The Experts below are selected from a list of 31590 Experts worldwide ranked by ideXlab platform
Douglas T. Carrell - One of the best experts on this subject based on the ideXlab platform.
-
Protamine extraction and analysis of human sperm Protamine 1/Protamine 2 ratio using Acid gel electrophoresis.
Methods in molecular biology (Clifton N.J.), 2012Co-Authors: Lihua Liu, Kenneth I. Aston, Douglas T. CarrellAbstract:Protamines, sperm-specific nuclear proteins, are essential for sperm chromatin condensation and DNA stabilization. They are small, highly basic, and rich in disulfide bonds. Under reducing conditions, Protamines, along with other basic proteins, are soluble in acid solutions. Because of their small and similar molecular weights, SDS-PAGE cannot resolve Protamine 1 and Protamine 2 well. Urea-acid gel electrophoresis separates proteins based on the level of the positive charge and is thus a suitable method for resolving Protamines 1 and 2. Here, we describe the commonly used Protamine extraction method and the Urea-acid gel electrophoresis for assessment of Protamine 1/Protamine 2 ratio.
-
The clinical utility of the Protamine 1/Protamine 2 ratio in sperm.
Protein and peptide letters, 2011Co-Authors: Laszlo Nanassy, Lihua Liu, Jeanine Griffin, Douglas T. CarrellAbstract:During spermiogenesis, human sperm undergo a dramatic reorganization of the chromatin in which canonical histones are replaced by two types of Protamines, Protamine 1 (P1) and Protamine (P2). P1 and P2 are expressed approximately at a 1:1 ratio in healthy men. Alteration of this ratio is associated with male infertility. Patients with an abnormal P1/P2 ratio generally exhibit diminished semen quality, lower fertilization ability, and lower pregnancy rates when undergoing in vitro fertilization. Many studies have reported an elevated incidence of abnormal P1/P2 ratios in infertile men compared to fertile controls, and have evaluated the relationship between infertility and abnormal protamination; however, no prospective study has investigated the normal range of the P1/P2 ratio in men from the general population. Here, we report a P1/P2 reference range of 0.54 to 1.43 in a fertile, normozoospermic population. This rather wide normal range of P1/P2 led us to the conclusion that abnormal protamination is more likely indicative of other perturbations during spermatogenesis than the underlying mechanism to cause infertility. Alternatively, Protamine expression may act as a checkpoint mechanism and thus be indirectly related to semen quality.
-
altered Protamine expression and diminished spermatogenesis what is the link
Human Reproduction Update, 2007Co-Authors: Douglas T. Carrell, Benjamin R Emery, Sue HammoudAbstract:During the elongating spermatid stage of spermiogenesis, human sperm chromatin undergoes a complex transition in which histones are extensively replaced by Protamines in a carefully regulated transition including histone modifications and intermediate and temporary replacement of the histones by sperm-specific transition proteins. The replacement of most histones by Protamines 1 and 2 facilitates a high order of chromatin packaging necessary for normal sperm function and may also be necessary for DNA silencing and imprinting changes within the sperm cell. Protamines 1 and 2 are usually expressed in nearly equal quantities, but elevated or diminished Protamine 1/Protamine 2 ratios are observed in some infertile men and is often associated with severe spermatogenesis defects. Human and animal studies demonstrate that expression of the Protamine proteins is uniquely regulated by transcription/translation factors, including storage of the mRNA in ribonucleoprotein (RNP) particles composed of the mRNA, transcription factors and a kinesin molecule necessary for transport of the RNP to the cytoplasm and removal of transcriptional activators from the nucleus. Recent studies indicate that most patients with abnormal Protamine protein levels have elevated levels of Protamine transcript in the mature sperm cell, indicating a possible defect in transcription or translation. The regulation of Protamine expression is unique and includes several possible mechanisms which may be responsible for dysregulation of Protamine expression and concurrent broad spectrum defects in spermatogenesis. We suggest two hypotheses: (i) that abnormal Protamine expression is indicative of a generalized defect in mRNA storage and/or translation which affects other mRNA transcripts or (ii) that Protamines may act as a checkpoint of spermatogenesis.
-
Protamine levels vary between individual sperm cells of infertile human males and correlate with viability and dna integrity
Journal of Andrology, 2006Co-Authors: Vincent W. Aoki, Lihua Liu, Benjamin R Emery, Douglas T. CarrellAbstract:Sperm Protamine deficiency has been associated with human male infertility. However, most studies have adopted a global approach to assessing sperm Protamine levels. Thus, it is not known whether sperm cells from individual human males possess variations in Protamine protein content. The objectives of this study were to evaluate variations in Protamine-1 (P1) and Protamine-2 (P2) content between individual sperm cells of fertile and infertile men and to correlate DNA integrity and sperm cell viability with Protamine levels in individual sperm cells. The semen samples of fertile and infertile men were evaluated globally for Protamine protein content using nuclear protein extraction, gel electrophoresis, and densitometry analysis. Individual sperm cell P1 and P2 levels were assessed using immunofluorescence microscopy in conjunction with automated image analysis. The terminal transferase dUTP nick end labeling (TUNEL) assay was performed simultaneously with Protamine immunostaining to assess the relationship between Protamine levels and DNA integrity in individual spermatozoa. Additionally, the relationship between sperm cell viability and Protamine levels was assessed via viability staining concomitant with Protamine staining. The Protamine fluorescence data demonstrate significant variations in Protamine content within individual sperm cells of human males. Overall population-based measures of DNA integrity and sperm cell viability correlate significantly with population-based measurements of Protamine levels. The data also demonstrate individual sperm cells displaying the lowest Protamine levels display diminished viability and increased sperm cell susceptibility to DNA damage.
-
sperm Protamine 1 Protamine 2 ratios are related to in vitro fertilization pregnancy rates and predictive of fertilization ability
Fertility and Sterility, 2006Co-Authors: Vincent W. Aoki, Lihua Liu, K P Jones, Harry H Hatasaka, Mark Gibson, Matthew C Peterson, Douglas T. CarrellAbstract:Objective To evaluate whether aberrant sperm P1/P2 ratios are predictive of abnormal fertilizing ability and are related to in vitro fertilization (IVF) outcome. Design Prospective case–control study. Setting University-based infertility and IVF clinic. Patient(s) Forty-three male infertility patients with an abnormally reduced P1/P2 ratio, 251 patients with a normal P1/P2 ratio, and 121 patients with an abnormally elevated P1/P2 ratio. Intervention(s) Human IVF, the sperm penetration assay (SPA), and sperm Protamine quantification via nuclear protein extraction, gel electrophoresis, and densitometry analysis. Main Outcome Measure(s) Sperm P1/P2 ratios; P1 and P2 quantities; SPA scores; and IVF-fertilization, embryo-quality, pregnancy, delivery, and spontaneous-abortion rates. Result(s) Standard IVF fertilization rates and SPA scores were significantly reduced in patients with abnormally low and high P1/P2 ratios. In vitro fertilization embryo quality was comparable between these groups, but pregnancy rates were significantly reduced in patients with abnormally reduced P1/P2 ratios. Conclusion(s) The P1/P2 ratio has a significant relationship to sperm fertilization ability. The relationship between Protamines and fertilization ability is not understood but may be either a reflection of generalized abnormalities during spermiogenesis or an indication of Protamine deficiency acting as a regulator or checkpoint of spermatogenesis.
Rafael Oliva - One of the best experts on this subject based on the ideXlab platform.
-
Mammalian Sperm Protamine Extraction and Analysis: A Step-By-Step Detailed Protocol and Brief Review of Protamine Alterations
Protein and Peptide Letters, 2018Co-Authors: Ada Soler-ventura, Judit Castillo, Ferran Barrachina, Meritxell Jodar, Josep Lluis Ballesca, Alberto De La Iglesia, Rafael OlivaAbstract:Protamines are the most abundant sperm nuclear proteins and pack approximately the 92-98% of the mammalian sperm DNA. In mammals, two types of Protamines have been described, the Protamine 1 (P1) and the Protamine 2 (P2) family. The deregulation of the relative P1/P2 ratio has been correlated to DNA damage, alterations in seminal parameters, and low success rate of assisted reproduction techniques. Additionally, the extraction and analysis of Protamines have been important to understand the fundamental aspects of the sperm chromatin structure and function, Protamine sequence conservation among species, and sperm chromatin alterations present in infertile males. However, Protamines show a particular chemical nature due to its special amino acid sequence, extremely rich in arginine and cysteine residues. Because of these peculiar characteristics of Protamines, their extraction and analysis is not as straightforward as the analysis of other chromatin-associated proteins, for which many detailed protocols are already available. A step-by-step protocol was needed to facilitate Protamine analysis to researchers interested in their implementation. Therefore, in order to contribute to fulfill this need, here we provide a detailed protocol, which should be useful to research teams and laboratories interested in the Protamine field. In addition, we also briefly review the different studies published so far on Protamine alterations and male infertility. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
-
Mammalian Sperm Protamine Extraction and Analysis: A Step-By-Step Detailed Protocol and Brief Review of Protamine Alterations
Protein and peptide letters, 2018Co-Authors: Ada Soler-ventura, Judit Castillo, Ferran Barrachina, Meritxell Jodar, Josep Lluis Ballesca, Alberto De La Iglesia, Rafael OlivaAbstract:BACKGROUND Protamines are the most abundant sperm nuclear proteins and pack approximately the 92-98% of the mammalian sperm DNA. In mammals, two types of Protamines have been described, the Protamine 1 (P1) and the Protamine 2 (P2) family. The deregulation of the relative P1/P2 ratio has been correlated to DNA damage, alterations in seminal parameters, and low success rate of assisted reproduction techniques. Additionally, the extraction and analysis of Protamines have been important to understand the fundamental aspects of the sperm chromatin structure and function, Protamine sequence conservation among species, and sperm chromatin alterations present in infertile males. However, Protamines show a particular chemical nature due to its special amino acid sequence, extremely rich in arginine and cysteine residues. Because of these peculiar characteristics of Protamines, their extraction and analysis is not as straightforward as the analysis of other chromatin-associated proteins, for which many detailed protocols are already available. CONCLUSION A step-by-step protocol was needed to facilitate Protamine analysis to researchers interested in their implementation. Therefore, in order to contribute to fulfill this need, here we provide a detailed protocol, which should be useful to research teams and laboratories interested in the Protamine field. In addition, we also briefly review the different studies published so far on Protamine alterations and male infertility.
-
Identification of a complex population of chromatin-associated proteins in the European sea bass (Dicentrarchus labrax) sperm
2018Co-Authors: Ferran Barrachina, Judit Castillo, Meritxell Jodar, Dafni Anastasiadi, Josep Maria Estanyol, Francesc Piferrer, Rafael OlivaAbstract:A very common conception about the function of the spermatozoon is that its unique role is to transmit the paternal genome to the next generation. Most of the sperm genome is known to be condensed in many species by Protamines, which are small and extremely positively charged proteins (50–70% arginine) with the functions of streamlining the sperm cell and protecting its DNA. However, more recently, it has been shown in mammals that 2–10% of its mature sperm chromatin is also associated to a complex population of histones and chromatin-associated proteins differentially distributed in the genome. These proteins are transferred to the oocyte upon fertilization and may be involved in the epigenetic marking of the paternal genome. However, little information is so far available on the additional potential sperm chromatin proteins present in other Protamine-containing non-mammalian vertebrates detected through high-throughput mass spectrometry. Thus, we started the present work with the goal of characterizing the mature sperm proteome of the European sea bass, with a particular focus on the sperm chromatin, chosen as a representative of non-mammalian vertebrate Protamine-containing species. Proteins were isolated by acidic extraction from purified sperm cells and from purified sperm nuclei, digested with trypsin, and subsequently the peptides were separated using liquid chromatography and identified through tandem mass spectrometry. A total of 296 proteins were identified. Of interest, the presence of 94 histones and other chromatin-associated proteins was detected, in addition to the Protamines. These results provide phylogenetically strategic information, indicating that the coexistence of histones, additional chromatin proteins, and Protamines in sperm is not exclusive of mammals, but is also present in other Protamine-containing vertebrates. Thus, it indicates that the epigenetic marking of the sperm chromatin, first demonstrated in mammals, could be more fundamental and conserved than previously thought. Abbreviations: AU-PAGE: acetic acid–urea polyacrylamide gel electrophoresis; CPC: chromosomal passenger complex; DTT: dithiothreitol; EGA: embryonic genome activation; FDR: false discovery rate; GO: Gene Ontology; IAA: iodoacetamide; LC: liquid chromatography; LC-MS/MS: liquid chromatography coupled to tandem mass spectrometry; MS: mass spectrometry; MS/MS: tandem mass spectrometry; MW: molecular weight; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate buffered saline; SDS: sodium dodecyl sulfate; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCA: trichloroacetic acid.
-
Protamine alterations in human spermatozoa.
Advances in experimental medicine and biology, 2013Co-Authors: Meritxell Jodar, Rafael OlivaAbstract:Protamines are the major nuclear proteins in sperm cells, having a crucial role in the correct packaging of the paternal DNA. The fact that Protamine haploinsufficiency in mice resulted in abnormal chromatin packaging and male infertility suggested that the Protamines could also be important candidates in explaining some of the idiopathic male infertility cases in humans. The first clinical studies focused on analyzing Protamines at the protein level. Various studies have found the presence of an altered amount of Protamines in some infertile patients, in contrast to the normal situation in fertile individuals where the two Protamines, Protamine 1 and Protamine 2, are both present in approximately equal quantities. Subsequently, the Protamine genes were the subject of various mutational genetic screening studies in search of variants that could be associated with deregulation in the Protamine expression observed. The results of these Protamine mutational studies showed that the presence of high penetrant mutations is a very rare cause of male infertility. However, some variants and some haplotypes described may behave as risk factors for male infertility. More recently, the presence of RNA in the mature sperm cell has also been investigated. The present chapter will introduce the basic aspects of Protamine evolution and function and review the various articles published to date on the relationship between the Protamines studied at the DNA, RNA, and protein levels and male infertility.
-
Protamines and male infertility.
Human reproduction update, 2006Co-Authors: Rafael OlivaAbstract:Protamines are the major nuclear sperm proteins. The human sperm nucleus contains two types of Protamine: Protamine 1 (P1) encoded by a single-copy gene and the family of Protamine 2 (P2) proteins (P2, P3 and P4), all also encoded by a single gene that is transcribed and translated into a precursor protein. The Protamines were discovered more than a century ago, but their function is not yet fully understood. In fact, different hypotheses have been proposed: condensation of the sperm nucleus into a compact hydrodynamic shape, protection of the genetic message delivered by the spermatozoa, involvement in the processes maintaining the integrity and repair of DNA during or after the nucleohistone-nucleoProtamine transition and involvement in the epigenetic imprinting of the spermatozoa. Protamines are also one of the most variable proteins found in nature, with data supporting a positive Darwinian selection. Changes in the expression of P1 and P2 Protamines have been found to be associated with infertility in man. Mutations in the Protamine genes have also been found in some infertile patients. Transgenic mice defective in the expression of Protamines also present several structural defects in the sperm nucleus and have variable degrees of infertility. There is also evidence that altered levels of Protamines may result in an increased susceptibility to injury in the spermatozoan DNA causing infertility or poor outcomes in assisted reproduction. The present work reviews the articles published to date on the relationship between Protamines and infertility.
Lihua Liu - One of the best experts on this subject based on the ideXlab platform.
-
Protamine extraction and analysis of human sperm Protamine 1/Protamine 2 ratio using Acid gel electrophoresis.
Methods in molecular biology (Clifton N.J.), 2012Co-Authors: Lihua Liu, Kenneth I. Aston, Douglas T. CarrellAbstract:Protamines, sperm-specific nuclear proteins, are essential for sperm chromatin condensation and DNA stabilization. They are small, highly basic, and rich in disulfide bonds. Under reducing conditions, Protamines, along with other basic proteins, are soluble in acid solutions. Because of their small and similar molecular weights, SDS-PAGE cannot resolve Protamine 1 and Protamine 2 well. Urea-acid gel electrophoresis separates proteins based on the level of the positive charge and is thus a suitable method for resolving Protamines 1 and 2. Here, we describe the commonly used Protamine extraction method and the Urea-acid gel electrophoresis for assessment of Protamine 1/Protamine 2 ratio.
-
The clinical utility of the Protamine 1/Protamine 2 ratio in sperm.
Protein and peptide letters, 2011Co-Authors: Laszlo Nanassy, Lihua Liu, Jeanine Griffin, Douglas T. CarrellAbstract:During spermiogenesis, human sperm undergo a dramatic reorganization of the chromatin in which canonical histones are replaced by two types of Protamines, Protamine 1 (P1) and Protamine (P2). P1 and P2 are expressed approximately at a 1:1 ratio in healthy men. Alteration of this ratio is associated with male infertility. Patients with an abnormal P1/P2 ratio generally exhibit diminished semen quality, lower fertilization ability, and lower pregnancy rates when undergoing in vitro fertilization. Many studies have reported an elevated incidence of abnormal P1/P2 ratios in infertile men compared to fertile controls, and have evaluated the relationship between infertility and abnormal protamination; however, no prospective study has investigated the normal range of the P1/P2 ratio in men from the general population. Here, we report a P1/P2 reference range of 0.54 to 1.43 in a fertile, normozoospermic population. This rather wide normal range of P1/P2 led us to the conclusion that abnormal protamination is more likely indicative of other perturbations during spermatogenesis than the underlying mechanism to cause infertility. Alternatively, Protamine expression may act as a checkpoint mechanism and thus be indirectly related to semen quality.
-
Protamine levels vary between individual sperm cells of infertile human males and correlate with viability and dna integrity
Journal of Andrology, 2006Co-Authors: Vincent W. Aoki, Lihua Liu, Benjamin R Emery, Douglas T. CarrellAbstract:Sperm Protamine deficiency has been associated with human male infertility. However, most studies have adopted a global approach to assessing sperm Protamine levels. Thus, it is not known whether sperm cells from individual human males possess variations in Protamine protein content. The objectives of this study were to evaluate variations in Protamine-1 (P1) and Protamine-2 (P2) content between individual sperm cells of fertile and infertile men and to correlate DNA integrity and sperm cell viability with Protamine levels in individual sperm cells. The semen samples of fertile and infertile men were evaluated globally for Protamine protein content using nuclear protein extraction, gel electrophoresis, and densitometry analysis. Individual sperm cell P1 and P2 levels were assessed using immunofluorescence microscopy in conjunction with automated image analysis. The terminal transferase dUTP nick end labeling (TUNEL) assay was performed simultaneously with Protamine immunostaining to assess the relationship between Protamine levels and DNA integrity in individual spermatozoa. Additionally, the relationship between sperm cell viability and Protamine levels was assessed via viability staining concomitant with Protamine staining. The Protamine fluorescence data demonstrate significant variations in Protamine content within individual sperm cells of human males. Overall population-based measures of DNA integrity and sperm cell viability correlate significantly with population-based measurements of Protamine levels. The data also demonstrate individual sperm cells displaying the lowest Protamine levels display diminished viability and increased sperm cell susceptibility to DNA damage.
-
sperm Protamine 1 Protamine 2 ratios are related to in vitro fertilization pregnancy rates and predictive of fertilization ability
Fertility and Sterility, 2006Co-Authors: Vincent W. Aoki, Lihua Liu, K P Jones, Harry H Hatasaka, Mark Gibson, Matthew C Peterson, Douglas T. CarrellAbstract:Objective To evaluate whether aberrant sperm P1/P2 ratios are predictive of abnormal fertilizing ability and are related to in vitro fertilization (IVF) outcome. Design Prospective case–control study. Setting University-based infertility and IVF clinic. Patient(s) Forty-three male infertility patients with an abnormally reduced P1/P2 ratio, 251 patients with a normal P1/P2 ratio, and 121 patients with an abnormally elevated P1/P2 ratio. Intervention(s) Human IVF, the sperm penetration assay (SPA), and sperm Protamine quantification via nuclear protein extraction, gel electrophoresis, and densitometry analysis. Main Outcome Measure(s) Sperm P1/P2 ratios; P1 and P2 quantities; SPA scores; and IVF-fertilization, embryo-quality, pregnancy, delivery, and spontaneous-abortion rates. Result(s) Standard IVF fertilization rates and SPA scores were significantly reduced in patients with abnormally low and high P1/P2 ratios. In vitro fertilization embryo quality was comparable between these groups, but pregnancy rates were significantly reduced in patients with abnormally reduced P1/P2 ratios. Conclusion(s) The P1/P2 ratio has a significant relationship to sperm fertilization ability. The relationship between Protamines and fertilization ability is not understood but may be either a reflection of generalized abnormalities during spermiogenesis or an indication of Protamine deficiency acting as a regulator or checkpoint of spermatogenesis.
-
dna integrity is compromised in Protamine deficient human sperm
Journal of Andrology, 2005Co-Authors: Vincent W. Aoki, Lihua Liu, Sergey I Moskovtsev, Jennifer Willis, Brendan Mullen, Douglas T. CarrellAbstract:The objective of this study was to examine the relationship between DNA integrity and Protamines in human sperm. One hundred forty-nine male infertility patients were included in an Institutional Review Board-approved study. Sperm were evaluated for DNA fragmentation using the DNA Integrity Assay, a test equivalent to the sperm chromatin structure assay (SCSA). Additionally, nuclear proteins were extracted and the Protamine-1/Protamine-2 ratio (P1/P2), Protamine-1 (P1), Protamine-2 (P2), and total Protamine concentrations were evaluated. We identified 37 patients with abnormally low P1/P2 ratios, 99 patients with normal P1/P2 ratios, and 13 patients with abnormally high P1/P2 ratios. DNA fragmentation was significantly elevated in patients with low P1/P2 ratios (37.1 +/- 6.02) vs those with normal and high P1/P2 ratios (26.7 +/- 1.9 and 23.8 +/- 3.2, respectively; P < .05) and was inversely correlated with the P1/P2 ratio (R(s) -0.18, P < .05), P1 concentration (R(s) -0.29, P < .001), P2 concentration (R(s) - 0.24, P < .005), and total Protamine concentration (R(s) -0.28, P < .001). Furthermore, chi2 analysis revealed a significant increase in the incidence of marked DNA fragmentation in patients with diminished levels of either P1 or P2. The present study is the first to report that human sperm Protamine content is significantly related to DNA fragmentation. In particular, sperm P1 and P2 concentrations inversely correlate with DNA fragmentation, indicating a protective role of the Protamines against sperm DNA damage. In light of recent studies highlighting the negative effect of sperm DNA damage on ART outcomes, these findings indicate a possible clinical significance for human sperm Protamine levels.
Klaus Steger - One of the best experts on this subject based on the ideXlab platform.
-
New monoclonal antibodies specific for mammalian Protamines P1 and P2
2018Co-Authors: Rod Balhorn, Klaus Steger, Martin Bergmann, Hans-christian Schuppe, Stefanie Neuhauser, Monique C. BalhornAbstract:The expression of Protamines and the binding of these small arginine-rich proteins to DNA complete the process of spermatid chromatin reorganization and the global inactivation of the male’s haploid genome that occurs during the final stages of sperm development in mammals. While a number of anti-Protamine antibodies have been created during the last 40 years, only a few have proven useful for detecting the presence of the Protamines, determining the timing of their expression and deposition in chromatin, and investigating their structure and function in both maturing spermatids and sperm. The aim of this effort was to develop an additional set of monoclonal antibodies (MAbs) that not only recognize new P1 and P2 Protamine epitopes but also work well as IHC reagents for detecting and identifying mammalian Protamines in testicular tissue and ejaculated sperm. Using a combination of native and synthetic human Protamines as antigens, 38 hybridoma clones recognizing human Protamine P1 or P2 were generated. Antibodies produced by the 12 best clones were screened for selectivity by enzyme-linked immunosorbent assay, and two were found to recognize only human Protamine P1 or P2, while a number of the others bound to both the human and mouse proteins. One MAb recognized every Protamine tested. All the antibodies, including one recognizing stallion P1 and another recognizing stallion P2, bound to the native Protamines in the chromatin of spermatids or sperm. While the majority labeled only elongating spermatids or sperm, several of the antibodies were found to also bind to the cytoplasm or nuclei of cells that lack Protamine, which indicates these MAbs must recognize epitopes present in the Protamines that are also found in other proteins. Thirteen overlapping human Protamine P1 peptides were synthesized and subsequently used to identify the epitopes recognized by the six best antibodies. Abbreviations: BSA: bovine serum albumin; ELISA: enzyme-linked immunosorbent assay; HCl: hydrochloric acid; IHC: immunohistochemistry; i.p: intraperitoneal; LIS: lithium diiodosalicylate; MAb: monoclonal antibody; PBS: phosphate buffered saline
-
Protamine mRNA ratio in stallion spermatozoa correlates with mare fecundity.
Journal of Andrology, 2014Co-Authors: Agnieszka Paradowska-dogan, Kikikipa Kretzer, Markus Vieweg, Przemyslaw Waliszewski, Michael Zitzmann, Wolfgang Weidner, Con Mallidis, Martin Bergmann, A. Fernandez, Klaus StegerAbstract:Summary Highly compacted sperm DNA in Protamine toroids and a minor fraction of nucleohistones are prerequisites for the efficient transmission of the paternal genome into the oocyte at fertilization. The objective of this study was to evaluate whether Protamines might serve as a prognostic factor for stallion fertility. In situ hybridization detected specific expression of P1 mRNA in the cytoplasm of stage I to VII spermatids, whereas comparable immunohistochemical stainings showed that protein expression was delayed till elongating spermatids in differentiation stages III to VIII. No staining was detectable in cryptorchid testis because of the lack of spermatids in the seminiferous tubules. Using quantitative real-time polymerase chain reaction, we identified mRNA transcripts of P1 and 2 variants of Protamine- 2 (P2, P3) in ejaculated spermatozoa from 45 thoroughbred stallions. According to the mare fertility descriptor (i.e. the ‘none-return-rate 28 percentage’ or NRR28%), stallions were divided into three groups (i.e. high, reduced and low fertility). The P2/P1 mRNA ratio was found to be significantly reduced in the group with lower fertility (p = 0.016) and was slightly correlated with sperm concentration (correlation coefficient r = 0.263). Furthermore, morphologically abnormal sperm count negatively correlated with P2/P1 mRNA ratio, indicating that spermatozoa carrying head defects display a diminished Protamine ratio (r = −0.348). Conversely, the P2/P1 ratio was positively correlated with mare fertility or NRR28% (r = 0.274). Interestingly, P3/P1 mRNA ratio remained unaltered in the investigated groups indicating that this variant plays a minor role in equine sperm chromatin compaction. Aberrant Protamine transcripts content in equine spermatozoa was not associated with DNA defragmentation rate as measured by flow cytometric acridine orange test. On the basis of these results, we suggest that, similar to human, equine Protamine expression constitutes a checkpoint of spermatogenesis and as a corollary the level of Protamine mRNA may reflect the quality of spermatogenesis and spermatozoa's fertilizing capacity.
-
endonuclease sensitive regions of human spermatozoal chromatin are highly enriched in promoter and ctcf binding sequences
Genome Research, 2009Co-Authors: Ali Arpanahi, Klaus Steger, Stephen A. Krawetz, Martin H Brinkworth, David Iles, Agnieszka Paradowska, Adrian E Platts, Myriam Saida, Philip Tedder, David MillerAbstract:During the haploid phase of mammalian spermatogenesis, nucleosomal chromatin is ultimately repackaged by small, highly basic Protamines to generate an extremely compact, toroidal chromatin architecture that is critical to normal spermatozoal function. In common with several species, however, the human spermatozoon retains a small proportion of its chromatin packaged in nucleosomes. As nucleosomal chromatin in spermatozoa is structurally more open than Protamine-packaged chromatin, we considered it likely to be more accessible to exogenously applied endonucleases. Accordingly, we have used this premise to identify a population of endonuclease-sensitive DNA sequences in human and murine spermatozoa. Our results show unequivocally that, in contrast to the endonuclease-resistant sperm chromatin packaged by Protamines, regions of increased endonuclease sensitivity are closely associated with gene regulatory regions, including many promoter sequences and sequences recognized by CCCTC-binding factor (CTCF). Similar differential packaging of promoters is observed in the spermatozoal chromatin of both mouse and man. These observations imply the existence of epigenetic marks that distinguish gene regulatory regions in male germ cells and prevent their repackaging by Protamines during spermiogenesis. The ontology of genes under the control of endonuclease-sensitive regulatory regions implies a role for this phenomenon in subsequent embryonic development.
-
transcriptional and translational regulation of gene expression in haploid spermatids
Anatomy and Embryology, 1999Co-Authors: Klaus StegerAbstract:During spermiogenesis, round spermatids undergo complex morphological, biochemical, and physiological modifications resulting in the formation of mature spermatozoa. While in round spermatids histones and non-histone proteins are replaced by transition proteins, in elongating spermatids, transition proteins are removed from the condensing chromatin and are replaced by Protamines, which are the principal basic nuclear proteins of mature spermatozoa. The tightly packed DNA-Protamine complexes cease transcription several days before the completion of spermiogenesis. Thus, major modifications in both nuclear and cytoplasmic structures continue throughout spermiogenesis, stringent temporal and stage-specific gene expression is a prerequisite for the correct differentiation of round spermatids into mature spermatozoa. The genes for transition proteins and Protamines are transcribed in round and elongating spermatids. Transcription is regulated via methylation and trans-acting factors that bind to the TATA-box, the CRE-box, or other specific DNA sequences in the promoter region. The transcripts are stored as ribonucleoprotein particles in a translationally repressed state for several days and are translated in elongating and elongated spermatids. It has been demonstrated that, in haploid spermatids, essentially every mRNA exhibits evidence of translational repression. Translational regulation involves protein repressors that bind to the poly-A tail or specific RNA sequences located in the 3′-UTR.
Vincent W. Aoki - One of the best experts on this subject based on the ideXlab platform.
-
Protamine levels vary between individual sperm cells of infertile human males and correlate with viability and dna integrity
Journal of Andrology, 2006Co-Authors: Vincent W. Aoki, Lihua Liu, Benjamin R Emery, Douglas T. CarrellAbstract:Sperm Protamine deficiency has been associated with human male infertility. However, most studies have adopted a global approach to assessing sperm Protamine levels. Thus, it is not known whether sperm cells from individual human males possess variations in Protamine protein content. The objectives of this study were to evaluate variations in Protamine-1 (P1) and Protamine-2 (P2) content between individual sperm cells of fertile and infertile men and to correlate DNA integrity and sperm cell viability with Protamine levels in individual sperm cells. The semen samples of fertile and infertile men were evaluated globally for Protamine protein content using nuclear protein extraction, gel electrophoresis, and densitometry analysis. Individual sperm cell P1 and P2 levels were assessed using immunofluorescence microscopy in conjunction with automated image analysis. The terminal transferase dUTP nick end labeling (TUNEL) assay was performed simultaneously with Protamine immunostaining to assess the relationship between Protamine levels and DNA integrity in individual spermatozoa. Additionally, the relationship between sperm cell viability and Protamine levels was assessed via viability staining concomitant with Protamine staining. The Protamine fluorescence data demonstrate significant variations in Protamine content within individual sperm cells of human males. Overall population-based measures of DNA integrity and sperm cell viability correlate significantly with population-based measurements of Protamine levels. The data also demonstrate individual sperm cells displaying the lowest Protamine levels display diminished viability and increased sperm cell susceptibility to DNA damage.
-
sperm Protamine 1 Protamine 2 ratios are related to in vitro fertilization pregnancy rates and predictive of fertilization ability
Fertility and Sterility, 2006Co-Authors: Vincent W. Aoki, Lihua Liu, K P Jones, Harry H Hatasaka, Mark Gibson, Matthew C Peterson, Douglas T. CarrellAbstract:Objective To evaluate whether aberrant sperm P1/P2 ratios are predictive of abnormal fertilizing ability and are related to in vitro fertilization (IVF) outcome. Design Prospective case–control study. Setting University-based infertility and IVF clinic. Patient(s) Forty-three male infertility patients with an abnormally reduced P1/P2 ratio, 251 patients with a normal P1/P2 ratio, and 121 patients with an abnormally elevated P1/P2 ratio. Intervention(s) Human IVF, the sperm penetration assay (SPA), and sperm Protamine quantification via nuclear protein extraction, gel electrophoresis, and densitometry analysis. Main Outcome Measure(s) Sperm P1/P2 ratios; P1 and P2 quantities; SPA scores; and IVF-fertilization, embryo-quality, pregnancy, delivery, and spontaneous-abortion rates. Result(s) Standard IVF fertilization rates and SPA scores were significantly reduced in patients with abnormally low and high P1/P2 ratios. In vitro fertilization embryo quality was comparable between these groups, but pregnancy rates were significantly reduced in patients with abnormally reduced P1/P2 ratios. Conclusion(s) The P1/P2 ratio has a significant relationship to sperm fertilization ability. The relationship between Protamines and fertilization ability is not understood but may be either a reflection of generalized abnormalities during spermiogenesis or an indication of Protamine deficiency acting as a regulator or checkpoint of spermatogenesis.
-
dna integrity is compromised in Protamine deficient human sperm
Journal of Andrology, 2005Co-Authors: Vincent W. Aoki, Lihua Liu, Sergey I Moskovtsev, Jennifer Willis, Brendan Mullen, Douglas T. CarrellAbstract:The objective of this study was to examine the relationship between DNA integrity and Protamines in human sperm. One hundred forty-nine male infertility patients were included in an Institutional Review Board-approved study. Sperm were evaluated for DNA fragmentation using the DNA Integrity Assay, a test equivalent to the sperm chromatin structure assay (SCSA). Additionally, nuclear proteins were extracted and the Protamine-1/Protamine-2 ratio (P1/P2), Protamine-1 (P1), Protamine-2 (P2), and total Protamine concentrations were evaluated. We identified 37 patients with abnormally low P1/P2 ratios, 99 patients with normal P1/P2 ratios, and 13 patients with abnormally high P1/P2 ratios. DNA fragmentation was significantly elevated in patients with low P1/P2 ratios (37.1 +/- 6.02) vs those with normal and high P1/P2 ratios (26.7 +/- 1.9 and 23.8 +/- 3.2, respectively; P < .05) and was inversely correlated with the P1/P2 ratio (R(s) -0.18, P < .05), P1 concentration (R(s) -0.29, P < .001), P2 concentration (R(s) - 0.24, P < .005), and total Protamine concentration (R(s) -0.28, P < .001). Furthermore, chi2 analysis revealed a significant increase in the incidence of marked DNA fragmentation in patients with diminished levels of either P1 or P2. The present study is the first to report that human sperm Protamine content is significantly related to DNA fragmentation. In particular, sperm P1 and P2 concentrations inversely correlate with DNA fragmentation, indicating a protective role of the Protamines against sperm DNA damage. In light of recent studies highlighting the negative effect of sperm DNA damage on ART outcomes, these findings indicate a possible clinical significance for human sperm Protamine levels.
-
The Genetics of Abnormal Protamine Expression
The Genetics of Male Infertility, 1Co-Authors: Vincent W. Aoki, Douglas T. CarrellAbstract:During spermiogenesis, the sperm chromatin undergoes dramatic remodeling. The testis-specific Protamine proteins facilitate these nuclear changes by replacing the somatic cell histones, a process that produces highly condensed, transcriptionally silent chromatin. In humans, there are two forms of sperm Protamine: Protamine 1 (P1) and Protamine 2 (P2), which occur in a strictly regulated 1:1 ratio. Sperm Protamine-deficiency and P1:P2 ratio deregulation have been implicated in male infertility. The details of the underlying genetic basis of abnormal Protamine expression are just emerging. This chapter summarizes our current knowledge of the sperm Protamines, their relationship with male infertility, and what is currently understood regarding the genetic basis of abnormal Protamine expression.
