Mechanism of liquefaction of the human ejaculate. II. Role of collagenase-like peptidase and seminal proteinase.

Collagenase-like peptidase and seminal proteinase were isolated from human testis and human seminal plasma. The effects of both enzymes upon proteins isolated from the human ejaculate were studied. Both enzymes degraded ejaculate proteins. The data suggest that collagenase-like peptidase is responsible for the first, and seminal proteinase for the second, phase of human ejaculate liquefaction in vitro.     

Transfer of molecules from ejaculate to females in Drosophila melanogaster and Drosophila pseudoobscura

Methionine in the form of free amino acids, small peptides and several proteins is transferred from ejaculate to females in Drosophila melanogaster and D. pseudoobscura. The male contribution is found mostly in the ovary and the sizes of the polypeptides transferred have been established. Some components of the ejaculate are rapidly transferred to the somatic tissues outside the ovary and reproductive system where they co-migrate with the yolk polypeptides. The incorporation of free methionine into polypeptides in non-reproductive tissues is higher in females that have been deprived of a source of protein prior to mating. The molecules transferred from ejaculate may have roles in regulating reproductive behaviour and may also be used as a nutrient source. We suggest that free amino acids in the ejaculate are rapidly utilised in protein synthesis in the somatic tissues outside the ovary and could provide a boost to yolk protein and hence egg production in females in nature, where nutrients may be in limited supply.     

Isolation and characterization of soybean waste-degrading microorganisms and analysis of fertilizer effects of the degraded products.

Two microorganisms which could degrade soybean lees efficiently were isolated and identified as Bacillus circulans and B. stearothermophilus. These two strains secreted thermostable proteases into the medium and could digest soybean lees rapidly and completely at 50 degrees C. Initially, the soybean lees were degraded to proteins in approximately 20 h by these two strains, after which time the concentrations of peptides in the medium gradually increased. The degraded products from soybean lees contained abundant nitrogen compounds, such as peptides, amino acids, and amides. Approximately 10 times more fresh plant weight was obtained (in the case of Brassica campestris) when these degraded products were applied than when water was applied for 42 days. These stimulatory effects of the soybean lees products were almost equal to those of a chemically synthesized fertilizer.     

Sexual selection and the adaptive evolution of mammalian ejaculate proteins

An elevated rate of substitution characterizes the molecular evolution of reproductive proteins from a wide range of taxa. Although the selective pressures explaining this rapid evolution are yet to be resolved, recent evidence implicates sexual selection as a potentially important explanatory factor. To investigate this hypothesis, we sought evidence of a high rate of adaptive gene evolution linked to postcopulatory sexual selection in muroid rodents, a model vertebrate group displaying a broad range of mating systems. Specifically, we sequenced 7 genes from diverse rodents that are expressed in the testes, prostate, or seminal vesicles, products of which have the potential to act in sperm competition. We inferred positive Darwinian selection in these genes by estimation of the ratio of nonsynonymous (d(N), amino acid changing) to synonymous (d(S), amino acid retaining) substitution rates (omega = d(N)/d(S)). Next, we tested whether variation in this ratio among lineages could be attributed to interspecific variation in mating systems, as inferred from the variation in these rodents' relative testis sizes (RTS). Four of the 7 genes examined (Prm1, Sva, Acrv1, and Svs2, but not Svp2, Msmb, or Spink3) exhibit unambiguous evidence of positive selection. One of these, the seminal vesicle-derived protein Svs2, also shows some evidence for a concentration of positive selection in those lineages in which sperm competition is common. However, this was not a general trend among all the rodent genes we examined. Using the same methods, we then reanalyzed previously published data on 2 primate genes, SEMG1 and SEMG2. Although SEMG2 also shows evidence of positive selection concentrated in lineages subject to high levels of sperm competition, no such trend was found for SEMG1. Overall, despite a high rate of positive selection being a feature of many ejaculate proteins, these results indicate that the action of sexual selection potentially responsible for elevated evolutionary rates may be difficult to detect on a gene-by-gene basis. Although the extreme diversity of reproductive phenotypes exhibited in nature attests to the power of sexual selection, the extent to which this force predominates in driving the rapid molecular evolution of reproductive genes therefore remains to be determined.     

Degradation of abnormal proteins in peptidase-deficient mutants of Salmonella typhimurium.

The degradation of abnormal proteins produced as a result of incorporation of the arginine analog L-canavanine or generated by exposure to puromycin was studied in wild-type and multiply peptidase-deficient strains of Salmonella typhimurium. Both types of abnormal protein were rapidly degraded during growth of Pep+ strains of this organism. Peptidase--deficient mutants (lacking peptidases N, A, B, and D) could also degrade these abnormal proteins, although the rate of production of trichloroacetic acid-soluble degradation products was slower in the mutant strain than in a strain carrying a normal complement of peptidases. Analysis of these trichloroacetic acid-soluble degradation products of ion-exchange chromatography showed that free amino acid was the major breakdown product produced by the wild-type strain. The acid-soluble degradation product produced by the mutant strain, however, was a complex mixture that contained a variety of small peptides as well as free amino acids. These results indicate that the same group of peptidases shown previously to function in the degradation of exogenously supplied peptides and in protein turnover during carbon starvation also lie on the pathway by which abnormal proteins are degraded.     

Concordance of mammalian ejaculate features.

Different measures of ejaculate characteristics, such as ejaculate volume, proportion of normal sperm, proportion of motile sperm, and total number of sperm per ejaculate, are directly related to the probability of fertilization both when females copulate with a single male and particularly with multiple males. Selection will therefore favour the evolution of ejaculate characteristics which enhance the probability of fertilization, and I predict positive relations between ejaculate parameters. I used a literature survey of mammalian ejaculate parameters to test this prediction. The data set was corrected for similarity between taxa which resulted from common ancestry, and was reduced to statistically independent standardized linear contrasts. The number of sperm per ejaculate and ejaculate volume were positively related to body mass, but when the confounding effect of body mass was controlled for, all four ejaculate characteristics showed positive relations, and five out of six were statistically significant. This suggests that the different measures of ejaculate quality in mammals have been improved simultaneously, apparently by a common selective force.     

The male ejaculate as inhibitor of female remating in two tephritid flies

The inhibition of female receptivity after copulation is usually related to the quality of the first mating. Males are able to modulate female receptivity through various mechanisms. Among these is the transfer of the ejaculate composed mainly by sperm and accessory gland proteins (AGPs). Here we used the South American fruit fly Anastrepha fraterculus (where AGP injections inhibit female receptivity) and the Mexican fruit fly Anastrepha ludens (where injection of AGPs failed to inhibit receptivity) as study organisms to test which mechanisms are used by males to prevent remating. In both species, neither the act of copulation without ejaculate transfer nor sperm stored inhibited female receptivity. Moreover, using multiply mated sterile and wild males in Mex flies we showed that the number of sperm stored by females varied according to male fertility status and number of previous matings, while female remating did not. We suggest female receptivity in both flies is inhibited by the mechanical and/or physiological effect of the full ejaculate. This finding brings us closer to understanding the mechanisms through which female receptivity can be modulated.     

The lack of effect of ejaculate sequence on sex ratio in rabbits.

The influence of the order of the ejaculate on the sex ratio was determined by ejaculating 1 of 5 experimental males 20 times over a period of 2 to 8 h. The sex ratio was noted for the offspring of ejaculates 1, 5, 10, 15 and 20 in a total of 32 litters. The sex ratio of these offspring did not deviate signigicantly from 50:50 (P greater than 0.1) for any ejaculate in the sequence.     

Condition-dependent ejaculate size and composition in a ladybird beetle

Sexually selected male ejaculate traits are expected to depend on the resource state of males. Theory predicts that males in good condition will produce larger ejaculates, but that ejaculate composition will depend on the relative production costs of ejaculate components and the risk of sperm competition experienced by low- and high-condition males. Under some conditions, when low condition leads to poorer performance in sperm competition, males in low condition may produce ejaculates with higher sperm content relative to their total ejaculate and may even transfer more sperm than high-condition males in an absolute sense. Previous studies in insects have shown that males in good condition transfer larger ejaculates or more sperm, but it has not been clear whether increased sperm content represents a shift in allocation or simply a larger ejaculate, and thus the condition dependence of ejaculate composition has been largely untested. We examined condition dependence in ejaculate by manipulating adult male condition in a ladybird beetle (Adalia bipunctata) in which males transfer three distinct ejaculate components during mating: sperm, non-sperm ejaculate retained within the female reproductive tract, and a spermatophore capsule that females eject and ingest following mating. We found that high condition males indeed transferred larger ejaculates, potentially achieved by an increased rate of ejaculate transfer, and allocated less to sperm compared with low-condition males. Low-condition males transferred ejaculates with absolutely and proportionally more sperm. This study provides the first experimental evidence for a condition-dependent shift in ejaculate composition.     

Biochemistry of the autolytic processes in Antarctic krill post mortem. Autoproteolysis.

1. Autoproteolysis post mortem was examined at 0 degree C by following the changes in the major classes of krill (Euphausia superba and Euphausia crystallorophias) proteins and by liberation of peptides and free amino acids, and was based on experiments conducted on board expedition vessels in the Antarctic. 2. Primarily salt-soluble proteins were broken down during the first week of incubation, whereas water-soluble and insoluble proteins were degraded to a much smaller extent. The enzymes responsible for the hydrolysis presumably originate primarily from the digestive apparatus of the krill. 3. In general, the individual amino acids were released at rates corresponding to their relative occurrence in the bulk protein of the krill. Alanine was liberated in larger amounts than would be expected from the composition of the krill protein, and was evidently formed also by reactions other than proteolysis. Glutamic acid, and certain amino acids which presumably occur with high frequency adjacent to glumatic acid residues in the krill protein, were liberated only to a limited extent, and accumulated in smaller peptides. 4. During proteolysis, arginine seemed to be converted to some degree into ornithine, and on prolonged incubation conversion of arginine and lysine into their corresponding decarboxylation products, agmatine and cadaverine, appeared to take place.     

Male age and strain affect ejaculate quality in the Mexican fruit fly

Aging in all organisms is inevitable. Male age can have profound effects on mating success and female reproduction, yet relatively little is known on the effects of male age on different components of the ejaculate. Furthermore, in mass‐reared insects used for the Sterile Insect Technique, there are often behavioral differences between mass‐reared and wild males, while differences in the ejaculate have been less studied. The ejaculate in insects is composed mainly of sperm and accessory gland proteins. Here, we studied how male age and strain affected (i) protein quantity of testes and accessory glands, (ii) the biological activity of accessory gland products injected into females, (iii) sperm viability, and (iv) sperm quantity stored by females in wild and mass‐reared Anastrepha ludens (Diptera: Tephritidae). We found lower protein content in testes of old wild males and lower sperm viability in females mated with old wild males. Females stored more sperm when mated to young wild males than with young mass‐reared males. Accessory gland injections of old or young males did not inhibit female remating. Knowledge of how male age affects different ejaculate components will aid our understanding on investment of the ejaculate and possible postcopulatory consequences on female behavior.     

Exposure to the seminal plasma of different portions of the boar ejaculate modulates the survival of spermatozoa cryopreserved in MiniFlatPacks

Spermatozoa present in the first collectable 10 mL of the sperm-rich fraction (SRF) of the boar ejaculate (portion 1, P1) have higher documented viability during and after cryopreservation than spermatozoa in the rest of the ejaculate (portion 2, P2), probably in relation to different features of the surrounding seminal plasma (SP). In the present study, we investigated whether the SP from these ejaculate portions (SP1 or SP2) was able to differently influence sperm viability and chromatin structure of the P1- or P2-contained spermatozoa from individual boars primarily or secondarily exposed (e.g., following cleansing and re-exposure) to pooled SP1 or SP2 from the same males during 60 min. Spermatozoa were subjected to controlled cooling and thawing in MiniFlatPacks (MFPs) and examined for motility (using computer-assisted sperm analysis, CASA) at selected stages of processing. Moreover, sperm plasma membrane intactness (investigated using SYBR-14/propidium iodide, PI), plasma membrane architecture (examined using Annexin-V-PI staining), and chromatin (deoxyribonucleic acid, DNA) integrity (tested using sperm chromatin structure assay, SCSA) were assessed post-thaw (PT). A higher proportion of P1 spermatozoa than of P2 spermatozoa incubated in their native SP portion were confirmed to be motile from collection to PT. When P1 spermatozoa were cleansed from their original SP and re-exposed to pooled P2-SP, sperm kinematics deteriorated from extension to PT. By contrast, cleansed P2 spermatozoa increased motility to P1 levels, especially PT when re-exposed to pooled P1-SP. Such differential effects on motility were not clearly accompanied by biologically related modifications of sperm membrane or chromatin structure. This influence of the SP on sperm kinematics was not sire-dependent and it was presumably related to different concentrations or either SP proteins or bicarbonate in the different ejaculate portions.     

Influence of the yeast strain on the changes of the amino acids,peptides and proteins during sparkling wine production by the traditional method

The influence of five yeast strains on the nitrogen fractions, amino acids, peptides and proteins, during 12 months of aging of sparkling wines produced by the traditional or Champenoise method, was studied. High-performance liquid chromatography (HPLC) techniques were used for analysis of the amino acid and peptide fractions. Proteins plus polypeptides were determined by the colorimetric Bradford method. Four main stages were detected in the aging of wines with yeast. In the first stage, a second fermentation took place; amino acids and proteins plus polypeptides diminished, and peptides were liberated. In the second stage, there was a release of amino acids and proteins, and peptides were degraded. In the third stage, the release of proteins and peptides predominated. In the fourth stage, the amino acid concentration diminished. The yeast strain used influenced the content of free amino acids and peptides and the aging time in all the nitrogen fractions. Received 25 March 2002/ Accepted in revised form 31 July 2002     

Purification and characterization of additional low-molecular-weight basic proteins degraded during germination of Bacillus megaterium spores.

Dormant spores Bacillus megaterium contained a group of low-molecular-weight (5,000 to 11,000) basic (pI greater than 9.4) proteins (termed D, E, F, and G proteins) which could be extracted from disrupted spores with strong acids. These proteins were distinct from the previously described A, B, and C proteins which are degraded during spore germination. However, the D, E, F, and G proteins were also rapidly degraded during spore germination, accounting for 10 to 15% of the protein degraded. Proteins similar to the D, E, F, and G species were also present in spores of other bacterial species. In B. megaterium, the D, E, F, and G proteins were low or absent (less than 15% of the spore level) in vegetative and young sporulating cells and appeared only late in sporulation. The D, E, F, and G proteins were purified to homogeneity, and all contained a high percentage of hydrophilic amino acids; one protein (G) contained 31% basic amino acids and also contained tryptophan. All four proteins were rapidly degraded in vitro by dormant spore extracts. Two proteins (D and F) were degraded in vitro by the previously described spore protease which initiates degradation of the A, B, and C proteins in vivo; the spore enzyme (s) degrading proteins E and G have not been identified.     

Abnormal proteins of shortened length are preferentially degraded in the cytosol of cultured MRC5 fibroblasts

Puromycyl peptides were degraded in MRC5 fibroblasts more rapidly than normal proteins labelled for the corresponding length of time for both long and short labelling periods. The degradation of the puromycyl peptides occurred almost exclusively in the cytosol of the cells. Even when the half-lives of normal and puromycyl peptides were manipulated to be similar, proportionally more of the normal proteins were degraded in the lysosomes. The rapid degradation of the puromycyl peptides was not due to the inhibition of protein synthesis brought about by puromycin but was due to the structure of the substrates themselves. The degree and intracellular site of degradation of puromycyl peptides closely mimic those of abnormal (missense) proteins containing amino acid analogues.     

N-terminal sequence analysis of polypeptide at the picomole level.

This paper describes a manual method for N-terminal sequence analysis of polypeptides at subnanomole sensitivity. The polypeptide is degraded stepwise by using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method, and the released dimethylaminoazobenzenethiohydantoins of amino acids were identified by reversed-phase high-pressure liquid chromatography. The dimethylaminoazobenzenethiohydantoins are coloured compounds and can be detected in the visible region with the sensitivity limit of 1 pmol (signal-to-baseline noise ratio 5). A high-pressure liquid-chromatographic method was developed for complete analysis of all amino acid dimethylaminoazobenzenethiohydantoin derivatives, including the by-products of serine and threonine. Thus, without use of an automatic sequenator or radioactive materials, it is possible to determine the complete sequence of peptides and N-terminal sequence of proteins with less than 1 nmol of material.     

Sperm competition, mating rate and the evolution of testis and ejaculate sizes: a population model

There are various ways to estimate ejaculate expenditure. Ejaculate size or sperm number (s) is an absolute number of units of ejaculate. Relative ejaculate expenditure (E) is the expenditure on the ejaculate as the proportion of the total expenditure on all aspects of the mating, including finding and acquiring a female, and so on. Relative testis size or gonadosomatic index (sigma) is testes mass divided by body mass; it is assumed to reflect the product of mating rate (M) and ejaculate mass (s). In a new model, where mating rate, sperm competition and sperm allocation interact, and where the female's inter-clutch interval is assumed to be independent of s or M, we show that sigma is directly proportional to the mean E for a species; across species sigma and E increase monotonically with sperm competition. However, the relation between s and sperm competition across species depends on the range of sperm competition (low risk or high intensity): s increases with sperm competition at low risk levels, but decreases with sperm competition at high intensity levels. This situation arises because s alpha E/M; both E and M increase with sperm competition, but E increases differently with sperm competition in its two ranges.     

Pathway for degradation of peptides generated by proteasomes: a key role for thimet oligopeptidase and other metallopeptidases

The degradation of cellular proteins by proteasomes generates peptides 2-24 residues long, which are hydrolyzed rapidly to amino acids. To define the final steps in this pathway and the responsible peptidases, we fractionated by size the peptides generated by proteasomes from beta-[14C]casein and studied in HeLa cell extracts the degradation of the 9-17 residue fraction and also of synthetic deca- and dodecapeptide libraries, because peptides of this size serve as precursors to MHC class I antigenic peptides. Their hydrolysis was followed by measuring the generation of smaller peptides or of new amino groups using fluorescamine. The 14C-labeled peptides released by 20 S proteasomes could not be degraded further by proteasomes. However, their degradation in the extracts and that of the peptide libraries was completely blocked by o-phenanthroline and thus required metallopeptidases. One such endopeptidase, thimet oligopeptidase (TOP), which was recently shown to degrade many antigenic precursors in the cytosol, was found to play a major role in degrading proteasome products. Inhibition or immunodepletion of TOP decreased their degradation and that of the peptide libraries by 30-50%. Pure TOP failed to degrade proteasome products 18-24 residues long but degraded the 9-17 residue fraction to peptides of 6-9 residues. When aminopeptidases in the cell extract were inhibited with bestatin, the 9-17 residue proteasome products were also converted to peptides of 6-9 residues, instead of smaller products. Accordingly, the cytosolic aminopeptidase, leucine aminopeptidase, could not degrade the 9-17 residue fraction but hydrolyzed the peptides generated by TOP to smaller products, recapitulating the process in cell extracts. Inactivation of both TOP and aminopeptidases blocked the degradation of proteasome products and peptide libraries nearly completely. Thus, degradation of most 9-17 residue proteasome products is initiated by endoproteolytic cleavages, primarily by TOP, and the resulting 6-9 residue fragments are further digested to amino acids by aminopeptidases.     

Resistance of proline-containing peptides to ruminal degradation in vitro.

Mixed ruminal bacteria utilized an enzymatic digest of casein at a rate faster than that for an enzymatic digest of gelatin, but neither amino acid source was completely utilized even when the incubation period was as long as 96 h. Since the reaction of ninhydrin with the residual nonammonia, nonprotein nitrogen was more than twofold stronger when the samples were hydrolyzed with 6 N HCl, it appeared that much of the residual nitrogen was from peptides. Approximately 66% of the nonammonia, nonprotein, ninhydrin-reactive material could not be recovered as amino acids, but there was a significant decrease in total amino acid nitrogen when the samples were pretreated with a C18 Sep-Pak column to remove peptides. The resistant peptides had an abundance of proline, and subsequent incubations showed that synthetic dipeptides which contained proline were hydrolyzed slowly. Lysine appears to be the amino acid which is most apt to limit ruminant production. Dipeptides containing proline and lysine were hydrolyzed at least fivefold slower than lysine-alanine. Methionine, another potentially limiting amino acid, was also degraded at a slower (2.5-fold) rate when it was present as part of a proline dipeptide.     

Resistance of proline-containing peptides to ruminal degradation in vitro.

Mixed ruminal bacteria utilized an enzymatic digest of casein at a rate faster than that for an enzymatic digest of gelatin, but neither amino acid source was completely utilized even when the incubation period was as long as 96 h. Since the reaction of ninhydrin with the residual nonammonia, nonprotein nitrogen was more than twofold stronger when the samples were hydrolyzed with 6 N HCl, it appeared that much of the residual nitrogen was from peptides. Approximately 66% of the nonammonia, nonprotein, ninhydrin-reactive material could not be recovered as amino acids, but there was a significant decrease in total amino acid nitrogen when the samples were pretreated with a C18 Sep-Pak column to remove peptides. The resistant peptides had an abundance of proline, and subsequent incubations showed that synthetic dipeptides which contained proline were hydrolyzed slowly. Lysine appears to be the amino acid which is most apt to limit ruminant production. Dipeptides containing proline and lysine were hydrolyzed at least fivefold slower than lysine-alanine. Methionine, another potentially limiting amino acid, was also degraded at a slower (2.5-fold) rate when it was present as part of a proline dipeptide.