Identification of sperm antigens as a first step towards the generation of a contraceptive vaccine to decrease fossorial water vole Arvicola terrestris Scherman proliferations

Immunocontraceptive strategies have proved to be efficient in controlling fertility of various mammalian species. In the present study we have made the first steps towards the identification of Arvicola terrestris sperm antigens that could be used as targets in the development of a contraceptive vaccine to limit the proliferations of this pest rodent. Rabbit-raised polyclonal antisera directed against complete A. terrestris spermatozoa were used to identify and characterize on 2D-gels coupled with a MALDI-TOF mass spectrometry analysis A. terrestris sperm proteins. Amongst the proteins pinpointed by this approach some were further investigated based on their tissue- and/or sperm-specific expression, and their relevance to fertility or sperm/egg interaction. In parallel, three proteins that have been already reported in the literature to be appropriate targets for the development of contraceptive vaccines in other mammalian species have also been looked for in A. terrestris. With the selected protein targets, a reverse-PCR approach using degenerate primers was employed to amplify corresponding A. terrestris cDNAs. After conceptual translation and sequence alignment, different proteins were studied to determine zones with sufficient sequence divergence and of antigenic/immunogenic nature that could be used in future assays to immunize animals.     

Sperm specific proteins-potential candidate molecules for fertility control

The increase in population growth rate warrants the development of additional contraceptive methods that are widely acceptable, free from side effects and less expensive. Immunocontraception, and in particular the targeting of antibodies to gamete-specific antigens implicated in sperm egg binding and fertilization, offers an attractive approach to control fertility. The development of a contraceptive vaccine based on sperm antigen represents a promising approach to contraception. In mammals, fertilization is completed by the direct interaction of sperm and egg, a process mediated primarily by sperm surface proteins. Sperm have proteins that are unique, cell specific, immunogenic and accessible to antibodies. A few of the sperm specific proteins have been isolated and characterized. The antibodies raised against the sperm specific antigens have proved to be extremely effective at reducing sperm-egg interaction in vitro; fertility trials in sub-human primates would eventually prove the effectiveness of the sperm antigens in terms of contraceptive efficacy.     

Effects of gossypol on the motility of mammalian sperm

The effects of the male contraceptive gossypol on the motility of mammalian spermatozoa are reviewed. The role of sperm motility in the processes of fertilization and the effect of the drug on these processes determine its effectiveness as a contraceptive. The promising male contraceptive potential of gossypol is discussed in the context of the serious adverse effects of the agent.     

Sperm competition,but not major histocompatibility divergence,drives differential fertilization success between alternative reproductive tactics in Chinook salmon

Post‐copulatory sexual selection processes, including sperm competition and cryptic female choice (CFC), can operate based on major histocompatibility (MH) genes. We investigated sperm competition between male alternative reproductive tactics [jack (sneaker) and hooknose (guard)] of Chinook salmon (Oncorhynchus tshawytscha). Using a full factorial design, we examined in vitro competitive fertilization success of paired jack and hooknose males at three time points after sperm activation (0, 15 and 60 s) to test for male competition, CFC and time effects on male fertilization success. We also examined egg‐mediated CFC at two MH genes by examining both the relationship between competitive fertilization success and MH divergence as well as inheritance patterns of MH alleles in resulting offspring. We found that jacks sired more offspring than hooknose males at 0 s post‐activation; however, jack fertilization success declined over time post‐activation, suggesting a trade‐off between sperm speed and longevity. Enhanced fertilization success of jacks (presumably via higher sperm quality) may serve to increase sneaker tactic competitiveness relative to dominant hooknose males. We also found evidence of egg‐mediated CFC (i.e. female × male interaction) influencing competitive fertilization success; however, CFC was not acting on the MH genes as we found no relationship between fertilization success and MH II β₁ or MH I α₁ divergence and we found no deviations from Mendelian inheritance of MH alleles in the offspring. Our study provides insight into evolutionary mechanisms influencing variation in male mating success within alternative reproductive tactics, thus underscoring different strategies that males can adopt to attain success.     

Human sperm centrosomal function during fertilization, a novel assessment for male sterility

In human fertilization, the sperm introduces the centrosome-the microtubule organizing center-and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, the sperm aster, the functioning of which is essential for pronuclear movement for the union of the male and female genomes. We established functional assay for human sperm centrosomal function, by using heterologus ICSI system with bovine and rabbit eggs. After human sperm incorporation into mammalian egg, we observed that the sperm aster was organized from sperm centrosome, and the sperm aster enlarged as the sperm nuclei underwent pronuclear formation. The normal human sperm aster formation rate at 6 h post-ICSI were 60.0% in bovine egg and 36.1% in rabbit egg, respectively. However, sperm aster formation rate following heterologus ICSI into bovine eggs with teratozoospermia (globozoospermia, dysplasia of fibrous sheath) were low. These data indicate that human sperm centrosomal function is low in abnormal shaped sperm. Wherus, elucidation of human sperm centrosomal function can lead us to find a new type of failure in "post ICSI events in fertilization".     

Current status of fertility control methods in India

Approximately 48.2% of couples of 15 to 49 years of age practice family planning methods in India. Female sterilization accounts for 34.2%, with male sterilization declining from 3·4% in 1992–93 to 1·9% in 1998–99. Use of the condom increased to 3·1% from 2·4%. There is an urgent need for research to develop new contraceptive modalities especially for men and also for women and to make existing methods more safe, affordable and acceptable. Current efforts in India to develop a male contraceptive are mainly directed towards (i) development of antispermatogenic agents to suppress sperm production, (ii) prevention of sperm maturation, (iii) prevention of sperm transport through vas deferens or rendering these sperm infertile and (iv) prevention of sperm deposition. Research work in the field of prevention of sperm transport through vas deferens has made significant advances. Styrene maleic anhydride (SMA) disturbed the electrical charge of spermatozoa leading to acrosome rupture and consequent loss in fertilizing ability of sperm. A multicentre phase-III clinical trial using SMA is continuing and it is hoped that the SMA approach would be available in the near future as an indigenously developed injectable intra-vasal male contraceptive. The safety and efficacy of available oral contraceptives were evaluated. An indigenously developed oral contraceptive ‘Centchorman’, which is a nonsteroidal, weakly estrogenic but potently antiestrogenic, was found to be safe and effective and is now being marketed in India since 1991 as a ‘once a week’ pill. Cyclofem and Mesigyna have been recommended as injectable contraceptives with proper counselling and service delivery by Indian studies. It has been recommended that these injectable contraceptives be added to the existing range of contraceptive methods available in the National Family Planning Programme. Based on the Indian studies CuT 200 was also recommended. Studies have indicated the advantage of intrauterine devices (IUD); they are long acting, relatively easily removed and fertility returns rapidly after their removal. Recent studies have recommended CuT 200 for use up to 5 years. The combination of some plant products i.e.Embelia ribes, Borax andPiper longum has been found to be safe and effective as a female contraceptive and the results of phase-I clinical trials are encouraging. Research work is going on in the country in various areas with special reference to hormonal contraceptive — a three monthly injectable contraceptive, immuno-contraceptives, antiprogestins, etc.     

Evidence for the involvement of testicular protein CRISP2 in mouse sperm-egg fusion

CRISP2, originally known as Tpx-1, is a cysteine-rich secretory protein specifically expressed in male haploid germ cells. Although likely to be involved in gamete interaction, evidence for a functional role of CRISP2 in fertilization still remains poor. In the present study, we used a mouse model to examine the subcellular localization of CRISP2 in sperm and its involvement in the different stages of fertilization. Results from indirect immunofluorescence and protein extraction experiments indicated that mouse CRISP2 is an intraacrosomal component that remains associated with sperm after capacitation and the acrosome reaction (AR). In vitro fertilization assays using zona pellucida-intact mouse eggs showed that an antibody against the protein significantly decreased the percentage of penetrated eggs, with a coincident accumulation of perivitelline sperm. The failure to inhibit zona pellucida penetration excludes a detrimental effect of the antibody on sperm motility or the AR, supporting a specific participation of CRISP2 at the sperm-egg fusion step. In agreement with this evidence, recombinant mouse CRISP2 (recCRISP2) specifically bound to the fusogenic area of mouse eggs, as previously reported for rat CRISP1, an epididymal protein involved in gamete fusion. In vitro competition investigations showed that incubation of mouse zona-free eggs with a fixed concentration of recCRISP2 and increasing amounts of rat CRISP1 reduced the binding of recCRISP2 to the egg, suggesting that the proteins interact with common complementary sites on the egg surface. Our findings indicate that testicular CRISP2, as observed for epididymal CRISP1, is involved in sperm-egg fusion through its binding to complementary sites on the egg surface, supporting the idea of functional cooperation between homologous molecules to ensure the success of fertilization.     

Sperm competition and the evolution of gamete morphology in frogs

Despite detailed knowledge of the ultrastructure of spermatozoa, there is a paucity of information on the selective pressures that influence sperm form and function. Theoretical models for both internal and external fertilizers predict that sperm competition could favour the evolution of longer sperm. Empirical tests of the external-fertilization model have been restricted to just one group, the fishes, and these tests have proved equivocal. We investigated how sperm competition affects sperm morphology in externally fertilizing myobatrachid frogs. We also examined selection acting on egg size, and covariation between sperm and egg morphology. Species were ranked according to probability of group spawning and hence risk of sperm competition. Body size, testis size and oviposition environment may also influence gamete traits and were included in our analyses. After controlling for phylogenetic relationships between the species examined, we found that an increased risk of sperm competition was associated with increased sperm head and tail lengths. Path analysis showed that sperm competition had its greatest direct effect on sperm tail length, as might be expected under selection resulting from competitive fertilization. Sperm competition did not influence egg size. Oviposition location had a strong influence on egg size and a weak influence on sperm length, with terrestrial spawners having larger gametes than aquatic spawners. Our analysis revealed significant correlated evolution between egg morphology and sperm morphology. These data provide a conclusive demonstration that sperm competition selects for increased sperm length in frogs, and evidence for evolutionary covariance between aspects of male and female gamete morphology.     

Breakdown of the sperm nuclear envelope is a prerequisite for male pronucleus formation: direct evidence from the gynogenetic crucian carp Carassius auratus langsdorfii

The gynogenetic fish, Carassius auratus langsdorfii (the ginbuna, a crucian carp), provides an interesting model for the study of the mechanisms controlling male pronucleus formation. When the sperm nucleus of a different subspecies (C. a. cuvieri) is incorporated into the gynogenetic egg, the nuclear envelope of the spermatozoon is not broken down, and the pronucleus fails to develop, although dispersion of the sperm chromatin occurs to some extent within the space limited by the nuclear envelope. When spermatozoa without plasma membranes and nuclear envelopes were microinjected into mature activated eggs, the sperm nuclei underwent chromatin dispersion, nuclear envelope formation, DNA synthesis, and transformation into male pronuclei. These results indicate that the failure of the male pronucleus to form in ginbuna is primarily due to the failure of sperm nuclear envelope breakdown. We conclude that sperm nuclear envelope breakdown is an indispensable step for the development of the male pronucleus.     

Gamete membrane microdomains and their associated molecules in fertilization signaling

Fertilization is the fundamental system of biological reproduction in many organisms, including animals, plants, and algae. A growing body of knowledge has emerged to explain how fertilization and activation of development are accomplished. Studies on the molecular mechanisms of fertilization are in progress for a wide variety of multicellular organisms. In this review, we summarize recent findings and debates about the long-standing questions concerning fertilization: how egg and sperm become competent for their interaction with each other, how the binding and fusion of these gamete cells are made possible, and how the fertilized eggs initiate development to a newborn. We will focus on the structure and function of the membrane microdomains (MDs) of egg and sperm that may serve as a platform or signaling center for the aforementioned cellular functions. In particular, we provide evidence that MDs of eggs from the African clawed frog, Xenopus laevis, play a pivotal role in receiving extracellular signals from fertilizing sperm and then transmitting them to the egg cytoplasm, where the tyrosine kinase Src is present and responsible for the subsequent signaling events collectively called egg activation. The presence of a new signaling axis involving uroplakin III, an MD-associated transmembrane protein, and Src in this system will be highlighted and discussed.     

Sperm-egg interaction at fertilization: glycans as recognition signals

This article first examines the events occurring in male and female genital tracts, which prepare human sperm to encounter the egg. Central is a glycoprotein, gp20, homologous to the leukocyte antigen CD52. This protein is secreted in the epididymal cells, inserted in the sperm plasma membrane and exposed in the equatorial region of the head at the end of the capacitation process. The mechanisms and molecules of the first interaction event between gametes in the mollusk bivalve Unio elongatulus and the current state of our knowledge of the same interaction in other species is then considered. The egg of Unio is very peculiar because it is highly polarized. Similar to other well-known egg models, the ligand for recognition is located on the egg coat which is a sort of fibrous network made up of very few glycoproteins, while the receptor is on the sperm surface. The difference is that in this egg, the ligand molecules are not uniformly distributed but are restricted to an area of the egg coat at the vegetal pole, the crater area. The role of carbohydrates in ligand function and of a specific type of oligosaccharide chain in particular, is discussed in the wider context of glycans acting as recognition signals.     

Initial evaluation of fertilin as an immunocontraceptive antigen and molecular cloning of the cynomolgus monkey fertilin β subunit

Fertilin (PH-30) is a sperm surface protein that functions in sperm adhesion and fusion with the egg plasma membrane. Because of its essential function in fertilization, fertilin is a potential target for novel contraceptive approaches. In a pilot fertility trial, immunization of male guinea pigs with purified guinea pig fertilin resulted in complete infertility. The contraceptive effect was partial (two out of six animals were infertile) when female guinea pigs were immunized with the antigen. These results suggest that fertilin or domains of fertilin may be effective as immunocontraceptive antigens. As a step toward achieving this goal, we communicate the cDNA and deduced amino acid sequence of the monkey fertilin β subunit. © 1996 Wiley Liss, Inc.     

Human sperm aster formation and pronuclear decondensation in bovine eggs following intracytoplasmic sperm injection using a Piezo-driven pipette: a novel assay for human sperm centrosomal function.

In human fertilization, the sperm introduces the centrosome; the microtubule-organizing center and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, called the sperm aster, the functioning of which is essential to pronuclear movement for union of male and female genome. The sperm centrosomal function is considered to be necessary for the normal human fertilization process. Therefore, the dysfunction of sperm centrosome is a possible cause of human fertilization failure. However, little information is available regarding human sperm centrosomal function during fertilization in clinically assisted reproductive technology. To assess the human sperm centrosomal function, we examined sperm aster formation and pronuclear decondensation following intracytoplasmic sperm injection (ICSI) with human sperm into the bovine egg using a Piezo-driven pipette and ethanol activation of eggs. After human sperm incorporation into bovine egg, we observed that the sperm aster was organized from sperm centrosome, and that the sperm aster was enlarged as the sperm nuclei underwent pronuclear formation. The sperm aster formation rate at 6 h post-ICSI and the male pronuclear formation rate at 8-12 h post-ICSI were 60.0% and 83.3%, respectively. No difference of the sperm aster formation rate and the male pronuclear formation rate was observed between eggs activated with ethanol and eggs without artificial activation. We concluded that this heterologous Piezo-ICSI system into bovine egg can be a novel assay for human sperm centrosomal function, and it is possible to explicate a course of fertilization failure that was unknown until now.     

Nontransitivity of paternity in a bird

In many animals reproductive success is determined after insemination by the interaction of male and female processes. While sperm competition is reasonably well understood in some taxa, other processes, such as cryptic female choice and differential early embryo mortality resulting from genetic incompatibilities, are less well understood. The relative importance of these different factors contributing to reproductive success is difficult to assess. Here we control for male-mediated effects (which are often considerable and can mask more subtle processes) through the artificial insemination of known numbers of sperm in the domestic fowl to reveal that male reproductive success is nontransitive across females: the success of a particular male depends on the background against which his sperm compete for fertilization. Two potential processes could account for this effect: cryptic female choice (sperm choice) or differential early embryo mortality. Regardless of the mechanism, nontransitivity of male reproductive success has important evolutionary consequences, including the maintenance of variation in male fitness.     

Epididymal approaches to male contraception

Today, a vast arsenal of contraceptive methods interfering at different levels of the female reproductive axis is available. This is not the case for men for whom, until now, there is no reliable male reversible method and for whom vasectomy, condom and withdrawal are the only options available. Despite this limited supply, more than one third of all contraceptive methods used worldwide rely on the cooperation of the male partner. Besides developing hormonal approaches to stop sperm production, there may be attractive approaches that will interfere with sperm functions rather than production. Sperm functions are primarily established during post-testicular maturation, with the epididymis accounting for the majority. The purpose of this review is to present some of the promising and/or already abandoned leads that emerge from research efforts targeting the epididymis and its activities as potential means to achieve male post-meiotic contraception.     

A nonsemen copulatory fluid influences the outcome of sperm competition in Japanese quail

Sperm competition is a powerful and widespread evolutionary force that drives the divergence of behavioural, physiological and morphological traits. Elucidating the mechanisms governing differential fertilization success is a fundamental question of sperm competition. Both sperm and nonsperm ejaculate components can influence sperm competition outcomes. Here, we investigate the role of a nonsemen copulatory fluid in sperm competition. Male Japanese quail possess a gland that makes meringue‐like foam. Males produce and store foam independent of sperm and seminal fluid, yet transfer foam to females during copulation. We tested whether foam influenced the outcome of sperm competition by varying foam state and mating order in competitive matings. We found that the presence of foam from one male decreased the relative fertilization success of a rival, and that foam from a given male increased the probability he obtained any fertilizations. Mating order also affected competitive success. Males mated first fertilized proportionally more eggs in a clutch and had more matings with any fertilizations than subsequent males. We conclude that the function of foam in sperm competition is mediated through the positive interaction of foam with a male's sperm, and we speculate whether the benefit is achieved through improving sperm storage, fertilizing efficiency or retention. Our results suggest males can evolve complex strategies to gain fertilizations at the expense of rivals as foam, a copulatory fluid not required for fertilization, nevertheless, has important effects on reproductive performance under competition.     

Male contraception: Another holy grail

The idea that men should participate in family planning by playing an active role in contraception has become more acceptable in recent years. Up to the present the condom and vasectomy have been the main methods of male contraception. There have been and continue to be efforts to develop an acceptable hormonal contraceptive involving testosterone (T) suppression. However the off target affects, delivery of the analogs and the need for T replacement have proven difficult obstacles to this technology. Research into the development of non-hormonal contraception for men is progressing in several laboratories and this will be the subject of the present review. A number of promising targets for the male pill are being investigated. These involve disruption of spermatogenesis by compromising the integrity of the germinal epithelium, interfering with sperm production at the level of meiosis, attacking specific sperm proteins to disrupt fertilizing ability, or interfering with the assembly of seminal fluid components required by ejaculated sperm for acquisition of motility. Blocking contractility of the vas deferens smooth muscle vasculature to prevent ejaculation is a unique approach that prevents sperm from reaching the egg. We shall note the lack of interest by big pharma with most of the support for male contraception provided by the NIH.     

Egg jelly influences sperm motility in the externally fertilizing frog,Crinia georgiana

Recent in vitro fertilization studies have revealed female and male × female interaction effects on the probability of fertilization. These findings suggest a mechanism of cryptic female choice via sperm–egg interactions. The egg jelly of anuran amphibians contains proteins that facilitate the chemoattraction and binding of sperm for fertilization. Here we show that egg jelly also influences the onset of motility and swimming velocity of motile sperm in the frog Crinia georgiana. Moreover, we found significant among female variation in the effects of egg jelly on sperm motility. We discuss this finding with respect to male and female effects on nonrandom fertilization observed in this species.     

Sexual selection in hermit crabs: a review and outlines of future research

The information currently available on sexual selection in hermit crabs is reviewed to identify the role of males and females before, during and after mating. According to this information, possible mechanisms of male–male competition, female choice and/or sexual conflict are suggested. Important male components that may affect mating success include dragging the female shell, rotations of the female's shell and male cheliped palpations, and male size and/or shell characteristics (species and size). Possible female determinants of male mating/fertilization success include size (as an indicator of egg production capacity), signalling of sexual receptivity to males, delay from mate guarding to copulation and mating duration. Avenues for deeper exploration in males include the role of the number and morphometry of male sexual tubes during sperm transfer, and whether ejaculate size and sperm number can be adjusted with variable situations of sperm competition intensity and risk. In females it would be interesting to investigate the chemical and behavioural mechanisms affecting spermatophore breakage for sperm release and the variable duration from sperm transfer to spawning. Given these possibilities, and that sperm is externally deposited on the female's body but inside her shell (except for those species that do not use shells, e.g. Birgus , or species where shells are rather small and do not cover the body totally, e.g. Parapagurus ), we conclude that hermit crabs are unique subjects for separating male and female effects, particularly with respect to the applicability of current ideas in sexual selection such as female choice and sexual conflict. Some practical ideas are provided to disentangle both hypotheses using these animals.     

Ocean acidification during prefertilization chemical communication affects sperm success

Ocean acidification (OA) poses a major threat to marine organisms, particularly during reproduction when externally shed gametes are vulnerable to changes in seawater pH. Accordingly, several studies on OA have focused on how changes in seawater pH influence sperm behavior and/or rates of in vitro fertilization. By contrast, few studies have examined how pH influences prefertilization gamete interactions, which are crucial during natural spawning events in most externally fertilizing taxa. One mechanism of gamete interaction that forms an important component of fertilization in most taxa is communication between sperm and egg‐derived chemicals. These chemical signals, along with the physiological responses in sperm they elicit, are likely to be highly sensitive to changes in seawater chemistry. In this study, we experimentally tested this possibility using the blue mussel, Mytilus galloprovincialis, a species in which females have been shown to use egg‐derived chemicals to promote the success of sperm from genetically compatible males. We conducted trials in which sperm were allowed to swim in gradients of egg‐derived chemicals under different seawater CO₂ (and therefore pH) treatments. We found that sperm had elevated fertilization rates after swimming in the presence of egg‐derived chemicals in low pH (pH 7.6) compared with ambient (pH 8.0) seawater. This observed effect could have important implications for the reproductive fitness of external fertilizers, where gamete compatibility plays a critical role in modulating reproduction in many species. For example, elevated sperm fertilization rates might disrupt the eggs' capacity to avoid fertilizations by genetically incompatible sperm. Our findings highlight the need to understand how OA affects the multiple stages of sperm‐egg interactions and to develop approaches that disentangle the implications of OA for female, male, and population fitness.