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1.
Gene ; 706: 201-210, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31085275

RESUMO

The functional sperm is the key factor for species continuation. The process spermatogenesis, to produce mature sperm is quite complex. It begins with the proliferation and differentiation of spermatogonia, which develop from primary spermatocytes to secondary spermatocytes and round spermatids, which eventually develop into fertile mature sperm. Spermiogenesis is the latest stage of spermatogenesis, where the round spermatids undergo a series of dramatic morphological changes and extreme condensation of chromatin to construct mature sperm with species-specific shape. During spermiogenesis, chromatin remodeling is a unique progress. It leads the nucleosome from a histone-based structure to a mostly protamine-based configuration. The main events of chromatin remodeling are the replacement of histone by histone variants, hyperacetylation, transient DNA strand breaks and repair, variants by transition proteins and finally by protamines. In this review, we synthesize and summarize the current knowledge on the progress of chromatin remodeling during spermiogenesis. We straighten out the chronological order of chromatin remodeling and illustrate the possible regulation mechanisms of each step.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/fisiologia , Espermatogênese/fisiologia , Animais , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , DNA/metabolismo , Histonas/metabolismo , Humanos , Masculino , Maturação do Esperma/genética , Espermátides/metabolismo , Espermatócitos/metabolismo , Espermatogênese/genética , Espermatozoides/metabolismo
2.
Cell Physiol Biochem ; 52(3): 421-434, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30845381

RESUMO

BACKGROUND/AIMS: The aim of this study was to evaluate the potential and significant applications of Sertoli cells (SCs) transplantation, and to explore the effect of transplantation on spermatogenesis process, in azospermic mice. METHODS: In this study, we utilized 18 adult mice (28‒30 g), divided into four experimental groups: (1) control, (2) vehicle (DMSO 2%) (10 µl) (3) busulfan and (4) busulfan+ SCs (1×104 cells/µL). SCs were isolated from the testis of 4-week-old mouse and after using anesthetics, 10 µl of SCs suspension (1×104 cells/µL) was injected over 3-5 min, into each testis and subsequently, sperm samples were collected from the tail of the epididymis. Afterward, the animals were euthanized and testis samples were taken for histopathology experiments, and RNA extraction, in order to examine the expression of c-kit, STRA8 and PCNA genes. RESULTS: Our data showed that SCs transplantation could notably increase the total sperm count and the number of testicular cells, such as spermatogonia, primary spermatocyte, round spermatid, SCs and Leydig cells, compared to the control, DMSO and busulfan groups. Furthermore, the result showed that the expression of c-kit and STRA8 were significantly decreased in busulfan and busulfan/SCs groups, at 8 weeks after the last injection (p<0.001), but no significant decrease was found for PCNA, compared to the control and DMSO groups (P<0.05). CONCLUSION: These findings suggest that SCs transplantation may be beneficial as a practical approach for therapeutic strategies in reproductive and regenerative medicine. We further highlighted the essential applications that might provide a mechanism for correcting fertility in males, suffering from cell deformity.


Assuntos
Células de Sertoli/transplante , Espermatogênese , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Bussulfano/farmacologia , Epididimo/citologia , Epididimo/metabolismo , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Medicina Regenerativa , Células de Sertoli/citologia , Motilidade Espermática , Espermátides/citologia , Espermátides/metabolismo , Espermatogênese/efeitos dos fármacos , Espermatogônias/citologia , Espermatogônias/fisiologia , Testículo/metabolismo , Testículo/patologia
3.
Gen Comp Endocrinol ; 279: 154-163, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-30902612

RESUMO

Dmrt1, doublesex- and mab-3-related transcription factor-1, has been suggested to play critical roles in male gonadogenesis, testicular differentiation and development, including spermatogenesis, among different vertebrates. Vasa is a putative molecular marker of germ cells in vertebrates. In this study, we cloned the full-length dmrt1 cDNA from Japanese eel, and the protein comprised 290 amino acids and presented an extremely conserved Doublesex and Mab-3 (DM) domain. Vasa proteins were expressed in gonadal germ cells in a stage-specific manner, and were expressed at high levels in PGC and spermatogonia, low levels in spermatocytes, and were absent in spermatids and spermatozoa of Japanese eels. Dmrt1 proteins were abundantly expressed in spermatogonia B cells, spermatocytes, spermatids, but not in spermatozoa, spermatogonia A and Sertoli cells. To our knowledge, this study is the first to show a restricted expression pattern for the Dmrt1 protein in spermatogonia B cells, but not spermatogonia A cells, of teleosts. Therefore, Dmrt1 might play vital roles at the specific stages during spermatogenesis from spermatogonia B cells to spermatids in the Japanese eel. Moreover, the Dmrt1 protein exhibited a restricted localization in differentiating oogonia in the early differentiating gonad (ovary-like structure) of male Japanese eels and in E2-induced feminized Japanese eels. We proposed that dmrt1 may be not only required for spermatogenesis but might also play a role in oogenesis in the Japanese eel.


Assuntos
Anguilla/crescimento & desenvolvimento , Anguilla/genética , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/crescimento & desenvolvimento , Gônadas/metabolismo , Espermatogênese , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Sequência de Bases , DNA Complementar/genética , Feminino , Masculino , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Filogenia , Espermátides/metabolismo , Espermatogênese/genética , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética
4.
Cells ; 8(2)2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30791486

RESUMO

During spermiogenesis, the proximal centriole forms a special microtubular structure: the centriolar adjunct. This structure appears at the spermatid stage, which is characterized by a condensed chromatin nucleus. We showed that the centriolar adjunct disappears completely in mature porcine spermatozoa. In humans, the centriolar adjunct remnants are present in a fraction of mature spermatids. For the first time, the structure of the centriolar adjunct in the cell, and its consequent impact on fertility, were examined. Ultrastructural analysis using transmission electron microscopy was performed on near 2000 spermatozoa per person, in two patients with idiopathic male sterility (IMS) and five healthy fertile donors. We measured the average length of the "proximal centriole + centriolar adjunct" complex in sections, where it had parallel orientation in the section plane, and found that it was significantly longer in the spermatozoa of IMS patients than in the spermatozoa of healthy donors. This difference was independent of chromatin condensation deficiency, which was also observed in the spermatozoa of IMS patients. We suggest that zygote arrest may be related to an incompletely disassembled centriolar adjunct in a mature spermatozoon. Therefore, centriolar adjunct length can be potentially used as a complementary criterion for the immaturity of spermatozoa in the diagnostics of IMS patients.


Assuntos
Centríolos/metabolismo , Fertilidade/fisiologia , Espermatogênese/fisiologia , Adulto , Animais , Centríolos/ultraestrutura , Cromatina/metabolismo , Humanos , Infertilidade Masculina/patologia , Masculino , Espermátides/metabolismo , Espermátides/ultraestrutura , Suínos , Doadores de Tecidos
5.
PLoS One ; 14(2): e0211739, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30707741

RESUMO

MicroRNAs (miRNAs) play a critical role in multiple aspects of biology. Dicer, an RNase III endonuclease, is essential for the biogenesis of miRNAs, and the germ cell-specific Dicer1 knockout mouse shows severe defects in gametogenesis. How miRNAs regulate germ cell development is still not fully understood. In this study, we identified germ cell-specific miRNAs (miR-741-3p, miR-871-3p, miR-880-3p) by analyzing published RNA-seq data of mouse. These miRNA genes are contiguously located on the X chromosome near other miRNA genes. We named them X chromosome-linked miRNAs (XmiRs). To elucidate the functions of XmiRs, we generated knockout mice of these miRNA genes using the CRISPR/Cas9-mediated genome editing system. Although no histological abnormalities were observed in testes of F0 mice in which each miRNA gene was disrupted, a deletion covering miR-871 and miR-880 or covering all XmiRs (ΔXmiRs) resulted in arrested spermatogenesis in meiosis in a few seminiferous tubules, indicating their redundant functions in spermatogenesis. Among candidate targets of XmiRs, we found increased expression of a gene encoding a WNT receptor, FZD4, in ΔXmiRs testis compared with that in wildtype testis. miR-871-3p and miR-880-3p repressed the expression of Fzd4 via the 3'-untranslated region of its mRNA. In addition, downstream genes of the WNT/ß-catenin pathway were upregulated in ΔXmiRs testis. We also found that miR-871, miR-880, and Fzd4 were expressed in spermatogonia, spermatocytes and spermatids, and overexpression of miR-871 and miR-880 in germ stem cells in culture repressed their increase in number and Fzd4 expression. Previous studies indicated that the WNT/ß-catenin pathway enhances and represses proliferation and differentiation of spermatogonia, respectively, and our results consistently showed that stable ß-catenin enhanced GSC number. In addition, stable ß-catenin partially rescued reduced GSC number by overexpression of miR-871 and miR-880. The results together suggest that miR-871 and miR-880 cooperatively regulate the WNT/ß-catenin pathway during testicular germ cell development.


Assuntos
MicroRNAs/genética , Espermatogênese/genética , Cromossomo X/genética , Animais , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Proliferação de Células , Células Germinativas , Masculino , Meiose , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/classificação , Espermátides/metabolismo , Espermatócitos/metabolismo , Espermatogônias/metabolismo , Testículo/metabolismo
6.
Biochem Biophys Res Commun ; 510(2): 309-314, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30704757

RESUMO

The aim of this study was to detect through immunohistochemistry the presence of the estrogen receptor alpha (ER-α) in different cell subpopulations, as well as to evaluate the histological and stereological changes of the testes of mature and aged chickens. Histological results demonstrated several changes in the tubular compartment and interstitial tissue in aged chicken testis as compared to mature chicken testis. Stereological results revealed that the mature chicken testis increases in volume as well as the total volume of the tubular compartment, whereas the total volume of the interstitial tissue and the total volume of Leydig cells diminishes as compared to aged chicken testis. The increase in the total volume of Leydig cells shown in aged chicken testis is due to the increase of cellular volume of Leydig cells, and not in their number, which decreases in aged chicken testis. Results also revealed positive ER-α immunostaining in mature and aged chicken testes, but cellular distribution of ER-α immunostaining changed according to the animal's age. In mature chicken testis, the ER-α was localized in the nuclei of germ and somatic cells. In contrast, in the aged chicken testis, only scarce spermatogonia presented ER-α immunoreactivity. This differential expression of ER-α may contribute to regulate the reproductive function in the mature chicken or the cessation of reproductive function in aged chickens.


Assuntos
Receptor alfa de Estrogênio/metabolismo , Testículo/metabolismo , Envelhecimento , Animais , Galinhas , Perfilação da Expressão Gênica , Imuno-Histoquímica , Masculino , Espermátides/metabolismo , Espermatócitos/citologia , Espermatogônias/metabolismo
7.
Genes (Basel) ; 10(1)2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30646585

RESUMO

The near complete replacement of somatic chromatin in spermatids is, perhaps, the most striking nuclear event known to the eukaryotic domain. The process is far from being fully understood, but research has nevertheless unraveled its complexity as an expression of histone variants and post-translational modifications that must be finely orchestrated to promote the DNA topological change and compaction provided by the deposition of protamines. That this major transition may not be genetically inert came from early observations that transient DNA strand breaks were detected in situ at chromatin remodeling steps. The potential for genetic instability was later emphasized by our demonstration that a significant number of DNA double-strand breaks (DSBs) are formed and then repaired in the haploid context of spermatids. The detection of DNA breaks by 3'OH end labeling in the whole population of spermatids suggests that a reversible enzymatic process is involved, which differs from canonical apoptosis. We have set the stage for a better characterization of the genetic impact of this transition by showing that post-meiotic DNA fragmentation is conserved from human to yeast, and by providing tools for the initial mapping of the genome-wide DSB distribution in the mouse model. Hence, the molecular mechanism of post-meiotic DSB formation and repair in spermatids may prove to be a significant component of the well-known male mutation bias. Based on our recent observations and a survey of the literature, we propose that the chromatin remodeling in spermatids offers a proper context for the induction of de novo polymorphism and structural variations that can be transmitted to the next generation.


Assuntos
Montagem e Desmontagem da Cromatina , Instabilidade Cromossômica , Espermátides/metabolismo , Animais , Masculino , Taxa de Mutação , Espermátides/citologia , Espermatogênese
8.
Eur J Pharmacol ; 846: 30-37, 2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30659824

RESUMO

Under the sustained hyperglycemic state, oxidative stress induces irreparable DNA double-strand breaks resulting in germ cell death and testicular atrophy. Although molecular mechanisms underlying DNA damage repair in testicular cells are gradually getting unraveled, the effects on DNA double-strand breaks sensing are not precisely known. In this study, using streptozotocin-induced type 1 diabetic rats, we report that hyperglycemic state for one month or three months does not increase the levels of ataxia telangiectasia mutated (ATM) protein- an upstream kinase responsible for the phosphorylation of histone 2AX (Ɣ-H2AX)- after the formation of DNA double-strand breaks. The ATM expression is seminiferous epithelial stage-dependent in spermatogonia and primary spermatocytes, and the pattern of stage-dependent expression varies in diabetic rats, especially after three-month-long diabetes. However, the levels of metastasis-associated protein-1 (MTA1), an essential protein for ATM function, increase although not in a time-dependent manner. The amount of DNA double-strand breaks increases in a time- and stage-dependent manner as indicated by increased Ɣ-H2AX levels, especially in spermatogonia and primary spermatocytes, and in late spermatids in some tubular stages. Although ATM levels do not increase in diabetic rats, protein is expressed more or less in same testicular cells in which Ɣ-H2AX is expressed indicating that ATM might play a vital role in the phosphorylation of the histone. We conclude that diabetes upregulates MTA1-Ɣ-H2AX signaling in diabetic rat testis as a response to time-dependent increases in DNA double-strand breaks.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Histonas/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Testículo/metabolismo , Animais , Quebras de DNA de Cadeia Dupla , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/genética , Masculino , Fosforilação , Ratos , Ratos Wistar , Transdução de Sinais , Espermátides/citologia , Espermátides/metabolismo , Espermatócitos/citologia , Espermatócitos/metabolismo , Espermatogônias/citologia , Espermatogônias/metabolismo , Estreptozocina , Regulação para Cima
9.
PLoS One ; 13(12): e0208835, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30571760

RESUMO

The fertility of men with neurofibromatosis 1 (NF1) is reduced. Despite this observation, gonadal function has not been examined in patients with NF1. In order to assess the role of reduced neurofibromin in the testes, we examined testicular morphology and function in an Nf1+/- mouse model. We found that although Nf1+/- male mice are able to reproduce, they have significantly fewer pups per litter than Nf1+/+ control males. Reduced fertility in Nf1+/- male mice is associated with disorganization of the seminiferous epithelium, with exfoliation of germ cells and immature spermatids into the tubule lumen. Morphometric analysis shows that these alterations are associated with decreased Leydig cell numbers and increased spermatid cell numbers. We hypothesized that hyper-activation of Ras in Nf1+/- males affects ectoplasmic specialization, a Sertoli-spermatid adherens junction involved in spermiation. Consistent with this idea, we found increased expression of phosphorylated ERK, a downstream effector of Ras that has been shown to alter ectoplasmic specialization, in Nf1+/- males in comparison to control Nf1+/+ littermates. These data demonstrate that neurofibromin haploinsufficiency impairs spermatogenesis and fertility in a mouse model of NF1.


Assuntos
Fertilidade , Haploinsuficiência , Neurofibromatose 1/metabolismo , Neurofibromina 1/metabolismo , Espermatogênese , Animais , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Masculino , Camundongos , Camundongos Mutantes , Neurofibromatose 1/genética , Neurofibromatose 1/patologia , Neurofibromina 1/genética , Epitélio Seminífero/metabolismo , Epitélio Seminífero/patologia , Espermátides/metabolismo , Espermátides/patologia , Proteínas ras/genética , Proteínas ras/metabolismo
10.
Biomed Res ; 39(4): 197-214, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30101840

RESUMO

Despite their pharmacologically opposite actions, long-acting depot formulations of both GnRH agonists and antagonists have been clinically applied for treatment of androgen-sensitive prostate cancer. Sustained treatment with GnRH analogues commonly suppresses both the synthesis and release of gonadotropins, leading to depletion of testicular testosterone. To clarify the underlying differences in the effects of GnRH agonists and antagonists on spermatogenesis, we compared histological changes in the seminiferous epithelium after administration of depot formulations of GnRH agonist leuprorelin and antagonist degarelix to male rats. Testicular weight had markedly declined by 28 days after administration of both GnRH analogues, although the testicular weight was decreased more promptly by leuprorelin compared with degarelix. Shortly after administration, massive exfoliation of premature spermatids and anomalous multinucleated giant cells was observed in seminiferous tubules of leuprorelin-treated rats, probably via the initial hyperstimulatory effects on the hypothalamic-pituitary-testicular axis, whereas no discernible changes were found in those of degarelix-treated rats. Long term treatment with both types of GnRH analogues similarly induced a marked reduction in the height of the epithelium and deformation of apical cytoplasm in Sertoli cells, resulting in premature detachment of spermatids from the epithelium. Lipid droplets had accumulated progressively in Sertoli cells, especially in those of degarelix-treated rats. These findings clearly demonstrate the differences in the effects of GnRH agonists and antagonists on the spermatogenic process. This study suggests that an appropriate choice of GnRH analogues is necessary to minimize their adverse effects on spermatogenesis when reproductive functions should be preserved in patients.


Assuntos
Hormônio Liberador de Gonadotropina , Leuprolida , Oligopeptídeos , Epitélio Seminífero/metabolismo , Espermatogênese/efeitos dos fármacos , Animais , Hormônio Liberador de Gonadotropina/agonistas , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Leuprolida/efeitos adversos , Leuprolida/farmacocinética , Leuprolida/farmacologia , Masculino , Oligopeptídeos/efeitos adversos , Oligopeptídeos/farmacocinética , Oligopeptídeos/farmacologia , Ratos , Ratos Wistar , Epitélio Seminífero/patologia , Células de Sertoli/metabolismo , Células de Sertoli/patologia , Espermátides/metabolismo , Espermátides/patologia
11.
Epigenetics Chromatin ; 11(1): 43, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-30068355

RESUMO

BACKGROUND: Linker histones establish and maintain higher-order chromatin structure. Eleven linker histone subtypes have been reported in mammals. HILS1 is a spermatid-specific linker histone, and its expression overlaps with the histone-protamine exchange process during mammalian spermiogenesis. However, the role of HILS1 in spermatid chromatin remodeling is largely unknown. RESULTS: In this study, we demonstrate using circular dichroism spectroscopy that HILS1 is a poor condenser of DNA and chromatin compared to somatic linker histone H1d. Genome-wide occupancy study in elongating/condensing spermatids revealed the preferential binding of HILS1 to the LINE-1 (L1) elements within the intergenic and intronic regions of rat spermatid genome. We observed specific enrichment of the histone PTMs like H3K9me3, H4K20me3 and H4 acetylation marks (H4K5ac and H4K12ac) in the HILS1-bound chromatin complex, whereas H3K4me3 and H3K27me3 marks were absent. CONCLUSIONS: HILS1 possesses significantly lower α-helicity compared to other linker histones such as H1t and H1d. Interestingly, in contrast to the somatic histone variant H1d, HILS1 is a poor condenser of chromatin which demonstrate the idea that this particular linker histone variant may have distinct role in histone to protamine replacement. Based on HILS1 ChIP-seq analysis of elongating/condensing spermatids, we speculate that HILS1 may provide a platform for the structural transitions and forms the higher-order chromatin structures encompassing LINE-1 elements during spermiogenesis.


Assuntos
Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , DNA/genética , Espermátides/metabolismo , Animais , Proteínas de Ligação a DNA/química , Histonas/metabolismo , Elementos Nucleotídeos Longos e Dispersos , Masculino , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Ratos , Espermátides/química
12.
Gene ; 673: 119-129, 2018 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-29890312

RESUMO

Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-ß superfamily, have been implicated in various biological and physiological processes, especially in the gonad development. However, scarce studies were focused on the roles of BMPs in the reproductive system of crustaceans. In this study, the whole gene encoding BMP7 protein was cloned and characterized firstly in Chinese mitten crab Eriocheir sinensis. The bioinformatics analysis of the deduced amino acid sequence showed that Es-BMP7 was composed of prodomain/latency-associated peptide and the TGF-ß characteristic domain. The sequence conservation and phylogenetic analysis were also conducted. Quantitative real-time PCR was conducted indifferent tissues. The highest expression in testis indicated the potential role of BMP7 to male gonad development. Western blot results showed the different translational levels of BMP7 in different tissues. In-situ hybridization revealed that the expression of es-bmp7 signals presented in a bimodal manner: highest in spermatogonia, decreased in spermatocytes and stage I spermatids, disappeared in stage II spermatids, and showed up again in stage III spermatids and mature sperm. To further verify the potential roles during spermatogenesis, immunofluorescence was conducted and results showed the similar expression tendency with in situ hybridization. The protein signal was highest in the cytoplasm of spermatogonia, continued to decline in the cytoplasm of spermatocytes and the following stages, and weak signal was found in the mature sperm. Taken together, our results revealed that Es-BMP7 might play a part in testis development in Eriocheir sinensis, presumably by maintaining the self-renewal of spermatogonia and promoting the germ cell differentiation/meiotic mitosis, or facilitating the successful fertilization.


Assuntos
Proteínas de Artrópodes/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Braquiúros/genética , Espermatogênese , Testículo/metabolismo , Animais , Proteínas de Artrópodes/genética , Proteína Morfogenética Óssea 7/genética , Braquiúros/metabolismo , Diferenciação Celular , Clonagem Molecular , Primers do DNA , Perfilação da Expressão Gênica , Células Germinativas/metabolismo , Masculino , Filogenia , RNA/metabolismo , Espermátides/metabolismo , Espermatócitos/metabolismo , Espermatogônias/metabolismo , Testículo/embriologia , Fatores de Tempo
13.
Development ; 145(13)2018 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-29866902

RESUMO

Transcription factors of the Sox protein family contain a DNA-binding HMG box and are key regulators of progenitor cell fate. Here, we report that expression of Sox30 is restricted to meiotic spermatocytes and postmeiotic haploids. Sox30 mutant males are sterile owing to spermiogenic arrest at the early round spermatid stage. Specifically, in the absence of Sox30, proacrosomic vesicles fail to form a single acrosomal organelle, and spermatids arrest at step 2-3. Although most Sox30 mutant spermatocytes progress through meiosis, accumulation of diplotene spermatocytes indicates a delayed or impaired transition from meiotic to postmeiotic stages. Transcriptome analysis of isolated stage-specific spermatogenic cells reveals that Sox30 controls a core postmeiotic gene expression program that initiates as early as the late meiotic cell stage. ChIP-seq analysis shows that Sox30 binds to specific DNA sequences in mouse testes, and its genomic occupancy correlates positively with expression of many postmeiotic genes including Tnp1, Hils1, Ccdc54 and Tsks These results define Sox30 as a crucial transcription factor that controls the transition from a late meiotic to a postmeiotic gene expression program and subsequent round spermatid development.


Assuntos
Regulação da Expressão Gênica/fisiologia , Meiose/fisiologia , Fatores de Transcrição SOX/metabolismo , Espermátides/metabolismo , Espermatogênese/fisiologia , Testículo/metabolismo , Iniciação da Transcrição Genética/fisiologia , Animais , Perfilação da Expressão Gênica , Masculino , Camundongos , Elementos de Resposta/fisiologia , Fatores de Transcrição SOX/genética , Espermátides/citologia , Testículo/citologia
14.
Development ; 145(11)2018 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-29848638

RESUMO

The postmeiotic development of male germ cells, also known as spermiogenesis, features the coordinated expression of a large number of spermatid-specific genes. However, only a limited number of key transcription factors have been identified and the underlying regulatory mechanisms remain largely unknown. Here, we report that SOX30, the most-divergent member of the Sry-related high-motility group box (SOX) family of transcription factors, is essential for mouse spermiogenesis. The SOX30 protein was predominantly expressed in spermatids, while its transcription was regulated by retinoic acid and by MYBL1 before and during meiosis. Sox30 knockout mice arrested spermiogenesis at step 3 round spermatids, which underwent apoptosis and abnormal chromocenter formation. We also determined that SOX30 regulated the expression of hundreds of spermatid-specific protein-coding and long non-coding RNA genes. SOX30 bound to the proximal promoter of its own gene and activated its transcription. These results reveal SOX30 as a novel key regulator of spermiogenesis that regulates its own transcription to enforce and activate this meiotic regulatory pathway.


Assuntos
Regulação da Expressão Gênica/genética , Fatores de Transcrição SOX/genética , Espermátides/metabolismo , Espermatogênese/fisiologia , Animais , Apoptose/fisiologia , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myb/genética , Transativadores/genética , Tretinoína/metabolismo
15.
Histochem Cell Biol ; 150(2): 173-185, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29797291

RESUMO

Spermatids are haploid differentiating cells that, in the meantime they differentiate, translocate along the seminiferous epithelium towards the tubule lumen to be just released as spermatozoa. The success of such a migration depends on dynamic of spermatid-Sertoli cell contacts, the molecular nature of which has not been well defined yet. It was demonstrated that the vascular endothelial cadherin (VEC) is expressed transitorily in the mouse seminiferous epithelium. Here, we evaluated the pattern of VEC expression by immunohistochemistry first in seminiferous tubules at different stages of the epithelial cycle when only unique types of germ cell associations are present. Changes in the pattern of VEC localization according to the step of spermatid differentiation were analysed in detail using testis fragments and spontaneously released germ cells. Utilizing the first wave of spermatogenesis as an in vivo model to have at disposal spermatids at progressive steps of differentiation, we checked for level of looser VEC association with the membrane by performing protein solubilisation under mild detergent conditions and assays through VEC-immunoblotting. Being changes in VEC solubilisation paralleled in changes in phosphotyrosine (pY) content, we evaluated if spermatid VEC undergoes Y658 phosphorylation and if this correlates with VEC solubilisation and spermatid progression in differentiation. Altogether, our study shows a temporally restricted pattern of VEC expression that culminates with the presence of round spermatids to progressively decrease starting from spermatid elongation. Conversely, pY658-VEC signs elongating spermatids; its intracellular polarized compartmentalization suggests a possible involvement of pY658-VEC in the acquisition of spermatid cell polarity.


Assuntos
Caderinas/metabolismo , Endotélio Vascular/metabolismo , Epitélio Seminífero/metabolismo , Células de Sertoli/metabolismo , Espermátides/metabolismo , Animais , Masculino , Camundongos , Espermátides/citologia
16.
Oncol Rep ; 40(2): 813-822, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29845259

RESUMO

Colorectal cancer is the third most common type of cancer and the fourth leading cause of cancer­related deaths worldwide. Although several genes have been identified to contribute to the pathogenesis of colorectal cancer, there are still many genes with unidentified functions in colorectal cancer. This study aimed to investigate the effect of shRNA­induced knockdown of the SPERT gene on the proliferation and apoptosis of human colorectal cancer RKO cells. SPERT was screened based on the TCGA dataset, and SPERT expression, cell growth, proliferation and apoptosis were detected in shSPERT­ and shCtrl­transfected RKO cells. In addition, the SPERT­related biological pathways were detected using a PathScan® Signaling Antibody Array Kit. We detected lower SPERT expression in shSPERT­transfected RKO cells than in shCtrl­transfected cells at both the translational and transcriptional levels (P<0.05), and an MTT assay revealed a clear­cut decrease in the proliferation of shSPERT­transfected RKO cells relative to shCtrl­transfected RKO cells (P<0.01). A Caspase­Glo® 3/7 assay detected an increase in the caspase­3/7 activity and the number of apoptotic cells in the shSPERT­transfected RKO cells than in the shCtrl­transfected cells (P<0.01), and flow cytometry detected a higher apoptotic rate in the shSPERT­transfected RKO cells than in the shCtrl­transfected cells (20.65±0.26 vs. 5.93±0.06%, respectively, P<0.01). Elevated levels of phosphorylated p44/42 MAPK (ERK1/2), Akt, Bad, HSP27, p38 MARK and Chk2, and elevated PARP and caspase­3 expression levels were detected in shSPERT­transfected RKO cells compared with the shCtrl­transfected cells (P<0.05). The results of the current study demonstrated that knockdown of SPERT suppresses colorectal cancer cell growth and promotes apoptosis. SPERT may serve as an oncogene and may be a potential target for the treatment of colorectal cancer.


Assuntos
Apoptose/genética , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , RNA Interferente Pequeno/genética , Espermátides/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Transdução de Sinais/genética , Transfecção/métodos
17.
Int J Mol Med ; 42(1): 53-60, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29620249

RESUMO

In vitro production of functional spermatids has special significance in the research of spermatogenesis and the treatment of male infertility. Primordial germ cells (PGCs) are the precursors of oocyte and sperm, which generate the totipotent cells. Studies have shown that PGCs have the potential ability to develop meiotic spermatids in vitro. Here we have shown that retinoic acid (RA) leads to PGC differentiation, and SCF can improve the efficiency of induction. We indicate an efficient approach to produce haploid spermatids from chicken PGCs in the presence of RA and stem cell factor (SCF). Real-time RT-PCR assays showed that RA and SCF induced a remarkable increase in expression of SYCP1, ACR, BOULE and DCM1 of meiotic germ cells and haploid germ cells, respectively. DNA content assays revealed that RA and SCF induced a remarkable increase of haploid cells. This study provides a theoretical basis and a great animal model for spermatogenesis study.


Assuntos
Haploidia , Espermátides/citologia , Animais , Biomarcadores/metabolismo , Forma Celular/efeitos dos fármacos , Embrião de Galinha , DNA/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Masculino , Meiose/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermátides/efeitos dos fármacos , Espermátides/metabolismo , Fator de Células-Tronco/farmacologia , Tretinoína/farmacologia
18.
PLoS One ; 13(3): e0193954, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29543876

RESUMO

Recently we have demonstrated the possibility to replace histones with protamine, through the heterologous expression of human protamine 1 (hPrm1) gene in sheep fibroblasts. Here we have optimized protaminization of somatic nucleus by adjusting the best concentration and exposure time to trichostatin A (TSA) in serum-starved fibroblasts (nuclear quiescence), before expressing Prm1 gene. To stop cell proliferation, we starved cells in 0.5% FBS in MEM ("starved"-ST group), whereas in the Control group (CTR) the cells were cultured in 10% FBS in MEM. To find the most effective TSA concentration, we treated the cells with increasing concentrations of TSA in MEM + 10% FBS. Our results show that combination of cell culture conditions in 50 nM TSA, is more effective in terminating cell proliferation than ST and CTR groups (respectively 8%, 17.8% and 90.2% p<0.0001). Moreover, nuclear quiescence marker genes expression (Dicer1, Smarca 2, Ezh1 and Ddx39) confirmed that our culture conditions kept the cells in a nuclear quiescent state. Finally, ST and 50 nM TSA jointly increased the number of spermatid-like cell (39.4%) at higher rate compared to 25 nM TSA (20.4%, p<0.05) and 100 nM TSA (13.7%, p<0.05). To conclude, we have demonstrated that nuclear quiescence in ST cells and the open nuclear structure conferred by TSA resulted in an improved Prm1-mediated conversion of somatic nuclei into spermatid-like structures. This finding might improve nuclear reprogramming of somatic cells following nuclear transfer.


Assuntos
Fibroblastos/metabolismo , Histonas/metabolismo , Protaminas/metabolismo , Ovinos/metabolismo , Acetilação/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , RNA Helicases DEAD-box/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Masculino , Camundongos , Técnicas de Transferência Nuclear , Ribonuclease III/metabolismo , Espermátides/metabolismo
19.
Mol Cell Biochem ; 447(1-2): 175-187, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29383560

RESUMO

SRY-related box (Sox) transcription factors are conserved among vertebrate species. These proteins regulate multiple processes including sex determination and testis differentiation of the male embryo. Members of the Sox family have been identified in pre- and postnatal testis and are known to play an important role in sex determination (Sry, Sox9), male gonadal development, and fertility (Sox4, Sox8, Sox30). However, their expression profiles per cell types remain elusive. The objectives of this research were to characterize the expression profiles of Sox family members within adult testes using publically available datasets and to determine whether these findings are consistent with literature as well as immunofluorescence and in situ hybridization results. We have found that Sox4, Sox8, Sox9, and Sox12 are highly expressed in Sertoli cells, whereas Sox5, Sox6, and Sox30 were typically expressed in spermatocytes and spermatids. Spermatogonia were characterized by the expressions of Sox3, Sox4, Sox12, Sox13, and Sox18. Hence, these results suggest that Sox transcription factors may play different roles according to cell types of the adult mammalian testis.


Assuntos
Regulação da Expressão Gênica/fisiologia , Fatores de Transcrição SOX/biossíntese , Células de Sertoli/metabolismo , Espermátides/metabolismo , Espermatócitos/metabolismo , Animais , Perfilação da Expressão Gênica , Masculino , Camundongos , Células de Sertoli/citologia , Espermátides/citologia , Espermatócitos/citologia
20.
Cell Mol Life Sci ; 75(15): 2859-2872, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29417179

RESUMO

De novo germline mutations arise preferentially in male owing to fundamental differences between spermatogenesis and oogenesis. Post-meiotic chromatin remodeling in spermatids results in the elimination of most of the nucleosomal supercoiling and is characterized by transient DNA fragmentation. Using three alternative methods, DNA from sorted populations of mouse spermatids was used to confirm that double-strand breaks (DSB) are created in elongating spermatids and repaired at later steps. Specific capture of DSB was used for whole-genome mapping of DSB hotspots (breakome) for each population of differentiating spermatids. Hotspots are observed preferentially within introns and repeated sequences hence are more prevalent in the Y chromosome. When hotspots arise within genes, those involved in neurodevelopmental pathways become preferentially targeted reaching a high level of significance. Given the non-templated DNA repair in haploid spermatids, transient DSBs formation may, therefore, represent an important component of the male mutation bias and the etiology of neurological disorders, adding to the genetic variation provided by meiosis.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Quebras de DNA de Cadeia Dupla , Fragmentação do DNA , Espermátides/metabolismo , Animais , Ensaio Cometa , DNA/genética , DNA/metabolismo , Reparo do DNA , Masculino , Meiose/genética , Camundongos Endogâmicos C57BL , Nucleossomos/genética
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