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1.
Semin Cell Dev Biol ; 121: 32-39, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34034987

RESUMO

Human spermatogonial stem cells (SSCs) and Sertoli cells might have the applications in reproduction and regenerative medicine. Abnormal spermatogenesis results in male infertility, which seriously affects human reproduction and health. Spermatogenesis depends on the epigenetic and genetic regulation of male germ cells and somatic cells. A number of microRNAs (miRNAs) have been identified in human testicular tissues, and they are closely related to male fertility. Significantly, we and peers have recently demonstrated that numerous miRNAs are essential for regulating the self-renewal and apoptosis of human SSCs and Sertoli cells through controlling their mRNA and lncRNA targets. In this review, we critically discuss these findings regarding the important functions and mechanisms of miRNAs in mediating the fate determinations of human SSCs and Sertoli cells. Meanwhile, we illustrate the regulatory networks for miRNAs by forming the upstream and downstream regulators of mRNAs and lncRNAs in human SSCs, and we address that miRNAs regulate the decisions of Sertoli cells by targeting genes and via N6-methyladenosine (m6A). We also point out the future directions for further studies on this field. This review could offer an update on novel molecular targets for treating male infertility and new insights into epigenetic regulation of human spermatogenesis.


Assuntos
MicroRNAs/genética , Células de Sertoli/metabolismo , Espermatogênese/imunologia , Espermatogônias/imunologia , Animais , Humanos , Masculino
2.
J Reprod Immunol ; 145: 103325, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33930667

RESUMO

Contraceptive vaccine (CV) is a valuable, non-invasive, and alternative method for purposeful contraception. Sperm antigens are useful targets for producing CVs due to their specialized expression in sperm. In this study, a recombinant protein containing three main sperm epitopes (IZUMO1, SACA3, and PH-20) was designed and evaluated as CV to control fertility in male mice. The chimeric recombinant protein was expressed and purified in E. coli. Male mice were immunized by 100 µg purified protein and sera were collected to assess IgG antibodies. Evaluating the reproductive performance, immunized male mice mated with normal-fertile female mice and mating rate and the number of newborns was studied. Immunized mice were sacrificed and necropsy and histopathology studies were conducted. The results revealed that the designed chimeric protein stimulated the immune system of the mice effectively. The level of IgG antibody was significantly higher in vaccinated mouse rather than control mouse. Eighty percent of the vaccinated mice became infertile and in the remaining ones, the number of children decreased to 4-6 offspring instead of 10-12 in normal mice. Histopathological studies showed that no organs including heart, brain, lung, liver, kidney and intestine were damaged. However, Normal spermatogenesis has been disrupted and necrotic spermatogonia cells were reported in Seminiferous tubules. We concluded that the designed chimeric protein containing IZUMO1, SACA3, and PH-20 epitopes can stimulate the immune system and cause male contraception without any side effects.


Assuntos
Anticoncepção Imunológica/métodos , Infertilidade Masculina/imunologia , Proteínas Recombinantes de Fusão/imunologia , Vacinas Anticoncepcionais/imunologia , Animais , Moléculas de Adesão Celular/administração & dosagem , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Modelos Animais de Doenças , Epitopos/administração & dosagem , Epitopos/genética , Epitopos/imunologia , Humanos , Hialuronoglucosaminidase/administração & dosagem , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/imunologia , Imunoglobulinas/administração & dosagem , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Infertilidade Masculina/patologia , Isoantígenos/administração & dosagem , Isoantígenos/genética , Isoantígenos/imunologia , Masculino , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas de Plasma Seminal/administração & dosagem , Proteínas de Plasma Seminal/genética , Proteínas de Plasma Seminal/imunologia , Túbulos Seminíferos/citologia , Túbulos Seminíferos/imunologia , Túbulos Seminíferos/patologia , Espermatogônias/imunologia , Espermatogônias/patologia , Vacinas Anticoncepcionais/administração & dosagem , Vacinas Anticoncepcionais/genética
3.
Fish Shellfish Immunol ; 112: 108-115, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33716110

RESUMO

Germ cell transplantation and testis graft represent promising biotechnologies that can be applied for the reproduction of commercial or endangered species. However, mechanisms of rejection from the host immune system might remove the transplanted donor cells/tissues and limit the surrogate production of gametes. In this work, we administered emulsion containing-immunosuppressants to verify whether they are capable to prevent immune rejection and promote survival of testis allografts in rainbow trout. In the first part of this study, we demonstrated in vitro that tacrolimus and cyclosporine were able to affect viability, inhibit leucocyte proliferation, and suppress il2 expression in vitro. In in vivo experiments, both doses of tacrolimus (0.5 and 1.5 mg/kg) and the lower dose of cyclosporine (20 mg/kg) significantly inhibited the expression of il2 in head kidney, three days post-injection. A higher dose of cyclosporine (40 mg/kg) was able to inhibit il2 expression for up to seven days post-injection. In the second part, testis allografts were conducted in fish treated weekly with emulsion containing-tacrolimus. Immunohistochemical, conventional histology, and qRT-PCR (vasa) analysis demonstrated the presence of spermatogonial cells by the fifth week, in animals treated with 0.5 mg/kg of tacrolimus similar as found in autografted group. In the group treated with the highest tacrolimus dose (1.5 mg/kg) and in the non-treated group (without immunosuppressant), no germ cells or their respective markers were detected. il2 expression in head kidney was also suppressed in grafted animals treated with tacrolimus compared to non-treated group. These results suggest that tacrolimus may be a promising immunosuppressant for testis allografts or germ cell transplantation in rainbow trout. Co-administration combining tacrolimus (at lower dose) with other immunosuppressive drugs for inhibiting other activation pathways of the immune system, as performed in human organ transplantation, could be an alternative approach to optimize the immunosuppressive effects in host organisms.


Assuntos
Aloenxertos/imunologia , Ciclosporina/farmacologia , Imunossupressores/farmacologia , Oncorhynchus mykiss/cirurgia , Espermatogônias/imunologia , Tacrolimo/farmacologia , Testículo/transplante , Transplante Homólogo/veterinária , Animais , Masculino
4.
Biol Reprod ; 101(2): 478-491, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31077286

RESUMO

We recently established a germ cell transplantation system in salmonids. Donor germ cells transplanted into the body cavity of recipient embryos migrate toward and are incorporated into the recipient gonad, where they undergo gametogenesis. Among the various types of testicular germ cells, only type A spermatogonia (A-SG) can be incorporated into the recipient gonads. Enriching for A-SG is therefore important for improving the efficiency of germ cell transplantation. To enrich for A-SG, an antibody against a cell surface marker is a convenient and powerful approach used in mammals; however, little is known about cell surface markers for A-SG in fish. To that end, we have produced novel monoclonal antibodies (mAbs) against cell-surface molecules of rainbow trout (Oncorhynchus mykiss) A-SG. We inoculated mice with living A-SG isolated from pvasa-GFP transgenic rainbow trout using GFP-dependent flow cytometry. By fusing lymph node cells of the inoculated mice with myeloma cells, we generated 576 hybridomas. To identify hybridomas that produce mAbs capable of labeling A-SG preferentially and effectively, we screened them using cell ELISA, fluorescence microscopy, and flow cytometry. We thereby identified two mAbs that can label A-SG. By using flow cytometry with these two antibodies, we could enrich for A-SG with transplantability to recipient gonads from amongst total testicular cells. Furthermore, one of these mAbs could also label zebrafish (Danio rerio) spermatogonia. Thus, we expect these monoclonal antibodies to be powerful tools for germ cell biology and biotechnology.


Assuntos
Anticorpos Monoclonais/imunologia , Oncorhynchus mykiss/fisiologia , Espermatogônias/fisiologia , Animais , Animais Geneticamente Modificados , Cruzamento , Ensaio de Imunoadsorção Enzimática , Epitopos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Espermatogênese/genética , Espermatogênese/fisiologia , Espermatogônias/classificação , Espermatogônias/imunologia
5.
Andrologia ; 50(11): e13083, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30569649

RESUMO

Male infertility is due to genetics, hormonal or environmental causes, or is idiopathic. Azoospermia is linked to local testicular microenvironment deregulation, with inflammatory cells present in the 15% of testicular biopsies of infertile patients. As widely reported, spermatogenesis and steroidogenesis are controlled by local immunoregulatory agents produced by immune and nonimmune cells. Moreover IL-6R, TNFR1, Fas and IL-1R are expressed on germ cells, indicating a direct action of pro-inflammatory agents on these cells. Beyond the known function of cytokines and nitric oxide on testicular function at the stable levels present in the normal testis, this review focalises on the effect of pro-inflammatory factors on germ cell survival and death when inflammatory conditions are established in the testis. As no cure for male infertility has been found up to the present, intracytoplasmic sperm injection is the therapeutic option for azoospermic patients who wish to achieve genetic parenthood. Therapies with antioxidant and anti-inflammatory agents in experimental models of testicular damage have been successful. However, clinical implementation is uncertain in cases with a prolonged inflammatory state of the testis. Therapies offering multiple approaches to treat infertility by restoring the spermatogonial stem cell niche and protecting germ cells from apoptosis should be considered.


Assuntos
Apoptose/imunologia , Azoospermia/imunologia , Orquite/imunologia , Espermatogônias/patologia , Testículo/patologia , Células-Tronco Germinativas Adultas/efeitos dos fármacos , Células-Tronco Germinativas Adultas/fisiologia , Animais , Apoptose/efeitos dos fármacos , Azoospermia/tratamento farmacológico , Azoospermia/patologia , Biópsia , Inibidores de Caspase/farmacologia , Inibidores de Caspase/uso terapêutico , Modelos Animais de Doenças , Humanos , Masculino , Orquite/complicações , Orquite/patologia , Espermatogênese/efeitos dos fármacos , Espermatogênese/imunologia , Espermatogônias/imunologia , Testículo/citologia , Testículo/imunologia , Agentes Urológicos/farmacologia , Agentes Urológicos/uso terapêutico
6.
Hum Antibodies ; 26(4): 209-218, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29889062

RESUMO

BACKGROUND: Generation and utilization of the specific monoclonal antibodies against testis antigens is reported to identify the antigens that are important in reproductive field. OBJECTIVE: Current study aimed to introduce a hybridoma that producing a specific anti-testis monoclonal antibody to identify the testis antigens that can be important in the reproduction field. METHODS: To make hybridoma against testis antigens, mice were immunized with testis cell lysate. After cell fusion, resulted hybridomas were screened by indirect ELISA, then cloned by limiting dilution and finally the produced monoclonal antibody were characterized by some of the molecular laboratory techniques such as immunohistochemistry, immunocytochemistry and western blot. RESULTS: By using hybridoma technique, cell fusion was performed and ten (8A11, 8D6, 8D7, 9F6, 9G11, 10C3, 10B3, 10B2, 10C2 and 10H7) antibodies specific to the testis antigens were produced finally. Among the produced antibodies, 10C3 was found to cross-react with testis, but not detected in other tissues. mAb 10C3 recognized the sperm and testis antigens, specifically the intertestitial tissue of testis, spermatogonia, primary and secondary spermatocyte antigens, so they were most likely the target of generated mAb. Also our mAb could totally detect the mouse sperm antigens and the specific antigens of head and tail of human sperm. In western blotting analysis, mAb 10C3 could recognize the specific protein bands of sperm and testis extracts. Also in this study the testis specific genes that were target of generated mAb, were selected according to the mouse EST profile available at UniGene part of NCBI. CONCLUSIONS: So this stable anti-testis mAb has a potential for laboratory researches and also for diagnostic procedures in fertilization. Thus it could be exploited as a suitable tool for target-specific diagnosis and research in several diseases.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Antígenos/análise , Fertilização/imunologia , Testículo/imunologia , Animais , Especificidade de Anticorpos , Fusão Celular , Clonagem Molecular , Reações Cruzadas , Hibridomas , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Cabeça do Espermatozoide/imunologia , Cauda do Espermatozoide/imunologia , Espermatócitos/imunologia , Espermatogônias/imunologia
7.
Proc Natl Acad Sci U S A ; 114(47): E10132-E10141, 2017 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-29109271

RESUMO

Mammalian spermatogenesis is an elaborately organized differentiation process, starting with diploid spermatogonia, which include germ-line stem cells, and ending with haploid spermatozoa. The process involves four pivotal transitions occurring in physical proximity: spermatogonial differentiation, meiotic initiation, initiation of spermatid elongation, and release of spermatozoa. We report how the four transitions are coordinated in mice. Two premeiotic transitions, spermatogonial differentiation and meiotic initiation, were known to be coregulated by an extrinsic signal, retinoic acid (RA). Our chemical manipulations of RA levels in mouse testes now reveal that RA also regulates the two postmeiotic transitions: initiation of spermatid elongation and spermatozoa release. We measured RA concentrations and found that they changed periodically, as also reflected in the expression patterns of an RA-responsive gene, STRA8; RA levels were low before the four transitions, increased when the transitions occurred, and remained elevated thereafter. We found that pachytene spermatocytes, which express an RA-synthesizing enzyme, Aldh1a2, contribute directly and significantly to RA production in testes. Indeed, chemical and genetic depletion of pachytene spermatocytes revealed that RA from pachytene spermatocytes was required for the two postmeiotic transitions, but not for the two premeiotic transitions. We conclude that the premeiotic transitions are coordinated by RA from Sertoli (somatic) cells. Once germ cells enter meiosis, pachytene spermatocytes produce RA to coordinate the two postmeiotic transitions. In combination, these elements underpin the spatiotemporal coordination of spermatogenesis and ensure its prodigious output in adult males.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Aldeído Desidrogenase/genética , Regulação da Expressão Gênica no Desenvolvimento , Espermatogênese/genética , Espermatozoides/metabolismo , Tretinoína/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aldeído Desidrogenase/metabolismo , Família Aldeído Desidrogenase 1 , Animais , Diferenciação Celular , Masculino , Meiose , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estágio Paquíteno , Retinal Desidrogenase , Transdução de Sinais , Espermátides/citologia , Espermátides/crescimento & desenvolvimento , Espermátides/metabolismo , Espermatócitos/citologia , Espermatócitos/crescimento & desenvolvimento , Espermatócitos/metabolismo , Espermatogônias/citologia , Espermatogônias/crescimento & desenvolvimento , Espermatogônias/imunologia , Espermatozoides/citologia , Espermatozoides/crescimento & desenvolvimento , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
8.
Bull Exp Biol Med ; 162(1): 146-152, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27878497

RESUMO

Using the model of hypogonadism in C57Bl/6 male mice, we showed that injection of streptozotocin to newborn animals and high-fat diet induced serum IFN-γ and IL-17 elevation, glucose metabolism disturbances, insulin resistance, destructive changes of the Langerhans islets (deficit of PDX1+ß cells), while the number of oligopotent ß cell precursors (CD45-TER119-CD133+CD49flow) increased. Diabetes played the role of an inducer of testicular tissue inflammation (pan-hemopoietic cell infiltration, increase of IL-2, IL-17, and IL-23 content) and reproductive system disturbances in mice (decrease in free testosterone concentration, suppression of spermatogenesis, and infertility). The development of hypogonadism was paralleled by an increase in the count of spermatogonial stem cells (CD117+CD29+CD90+), multipotent mesenchymal stromal cells (CD45-CD31-CD90+CD106+), hemangiogenesis precursors (CD45-CD117+Flk1+), and epithelial cells (CD45-CD31-CD49f+CD326+).


Assuntos
Diabetes Mellitus Experimental/patologia , Hipogonadismo/patologia , Pâncreas/patologia , Regeneração/imunologia , Testículo/patologia , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Glicemia/imunologia , Glicemia/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/imunologia , Dieta Hiperlipídica , Feminino , Expressão Gênica , Hipogonadismo/induzido quimicamente , Hipogonadismo/genética , Hipogonadismo/imunologia , Imunofenotipagem , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Interferon gama/genética , Interferon gama/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-23/genética , Interleucina-23/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Pâncreas/imunologia , Regeneração/genética , Espermatogênese/genética , Espermatogônias/imunologia , Espermatogônias/patologia , Células-Tronco/imunologia , Células-Tronco/patologia , Estreptozocina , Testículo/imunologia
9.
Fertil Steril ; 106(6): 1539-1549.e8, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27490045

RESUMO

OBJECTIVE: To study the ability of human spermatogonial stem cells (hSSCs) to proliferate in vitro under mouse spermatogonial stem cell (mSSC) culture conditions. DESIGN: Experimental basic science study. SETTING: Reproductive biology laboratory. PATIENT(S): Cryopreserved testicular tissue with normal spermatogenesis obtained from three donors subjected to orchiectomy due to a prostate cancer treatment. INTERVENTION(S): Testicular cells used to create in vitro cell cultures corresponding to the following groups: [1] unsorted human testicular cells, [2] differentially plated human testicular cells, and [3] cells enriched with major histocompatibility complex class 1 (HLA-)/epithelial cell surface antigen (EPCAM+) in coculture with inactivated testicular feeders from the same patient. MAIN OUTCOME MEASURE(S): Analyses and characterization including immunocytochemistry and quantitative reverse-transcription polymerase chain reaction for somatic and germ cell markers, testosterone and inhibin B quantification, and TUNEL assay. RESULT(S): Putative hSSCs appeared in singlets, doublets, or small groups of up to four cells in vitro only when testicular cells were cultured in StemPro-34 medium supplemented with glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Fluorescence-activated cell sorting with HLA-/EPCAM+ resulted in an enrichment of 27% VASA+/UTF1+ hSSCs, compared to 13% in unsorted controls. Coculture of sorted cells with inactivated testicular feeders gave rise to an average density of 112 hSSCs/cm2 after 2 weeks in vitro compared with unsorted cells (61 hSSCs/cm2) and differentially plated cells (49 hSSCS/cm2). However, putative hSSCs rarely stained positive for the proliferation marker Ki67, and their presence was reduced to the point of almost disappearing after 4 weeks in vitro. CONCLUSION(S): We found that hSSCs show limited proliferation in vitro under mSSC culture conditions. Coculture of HLA-/EPCAM+ sorted cells with testicular feeders improved the germ cell/somatic cell ratio.


Assuntos
Proliferação de Células , Espermatogônias/fisiologia , Animais , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Sobrevivência Celular , Células Cultivadas , Molécula de Adesão da Célula Epitelial/imunologia , Células Alimentadoras , Regulação da Expressão Gênica , Antígenos HLA/imunologia , Humanos , Masculino , Camundongos , Fenótipo , Espermatogônias/imunologia , Espermatogônias/metabolismo , Fatores de Tempo
10.
Tsitologiia ; 58(5): 356-63, 2016.
Artigo em Inglês, Russo | MEDLINE | ID: mdl-30188629

RESUMO

It is assumed that tissues with different degree of imminological privileges have a number of distinctions in the processes of reparation. This may be associated with mast cells, which are found in all body tissues of an organism, secrete a wide range of biologically active substances and are important in the regulation of repair processes. This paper present the results of investigations of morphometric parameners and functional activity of mast cells in tissues with varying degrees of immune privilege (skin, testis). It has been shown that migration of must cells in the skin is observed early after the injury and followed by a slow increase of synthetic activity and index of degranulation of mastocytes within 30 days. Index of degranulation of mast cells in the testes increased immediately after the injury in the absence of their pronounced migration. Stabilizing membranes of mast cells using the drug ketotifen led to inhibition of the repair of the skin, which was manifested itself in the absence of increasing the thickness of dermis, epidermis, the number of fibroblasts and collagen fibers, and also in slowing down the formation of scar. At the same time, the inactivation of mast cells promoted reparative regeneration of testes as was indicated by increase in the number of normal spermatogonia which are proliferative pool for all subsequent stages of spermatogenesis, and by considerable decrease in the number of non-functioning tubules. Thus, the number and the functional sate of the mast cells have an impact on the course of reparative processes in tissues with varying degrees of immune privileges.


Assuntos
Mastócitos/imunologia , Regeneração/imunologia , Fenômenos Fisiológicos da Pele/imunologia , Pele/imunologia , Espermatogênese/imunologia , Espermatogônias/imunologia , Testículo/fisiologia , Animais , Degranulação Celular/imunologia , Movimento Celular/imunologia , Masculino , Mastócitos/citologia , Especificidade de Órgãos , Ratos , Ratos Wistar , Pele/citologia , Espermatogônias/citologia , Testículo/citologia
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