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1.
Int J Nanomedicine ; 15: 9499-9514, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33281445

RESUMO

Background: Gold nanoparticles (AuNPs) have shown great promise in various biomedical applications, but their effects on male reproductive function remain to be ascertained. The aim of this study was to investigate the uptake, cytotoxicity and testosterone production inhibition of AuNPs in mouse Leydig cells, as well as their accumulation in the testes of male mice and their effects on male reproductive function. Results: AuNPs (5 nm) were able to be internalized into the endosomes/lysosomes of TM3 Leydig cells, induce the formation of autophagosomes, increase the production of reactive oxygen species (ROS), and disrupt the cell cycle in S phase, resulting in concentration-dependent cytotoxicity and DNA damage. Interestingly, AuNPs significantly reduced testosterone production in TM3 cells by inhibiting the expression of 17α-hydroxylase, an important enzyme in androgen synthesis. After repeated intravenous injection, AuNPs gradually accumulated and retained in the testes of male BALB/c mice in a dose-dependent manner. One week after withdrawal, the level of plasma testosterone in the 0.5 mg/kg AuNPs group was significantly reduced compared to that in the PBS control group, accompanied by the decreased expression of 17α-hydroxylase in the testes. In addition, AuNPs treatment significantly increased the rate of epididymal sperm malformation, but without affecting fertility. Conclusion: Our results suggest that AuNPs can accumulate in the testes and reduce testosterone production in Leydig cells by down-regulating the expression of 17α-hydroxylase, thus affecting the quality of epididymal sperm.


Assuntos
Ouro/química , Ouro/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Nanopartículas Metálicas/química , Reprodução/efeitos dos fármacos , Animais , Ciclo Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/metabolismo , Testículo/citologia , Testículo/metabolismo , Testículo/fisiologia , Testosterona/biossíntese
2.
Anim Sci J ; 91(1): e13479, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33331680

RESUMO

The Agu is the only indigenous pig breed in Japan but its population is very small. In order to estimate the efficacy of testicular xenografting for the conservation of Agu pigs, we investigated whether neonatal testicular fragments would acquire the capacity to produce sperm after they had been cryopreserved and grafted into nude mice. Although on day 180 (day 0 = xenografting), grafts showed a low proportion of seminiferous tubule cross-sections containing sperm (0.1 ± 0.1%, mean ± SEM for four mice), the proportion reached 36.9 ± 16.7% (n = 4 mice) by day 240. When single sperm obtained on day 240 was injected into individual porcine oocytes, 28.2% of the oocytes were found to contain one male and one female pronuclei with the second polar body. Moreover, the blastocyst formation rate after injection of the xenogeneic sperm was 28.4%, whereas that in the absence of sperm injection (attributable to parthenogenesis) was 13.3%. These findings suggest that more than half of the blastocysts resulted from fertilization. Thus, testicular xenografting could assist the conservation of Agu pigs by salvaging germ cells present in neonatal testes even after cryopreservation.


Assuntos
Animais Recém-Nascidos , Blastocisto , Conservação dos Recursos Naturais , Criopreservação/métodos , Criopreservação/veterinária , Embrião de Mamíferos , Espécies em Perigo de Extinção , Injeções de Esperma Intracitoplásmicas/métodos , Injeções de Esperma Intracitoplásmicas/veterinária , Espermatogênese , Espermatozoides/transplante , Suínos , Testículo/citologia , Preservação de Tecido/métodos , Preservação de Tecido/veterinária , Animais , Feminino , Japão , Masculino , Camundongos Nus , Transplante Heterólogo/métodos , Transplante Heterólogo/veterinária
3.
Nat Commun ; 11(1): 5656, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-33168808

RESUMO

Establishment of spermatogonia throughout the fetal and postnatal period is essential for production of spermatozoa and male fertility. Here, we establish a protocol for in vitro reconstitution of human prospermatogonial specification whereby human primordial germ cell (PGC)-like cells differentiated from human induced pluripotent stem cells are further induced into M-prospermatogonia-like cells and T1 prospermatogonia-like cells (T1LCs) using long-term cultured xenogeneic reconstituted testes. Single cell RNA-sequencing is used to delineate the lineage trajectory leading to T1LCs, which closely resemble human T1-prospermatogonia in vivo and exhibit gene expression related to spermatogenesis and diminished proliferation, a hallmark of quiescent T1 prospermatogonia. Notably, this system enables us to visualize the dynamic and stage-specific regulation of transposable elements during human prospermatogonial specification. Together, our findings pave the way for understanding and reconstructing human male germline development in vitro.


Assuntos
Células Germinativas Embrionárias/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Espermatogênese/genética , Espermatogênese/fisiologia , Animais , Diferenciação Celular , Epigenômica , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Elementos Reguladores de Transcrição , Análise de Sequência de RNA , Espermatogônias/citologia , Espermatozoides , Testículo/citologia , Transcriptoma
4.
Nat Commun ; 11(1): 5683, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33173058

RESUMO

Clinical efficacy of treatments against non-obstructive azoospermia (NOA), which affects 1% of men, are currently limited by the incomplete understanding of NOA pathogenesis and normal spermatogenic microenvironment. Here, we profile >80,000 human testicular single-cell transcriptomes from 10 healthy donors spanning the range from infant to adult and 7 NOA patients. We show that Sertoli cells, which form the scaffold in the testicular microenvironment, are severely damaged in NOA patients and identify the roadmap of Sertoli cell maturation. Notably, Sertoli cells of patients with congenital causes (Klinefelter syndrome and Y chromosome microdeletions) are mature, but exhibit abnormal immune responses, while the cells in idiopathic NOA (iNOA) are physiologically immature. Furthermore, we find that inhibition of Wnt signaling promotes the maturation of Sertoli cells from iNOA patients, allowing these cells to regain their ability to support germ cell survival. We provide a novel perspective on the development of diagnostic methods and therapeutic targets for NOA.


Assuntos
Azoospermia , Células de Sertoli/patologia , Espermatozoides/patologia , Adulto , Azoospermia/etiologia , Azoospermia/metabolismo , Azoospermia/patologia , Perfilação da Expressão Gênica , Humanos , Masculino , Análise de Célula Única , Espermatogênese , Testículo/citologia
5.
PLoS One ; 15(10): e0240844, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33079963

RESUMO

Cruciform DNA is a causing factor of genome instability and chromosomal translocation, however, most studies about cruciform DNA in mammalian cells were based on palindromic sequences containing plasmids and reports about endogenous cruciform DNA are rare. In this study we observed the dynamics of endogenous cruciform DNA in mouse growing oocytes using immunofluorescence labeling method. We found cruciform DNA foci exist in transcription active growing oocytes but not in transcription inactive fully grown oocytes and colocalized with Parp1 but not with DNA damage marker γH2A.X. By analyzing the Genotype-Tissue Expression data, we found cruciform DNA-mediated chromosomal translocation in human spermatocytes is associated with the specific DNA transcription in testis. When inhibiting the transcription with α-amanitin in mouse oocytes, we found oocyte cruciform DNA foci decreased significantly. In summary, we observed the endogenous cruciform DNA in growing oocytes and our results showed that the cruciform DNA formation is transcription-dependent.


Assuntos
DNA Cruciforme/metabolismo , Oócitos , Transcrição Genética/fisiologia , Alfa-Amanitina/efeitos adversos , Animais , Imunofluorescência/métodos , Histonas/metabolismo , Masculino , Camundongos , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oogênese , Poli(ADP-Ribose) Polimerase-1/metabolismo , Espermatogênese , Testículo/citologia , Testículo/metabolismo
6.
J Vis Exp ; (162)2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32925873

RESUMO

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm development. However, immunolabeling of the whole Drosophila testis is often associated with significant non-uniformity of staining due to antibody penetration. Squashed preparations only partially overcome the problem since it decreases the 3D quality of the analyses. Herein, we describe a whole-mount protocol using NP40 and heptane during fixation together with immunolabeling in liquid media. It preserves the volume suitable for confocal microscopy together with reproducible and reliable labeling. We show different examples of 3D reconstitution of spermatocyte nuclei from confocal sections. The intra- and inter-testes reproducibility allows 3D quantification and comparison of fluorescence between single cells from different genotypes. We used different components of the intranuclear MINT structure (Mad1-containing Intra Nuclear Territory) as well as two components associated with the nuclear pore complex to illustrate this protocol and its applications on the largest cells of the testis, the S4-S5 spermatocytes.


Assuntos
Drosophila melanogaster/citologia , Imageamento Tridimensional , Microscopia Confocal/métodos , Espermatócitos/citologia , Testículo/citologia , Animais , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Imunofluorescência , Masculino , Interferência de RNA , Reprodutibilidade dos Testes , Espermatócitos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fixação de Tecidos
7.
Proc Natl Acad Sci U S A ; 117(38): 23751-23761, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32917815

RESUMO

Mast cell (MC)-associated diseases, including allergy/anaphylaxis and neuroinflammatory pain disorders, exhibit a sex bias, with females at increase risk. While much attention has been directed toward adult sex hormones as drivers of sex differences, that female sex bias in MC-associated diseases is evident in prepubertal children, suggesting early-life origins of sex differences which have yet to be explored. Utilizing rodent models of MC-mediated anaphylaxis, our data here reveal that, 1) compared with females, males exhibit significantly reduced severity of MC-mediated anaphylactic responses that emerge prior to puberty and persist into adulthood, 2) reduced severity of MC-mediated anaphylaxis in males is linked with the naturally high level of perinatal androgens and can be recapitulated in females by perinatal exposure to testosterone proprionate, 3) perinatal androgen exposure guides bone marrow MC progenitors toward a masculinized tissue MC phenotype characterized by decreased concentration of prestored MC granule mediators (e.g., histamine, serotonin, and proteases) and reduced mediator release upon degranulation, and 4) engraftment of MC-deficient Kit W-sh/W-sh mice with adult male, female, or perinatally androgenized female MCs results in MC-mediated anaphylaxis response that reflects the MC sex and not host sex. Together, these data present evidence that sex differences in MC phenotype and resulting disease severity are established in early life by perinatal androgens. Thus, factors affecting levels of perinatal androgens could have a significant impact on MC development and MC-associated disease risk across the life span.


Assuntos
Anafilaxia , Androgênios/farmacologia , Mastócitos/efeitos dos fármacos , Fatores Sexuais , Animais , Modelos Animais de Doenças , Feminino , Inflamação , Masculino , Mastócitos/fisiologia , Camundongos , Camundongos Transgênicos , Testículo/citologia , Testículo/efeitos dos fármacos
8.
Fertil Steril ; 114(1): 33-43, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32622411

RESUMO

OBJECTIVE: To identify cell types in the male and female reproductive systems at risk for SARS-CoV-2 infection because of the expression of host genes and proteins used by the virus for cell entry. DESIGN: Descriptive analysis of transcriptomic and proteomic data. SETTING: Academic research department and clinical diagnostic laboratory. PATIENT(S): Not applicable (focus was on previously generated gene and protein expression data). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Identification of cell types coexpressing the key angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) genes and proteins as well as other candidates potentially involved in SARS-CoV-2 cell entry. RESULT(S): On the basis of single-cell RNA sequencing data, coexpression of ACE2 and TMPRSS2 was not detected in testicular cells, including sperm. A subpopulation of oocytes in nonhuman primate ovarian tissue was found to express ACE2 and TMPRSS2, but coexpression was not observed in ovarian somatic cells. RNA expression of TMPRSS2 in 18 samples of human cumulus cells was shown to be low or absent. There was general agreement between publicly available bulk RNA and protein datasets in terms of ACE2 and TMPRSS2 expression patterns in testis, ovary, endometrial, and placental cells. CONCLUSION(S): These analyses suggest that SARS-CoV-2 infection is unlikely to have long-term effects on male and female reproductive function. Although the results cannot be considered definitive, they imply that procedures in which oocytes are collected and fertilized in vitro are associated with very little risk of viral transmission from gametes to embryos and may indeed have the potential to minimize exposure of susceptible reproductive cell types to infection in comparison with natural conception.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Fertilidade/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Pneumonia Viral/metabolismo , Reprodução/fisiologia , Internalização do Vírus , Adolescente , Adulto , Animais , Betacoronavirus/genética , Linhagem Celular , Infecções por Coronavirus/genética , Feminino , Humanos , Macaca fascicularis , Masculino , Ovário/citologia , Ovário/metabolismo , Ovário/virologia , Pandemias , Peptidil Dipeptidase A/biossíntese , Peptidil Dipeptidase A/genética , Pneumonia Viral/genética , Gravidez , Proteômica/métodos , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Testículo/citologia , Testículo/metabolismo , Testículo/virologia , Transcriptoma/fisiologia , Adulto Jovem
9.
Sci Rep ; 10(1): 11674, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669615

RESUMO

Stem cell activity and cell differentiation is robustly influenced by the nutrient availability in the gonads. The signal that connects nutrient availability to gonadal stem cell activity remains largely unknown. In this study, we show that tumor necrosis factor Eiger (Egr) is upregulated in testicular smooth muscles as a response to prolonged protein starvation in Drosophila testis. While Egr is not essential for starvation-induced changes in germline and somatic stem cell numbers, Egr and its receptor Grindelwald influence the recovery dynamics of somatic cyst stem cells (CySCs) upon protein refeeding. Moreover, Egr is also involved in the refeeding-induced, ectopic expression of the CySC self-renewal protein and the accumulation of early germ cells. Egr primarily acts through the Jun N-terminal kinase (JNK) signaling in Drosophila. We show that inhibition of JNK signaling in cyst cells suppresses the refeeding-induced abnormality in both somatic and germ cells. In conclusion, our study reveals both beneficial and detrimental effects of Egr upregulation in the recovery of stem cells and spermatogenesis from prolonged protein starvation.


Assuntos
Proteínas na Dieta/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas de Membrana/genética , Espermatozoides/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular , Proteínas na Dieta/administração & dosagem , Proteínas de Drosophila/agonistas , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Ingestão de Alimentos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Homeostase/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Proteínas de Membrana/agonistas , Proteínas de Membrana/metabolismo , Espermatogênese/efeitos dos fármacos , Espermatogênese/genética , Espermatozoides/citologia , Espermatozoides/efeitos dos fármacos , Inanição/genética , Inanição/metabolismo , Nicho de Células-Tronco/genética , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Testículo/citologia , Testículo/efeitos dos fármacos , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
10.
Mol Cell ; 79(4): 645-659.e9, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32692974

RESUMO

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.


Assuntos
Grânulos Citoplasmáticos/genética , DNA Helicases/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Biossíntese de Proteínas , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Estresse Fisiológico/genética , Regiões 5' não Traduzidas , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/patologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Feminino , Células HCT116 , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Espermatogônias/citologia , Espermatogônias/patologia , Testículo/citologia , Testículo/metabolismo
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.
Tissue Cell ; 64: 101342, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32473707

RESUMO

The developmental changes of Sertoli cells were examined and described in the freshwater pearl mussel Margaritifera laevis using light and transmission electron microscopy. Sertoli cells, which are located on the basal lamina of acini in the testis, include a large number of glycogen granules, electron-dense globules, lipid droplets, and sperm morulae. Electron-dense globules are the vacuoles into which the electron-dense material is condensed. In aging Sertoli cells, the content of the globules leaks out to the extracellular area. Large lipid droplets are formed by the deposition of smaller lipid droplets into a vacuole. After the disruption of the Sertoli cell, the lipid droplets are discharged to the extracellular area and fuse with to form a larger mass. The spermatogonia which were engulfed by the Sertoli cells begin to condense their chromatin and transform themselves into sperm morulae. The constituent cells of the sperm morulae proliferate and finally differentiate into the spermatozoa. After the disruption of the Sertoli cell, the spermatozoa produced from the sperm morulae are released into the acinus lumen. Numerous matured spermatozoa in the acini gather around the large lipid droplet, to form the sperm sphere. The completed sperm spheres are subsequently released through the exhalant siphon into the stream.


Assuntos
Bivalves/citologia , Células de Sertoli/ultraestrutura , Espermatogênese/fisiologia , Animais , Masculino , Células de Sertoli/citologia , Espermatogônias/citologia , Espermatogônias/ultraestrutura , Testículo/citologia , Testículo/ultraestrutura
13.
Gene ; 754: 144848, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32522697

RESUMO

The TGF-beta superfamily is widely involved in cell events such as cell division and differentiation, while bone morphogenetic proteins (BMPs) belong to one of the subgroups. Their functions in crustacean spermatogenesis are still unknown. In this study, we first identified the bone morphogenetic protein 2 (bmp2) from Eriocheir sinensis (E. sinensis) testis. The es-BMP2 shows high expression in E. sinensis testis. We found that es-BMP2 is expressed in spermatids. The successfully knockdown of es-BMP2 through in vivo RNAi are used for functional analysis. Compared with the control group, the proportion of abnormal nuclear cup morphology in mature spermatozoa increased significantly after es-bmp2 RNAi, suggesting that es-BMP2 plays an important role in mature sperm morphogenesis. Immunofluorescence results confirm this finding. In order to study the specific mechanism of es-BMP2 involved in spermiogenesis, we tested kinesin-14 KIFC1, which functions in the nucleus formation of spermatozoa in E. sinensis. The results showed that knockdown of es-BMP2 caused a significant decrease of es-KIFC1 expression. We further performed es-bmp2 knockdown in vitro in primary cultured testis cells. es-KIFC1 expression was significantly reduced after es-bmp2 RNAi. The above results indicate that es-BMP2 participates in maintaining the spermiogenesis of E. sinensis by regulating es-KIFC1 expression.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Células Germinativas/citologia , Cinesina/metabolismo , Espermatogênese , Testículo/citologia , Animais , Proteína Morfogenética Óssea 2/genética , Braquiúros , Regulação da Expressão Gênica , Células Germinativas/metabolismo , Cinesina/genética , Masculino , Testículo/metabolismo
14.
J Vis Exp ; (159)2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32510475

RESUMO

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes' large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.


Assuntos
Divisão Celular/fisiologia , Drosophila/patogenicidade , Larva/patogenicidade , Microscopia Confocal/métodos , Pupa/patogenicidade , Testículo/metabolismo , Animais , Masculino , Testículo/citologia
15.
Gen Comp Endocrinol ; 295: 113525, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32502497

RESUMO

We aim to establish a small-bodied surrogate broodstock, such as mackerel, which produces functional bluefin tuna gametes by spermatogonial transplantation. When reproductively fertile fish are used as recipients, endogenous gametogenesis outcompetes donor-derived gametogenesis, and recipient fish predominantly produce their gametes. In this study, we assessed fertility of hybrid mackerel, Scomber australasicus × S. japonicus, and its suitability as a recipient for transplantation of bluefin tuna germ cells. Hybrid mackerel were produced by artificially inseminating S. australasicus eggs with S. japonicus spermatozoa. Cellular DNA content and PCR analyses revealed that F1 offspring were diploid carrying both paternal and maternal genomes. Surprisingly, histological observations found no germ cells in hybrid mackerel gonads at 120 days post-hatch (dph), although they were present in the gonad of 30- and 60-dph hybrid mackerel. The frequency of germ cell-less fish was 100% at 120-dph, 63.1% at 1-year-old, and 81.8% at 2-year-old. We also confirmed a lack of expression of germ cell marker (DEAD-box helicase 4, ddx4) in the germ cell-less gonads of hybrid mackerel. By contrast, expression of Sertoli cell marker (gonadal soma-derived growth factor, gsdf) and of Leydig cell marker (steroid 11-beta-hydroxlase, cyp11b1) were clearly detected in hybrid mackerel gonads. Together these results showed that most of the hybrid gonads were germ cell-less sterile, but still possessed supporting cells and steroidogenic cells, both of which are indispensable for nursing donor-derived germ cells. To determine whether hybrid gonads could attract and incorporate donor bluefin tuna germ cells, testicular cells labeled with PKH26 fluorescent dye were intraperitoneally transplanted. Fluorescence observation of hybrid recipients at 14 days post-transplantation revealed that donor cells had been incorporated into the recipient's gonads. This suggests that hybrid mackerel show significant promise for use as a recipient to produce bluefin tuna gametes.


Assuntos
Cruzamentos Genéticos , Células Germinativas/citologia , Células Germinativas/transplante , Gônadas/metabolismo , Hibridização Genética , Infertilidade/genética , Atum/genética , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Reprodução , Testículo/citologia , Testículo/metabolismo , Testículo/transplante
16.
PLoS One ; 15(5): e0233322, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469895

RESUMO

The importance of dietary lipids in male reproduction are not as well understood as in females, in which dietary lipids, such as phospholipids (PL) and associated fatty acids (FA), are important structural components of the eggs and provide energy for their offspring. In mammals, lipids are suggested to be important for spermatogenesis and to structural components of the spermatozoa that could improve fertilization rates. New knowledge of how lipids affect sexual maturation in male Atlantic salmon (Salmo salar), an important global aquaculture species, could provide tools to delay maturation and/or improve reproductive success. Therefore, changes in testicular composition of lipids and gene transcripts associated with spermatogenesis and lipid metabolism were studied in sexually maturing male salmon compared to immature males and females. An increase in total testis content of FA and PL, and a shift to higher PL composition was observed in maturing males, concomitant with increases in mRNA levels for genes involved in spermatogenesis, FA uptake and synthesis, and production of long chain-polyunsaturated fatty acids (LC-PUFA) and PL. A particularly interesting finding was elevated testis expression of acyl-CoA synthetase 4 (acsl4), and acyl-CoA thioesterase 2 (acot2), critical enzymes that regulate intra-mitochondrial levels of 20:4n-6 FA (arachidonic acid), which have been associated with improved cholesterol transport during steroidogenesis. This suggested that FA may have direct effects on sex steroid production in salmon. Furthermore, we observed increased testis expression of genes for endogenous synthesis of 16:0 and elongation/desaturation to 22:6n-3 (docosahexaenoic acid) in sexually maturing males relative to immature fish. Both of these FA are important structural components of the PL, phosphatidylcholine (PC), and were elevated concomitant with increases in the content of phosphatidic acid, an important precursor for PC, in maturing males compared to immature fish. Overall, this study suggests that, similar to mammals, lipids are important to spermatogenesis and serve as structural components during testicular growth and maturation in Atlantic salmon.


Assuntos
Ácidos Graxos Insaturados/metabolismo , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Fosfolipídeos/metabolismo , Salmo salar/metabolismo , Maturidade Sexual , Testículo/metabolismo , Animais , Ácidos Graxos Dessaturases/genética , Feminino , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Masculino , Salmo salar/genética , Testículo/citologia
17.
Sci China Life Sci ; 63(7): 1006-1015, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32361911

RESUMO

Being infected by SARS-CoV-2 may cause damage to multiple organs in patients, such as the lung, liver and heart. Angiotensin-converting enzyme 2 (ACE2), reported as a SARS-CoV-2 receptor, is also expressed in human male testes. This suggests a potential risk in human male reproductive system. However, the characteristics of ACE2-positive cells and the expression of other SARS-CoV-2 process-related genes are still worthy of further investigation. Here, we performed singlecell RNA seq (scRNA-seq) analysis on 853 male embryo primordial germ cells (PGCs) and 2,854 normal testis cells to assess the effects of the SARS-CoV-2 virus on the male reproductive system from embryonic stage to adulthood. We also collected and constructed the scRNA-seq library on 228 Sertoli cells from three non-obstructive azoospermia (NOA) patients to assess the effects at disease state. We found that ACE2 expressing cells existed in almost all testis cell types and Sertoli cells had highest expression level and positive cells ratio. Moreover, ACE2 was also expressed in human male PGCs. In adulthood, the level of ACE2 expression decreased with the increase of age. We also found that ACE2 positive cells had high expressions of stress response and immune activation-related genes. Interestingly, some potential SARS-CoV-2 process-related genes such as TMPRSS2, BSG, CTSL and CTSB had different expression patterns in the same cell type. Furthermore, ACE2 expression level in NOA donors' Sertoli cells was significantly decreased. Our work would help to assess the risk of SARS-CoV-2 infection in the male reproductive system.


Assuntos
Azoospermia/genética , Betacoronavirus/patogenicidade , Infecções por Coronavirus , Pandemias , Peptidil Dipeptidase A/genética , Pneumonia Viral , Testículo/metabolismo , Testículo/virologia , Adulto , Azoospermia/complicações , Azoospermia/metabolismo , Betacoronavirus/metabolismo , Infecções por Coronavirus/complicações , Infecções por Coronavirus/fisiopatologia , Infecções por Coronavirus/virologia , Células Germinativas Embrionárias/metabolismo , Células Germinativas Embrionárias/virologia , Expressão Gênica , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Masculino , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/complicações , Pneumonia Viral/fisiopatologia , Pneumonia Viral/virologia , Receptores Virais/genética , Receptores Virais/metabolismo , Células de Sertoli/metabolismo , Células de Sertoli/virologia , Análise de Célula Única , Espermatogênese/genética , Espermatogênese/fisiologia , Testículo/citologia
18.
Cells ; 9(4)2020 04 09.
Artigo em Inglês | MEDLINE | ID: covidwho-45956

RESUMO

In December 2019, a novel coronavirus (SARS-CoV-2) was identified in COVID-19 patients in Wuhan, Hubei Province, China. SARS-CoV-2 shares both high sequence similarity and the use of the same cell entry receptor, angiotensin-converting enzyme 2 (ACE2), with severe acute respiratory syndrome coronavirus (SARS-CoV). Several studies have provided bioinformatic evidence of potential routes of SARS-CoV-2 infection in respiratory, cardiovascular, digestive and urinary systems. However, whether the reproductive system is a potential target of SARS-CoV-2 infection has not yet been determined. Here, we investigate the expression pattern of ACE2 in adult human testes at the level of single-cell transcriptomes. The results indicate that ACE2 is predominantly enriched in spermatogonia and Leydig and Sertoli cells. Gene Set Enrichment Analysis (GSEA) indicates that Gene Ontology (GO) categories associated with viral reproduction and transmission are highly enriched in ACE2-positive spermatogonia, while male gamete generation related terms are downregulated. Cell-cell junction and immunity-related GO terms are increased in ACE2-positive Leydig and Sertoli cells, but mitochondria and reproduction-related GO terms are decreased. These findings provide evidence that the human testis is a potential target of SARS-CoV-2 infection, which may have significant impact on our understanding of the pathophysiology of this rapidly spreading disease.


Assuntos
Infecções por Coronavirus/transmissão , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/transmissão , Receptores Virais/genética , Receptores Virais/metabolismo , Testículo/metabolismo , Testículo/virologia , Infecções por Coronavirus/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Pandemias , Pneumonia Viral/virologia , RNA Citoplasmático Pequeno/química , RNA Citoplasmático Pequeno/genética , Testículo/citologia , Replicação Viral/genética
19.
Int J Mol Sci ; 21(7)2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32244802

RESUMO

The double sex and mab-3-related transcription factors like family C2 (DMRTC2) gene is indispensable for mammalian testicular function and spermatogenesis. Despite its importance, what expression and roles of DMRTC2 possesses and how it regulates the testicular development and spermatogenesis in sheep, especially in Tibetan sheep, remains largely unknown. In this study, DMRTC2 cDNA from testes of Tibetan sheep was firstly cloned by the RT-PCR method, and its molecular characterization was identified. Subsequently, the expression and localization patterns of DMRTC2 were evaluated by quantitative real-time PCR (qPCR), Western blot, and immunofluorescence. The cloning and sequence analysis showed that the Tibetan sheep DMRTC2 cDNA fragment contained 1113 bp open reading frame (ORF) capable of encoding 370 amino acids, and displayed high identities with some other mammals, which shared an identical DM domain sequence of 47 amino acids ranged from residues 38 to 84. qPCR and Western blot results showed that DMRTC2 was expressed in testes throughout the development stages while not in epididymides (caput, corpus, and cauda), with higher mRNA and protein abundance in Tibetan sheep testes of one- and three-year-old (post-puberty) compared with that of three-month-old (pre-puberty). Immunofluorescence results revealed that immune staining for DMRTC2 protein was observed in spermatids and spermatogonia from post-puberty Tibetan sheep testes, and gonocytes from pre-puberty Tibetan sheep testes. Together, these results demonstrated, for the first time, in sheep, that DMRTC2, as a highly conserved gene in mammals, is essential for sheep spermatogenesis by regulating the proliferation or differentiation of gonocytes and development of spermatids in ram testes at different stages of maturity.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Ovinos/genética , Espermátides/metabolismo , Espermatogênese/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Tibet
20.
Sci Rep ; 10(1): 6751, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317665

RESUMO

SOX3 is a transcription factor expressed within the developing and adult nervous system where it mostly functions to help maintain neural precursors. Sox3 is also expressed in other locations, notably within the spermatogonial stem/progenitor cell population in postnatal testis. Independent studies have shown that Sox3 null mice exhibit a spermatogenic block as young adults, the mechanism of which remains poorly understood. Using a panel of spermatogonial cell marker genes, we demonstrate that Sox3 is expressed within the committed progenitor fraction of the undifferentiated spermatogonial pool. Additionally, we use a Sox3 null mouse model to define a potential role for this factor in progenitor cell function. We demonstrate that Sox3 expression is required for transition of undifferentiated cells from a GFRα1+ self-renewing state to the NGN3 + transit-amplifying compartment. Critically, using chromatin immunoprecipitation, we demonstrate that SOX3 binds to a highly conserved region in the Ngn3 promoter region in vivo, indicating that Ngn3 is a direct target of SOX3. Together these studies indicate that SOX3 functions as a pro-commitment factor in spermatogonial stem/progenitor cells.


Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/genética , Espermatogônias/metabolismo , Testículo/metabolismo , Células-Tronco Germinativas Adultas/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Ligação Proteica , Fatores de Transcrição SOXB1/deficiência , Transdução de Sinais , Espermatogênese/genética , Espermatogônias/citologia , Espermatogônias/crescimento & desenvolvimento , Testículo/citologia , Testículo/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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