Molecular and Functional Characterization of Human Sex-Determining Region on the Y Chromosome Variants Using Protamine 1 Promoter.

The male sex-determining gene, sex-determining region on the Y chromosome (SRY), is expressed in adult testicular germ cells; however, its role in regulating spermatogenesis remains unclear. The role of SRY in the postmeiotic gene expression was investigated by determining the effect of SRY on the promoter of the haploid-specific Protamine 1 (PRM1) gene, which harbors five distinct SRY-binding motifs. In a luciferase reporter assay system, SRY upregulates PRM1 promoter activity in vitro in a dose-dependent manner. Through a gel-shift assay involving a 31-bp DNA fragment encompassing the SRY element within the PRM1 promoter, the third SRY-binding site on the sense strand (-373/-367) was identified as crucial for PRM1 promoter activation. This assay was extended to analyze 9 SRY variants found in the testicular DNA of 44 azoospermia patients. The findings suggest that SRY regulates PRM1 promoter activity by directly binding to its specific motif within the PRM1 promoter.

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)

Identification of heat shock protein70-2 and protamine-1 mRNA, proteins, and analyses of their association with fertility using frozen-thawed sperm in Madura bulls.

OBJECTIVE: This study aims to identify heat shock protein70-2 (HSP70-2) and protamine-1 (PRM1) mRNA and protein in Madura bull sperm and demonstrate their relation as bull fertility biomarkers. METHODS: The Madura bull fertility rates were grouped based on the percentage of first service conception rate (%FSCR) as high fertility (HF) (79.04%; n = 4), and low fertility (LF) (65.84%; n = 4). mRNA of HSP70-2 and PRM1 with peptidylprolyl isomerase A (PPIA) as a housekeeping gene were determined by quantitative real-time polymerase chain reaction, while enzyme-linked immunoassay was used to measure protein abundance. In the post-thawed semen samples, sperm motility, viability, acrosome integrity, and sperm DNA fragmentation index were analyzed. Data analysis was performed on the measured parameters of semen quality, relative mRNA expression, and protein abundance of HSP70-2 and PRM1, among the bulls with various fertility levels (HF and LF) in a one-way analysis of variance analysis. The Pearson correlation was used to analyze the relationship between semen quality, mRNA, proteins, and fertility rate. RESULTS: Relative mRNA expression and protein abundance of HSP70-2 and PRM1 were detected and were found to be highly expressed in bulls with HF (p<0.05) and were associated with several parameters of semen quality. CONCLUSION: HSP70-2 and PRM1 mRNA and protein molecules have great potential to serve as molecular markers for determining bull fertility.

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.

Sperm mRNA screening by reverse transcription-recombinase polymerase amplification (RT-RPA) assay: Decision-making for differential extraction.

The presence of sperm cells is an indicator for differential extraction on sexual assault samples. In general, sperm cells are identified by microscopic analysis; however, this conventional method takes time and effort, even for trained personnel. Here, we present a reverse transcription-recombinase polymerase amplification (RT-RPA) assay targeting sperm mRNA marker (PRM1). The RT-RPA assay requires only 40 min for PRM1 detection and demonstrates a sensitivity of 0.1 µL of semen. Our results indicate that the RT-RPA assay may be a rapid, simple, and specific strategy for screening sperm cells in sexual assault samples.

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.

Loss of Prm1 leads to defective chromatin protamination, impaired PRM2 processing, reduced sperm motility and subfertility in male mice.

One of the key events during spermiogenesis is the hypercondensation of chromatin by substitution of the majority of histones by protamines. In humans and mice, protamine 1 (PRM1/Prm1) and protamine 2 (PRM2/Prm2) are expressed in a species-specific ratio. Using CRISPR-Cas9-mediated gene editing, we generated Prm1-deficient mice and demonstrated that Prm1+/- mice were subfertile, whereas Prm1-/- mice were infertile. Prm1-/- and Prm2-/- sperm showed high levels of reactive oxygen species-mediated DNA damage and increased histone retention. In contrast, Prm1+/- sperm displayed only moderate DNA damage. The majority of Prm1+/- sperm were CMA3 positive, indicating protamine-deficient chromatin, although this was not the result of increased histone retention in Prm1+/- sperm. However, sperm from Prm1+/- and Prm1-/- mice contained high levels of incompletely processed PRM2. Furthermore, the PRM1:PRM2 ratio was skewed from 1:2 in wild type to 1:5 in Prm1+/- animals. Our results reveal that PRM1 is required for proper PRM2 processing to produce mature PRM2, which, together with PRM1, is able to hypercondense DNA. Thus, the species-specific PRM1:PRM2 ratio has to be precisely controlled in order to retain full fertility.

PRM1 Gene Expression and Its Protein Abundance in Frozen-Thawed Spermatozoa as Potential Fertility Markers in Breeding Bulls.

Functional genes and proteins in sperm play an essential role in bulls' reproductive processes. They are more accurate in determining bull fertility than conventional semen quality tests. Protamine-1 (PRM1) is a gene or protein crucial for packaging and protecting sperm DNA until fertilization affects normal sperm function. This study analyzes the genes and proteins potential from PRM1 as fertility markers for different breeds of bulls utilized in the artificial insemination programs, expected to be an accurate tool in interpreting bull fertility in Indonesia. This study used Limousin, Holstein, and Ongole Grade bulls divided into two groups based on fertility, high-fertility (HF) and low fertility (LF). The semen quality assessment included progressive motility (computer-assisted semen analysis), viability (eosin-nigrosine), and plasma membrane integrity (HOS test). Sperm DNA fragmentation (SDF) was assessed using the acridine orange staining and the Halomax test. Sperm PRM deficiency was evaluated with the chromomycin A3 method. Moreover, PRM1 gene expression was measured using qRT-PCR, and the PRM1 protein abundance was measured with the enzyme immunoassay method. Semen quality values, relative expression of PRM1 gene, and quantity of PRM1 protein were significantly higher (p < 0.05) in HF bulls than in LF bulls. The SDF and PRM deficiency values in LF bulls were significantly higher (p < 0.05) than HF bulls. Additionally, PRM1 at the gene and protein levels correlated significantly (p < 0.01) with fertility. Therefore, PRM1 is a potential candidate for fertility markers in bulls in Indonesia.

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.

The expression, function, and utilization of Protamine1: a literature review.

OBJECTIVE: Protamine 1 (PRM1) is specific in sperm and plays essential roles in fertilization, also a member of cancer testis antigen (CTA) family. This study aims to summarize the expression and function of PRM1 in spermatogenesis, and to broaden the current knowledge and inspire future development of PRM1-based therapeutic strategies in cancer treatment and nanomedicine. BACKGROUND: The protamine proteins, are characterized by an arginine-rich core and cysteine residues. Humans express two types of protamine: PRM1 and PRM2. The abnormal expression or proportion of PRM1 and PRM2 is known to be associated with subfertility and infertility, especially for PRM1 which is highly evolutionary conserved in mammalians and expressed in all vertebrates. Biological functions of PRM1 have been unveiled in diverse cellular processes, such as tumorigenesis, somatic cell nucleus transfer, and drug delivery systems. Moreover, PRM1 is identified as a CTA in chronic leukemia (CLL) and colorectal cancer (CRC). METHODS: Literature was obtained using PubMed and the keywords protamine 1, PRM1, or P1, from January 1, 1980, through July 20, 2021. We also collect the additional evidence through screening references of articles identified through the PubMed searches. CONCLUSIONS: PRM1 is well-studied in male infertility, and further researches and attempts to develop PRM1 as novel tumor marker, as well as drug delivery vector, will be of important clinical significance.

Freezing-thawing induces deprotamination, cryocapacitation-associated changes; DNA fragmentation; and reduced progesterone sensitivity in buck spermatozoa.

In the present study, there was evaluation of cryocapacitation-associated changes, apoptotic-like changes, deprotamination, total antioxidant capacity (TAC), and in vitro sperm functional attributes in Barbari bucks after freezing-thawing. The correlation between deprotamination and sperm functional characteristics was established. Using immunoblotting procedures, there was detection of the presence of a single 28-kDa protein band corresponding to protamine-1. The localization in the head region of the spermatozoa was further validated by an immunofluorescence test. Capacitated (B-) and acrosome-reacted (AR-) pattern spermatozoa, spermatozoa with the externalization of phosphatidylserine and a relatively lesser mitochondrial transmembrane potential, and deprotamination and DNA fragmentation was greater (P < 0.05) after freezing-thawing and indicated there were cryocapacitation- and apoptotic-like changes, respectively. Furthermore, the detection of phosphorylation of tyrosine-containing proteins with use of immunoblotting and immunofluorescence procedures confirmed there were cryocapacitation-like changes in the buck spermatozoa after freezing-thawing. Total antioxidant capacity (TAC), in vitro thermal resistance response, Vanguard distance, progesterone sensitivity, and in vitro capacitation response were less (P < 0.05) in the spermatozoa after freezing-thawing compared with spermatozoa after initial dilution and equilibration. Deprotamination (chromomycin A3-positive cells, CMA3+) and DNA fragmentation (TUNEL+ve) were positively correlated with B- and AR-pattern spermatozoa, while other values for other variables were negatively correlated. In conclusion, the results of this study indicated there was protamine-1 in buck spermatozoa and after freezing-thawing there was a loss of protamine-1 combined with cryocapacitation-associated changes and apoptotic-like changes in buck spermatozoa. Spermatozoa deprotamination might be attributed to increased DNA fragmentation, resulting in compromised fertilizing capacity of buck spermatozoa.

The concentration of testosterone, pituitary adenylate cyclase-activating polypeptide, and protamine 1 in the serum of male chicken following administration of epididymis and testicular extracts and their combination.

BAKCGROUND AND AIM: Testis and epididymis are male reproductive organs that play an important role in spermatogenesis. These two organs are rich in the content of hormones and other molecules needed in the process of spermatogenesis which affect the quality of the spermatozoa. The objective of this study was to examine the effect of the administration of epididymis and testicular extracts and their combination on testosterone, pituitary adenylate cyclase-activating polypeptide (PACAP), and protamine 1 (PRM1) concentrations in the serum of male chicken. MATERIALS AND METHODS: Twenty male chickens (broiler strain Cp707), aged 3 weeks and weighing 800-1000 g, were randomly divided into four different groups including a control group (T0) = injected with 1 ml normal saline and treatment groups: T1 = injected with 1 ml epididymis extract, T2 = injected with 1 ml testicular extract, and T3 = injected with a combination of 1 ml epididymis + 1 ml testicular extract. The experiment was conducted for 13 days and at the end of the study (day 14), the chickens were sacrificed to obtain the serum. Furthermore, the concentrations of testosterone, PACAP, and PRM1 were then measured by using an enzyme-linked immunosorbent assay technique. RESULTS: The concentrations of PACAP and PRM1 did not show a significant difference between treatment groups (T1, T2, and T3) and control group (T0) (p>0.05). However, the concentration of testosterone showed a significantly higher difference in a group injected with a combination of 1 ml epididymis and 1 ml testicular extracts (T3) compared to the control group (T0) (p<0.05). CONCLUSION: The administration of epididymis and testicular extracts and their combination did not affect the increase of PACAP and PRM1 concentration. However, a combination of these extracts significantly affects the increase of testosterone concentration in the serum of male chicken.

Screening of the Single Nucleotide Polymorphisms in the Protamine 1 and 2 Genes of Korean Infertile Men / 대한불임학회지

OBJECTIVE: Although several genetic factors have been associated with defects in human spermatogenesis, the unambiguous causative genes have not been elucidated. The male infertility by haploinsufficiency of PRM1 or PRM2 has been reported in mouse model. The aim of this study was to identify the single nucleotide polymorphisms (SNPs) of PRM1 and PRM2, related to the genotype of Korean infertile men. METHODS: Genomic DNAs were extracted from peripheral bloods of infertile men with oligozoospermia or azoospermia, and analyzed using polymerase chain reaction (PCR) and direct sequencing. We carried out the direct sequencing analysis of amplified fragments in PRM1 (557 nucleotides from -42 to 515) and PRM2 (599 nucleotides from 49 to 648) genes, respectively. RESULTS: Three SNPs of coding region in the PRM1 gene was found in the analysis of 130 infertile men. However, the SNPs at a133g (aa 96.9%, ag 3.1% and gg 0.0%), c160a (cc 99.2%, ca 0.8% and aa 0.0%) and c321a (cc 56.9%, ca 35.4% and aa 7.7%) coded the same amino acids, in terms of silence phenotypes. On the other hand, as results of the PRM2 gene sequencing in 164 infertile men, only two SNPs, g398c (gg 62.2%, gc 31.1% and ga 6.7%) and a473c (aa 63.4%, ac 29.9% and cc 6.7%), were identified in the intron of the PRM2 gene. CONCLUSIONS: There was no mutation and significant SNPs on PRM1 and PRM2 gene in Korean infertile men. These results suggest that the PRM1 and PRM2 genes are highly conserved and essential for normal fertility of men.