Sperm aging in the male and cytogenetic anomalies. An animal model

Summary Superovulated females, outbred ICR Swiss, were inseminated naturally with spermatozoa aged 6–20 days in the male genital tract by bilateral ligation of the corpus epididymis. Females inseminated by the males (also ICR Swiss) mating at 3-day intervals prior to ligation and those mated to sham-operated males served as controls. A total of 1167 fertilized one-cell zygotes of which 868 were at metaphase first cleavage were recovered 33–35 h post-HCG. First-cleavage divisions were analyzed in 631, or 72%, of all zygotes at metaphase and late prophase. The gametic origin of chromosome anomalies was determined on the basis of differential condensation of the chromosomes. The sex ratio of the zygotes was unaffected by aging sperm. In 321 zygotes from the controls the frequency of trisomy was 3.1%, monosomy 2.2%, triploidy 0.93%, and structural rearrangements 0.31%. Aging of sperm for 6 days did not significantly alter the number of heteroploid zygotes recovered. For the periods beyond day 6 and in the combined experimental series there was a highly significant increase in the number of all chromosome anomalies compared with controls. In the experimental zygotes, the incidence of trisomy was 9.7%, monosomy 7.4%, triploidy 3.9%, and structural anomalies 2.6%. There were also significant differences between control and experimental in the origin of the anomalies. The male genome was implicated significantly more often than the female in the origin of trisomies in the experimental series compared with the controls. It was also the direct source of all the structural anomalies and the majority of the triploids in the experimentals, where 8 of 9 were a result of diploid sperm. Therefore, the over-riding mechanism involved in the production of chromosomally anomalous offspring from aging sperm seems to be altered conditions of competition between chromosomally balanced and unbalanced sperm. Additionally, chromosomally normal sperm in an aging population may be affected in some aspects of their physiology so that they create preferential loss of maternally derived chromosomes leading to monosomy or nuclear fragmentation. The implications of these findings for the etiology of human chromosome anomalies are discussed.     

Analysis of the chromosome complement in outbred mouse sperm fertilizing in vitro

The chromosome complements in a population of mouse sperm from random-bred ICR donors were analyzed at first-cleavage metaphase after in vitro fertilization (IVF) of oocytes from females of the same strain. The sperm were aged as donations occurred within an average of 31 days, either since last mating or at arrival at the animal facility in the case of virgin males. Of a total of 598 sperm complements studied from 22 sexually mature males aged 10–26 weeks old, there was one diploid complement (0.17%). The frequencies of hyperhaploidy and structural aberrations that were studied in 338 complements were 4.4% and 3.6%, respectively, giving an overall frequency of 8.0%. The hyperhaploid complements consisted of n + 1, n + 2, n + 3, and n + 7 counts, while the structural abnormalities were of the chromosome type and included large and small fragments and a possible translocation. This is the highest frequency of sperm chromosome abnormalities reported for mouse sperm obtained from males under physiological conditions and fertilized in vitro or in vivo. Sperm aging, strain, and/or technique differences are among the factors that may be responsible for this high frequency. Since the 8.0% frequency of hyperhaploidy and structural abnormalities is similar to the frequency reported for human sperm after IVF, the outbred murine in vitro fertilization system may be a useful model to study the origin of human sperm chromosome abnormalities.     

Sexual rest and post‐meiotic sperm ageing in house mice

Fertilization by aged sperm can result in adverse fitness consequences for both males and females. Sperm storage during male sexual rest could provide an environment for post‐meiotic sperm senescence causing a deterioration in the quality of stored sperm, possibly impacting on both sperm performance (e.g. swimming ability) and DNA quality. Here, we compared the proportion of sperm with fragmented DNA, an indicator of structural damage of DNA within the sperm cell, among males that had been sexually rested for approximately 2 months, to that of males that had mated recently. We found no evidence of intra‐epididymal sperm DNA damage or any impairment in sperm performance, and consequently no evidence of post‐meiotic sperm senescence. Our results suggest that male house mice are likely to possess mechanisms that function to ensure that their sperm reserves remain stocked with ‘young’, viable sperm during periods of sexual inactivity. We also discuss the possibility that our experimental design leads to no difference in the age of sperm among males from the two mating treatments. Post‐meiotic sperm senescence is especially relevant under sperm competition. Thus, we sourced mice from populations that differed in their levels of post‐copulatory sexual selection, enabling us to gain insight into how selection for higher sperm production influences the rate of sperm ageing and levels of DNA fragmentation. We found that males from the population that produced the highest number of sperm also had the smallest proportion of DNA‐fragmented sperm and discuss this outcome in relation to selection acting upon males to ensure that they produce ejaculates with high‐quality sperm that are successful in achieving fertilizations under competitive conditions.     

Evidence for differential maturation of reciprocal sperm segregants in the murine Rb(6.16) translocation heterozygote.

The fertilizing ability of unaged sperm and those aged experimentally in the cauda by surgically ligating the corpus epididymis in males carrying the Rb(6.16) translocation was studied. Chromosomally normal females were inseminated with unaged sperm delivered by males mating at 3-day intervals, and aged sperm were studied after matings on 6-14 postoperative days. The sperm chromosome complement was analyzed in first-cleavage metaphase zygotes after sequential G- and C-banding of the chromosomes. Of 283 metaphasic zygotes in the control group, 183 (or 64.7%) were analyzed and showed a ratio of 2.7:1 for chromosomally normal and balanced segregants of the translocation, deviating significantly (P less than 0.001) from the expected 1:1. The ratio of X- to Y-bearing sperm also deviated from expected (P less than 0.01) mostly due to a significant deficiency (P less than 0.05) of balanced sperm that were X-bearing. Fertilized oocytes were recovered from matings of 10 males on days 6-8 postoperatively, and, of 139 metaphasic one-cell zygotes, 101 (or 72.3%) were analyzed. These showed a Mendelian ratio of 1:1 for normal and balanced segregants. The sex ratio in the aged group (57Y:41X) also showed no deviation from 1:1. The results, which reveal significant physiological distortions for both the segregation and the sex ratios in males heterozygous for the Rb(6.16) translocation, suggest that differential maturation of the translocation-bearing sperm and the chromosomally normal reciprocal exists. The findings further support the concept that sperm chromosomal complement affects their maturation and function, and that factors on chromosome 6 and the X or Y chromosome additively affect sperm function.     

Sperm age, sex ratio, and hyperhaploidy frequency in mice.

Physiologically aged and unaged sperm from each of 12 sexually mature B6SJLF1/J mice were used to fertilize oocytes from females of the same strain, with each male serving as its own control. Male genomes in 323 and 307 first-cleavage metaphases obtained by in vivo and in vitro fertilization, respectively, were analyzed cytogenetically, using C-banding for detection of the Y chromosome. The sex (X:Y) ratio among all zygotes resulting from in vivo fertilization was 1.18; in zygotes resulting from in vivo fertilization by aged (14-d mating intervals) sperm, however, the ratio was 1.53, which differed significantly (chi 2 = 6.72, P less than 0.01) from the theoretical value of 1.00. Comparison of the sex ratio in zygotes resulting from in vivo fertilization by unaged sperm (3-d mating intervals), 0.94, with that in zygotes resulting from fertilization by aged sperm (using a 2 x 2 contingency table) showed a significant (chi c2 = 4.19, P less than 0.05) relationship between sex ratio and sperm age. In vitro neither the combined nor the individual 3- and 14-d data deviated significantly from the expected sex ratio of 1.00. The frequency of sperm-derived hyperhaploidy did not differ significantly between the in vivo (3.4%) and in vitro (5.9%) populations, but did between unaged (2.5%) and aged (6.8%) sperm (chi c2 = 5.74, P less than 0.01). All hyperhaploid zygotes had a complement of n + 1 chromosomes, except the 14-d in vitro group, where complements of n + 2 and n + 3 chromosomes were seen. Sperm-derived polyploidy, which was observed only in the in vitro group, was independent of sperm age and occurred in 6.8% of the zygotes. These data provide support for the sperm-aging hypothesis and indicate, for the first time, an influence of sperm aging in the male genital tract on the X:Y ratio of conceptuses resulting from natural matings of chromosomally normal males.     

Chromosomal analysis in mouse eggs fertilized in vitro with sperm exposed to ultraviolet light (UV) and methyl and ethyl methanesulfonate (MMS and EMS)

Chromosome aberrations were analyzed at the first-cleavage metaphase of mouse eggs fertilized in vitro with sperm exposed to ultraviolet light (UV) as well as to methyl and ethyl methanesulfonate (MMS and EMS). The frequencies of chromosome aberrations markedly increased with dose of UV as well as with concentration of MMS and EMS. In the UV-irradiation group, the frequency of chromosome-type aberrations was much higher than that of chromatid-type aberrations. About 90% of chromosome aberrations observed in the eggs following MMS and EMS treatment to sperm were chromosome type in which the frequency of chromosome fragments was the highest. The effects of UV on the induction of chromosome aberrations were clearly potentiated by post-treatment incubation of fertilized eggs in the presence of Ara-C or caffeine, but the effects of MMS and EMS were not pronounced by post-treatment of Ara-C or caffeine. The results indicate a possibility that UV damage induced in mouse sperm DNA is reparable in the eggs during the period between the entry of sperm into the egg cytoplasm and the first-cleavage metaphase.     

Males' evolutionary responses to experimental removal of sexual selection

We evaluated the influence of pre- and post-copulatory sexual selection upon male reproductive traits in a naturally promiscuous species, Drosophila melanogaster. Sexual selection was removed in two replicate populations through enforced monogamous mating with random mate assignment or retained in polyandrous controls. Monogamous mating eliminates all opportunities for mate competition, mate discrimination, sperm competition, cryptic female choice and, hence, sexual conflict. Levels of divergence between lines in sperm production and male fitness traits were quantified after 38-81 generations of selection. Three a priori predictions were tested: (i) male investment in spermatogenesis will be lower in monogamy-line males due to the absence of sperm competition selection, (ii) due to the evolution of increased male benevolence, the fitness of females paired with monogamy-line males will be higher than that of females paired with control-line males, and (iii) monogamy-line males will exhibit decreased competitive reproductive success relative to control-line males. The first two predictions were supported, whereas the third prediction was not. Monogamy males evolved a smaller body size and the size of their testes and the number of sperm within the testes were disproportionately further reduced. In contrast, the fitness of monogamous males (and their mates) was greater when reproducing in a non-competitive context: females mated once with monogamous males produced offspring at a faster rate and produced a greater total number of surviving progeny than did females mated to control males. The results indicate that sexual selection favours the production of increased numbers of sperm in D. melanogaster and that sexual selection favours some male traits conferring a direct cost to the fecundity of females.     

Factors affecting female sexual receptivity in the planthopper, Prokelisia dolus

Abstract. The mating system of Prokelisia dolus Wilson (Homoptera: Delphacidae) was characterized by determining: if males and females multiply mate; when transitions occur in female sexual receptivity, what triggers sexual refractoriness; and what behaviours characterize unreceptive virgins, receptive virgins, and unreceptive mated females. Males copulated with up to six females in less than 1 h, but completely inseminate, on average, only the first four females. Females rarely mated more than once, unless males were depleted of sperm due to previous copulations or if copulation was interrupted (if duration was<2 min). Male and female calling was associated (100% and 91%, respectively) with sexual receptivity and resultant matings. The transition from unreceptive virgin to receptive (calling) mature virgin occurred 48 h posteclosion, and all were mated by day 4. Females that were sexually immature and those completely inseminated did not call. Rejection of males by females included walking away from approaching males (65%), female kicking (7%), and abdominal lifting (5%). Rejection of males was observed by immature, mature and calling, and mated females. Sexual refractoriness was not triggered by acoustic and visual stimuli or mechanical stimulation of genitalia. Refractoriness was also not triggered by reception of small quantities of sperm because some females laid a few viable eggs yet calling was not terminated. Sexual refractoriness was activated by a substance in the ejaculate as demonstrated by injection into the haemocoel of male accessory glands or testes and homogenates of seminal vesicles. This is the first study that documents the role of male ejaculate in inhibiting female sexual receptivity in Hemiptera (Homoptera).     

Sperm storage mediated by cryptic female choice for nuptial gifts

Polyandrous females are expected to discriminate among males through postcopulatory cryptic mate choice. Yet, there is surprisingly little unequivocal evidence for female-mediated cryptic sperm choice. In species in which nuptial gifts facilitate mating, females may gain indirect benefits through preferential storage of sperm from gift-giving males if the gift signals male quality. We tested this hypothesis in the spider Pisaura mirabilis by quantifying the number of sperm stored in response to copulation with males with or without a nuptial gift, while experimentally controlling copulation duration. We further assessed the effect of gift presence and copulation duration on egg-hatching success in matings with uninterrupted copulations with gift-giving males. We show that females mated to gift-giving males stored more sperm and experienced 17% higher egg-hatching success, compared with those mated to no-gift males, despite matched copulation durations. Uninterrupted copulations resulted in both increased sperm storage and egg-hatching success. Our study confirms the prediction that the nuptial gift as a male signal is under positive sexual selection by females through cryptic sperm storage. In addition, the gift facilitates longer copulations and increased sperm transfer providing two different types of advantage to gift-giving in males.     

Impact of male mating history on the postmating resumption of sexual receptivity and lifetime reproductive success in Choristoneura rosaceana females

Abstract.  The first objective of the present study is to test the hypothesis that the decrease in the number of eupyrene spermatozoa in the spermatheca is directly associated with the resumption of sexual receptivity in female moths, an aspect that has not been examined in previous studies. The obliquebanded leafroller, Choristoneura rosaceana , is used and females mated with previously mated males have a shorter refractory period than those mated with virgins. This difference is associated with a faster rate of movement of sperm from the spermatheca. Overall, the length of the female refractory period coincides with the mean time required for the number of eupyrene sperm in the spermatheca to drop to approximately 3000, regardless of male mating history. Although such a decline in sperm numbers may be a factor responsible for the resumption of sexual receptivity, this is clearly not the only one because more than 40% of females remate even though sperm numbers in the spermatheca are well above this threshold. Virgin males do not vary the mass or the content of their ejaculate as a function of the female's reproductive status and this may increase the risk of sperm competition if the female is previously mated. The second objective of this study is to examine the effect of previous male mating history on female reproductive potential. Females mated with previously mated males have a significantly lower fecundity than those mated with virgin males. However, in all treatments, remating increases both female longevity and lifetime fecundity. There is also a significant effect of female mass on the length of the refractory period and on lifetime fecundity, with large females resuming sexual receptivity sooner and laying more eggs than small ones, regardless of male mating history.     

A species‐specific multigene family mediates differential sperm displacement in Drosophila melanogaster

Sperm competition is a postcopulatory sexual selection mechanism in species in which females mate with multiple males. Despite its evolutionary relevance in shaping male traits, the genetic mechanisms underlying sperm competition are poorly understood. A recently originated multigene family specific to Drosophila melanogaster, Sdic, is important for the outcome of sperm competition in doubly mated females, although the mechanistic nature of this phenotype remained unresolved. Here, we compared doubly mated females, second mated to either Sdic knockout or nonknockout males, and directly visualize sperm dynamics in the female reproductive tract. We found that a less effective removal of first‐to‐mate male's sperm within the female's sperm storage organs is consistent with a reduced sperm competitive ability of the Sdic knockout males. Our results highlight the role young genes can play in driving the evolution of sperm competition.     

Discriminating Males and Unpredictable Females: Males Bias Sperm Allocation in Favor of Virgin Females

When both sexes mate with multiple partners, theory predicts that males should adjust their investment in ejaculates in response to the risk and/or intensity of sperm competition. Here, we demonstrate that, in the harlequin beetle riding pseudoscorpion, Cordylochernes scorpioides, males use cues deposited on females by previous males to distinguish between virgin, once‐mated, and multiply‐mated females and adjust sperm allocation accordingly. Sperm number declined in direct proportion to the number of previous males, with virgin females receiving nearly three times more sperm than females exposed to three previous males. Given the lack of first‐male sperm precedence in C. scorpioides, this pattern is not consistent with current sperm competition models and appears best explained by a significant risk of wasting ejaculates on deceitful, mated females. In C. scorpioides, males transfer sperm indirectly to females via a stalked spermatophore deposited on the substrate. Mated females often feign sexual receptivity and cooperate throughout mating, only to reject the sperm packet produced by the male. While indirect sperm transfer facilitates a high level of female deceit and control, females of many species are able to influence the number and fate of sperm transferred during copulation and are likely to conceal their sexual unreceptivity to minimize male retaliation. If males cannot accurately assess female receptivity, increased risk of sperm rejection by mated females could outweigh the risk of sperm competition and favor greater sperm allocation to virgin females.     

Females Mated without Sperm Transfer Maintain High Sexual Receptivity in the Wolf Spider Schizocosa malitiosa

The mating of the Neotropical lycosid Schizocosa malitiosa is long and complex, involving intense genital stimulation and copulatory courtship. This suggests functions other than just insemination. Previous data indicated that mated females of this species are less sexually receptive than virgins. We hypothesise that copulatory characteristics presented by males during prolonged copulations could be responsible for subsequent female sexual reluctance, and may be selected by cryptic female choice. Our objective was to examine the influence of copulatory behaviour on subsequent female sexual receptivity in S. malitiosa, isolating it from the effects of sperm transfer per se. For this purpose, we obtained males without sperm in their copulatory organs (palpal bulbs), and prevented them from charging their palps by sealing their genital pores immediately after their last moult (treated males). Virgin females were separated into three groups: (i) females exposed once to normal males, (ii) females exposed twice to normal males, and (iii) females exposed first to treated and second to normal males. The results showed that, 3 d after their first mating, females first mated with untreated males were frequently refractory to remating, whereas all those first mated with treated males were receptive. Copulations performed by treated males showed some differences from those performed by normal males, but maintained the basic behavioural pattern with abundant sexual stimulation. The presence of sperm fluids in the female receptacles appears to be the most likely factor generating female remating reluctance. Males may manipulate female responses using receptivity inhibiting substances in their sperm, like those described for insects. Females would first ensure sperm supply, becoming more choosy afterwards. All females, whether mated once or twice, generated similar numbers of progeny, indicating no relationship between number of matings and number of spiderlings.     

Number of mating males and mating interval affect last-male sperm precedence in Tenebrio molitor L.

Polyandrous females often mate with more than two males, and yet most studies of postcopulatory sexual selection involve only two males. In insects, second-male sperm precedence is usually taken as evidence of overall last-male sperm precedence despite some studies to the contrary. Furthermore, the processes or mechanisms causing the patterns are often unknown and yet are important when estimating how postcopulatory sexual selection might act on males. Whether the patterns and processes change in normal sperm competitive situations and the effects of other factors besides mating order need to be examined to better assess the evolutionary potential of postcopulatory sexual selection. In this study, I assessed the effects of mating interval and number of mating males on sperm precedence patterns and their causal mechanisms in the mealworm beetle, Tenebrio molitor. Last-male sperm precedence was the same when two or three males mated, but also depended on mating intervals and hence mechanisms of paternity bias. However, when females mated with many males, one of the mechanisms no longer created last-male sperm precedence. This example illustrates the importance of knowing both the patterns and mechanisms of paternity bias and whether they change depending on female mating frequency to make reasonable inferences about the potential for postcopulatory sexual selection on males. Copyright 2003 Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour.      

Remating inhibition in female Queensland fruit flies: effects and correlates of sperm storage

Reproductive success of male insects commonly hinges both on their ability to secure copulations with many mates and also on their ability to inseminate and inhibit subsequent sexual receptivity of their mates to rival males. We here present the first investigation of sperm storage in Queensland fruit flies (Tephritidae: Bactrocera tryoni; a.k.a. 'Q-flies') and address the question of whether remating inhibition in females is directly influenced by or correlated with number of sperm stored from their first mates. We used irradiation to disrupt spermatogenesis and thereby experimentally reduce the number of sperm stored by some male's mates while leaving other aspects of male sexual performance (mating probability, latency until copulating, copula duration) unaffected. Females that mated with irradiated rather than normal males were less likely to store any sperm at all (50% vs. 89%) and, if some sperm were stored, the number was greatly reduced (median 11 vs. 120). Despite the considerable differences in sperm storage, females mated by normal males and irradiated males were similarly likely to remate at the next opportunity, indicating (1) number of sperm stored does not directly drive female remating inhibition and (2) factors actually responsible for remating inhibition are similarly expressed in normal and irradiated males. While overall levels of remating were similar for mates of normal and irradiated males, factors responsible for female remating inhibition were positively associated with presence and number of sperm stored by mates of normal but not irradiated males. We suggest seminal fluids as the most likely factor responsible for remating inhibition in female Q-flies, as these are likely to be transported in proportion to number of sperm in normal males, be uninfluenced by irradiation, and be transported without systematic relation to sperm number in irradiated males.     

Potential fitness benefits from mate selection in the Atlantic cod (Gadus morhua)

Little evidence of benefits from female mate choice has been found when males provide no parental care or resources. Yet, good genes models of sexual selection suggest that elaborated male sexual characters are reliable signals of mate quality and that the offspring of males with elaborate sexual ornaments will perform better than those of males with less elaborate ornaments. We used cod (Gadus morhua L.), an externally fertilizing species where males provide nothing but sperm, to examine the potential of optimal mate selection with respect to offspring survival. By applying in vitro fertilizations, we crossed eight females with nine males in all possible combinations and reared each of the 72 sib groups. We found that offspring survival was dependent on which female was mated with which male and that optimal mate selection has the potential to increase mean offspring survival from 31.9 to 55.6% (a 74% increase). However, the size of the male sexual ornaments and sperm quality (i.e. sperm velocity and sperm density) could not predict offspring survival. Thus, even if there may be large fitness benefits of mate selection, we might not yet have identified the male characteristics generating high offspring survival.     

Female choice and the relatedness of mates in the guppy (<Emphasis Type="Italic">Poecilia reticulata</Emphasis>)

Several studies suggest that females may offset the costs of genetic incompatibility by exercising pre-copulatory or post-copulatory mate choice to bias paternity toward more compatible males. One source of genetic incompatibility is the degree of relatedness among mates; unrelated males are expected to be genetically more compatible with a female than her relatives. To address this idea, we investigated the potential for inbreeding depression and paternity biasing mechanisms (pre- and post-copulatory) of inbreeding avoidance in the guppy, Poecilia reticulata. Inbreeding resulted in a reduction in offspring number and quality. Females mated to siblings gave birth to significantly fewer offspring compared to females mated to non-siblings and inbred male offspring took longer to reach sexual maturity. There was no evidence of inbreeding avoidance in pre-copulatory behaviors of females or males. Sexual responsiveness of females to courting males and the number of sexual behaviors males directed at females did not decrease as a function of the relatedness of the two individuals. We also tested whether female guppies can use post-copulatory mechanisms to bias sperm usage toward unrelated males by comparing the number of offspring produced by females mated to two of their siblings (SS), two males unrelated to the female (NN), or to one unrelated male and a sibling male (NS). We found that NS females produced a number of offspring not significantly different than what would be expected if fertilization success were halfway between completely outbreeding (NN) and completely inbreeding (SS) females. This suggests that there is no significant improvement in the number of offspring produced by females mating to both related and unrelated males, relative to that which would be expected if sperm from both males were used equally. Our results suggest that female guppies do not discriminate against closely related males or their sperm.     

Sperm storage and viability in Photinus fireflies

In many species females mate with and store sperm from multiple males, and some female insects have evolved multiple compartments for sperm storage. Sperm storage and sperm viability were investigated in two firefly species, Photinus greeni and P. ignitus, which differ in the morphology of the female reproductive tract. Although the primary spermatheca is similar in both species, P. greeni females have an additional, conspicuous outpocketing within the bursa copulatrix whose potential role in sperm storage was investigated in this study. An assay that distinguishes between live and dead sperm was used to examine sperm viability in male seminal vesicles and sperm storage sites within the female reproductive tract. For both Photinus species, sperm from male seminal vesicles showed significantly higher viability compared to sperm from the primary spermatheca of single mated females. In single mated P. greeni females, sperm taken from the channel outpocketing (secondary spermatheca) showed significantly higher viability compared to sperm from the primary spermatheca. This sperm viability difference was not evident in double mated females. There were no significant differences between P. greeni and P. ignitus females in the viability of sperm from the primary spermatheca. These studies contribute to our understanding of post-mating processes that may influence paternity success, and suggest that sexual conflict over control of fertilizations may occur in multiply mated firefly females.     

Female Remating Behavior in a Lekking Moth

In polyandrous species, male reproductive success will at least partly be determined by males' success in sperm competition. To understand the potential for post‐mating sexual selection, it is therefore important to assess the extent of female remating. In the lekking moth Achroia grisella, male mating success is strongly determined by female choice based on the attractiveness of male ultrasonic songs. Although observations have indicated that some females will remate, only little is known about the level of sperm competition. In many species, females are more likely to remate if their first mating involved an already mated male than if the first male was virgin. Potentially, this is because mated males are less well able to provide an adequate sperm supply, nutrients, or substances inhibiting female remating. This phenomenon will effectively reduce the strength of pre‐copulatory sexual selection because attractive males with high mating success will be more susceptible to sperm competition. We therefore performed an experiment designed both to provide a more precise estimate of female remating probability and simultaneously to test the hypothesis that female remating is influenced by male mating history. Overall, approximately one of five females remated with a second male. Yet, although females mated to non‐virgin males were somewhat more prone to remate, the effect of male mating history was not significant. The results revealed, however, that heavier females were more likely to remate. Furthermore, we found that females' second copulations were longer, suggesting that, in accordance with theory, males may invest more sperm in situations with an elevated risk of sperm competition.     

Effects of oil pollution on the development of sex cells in sea urchins

The sea urchinStrongylocentrotus nudus is highly sensitive to oil pollution. Experiments were performed in winter, spring and summer over periods of 15 to 45 days. Experimental urchins were kept in water with hydrocarbon concentrations of 10 to 30 mg l⁻¹, and control urchins in pure sea water. Thermal stimulation by Evdokimov's method was applied to obtain mature sexual products during winter and spring tests. Summer investigations were conducted at temperatures of 17 to 18 °C. The gonads were studied histologically and morphometrically, and the sexual cells obtained were analyzed at the embryological level. No histological and morphometrical differences were recorded between sexual cells of controls and experimentals. However, marked hydrocarbon effects were observed in the embryonic development of artificially fertilized cells from experimental urchins. Control embryos developed normally. Embryogenesis of artificially fertilized gametes from control females and experimental males, and vice versa, was found to be distinctly abnormal. Many abnormalities were identified at the first cleavage stage, as well as in blastula, gastrula and pluteus. Fertilization of experimental eggs with experimental sperm resulted in serious disturbances of embryos, followed by the development of non-viable larvae. On the whole, embryogenesis of sexual cells from experimental urchins was characterized by prominent delay, asynchronism and presence of abnormal non-viable larvae. Consequently, long-term effects of sublethal hydrocarbon concentrations resulted in the formation of defective sex cells and high larval mortality.