Hyaluronic acid-alginate hydrogel stimulates the differentiation of neonatal mouse testicular cells into hepatocyte-like and other cell lineages in three-dimensional culture.

BACKGROUND INFORMATION: Extracellular matrix (ECM)-derived hydrogels are frequently used in three-dimensional (3D) cell culture and organoid formation in several tissues. However, in the 3D cultivation of testicular cells, the hyaluronic acid (HA) hydrogel has not received as much attention. This study examined the effects of three distinct composites, including HA-alginate (HA-Alg), HA-alginate-collagen (HA-Alg-Col), and HA-alginate-decellularized ECM (HA-Alg-dECM), on mouse testicular cell culture and in vitro spermatogenesis. METHODS: For the creation of composites, the concentration of biomaterials used was 0.5% HA, 1% alginate, 2.5 mg/mL collagen, and 25 mg/mL dECM derived from the testicles of Rams. After 3D culture of 5 days post-partum (dpp) mouse testicular cells for 14 days, HA-Alg was selected as a superior composite due to the greater number and size of the produced organoids. Then, cell culture was rerun by HA-Alg for 14 days, which was later extended for an additional 28 days. In addition, the 3D culture of 10 dpp mouse testicular cells was used to compare with 5 dpp mice on day 14. The morphology and gene expression were analyzed using appropriate techniques. RESULTS: On day 14, the HA-Alg hydrogel showed significantly more organoids in terms of size and number than the other two groups (p < 0.05); nevertheless, none of the groups showed the expected signs of testis organoids. Remarkably, on day 14, the histology and immunostaining tests revealed features of hepatocyte-like cells (HLCs) and albumin production as a marker of HLC functionality. Furthermore, the analysis of gene expression verified the significant expression of angiogenesis markers (p < 0.01). After the extended culture to 28 days, 5 dpp testicular cells once more differentiated into erythrocytes and HLCs, while a small number of organoids showed the characteristic of renal cells. Cell culture of 10 dpp mice for 14 days showed a wide range of cell lineages, including renal, glandular, chondrocyte, and hepatocyte-like cells in comparison to the 5 dpp mice. CONCLUSION AND SIGNIFICANCE: While the HA-Alg composite did not support spermatogenesis in the 3D culture of mouse testicular cells, it demonstrated an unpredicted potential for promoting the differentiation of neonate mouse testicular cells into HLC, erythrocytes, and other cell lineages.

The anti-oxidant roles of Taurine and Hypotaurine on acrosome integrity, HBA and HSPA2 of the human sperm during vitrification and post warming in two different temperature.

Despite advances in vitrification techniques for sperm cryopreservation, cryo-damages of sperm caused by generation of reactive oxygen species (ROS) continue to impede implementation of this technique. This study analyses the effects of taurine and hypotaurine as anti-oxidants during vitrification of human sperms. The study was performed in two steps. In the first step, 20 normospermic semen samples were vitrified in the presence of varying concentrations of taurine and hypotaurine, and their effects as anti-oxidant agents on classical sperm parameters, hyaluronan-binding assay (HBA), lipid peroxidation (LPO) and acrosome reaction (AR) were studied. Statistical analyses showed that the sperm parameters in all vitrified groups decreased significantly (P < 0.05) compared to the fresh group. However, HBA and acrosome integrity in vitrified groups containing taurine and 50 mM of hypotaurine were better than in the control group (P < 0.05). The morphology of the vitrified group was good only in the group that contained 50 mM of hypotaurine (P < 0.05). Based on the results from the first step, 50 mM of hypotaurine was considered the ideal anti-oxidant formulation and further tests were carried out on 10 normospermic semen samples with this protecting agent. In addition to the mentioned parameters, the expression of heat shock proteinA2 (HSPA2) was studied in the vitrified group with 50 mM hypotaurine, warmed under two different warming temperatures 37 and 42 °C. 50 mM Hypotaurine was found to equally improve motility, morphology, HBA, and AR after warming at 37 °C and 42 °C (P < 0.05). However, at both warming temperatures, the expression of HSPA2 was reduced in all vitrified groups comparing to the fresh group (P < 0.05). In conclusion, taurine and hypotaurine antioxidants, especially 50 mM hypotaurine, are able to reduce deleterious cryo-injuries on morphology, acrosome and HBA and improve sperm recovery at both warming temperatures (37 and 42 °C). However, they do not have any protective action on expression of HSPA2.

Evaluation of conventional semen parameters, intracellular reactive oxygen species, DNA fragmentation and dysfunction of mitochondrial membrane potential after semen preparation techniques: a flow cytometric study.

PURPOSE: The aim of this study was to evaluate the conventional sperm parameters, level of intracellular reactive oxygen species (ROS), DNA fragmentation (DF) and dysfunction of mitochondrial membrane potential (MMP) after semen preparation techniques with flow cytometry. METHODS: Semen samples were obtained from 28 men with normal semen analysis according to WHO (world health organization). Each was divided into three equal parts for processing with routine techniques: conventional swim up (CSW), direct swim up (DSW) and density gradient centrifugation (DGC). The conventional sperm parameters were evaluated with computer-assisted sperm analyzer (CASA) and the level of intracellular ROS, dysfunction of MMP and DF were determined with flow cytometry procedure. RESULTS: Conventional sperm parameters such as motility, progressive motility and normal morphology increased after sperm processing by CSW and DGC compared to DSW. A significant increase in intracellular H2O2 (p < 0.05) was demonstrated in the CSW versus DSW technique, while processed sperm by the DSW procedure showed a significant increase in the percentage of dysfunction of MMP and intracellular O2(•-) (p < 0.05) when compared with CSW and DGC techniques. Additionally, a high mean of DF (p < 0.05) was observed in the DGC technique as compared to CSW. CONCLUSION: Data from flow cytometry study demonstrated that intracellular H2O2 and DF increased after CSW and DGC processing techniques, respectively, whereas the level of intracellular O2(•-) and dysfunction of MMP only increased after the DSW processing technique.

Epidemiology of sperm DNA fragmentation in a retrospective cohort of 1191 men.

BACKGROUND: The scientific and clinical communities now recognize that sperm DNA integrity is crucial for successful fertilization, good embryo development, and offspring quality of life. Despite the apparent unanimity, this criterion is rarely evaluated in clinical practice. We evaluated the sperm DNA fragmentation index of nearly 1200 sperm samples and its connections based on the patient's age, body mass index, the season of sperm collection, geographical location, medical history, and addictive behaviors. METHODS: A cohort of 1503 patients who were referred to the Royan Institute between July 2018 and March 2020 was examined. Only 1191 patient records with demographic data, complete semen analysis, and DNA fragmentation index measurements were included in the final cohort. Documents were classified, incorporated into statistical models, and analyzed. RESULTS: The results confirmed previous findings that the sperm DNA fragmentation index was significantly higher in aging men. The sperm DNA fragmentation index and high DNA stainability levels were significantly higher in spring and summer samples than in those of other seasons. No correlation was found between semen DNA fragmentation index and patient body mass index, although the study cohort was significantly overweight. Contrary to what might be expected, we observed that the sperm DNA fragmentation index was higher in rural than in urban patients. Intriguingly, epileptic patients exhibited significantly higher sperm DNA fragmentation index levels. DISCUSSION AND CONCLUSION: Age is the factor that is most strongly associated with sperm DNA fragmentation index levels. Our analysis of 1191 samples indicates that between the ages of 19 and 59, the sperm DNA fragmentation index increases by an average of 2% each year. Intriguingly, from an epidemiological perspective, the warm season (spring/summer) is associated with a higher sperm DNA fragmentation index in the study population, possibly due to the deleterious effect of temperature on sperm quality. Some neurological diseases, such as epilepsy, are associated with decreased sperm DNA integrity. This observation could be related to the iatrogenic effects of associated therapies. In the study cohort, body mass index did not appear to be correlated with the DNA fragmentation index.

The Relationship of Mitochondrial Membrane Potential, Reactive Oxygen Species, Adenosine Triphosphate Content, Sperm Plasma Membrane Integrity, and Kinematic Properties in Warmblood Stallions.

Equine sperm possesses a unique physiology because its energy supply is mostly dependent on oxidative phosphorylation of mitochondria as an aerobic source of adenosine triphosphate (ATP) generation. The present study was, therefore, conducted to investigate the relationship between sperm kinematic and functional variables in stallions. Semen samples were collected from five warmblood stallions (three ejaculates from each stallion), diluted with INRA96 and transferred to the laboratory. Next, sperm motility, mitochondrial membrane potential (MMP), production of superoxide anion (as a reactive oxygen species; ROS), ATP content, and plasma membrane integrity were assessed. Motion and functional characteristics differed among investigated stallions (P < .05). In addition, it was revealed MMP was positively correlated with the level of ROS and ATP content and progressive motility (P < .05). The level of ROS was positively correlated with ATP content and negatively correlated with plasma membrane integrity and straightness (P < .05). Adenosine triphosphate content was positively correlated with progressive motility, curvilinear velocity, average path velocity, and beat cross frequency and reversely correlated with plasma membrane integrity and straightness (P < .05). Plasma membrane integrity was positively correlated with straight line velocity, linearity, and straightness and negatively correlated with curvilinear velocity (P < .01). In conclusion, the present study substantiated that kinematic and functional characteristics varied among various warmblood stallions. Furthermore, the present study implicated although higher mitochondrial activity increases ATP synthesis, it leads to elevated superoxide anion production, which could culminate in disintegration of the sperm plasma membrane, thereby altering motion characteristics and swimming pattern of sperm.

The Relationship between Seminal Melatonin with Sperm Parameters, DNA Fragmentation and Nuclear Maturity in Intra-Cytoplasmic Sperm Injection Candidates.

OBJECTIVE: Melatonin, the chief secretory product of the pineal gland, regulates dynamic physiological adaptations that occur in seasonally breeding mammals as a response to changes in daylight hours. Because of the presence of melatonin in semen and the mem- brane melatonin receptor in spermatozoa, the impact of melatonin on the regulation of male infertility is still questionable. The aim of this study was to determine the effects of endogenous melatonin on human semen parameters (sperm concentration, motility and normal morphology), DNA fragmentation (DF) and nuclear maturity. MATERIALS AND METHODS: In this clinical prospective study, semen samples from 75 infer- tile men were routinely analyzed and assessed for melatonin and total antioxidant capac- ity (TAC) levels using the enzyme-linked immunosorbent assay (ELISA) and colorimetric assay kits, respectively. DF was examined by the sperm chromatin dispersion (SCD) test. Acidic aniline blue staining was used to detect chromatin defects in the sperm nuclei. RESULTS: There was no significant correlation between seminal plasma melatonin and TAC with sperm parameters and nuclear maturity. However, we observed a positive significant correlation between DF and melatonin level (r=0.273, P<0.05). CONCLUSION: Melatonin in seminal plasma is positively correlated with damaged sperm DNA of infertile patients. The mechanism of this phenomenon needs further study.