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.     

Secondary structure of protamine in sperm nuclei: an infrared spectroscopy study

Background  

Protamines are small basic proteins that condense the DNA in mature spermatozoa. Typical protamines are of simple composition and very arginine-rich, usually in the range of 60-80%. Arginine residues are distributed in a number of stretches separated by neutral amino acids. We have used Fourier transform infrared spectroscopy (FTIR) to gain access for the first time to the secondary structure of protamines in sperm nuclei. This technique is particularly well suited to the study of DNA-bound protamine in whole nuclei since it is not affected by turbidity.     

Primary structure of the ram (Ovis aries) protamine

The amino acid sequence of the protamine isolated from mature sperm nuclei of the ram (Ovis aries) has been established from automated sequence analysis of the S-carboxymethylated protamine. Ram and bull protamines differ only by two point changes and the deletion in bull protamine of the tripeptide Cys39-Arg-Arg41. In mammalian protamines the central region (residues 13-36) consisting mainly of arginine clusters appears to be conserved whereas the N-terminal and C-terminal regions are more variable.     

Primary structure of protamine from the Northern pike Esox lucius

The basic nuclear protein in the sperm of the Northern pike is a protamine, 32-residues long, which behaved as a single component during ion-exchange chromatography and gel electrophoresis. Amino acid analysis gave close to molar ratios for the eight different residues with no evidence of microheterogeneity. However, the presence of sequence variants was revealed following a combination of automated protein sequencing and cleavage of the protamine by CNBr, endoproteinase Lys-C and thermolysin. At position 28 there is an equal probability of having serine or glycine. At position 9 glycine is found more frequently than serine. The reciprocal nature of the substitutions results in glycine and serine contents which are close to a 4:2 ratio. Pike protamines are homologous to those of the trout but show less sequence variation between components.     

Factors which may affect removal of protamine from sperm DNA during fertilization in the rabbit

In order to derive information about possible mechanisms by which the sperm head is converted into the male pronucleus during fertilization in the rabbit, unfertilized egg homogenate was assayed for two enzyme activities. Protamine was extracted from rabbit sperm, purified, and labelled with [¹⁴C] in an in vitro reaction and used as a probe to assay for a protein kinase which could transfer [³²P]PO₄ from [γ-³²P]ATP onto the substrate. A kinase with a pH optimum of approximately 8.0 to 8.5 is described. Assays for the enzyme glutathione reductase were performed using homogenates from eggs or embryos at three early stages of development. Results suggest that oocytes can oxidize 2.58 × 10^?6 μmol NADPH per minute per oocyte, unfertilized eggs 5.16 × 10^?7 μmol NADPH per minute per ovum, and 20- to 24-hour postcoitus fertilized eggs 2.30 × 10^?6 μmol NADPH per minute per ovum. The relevance of these observations to male pronuclear formation is discussed.     

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.     

Primary structure of sperm whale luteinizing hormone

Luteinizing hormone was extracted from sperm whale pituitaries and separated into alpha- and beta-subunits. These subunits were cleaved with cyanogen bromide, and digested with trypsin and chymotrypsin. The fragments obtained were separated and purified by gel filtration on Sephadex and by ion exchange chromatography, reversed phase chromatography and chromatoelectrophoresis. The amino acid sequence of peptides obtained was studied by dansyl-Edman's method and Edman's modification of Chang et al. The study made it possible to establish the complete amino acid sequence of sperm whale LH alpha- and beta-subunits.     

Primary structure of rabbit alpha-lactalbumin.

Rabbit alpha-lactalbumin was purified from the milk of New Zealand White rabbits. It was found to exist predominantly as a glycoprotein, containing 5 mol of glucosamine per mol of protein, as well as other sugars. The amino acid sequence of the protein was determined by sequenator analysis and carboxypeptidase digestion. There are 122 amino acids in the protein and a single carbohydrate moiety, probably attached to an asparagine residue at position 45. The C terminus of rabbit alpha-lactalbumin is one residue shorter than that of the other alpha-lactalbumins. Sequence comparisons indicate that the alpha-lactalbumin gene has been undergoing more frequent mutation than had previously been thought. A new method of preparative peptide mapping using 2,5-diphenyloxazole (PPO) fluor to detect peptides is described.     

Factors affecting the respiration of rabbit sperm measured with an oxygen electrode

The respiration of rabbit sperm was measured by a Clarke type electrode which has two advantages over the conventional Warburg technique, greater sensitivity, and no necessity for a carbon dioxide-free atmosphere. It was not necessary to resaturate the sample chamber of the oxygen monitor with air, down to about 30% desaturation.Rabbit seminal plasma had a measurable oxygen uptake (0.6 μl/hr/ml) but this was much less than for human seminal plasma (4.3 μl/hr/ml). Hoderate dilution of the sperm and storing the semen at 0°C after slow cooling had no effect on oxygen uptake. Unlike those of most other species, rabbit sperm were also little affected by deliberate exposure to cold shock and the respiration before and after rapid cooling to 0°C was about the same. On the other hand very brief exposure of rabbit sperm to 65°C abolished motility and greatly reduced the respiration rate. Bicarbonate (6 mM) stimulated the oxygen uptake of freshly collected samples of rabbit sperm after washing. Increasing the phosphate concentration of the medium to 80 mM did not greatly depress the oxygen uptake.     

Primary structure of rabbit lens α-crystallins

The primary structure and posttranslational modifications of rabbit lens α-crystallins were examined using electrospray ionization mass spectrometry to determine the molecular weights of the intact proteins and fast atom bombardment mass spectrometry to analyze proteolytic digests of the αA- and αB-crystallins. The previously determined primary structure of αA-crystallin was confirmed. Posttranslational modifications detected included one phosphorylation site and the presence of a truncated form minus the five C-terminal residues. The previously undetermined amino acid sequence of rabbit αB-crystallin was determined to be the same as the bovine αB-crystallin sequence except at three residues: Thr 40, Thr 132, and Pro 153. Rabbit αB-crystallin showed evidence of phosphorylation at the same three sites as bovine αB-crystallin. The molecular weights of the intact proteins indicated that any one molecule had a maximum of two phosphorylations. Also, there was a truncated form which did not include the five C-terminal residues.     

Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion

A variety of testicular insults can induce changes in the structure of spermatozoal chromatin, resulting in spermatozoal DNA that is more susceptible to acid-induced denaturation. The degree of change in the DNA can be measured using the sperm chromatin structure assay (SCSA). The SCSA measures the relative amounts of single- and double-stranded DNA after staining with the metachromatic dye, acridine orange. Here we used a stallion model (n = 4) to study the effects of scrotal heat stress on spermatozoal DNA. This model was created by insulating stallion testes for 48 h and collecting sperm daily thereafter for 60 days. Changes in the SCSA were then correlated with protamine disulfide content and protamine types and levels. Results of the SCSA indicated that the susceptibility of spermatozoal DNA to denaturation was dependent on the spermatogenic cell stage that the ejaculated sperm was in at the time of the heat stress. Spermatozoa with altered DNA had a decrease in the extent of disulfide bonding that was associated with an increase in the susceptibility of DNA to denaturation. However, there were no detectable changes in either the protamine type or level. Thus, in this model, decreased disulfide bonding is associated with an increased susceptibility of spermatozoal DNA to denaturation in the absence of protamine changes.     

Formation of native-like mammalian sperm cell chromatin with folded bull protamine

The DNA of most vertebrate sperm cells is packaged by protamines. The primary structure of mammalian protamine I can be divided into three domains, a central DNA binding domain that is arginine-rich and amino- and carboxyl-terminal domains that are rich in cysteine residues. In native bull sperm chromatin, intramolecular disulfide bonds hold the terminal domains of bull protamine folded back onto the central DNA binding domain, whereas intermolecular disulfide bonds between DNA-bound protamines help stabilize the chromatin of mature mammalian sperm cells. Folded bull protamine was used to condense DNA in vitro under various solution conditions. Using transmission electron microscopy and light scattering, we show that bull protamine forms particles with DNA that are morphologically similar to the subunits of native bull sperm chromatin. In addition, the stability provided by intermolecular disulfide bonds formed between bull protamine molecules within in vitro DNA condensates is comparable with that observed for native bull sperm chromatin. The importance of the bull protamine terminal domains in controlling the bull sperm chromatin morphology is indicated by our observation that DNA condensates formed under identical conditions with a fish protamine, which lacks cysteine-rich terminal domains, do not produce as uniform structures as bull protamine. A model is also presented for the bull protamine.DNA complex in native sperm cell chromatin that provides an explanation for the positions of the cysteine residues in bull protamine that form intermolecular disulfide bonds.     

Primary structure of the basic nuclear protein from spermatozoa of the mollusk Illex argentinus and its comparison with the structure of sperm proteins in other animals

The spermatic protein of chromatin I2 of squid Illex argentinus was separated by HPLC into two components I2-1 and I2-2. Amino acid sequences of the major portion of protein I2-1 (52 residues) and the N-terminal sequence of protein I2-2 (21 residues) were determined. Arginines in protein I2-1 are arranged in clusters typical of protamines; the first cluster is in the N-terminus, the longest heterogeneous basic cluster is in the central part of the protein chain, the C-terminal part of the molecule contains two clusters of three hydroxyamino acids each. The N-terminal sequences of illexins I2-1 and I2-2 (1-14 residues) are highly homologous. Homologous regions were found in illexin I2-1, tunnin of tuna fish and avian gallin thus defining the notion of proteins of an intermediate type from mollusc spermatozoa chromatin exemplified by the squid protamine-like protein.