RESUMEN
Recently, it was reported that a testicular organ culture system (TOCS) using polydimethylsiloxane (PDMS) chips with excellent oxygen permeability and biocompatibility, called the PDMS-chip ceiling (PC) method, enables improved spermatogenesis efficiency. We investigated whether this PC method is useful for detecting impaired spermatogenesis caused by busulfan (Bu), a typical testicular toxicant. In this study, testicular tissue fragments from Acro3-EGFP mice, which express the green fluorescent protein (GFP) and reflect the progression of spermatogenesis, were subjected to the PC method. When treated with Bu, cultured tissues shrank in volume, and their GFP-expressing area decreased or disappeared. Histological examination confirmed the regression of spermatogenesis. In addition, immunohistochemical examination revealed that spermatogonia, including spermatogonial stem cells (SSCs), were the primary targets of Bu toxicity. Time-course analysis demonstrated that the recovery of spermatogenesis, dependent on Bu concentration, correlated closely with the severity of damage to these target cells. These results suggest that the PC method is a useful approach for detecting spermatogenesis impairment accurately through faithful recapitulation of spermatogenesis in vivo.
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Busulfano , Técnicas de Cultivo de Órganos , Espermatogénesis , Testículo , Animales , Masculino , Espermatogénesis/efectos de los fármacos , Testículo/efectos de los fármacos , Testículo/citología , Técnicas de Cultivo de Órganos/métodos , Ratones , Busulfano/farmacología , Espermatogonias/efectos de los fármacos , Espermatogonias/citología , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genéticaRESUMEN
Background: The mammalian testicular interstitial cells are not well-defined. The present study characterized the interstitial cell types and their turnover dynamics in adult rats. Additionally, the heterogeneity of the mesenchymal population and the effects of Leydig cell elimination on interstitial homeostasis were further analyzed by scRNA-seq datasets and immunocytochemical techniques. Methods: Interstitial cells were defined at the transcriptomic level by scRNA-seq and then confirmed and quantified with protein markers. The dividing activity of the major cell types was determined by continuous EdU labeling of the animals for one week. Some of the rats were also treated with a dose of ethylenedimethylsulfonate (EDS) to examine how the loss of Leydig cells (LCs) could affect interstitial homeostasis for three weeks. Results: Seven interstitial cell types were identified, including cell types (percentage of the whole interstitial population) as follows: Leydig (44.6%), macrophage and dendritic (19.1%), lymphoid (6.2%), vascular endothelial (7.9%), smooth muscle (10.7%), and mesenchymal (11.5%) cells. The EdU experiment indicated that most cell types were dividing at relatively low levels (<9%) except for the mesenchymal cells (MCs, 17.1%). Further analysis of the transcriptome of MCs revealed 4 subgroups with distinct functions, including 1) glutathione metabolism and xenobiotic detoxification, 2) ROS response and AP-1 signaling, 3) extracellular matrix synthesis and binding, and 4) immune response and regulation. Stem LCs (SLCs) are primarily associated with subgroup 3, expressing ARG1 and GAP43. EDS treatment not only eliminated LCs but also increased subgroup 3 and decreased subgroups 1 and 2 of the mesenchymal population. Moreover, EDS treatment increased the division of immune cells by more than tenfold in one week. Conclusion: Seven interstitial cell types were identified and quantified for rat testis. Many may play more diversified roles than previously realized. The elimination of LCs led to significant changes in MCs and immune cells, indicating the importance of LCs in maintaining testicular interstitial homeostasis.
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Células Intersticiales del Testículo , Masculino , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/efectos de los fármacos , Animales , Ratas , Inmunohistoquímica , Testículo/metabolismo , Testículo/citología , Ratas Sprague-Dawley , RNA-Seq , Transcriptoma , ARN Citoplasmático Pequeño/metabolismo , ARN Citoplasmático Pequeño/genética , Análisis de Expresión Génica de una Sola CélulaRESUMEN
Cultured mammalian spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), hold great promise for applications such as fertility preservation, gene therapy, and animal breeding, particularly in conjunction with accurate gene editing. Although the in vitro development of mouse GSC (mGSC) lines, and gene-targeting procedures for such lines, were initially established about two decades ago, it remains challenging for beginners to efficiently accomplish these tasks, partly because mGSCs proliferate more slowly and are more resistant to lipid-mediated gene transfection than pluripotent stem cells (PSCs). Meanwhile, methods for mGSC culture and gene editing have been evolving constantly to become simpler and more efficient. Here, we describe how to develop mGSC lines from small mouse testis samples and how to carry out gene knock-in in these cells using CRISPR/Cas9 technology, detailing three basic protocols that constitute a streamlined procedure. Using these simple and efficient procedures, site-specific knock-in mGSC lines can be obtained in 3 months. We hope that these protocols will help researchers use genetically modified GSCs to explore scientific questions of interest and to accumulate experience for application to GSC research in other mammalian species. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Establishment of mouse GSCs lines from small testicular samples Basic Protocol 2: Preparation of plasmids for gene knock-in using the CRISPR/Cas9 system Basic Protocol 3: Establishment of gene knock-in mGSC lines by electroporation gene delivery.
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Sistemas CRISPR-Cas , Técnicas de Sustitución del Gen , Animales , Sistemas CRISPR-Cas/genética , Ratones , Masculino , Técnicas de Sustitución del Gen/métodos , Línea Celular , Testículo/citología , Testículo/metabolismo , Edición Génica/métodos , Técnicas de Cultivo de Célula/métodos , Células Madre Germinales Adultas/metabolismo , Células Germinativas/metabolismo , Células Germinativas/citologíaRESUMEN
Spermatogonial stem cells (SSCs) possess the characteristics of self-renewal and differentiation, as well as the ability to generate functional sperm. Their unique stemness has broad applications in male infertility treatment and species preservation. In rodents, research on SSCs has been widely reported, but progress is slow in large livestock such as cattle and pigs due to long growth cycles, difficult proliferation in vitro, and significant species differences. Previously, we showed that histone 3 (H3) lysine 9 (K9) trimethylation (H3K9me3) is associated with the proliferation of bovine SSCs. Here, we isolated and purified SSCs from calf testicular tissues and investigated the impact of different H3K9me3 levels on the in vitro proliferation of bovine SSCs. The enriched SSCs eventually formed classical stem cell clones in vitro in our feeder-free culture system. These clones expressed glial cell-derived neurotrophic factor family receptor alpha-1 (GFRα1, specific marker for SSCs), NANOG (pluripotency protein), C-KIT (germ cell marker), and strong alkaline phosphatase (AKP) positivity. qRT-PCR analysis further showed that these clones expressed the pluripotency genes NANOG and SOX2, and the SSC-specific marker gene GFRα1. To investigate the dynamic relationship between H3K9me3 levels and SSC proliferation, H3K9me3 levels in bovine SSCs were first downregulated using the methyltransferase inhibitor, chaetocin, or transfection with the siRNA of H3K9 methyltransferase suppressor of variegation 3-9 homologue 1 (SUV39H1). The EDU (5-Ethynyl-2'-deoxyuridine) assay revealed that SSC proliferation was inhibited. Conversely, when H3K9me3 levels in bovine SSCs were upregulated by transfecting lysine demethylase 4D (KDM4D) siRNA, the EDU assay showed a promotion of cell proliferation. In summary, this study established a feeder-free culture system to obtain bovine SSCs and explored its effects on the proliferation of bovine SSCs by regulating H3K9me3 levels, laying the foundation for elucidating the regulatory mechanism underlying histone methylation modification in the proliferation of bovine SSCs.
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Células Madre Germinales Adultas , Proliferación Celular , Histonas , Animales , Bovinos , Masculino , Histonas/metabolismo , Células Madre Germinales Adultas/metabolismo , Células Madre Germinales Adultas/citología , Células Cultivadas , Espermatogonias/metabolismo , Espermatogonias/citología , Metilación , Diferenciación Celular , Testículo/metabolismo , Testículo/citologíaRESUMEN
Testicular organoids have great potential for maintaining male fertility and even restoring male infertility. However, existing studies on generating organoids with testis-specific structure and function are scarce and come with many limitations. Research on cryopreservation of testicular organoids is even more limited, and inappropriate cryopreservation methods may result in the loss of properties in resuscitated or regenerated organoids, rendering them unsuitable for clinical or research needs. In this paper, we investigated the effects of mouse age and cell number on the self-aggregation of testicular cells into spheres in low-adsorption plates. Various media compositions, culture systems, and cell numbers were used to culture cell spheres for 14 days to form testicular organoids, and the self-organization of the organoids was assessed by histological and immunofluorescence staining. We determined the appropriate cryopreservation conditions for testicular cells, cell spheres, and tissues. Subsequently, organoids derived from cryopreserved testicular tissues, testicular cells, and testicular cell spheres were compared and evaluated by histological and immunofluorescence staining. The results indicate that testicular cell spheres consisting of 30 × 104 testicular cells from 2-week-old mice were able to form organoids highly similar to the luminal structure and cell distribution of natural mouse testicular tissues. This transformation occurred over 14 days of incubation in α-MEM medium containing 10 % knockout serum replacer (KSR) using an agarose hydrogel culture system. Additionally, the Sertoli cells were tightly connected to form a blood-testis barrier. The relative rates of tubular area, germ cells, Sertoli cells, and peritubular myoid cells were 36.985 % ± 0.695, 13.347 % ± 3.102, 47.570 % ± 0.379, and 27.406 % ± 1.832, respectively. The optimal cryopreservation protocol for primary testicular cells involved slow freezing with a cryoprotectant consisting of α-MEM with 10 % dimethyl sulfoxide (DMSO). Slow freezing with cryoprotectants containing 5 % DMSO and 5 % ethylene glycol (EG) was optimal for all different volumes of testicular cell spheres. Compared to testicular organoids generated from frozen testicular tissue and cell spheres, freezing testicular cells proved most effective in maintaining organoid differentiation characteristics and cell-cell interactions. The findings of this study contribute to a "universal" testicular organoid in vitro culture protocol with promising applications for fertility preservation and restoration in prepubertal cancer patients and adult infertile patients.
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Criopreservación , Organoides , Testículo , Animales , Masculino , Criopreservación/métodos , Organoides/citología , Ratones , Testículo/citología , Células de Sertoli/citología , Ratones Endogámicos C57BL , Técnicas de Cultivo de Célula/métodos , Barrera HematotesticularRESUMEN
Sperm, a crucial gamete for reproduction in sexual reproduction, is generated through the proliferation, differentiation, and morphological transformations of spermatogonial stem cells within the specialized microenvironment of the testes. Replicating this environment artificially presents challenges. However, interdisciplinary advancements in physics, materials science, and cell engineering have facilitated the utilization of innovative materials, technologies, and structures for inducing in vitro sperm production. This article offers a comprehensive overview of research progress on inducing in vitro sperm production by categorizing techniques into two major systems based on matrix-based and non-matrix-based approaches, respectively. Detailed discussions are provided for both types of technology systems through comparisons of their similarities and differences, as well as research advancements. The aim is to provide researchers in this field with a comprehensive panoramic view while presenting our own perspectives and prospects.
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Espermatogénesis , Humanos , Masculino , Animales , Diferenciación Celular , Espermatozoides/fisiología , Espermatozoides/citología , Espermatozoides/metabolismo , Testículo/citologíaRESUMEN
Mammalian meiosis is a highly specialized cell division process, resulting in the production of genetically unique haploid cells. However, the molecular mechanisms governing meiosis remain largely unknown, primarily due to the difficulty in isolating pure sub-populations of spermatocytes. Definitive molecular, biochemical, and functional investigations of the meiosis process require the isolation of these individual homogeneous sub-populations of spermatocytes. Here, we present an approach that enables the purification of homogeneous spermatocytes from mouse testis at desired sub-stages. This approach consists of two strategic steps. The first is to synchronize spermatogenesis, aiming to minimize the diversity and complexity of testicular germ cells. The second involves utilizing mouse models with germ cell-specific fluorescent markers to differentiate the desired subtype from other cells in the testis. By employing fluorescence-activated cell sorting (FACS), this approach yields highly pure populations of spermatocytes at each sub-stage. When combined with other massively parallel sequencing techniques and in vitro cell culture methods, this approach will enhance our comprehension of the molecular mechanisms underlying mammalian meiosis and promote in vitro gametogenesis.
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Separación Celular , Citometría de Flujo , Meiosis , Espermatocitos , Espermatogénesis , Testículo , Animales , Masculino , Espermatocitos/citología , Espermatocitos/metabolismo , Ratones , Testículo/citología , Testículo/metabolismo , Citometría de Flujo/métodos , Separación Celular/métodosRESUMEN
The horned octopod Eledone cirrhosa, a medium-sized species found in Arabian Gulf off Saudi Arabia, was collected monthly from the Arabian Gulf off Dammam city during January to December 2022. Samples were dissected and prepared for examination using transmission electron microscopy. During genital maturation, seminiferous tubules are established in the testis, with active spermatogonia dividing. Spermatocytes 1 are observed in the tubule, followed by an increase in spermatogonia and spermatocytes in August. Spermiogenesis begins, with spherical spermatids evolving into elongated spermatids. In September, active spermatogonia, meiotic divisions, and increased spermiogenesis continue. Spermatozoa appear in Needham's pouch, indicating sexual maturity. The ovary undergoes various stages of development, with oocytes at stage I in June and July, followed by stage II in October and November. In stage III, follicular cords invade the oocyte's cytoplasm, forming numerous lipid inclusions and protein granules. The cytoplasm contains cisternae of endoplasmic reticulum and a poorly developed Golgi apparatus. Stage IV occurs in November, characterized by the maximum development of follicular cords and the beginning of vitellogenesis. The ooplasm contains numerous lipid inclusions, a syncytium, and secretory cells. From December, stage V oocytes are mainly present, indicating the activity phase of maximum secretion. Yolk platelets accumulate in the oocyte ooplasm, and chorion forms at the zona pellucida. In January, the first smooth eggs are found in some octopuses' ovary, with their proportion increasing steadily. This study aimed to investigate the mitogenic action of gonadotropin and identify the periods of intense cell multiplication during the sexual cycle using cytological methods.
Asunto(s)
Microscopía Electrónica de Transmisión , Octopodiformes , Ovario , Maduración Sexual , Animales , Femenino , Masculino , Maduración Sexual/fisiología , Ovario/ultraestructura , Ovario/anatomía & histología , Ovario/citología , Octopodiformes/anatomía & histología , Estaciones del Año , Testículo/ultraestructura , Testículo/citología , Espermatogénesis/fisiología , Oocitos/ultraestructuraRESUMEN
This study explores the effects of Trib3 gene knockout on adult male rat spermatogenesis. Using CRISPR/Cas9, we knocked out the Trib3 gene in Wistar rats. Results indicate altered expression of PLZF, ID4, and c-KIT in knockout rats, suggesting impaired spermatogonial stem cell proliferation and differentiation. Histological analysis reveals reduced seminiferous tubule area and decreased spermatocyte numbers. Mating experiments demonstrate reduced offspring rates after the second self-mating in knockout rats. SYCP3, a meiosis marker, shows elevated expression in knockout rat testes at 14 days postpartum, suggesting an impact on reproductive processes. ELISA results indicate decreased testosterone, FSH, and FGF9 levels in knockout rat testicular tissues. In conclusion, Trib3 gene deletion may impede spermatogonial self-renewal and promote differentiation through the FSH-FGF9- c-KIT interaction and p38MAPK pathway, affecting reproductive capacity. These findings contribute to understanding the molecular mechanisms regulating spermatogenesis.
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Células Madre Germinales Adultas , Diferenciación Celular , Proliferación Celular , Espermatogénesis , Animales , Masculino , Ratas , Células Madre Germinales Adultas/fisiología , Células Madre Germinales Adultas/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Técnicas de Inactivación de Genes , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas Wistar , Espermatogénesis/fisiología , Espermatogonias/fisiología , Espermatogonias/metabolismo , Espermatogonias/citología , Testículo/citología , Testículo/metabolismoRESUMEN
This study presents a biphasic approach to overcome the limitations of current testicular organoid (TO) cultures, including histological heterogeneity, germ cell loss and absence of spermatogenesis. Agarose microwells were utilized to create TOs from prepubertal C57BL/6 J testicular cells. First emphasis was on improving germ cell survival during the initial 2-week reorganization phase by comparingα-MEM + 10% knockout serum replacement (KSR) medium, known to support TO generation in mice, to three optimized media (1-3). Cell densities and culture dynamics were also tested to recreate histological resemblance to testes. After optimizing germ cell survival and cell organization, the effect of growth factors and immunomodulation through CD45+immune cell depletion or dexamethasone (DEX) supplementation were assessed for enhancing spermatogenesis during the subsequent differentiation phase. Testicular cells self-reorganized into organoids resembling the testicular anatomical unit, characterized by one tubule-like structure surrounded by interstitium. Media 1-3 proved superior for organoid growth during the reorganization phase, with TOs in medium 3 exhibiting germ cell numbers (7.4% ± 4.8%) comparable to controls (9.3% ± 5.3%). Additionally, 37% ± 30% demonstrated organized histology from 32 × 103cells under static conditions. Switching toα-MEM + 10% KSR during the differentiation phase increased formation efficiency to 85 ± 7%, along with elevated germ cell numbers, testosterone production (3.1 ± 0.9 ng ml-1) and generation ofγ-H2AX+spermatid-like cells (steps 8-11, 1.2% ± 2.2% of the total). Adding differentiation factors to theα-MEM increased spermatid-like cell numbers to 2.9% ± 5.9%, confirmed through positive staining for CREM, transition protein 1, and peanut agglutinin. Although, these remained diploid with irregular nuclear maturation. DEX supplementation had no additional effect, and immune cell depletion adversely impacted TO formation. The manipulability of TOs offers advantages in studying male infertility and exploring therapies, with scalability enabling high-throughput chemical screening and reducing animal usage in reproductive toxicity and drug discovery studies.
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Supervivencia Celular , Ratones Endogámicos C57BL , Organoides , Espermatogénesis , Testículo , Testosterona , Masculino , Animales , Organoides/citología , Organoides/metabolismo , Organoides/efectos de los fármacos , Testículo/citología , Testículo/efectos de los fármacos , Testículo/metabolismo , Testosterona/farmacología , Espermatogénesis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ratones , Diferenciación Celular/efectos de los fármacos , Células Germinativas/citología , Células Germinativas/efectos de los fármacos , Células Germinativas/metabolismo , Dexametasona/farmacologíaRESUMEN
We evaluated the influence of different media plus various concentrations of Glial cell line-derived neurotrophic factor (GDNF) during the in vitro culture (IVC) of testicular tissues from prepubertal collared peccary. Testes from 5 individuals were collected, fragmented and cultured for 28 days (34°C and 5% CO2). Culture media were Dulbecco's modified essential medium (DMEM) or stem cell serum free media (StemPro-34™ SFM), both supplemented with various concentrations of GDNF (0, 10, or 20 ng/mL). Fragments were cultured on the flat surface of 0.75% agarose gel and were evaluated every 7 days for fragment area, histomorphology, cellular viability, and proliferative activity. Data were expressed as mean ± standard error and analyzed by Kruskal-Wallis's and Tukey test. Fragments area decreased over the 28 days-culture, regardless of the treatment. For morphology, the StemPro-37 SFM medium plus 10 ng/mL GDNF provided higher scores at all time points in comparison to DMEM using any GDNF concentration (p < .05). After 28 days, similar cellular viability (~70%) was observed in all treatments (p > .05). For proliferating cell nuclear antigen assay, only DMEM plus 10 ng/mL GDNF improved (p < .05) cellular proliferation on Days 14 and 28. Looking at argyrophilic nucleolar organizing regions, after 28 days, there were no differences among treatments regarding cell proliferative capacity for both spermatogonia and Sertoli cells (p > .05). In summary, the DMEM and StemPro-34 SFM are adequate medium for IVC of prepubertal peccary testicular tissue. Supplementation with GDNF, especially at a 10 ng/mL concentration, appears to be essential for the maintenance of cell survival and proliferation.
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Supervivencia Celular , Medios de Cultivo , Factor Neurotrófico Derivado de la Línea Celular Glial , Testículo , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Masculino , Testículo/citología , Testículo/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo/farmacología , Medios de Cultivo/química , Proliferación Celular/efectos de los fármacos , Carica , Técnicas de Cultivo de Tejidos/métodosRESUMEN
Spermatogonia cryopreservation can be a strategy for future conservation actions. The neotropical Siluriformes Pseudopimelodus mangurus was already classified as vulnerable on the Red List of Threatened Species. P. mangurus spermatogonial cells were isolated, assessed, and cryopreserved. Fragments of the testis were enzymatically dissociated, purified using Percoll density gradient, and submitted to differential plating. Fractionated cells were evaluated by microscopy, ddx4 (vasa) relative expression, and alkaline phosphatase activity. Cryopreservation was conducted using ethylene glycol, glycerol, dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), and propanediol at 1 M, 1.5 M, and 2 M. Cell viability was evaluated and cell concentration was determined. Cell fractions from 20 % and 30 % Percoll gradient bands showed the highest concentrations of spermatogonia. The fraction mix showed 54 % purity and 93 % viability. After differential plating, 60 % purity and 92 % viability were obtained. Spermatogonial cells showed high alkaline phosphatase activity compared to spermatocytes and spermatids. The relative spermatogonial ddx4 expression from the Percoll density gradient was about twice as high as in samples from the testis and the differential plating. The increased ddx4 expression indicated the enrichment of spermatogonial cells by density gradient step and dead cells expressing ddx4 in differential plating, or ddx4 decreasing expression during cell culture. For this reason, cells from the Percoll gradient were chosen for cryopreservation. Propanediol at 1 M demonstrated the best condition for spermatogonial cell cryopreservation, presenting 98 % viability, while dimethylacetamide at 2 M represented the least favorable condition, with approximately 47 % viability. These findings are essential for P. mangurus spermatogonial cell cryopreservation, aiming to generate a spermatogonia cryobank for future conservation efforts.
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Bagres , Supervivencia Celular , Criopreservación , Espermatogonias , Animales , Masculino , Criopreservación/métodos , Criopreservación/veterinaria , Espermatogonias/citología , Crioprotectores/farmacología , Testículo/citología , Dimetilsulfóxido/farmacología , Acetamidas/farmacología , Acetamidas/química , Glicol de Etileno/farmacología , ARN Helicasas DEAD-box/metabolismo , Glicerol/farmacología , Glicerol/metabolismo , Fosfatasa Alcalina/metabolismo , Propilenglicol/farmacología , Separación Celular/métodosRESUMEN
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.
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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
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
BACKGROUND: Exploring the molecular mechanisms of primordial germ cell (PGC) migration and the involvement of gonadal somatic cells in gonad development is valuable for comprehending the origins and potential treatments of reproductive-related diseases. METHODS: Diaphanous related formin 1 (Diaph1, also known as mDia1) was screened by analyzing publicly available datasets (ATAC-seq, DNase-seq, and RNA-seq). Subsequently, the CRISPR-Cas9 technology was used to construct Diaph1 knockout mice to investigate the role of Diaph1 in gonad development. RESULTS: Based on data from public databases, a differentially expressed gene Diaph1, was identified in the migration of mouse PGC. Additionally, the number of PGCs was significantly reduced in Diaph1 knockout mice compared to wild type mice, and the expression levels of genes related to proliferation (Dicer1, Mcm9), adhesion (E-cadherin, Cdh1), and migration (Cxcr4, Hmgcr, Dazl) were significantly decreased. Diaph1 knockout also inhibited Leydig cell proliferation and induced apoptosis in the testis, as well as granulosa cell apoptosis in the ovary. Moreover, the sperm count in the epididymal region and the count of ovarian follicles were significantly reduced in Diaph1 knockout mice, resulting in decreased fertility, concomitant with lowered levels of serum testosterone and estradiol. Further research found that in Diaph1 knockout mice, the key enzymes involved in testosterone synthesis (CYP11A1, 3ß-HSD) were decreased in Leydig cells, and the estradiol-associated factor (FSH receptor, AMH) in granulosa cells were also downregulated. CONCLUSIONS: Overall, our findings indicate that the knockout of Diaph1 can disrupt the expression of factors that regulate sex hormone production, leading to impaired secretion of sex hormones, ultimately resulting in damage to reproductive function. These results provide a new perspective on the molecular mechanisms underlying PGC migration and gonadal development, and offer valuable insights for further research on the causes, diagnosis, and treatment of related diseases.
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Proliferación Celular , Forminas , Células Germinativas , Gónadas , Ratones Noqueados , Animales , Ratones , Femenino , Masculino , Forminas/genética , Forminas/metabolismo , Proliferación Celular/genética , Gónadas/metabolismo , Células Germinativas/metabolismo , Apoptosis/genética , Testículo/metabolismo , Testículo/crecimiento & desarrollo , Testículo/citología , Movimiento Celular/genética , Ovario/metabolismo , Ovario/crecimiento & desarrollo , Ratones Endogámicos C57BLRESUMEN
The cryopreservation of teleost eggs and embryos remains challenging, and there are no previous reports that demonstrate successful cryopreservation in medaka (Oryzias latipes). We have reported egg and sperm production, followed by the generation of donor-derived offspring by transplanting vitrified whole testes-derived testicular cells into surrogate fish. The vitrification solutions contained ethylene glycol, sucrose, and ficoll. In this study, we replaced sucrose with trehalose in the vitrification solution and medaka whole testes were vitrified with the solution. The post-vitrification survival (72.8 ± 3.5 %) was markedly improved compared with that achieved using the sucrose-containing solution (44.7 ± 4.2 %). Moreover, we demonstrated the production of eggs, sperm, and donor-derived offspring from testicular cells transplanted into surrogate recipients. The phenotype of donor-derived offspring was identical to that of transplanted testicular cells. These findings suggest that trehalose is effective for the vitrification of medaka whole testis and can be considered an effective and reliable method for the long-term preservation of their genetic resources.
Asunto(s)
Criopreservación , Crioprotectores , Oryzias , Testículo , Trehalosa , Vitrificación , Animales , Trehalosa/farmacología , Masculino , Criopreservación/métodos , Testículo/citología , Testículo/metabolismo , Crioprotectores/farmacología , Femenino , Glicol de Etileno/farmacología , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Sacarosa/farmacología , Sacarosa/metabolismo , Supervivencia Celular/efectos de los fármacosRESUMEN
Organoid systems have revolutionised various facets of biological research by offering a three-dimensional (3D), physiologically relevant in vitro model to study complex organ systems. Over recent years, testicular organoids have been publicised as promising platforms for reproductive studies, disease modelling, drug screening, and fertility preservation. However, the full potential of these systems has yet to be realised due to inherent limitations. This paper offers a comprehensive analysis of the current challenges associated with testicular organoid models. Firstly, we address the inability of current organoid systems to fully replicate the intricate spatial organisation and cellular diversity of the in vivo testis. Secondly, we scrutinise the fidelity of germ cell maturation within the organoids, highlighting incomplete spermatogenesis and epigenetic inconsistencies. Thirdly, we consider the technical challenges faced during organoid culture, including nutrient diffusion limits, lack of vasculature, and the need for specialised growth factors. Finally, we discuss the ethical considerations surrounding the use of organoids for human reproduction research. Addressing these limitations in combination with integrating complementary approaches, will be essential if we are to advance our understanding of testicular biology and develop novel strategies for addressing reproductive health issues in males.
Asunto(s)
Organoides , Espermatogénesis , Testículo , Organoides/fisiología , Organoides/citología , Masculino , Testículo/citología , Humanos , Espermatogénesis/fisiología , AnimalesRESUMEN
In brief: Atrazine, like oestrogen, disorganises laminin formation and reduces the number of germ cells and Sertoli cells in the developing testes of the tammar wallaby. This study suggests that interfering with the balance of androgen and oestrogen affects the integrity of laminin structure and testis differentiation. Abstract: The herbicide atrazine was banned in Europe in 2003 due to its endocrine disrupting activity but remains widely used. The integrity of the laminin structure in fetal testis cords requires oestrogen signalling but overexposure to xenoestrogens in the adult can cause testicular dysgenesis. However, whether xenoestrogens affect laminin formation in developing testes has not been investigated. Here we examined the effects of atrazine in the marsupial tammar wallaby during early development and compare it with the effects of the anti-androgen flutamide, oestrogen, and the oestrogen degrader fulvestrant. The tammar, like all marsupials, gives birth to altricial young, allowing direct treatment of the developing young during the male programming window (day 20-40 post partum (pp)). Male pouch young were treated orally with atrazine (5 mg/kg), flutamide (10 mg/kg), 17ß-oestradiol (2.5 mg/kg) and fulvestrant (1 mg/kg) daily from day 20 to 40 pp. Distribution of laminin, vimentin, SOX9 and DDX4, cell proliferation and mRNA expression of SRY, SOX9, AMH, and SF1 were examined in testes at day 50 post partum after the treatment. Direct exposure to atrazine, flutamide, 17ß-oestradiol, and fulvestrant all disorganised laminin but had no effect on vimentin distribution in testes. Atrazine reduced the number of germ cells and Sertoli cells when examined at day 40-50 pp and day 20 to 40 pp, respectively. Both flutamide and fulvestrant reduced the number of germ cells and Sertoli cells. Atrazine also downregulated SRY expression and impaired SOX9 nuclear translocation. Our results demonstrate that atrazine can compromise normal testicular differentiation during the critical male programming window.
Asunto(s)
Atrazina , Diferenciación Celular , Herbicidas , Laminina , Testículo , Masculino , Animales , Testículo/efectos de los fármacos , Testículo/metabolismo , Testículo/citología , Atrazina/farmacología , Laminina/metabolismo , Diferenciación Celular/efectos de los fármacos , Herbicidas/farmacología , Macropodidae/metabolismo , Células de Sertoli/efectos de los fármacos , Células de Sertoli/metabolismo , Células de Sertoli/citología , Estrógenos/farmacología , Estrógenos/metabolismo , Disruptores Endocrinos/farmacología , Recuento de Células , Antagonistas de Andrógenos/farmacología , Flutamida/farmacologíaRESUMEN
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.
Asunto(s)
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
OBJECTIVE: To investigate the mechanism of DNA-damage-inducible transcript 4(DDIT4)targeting miR-221-3p in microRNA(miRNA) on cadmium-induced apoptosis of mouse testicular stromal cells. METHODS: The activity of mouse testicular interstitial cells(TM3) was detected by CCK-8 after exposure to different concentrations of cadmium(0, 10, 20, 30, 40 µmol/L). Total RNA was extracted from cadmium-treated TM3 cells, and the significantly differentially expressed miRNA was screened with fold change(FC)>1.2 and P<0.05 as the criterion. TM3 cells were divided into blank control group, negative control group, cadmium exposure group(CdCl_2, 20 µmol/L), and cadmium+miR-221-3p mimic group. miR-221-3p mimic group was transfected into TM3 cells first, combined with cadmium exposure for 24 hours. The cell morphology was detected by Hoechst staining, and the apoptosis rate was analyzed by flow cytometry. Quantitative real-time PCR(qRT-PCR) and Western blot were used to detect DDIT4 expression. Dual luciferase reporter gene assay verified the binding of miR-221-3p to DDIT4. The function of DDIT4 and its relationship with apoptosis were analyzed by bioinformatics. The expression levels of B-cell lymphoma-2(Bcl-2) and Bcl-2 associated X protein(BAX) were observed after overexpression of miR-221-3p. RESULTS: Cadmium treatment of TM3 cells could reduce cell activity and there was a dose-effect relationship. The cell morphology showed that compared with the control group, the cells were wrinkled and the nuclei were heavily stained, and the apoptosis rate increased to 19.66%±0.45%(P<0.01). Compared with the cadmium exposure group, the normal morphologic cells increased in the cadmium exposure +miR-221-3p mimic group, and the apoptosis rate decreased to 13.76%±0.37%(P<0.05). The expression level of miR-221-3p was down-regulated(P<0.01), and the expression level of DDIT4 was up-regulated(P<0.05). Bioinformatics analysis and dual luciferase report analysis showed that DDIT4 was one of the target genes of miR-221-3p. Compared with the cadmium exposure group, the expression level of DDIT4 in the cadmium+miR-221-3p mimic group was down-regulated(P<0.05), and the ratio of Bcl-2/BAX was increased from 0.54±0.03 to 0.71±0.04. CONCLUSION: miR-221-3p inhibits cadmium-induced apoptosis of TM3 cells by targeting DDIT4.