Male-derived cuticular hydrocarbons signal sperm competition intensity and affect ejaculate expenditure in crickets

Female sexual promiscuity can have significant effects on male mating decisions because it increases the intensity of competition between ejaculates for fertilization. Because sperm production is costly, males that can detect multiple matings by females and allocate sperm strategically will have an obvious fitness advantage. The presence of rival males is widely recognized as a cue used by males to assess sperm competition. However, for species in which males neither congregate around nor guard females, other more cryptic cues might be involved. Here, we demonstrate unprecedented levels of sperm competition assessment by males, which is mediated via the use of chemical cues. Using the cricket Teleogryllus oceanicus, we manipulated male perception of sperm competition by experimentally coating live unmated females with cuticular compounds extracted from males. We found that males adjusted their ejaculate allocation in response to these compounds: the viability of sperm contained within a male's ejaculate decreased as the number of male extracts applied to his virgin female partner was increased. We further show that males do not respond to the relative concentration of male compounds present on females, but rather to the number of distinct signature odours of individual males. Our results conform to sperm competition theory, and show for the first time, to our knowledge, that males can detect different intensities of sperm competition by using distinct chemical cues of individual males present on females.     

Sperm length is not influenced by haploid gene expression in the flies Drosophila melanogaster and Scathophaga stercoraria

Recent theoretical models have postulated a role for haploid–diploid conflict and for kin selection favouring sperm cooperation and altruism in the diversification and specialization of sperm form. A critical assumption of these models—that haploid gene expression contributes to variation in sperm form—has never been demonstrated and remains contentious. By quantifying within-male variation in sperm length using crosses between males and females from populations that had been subjected to divergent experimental selection, we demonstrate that haploid gene expression does not contribute to variation in sperm length in both Drosophila melanogaster and Scathophaga stercoraria. This finding casts doubt on the importance of haploid–diploid conflict and kin selection as evolutionary influences of sperm phenotypes.     

Adaptive modulation of sperm production rate in Drosophila bifurca, a species with giant sperm

Sperm have traditionally been regarded as energetically cheap and effectively limitless in supply, although evidence conflicting with this view has become increasingly abundant. For instance, males from a variety of taxa have been shown to strategically partition sperm across ejaculates in response to perceived sperm competition risk. It follows that males might also be predicted to adaptively modulate the rate at which sperm are produced. Here we show that, in the giant sperm producing fruitfly Drosophila bifurca, solitary males with infrequent access to females produce sperm at a much lower rate than males raised in association with females and other males. Our results support the prediction that males with little risk of sperm competition risk or few mating opportunities should divert resources away from gamete production.     

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.     

Ejaculate economics: an experimental test in a moth

Increasing evidence shows that spermatogenesis is costly. As a consequence, males should optimize the use of their sperm to maximize their reproductive outputs in their lifetime. However, experimental evidence on this prediction is largely lacking. Here, we examine how a male moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) responds to the presence of rivals or additional mates and how such response influences his lifetime reproductive fitness. We show that when rival males are present around a copulating pair, the male ejaculates more sperm to win a sperm competition battle but in such an environment he inseminates fewer females, sires fewer offspring and lives shorter. The opposite is the case when additional females are present during copulation. These findings reveal that elevated reproductive expenditure owing to sperm competition intensity is made at the expense of longevity and future reproduction.     

Pheromonostasis is not directly associated with post-mating sperm dynamics in Choristoneura fumiferana and C. rosaceana females

In Lepidoptera, a number of humoral and neural cues are involved in post-mating pheromonostasis, including the presence of sperm in the spermatheca. However, as there are two types of sperm, apyrene and eupyrene, they may play different roles in pheromonostasis, an aspect not considered in previous studies. As a first step to examine this possibility, we determined the quantity of sperm transferred by the male at the time of mating and the temporal migration of both sperm types from the bursa copulatrix to the spermatheca in the spruce budworm, Choristoneura fumiferana, and the obliquebanded leafroller, C. rosaceana. While the mass of the ejaculate was positively correlated to male body mass, there was no relation between ejaculate mass and sperm numbers. In both species, the migration of the two sperm types was asynchronous, with the apyrene sperm migrating before the eupyrene type. There were, however, some interspecific temporal differences in the migration of both sperm types. Eupyrene sperm would not serve as a direct signal for pheromonostasis in either species as it does not reach the spermatheca for at least 7 h while the neural message for pheromonostasis in both tortricids occurs within 3 h of mating. Given the time apyrene sperm arrives in the spermatheca (between 3 and 5 h post-mating), it could serve as a direct cue for pheromonostasis in C. fumiferana but not in C. rosaceana. However, considering that these two Choristoneura species have similar pheromone physiologies, it seems somewhat unlikely that apyrene sperm would be involved in one species and not the other.     

Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization

Since the first successful reports into oocyte freezing, many papers concerning the cryopreservation of mouse oocytes have been published. However, a simple and practical cryopreservation method for unfertilized C57BL/6 mouse oocytes, and an IVF system using these cryopreserved oocytes have yet to be established, in spite of the fact that C57BL/6 is the prevalent inbred strain and is used for large-scale knockout programs. In this study, unfertilized C57BL/6 mouse oocytes were cryopreserved via a simple vitrification method. After warming, IVF was performed using cryopreserved unfertilized oocytes and fresh sperm, cryopreserved unfertilized oocytes and cold-stored sperm, cryopreserved unfertilized oocytes and frozen sperm (C57BL/6 strain sperm), and cryopreserved unfertilized oocytes and frozen sperm derived from GEM strains (C57BL/6 background GEM strains). Nearly all of the cryopreserved oocytes were recovered, of which over 90% were morphologically normal. Those oocytes were then used for in vitro fertilization, resulting in 72–97% of oocytes developing into 2-cell embryos. A portion of the 2-cell embryos were transferred to recipients, resulting in live young being produced from 32–49% of the embryos. In summary, we established the simple and practical method of mouse oocyte vitrification with high survivability and developmental ability and the IVF using the vitrified-warmed oocytes with fresh, cold-stored or cryopreserved sperm with high fertility.     

Acrosome reaction in sperm of the frog, Xenopus laevis: its detection and induction by oviductal pars recta secretion

Previous electron microscopic observations have shown that the acrosome of the sperm of the frog, Xenopus laevis, comprises a membrane-bounded vesicle covering the anterior-most position of the head. We obtained a sperm suspension from the testes and stained it with LysoSensor Green for observation under a confocal laser scanning microscope and found a bright fluorescence reflecting the presence of the acrosomes at the top of the sperm head in about 64% of the sperm, with no deterioration of their capacity to fertilize. About 40% of the sperm with an acrosome underwent an acrosome reaction in response to Ca(2+) ionophore A23187, as evidenced by a loss of LysoSensor Green stainability, accompanied by breakdown of the acrosomal vesicle. About 53% of the sperm bound to isolated vitelline envelopes underwent an acrosome reaction, whereas both jelly water and solubilized vitelline envelopes weakly induced an acrosome reaction. When the sperm were treated with an oviductal extract obtained from the pars recta, but not the pars convoluta region, about 40% of the sperm with acrosomes underwent an acrosome reaction. The substance containing acrosome reaction-inducing activity in the pars recta extract seemed to be a heat-unstable substance with a molecular weight of greater than 10 kDa. The activity was not inhibited by protease inhibitors but required extracellular Ca(2+) ions. These results indicate that the acrosome reaction occurs on the vitelline envelopes in response to the substance deposited from the pars recta during the passage of the oocytes through the oviduct.     

Impact of male mating history on the temporal sperm dynamics of Choristoneura rosaceana and C. fumiferana females

In the oblique-banded leafroller, Choristoneura rosaceana, and the spruce budworm, C. fumiferana, male reproductive performance decreases with consecutive matings. While the onset time of mating did not vary, the time spent mating was longer in mated than in virgin males. Furthermore, a decline observed in the spermatophore mass with successive matings was associated with a concomitant decline in its apyrene and eupyrene spermatozoa content. In the hours following mating, spermatozoa migrate from the spermatophore, located in the bursa copulatrix, to the spermatheca. Regardless of the male's previous mating history, the number of apyrene sperm dropped rapidly in the days following mating whereas the number of eupyrene spermatozoa declined gradually. As the temporal pattern of sperm movement was similar in all treatments, females mated with previously-mated males would suffer from sperm shortage sooner than those mated with virgins. Large C. rosaceana females stored more apyrene spermatozoa in their spermatheca than small ones, irrespective of the time after mating or male mating history, while only large females mated with once-mated males received more apyrene sperm and accessory gland secretions than small ones mated with virgin or twice-mated males. The results obtained in this study are discussed in relation with their potential impact on the reproductive success of both sexes.     

Cholesterol and phospholipid levels of washed and percoll gradient centrifuged mouse sperm: Presence of lipids possessing inhibitory effects on sperm motility

Levels of DNA, cholesterol, and phospholipids of mouse caudal epididymal and vas deferens sperm that were processed through simple washing and Percoll gradient centrifugation were measured. The DNA and cholesterol contents of washed sperm and Percoll gradient centrifuged (PGC) sperm (DNA = 3.6 ± 0.3 pg/sperm and 3.4 ± 0.3 pg/sperm, respectively; cholesterol = 0.219 ± 0.057 nmole/μg DNA and 0.224 ± 0.030 nmole/μg DNA, respectively, for washed and PGC sperm) were not significantly different from each other; however, the phospholipid level of PGC sperm was only one half of that of washed sperm (0.315 ± 0.071 nmole/μg DNA versus 0.720 ± 0.075 nmole/μg DNA, respectively). The presence of 0.3% bovine serum albumin (BSA) in the culture medium used in sperm washing did not change the cholesterol and phospholipid contents of washed sperm. Similarly, the cholesterol and phospholipid levels of washed sperm and PGC sperm that were further incubated in BSA-containing medium for 30 min remained the same. Interestingly, substantial amounts of lipids, as determined by the cholesterol and phospholipid levels, were released into the supernatants of the sperm washes, and sperm needed to be washed at least twice to ensure their stable levels of cholesterol and phospholipids. The lipid mixture in the first sperm wash supernatant was shown to have inhibitory effects on PGC sperm motility. © 1996 Wiley-Liss, Inc.     

Production of female bovine embryos with sex-sorted sperm using intracytoplasmic sperm injection: Efficiency and in vitro developmental competence

The production of embryos with a preselected sex sperm is important in the livestock industry. In this study, we examined the efficiency of producing female embryos by intracytoplasmic sperm injection (ICSI) with flow cytometry sorted (ssICSI) and unsorted (usICSI) bovine sperm, and their developmental competence in vitro. For comparison, bovine embryos were also produced by in vitro fertilization (IVF) with sorted (ssIVF) and unsorted (usIVF) bovine sperm. The semen used in this study was from a bull selected for its high fertility and blastocyst developmental competence among four bulls. We first examined and compared pronuclear (PN) formation and cleavage rates of the produced embryos among the treatment groups. Our results demonstrated that PN formation rates (judged by two pronucleus [2PN]) and cleavage rates in ssIVF group (23.1% and 43.6%) were lower than those in the usIVF (71.1% and 71.6%), usICSI (73.1% and 92.8%) and ssICSI (75% and 79.1%) groups, respectively (P < 0.05). Moreover, the blastocyst formation rate in the ssIVF group was less than those in the usIVF, usICSI, and ssICSI groups (2.7% vs. 30.2%, 28.7% and 24.7%, respectively; P < 0.05). Importantly, we reported that the blastocyst formation rate in the ssICSI group was similar to that in the usICSI group, which indicated that ICSI can rescue the damage introduced to sperm by flow cytometry–mediated sex-sorting. Of note, we achieved a blastocyst formation rate in the ssICSI group to be comparable with the usIVF group. We then examined embryo quality by counting the number of normal and apoptotic cells in blastocysts. It was found that, despite the fact that blastocyst formation rate in the ssIVF group was significantly lower than those in the usIVF, usICSI and ssICSI groups, there was no difference in total and apoptotic cell numbers among these groups (P > 0.05). Finally, karyotyping analysis demonstrated that the proportion of female embryos in the ssICSI and ssIVF groups was 100%, whereas it was 58.8% and 57.8% in the usIVF and usICSI groups, respectively. In conclusion, ICSI with flow cytometry sorted bovine sperm provides an alternative approach to produce embryos with predetermined sex.