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BACKGROUND: Interspecific hybridization plays vital roles in enriching animal diversity, while male hybrid sterility (MHS) of the offspring commonly suffered from spermatogenic arrest constitutes the postzygotic reproductive isolation. Cattle-yak, the hybrid offspring of cattle (Bos taurus) and yak (Bos grunniens) can serve as an ideal MHS animal model. Although meiotic arrest was found to contribute to MHS of cattle-yak, yet the cellular characteristics and developmental potentials of male germline cell in pubertal cattle-yak remain to be systematically investigated. RESULTS: Single-cell RNA-seq analysis of germline and niche cell types in pubertal testis of cattle-yak and yak indicated that dynamic gene expression of developmental germ cells was terminated at late primary spermatocyte (meiotic arrest) and abnormal components of niche cell in pubertal cattle-yak. Further in vitro proliferation and differentially expressed gene (DEG) analysis of specific type of cells revealed that undifferentiated spermatogonia of cattle-yak exhibited defects in viability and proliferation/differentiation potentials. CONCLUSION: Comparative scRNA-seq and in vitro proliferation analysis of testicular cells indicated that not only meiotic arrest contributed to MHS of cattle-yak. Spermatogenic arrest of cattle-yak may originate from the differentiation stage of undifferentiated spermatogonia and niche cells of cattle-yak may provide an adverse microenvironment for spermatogenesis.
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Infertilidad Masculina , Testículo , Animales , Masculino , Humanos , Bovinos , Testículo/metabolismo , Análisis de Expresión Génica de una Sola Célula , Infertilidad Masculina/genética , Infertilidad Masculina/veterinaria , Espermatogénesis/genética , EspermatogoniasRESUMEN
PURPOSE: Development of organoids using human primary testicular cells has remained a challenge due to the complexity of the mammalian testicular cytoarchitecture and culture methods. In this study, we generated testicular organoids derived from human primary testicular cells. Then, we evaluated the effect of stem cell factor (SCF) on cell differentiation and apoptosis in the testicular organoid model. METHODS: The testicular cells were harvested from the three brain-dead donors. Human spermatogonial stem cells (SSCs) were characterized using immunocytochemistry (ICC), RT-PCR and flow cytometry. Testicular organoids were generated from primary testicular cells by hanging drop culture method and were cultured in three groups: control group, experimental group 1 (treated FSH and retinoic acid (RA)), and experimental group 2 (treated FSH, RA and SCF), for five weeks. We assessed the expression of SCP3 (Synaptonemal Complex Protein 3) as a meiotic gene, PRM2 (Protamine 2) as a post-meiotic marker and apoptotic genes of Bax (BCL2-Associated X Protein) and Bcl-2 (B-cell lymphoma 2), respectively by using RT-qPCR. In addition, we identified the expression of PRM2 by immunohistochemistry (IHC). RESULTS: Relative expression of SCP3, PRM2 and Bcl-2 were highest in group 2 after five weeks of culture. In contrast, BAX expression level was lower in experimental group 2 in comparison with other groups. IHC analyses indicated the highest expression of PRM2 as a postmeiotic marker in group 2 in comparison to 2D culture and control groups but not find significant differences between experimental group 1 and experimental group 2 groups. Morphological evaluations revealed that organoids are compact spherical structures and in the peripheral region composed of uncharacterized elongated fibroblast-like cells. CONCLUSION: Our findings revealed that the testicular organoid culture system promote the spermatogonial stem cell (SSC) differentiation, especially in presence of SCF. Developed organoids are capable of recapitulating many important properties of a stem cell niche.
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Espermatogonias , Factor de Células Madre , Masculino , Animales , Humanos , Factor de Células Madre/farmacología , Factor de Células Madre/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Proteína X Asociada a bcl-2/farmacología , Espermatogonias/metabolismo , Espermatogénesis/genética , Diferenciación Celular , Organoides , Hormona Folículo Estimulante/metabolismo , Hormona Folículo Estimulante/farmacología , Células Cultivadas , MamíferosRESUMEN
Undifferentiated germ cells, including the spermatogonial stem cell subpopulation required for fertility restoration using human immature testicular tissue (ITT), are difficult to recover as they do not easily adhere to plastics. Due to the scarcity of human ITT for research, we used neonatal porcine ITT. Strategies for maximizing germ cell recovery, including a comparison of two enzymatic digestion protocols (P1 and P2) of ITT fragment sizes (4 mm3 and 8 mm3) and multi-step differential plating were explored. Cellular viability and yield, as well as numbers and proportions of DDX4+ germ cells, were assessed before incubating the cell suspensions overnight on uncoated plastics. Adherent cells were processed for immunocytochemistry (ICC) and floating cells were further incubated for three days on Poly-D-Lysine-coated plastics. Germ cell yield and cell types using ICC for SOX9, DDX4, ACTA2 and CYP19A1 were assessed at each step of the multi-step differential plating. Directly after digestion, cell suspensions contained >92% viable cells and 4.51% DDX4+ germ cells. Pooled results for fragment sizes revealed that the majority of DDX4+ cells adhere to uncoated plastics (P1; 82.36% vs. P2; 58.24%). Further incubation on Poly-D-Lysine-coated plastics increased germ cell recovery (4.80 ± 11.32 vs. 1.90 ± 2.07 DDX4+ germ cells/mm2, respectively for P1 and P2). The total proportion of DDX4+ germ cells after the complete multi-step differential plating was 3.12%. These results highlight a reduced proportion and number of germ cells lost when compared to data reported with other methods, suggesting that multi-step differential plating should be considered for optimization of immature germ cell recovery. While Poly-D-Lysine-coating increased the proportions of recovered germ cells by 16.18% (P1) and 28.98% (P2), future studies should now focus on less cell stress-inducing enzymatic digestion protocols to maximize the chances of fertility restoration with low amounts of cryo-banked human ITT.
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Neoplasias , Polilisina , Humanos , Animales , Porcinos , Células Germinativas , Fertilidad , Lisina , Plásticos , Poli A , DigestiónRESUMEN
The present study was aimed to elucidate the host-virus interactions using RNA-Seq analysis at 1 h and 8 h of post-infection of sheeppox virus (SPPV) in lamb testis cell. The differentially expressed genes (DEGs) and the underlying mechanisms linked to the host immune responses were obtained. The protein-protein interaction (PPI) network analysis and ingenuity pathway analysis (IPA) illustrated the interaction between the DEGs and their involvement in cell signalling responses. Highly connected hubs viz. AURKA, CHEK1, CCNB2, CDC6 and MAPK14 were identified through PPI network analysis. IPA analysis showed that IL-6- and ERK5-mediated signalling pathways were highly enriched at both time points. The TP53 gene was identified to be the leading upstream regulator that directly responded to SPPV infection, resulting in downregulation at both time points. The study provides an overview of how the lamb testis genes and their underlying mechanisms link to growth and immune response during SPPV infection.
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Capripoxvirus , Infecciones por Poxviridae , Enfermedades de las Ovejas , Masculino , Ovinos , Animales , Testículo , Infecciones por Poxviridae/veterinaria , Capripoxvirus/genética , Transcriptoma , Perfilación de la Expresión GénicaRESUMEN
At present, there is still a lack of attention to male infertility and fertility impairment. Indeed, the pathologies affecting the reproductive area in man are derived from anatomical or functional alterations of neuroendocrine system; thus, the study of these dysfunctions is necessary for a correct aetiopathogenetic and therapeutic framing of infertile patients. In this article, we underline the importance of the study of the molecular mechanisms regulated by the most common therapy used to treat infertile men, with the aim to highlight the necessity to avoid the administration of the wrong posology or, even more important, the wrong therapy to the patient. Accordingly, we present some pioneer data obtained on primary testicular cells cultured in vitro and treated with human chorionic gonadotropin (hCG). These data pave the way on the possibility to preliminarily test the effectiveness of the therapy in vitro, in order to identify the responsiveness of patient-derived cells to the treatment and its effectiveness in each subject, in order to identify the correct dosage in a personalised way.
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Fertilidad , Infertilidad Masculina , Gonadotropina Coriónica/farmacología , Humanos , Infertilidad Masculina/terapia , Masculino , Medicina de Precisión , TestículoRESUMEN
Bull fertility is pivotal to the prosperity of the cattle industry worldwide. miR-202 has been shown to be gonad specific and to have key roles in gonad function in different species. To further understand the involvement of miR-202 in bull reproduction, this study aimed to establish its localization in bovine testicular tissue and to identify putative biological functions using bioinformatics approaches. We assessed the miR-202 expression in paraffin-embedded tissue samples collected form an abattoir using in situ hybridization. miR-202 was present in Sertoli cells and in germ cells at different stages of development. Using available databases, a total of 466 predicted gene targets of miR-202 were identified. Functional annotation revealed that miR-202 target genes were mainly associated with protein modification and phosphorylation processes as well as longevity regulating pathway. Moreover, genes in the longevity regulating pathway mapped to PI3K/Akt/mTOR pathway which is involved in promoting proliferation of testicular cells and spermatogenesis. These findings suggest that miR-202 plays important roles in regulating proliferation and viability of testicular cells including somatic and germ cells.
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MicroARNs , Testículo , Animales , Bovinos/genética , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Células de Sertoli/metabolismo , Espermatogénesis/genética , Testículo/metabolismoRESUMEN
Testicular oxidative stress is one of the most common factors underlying male infertility. Welted thistle, Carduus crispus Linn., and its bioactive principles are attracting scientific interest in treating male reproductive dysfunctions. Here, the protective effects of apigenin isolated from C. crispus against oxidative damage induced by hydrogen peroxide (H2O2) and dysregulation in spermatogenesis associated parameters in testicular sperm cells was investigated. Cell viabilities, ROS scavenging effects, and spermatogenic associated molecular expressions were measured by MTT, DCF-DA, Western blotting and real-time RT-PCR, respectively. A single peak with 100% purity of apigenin was obtained in HPLC conditions. Apigenin treated alone (2.5, 5, 10 and 20 µM) did not exhibit cytotoxicity, but inhibited the H2O2-induced cellular damage and elevated ROS levels significantly (p < 0.05 at 5, 10 and 20 µM) and dose-dependently. Further, H2O2-induced down-regulation of antioxidant (glutathione S-transferases m5, glutathione peroxidase 4, and peroxiredoxin 3) and spermatogenesis-associated (nectin-2 and phosphorylated-cAMP response element-binding protein) molecular expression in GC-2spd cells were attenuated by apigenin at both protein and mRNA levels (p < 0.05). In conclusion, our study showed that apigenin isolated from C. crispus might be an effective agent that can protect ROS-induced testicular dysfunctions.
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Apigenina , Carduus , Apigenina/metabolismo , Apigenina/farmacología , Carduus/metabolismo , Peróxido de Hidrógeno/farmacología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Espermatogénesis , Espermatozoides/metabolismoRESUMEN
The serious coronavirus disease-2019 (COVID-19) was first reported in December 2019 in Wuhan, China. COVID-19 is an infectious disease caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). Angiotensin converting enzyme 2(ACE2) is the cellular receptor for SARS-CoV-2. Considering the critical roles of testicular cells for the transmission of genetic information between generations, we analyzed single-cell RNA-sequencing (scRNA-seq) data of adult human testis. The mRNA expression of ACE2 was expressed in both germ cells and somatic cells. Moreover, the positive rate of ACE2 in testes of infertile men was higher than normal, which indicates that SARS-CoV-2 may cause reproductive disorders through pathway activated by ACE2 and the men with reproductive disorder may easily to be infected by SARS-CoV-2. The expression level of ACE2 was related to the age, and the mid-aged with higher positive rate than young men testicular cells. Taken together, this research provides a biological background of the potential route for infection of SARS-CoV-2 and may enable rapid deciphering male-related reproductive disorders induced by COVID-19.
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Enzima Convertidora de Angiotensina 2/metabolismo , Infertilidad Masculina/metabolismo , Receptores Virales/metabolismo , Células de Sertoli/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Adulto , Enzima Convertidora de Angiotensina 2/genética , COVID-19/complicaciones , Humanos , Infertilidad Masculina/genética , Infertilidad Masculina/virología , Masculino , Persona de Mediana Edad , RNA-Seq , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Receptores de Serotonina 5-HT3/genética , Receptores de Serotonina 5-HT3/metabolismo , Análisis de la Célula IndividualRESUMEN
Testicular cell suspension (TCS) can be cryopreserved for male germ-line preservation and fertility restoration. We aimed to validate a cryopreservation protocol for TCS of domestic cat to be applied in endangered felids species. Testis tissue from adult domestic cats was enzymatically dissociated and spermatogenic cells were enriched. The resulting TCS was diluted in 7.5% or 15% Me2SO based medium. Slow and fast freezing methods were tested. We examined the effects of freezing approaches using two combinations of fluorescent dyes: Calcein-AM with Propidium iodide (C/PI) and SYBR14 with Propidium iodide (S/PI). Ploidy analysis of domestic cat fresh TCS revealed that the majority of testicular cells were haploid cells. Based on microscopic observation, two size populations (12.3 ± 2.3 µm and 20.5 ± 4 µm in diameter) were identified and presumed to be mainly spermatids and spermatocytes, respectively. Both evaluation methods proved higher viability of aggregated cells before and after cryopreservation compared with single cells, and superiority of low concentration of Me2SO (7.5%) in association with slow freezing to preserve viability of testicular cells. However, S/PI resulted in a more precise evaluation compared with the C/PI method. The combination of 7.5% Me2SO-based medium with slow freezing yielded post thaw viability of S/PI labeled aggregated (49.8 ± 20%) and single cells (31.5 ± 8.1%). Comparable results were achieved using testes of a Cheetah and an Asiatic golden cat. In conclusion, TCS from domestic cat can be successfully cryopreserved and has the potential to support fertility restoration of endangered felids species.
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Criopreservación , Testículo , Animales , Gatos , Supervivencia Celular , Criopreservación/métodos , Congelación , Masculino , EspermatozoidesRESUMEN
The spermatogenesis is temperature-dependent and heat stress have destructive effects on spermatogenesis and reduces sperm quality. Sixteen adult mice were allocated to two groups: hyperthermia and control groups. Scrotal hyperthermia was induced by water bath with 43°C for 30 min. Then, the spermatozoon was isolated through the tail region of epididymis for sperm parameters analysis. The testicular tissues were taken for stereological studies, hormonal assay, TUNEL assay and molecular studies. We found a marked decrease in sperm parameters and serum testosterone level in mice induced by scrotal hyperthermia as well as stereological analysis indicated a significant reduction in testicular cells and changes in the spatial arrangement of testicular cells in the scrotal hyperthermia groups compared to the control groups. Moreover, the TUNEL assay results showed that apoptotic cells were enhanced significantly in the group of scrotal hyperthermia compared to the control groups. Furthermore, scrotal hyperthermia caused a reduction in the expression of retinoic acid 8 (STRA8), c-kit and proliferating cell nuclear antigen (PCNA) genes in the scrotal hyperthermia groups compared to the control. According to results, induction of transient scrotal hyperthermia leads to a fluctuation in the spatial arrangement of testicular cells, which finally influences the normal function of spermatogenesis.
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Calor , Hipertermia , Animales , Masculino , Ratones , Escroto , Espermatogénesis , Espermatozoides , TestículoRESUMEN
Porcine deltacoronavirus (PDCoV) is a recently identified coronavirus that causes intestinal diseases in neonatal piglets with diarrhea, vomiting, dehydration, and post-infection mortality of 50-100%. Currently, there are no effective treatments or vaccines available to control PDCoV. To study the potential of RNA interference (RNAi) as a strategy against PDCoV infection, two short hairpin RNA (shRNA)-expressing plasmids (pGenesil-M and pGenesil-N) that targeted the M and N genes of PDCoV were constructed and transfected separately into swine testicular (ST) cells, which were then infected with PDCoV strain HB-BD. The potential of the plasmids to inhibit PDCoV replication was evaluated by cytopathic effect, virus titers, and real-time quantitative RT-PCR assay. The cytopathogenicity assays demonstrated that pGenesil-M and pGenesil-N protected ST cells against pathological changes with high specificity and efficacy. The 50% tissue culture infective dose showed that the PDCoV titers in ST cells treated with pGenesil-M and pGenesil-N were reduced 13.2- and 32.4-fold, respectively. Real-time quantitative RT-PCR also confirmed that the amount of viral RNA in cell cultures pre-transfected with pGenesil-M and pGenesil-N was reduced by 45.8 and 56.1%, respectively. This is believed to be the first report to show that shRNAs targeting the M and N genes of PDCoV exert antiviral effects in vitro, which suggests that RNAi is a promising new strategy against PDCoV infection.
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Infecciones por Coronavirus/genética , Coronavirus/genética , Proteínas Virales/genética , Replicación Viral/genética , Animales , Coronavirus/patogenicidad , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Diarrea/genética , Diarrea/patología , Diarrea/veterinaria , Diarrea/virología , Masculino , Plásmidos/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Viral/genética , Porcinos/virología , Enfermedades de los Porcinos/genética , Enfermedades de los Porcinos/virología , Testículo/crecimiento & desarrollo , Testículo/virologíaRESUMEN
Cryopreservation of immature testicular tissue (ITT) prior to chemo/radiotherapy is now ethically accepted and is currently the only way to preserve fertility of prepubertal boys about to undergo cancer therapies. So far, three-dimensional culture of testicular cells isolated from prepubertal human testicular tissue was neither efficient nor reproducible to obtain mature spermatozoa, and ITT transplantation is not a safe option when there is a risk of cancer cell contamination of the testis. Hence, generation of testicular organoids (TOs) after cell selection is a novel strategy aimed at restoring fertility in these patients. Here, we created TOs using hydrogels developed from decellularized porcine ITT and compared cell numbers, organization and function to TOs generated in collagen only hydrogel. Organotypic culture of porcine ITT was used as a control. Rheological and mass spectrometry analyses of both hydrogels highlighted differences in terms of extracellular matrix stiffness and composition, respectively. Sertoli cells (SCs) and germ cells (GCs) assembled into seminiferous tubule-like structures delimited by a basement membrane while Leydig cells (LCs) and peritubular cells localized outside. TOs were maintained for 45 days in culture and secreted stem cell factor and testosterone demonstrating functionality of SCs and LCs, respectively. In both TOs GC numbers decreased and SC numbers increased. However, LC numbers decreased significantly in the collagen hydrogel TOs (p < 0.05) suggesting a better preservation of growth factors within TOs developed from decellularized ITT and thus a better potential to restore the reproductive capacity.
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Criopreservación/métodos , Matriz Extracelular/metabolismo , Preservación de la Fertilidad/métodos , Hidrogeles/metabolismo , Organoides/citología , Testículo/citología , Animales , Proliferación Celular , Humanos , Células Intersticiales del Testículo/citología , Células Intersticiales del Testículo/metabolismo , Masculino , Técnicas de Cultivo de Órganos , Organoides/metabolismo , Túbulos Seminíferos/citología , Células de Sertoli/citología , Células de Sertoli/metabolismo , Solubilidad , Espermatogonias/citología , Factor de Células Madre/metabolismo , Porcinos , Testosterona/metabolismoRESUMEN
miRNAs, a type of small RNA, play critical roles in mammalian spermatogenesis. Spermatogonia are the foundation of spermatogenesis and are valuable for the study of spermatogenesis. However, the expression profiling of the miRNAs in spermatogonia of dairy goats remains unclear. CD49f has been one of the surface markers used for spermatogonia enrichment by magnetic activated cell sorting (MACS). Therefore, we used a CD49f microbead antibody to purify CD49f-positive and -negative cells of dairy goat testicular cells by MACS and then analysed the miRNA expression in these cells in depth using Illumina sequencing technology. The results of miRNA expression profiling in purified CD49f-positive and -negative testicular cells showed that 933 miRNAs were upregulated in CD49f-positive cells and 916 miRNAs were upregulated in CD49f-negative cells with a twofold increase, respectively; several miRNAs and marker genes specific for spermatogonial stem cells (SSCs) in testis had a higher expression level in CD49f-positive testicular cells, including miR-221, miR-23a, miR-29b, miR-24, miR-29a, miR-199b, miR-199a, miR-27a, and miR-21 and CD90, Gfra1, and Plzf. The bioinformatics analysis of differently expressed miRNAs indicated that the target genes of these miRNAs in CD49f-positive cells were involved in cell-cycle biological processes and the cell-cycle KEGG pathway. In conclusion, our comparative miRNAome data provide useful miRNA profiling data of dairy goat spermatogonia cells and suggest that CD49f could be used to enrich dairy goat spermatogonia-like cells, including SSCs.
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Integrina alfa6/metabolismo , MicroARNs/genética , Espermatogénesis/genética , Testículo/citología , Testículo/metabolismo , Animales , Secuencia de Bases , Ciclo Celular/genética , Diferenciación Celular/genética , Separación Celular/métodos , Citometría de Flujo , Cabras , Masculino , MicroARNs/biosíntesis , Análisis de Secuencia de ARN , Espermatogonias/citologíaRESUMEN
INTRODUCTION: The suppressing effects of chronic stress on sexual desire have long been noted. Yet the biological mechanisms underlying such effects, especially at the level of cellular biology of testicular cells, have not been fully investigated. AIM: In the present study, we used a chronic unpredictable mild stress model to examine the association between chronic stress and structural alterations in the male reproductive system. MAIN OUTCOME MEASURES: The main outcome measures were the structural changes in sperm cells and Leydig cells of male rats. We used Agmo and Ellingsen's procedure to study partner preference behavior and observed the morphology of Leydig cells and germ cells in the control and stress groups. METHODS: Our methods included histology, electron microscopy, and animal behavior tests. RESULTS: The results showed that after 5 weeks of chronic stress exposure, partner preference behavior was impaired, the total surface area of Leydig cells and the number and diameter of seminiferous tubules decreased significantly, and the number and size of Leydig cells, as well as the number and the short-axis diameter of spermatogenic cells, also decreased. At the ultrastructural level, transmission electron microscopy revealed that the basement membranes of seminiferous tubules in stressed rats was far thinner, had a low density, and was uneven in thickness compared with the normal group, with enhanced apoptosis in germ cells. CONCLUSION: We conclude that chronic stress can trigger organic damage to testicular cells in male rats.
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Enfermedades de los Genitales Masculinos/patología , Células Intersticiales del Testículo/patología , Motivación/fisiología , Túbulos Seminíferos/patología , Conducta Sexual Animal/fisiología , Estrés Psicológico/psicología , Animales , Apoptosis/efectos de los fármacos , Estro/fisiología , Femenino , Enfermedades de los Genitales Masculinos/psicología , Humanos , Masculino , Microscopía Electrónica de Transmisión , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Espermatozoides/patología , Estrés Psicológico/patologíaRESUMEN
Male gonad development is initiated by the aggregation of pre-Sertoli cells (SCs), which surround germ cells to form cords. Several attempts to reconstruct testes from dissociated testicular cells have been made; however, only very limited morphogenesis beyond seminiferous cord formation has been achieved. Therefore, we aimed to reconstruct seminiferous tubules using a 3-dimensional (D) re-aggregate culture of testicular cells, which were dissociated from 6-dpp neonatal mice, inside a collagen matrix. We performed a short-term culture (for 3 days) and a long-term culture (up to 3 wks). The addition of KnockOut Serum Replacement (KSR) promoted (1) the enlargement of SC re-aggregates; (2) the attachment of peritubular myoid (PTM) cells around the SC re-aggregates; (3) the sorting of germ cells inside, and Leydig cells outside, seminiferous cord-like structures; (4) the alignment of SC polarity inside a seminiferous cord-like structure relative to the basement membrane; (5) the differentiation of SCs (the expression of the androgen receptor); (6) the formation of a blood-testis-barrier between the SCs; (7) SC elongation and lumen formation; and (8) the proliferation of SCs and spermatogonia, as well as the differentiation of spermatogonia into primary spermatocytes. Eventually, KSR promoted the formation of seminiferous tubule-like structures, which accompanied germ cell differentiation. However, these morphogenetic events did not occur in the absence of KSR. This in vitro system presents an excellent model with which to identify the possible factors that induce these events and to analyze the mechanisms that underlie cellular interactions during testicular morphogenesis and germ cell differentiation.
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Técnicas de Cultivo de Célula/métodos , Colágeno/farmacología , Túbulos Seminíferos/citología , Animales , Animales Recién Nacidos , Agregación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular , Células Cultivadas , Humanos , Células Intersticiales del Testículo/citología , Células Intersticiales del Testículo/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Modelos Biológicos , Receptores Androgénicos/metabolismo , Túbulos Seminíferos/efectos de los fármacos , Túbulos Seminíferos/metabolismo , Túbulos Seminíferos/ultraestructura , Espermatocitos/citología , Espermatocitos/efectos de los fármacos , Uniones Estrechas/metabolismoRESUMEN
Superparamagnetic iron oxide nanoparticles (SPIONs) have gained significant attention in biomedical research due to their potential applications. However, little is known about their impact and toxicity on testicular cells. To address this issue, we conducted an in vitro study using primary mouse testicular cells, testis fragments, and sperm to investigate the cytotoxic effects of sodium citrate-coated SPIONs (Cit_SPIONs). Herein, we synthesized and physiochemically characterized the Cit_SPIONs and observed that the sodium citrate diminished the size and improved the stability of nanoparticles in solution during the experimental time. The sodium citrate (measured by thermogravimetry) was biocompatible with testicular cells at the used concentration (3%). Despite these favorable physicochemical properties, the in vitro experiments demonstrated the cytotoxicity of Cit_SPIONs, particularly towards testicular somatic cells and sperm cells. Transmission electron microscopy analysis confirmed that Leydig cells preferentially internalized Cit_SPIONs in the organotypic culture system, which resulted in alterations in their cytoplasmic size. Additionally, we found that Cit_SPIONs exposure had detrimental effects on various parameters of sperm cells, including motility, viability, DNA integrity, mitochondrial activity, lipid peroxidation (LPO), and ROS production. Our findings suggest that testicular somatic cells and sperm cells are highly sensitive and vulnerable to Cit_SPIONs and induced oxidative stress. This study emphasizes the potential toxicity of SPIONs, indicating significant threats to the male reproductive system. Our findings highlight the need for detailed development of iron oxide nanoparticles to enhance reproductive nanosafety.
RESUMEN
Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are widely used in a variety of industrial processes and manufacturing of consumer products. Current efforts by the manufacturing industry will limit use of long-chain or legacy PFAS represented by perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) and replace with short-chain or emerging PFAS such as perfluorobutanoic acid (PFBA) and perfluorobutane sulfonic acid (PFBS). However, there is little to no information on the toxicity of new and emerging PFAS. Therefore, we performed experiments in growing Long-Evans male rats to investigate effects of low-dose prepubertal and pubertal exposures to PFAS on gonadal steroid hormone secretion. The results demonstrated that both legacy and emerging PFAS have the capacity to regulate testicular steroidogenesis. For instance, prepubertal exposures to PFOS, PFBA, and PFBS increased serum and testicular testosterone concentrations. Exposure to PFBA increased testicular 17ß-estradiol (E2) concentrations, and PFOS and PFBS both decreased serum E2 concentrations while stimulating testicular E2 secretion. The data also demonstrated additive effects due to legacy and emerging PFAS mixtures compared with the individual chemicals. The gonadal effects due to PFAS exposures occurred at nanomolar concentrations, which approximate PFAS levels in the environment. Taken together, the present study supports the need for development of cost-effective and sustainable filtration media for different processes to remove PFAS from water and other sources of exposure. Current action by regulatory agencies such as the US Environmental Protection Agency to limit use of PFAS in the manufacture of consumer products will protect public health.
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Ácidos Alcanesulfónicos , Fluorocarburos , Ratas , Animales , Masculino , Ratas Long-Evans , Ácidos Alcanesulfónicos/toxicidad , Fluorocarburos/toxicidad , GónadasRESUMEN
Zinc (Zn) is a critical microelement for physiological process, but excess exposure can cause testicular dysfunction. However, the underlying mechanism of Zn-induced ferroptosis via regulating mitophagy is unknown. In this study, a total of 60 male weaned pigs were randomly divided into three groups and the content of Zn were 75 mg/kg (control), 750 mg/kg (Zn-I), 1500 mg/kg (Zn-II). Meanwhile, testicular cells were treated with ZnSO4 (0, 50 and 100 µM), and in combination of ZnSO4 (100 µM) and ferrostation-1, ML-210, or 3-methyladenine for 24 h. Our results verified that Zn could cause ferroptosis and lipid peroxidation, which were characterized by down-regulating level of SLC7A11, GPX4, and ferritin, and up-regulating levels of MDA, CD71, TF, and HMGB1 by Western blot, immunohistochemistry, immunofluorescence, peroxidase assay, et.ac. The opposite effect was shown after treatment with ferrostation-1 or ML-210. Meanwhile, the mitophagy-related proteins (PINK, Parkin, ATG5, LC3-II/LC3-I) were significantly upregulated in vivo and in vitro. Most importantly, 3-methyladenine observably relieved ferroptosis under Zn treatment through inhibiting mitophagy. Collectively, we demonstrated that mitophagy contributes to Zn-induced ferroptosis in porcine testis cells, providing a new insight into Zn toxicology.
Asunto(s)
Ferroptosis , Zinc , Masculino , Animales , Porcinos , Zinc/farmacología , Testículo , Mitofagia , Peroxidación de LípidoRESUMEN
Molecular characterisation of testicular cells is a pivotal step towards a profound understanding of spermatogenesis and developing assisted reproductive techniques (ARTs) based on germline preservation. To enable the identification of testicular somatic and spermatogenic cell types in felids, we investigated the expression of five molecular markers at the protein level in testes from domestic cats (Felis catus) at different developmental phases (prepubertal, pubertal I and II, postpubertal I and II) classified by single-cell ploidy analysis. Our findings indicate a prominent co-labelling for two spermatogonial markers, UCHL1 and FOXO1, throughout postnatal testis development. Smaller subsets of UCHL1 or FOXO1 single-positive spermatogonia were also evident, with the FOXO1 single-positive spermatogonia predominantly observed in prepubertal testes. As expected, DDX4+ germ cells increased in numbers beginning in puberty, reaching a maximum at adulthood (post-pubertal phase), corresponding to the sequential appearance of labelled spermatogonia, spermatocytes and spermatids. Furthermore, we identified SOX9+ Sertoli cells and CYP17A1+ Leydig cells in all of the developmental groups. Importantly, testes of African lion (Panthera leo), Sumatran tiger (Panthera tigris sumatrae), Chinese leopard (Panthera pardus japonesis) and Sudan cheetah (Acinonyx jubatus soemmeringii) exhibited conserved labelling for UCHL1, FOXO1, DDX4, SOX9 and CYP17A1. The present study provides fundamental information about the identity of spermatogenic and somatic testicular cell types across felid development that will be useful for developing ART approaches to support endangered felid conservation.
RESUMEN
The threat posed by increased surface temperatures worldwide has attracted the attention of researchers to the reaction of animals to heat stress. Spermatogenesis in animals such as stallions is a temperature-dependent process, ideally occurring at temperatures slightly below the core body temperature. Thus, proper thermoregulation is essential, especially because stallion spermatogenesis and the resulting spermatozoa are negatively affected by increased testicular temperature. Consequently, the failure of thermoregulation resulting in heat stress may diminish sperm quality and increase the likelihood of stallion infertility. In this review, we emphasize upon the impact of heat stress on spermatogenesis and the somatic and germ cells and describe the subsequent testicular alterations. In addition, we explore the functions and molecular responses of heat shock proteins, including HSP60, HSP70, HSP90, and HSP105, in heat-induced stress conditions. Finally, we discuss the use of various therapies to alleviate heat stress-induced reproductive harm by modulating distinct signaling pathways.