of protein expression patterns. STAGETOOL is the first fluorescent labeling-based automated method for mouse testis histological analysis that enables both stage and cell-type recognition. While STAGETOOL qualitatively parallels an experienced human histologist, it outperforms humans time-wise, therefore representing a major advancement in male reproductive biology research.


Assuntos
Túbulos Seminíferos , Testículo , Masculino , Camundongos , Humanos , Animais , Espermatogênese , Epitélio Seminífero , Células Epiteliais
6.
Nucleic Acids Res ; 50(20): 11470-11491, 2022 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-36259644

RESUMO

Nonsense-mediated RNA decay (NMD) is a highly conserved and selective RNA turnover pathway that depends on the endonuclease SMG6. Here, we show that SMG6 is essential for male germ cell differentiation in mice. Germ-cell conditional knockout (cKO) of Smg6 induces extensive transcriptome misregulation, including a failure to eliminate meiotically expressed transcripts in early haploid cells, and accumulation of NMD target mRNAs with long 3' untranslated regions (UTRs). Loss of SMG6 in the male germline results in complete arrest of spermatogenesis at the early haploid cell stage. We find that SMG6 is strikingly enriched in the chromatoid body (CB), a specialized cytoplasmic granule in male germ cells also harboring PIWI-interacting RNAs (piRNAs) and the piRNA-binding protein PIWIL1. This raises the possibility that SMG6 and the piRNA pathway function together, which is supported by several findings, including that Piwil1-KO mice phenocopy Smg6-cKO mice and that SMG6 and PIWIL1 co-regulate many genes in round spermatids. Together, our results demonstrate that SMG6 is an essential regulator of the male germline transcriptome, and highlight the CB as a molecular platform coordinating RNA regulatory pathways to control sperm production and fertility.


Assuntos
Endorribonucleases , Grânulos de Ribonucleoproteínas de Células Germinativas , Espermatogênese , Transcriptoma , Animais , Masculino , Camundongos , Células Germinativas/metabolismo , RNA Interferente Pequeno/genética , Espermátides/metabolismo , Espermatogênese/genética , Endorribonucleases/metabolismo
7.
Biomedicines ; 10(4)2022 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-35453658

RESUMO

Excessive adiposity caused by high-fat diets (HFDs) is associated with testicular metabolic and functional abnormalities up to grand-offspring, but the mechanisms of this epigenetic inheritance are unclear. Here we describe an association of sperm small non-coding RNA (sncRNA) with testicular "inherited metabolic memory" of ancestral HFD, using a transgenerational rodent model. Male founders were fed a standard chow for 200 days (CTRL), HFD for 200 days (HFD), or standard chow for 60 days followed by HFD for 140 days (HFDt). The male offspring and grand-offspring were fed standard chow for 200 days. The sncRNA sequencing from epidydimal spermatozoa revealed signatures associated with testicular metabolic plasticity in HFD-exposed mice and in the unexposed progeny. Sperm tRNA-derived RNA (tsRNA) and repeat-derived small RNA (repRNA) content were specially affected by HFDt and in the offspring of HFD and HFDt mice. The grand-offspring of HFD and HFDt mice showed lower sperm counts than CTRL descendants, whereas the sperm miRNA content was affected. Although the causality between sperm sncRNAs content and transgenerational epigenetic inheritance of HFD-related traits remains elusive, our results suggest that sperm sncRNA content is influenced by ancestral exposure to HFD, contributing to the sperm epigenome up to the grand-offspring.

8.
Front Immunol ; 12: 719979, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34381461

RESUMO

Adipose tissue macrophages (ATMs) regulate homeostasis and contribute to the metabolically harmful chronic inflammation in obese individuals. While evident heterogeneity of resident ATMs has been described previously, their phenotype, developmental origin, and functionality remain inconsistent. We analyzed white adipose tissue (WAT) during homeostasis and diet interventions using comprehensive and unbiased single-cell mass cytometry and genetic lineage tracking models. We now provide a uniform definition of individual subsets of resident ATMs. We show that in lean mice, WAT co-harbors eight kinetically evolving CD206+ macrophage subpopulations (defined by TIM4, CD163, and MHC II) and two CD206- macrophage subpopulations. TIM4-CD163+, TIM4-CD163- and CD206- macrophage populations are largely bone marrow-derived, while the proliferating TIM4+CD163+ subpopulation is of embryonic origin. All macrophage subtypes are active in phagocytosis, endocytosis, and antigen processing in vitro, whereas TIM4+CD163+ cells are superior in scavenging in vivo. A high-fat diet induces massive infiltration of CD206- macrophages and selective down-regulation of MHC II on TIM4+ macrophages. These changes are reversed by dietary intervention. Thus, the developmental origin and environment jointly regulate the functional malleability of resident ATMs.


Assuntos
Tecido Adiposo Branco/metabolismo , Macrófagos/metabolismo , Proteoma/metabolismo , Proteômica , Análise de Célula Única , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Tecido Adiposo Branco/imunologia , Animais , Biomarcadores , Diferenciação Celular , Plasticidade Celular/genética , Plasticidade Celular/imunologia , Reprogramação Celular , Biologia Computacional , Metabolismo Energético , Imuno-Histoquímica , Imunofenotipagem , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Fagocitose , Proteômica/métodos , Análise de Célula Única/métodos
9.
Genome Res ; 31(7): 1174-1186, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34158368

RESUMO

The testis transcriptome is highly complex and includes RNAs that potentially hybridize to form double-stranded RNA (dsRNA). We isolated dsRNA using the monoclonal J2 antibody and deep-sequenced the enriched samples from testes of juvenile Dicer1 knockout mice, age-matched controls, and adult animals. Comparison of our data set with recently published data from mouse liver revealed that the dsRNA transcriptome in testis is markedly different from liver: In testis, dsRNA-forming transcripts derive from mRNAs including promoters and immediate downstream regions, whereas in somatic cells they originate more often from introns and intergenic transcription. The genes that generate dsRNA are significantly expressed in isolated male germ cells with particular enrichment in pachytene spermatocytes. dsRNA formation is lower on the sex (X and Y) chromosomes. The dsRNA transcriptome is significantly less complex in juvenile mice as compared to adult controls and, possibly as a consequence, the knockout of Dicer1 has only a minor effect on the total number of transcript peaks associated with dsRNA. The comparison between dsRNA-associated genes in testis and liver with a reported set of genes that produce endogenous siRNAs reveals a significant overlap in testis but not in liver. Testis dsRNAs also significantly associate with natural antisense genes-again, this feature is not observed in liver. These findings point to a testis-specific mechanism involving natural antisense transcripts and the formation of dsRNAs that feed into the RNA interference pathway, possibly to mitigate the mutagenic impacts of recombination and transposon mobilization.

10.
J Vis Exp ; (164)2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33104058

RESUMO

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I-XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.


Assuntos
Células Epiteliais/citologia , Túbulos Seminíferos/citologia , Coloração e Rotulagem , Transiluminação , Acrossomo/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Microdissecção , Células de Sertoli/citologia , Espermatogênese , Espermatozoides/citologia
11.
Nucleic Acids Res ; 48(13): 7135-7153, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32484548

RESUMO

Constitutive heterochromatin at the pericentric regions of chromosomes undergoes dynamic changes in its epigenetic and spatial organization during spermatogenesis. Accurate control of pericentric heterochromatin is required for meiotic cell divisions and production of fertile and epigenetically intact spermatozoa. In this study, we demonstrate that pericentric heterochromatin is expressed during mouse spermatogenesis to produce major satellite repeat (MSR) transcripts. We show that the endonuclease DICER localizes to the pericentric heterochromatin in the testis. Furthermore, DICER forms complexes with MSR transcripts, and their processing into small RNAs is compromised in Dicer1 knockout mice leading to an elevated level of MSR transcripts in meiotic cells. We also show that defective MSR forward transcript processing in Dicer1 cKO germ cells is accompanied with reduced recruitment of SUV39H2 and H3K9me3 to the pericentric heterochromatin and meiotic chromosome missegregation. Altogether, our results indicate that the physiological role of DICER in maintenance of male fertility extends to the regulation of pericentric heterochromatin through direct targeting of MSR transcripts.


Assuntos
RNA Helicases DEAD-box/fisiologia , Ribonuclease III/fisiologia , Espermátides , Espermatócitos , Espermatogênese , Testículo/metabolismo , Animais , Centrômero/metabolismo , Segregação de Cromossomos , Fertilidade , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Masculino , Meiose/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espermátides/citologia , Espermátides/metabolismo , Espermatócitos/citologia , Espermatócitos/metabolismo , Sequências de Repetição em Tandem/genética , Testículo/citologia
12.
J Vis Exp ; (151)2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31609338

RESUMO

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.


Assuntos
Separação Celular/instrumentação , Espermátides/citologia , Espermatócitos/citologia , Testículo/citologia , Animais , Laboratórios , Masculino , Camundongos , Espermatogênese
13.
Endocrinology ; 160(5): 1119-1136, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30759202

RESUMO

A prerequisite for lifelong sperm production is that spermatogonial stem cells (SSCs) balance self-renewal and differentiation, yet factors required for this balance remain largely undefined. Using mouse genetics, we now demonstrate that the ubiquitously expressed transcription factor upstream stimulatory factor (USF)1 is critical for the maintenance of SSCs. We show that USF1 is not only detected in Sertoli cells as previously reported, but also in SSCs. Usf1-deficient mice display progressive spermatogenic decline as a result of age-dependent loss of SSCs. According to our data, the germ cell defect in Usf1-/- mice cannot be attributed to impairment of Sertoli cell development, maturation, or function, but instead is likely due to an inability of SSCs to maintain a quiescent state. SSCs of Usf1-/- mice undergo continuous proliferation, which provides an explanation for their age-dependent depletion. The proliferation-coupled exhaustion of SSCs in turn results in progressive degeneration of the seminiferous epithelium, gradual decrease in sperm production, and testicular atrophy. We conclude that the general transcription factor USF1 is indispensable for the proper maintenance of mammalian spermatogenesis.


Assuntos
Diferenciação Celular/genética , Proliferação de Células/genética , Espermatozoides/metabolismo , Células-Tronco/metabolismo , Fatores Estimuladores Upstream/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Espermatogênese/genética , Espermatogônias/citologia , Espermatogônias/metabolismo , Espermatozoides/citologia , Células-Tronco/citologia , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Testosterona/metabolismo , Fatores Estimuladores Upstream/metabolismo
14.
Nat Commun ; 10(1): 777, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30770815

RESUMO

Androgen receptor (AR) is regulated by SUMOylation at its transactivation domain. In vitro, the SUMOylation is linked to transcriptional repression and/or target gene-selective regulation. Here, we generated a mouse model (ArKI) in which the conserved SUMO acceptor lysines of AR are permanently abolished (ArK381R, K500R). ArKI males develop normally, without apparent defects in their systemic androgen action in reproductive tissues. However, the ArKI males are infertile. Their spermatogenesis appears unaffected, but their epididymal sperm maturation is defective, shown by severely compromised motility and fertilization capacity of the sperm. Fittingly, their epididymal AR chromatin-binding and gene expression associated with sperm maturation and function are misregulated. AR is SUMOylated in the wild-type epididymis but not in the testis, which could explain the tissue-specific response to the lack of AR SUMOylation. Our studies thus indicate that epididymal AR SUMOylation is essential for the post-testicular sperm maturation and normal reproductive capability of male mice.


Assuntos
Epididimo/metabolismo , Epididimo/fisiopatologia , Infertilidade Masculina/metabolismo , Infertilidade Masculina/fisiopatologia , Receptores Androgênicos/metabolismo , Espermatogênese/fisiologia , Animais , Epididimo/patologia , Humanos , Infertilidade Masculina/patologia , Masculino , Camundongos , Receptores Androgênicos/genética , Espermatogênese/genética , Sumoilação/genética , Sumoilação/fisiologia
15.
Cell Rep ; 24(13): 3423-3432.e4, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30257204

RESUMO

PIWI proteins and their associated small RNAs, called PIWI-interacting RNAs (piRNAs), restrict transposon activity in animal gonads to ensure fertility. Distinct biogenesis pathways load piRNAs into the PIWI proteins MILI and MIWI2 in the mouse male embryonic germline. While most MILI piRNAs are derived via a slicer-independent pathway, MILI slicing loads MIWI2 with a series of phased piRNAs. Tudor domain-containing 12 (TDRD12) and its interaction partner Exonuclease domain-containing 1 (EXD1) are required for loading MIWI2, but only Tdrd12 is essential for fertility, leaving us with no explanation for the physiological role of Exd1. Using an artificial piRNA precursor, we demonstrate that MILI-triggered piRNA biogenesis is greatly reduced in the Exd1 mutant. The situation deteriorates in the sensitized Exd1 mutant (Exd1-/-;Tdrd12+/-), where diminished MIWI2 piRNA levels de-repress LINE1 retrotransposons, leading to infertility. Thus, EXD1 enhances MIWI2 piRNA biogenesis via a functional interaction with TDRD12.


Assuntos
Proteínas de Transporte/metabolismo , Infertilidade Masculina/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas Argonautas/metabolismo , Masculino , Camundongos , Ligação Proteica , Processamento Pós-Transcricional do RNA , RNA Interferente Pequeno/genética
16.
FASEB J ; 32(6): 3229-3241, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29401623

RESUMO

The pituitary gonadotrophins and testosterone are the main hormonal regulators of spermatogenesis, but estradiol is also known to play a role in the process. The hormonal responses in the testis are partially mediated by somatic Sertoli cells that provide nutritional and physical support for differentiating male germ cells. Hydroxysteroid (17ß) dehydrogenase 1 (HSD17B1) is a steroidogenic enzyme that especially catalyzes the conversion of low potent 17keto-steroids to highly potent 17ß-hydroxysteroids. In this study, we show that Hsd17b1 is highly expressed in Sertoli cells of fetal and newborn mice, and HSD17B1 knockout males present with disrupted spermatogenesis with major defects, particularly in the head shape of elongating spermatids. The cell-cell junctions between Sertoli cells and germ cells were disrupted in the HSD17B1 knockout mice. This resulted in complications in the orientation of elongating spermatids in the seminiferous epithelium, reduced sperm production, and morphologically abnormal spermatozoa. We also showed that the Sertoli cell-expressed HSD17B1 participates in testicular steroid synthesis, evidenced by a compensatory up-regulation of HSD17B3 in Leydig cells. These results revealed a novel role for HSD17B1 in the control of spermatogenesis and male fertility, and that Sertoli cells significantly contribute to steroid synthesis in the testis.-Hakkarainen, J., Zhang, F.-P., Jokela, H., Mayerhofer, A., Behr, R., Cisneros-Montalvo, S., Nurmio, M., Toppari, J., Ohlsson, C., Kotaja, N., Sipilä, P., Poutanen, M. Hydroxysteroid (17ß) dehydrogenase 1 expressed by Sertoli cells contributes to steroid synthesis and is required for male fertility.


Assuntos
17-Hidroxiesteroide Desidrogenases/biossíntese , Fertilidade/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Células de Sertoli/enzimologia , Espermatogênese/fisiologia , Esteroides/biossíntese , 17-Hidroxiesteroide Desidrogenases/genética , Animais , Masculino , Camundongos , Camundongos Knockout , Epitélio Seminífero/citologia , Epitélio Seminífero/enzimologia , Células de Sertoli/citologia , Espermátides/citologia , Espermátides/enzimologia
17.
Sci Rep ; 8(1): 859, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29339787

RESUMO

Sperm flagellar protein 2 (SPEF2) is essential for motile cilia, and lack of SPEF2 function causes male infertility and primary ciliary dyskinesia. Cilia are pointing out from the cell surface and are involved in signal transduction from extracellular matrix, fluid flow and motility. It has been shown that cilia and cilia-related genes play essential role in commitment and differentiation of chondrocytes and osteoblasts during bone formation. Here we show that SPEF2 is expressed in bone and cartilage. The analysis of a Spef2 knockout (KO) mouse model revealed hydrocephalus, growth retardation and death prior to five weeks of age. To further elucidate the causes of growth retardation we analyzed the bone structure and possible effects of SPEF2 depletion on bone formation. In Spef2 KO mice, long bones (tibia and femur) were shorter compared to wild type, and X-ray analysis revealed reduced bone mineral content. Furthermore, we showed that the in vitro differentiation of osteoblasts isolated from Spef2 KO animals was compromised. In conclusion, this study reveals a novel function for SPEF2 in bone formation through regulation of osteoblast differentiation and bone growth.


Assuntos
Diferenciação Celular , Proteínas/genética , Animais , Densidade Óssea , Cartilagem/metabolismo , Cartilagem/patologia , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fêmur/diagnóstico por imagem , Fêmur/patologia , Fêmur/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese , Proteínas/metabolismo , Tíbia/diagnóstico por imagem , Tíbia/patologia , Tíbia/fisiologia , Microtomografia por Raio-X
18.
Reproduction ; 155(2): R77-R91, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29038333

RESUMO

Germ cells have exceptionally diverse transcriptomes. Furthermore, the progress of spermatogenesis is accompanied by dramatic changes in gene expression patterns, the most drastic of them being near-to-complete transcriptional silencing during the final steps of differentiation. Therefore, accurate RNA regulatory mechanisms are critical for normal spermatogenesis. Cytoplasmic germ cell-specific ribonucleoprotein (RNP) granules, known as germ granules, participate in posttranscriptional regulation in developing male germ cells. Particularly, germ granules provide platforms for the PIWI-interacting RNA (piRNA) pathway and appear to be involved both in piRNA biogenesis and piRNA-targeted RNA degradation. Recently, other RNA regulatory mechanisms, such as the nonsense-mediated mRNA decay pathway have also been associated to germ granules providing new exciting insights into the function of germ granules. In this review article, we will summarize our current knowledge on the role of germ granules in the control of mammalian male germ cell's transcriptome and in the maintenance of fertility.


Assuntos
Regulação da Expressão Gênica , Células Germinativas/fisiologia , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Espermatogênese , Animais , Células Germinativas/citologia , Humanos , Masculino
19.
Development ; 144(14): 2683-2693, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28619825

RESUMO

Sperm differentiation requires specific protein transport for correct sperm tail formation and head shaping. A transient microtubular structure, the manchette, appears around the differentiating spermatid head and serves as a platform for protein transport to the growing tail. Sperm flagellar 2 (SPEF2) is known to be essential for sperm tail development. In this study we investigated the function of SPEF2 during spermatogenesis using a male germ cell-specific Spef2 knockout mouse model. In addition to defects in sperm tail development, we observed a duplication of the basal body and failure in manchette migration resulting in an abnormal head shape. We identified cytoplasmic dynein 1 and GOLGA3 as novel interaction partners for SPEF2. SPEF2 and dynein 1 colocalize in the manchette and the inhibition of dynein 1 disrupts the localization of SPEF2 to the manchette. Furthermore, the transport of a known SPEF2-binding protein, IFT20, from the Golgi complex to the manchette was delayed in the absence of SPEF2. These data indicate a possible novel role of SPEF2 as a linker protein for dynein 1-mediated cargo transport along microtubules.


Assuntos
Proteínas/fisiologia , Espermátides/crescimento & desenvolvimento , Espermátides/fisiologia , Espermatogênese/fisiologia , Animais , Proteínas de Transporte/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Dineínas do Citoplasma/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/fisiologia , Transporte Proteico/genética , Transporte Proteico/fisiologia , Proteínas/genética , Cauda do Espermatozoide/fisiologia , Cauda do Espermatozoide/ultraestrutura , Espermátides/citologia , Espermatogênese/genética
20.
Autophagy ; 13(2): 302-321, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27929729

RESUMO

Ribonucleoprotein (RNP) granules play a major role in compartmentalizing cytoplasmic RNA regulation. Haploid round spermatids that have exceptionally diverse transcriptomes are characterized by a unique germ cell-specific RNP granule, the chromatoid body (CB). The CB shares many characteristics with somatic RNP granules but also has germline-specific features. The CB appears to be a central structure in PIWI-interacting RNA (piRNA)-targeted RNA regulation. Here, we identified a novel CB component, FYCO1, which is involved in the intracellular transport of autophagic vesicles in somatic cells. We demonstrated that the CB is associated with autophagic activity. Induction of autophagy leads to the recruitment of lysosomal vesicles onto the CB in a FYCO1-dependent manner as demonstrated by the analysis of a germ cell-specific Fyco1 conditional knockout mouse model. Furthermore, in the absence of FYCO1, the integrity of the CB was affected and the CB was fragmented. Our results suggest that RNP granule homeostasis is regulated by FYCO1-mediated autophagy.


Assuntos
Autofagia , Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Haploidia , Proteínas do Tecido Nervoso/metabolismo , Ribonucleoproteínas/metabolismo , Espermatozoides/citologia , Espermatozoides/metabolismo , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Grânulos Citoplasmáticos/ultraestrutura , Proteínas do Citoesqueleto , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Masculino , Camundongos Knockout , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Especificidade de Órgãos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ligação Proteica , Transporte Proteico , Espermátides/metabolismo , Espermátides/ultraestrutura , Espermatogênese , Espermatozoides/ultraestrutura , Testículo/metabolismo
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