The clinical utility of the protamine 1/protamine 2 ratio in sperm

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.     

Involvement of an acrosinlike proteinase in the sulfhydryl-induced degradation of rabbit sperm nuclear protamine

Previous studies demonstrated that proteolytic activity is associated with isolated rabbit sperm nuclei and is responsible for the degradation of nuclear protamine that occurs during thiol-induced in vitro decondensation of the nuclei (Zirkin and Chang, 1977; Chang and Zirkin, 1978). In this study, we present the results of experiments designed to characterize this proteolytic activity. Basic protein isolated from rabbit sperm nuclei incubated with 5 mM dithiothreitol (DTT) and 1 percent Triton X-100 for increasing periods of time exhibited progressively faster migrating bands on acid-urea polyacrylamide gels, reflection the progressive degradation of protamine. Ultimately, a specific and characteristic peptide banding pattern resulted. When sperm nuclei were treated with the esterase inhibitor nitrophenyl-p-guanidino benzoate (NPGB) to inhibit the nuclear-associated proteolytic activity and then incubated with one of several exogenous proteinases in addition to DTT and Triton X-100, characteristic peptide banding patterns were seen for each exogenous proteinase employed. For trypsin, chymotrypsin, pronase, and papain, the peptide banding patterns differed from one another and from the pattern characteristic of protamine degradation by the nuclear-associated proteinase. By contrast, when rabbit acrosin served as the exogenous proteinase, the peptide banding pattern seen was identical to the pattern characteristic of the nuclear-associated proteinase. These results demonstrate directly that the proteinase associated with rabbit sperm nuclei and involved in sperm nuclear decondensation in vitro is acrosinlike.     

The amino acid sequence of human sperm protamine P1

Human sperm protamines have been extracted from spermatozoa pooled from several donors, converted to their S-pyridylethylated derivatives and resolved into two major components, P1 and PI, by Bio-Rex 70 chromatography. Protamine P1 was further purified by Bio-Gel P-10 chromatography and sequenced directly on a gas phase protein sequencer for 43 residues. To complete the sequence, P1 was cleaved at methionine 36 and the C-terminal tetradecapeptide was purified by h.p.i.c , and sequenced completely. The 50 residue sequence is: 10 20 30 40 ARYRC CRSQS RSRYY RQRQR SRRRR RRSCQ TRRRA MRCCR 50 PRYRP RCRRH. This sequence has a calculated molecular weight of 6674 and is homologous with four other published mammalian protamine sequences.     

Interaction of the nuclear proteins protamine and histone with charged bilayer membranes]

The interaction of nuclear proteins of protamine and histone with neutral and charged BLM was studied. Anion and cation detergents were used to create the surface charge. The surface density of charges in BLM was comparable with that in biomembranes. Protamine and histone increased the electroconductivity of negatively charged BLM for anions and cations correspondingly. It is suggested that the surface charge of the membrane may influence the ion transport directly and indirectly due to the interaction of the membrane structures with charged proteins present in the surrounding medium.     

Purification and characterization of nuclear basic proteins of human sperm

High purified nuclei were obtained from human sperm without protein loss through the use of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), a newly available detergent. The basic protein complement of these nuclei is highly heterogeneous and comprises histones (some of which are testis-specific), protamines and proteins of intermediate basicity and molecular size. The protamines belong to two different classes of protein. Microheterogeneity observed in some of these protamines originates from slight variations in their amino acid composition as well as from post-synthetic modifications. Two of these protamines previously considered as two different proteins as in fact the same protein with different degrees of phophorylation. All these protamines and intermediate basic proteins are characterized by high amounts of arginine and cysteine. Three of the protamines and all five intermediate basic proteins are also histidine-rich.     

Relationships between bacteriospermia,DNA integrity,nuclear protamine alteration,sperm quality and ICSI outcome

The Aim of this study was to evaluate the effects of bacteriospermia on human sperm parameters, nuclear protamines, DNA integrity and ICSI outcome in patients enrolled for ICSI treatment. 84 unselected couples consulting in infertility and obstetrics clinic and enrolled for ICSI treatment were included in this study. The semen specimens were screened bacteriologically; semen and sperm parameters were also evaluated according to WHO guidelines. DNA integrity, protamines concentration and protamine deficiency were estimated by TUNEL assay, AU-PAGE and Chromomycin (CMA3) respectively. The results of this study revealed that 34.52% of studied semen samples were infected with bacteria. The isolated bacteria were identified as Staphylococcus aureus, Staph. epidermidis, Staph. haemolyticus, Escherichia coli, Enterococcus faecalis and Streptococcus agalactiae. Bacteriospermia had a significant (p?<?.010) negative effect on sperm parameters; concentration, motility, progressive motility and chromatin condensation. Moreover, high DNA fragmentation with low P1 and P2 concentrations were noticed in infected patients in comparison to non-infected patients but non-significant. Also, the fertilization rate decreased significantly (p?<?.05) with infected patients. In conclusion: bacteriospermia has significant negative effect on sperm quality and fertilization rate in patients who underwent ICSI treatment.     

Further evidence that sperm nuclear proteins are necessary for embryogenesis

We have recently presented evidence that the structural integrity of the mouse sperm nuclear matrix may be necessary for the proper unpackaging of sperm DNA for participation in embryogenesis. It is likely that the sperm nuclear matrix contributes to the organisation of the sperm DNA and its disturbance can seriously damage the paternal genome or its expression. In this work, we confirm our previous data and further suggest that even very subtle changes in the sperm nuclear structure may have a significant impact on embryo development. As reported previously, dithiothreitol (DTT) in the presence of an ionic detergent, ATAB, destabilized the nuclear matrix as measured by the halo assay, and oocytes injected with these nuclei failed to develop. We also discovered that omitting the protease inhibitor PMSF from the buffers used to extract spermatozoa prevented sperm injected into oocytes from participating in development. The organization of DNA into loop domains by the nuclear matrix in these nuclei appeared normal, as measured by the halo assay. Oocytes injected with sperm nuclei that had been washed with ATAB in the presence of phenylmethylsulphonyl fluoride (PMSF) but in the absence of DTT resulted in live births. Neither DTT treatment nor the absence of PMSF would be expected to disrupt the integrity of the paternal DNA. The data therefore suggest that even very subtle alterations in the structural proteins of the nucleus are enough to deprive sperm DNA of the ability to contribute to embryonic development.     

The PSI family of nuclear proteins is required for growth in arabidopsis

PSI1 was identified as a gene that is co-expressed with the phytosulfokine (PSK) receptor genes PSKR1 and PSKR2 in Arabidopsis thaliana. It represents a plant-specific protein family of unknown function with six members in two clades. Clade 1 members PSI1, PSI2 and PSI3 were characterized in this study. All three are nuclear localized. A predicted N-terminal myristoylation site was functionally analyzed. psi1-1 seedlings have shorter roots and hypocotyls. This growth-retarded phenotype was restored by expression of either wildtype PSI1 or PSI1 _G2A with a mutated myristate attachment site in the psi1-1 background suggesting that myristate attachment was not essential for PSI1 function. psi2-1 and psi3-1 seedlings have a wildtype phenotype but overexpression of PSI1 or PSI2 promoted seedling growth. PSI2 activity appears to be linked to PSK signaling as psi2-1 and psi2-1 psi3-1 roots are unresponsive to PSK. PSI3 functions in vegetative plant growth synergistic with PSI2. psi3-1 and particularly psi2-1 psi3-1 rosettes are small. Overexpression of PSI3 promoted plant growth indicating that PSI3 is limiting at the vegetative stage. Severe dwarfism of psi2-1 psi3-1 plants results from reduced cell growth and proliferation and premature leaf growth arrest. Plants further display reduced fertility and premature senescence revealing a crucial function of PSI proteins in vegetative growth and reproduction. Psi single and double knock-out plants have less and PSI3ox plants have more starch compared to wt and growth retardation is partially rescued by sucrose. Our studies reveal a crucial function of the nuclear-localized PSI proteins in growth possibly through metabolic control.     

The major protamine from stallion sperm. Isolation and amino-acid sequence

The major stallion protamine was isolated from sperm cell nuclei by extraction with 6M guanidine/5% mercaptoethanol, alkylation with 4-vinylpyridine and subsequent reversed-phase high-performance liquid chromatography. The primary structure of stallion protamine was determined by N-terminal sequencing of the intact protein and of the fragments obtained from thermolysin cleavage of the S-pyridylethylated and from endoproteinase Lys-C cleavage of the S-aminoethylated protein. Stallion protamine consists of 49 amino-acid residues and shows 49% identity with all other sequenced mammalian type 1 protamines.     

A new family of yeast nuclear pore complex proteins

We have identified a novel family of yeast nuclear pore complex proteins. Three individual members of this family, NUP49, NUP100, and NUP116, have been isolated and then characterized by a combination of molecular genetics and immunolocalization. Employing immunoelectron and immunofluorescence microscopy on yeast cells, we found that the binding of a polyspecific monoclonal antibody recognizing this family was predominantly at the nuclear pore complexes. Furthermore, the tagging of NUP49 with a unique epitope enabled the immunolocalization of this protein to the nuclear pore complex by both fluorescence and electron microscopy. DNA sequence analysis has shown that the amino-terminal regions of NUP49, NUP100, and NUP116 share repeated "GLFG" motifs separated from each other by glutamine, asparagine, serine and threonine rich spacers. All three proteins lack a repetitive domain found in the two precisely described yeast nuclear pore complex proteins. Only NUP49 is essential for cell viability. NUP116-deficient cells grow very slowly and are temperature sensitive, whereas the lack of NUP100 has no detectable phenotype. NUP100 and NUP116 are homologous over their entire lengths. Interestingly, NUP100 and NUP116 are both flanked by a histidine tRNA gene and a transposon element suggesting that they may have arisen by gene duplication. We propose that subfamilies of pore complex proteins can be defined by their characteristic combinations of different modular domains.     

Rat sperm protamine. Isolation and sequence analysis

Rat protamine was extracted from S-(pyridylethylated) epididymal sperm cell nuclei with dilute hydrochloric acid. The final purification was achieved by reversed-phase high-performance liquid chromatography. The primary structure was determined by N-terminal sequencing of the total S-(pyridylethylated) protein, and of endoproteinase Lys-C- and thermolysin-derived fragments. Rat protamine consists of 50 amino-acid residues. It is a typical type 1 protamine and differs in two and ten positions from the corresponding mouse and rabbit protamine, respectively. Only 26 positions are invariant in all type 1 mammalian protamines.     

Primary structure of rabbit sperm protamine, the first protamine of its type with an aberrant N-terminal

Rabbit protamine was extracted from S-(pyridylethylated) sperm cell nuclei with hydrochloric acid and then isolated by reversed-phase HPLC. The primary structure was determined by amino acid sequence analysis of the total protein and of fragments obtained by digestion with endoproteinase Lys-C and thermolysin. The protamine contains 49 amino acid residues and is clearly homologous with mammalian type 1 protamines, 47% of the positions being invariant. Surprisingly, rabbit protamine possesses an N-terminal valine residue, whereas all mammalian and several non-mammalian protamine sequences of this type start with alanine, the N-terminal region being remarkably conserved during evolution.     

A comparative study of the basic nuclear proteins from sperm of bivalve molluscs

• 1.1. Basic nuclear proteins from spermatozoa of the three mollusc species belonging to the class Bivalvia have been analyzed using one- and two-dimensional electrophoresis.
• 2.2. Four nuclear basic proteins have been purified and their amino acid compositions determined.
• 3.3. In these spermatozoa histone-type proteins coexist with protamine-like proteins.
• 4.4. The protamine-like proteins that have been studied show different electrophoretic behavior but in general are similar, with a high content of lysine, arginine, alanine and serine.
• 5.5. Interspecific variability has been found for the H1-like histone.

     

The tubby family proteins

The tubby mouse shows a tripartite syndrome characterized by maturity-onset obesity, blindness and deafness. The causative gene Tub is the founding member of a family of related proteins present throughout the animal and plant kingdoms, each characterized by a signature carboxy-terminal tubby domain. This domain consists of a β barrel enclosing a central α helix and binds selectively to specific membrane phosphoinositides. The vertebrate family of tubby-like proteins (TULPs) includes the founding member TUB and the related TULPs, TULP1 to TULP4. Tulp1 is expressed in the retina and mutations in TULP1 cause retinitis pigmentosa in humans; Tulp3 is expressed ubiquitously in the mouse embryo and is important in sonic hedgehog (Shh)-mediated dorso-ventral patterning of the spinal cord. The amino terminus of these proteins is diverse and directs distinct functions. In the best-characterized example, the TULP3 amino terminus binds to the IFT-A complex, a complex important in intraflagellar transport in the primary cilia, through a short conserved domain. Thus, the tubby family proteins seem to serve as bipartite bridges through their phosphoinositide-binding tubby and unique amino-terminal functional domains, coordinating multiple signaling pathways, including ciliary G-protein-coupled receptor trafficking and Shh signaling. Molecular studies on this functionally diverse protein family are beginning to provide us with remarkable insights into the tubby-mouse syndrome and other related diseases.     

The Homer family proteins

The Homer family of adaptor proteins consists of three members in mammals, and homologs are also known in other animals but not elsewhere. They are predominantly localized at the postsynaptic density in mammalian neurons and act as adaptor proteins for many postsynaptic density proteins. As a result of alternative splicing each member has several variants, which are classified primarily into the long and short forms. The long Homer forms are constitutively expressed and consist of two major domains: the amino-terminal target-binding domain, which includes an Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) homology 1 (EVH1) domain, and the carboxy-terminal self-assembly domain containing a coiled-coil structure and leucine zipper motif. Multimers of long Homer proteins, coupled through their carboxy-terminal domains, are thought to form protein clusters with other postsynaptic density proteins, which are bound through the amino-terminal domains. Such Homer-mediated clustering probably regulates or facilitates signal transduction or cross-talk between target proteins. The short Homer forms lack the carboxy-terminal domain; they are expressed in an activity-dependent manner as immediate-early gene products, possibly disrupting Homer clusters by competitive binding to target proteins. Homer proteins are also involved in diverse non-neural physiological functions.     

Interactions of nuclear proteins with DNA,during sperm differentiation in the ram

Ram spermatid nuclei and caput epididymal sperm nuclei were prepared and treated with DTT under conditions avoiding proteolysis. Whole-mount preparations for the electron microscope were made in the presence or absence of the detergent Joy. The chromatin of the less mature, non-round spermatid nuclei displayed a nucleosomal organization that gradually disappeared at the time the histones leave the nuclei (elongating spermatids). Digestion with micrococcal nuclease suggests that polynucleosome arrays are scarcer and more accessible to nuclease in the elongating than in the round nuclei, with increasing amounts of DNA becoming devoid of nuleosomes. In the protamine-containing nuclei (elongated spermatids), only smooth filaments were observed, which formed thick fibers by parallel aggregation. The change from a nucleosomal organization to bundles of smooth filaments appeared to result from a complex process involving the transitory presence of conspicuous knobby fibers that suggest a periodicity in the organization of the spermatidal proteins along the DNA molecules. X-ray diffraction patterns obtained with protamine-containing spermatid nuclei and with sperm nuclei confirm that the DNA is arranged in smoothly bent bundles of parallel molecules. No higher-order reflections that might correspond to nucleosome structures were detected in the 30–200 Å region.