RESUMEN
Extracellular membrane proteins are crucial for mediating cell attachment, recognition, and signal transduction in the testicular microenvironment, particularly germline stem cells. Cadherin 18 (CDH18), a type II classical cadherin, is primarily expressed in the nervous and reproductive systems. Here, we investigated the expression of CDH18 in neonatal porcine prospermatogonia (ProSGs) and murine spermatogonial stem cells (SSCs). Disruption of CDH18 expression did not adversely affect cell morphology, proliferation, self-renewal, or differentiation in cultured porcine ProSGs, but enhanced cell adhesion and prolonged cell maintenance. Transcriptomic analysis indicated that the down-regulation of CDH18 in ProSGs significantly up-regulated genes and signaling pathways associated with cell adhesion. To further elucidate the function of CDH18 in germ cells, Cdh18 knockout mice were generated, which exhibited normal testicular morphology, histology, and spermatogenesis. Transcriptomic analysis showed increased expression of genes associated with adhesion, consistent with the observations in porcine ProSGs. The interaction of CDH18 with ß-catenin and JAK2 in both porcine ProSGs and murine SSCs suggested an inhibitory effect on the canonical Wnt and JAK-STAT signaling pathways during CDH18 deficiency. Collectively, these findings highlight the crucial role of CDH18 in regulating cell adhesion in porcine ProSGs and mouse SSCs. Understanding this regulatory mechanism provides significant insights into the testicular niche.
Asunto(s)
Cadherinas , Adhesión Celular , Animales , Masculino , Porcinos , Adhesión Celular/fisiología , Ratones , Cadherinas/metabolismo , Cadherinas/genética , Ratones Noqueados , Espermatogonias/metabolismo , Espermatogonias/fisiología , Testículo/metabolismo , Testículo/fisiología , Células Madre Germinales Adultas/metabolismo , Células Madre Germinales Adultas/fisiología , Regulación de la Expresión Génica , Células Madre/fisiología , Células Madre/metabolismoRESUMEN
Context A population of sperm progenitor cells, known as Asingle spermatogonia, has been described in mammalian testes. During division cycles in spermatogenesis, some cells will form part of the Asingle spermatogonia group, while others form primary spermatocytes. Thus, during spermatogenesis, spermatogonia are the progenitor cells of spermatozoa. Aims In this study, we characterise the spermatogonial stem cells (SSCs) in the testicles of Artibeus jamaicensis and Sturnira lilium bats. The knowledge generated from this will contribute to the understanding of the biology of germ cells and the mechanisms of spermatogenesis in mammals, generating information on wildlife species that are important for biodiversity. Methods Testes were analysed by light and electron microscopy. Likewise, the expression of specific factors of stem cells (Oct4 and C-kit), germ cells (Vasa), cell proliferation (pH3 and SCP1) and testicular somatic cells (MIS, 3ßHSD and Sox9) was characterised by immunofluorescence and western blot. Key results The histological analysis enabled the location of type Asingle, Apaired and Aaligned spermatogonia in the periphery of the seminiferous tubules adjacent to Sertoli cells. The expression of genes of stem and germ cells made it possible to corroborate the distribution of the SSCs. Conclusions Results indicate that type Asingle spermatogonia were not randomly distributed, since proliferative activity was detected in groups of cells adjacent to the seminiferous tubules membrane, suggesting the localisation of spermatogonial niches in a specific region of testes. Implications This study provides evidence for the existence of SSCs in the testis of chiropterans that contribute to the renewal of germline progenitor cells to maintain the reproduction of the organisms.
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Quirópteros , Espermatogénesis , Espermatogonias , Testículo , Animales , Masculino , Testículo/citología , Testículo/metabolismo , Espermatogonias/citología , Espermatogénesis/fisiología , Células Madre/citología , Proliferación Celular , Células Madre Germinales Adultas/metabolismo , Células Madre Germinales Adultas/citologíaRESUMEN
Spermatogonial stem cells (SSCs) comprise the foundation of spermatogenesis and hence have great potential for fertility preservation of rare or endangered species and the development of transgenic animals and birds. Yet, developing optimal conditions for the isolation, culture, and maintenance of SSCs in vitro remains challenging, especially for chicken. The objectives of this study were to (1) find the optimal age for SSC isolation in Huaixiang chicken, (2) develop efficient protocols for the isolation, (3) enrichment, and (4) culture of isolated SSCs. In the present study, we first compared the efficiency of SSC isolation using 11 different age groups (8-79 days of age) of Huaixiang chicken. We found that the testes of 21-day-old chicken yielded the highest cell viability. Next, we compared two different enzymatic combinations for isolating SSCs and found that 0.125% trypsin and 0.02 g/L EDTA supported the highest number and viability of SSCs. This was followed by investigating optimal conditions for the enrichment of SSCs, where we observed that differential plating had the highest enrichment efficiency compared to the Percoll gradient and magnetic-activated cell sorting methods. Lastly, to find the optimal culture conditions of SSCs, we compared adding different concentrations of foetal bovine serum (FBS; 2%, 5%, 7%, and 10%) and different concentrations of GDNF, bFGF, or LIF (5, 10, 20, or 30 ng/mL). We found that a combination of 2% FBS and individual growth factors, including GDNF (20 ng/mL), bFGF (30 ng/mL), or LIF (5 ng/mL), best supported the proliferation and colony formation of SSCs. In conclusion, SSCs can be optimally isolated through enzymatic digestion from testes of 21-day-old chicken, followed by enrichment using differential plating. Furthermore, adding 2% FBS and optimized concentrations of GFNF, bFGF, or LIF in the culture promotes the proliferation of chicken SSCs.
Asunto(s)
Células Madre Germinales Adultas , Técnicas de Cultivo de Célula , Separación Celular , Pollos , Animales , Masculino , Técnicas de Cultivo de Célula/veterinaria , Separación Celular/métodos , Separación Celular/veterinaria , Testículo/citología , Espermatogonias/citología , Supervivencia Celular , Células CultivadasRESUMEN
Spermatogonial stem cells (SSCs) undergo self-renewal division to sustain spermatogenesis. Although it is possible to derive SSC cultures in most mouse strains, SSCs from a 129 background never proliferate under the same culture conditions, suggesting they have distinct self-renewal requirements. Here, we established long-term culture conditions for SSCs from mice of the 129 background (129 mice). An analysis of 129 testes showed significant reduction of GDNF and CXCL12, whereas FGF2, INHBA and INHBB were higher than in testes of C57BL/6 mice. An analysis of undifferentiated spermatogonia in 129 mice showed higher expression of Chrna4, which encodes an acetylcholine (Ach) receptor component. By supplementing medium with INHBA and Ach, SSC cultures were derived from 129 mice. Following lentivirus transduction for marking donor cells, transplanted cells re-initiated spermatogenesis in infertile mouse testes and produced transgenic offspring. These results suggest that the requirements of SSC self-renewal in mice are diverse, which has important implications for understanding self-renewal mechanisms in various animal species.
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Ratones Endogámicos C57BL , Espermatogénesis , Espermatogonias , Testículo , Animales , Masculino , Ratones , Espermatogonias/citología , Espermatogonias/metabolismo , Espermatogénesis/genética , Espermatogénesis/fisiología , Testículo/metabolismo , Testículo/citología , Autorrenovación de las Células , Células Madre Germinales Adultas/metabolismo , Células Madre Germinales Adultas/citología , Células Cultivadas , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/genética , Ratones Endogámicos , Diferenciación Celular , Proliferación Celular , Células Madre/citología , Células Madre/metabolismo , Ratones TransgénicosRESUMEN
Normal testicular development ensures the process of spermatogenesis, which is a complex biological process. The sustained high productivity of spermatogenesis throughout life is predominantly attributable to the constant proliferation and differentiation of spermatogonial stem cells (SSCs). The self-renewal and differentiation processes of SSCs are strictly regulated by the SSC niche. Therefore, understanding the developmental pattern of SSCs is crucial for spermatogenesis. The Shaziling pig is a medium-sized indigenous pig breed originating from central China. It is renowned for its superior meat quality and early male sexual maturity. The spermatogenic ability of the boars is of great economic importance to the pig industry. To investigate testicular development, particularly the pattern of SSC development in Shaziling pigs, we used single-cell transcriptomics to identify gene expression patterns in 82,027 individual cells from nine Shaziling pig testes at three key postnatal developmental stages. We generated an unbiased cell developmental atlas of Shaziling pig testicular tissues. We elucidated the complex processes involved in the development of SSCs within their niche in the Shaziling pig. Specifically, we identified potential marker genes and cellular signaling pathways that regulate SSC self-renewal and maintenance. Additionally, we proposed potential novel marker genes for SSCs that could be used for SSC isolation and sorting in Shaziling pigs. Furthermore, by immunofluorescence staining of testicular tissues of different developmental ages using marker proteins (UCHL1 and KIT), the developmental pattern of the spermatogonia of Shaziling pigs was intensively studied. Our research enhances the comprehension of the development of SSCs and provides a valuable reference for breeding Shaziling pigs.
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RNA-Seq , Espermatogonias , Testículo , Animales , Masculino , Porcinos/genética , Espermatogonias/metabolismo , Espermatogonias/citología , Testículo/metabolismo , Testículo/citología , Testículo/crecimiento & desarrollo , Células Madre Germinales Adultas/metabolismo , Células Madre Germinales Adultas/citología , Análisis de la Célula Individual , Diferenciación Celular/genética , Espermatogénesis/genética , Células Madre/metabolismo , Células Madre/citología , Transcriptoma/genéticaRESUMEN
Infertility is intricately linked with alterations in circadian rhythms along with physiological decline and stem cell senescence. Yet, the direct involvement of circadian mechanisms in nicotine-induced injury to the testes, especially the senescence of spermatogonia stem cells (SSCs), is not well comprehended. This study revealed that nicotine exposure induced testis injury by triggering SSCs senescence along with the upregulation of senescence marker genes and senescence-associated secretory phenotype components. Moreover, nicotine treatment caused mitochondrial hyper-fusion, increased oxidative stress, and DNA damage. Exposure to nicotine was found to suppress the expression of sirtuin 6 (SIRT6), which accelerated the senescence of spermatogonia stem cells (SSCs). This acceleration led to increased acetylation of brain and muscle ARNT-like protein (Bmal1), consequently reducing the expression of Bmal1 protein. Conversely, the overexpression of Bmal1 alleviated mitochondrial hyper-fusion and senescence phenotypes induced by nicotine. Overall, this study unveiled a novel molecular mechanism behind nicotine-induced disorders in spermatogenesis and highlighted the SIRT6/Bmal1 regulatory pathway as a potential therapeutic target for combating nicotine-associated infertility.
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Factores de Transcripción ARNTL , Senescencia Celular , Ritmo Circadiano , Dinámicas Mitocondriales , Nicotina , Sirtuinas , Sirtuinas/metabolismo , Sirtuinas/genética , Masculino , Animales , Nicotina/farmacología , Nicotina/efectos adversos , Senescencia Celular/efectos de los fármacos , Ritmo Circadiano/efectos de los fármacos , Dinámicas Mitocondriales/efectos de los fármacos , Ratones , Factores de Transcripción ARNTL/metabolismo , Factores de Transcripción ARNTL/genética , Espermatogonias/efectos de los fármacos , Espermatogonias/metabolismo , Homeostasis/efectos de los fármacos , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células Madre Germinales Adultas/metabolismo , Células Madre Germinales Adultas/efectos de los fármacosRESUMEN
PURPOSE: This study examined SSC proliferation on an epididymosome-enriched decellularized testicular matrix (DTM) hydrogel and spermatogenesis induction in azoospermic mice. METHODS: Epididymosomes were extracted and characterized using SEM and western blotting. After cryopreservation, thawed SSCs were cultured in a hydrogel-based three-dimensional (3D) culture containing 10 ng/mL GDNF or 20 µg/mL epididymosomes. SSCs were assessed using the MTT assay, flow cytometry, and qRT-PCR after two weeks of culture. The isolated SSCs were microinjected into the efferent ducts of busulfan-treated mice. DiI-labeled SSCs were followed, and cell homing was assessed after two weeks. After 8 weeks, the testes were evaluated using morphometric studies and immunohistochemistry. RESULTS: The expression of PLZF, TGF-ß, and miR-10b did not increase statistically significantly in the 3D + GDNF and 3D + epididymosome groups compared to the 3D group. Among the groups, the GDNF-treated group exhibited the highest expression of miR-21 (*P < 0.05). Caspase-3 expression was lower in the epididymosome-treated group than in the other groups (***P < 0.001). Compared to the 3D and negative control groups, the 3D + epididymosomes and 3D + GDNF groups showed an increase in spermatogenic cells. Immunohistochemical results confirmed the growth and differentiation of spermatogonial cells into spermatids in the treatment groups. CONCLUSION: The DTM hydrogel containing 20 µg/mL epididymosomes or 10 ng/mL GDNF is a novel and safe culture system that can support SSC proliferation in vitro to obtain adequate SSCs for transplantation success. It could be a novel therapeutic agent that could recover deregulated SSCs in azoospermic patients.
Asunto(s)
Azoospermia , Criopreservación , Epidídimo , Espermatogénesis , Testículo , Animales , Masculino , Ratones , Azoospermia/patología , Testículo/crecimiento & desarrollo , Criopreservación/métodos , Epidídimo/metabolismo , Espermatogonias/citología , Andamios del Tejido/química , Células Madre Germinales Adultas/metabolismo , Proliferación CelularRESUMEN
Spermatogenesis involves a complex process of cellular differentiation maintained by spermatogonial stem cells (SSCs). Being critical to male reproduction, it is generally assumed that spermatogenesis starts and ends in equivalent transcriptional states in related species. Based on single-cell gene expression profiling, it has been proposed that undifferentiated human spermatogonia can be subclassified into four heterogenous subtypes, termed states 0, 0A, 0B, and 1. To increase the resolution of the undifferentiated compartment and trace the origin of the spermatogenic trajectory, we re-analysed the single-cell (sc) RNA-sequencing libraries of 34 post-pubescent human testes to generate an integrated atlas of germ cell differentiation. We then used this atlas to perform comparative analyses of the putative SSC transcriptome both across human development (using 28 foetal and pre-pubertal scRNA-seq libraries) and across species (including data from sheep, pig, buffalo, rhesus and cynomolgus macaque, rat, and mouse). Alongside its detailed characterisation, we show that the transcriptional heterogeneity of the undifferentiated spermatogonial cell compartment varies not only between species but across development. Our findings associate 'state 0B' with a suppressive transcriptomic programme that, in adult humans, acts to functionally oppose proliferation and maintain cells in a ready-to-react state. Consistent with this conclusion, we show that human foetal germ cells-which are mitotically arrested-can be characterised solely as state 0B. While germ cells with a state 0B signature are also present in foetal mice (and are likely conserved at this stage throughout mammals), they are not maintained into adulthood. We conjecture that in rodents, the foetal-like state 0B differentiates at birth into the renewing SSC population, whereas in humans it is maintained as a reserve population, supporting testicular homeostasis over a longer reproductive lifespan while reducing mutagenic load. Together, these results suggest that SSCs adopt differing evolutionary strategies across species to ensure fertility and genome integrity over vastly differing life histories and reproductive timeframes.
Asunto(s)
Espermatogonias , Humanos , Animales , Masculino , Espermatogonias/citología , Espermatogonias/metabolismo , Células Madre Germinales Adultas/metabolismo , Células Madre Germinales Adultas/citología , Diferenciación Celular/genética , Espermatogénesis/genética , Transcriptoma/genética , Adulto , Ratones , Feto/citología , Testículo/citología , Testículo/metabolismo , Roedores , Ratas , Análisis de la Célula IndividualRESUMEN
Spermatogonial stem cells (SSCs) are essential for the maintenance of male fertility and survival of species. Environmental conditions, notably heat stress, have been identified as important causes of male infertility and have a negative impact on SSCs. Animals with cryptorchid testes (CT) are optimal models for the study of long-term heat stress-related changes in germ cells. The effect of heat stress on germ cells differs depending on the spermatogenesis stage. Thus, verifying whether the specific phase of spermatogenesis is dependent or independent of heat stress in stallions is important. We evaluated the heat stress-related response of SSCs by comparing the relative abundance of mRNA transcripts and expression patterns of the undifferentiated embryonic cell transcription factor 1 (UTF-1) and deleted in azoospermia-like (DAZL) in the seminiferous tubules of CT and normal testes (NT) of stallions using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and western blotting. We also analyzed the relative abundance of mRNA of different proliferative markers, including minichromosome maintenance 2 (MCM2), marker of proliferation Ki-67 (MKI-67), and proliferating cell nuclear antigen (PCNA). Testicular tissues from four Thoroughbred unilateral cryptorchid postpubertal stallions were used in this study during the breeding season. The relative abundance of the mRNA transcripts of UTF-1 and MCM2 was significantly upregulated in the CT group than that of those in the NT group. In contrast, the relative abundance of the mRNA transcripts of DAZL was significantly downregulated in the CT group than that of those in the NT group. Western blot quantification showed that the relative intensity of UTF-1 protein bands was significantly higher, while that of DAZL protein bands was significantly lower in the CT group than in the NT group. Immunofluorescence studies showed that the number of germ cells immunostained with UTF-1 was significantly higher while immunostained with DAZL was significantly lower in the CT group than that in the NT group. The higher expression level of UTF-1 in the CT group shows that undifferentiated SSCs are not affected by long-term exposure to heat stress. These results also indicate that germ cells after differentiation phase are directly affected by heat-stress conditions, such as cryptorchidism, in stallions.
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Células Madre Germinales Adultas , Animales , Masculino , Caballos/fisiología , Células Madre Germinales Adultas/metabolismo , Células Madre Germinales Adultas/fisiología , Respuesta al Choque Térmico/fisiología , Regulación de la Expresión Génica , Testículo/metabolismo , Espermatogonias/metabolismo , Calor , Espermatogénesis/fisiología , ARN Mensajero/metabolismo , ARN Mensajero/genéticaRESUMEN
Spermatogonial stem cells (SSCs) are capable of transmitting genetic information to the next generations and they are the initial cells for spermatogenesis. Nevertheless, it remains largely unknown about key genes and signaling pathways that regulate fate determinations of human SSCs and male infertility. In this study, we explored the expression, function, and mechanism of USP11 in controlling the proliferation and apoptosis of human SSCs as well as the association between its abnormality and azoospermia. We found that USP11 was predominantly expressed in human SSCs as shown by database analysis and immunohistochemistry. USP11 silencing led to decreases in proliferation and DNA synthesis and an enhancement in apoptosis of human SSCs. RNA-sequencing identified HOXC5 as a target of USP11 in human SSCs. Double immunofluorescence, Co-immunoprecipitation (Co-IP), and molecular docking demonstrated an interaction between USP11 and HOXC5 in human SSCs. HOXC5 knockdown suppressed the growth of human SSCs and increased apoptosis via the classical WNT/ß-catenin pathway. In contrast, HOXC5 overexpression reversed the effect of proliferation and apoptosis induced by USP11 silencing. Significantly, lower levels of USP11 expression were observed in the testicular tissues of patients with spermatogenic disorders. Collectively, these results implicate that USP11 regulates the fate decisions of human SSCs through the HOXC5/WNT/ß-catenin pathway. This study thus provides novel insights into understanding molecular mechanisms underlying human spermatogenesis and the etiology of azoospermia and it offers new targets for gene therapy of male infertility.
Asunto(s)
Apoptosis , Proliferación Celular , Espermatogénesis , Tioléster Hidrolasas , Vía de Señalización Wnt , Humanos , Masculino , Células Madre Germinales Adultas/metabolismo , Apoptosis/genética , Azoospermia/metabolismo , Azoospermia/genética , Azoospermia/patología , beta Catenina/metabolismo , beta Catenina/genética , Proliferación Celular/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Espermatogénesis/genética , Espermatogonias/metabolismo , Espermatogonias/citología , Testículo/metabolismo , Testículo/citología , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/metabolismo , Vía de Señalización Wnt/genéticaRESUMEN
The continuous regeneration of spermatogonial stem cells (SSCs) underpins spermatogenesis and lifelong male fertility, but the developmental origins of the SSC pool remain unclear. Here, we document that hnRNPU is essential for establishing the SSC pool. In male mice, conditional loss of hnRNPU in prospermatogonia (ProSG) arrests spermatogenesis and results in sterility. hnRNPU-deficient ProSG fails to differentiate and migrate to the basement membrane to establish SSC pool in infancy. Moreover, hnRNPU deletion leads to the accumulation of ProSG and disrupts the process of T1-ProSG to T2-ProSG transition. Single-cell transcriptional analyses reveal that germ cells are in a mitotically quiescent state and lose their unique identity upon hnRNPU depletion. We further show that hnRNPU could bind to Vrk1, Slx4, and Dazl transcripts that have been identified to suffer aberrant alternative splicing in hnRNPU-deficient testes. These observations offer important insights into SSC pool establishment and may have translational implications for male fertility.
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Espermatogénesis , Espermatogonias , Animales , Masculino , Ratones , Células Madre Germinales Adultas/metabolismo , Empalme Alternativo/genética , Diferenciación Celular , Espermatogénesis/genética , Espermatogonias/metabolismo , Espermatogonias/citología , Células Madre/metabolismo , Células Madre/citología , Testículo/metabolismo , Testículo/citología , Ribonucleoproteína Heterogénea-Nuclear Grupo U/metabolismoRESUMEN
In brief: Oogonial stem cells in the adult ovary can generate oocytes, but they are usually quiescent. TGFB1 is key in stimulating the proliferation of OSC, thereby ensuring the sustained reproductive potential in poultry species. Abstract: Oogonial stem cells (OSCs) are a type of germ stem cell present in the adult ovary. They have the ability to self-renew through mitosis and differentiate into oocytes through meiosis. We have previously identified a population of OSCs in the chicken ovary, but the underlying mechanisms controlling their activation and proliferation were unclear. In this study, we observed that OSCs showed robust proliferation when cultured on a layer of chicken embryo fibroblasts (CEF), suggesting that CEF may secrete certain crucial factors that activate OSC proliferation. We further detected TGFB1 as a potent signaling molecule to promote OSC proliferation. Additionally, we revealed the signaling pathways that play important roles downstream of TGFB1-induced OSC proliferation. These findings provide insights into the mechanisms underlying OSC proliferation in chickens and offer a foundation for future research on in situ activation of OSC proliferation in ovary and improvement of egg-laying performance in chickens.
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Proliferación Celular , Pollos , Factor de Crecimiento Transformador beta1 , Animales , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Femenino , Células Cultivadas , Embrión de Pollo , Oogonios/citología , Oogonios/metabolismo , Oogonios/fisiología , Ovario/citología , Ovario/metabolismo , Transducción de Señal , Fibroblastos/citología , Fibroblastos/metabolismo , Células Madre Germinales Adultas/citología , Células Madre Germinales Adultas/metabolismo , Células Madre Germinales Adultas/fisiologíaRESUMEN
RNA-binding proteins (RBPs), as key regulators of mRNA fate, are abundantly expressed in the testis. However, RBPs associated with human male infertility remain largely unknown. Through bioinformatic analyses, we identified 62 such RBPs, including an evolutionarily conserved RBP, DEAD-box helicase 20 (DDX20). Male germ-cell-specific inactivation of Ddx20 at E15.5 caused T1-propsermatogonia (T1-ProSG) to fail to reenter cell cycle during the first week of testicular development in mice. Consequently, neither the foundational spermatogonial stem cell (SSC) pool nor progenitor spermatogonia were ever formed in the knockout testes. Mechanistically, DDX20 functions to control the translation of its target mRNAs, many of which encode cell-cycle-related regulators, by interacting with key components of the translational machinery in prospermatogonia. Our data demonstrate a previously unreported function of DDX20 as a translational regulator of critical cell-cycle-related genes, which is essential for cell-cycle reentry of T1-ProSG and formation of the SSC pool.
Asunto(s)
Ciclo Celular , ARN Helicasas DEAD-box , Espermatogénesis , Espermatogonias , Testículo , Animales , Masculino , Ratones , Células Madre Germinales Adultas/metabolismo , Células Madre Germinales Adultas/citología , Ciclo Celular/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Regulación del Desarrollo de la Expresión Génica , Ratones Noqueados , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Espermatogénesis/genética , Espermatogénesis/fisiología , Espermatogonias/metabolismo , Espermatogonias/citología , Testículo/metabolismo , Testículo/citologíaRESUMEN
Preservation of human spermatogonial stem cells (SSCs) may be suitable for young male patients at risk of male infertility due to various causes, such as gonadotoxic treatment or genetic diseases. With optimal cryopreservation, cell viability can be retained to reestablish spermatogenesis in the future through autologous transplantation or in vitro differentiation of SSCs. This protocol outlines techniques to optimize the SSCs isolation and in vitro culture. With particular emphasis on the microscopic characteristics encountered, this protocol outlines a broader approach to processing tissues with varying morphologies among patients.
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Células Madre Germinales Adultas , Infertilidad Masculina , Humanos , Masculino , Espermatogonias , Espermatogénesis , Criopreservación/métodos , TestículoRESUMEN
Continuous spermatogenesis depends on the self-renewal and differentiation of spermatogonial stem cells (SSCs). SSCs, the only male reproductive stem cells that transmit genetic material to subsequent generations, possess an inherent self-renewal ability, which allows the maintenance of a steady stem cell pool. SSCs eventually differentiate to produce sperm. However, in an in vitro culture system, SSCs can be induced to differentiate into various types of germ cells. Rodent SSCs are well defined, and a culture system has been successfully established for them. In contrast, available information on the biomolecular markers and a culture system for livestock SSCs is limited. This review summarizes the existing knowledge and research progress regarding mammalian SSCs to determine the mammalian spermatogenic process, the biology and niche of SSCs, the isolation and culture systems of SSCs, and the biomolecular markers and identification of SSCs. This information can be used for the effective utilization of SSCs in reproductive technologies for large livestock animals, enhancement of human male fertility, reproductive medicine, and protection of endangered species.
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Animales , Masculino , Células Madre Germinales Adultas , Diferenciación Celular , Espermatogénesis , Espermatogonias , Células MadreRESUMEN
Azoospermia patients who carry a monogenetic mutation that causes meiotic arrest may have their biological child through genetic correction in spermatogonial stem cells (SSCs). However, such therapy for infertility has not been experimentally investigated yet. In this study, a mouse model with an X-linked testis-expressed 11 (TEX11) mutation (Tex11
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Animales , Masculino , Ratones , Células Madre Germinales Adultas/metabolismo , Azoospermia/genética , Infertilidad Masculina/terapia , Mutación/genética , Espermatogénesis/genéticaRESUMEN
An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell (SSC) transplantation. Busulfan has been commonly used to develop such a model, but 30%-87% of mice die when administered an intraperitoneal injection of 40 mg kg-1. In the present study, hematoxylin and eosin staining, Western blot, immunofluorescence, and quantitative real-time polymerase chain reaction were used to test the effects of busulfan exposure in a mouse model that received two intraperitoneal injections of busulfan at a 3-h interval at different doses (20, 30, and 40 mg kg-1) on day 36 or a dose of 40 mg kg-1 at different time points (0, 9, 18, 27, 36, and 63 days). The survival rate of the mice was 100%. When the mice were treated with 40 mg kg-1 busulfan, dramatic SSC depletion occurred 18 days later and all of the germ cells were cleared by day 36. In addition, the gene expressions of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), chemokine (C-X-C Motif) ligand 12 (CXCL12), and colony-stimulating factor 1 (CSF1) were moderately increased by day 36. A 63-day, long-term observation showed the rare restoration of endogenous germ cells in the testes, suggesting that the potential period for SSC transplantation was between day 36 and day 63. Our results demonstrate that the administration of two intraperitoneal injections of busulfan (40 mg kg-1 in total) at a 3-h interval to mice provided a nonlethal and efficient method for recipient preparation in SSC transplantation and could improve treatments for infertility and the understanding of chemotherapy-induced gonadotoxicity.
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Animales , Masculino , Ratones , Células Madre Germinales Adultas/trasplante , Azoospermia/inducido químicamente , Busulfano/toxicidad , Modelos Animales de Enfermedad , Infertilidad Masculina/inducido químicamente , Inyecciones Intraperitoneales , Espermatogénesis/efectos de los fármacos , Espermatogonias/efectos de los fármacos , Trasplante de Células Madre/métodosRESUMEN
While it is known that spermatogonial stem cells (SSCs) initiate the production of male germ cells, the mechanisms of SSC self-renewal, proliferation, and differentiation remain poorly understood. We have previously identified Strawberry Notch 1 (SBNO1), a vertebrate strawberry notch family protein, in the proteome profile for mouse SSC maturation and differentiation, revealing SBNO1 is associated with neonatal testicular development. To explore further the location and function of SBNO1 in the testes, we performed Sbno1 gene knockdown in mice to study the effects of SBNO1 on neonatal testicular and SSC development. Our results revealed that SBNO1 is required for neonatal testicular and SSC development in mice. Particularly, in vitro Sbno1 gene knockdown with morpholino oligonucleotides caused a reduction of SSCs and inactivation of the noncanonical Wnt pathway, through Jun N-terminal kinases. Our study suggests SBNO1 maintains SSCs by promoting the noncanonical Wnt pathway.
Asunto(s)
Animales , Masculino , Ratones , Células Madre Germinales Adultas/metabolismo , Proliferación Celular/fisiología , Técnicas de Silenciamiento del Gen , Proteoma , Proteínas Represoras/metabolismo , Testículo/metabolismo , Vía de Señalización Wnt/fisiologíaRESUMEN
Stem cell niche is a specialized microenvironment crucial to self-renewal. The testis in Drosophila contains two different types of stem cells, the germline stem cells and the somatic cyst stem cells that are sustained by their respective niche signals, thus is a good system for studying the interaction between the stem cells and their hosting niche. The JAK-STAT and BMP pathways are known to play critical roles in the self-renewal of different kinds of stem cells, but the roles of several other pathways have emerged recently in a complex signaling network in the testis niche. Reports of independent observations from three research groups have uncovered an important role of Hedgehog (Hh) in the Drosophila testis niche. In this review, we summarize these recent findings and discuss the interplay between the Hh signaling mechanisms and those of the JAK-STAT and BMP pathways. We also discuss directions for further investigation.