Resveratrol ameliorates spermatogenesis by increasing protamine 1, 2 and HSPA2 expression in experimental varicocele rat model / El resveratrol mejora la espermatogénesis al aumentar la expresión de protamina 1, 2 y HSPA2 en un modelo experimental de ratas con varicocele

Objectives: Varicocele is a common cause of male infertility associated with an elevated testicular temperature that induces apoptosis, spermatogenesis dysfunction, and affects sperm parameters. In this study, we investigate the probable therapeutic effects of resveratrol (RES), a natural phytoalexin, against varicocele. Materials and methods: In this study, 48 male Wistar rats randomly divided into 8 groups: normal, sham, normal+RES (20 and 50mg/kg), varicocele, varicocele+ethanol and varicocele+RES (20 and 50mg/kg). Incomplete closure of the left renal vein was used for varicocele induction and two months later, RES was administrated orally for 60 days. Then, sperm parameters, DNA fragmentations, chromatin density, and testis histopathology were analyzed. In addition, HSPA2, protamine 1, and 2 expression levels were evaluated using real-time PCR. Results: According to our results, resveratrol treatment improved sperm parameters, testis histopathology, DNA fragmentation, and chromatin maturation which damaged follow varicocele (p≤0.05). Also, it increased HSPA2, protamine 1, and 2 expression levels significantly in both doses (p≤0.05). Conclusion: Resveratrol potentially attenuates varicocele-induced spermatogenic impairments by its antioxidant features and regulates spermatogenic gene expression undergoing DNA fragmentation, so leads histopathological properties of tissues to physiological parameters. (AU)

Objetivos: El varicocele es una causa común de infertilidad masculina asociada con una temperatura testicular elevada que induce apoptosis, disfunción de la espermatogénesis y afecta los parámetros espermáticos. En este estudio, investigamos los probables efectos terapéuticos del resveratrol (RES), una fitoalexina natural, contra el varicocele. Materiales y métodos: En este estudio, 48 ratas Wistar macho se dividieron aleatoriamente en 8 grupos: normal, simulado, normal+ RES (20 y 50mg/kg), varicocele, varicocele+ etanol y varicocele+ RES (20 y 50mg/kg). Se utilizó el cierre incompleto de la vena renal izquierda para la inducción del varicocele y dos meses después se administró RES por vía oral durante 60 días. Luego se analizaron los parámetros espermáticos, las fragmentaciones de ADN, la densidad de cromatina y la histopatología testicular. Además, los niveles de expresión de HSPA2, protamina 1 y 2 se evaluaron mediante PCR en tiempo real. Resultados: Según nuestros resultados, el tratamiento con resveratrol mejoró los parámetros espermáticos, la histopatología testicular, la fragmentación del ADN y la maduración de la cromatina que dañó el varicocele posterior (p≤0,05). Además, aumentó significativamente los niveles de expresión de HSPA2, protamina 1 y 2 en ambas dosis (p≤0,05). Conclusión: El resveratrol atenúa potencialmente las deficiencias espermatogénicas inducidas por varicocele por sus características antioxidantes y regula la expresión de genes espermatogénicos que sufren fragmentación del ADN, por lo que conduce las propiedades histopatológicas de los tejidos a parámetros fisiológicos. (AU)

Resveratrol ameliorates spermatogenesis by increasing protamine 1, 2 and HSPA2 expression in experimental varicocele rat model.

OBJECTIVES: Varicocele is a common cause of male infertility associated with an elevated testicular temperature that induces apoptosis, spermatogenesis dysfunction, and affects sperm parameters. In this study, we investigate the probable therapeutic effects of resveratrol (RES), a natural phytoalexin, against varicocele. MATERIALS AND METHODS: In this study, 48 male Wistar rats randomly divided into 8 groups: normal, sham, normal+RES (20 and 50mg/kg), varicocele, varicocele+ethanol and varicocele+RES (20 and 50mg/kg). Incomplete closure of the left renal vein was used for varicocele induction and two months later, RES was administrated orally for 60 days. Then, sperm parameters, DNA fragmentations, chromatin density, and testis histopathology were analyzed. In addition, HSPA2, protamine 1, and 2 expression levels were evaluated using real-time PCR. RESULTS: According to our results, resveratrol treatment improved sperm parameters, testis histopathology, DNA fragmentation, and chromatin maturation which damaged follow varicocele (p≤0.05). Also, it increased HSPA2, protamine 1, and 2 expression levels significantly in both doses (p≤0.05). CONCLUSION: Resveratrol potentially attenuates varicocele-induced spermatogenic impairments by its antioxidant features and regulates spermatogenic gene expression undergoing DNA fragmentation, so leads histopathological properties of tissues to physiological parameters.

Possible Harmful Effects of Smoking Hookah on Sperm DNA Fragmentation Index and Protamine Genes Expression in Normozoospermic Men.

Objectives: In recent years, smoking water pipes or hookah has increased among adolescents in most countries. Although there is evidence in support of the negative effects of this type of smoking on human health, such as the increased risk of lung disease, little is known about the potential effects of hookah smoking on the male reproductive system, especially on the molecular aspects of sperm. Patients and methods: This cross-sectional study examined sperm DNA fragmentation index, protamine 1 and 2 (PRM1 and PRM2) genes expression, and oxidant status in normozoospermic hookah smokers in comparison with non-smoker controls. Results: Our results showed significantly higher rates of DNA fragmentation, protamine deficiency, and abnormal chromatin condensation in the spermatozoa of hookah smokers (P < .0001). Also, protamine gene expression showed a remarkable decrease in hookah smokers (1.55 ± 2.54 and 0.33 ± 0.54) compared to the controls (3.49 ± 5.41 and 1.22 ± 1.96), although the reduction was not statistically significant (P = .155 and P = .066, respectively). Moreover, a significantly higher level of semen MDA was observed in the case group compared to the controls (0.39 ± 1.04 vs 0.15 ± 0.21; P = .013). Conclusion: According to our study, although hookah smoking does not have a significant effect on sperm parameters, it may have deleterious effects on DNA integrity, oxidative status, and nuclear protein levels of spermatozoa.

Direct visualization of pre-protamine 2 detects protamine assembly failures and predicts ICSI success.

Histone-to-protamine transition is an essential step in the generation of fully functional spermatozoa in various mammalian species. In human and mouse, one of the two protamine-encoding genes produces a precursor pre-protamine 2 (pre-PRM2) protein, which is then processed and assembled. Here, we design an original approach based on the generation of pre-PRM2-specific antibodies to visualize the unprocessed pre-PRM2 by microscopy, flow cytometry and immunoblotting. Using mouse models with characterized failures in histone-to-protamine replacement, we show that pre-PRM2 retention is tightly linked to impaired nucleosome disassembly. Additionally, in elongating/condensing spermatids, we observe that pre-PRM2 and transition protein are co-expressed spatiotemporally, and their physical interaction suggests that these proteins act simultaneously rather than successively during histone replacement. By using our anti-human pre-PRM2 antibody, we also measured pre-PRM2 retention rates in the spermatozoa from 49 men of a series of infertile couples undergoing ICSI, which shed new light on the debated relation between pre-PRM2 retention and sperm parameters. Finally, by monitoring 2-pronuclei embryo formation following ICSI, we evaluated the fertilization ability of the sperm in these 49 patients. Our results suggest that the extent of pre-PRM2 retention in sperm, rather than pre-PRM2 accumulation per se, is associated with fertilization failure. Hence, anti-pre-PRM2 antibodies are valuable tools that could be used in routine monitoring of sperm parameters in fertility clinics, as well as in experimental research programmes to better understand the obscure process of histone-to-protamine transition.

An improved acetic acid-urea polyacrylamide electrophoresis method to evaluate bovine sperm protamines.

The acetic acid-urea polyacrylamide gel electrophoresis system could separate very similar basic proteins on differences in size and effective charge. This system has been used for many years to analyse histones and their post-translational modifications and widely used in the study of mammal protamines. Two types of protamine have been described, the protamine 1 (P1) and the protamine 2 (P2) family members, which are synthetized by PRM1 and PRM2 genes. The ratio of P1 and P2 is important for predicting fertility in humans and mice. Therefore, the quantification of protamines is a fundamental step in order to establish the ratio between P1 and P2 in these species. In other mammals, studies linking sperm protamination and the protamine ratio with fertility are increasing. So, the use of an effective technique to separate and quantify protamines is important to study sperm P1/P2 ratio. Therefore, this article describes in detail a feasible and useful procedure to isolate bovine sperm protamines, to perform pre-electrophoresis with PEG solution and finally to carry out acid-urea polyacrylamide gel electrophoresis in reverse polarity. This technique allows a clear separation and efficient detection of bovine sperm protamines.

Protamine 1/Protamine 2 mRNA ratio in nonobstructive azoospermic patients.

Altered protamine 1 (PRM1)/ protamine 2 (PRM2) mRNA ratio in testicular biopsy samples correlates with sperm quality and its fertilising ability. This study is planned to assess PRM1/ PRM2 mRNA ratio in subgroups of azoospermia to suggest a more reliable and accurate marker for assessing sperm quality in nonobstructive azoospermia (NOA). A cross-sectional study was done on testicular biopsy samples, taken from 106 azoospermic patients. Samples were histologically classified into subgroups: 36 obstructive azoospermia (OA), and two groups of NOA: 41 round spermatid maturation arrest (SMA) and 29 Sertoli cell-only syndrome (SCOS). OA samples showed histologically normal spermatogenesis and serve as a positive control. mRNA expression of jumonji domain-containing 1A (JMJD1A), PRM1, PRM2 and transition nuclear proteins (TNP1, TNP2) genes was determined, by RT-qPCR. Significantly lower expression of JMJD1A (p < .001), PRM1 (p = .0265) and PRM2 (p = .0032) has been seen in the SCOS group of NOA. We found significant (p < .001) increase in PRM1/PRM2 mRNA ratio in testicular biopsy samples of SCOS group of NOA patients and significant negative correlation of PRM1/PRM2 mRNA ratio with JMJD1A. Hence, PRM1/PRM2 mRNA ratio may represent a more reliable and accurate marker to assess sperm quality in NOA in addition to standard semen parameters.

Detection of protamine 2 in bovine spermatozoa and testicles.

BACKGROUND: Sperm DNA integrity is crucial for transmission of genetic information to future generations and DNA damage can occur during chromatin packaging. Chromatin packaging involves the replacement of somatic nucleosomal histones by nuclear proteins called protamines. Protamine 1 (PRM1) is transcribed and translated in spermatids of all mammals; however, protamine 2 (PRM2) is transcribed in low levels in spermatids and it is not yet described in bull mature spermatozoa. OBJECTIVES: The aim of this study was to assess gene and protein expression of PRM2 and corroborate gene and protein expression of PRM1 in bull spermatozoa and testis. MATERIALS AND METHODS: For this purpose, absolute q-RT-PCR was performed to calculate the number of copies of PRM1 and PRM2 mRNAs in bovine epididymal spermatozoa and testicular tissue. Western blot and mass spectrometry were performed to identify PRM1 and PRM2 in samples of bovine epididymal spermatozoa. Samples of bovine testicular tissue were collected to identify PRM1 and PRM2 by immunohistochemistry. RESULTS: We evaluated that the number of PRM1 mRNA copies was about hundred times higher than PRM2 mRNA copies in sperm and testicular samples (p < 0.0001). In addition, we estimated the PRM1: PRM2 ratio based on mRNA number of copies. In spermatozoa, the ratio was 1: 0.014, and in testicle, the ratio was 1: 0.009. We also evaluated the immunolocalization for PRM1 and PRM2 in bovine testis, and both proteins were detected in spermatids. Western blot and mass spectrometry in bovine epididymal spermatozoa confirmed these results. CONCLUSION: Our work identifies, for the first time, PRM2 in bovine epididymal spermatozoa and in testis. Further studies are still needed to understand the role of PRM2 on the chromatin of the spermatozoa and to verify how possible changes in PRM2 levels may influence the bull fertility.

Detection of protamine 2 in bovine spermatozoa and testicles

Background Sperm DNA integrity is crucial for transmission of genetic information to future generations and DNA damage can occur during chromatin packaging. Chromatin packaging involves the replacement of somatic nucleosomal histones by nuclear proteins called protamines. Protamine 1 (PRM1) is transcribed and translated in spermatids of all mammals; however, protamine 2 (PRM2) is transcribed in low levels in spermatids and it is not yet described in bull mature spermatozoa. Objectives The aim of this study was to assess gene and protein expression of PRM2 and corroborate gene and protein expression of PRM1 in bull spermatozoa and testis. Materials and methods For this purpose, absolute q-RT-PCR was performed to calculate the number of copies of PRM1 and PRM2 mRNAs in bovine epididymal spermatozoa and testicular tissue. Western blot and mass spectrometry were performed to identify PRM1 and PRM2 in samples of bovine epididymal spermatozoa. Samples of bovine testicular tissue were collected to identify PRM1 and PRM2 by immunohistochemistry. Results We evaluated that the number of PRM1 mRNA copies was about hundred times higher than PRM2 mRNA copies in sperm and testicular samples (p < 0.0001). In addition, we estimated the PRM1: PRM2 ratio based on mRNA number of copies. In spermatozoa, the ratio was 1: 0.014, and in testicle, the ratio was 1: 0.009. We also evaluated the immunolocalization for PRM1 and PRM2 in bovine testis, and both proteins were detected in spermatids. Western blot and mass spectrometry in bovine epididymal spermatozoa confirmed these results. Conclusion Our work identifies, for the first time, PRM2 in bovine epididymal spermatozoa and in testis. Further studies are still needed to understand the role of PRM2 on the chromatin of the spermatozoa and to verify how possible changes in PRM2 levels may influence the bull fertility.

Association study of PRM2 gene polymorphism with male infertility in the 386 Chinese Han population / 重庆医学

Objective To determine the association of one single nucleotide polymorphism loci G 398C in PRM2 with male in‐fertility in Chinese Han population .Methods A total of 386 infertile men were recruited as the observation group and 255 fertile men were recruited as the control group .Routine semen analysis as well as sperm functional parameters such as DNA integrity and nucleoprotein maturity rates were analyzed .Direct sequencing of G398C in PRM2 gene of infertile and fertile men was also conduc‐ted to evaluate the association of G398C SNP loci with male infertility .Results Statistical analysis showed that the frequencies of CC genotype of PRM2 G398C was significantly different between the infertile (11 .92% ) and fertile men (6 .67% ) and it was asso‐ciated with increased risk of male infertility (OR= 2 .002 ,95% CI= 1 .097 -3 .653 ,P< 0 .05) .Moreover ,it was discovered that sperm DNA integrity as well as nucleoprotein maturity rate of CC genotype were dramatically decreased compared with other geno ‐types (P<0 .05) ,which would probably lead to infertility .Conclusion Our results gave the first evidence that PRM 2 G398C poly‐morphism was associated with male infertility in Chinese Han population .