Protamine 1 Research Articles

MicroRNA-1307-3p contributes to breast cancer progression through PRM2.

Despite advances in screening and therapy, breast cancer (BC) remains the predominant cancer in women globally. Dysregulation of microRNAs (miRNAs) is pivotal in carcinogenesis across various cancers, including BC. Evidence indicates that miR-1307-3p is upregulated in BC tumors, yet its target genes are not fully elucidated. This study aimed to explore how miR-1307-3p regulates BC proliferation, migration, invasion, and angiogenesis and to identify potential target genes. Basal miR-1307-3p levels were quantified in BC cell lines MDA-MB-231 and MCF-7, as well as MCF-10A using quantitative real-time reverse transcription-PCR (RT-qPCR). The impact of miR-1307-3p inhibition on BC cell proliferation, migration, invasion, and angiogenesis was assessed. Nine miRNA-target prediction databases identified potential miR-1307-3p targets. Target expression was validated using RT-qPCR, Western blot, and dual-luciferase reporter assays. MiR-1307-3p was overexpressed in MDA-MB-231 and MCF-7 compared to MCF-10A. Inhibiting miR-1307-3p significantly reduced BC cell proliferation, migration, invasion, and angiogenesis. Bioinformatics analysis identified 17 potential miR-1307-3p targets, with protamine 2 (PRM2) overexpression confirmed via Western blot and dual-luciferase assays. MiR-1307-3p overexpression in BC promotes proliferation, migration, invasion, and angiogenesis. PRM2 emerges as a novel miR-1307-3p target in BC.

Adverse Effects of Nicotine on Human Sperm Nuclear Proteins.

The effects of smoking on human health have long been documented. However, only a few studies have highlighted the direct effects of nicotine on sperm function. Nicotine, as a chemical compound found in tobacco, has been shown to modulate different aspects of spermatogenesis and sperm functions. Nicotine can lead to a reduction in the number of sperm, their motility and functionality. It can change the molecular expressions involved in sperm function, including genes encoding sperm nuclear proteins. The most important nuclear proteins that play a critical role in sperm function are known as H2B histone family, member W, testis-specific (H2BFWT), transition protein 1 (TNP1), transition protein 2 (TNP2), protamine-1 (PRM1), and protamine-2 (PRM2). These proteins are involved in sperm chromatin condensation, which in turn affects fertilization and embryonic development. Any alteration in the expression of these genes due to nicotine exposure/usage may lead to adverse implications in couples' fertility and the health of future generations. Since research in this area is still relatively new, it underscores the importance of understanding the potential side effects of environmental factors such as nicotine on reproductive health.

Study of the impact of organic minerals on spermatozoal gene expression in Osmanabadi bucks

Sperm transcripts aside from providing genetic material have an eminent role in post-fertilization events and embryonic development. The current study was carried out to study the gene expression in Osmanabadi buck (Capra hircus) supplemented with organic minerals. Organic minerals alone or in combination with different concentrations were fed to the treatment groups (T2- Zn 20 mg, T3- Zn 40 mg, T4- Zn 60 mg, T5- Cu 12.5 mg, T6- Cu 25 mg, T7- Cu 37.5 mg, T8- Zn 20 mg + Cu 12.5 mg, T9- Zn 40 mg + Cu 25 mg, T10- Zn 60 mg + Cu 37.5 mg) along with the concentration mixture and roughages; whereas control (T1) group was fed concentrate mixture and roughages. The sperm samples were processed for gene expression studies of Ras Homolog Family Member A (RHOA), Mesoderm Specific Transcript (MEST), Nucleoside diphosphate-linked moiety X motif 6 (NUDT6), Protamine 1 (PRM1), Protamine 2 (PRM2) and Heat shock protein 90 alpha family class A member 1 (HSP90AA) using qRT-PCR and the protein-protein interaction analysis was carried out through String database version 11.0. Supplementation of trace minerals has enhanced the gene expression at a significant level (P<0.05) in the supplemented groups. The expression of MEST, RHOA, and PRM1 was higher in Cu 25-supplemented groups; whereas, NUDT6 in Zn 20, PRM2 and HSP90AA in Zn 40 showed higher expressions. Osmanabadi bucks supplemented with Cu 25 mg have shown promising results in the present and earlier studies so it can be concluded that the field-level application of Cu 25 mg would lead to promising results.

Effect of Testosterone and Antioxidant-Loaded Nanoliposomes on the Performance of Normozoospermic Samples: A Case-Control Study

Introduction: Antioxidants reduce the harmful effects of reactive oxygen species (ROS) in sperm. Additionally, nanoliposomes can be used as an efficient new drug delivery system that mitigates the adverse effects of drugs. This study aimed to investigate the use of nanoliposomes for delivering drugs such as resveratrol, catalase, resveratrol-catalase, and testosterone to normozoospermic samples to minimize the side effects of ROS. Methods: This study took place at Yazd Reproductive Sciences Institute in Iran from October 2018 to December 2019. It involved 450 samples divided into three groups. Each of the 15 normozoospermic sperm samples collected from men was divided into 10 parts and analyzed in three groups: free drugs, nanoliposomes, and a control group. The study analyzed sperm parameters, PRM2 (Protamine 2) and HSP70 (heat shock proteins) expression, as well as sperm acrosome reaction and DNA fragmentation before and after freezing. Results: Nanoliposomes improved sperm motility and viability before freezing (P<0.05). Free resveratrol-catalase reduced motility before freezing (P<0.05). PRM2 and HSP70 expression were lower in the blank and testosterone nanoliposome groups (P<0.0001). After freezing, nanocarriers and free testosterone improved sperm motility (P<0.05). Resveratrol and testosterone-loaded nanoliposomes increased sperm viability (P<0.01). Nanoliposomes caused less sperm DNA damage (P>0.05). Free catalase reduced PRM2 and HSP70 expression (P<0.0001). Conclusion: Nanoliposomes can be used as an efficient drug delivery system to decrease ROS production or their side effects. Additionally, during freezing, they improve sperm viability and reduce DNA damage.

CircANXA4 (hsa_circ_0055087) regulates the miR-1256/PRM1 axis to promote tumor progression in colorectal cancer

Colorectal cancer (CRC) incidence ranks third among malignant cancers with a high propensity for distant metastasis. Despite continuous efforts to improve treatment, the prognosis especially in patients with advanced distant metastasis is low. The mechanism of development and progression of CRC is not fully understood. Non-coding RNAs (ncRNAs) have emerged as essential regulators in cancer progression. Here, we aim to dissect the role of one critical ncRNA, circANXA4, in CRC progression. CircANXA4 expression was analyzed by the GEO database. Differentially expressed circRNAs were identified by the Limma package R software. Expression of circANXA4 and miR-1256 was detected by qRT-PCR. The regulation of circANXA4 on cell proliferation and progression was confirmed with the cell viability assay using cell counting kit-8 (CCK-8) and transwell migration assay. RNA pull-down assay, RNA immunoprecipitation (RIP), and western blot were used to determine the interaction between circANXA4, miR-1256, and protamine1 (PRM1). CircANXA4 was upregulated in both CRC tissues and cell lines. Knockdown of circANXA4 effectively reduced cell proliferation, progression, and migration. Additionally, silencing circANXA4 remarkably increased miR-1256 expression, while reducing PRM1 expression, thereby demonstrating that circANXA4 downregulates miR-1256 expression through a complementary binding site. Rescue experiments revealed the interactions between circANXA4, miR-1256, and PRM1. Pearson correlation analysis revealed that circANXA4 expression positively correlated with PRM1 expression and miR-1256 expression inversely correlated with PRM1 expression. In sum, we demonstrated that circANXA4 promotes cancer cell proliferation and progression by sponging miR-1256 and upregulating PRM1 in CRC.

A novel sorting method for the enrichment of early human spermatocytes from clinical biopsies

To determine if early spermatocytes can be enriched from a human testis biopsy using fluorescence-activated cell sorting (FACS). Potential surface markers for early spermatocytes were identified using bioinformatics analysis of single-cell RNA-sequenced human testis tissue. Testicular sperm extraction samples from three participants with normal spermatogenesis were digested into single-cell suspensions and cryopreserved. Two to four million cells were obtained from each and sorted by FACS as separate biologic replicates using antibodies for the identified surface markers. A portion from each biopsy remained unsorted to serve as controls. The sorted cells were then characterized for enrichment of early spermatocytes. A laboratory study. Three men with a diagnosis of obstructive azoospermia (age range, 30-40 years). None. Sorted cells were characterized for RNA expression of markers encompassing the stages of spermatogenesis. Sorting markers were validated by their reactivity on human testis formalin-fixed paraffin-embedded tissue. Serine protease 50 (TSP50) and SWI5-dependent homologous recombination repair protein 1 were identified as potential surface proteins specific for early spermatocytes. After FACS sorting, the TSP50-sorted populations accounted for 1.6%-8.9% of total populations and exhibited the greatest average-fold increases in RNA expression for the premeiotic marker stimulated by retinoic acid (STRA8), by 23-fold. Immunohistochemistry showed the staining pattern for TSP50 to be strong in premeiotic undifferentiated embryonic cell transcription factor 1-/doublesex and Mab-3 related transcription factor 1-/STRA8+ spermatogonia as well as SYCP3+/protamine 2- spermatocytes. This work shows that TSP50 can be used to enrich early STRA8-expressing spermatocytes from human testicular biopsies, providing a means for targeted single-cell RNA sequencing analysis and invitro functional interrogation of germ cells during the onset of meiosis. This could enable investigation into details of the regulatory pathways underlying this critical stage of spermatogenesis, previously difficult to enrich from whole tissue samples.

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.

Differentiation of human primary testicular cells in the presence of SCF using the organoid culture system.

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. 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). 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. 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.

Development of a multiplex reverse transcription qPCR assay for identification of saliva, blood, and semen

ABSTRACT The identification of body fluids can provide important information for case processing in forensic investigations. Accurate identification through messenger RNA (mRNA) is effective, especially in cases of sexual assault, where body fluids from one or more contributors are mixed. In this study, a saliva, blood, and semen simultaneous identification reverse transcription-quantitative polymerase chain reaction (SBS RT-qPCR) assay using mRNA from a DNA/RNA co-extraction method was developed. The body fluid-specific mRNA markers histatin 3 (HTN3), haemoglobin subunit beta (HBB), and protamine 1 (PRM1) were selected for identification of saliva, blood, and sperm cells, respectively. The assay accurately identified saliva, blood, and semen without cross-reaction with 10 body fluids. The limits of detection (LoD) were 1/102-, 1/105-, and 1/103-fold dilutions of total RNA in saliva, blood, and semen, respectively. Furthermore, the assay accurately identified trace amounts of body fluids in mixed samples at a 58:1:1 ratio. Finally, it was possible to analyse the origin of body fluids by obtaining an STR profile using the DNA/RNA co-extraction method. The SBS RT-qPCR assay was able to simultaneously identify each body fluid with efficiency in sample consumption. Therefore, it is advisable to use the DNA/RNA co-extraction method to identify mixed body fluids in sexual assault cases.

Sperm chromatin structure and reproductive fitness are altered by substitution of a single amino acid in mouse protamine 1.

Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by electrostatic interactions between DNA and the arginine-rich core of protamines. Phylogenetic analysis reveals several non-arginine residues conserved within, but not across species. The significance of these residues and their post-translational modifications are poorly understood. Here, we investigated the role of K49, a rodent-specific lysine residue in protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In sperm, alanine substitution (P1(K49A)) decreases sperm motility and male fertility-defects that are not rescued by arginine substitution (P1(K49R)). In zygotes, P1(K49A) leads to premature male pronuclear decompaction, altered DNA replication, and embryonic arrest. In vitro, P1(K49A) decreases protamine-DNA binding and alters DNA compaction and decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential for reproductive fitness.

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.

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. 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. 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. HSP70-2 and PRM1 mRNA and protein molecules have great potential to serve as molecular markers for determining bull fertility.

Decabromodiphenyl ether induces the chromosome association disorders of spermatocytes and deformation failures of spermatids in mice

The environmental presence of decabromodiphenyl ether (BDE-209), which is toxic to the male reproductive system, is widespread. The current study investigated its mechanism of toxicity in mice. The results showed, that BDE-209 induced DNA damage, decreased the expression of the promoter of meiosis spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 (Sohlh1), meiosis related-factors Lethal (3) malignant brain tumor like 2 (L3MBTL2), PIWI-like protein 2 (MILI), Cyclin-dependent kinase 2 (CDK2), Cyclin A, synaptonemal complex protein 1 (SYCP1) and synaptonemal complex protein 3 (SYCP3), and caused spermatogenic cell apoptosis, resulting in a decrease in sperm quantity and quality. Furthermore, BDE-209 downregulated the levels of anaphase-promoting complex/cyclosome (APC/C), increased the expression of PIWI-like protein 1 (MIWI) in the cytoplasm of elongating spermatids, and decreased the nuclear levels of RING finger protein 8 (RNF8), ubiquitinated (ub)-H2A/ub-H2B, and Protamine 1 (PRM1)/Protamine 2 (PRM2), while increasing H2A/H2B nuclear levels in spermatids. The reproductive toxicity was persistent for 50 days following the withdrawal of BDE-209 exposure. The results suggested that BDE-209 inhibits the initiation of meiosis by decreasing the expression of Sohlh1. Furthermore, the reduced expression of L3MBTL2 inhibited the formation of chromosomal synaptonemal complexes by depressing the expression of meiosis regulators affecting the meiotic progression and also inhibited histone ubiquitination preventing the replacement of histones by protamines, by preventing RNF8 from entering nuclei, which affected the evolution of spermatids into mature sperm.

Protamine 1 as a secreted colorectal cancer-specific antigen facilitating G1/S phase transition under nutrient stress conditions

PurposeCancer testis antigens (CTAs) are optimal tumor diagnostic markers and involved in carcinogenesis. However, colorectal cancer (CRC) related CTAs are less reported with impressive diagnostic capability or relevance with tumor metabolism rewiring. Herein, we demonstrated CRC-related CTA, Protamine 1 (PRM1), as a promising diagnostic marker and involved in regulation of cellular growth under nutrient deficiency.MethodsTranscriptomics of five paired CRC tissues was used to screen CRC-related CTAs. Capability of PRM1 to distinguish CRC was studied by detection of clinical samples through enzyme linked immunosorbent assay (ELISA). Cellular functions were investigated in CRC cell lines through in vivo and in vitro assays.ResultsBy RNA-seq and detection in 824 clinical samples from two centers, PRM1 expression were upregulated in CRC tissues and patients` serum. Serum PRM1 showed impressive accuracy to diagnose CRC from healthy controls and benign gastrointestinal disease patients, particularly more sensitive for early-staged CRC. Furthermore, we reported that when cells were cultured in serum-reduced medium, PRM1 secretion was upregulated, and secreted PRM1 promoted CRC growth in culture and in mice. Additionally, G1/S phase transition of CRC cells was facilitated by PRM1 protein supplementation and overexpression via activation of PI3K/AKT/mTOR pathway in serum deficient medium.ConclusionsIn general, our research presented PRM1 as a specific CRC antigen and illustrated the importance of PRM1 in CRC metabolism rewiring. The new vulnerability of CRC cells was also provided with the potential to be targeted in future.Graphical abstractDiagnostic value and grow factor-like biofunction of PRM1 A represents the secretion process of PRM1 regulated by nutrient deficiency. B represents activation of PI3K/AKT/mTOR pathway of secreted PRM1.

Differential Expressions of Protamine 1 (PRM1) and Protamine 2 (PRM2) Genes as Markers of Semen Quality in Pasundan Bulls

Several gene expressions are related to the success of spermatogenesis. Protamine plays an important role in sperm DNA packaging, spermatogenesis, and sperm quality. This study aimed to isolate RNA from spermatozoa and determine the gene expression profiles of protamine 1 (PRM1) and protamine 2 (PRM2). Six Pasundan bulls aged 5-8 years with a body weight of 380 kg-430 kg were used for this study. The Pasundan bulls were classified into group A (70%-79%) and group B (80%-89%) based on their sperm motility in fresh semen. In this study, correlation analysis was performed between fresh semen characteristics (volume, motility, concentration, abnormality, viability, intact plasma membrane (IPM), DNA integrity) and frozen semen characteristics after thawing (motility, viability, IPM, and DNA integrity). Total RNA was isolated from the frozen sperm pellet, and cDNA was synthesized. Specific PCR primers were used for the transcription of PRM1 and PRM2 from sperm. PRM1 and PRM2 gene expressions were evaluated by qRT-PCR, and ACTB was used as a control. The results showed that progressive sperm motility in the fresh sperm positively correlated with sperm viability and IPM. PRM1 and PRM2 were higher (p<0.05) in group B than in group A. This condition indicates the existence and influence of protamine on the parameters of progressive sperm motility. The expression of PRM1 and PRM2 genes could use as markers of semen quality in Pasundan bulls.

Effects of freeze-drying on the quality and fertilising ability of goat sperm recovered from different parts of the epididymis

Lyophilisation is an alternative method for sperm preservation. The aim of this study was to evaluate the effects of freeze-thawing (F/T) and freeze-drying (F/D) on the quality of epididymal goat sperm. Sperm from each region of the epididymis (caput, corpus and cauda) were collected and evaluated for the expression of phospholipase C zeta (PLC-ζ), protamine 1 (PRM1), transition protein 1 (TNP1) and 2 (TNP2). The effects of F/T and F/D on sperm quality in terms of PLC-ζ expression, chromatin stability (Chromomycin A3; CMA3) and DNA integrity were examined. The fertilising ability after intracytoplasmic sperm injection (ICSI) was also tested. Fresh sperm existed PLC-ζ, PRM1, TNP1 and TNP2, irrespective of the regions of the epididymis. However, different patterns of PLC-ζ expression were found. Although PRM1, TNP1, TNP2 were still expressed after F/T or F/D, only F/T could preserve the presence of PLC-ζ. For fresh sperm, caput epididymal sperm had the lowest evidence of chromatin stability when compared to sperm harvested from other regions of the epididymis. The F/T and F/D further increased the numbers of CMA3-positive sperm (P < 0.001). In all cases, no CMA3 staining was observed in caudal epididymal sperm. The caudal epididymal sperm had significantly greater proportions of sperm with intact DNA compared with caput and corpus epididymal sperm, especially when F/T and F/D were performed. The fertilisation rates of F/D sperm tended to decrease when compared with F/T sperm (4.2 ± 3.2 vs. 13.6 ± 9.0, P = 0.08). It is concluded that the sperm recovered from the caudal epididymis is suitable for freezing and lyophilisation. However, poor fertilisation rates of F/D sperm were coincidently observed, with a deficit demonstration of PLC-ζ.

The Effect of Temperature on Age Estimation of Semen Stains on Porous Versus Non-porous Surfaces Using Messenger Ribonucleic Acid Measurement

Background: While messenger Ribonucleic Acid (mRNA) can be used to identify the type of body fluid, its degradation can also indicate the time interval since it was deposited. This study was conducted to evaluate the effect of temperature on the estimation of the age of human semen stains using mRNA deposited on porous versus non-porous surfaces at different time intervals. Methods: Ten semen samples were applied on two different media (glass and cotton) and exposed to three different temperatures (4°C, room temperature, 40°C) and examined at threetime intervals (0, 45, and 90 days). The semen-specific mRNA markers protamine 1 (PRM1) and protamine 2 (PRM2) were quantitatively assessed along with a reference gene, beta-actin, using a reverse transcription-quantitative polymerase chain reaction. Results: Mean Cq values of mRNA markers (PRM1 and PRM2) and the reference gene (betaactin) increased with time of storage at different temperatures in both examined media. The mean quantification cycle (Cq) values of PRM2 were lower than PRM1, indicating that the levels of PRM2 marker in semen stain were higher than those of PRM1 marker. However, the mean Cq values of PRM2 at each time interval were not significantly different between temperatures, while PRM1 showed statistically significant differences in mean Cq values between temperatures at day 45 in both media. Conclusion: These results indicate that PRM2 can act as a reliable mRNA marker to estimate the time of deposition of semen stain at different temperatures on two different media.

Impact of sperm protamine on semen quality and fertility

Protamines are the nuclear proteins essential for chromatin compaction during spermatogenesis. During chromatin compaction, histones are replaced by transition proteins, which are then replaced by protamines. This process is essential for DNA stability. Protamines are rapidly evolved proteins with high evolutionary variation and encompass positively charged amino acids, especially 48% of arginine. Cysteines present in their sequence allow the formation of disulfide bonds between adjacent protamine molecules. Protamine 1 (PRM1), Protamine 2 (PRM2), and Protamine 3 (PRM3) are reported in mammals. Among these, PRM1 and PRM2 were extensively studied. The normal PRM1 and PRM2 ratios in men, stallions, and mice are 1:1, 3:1, and 1:2, respectively. However, in infertile males, the PRM1: PRM2 ratio is altered due to decreased PRM2 expression, which, in turn, is due to incomplete PRM2 precursor processing and zinc deficiency. In bull, ram, and buck, PRM2 mRNA is present but not PRM2 protein. In mice, rats, bulls, and men, the protamine cluster contains an open reading frame called protamine 3 (gene-4 or protamine-3). The proportion of protamine deficient sperm in the sample is indicative of problems in protamination. Recently, omics technologies, RT-qPCR, and gene knockout-based studies also reported the presence of protamine in sperm. All these semen quality and knockout studies envisage that protamines are indispensable for fertility. Henceforth, protamine-like biomolecules also may be evaluated for fertility prediction or markers in addition to the existing structural and functional attributes of sperm.

The Activation of Protamine 1 Using Epigenome Editing Decreases the Proliferation of Tumorigenic Cells.

DNA methyltransferases (DNMT) and histone deacetylases (HDAC) inhibitors are used as cancer epigenome drugs. However, these epigenetic drugs lack targeting specificity and could risk inducing genome instability and the expression of oncogenes. Therefore, there is a need to develop new therapeutic strategies where specific cancer genes can be targeted for silencing or activation. The CRISPR/dCas9 system represents a promising, powerful therapeutic tool because of its simplicity and specificity. Protamine 1 (PRM1) is exclusively expressed in sperm and has a vital role in the tight packaging of DNA, thus inducing transcriptional silencing in sperm cells. We hypothesized that the activation of the PRM1 gene in tumorigenic cells would lead to DNA condensation and reduce the proliferation of these cells. To test our hypothesis, we transfected human embryonic kidney cells 293T with a dCas9-P300 plasmid that adds acetyl groups to the promoter region of PRM1 via specific gRNAs plasmids. RNA-Seq analysis of transfected cells revealed high specificity of targeted gene activation. PRM1 expression resulted in a significant decrease in cell proliferation as measured by the BrdU ELISA assay. To confirm that the activation of PRM1 was due to acetyl groups deposited to H3K27, a ChIP-qPCR was performed. The acetylation of the PRM1 promoter region targeted by dCas9-p300 in transfected cells was higher than that of the control cells. Interestingly, the targeted promoter region for acetylation showed reduced DNA methylation. These findings demonstrate the efficacy of epigenome editing in activating PRM1 in non-expressing tumorigenic cells, which could be used as a promising therapeutic strategy in cancer treatment.

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.

Rapid detection of blood and semen mRNA markers by reverse transcription-recombinase polymerase amplification

Body fluid identification is crucial for crime scene reconstruction. Recently, messenger RNA (mRNA) profiling has been an effective approach for body fluid identification. In general, mRNA is detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) or end-point RT-PCR; however, these conventional methods are time-consuming and require extensive sample processing. Therefore, we developed a rapid and simple method for the detection of blood and semen mRNA markers by reverse transcription-recombinase polymerase amplification (RT-RPA). First, we screened mRNA markers for blood and semen and selected hemoglobin beta (HBB) and protamine 1 (PRM1), respectively, based on amplification specificity. Under optimized conditions, our RT-RPA assay detected HBB and PRM1 mRNAs within 20 min at a constant temperature of 42 °C. The detection limits for the assay were 0.01 ng/µL leukocyte RNA for HBB and 0.2 ng/µL semen RNA for PRM1. In addition, our RT-RPA assay exhibited high specificity and accuracy for HBB and PRM1 mRNA detection from mixed samples. Furthermore, as RPA has been reported to possess inhibitor tolerance, we evaluated the feasibility of direct RT-RPA for HBB mRNA detection. This direct approach reduced the number of processing steps and time required for template preparation and enabled the successful detection of HBB mRNA within 45 min from sample preparation. These findings suggest that RT-RPA is a useful method for mRNA-based blood and semen identification.