Egg Penetration Research Articles

Recombinant Mouse Sperm ZP3-binding Protein (ZP3R/sp56) Forms a High Order Oligomer That Binds Eggs and Inhibits Mouse Fertilization in Vitro

Many candidates have been proposed as zona pellucida-binding proteins. Without precluding a role for any of those candidates, we focused on mouse sperm protein ZP3R/sp56, which is localized in the acrosomal matrix. The objective of this study was to analyze the role of ZP3R/sp56 in mouse fertilization. We expressed recombinant ZP3R/sp56 as a secreted protein in HEK293 cells and purified it from serum-free, conditioned medium. In the presence of reducing agents, the recombinant ZP3R/sp56 exhibited a molecular weight similar to that observed for the native ZP3R/sp56. Reminiscent of the native protein, recombinant ZP3R/sp56 formed a high molecular weight, disulfide cross-linked oligomer consisting of six or more monomers under non-reducing conditions. Recombinant ZP3R/sp56 bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos, indicating that the changes that take place in the zona pellucida at fertilization affected the interaction of this protein with the zona pellucida. The extent of in vitro fertilization was reduced in a dose-dependent manner when unfertilized eggs were preincubated with recombinant ZP3R/sp56 (74% drop at the maximum concentrations assayed). Eggs incubated with the recombinant protein showed an absence of or very few sperm in the perivitelline space, suggesting that the reduction in the fertilization rate is caused by the inhibition of sperm binding and/or penetration through the zona pellucida. These results indicate that sperm ZP3R/sp56 is important for sperm-zona interactions during fertilization and support the concept that the acrosomal matrix plays an essential role in mediating the binding of sperm to the zona pellucida.

Sperm cell architecture, insemination, and fertilization in the model fern, Ceratopteris richardii

Motile sperm cells of land plants are released directly into the environment and encounter numerous constraints on their way to the egg. Sperm cell organization, shape, size, and plasticity are crucial to the processes associated with fertilization. We conducted an ultrastructural investigation to detail insemination (sperm release, swimming and movement within the archegonium) and fertilization in the model fern Ceratopteris richardii. Gametophytes were grown from spores using sterile culture techniques and flooded in water when sexually mature. Materials were examined at different stages post-flooding. During insemination in C. richardii, the sperm cytoskeleton and flagella rearrange, and the coils of the cell extend while entering the neck canal. In this nearly linear configuration, the dense ridge, a densely compacted band of filaments presumed to be actin, expands to surround the leading edge of the sperm cell. This ridge fuses with the receptive site on the female gamete and is the sperm component that initiates contact with the egg nuclear envelope. All cellular components, except plastids, enter the egg cytoplasm. Sperm mitochondria are distinguishable from those of the egg because they are encased by two or three additional membranes and are sequestered from the zygote cytoplasm. During karyogamy, the sperm components, including the microtubule cytoskeleton (spline) and flagella, maintain their spatial integrity. Microtubules play key roles not only in sperm cell structure but also in facilitating karyogamy in this fern. After karyogamy is completed, microtubule arrays of the sperm cell and the components of the locomotory apparatus are disassembled. We provide the first demonstration of the likely involvement of sperm actin in egg penetration in land plants and new insights into the fate of paternal organelles. This study points to the roles sperm cell structure and dynamics play in the intricate processes of insemination and fertilization in land plants.

Ability of Abnormally‐Shaped Human Spermatozoa to Adhere to and Penetrate Zona‐Free Hamster Eggs: Correlation With Sperm Morphology and Postincubation Motility

A body of evidence indicates that morphologically abnormal human spermatozoa may exhibit impaired ability to fertilize. Yet teratospermia has widely varying etiologies, including associations with varicoceles, following fever, cigarette smoking, and exposure to polychlorinated biphenyls. Abnormalities of sperm shape in mice have also been shown to be associated with autosomal gene mutations. These varying causes of teratospermia could have different molecular consequences reflected in altered sperm function. We studied the ability of morphologically abnormal human sperm to penetrate zona-free hamster eggs as a measure of their ability to undergo an acrosome reaction and gamete membrane fusion. Motile sperm from ejaculates containing 15% normal sperm or less, as judged by World Health Organization (1999) criteria, were recovered by ISolate density centrifugation and capacitated by overnight incubation. Zona-free hamster eggs were inseminated with 1 x 10(6) motile capacitated cells and scored for sperm penetration after 3 hours of coincubation. A significant trend was found between the percent of abnormal spermatozoa within the ejaculate and impaired egg-penetrating ability, reflected in the percent of eggs penetrated, the number of penetrating sperm per egg, and the number of sperm adherent to the oolemma. Because only acrosome-reacted human spermatozoa adhere to the oolemma, these results support the notion that abnormally shaped sperm may exhibit an impaired ability to undergo an acrosome reaction. A correlation was also noted between the loss of motility of sperm following overnight incubation and impairment of their ability to undergo gamete membrane fusion. These results confirm prior findings at the level of the zona pellucida that abnormally shaped sperm exhibit functional abnormalities. However, a wide variation was observed between men in the behavior of such sperm, including occasionally high rates of egg penetration. These observations suggest that assessment of morphology may be an unreliable measure, for the individual, of sperm fertilizing ability and emphasize that sperm function testing is an important part of the evaluation of teratospermia.

Final Report on the Safety Assessment of Maleic Acid1

Maleic Acid is a dicarboxylic acid that functions as a fragrance ingredient and pH adjuster in cosmetics - it is used in a few cosmetic product formulations at low concentrations. Maleic Acid is commonly used in research studies to induce Fanconi syndrome in rats and dogs in an attempt to study the mechanism of this disease. One such study found decreased glomerular filtration rate in rats given 9.0 mmol/kg, but not with 1.5 mmol/kg, Maleic Acid intraperitoneally. Preincubation with 0.75 mmol/L of Maleic Acid reduced sperm penetration of golden hamster eggs to zero. Maleic Acid failed to induce any significant increases in revertant count in strains TA1535, TA1537, TA98, and TA100 at concentrations up to 7500 mu g/plate. A concentration of 2.0 x 10(-2) M Maleic Acid did show a positive pattern in a DNA synthesis inhibition test. Maleic Acid at 10%, pH 1.0, applied for 30 s on rabbit eyes, caused permanent opacity. A 1% solution, pH 1.0, applied for 2 min caused cloudiness of the cornea, but no lasting injury, and a 5% solution, also at pH 1.0, had a similar but more intense effect, with recovery delayed 6 to 7 days. Application of 10 mu l Maleic Acid (pH not stated) to the volar forearm and labia majora of 21 female Caucasians produced an inflammatory response at 24 and 48 h, which varied from minimal erythema to marked erythema with marked vesiculation. Maleic Acid at 20% (pH not stated) applied to one forearm daily for a period of 6 weeks to 50 human subjects produced acute vesicular dermatitis in 17 subjects, who were dropped from the study. Only five of the remaining subjects accommodated to the treatment, the rest had varying degrees of inflammation or hyperirritable skin. Although Maleic Acid itself may be a dermal and/or ocular irritant, its use as a pH adjustor in cosmetic formulations dictates that most of the acid will be neutralized into various maleate salts. Therefore, the concentration of free Maleic Acid is expected to be low, and dermal or systemic toxicity is not expected to be a concern. The safety of Maleic Acid as a pH adjustor should not be based on the concentration of use, but on the amount of free Maleic Acid that remains after neutralizing the formulation. There is no reason to expect this ingredient to induce any toxicity when used for this purpose. The Cosmetic Ingredient Review (CIR) Expert Panel concluded that Maleic Acid is safe for use in cosmetic formulations as a pH adjustor in the practices of use as described in this safety assessment.

Correlation Between Semen Parameters and the Hamster Egg Penetration Test (HEPT) Among Fertile and Subfertile Men in Singapore

The objective of this retrospective study was to distinguish between fertile and subfertile men based on their semen parameters and hamster egg penetration test (HEPT) outcome. This study involved 110 subfertile men recruited from an infertility clinic and 48 fertile men attending an antenatal clinic in Singapore. The men were required to donate a semen specimen for semen analysis and HEPT assay. The results indicated that the subfertile group had significantly lower normal sperm morphology according to the Tygerberg strict criteria, and lower progressive motility (P < .05). Semen volume, density, HEPT decondensation rate, and sperm penetration index were not significantly different between the 2 groups. Receiver operating characteristic curve analysis indicated that sperm morphology had the highest predictive power of 65.7% with a threshold value of 7%, and progressive motility had a predictive power of 61.8% with a threshold value of 50%. Using the tenth percentile of the fertile population as the cutoff, lower adjusted thresholds of 3% for sperm morphology and 28% for progressive motility were obtained, giving higher positive predictive values of 81.8% and 84.4%, respectively. This study shows that these new cutoff values can be used to screen the general population to identify subfertile men. In contrast, the HEPT proved to be an insensitive and unreliable assay in identifying subfertile males. To our knowledge the comparison of HEPT and semen parameters between subfertile and fertile men has not been previously reported in an Asian population.

Eggshell penetration of hen's eggs by Salmonella enterica serovar Enteritidis upon various storage conditions

1. The survival and penetration of Salmonella enterica serovar Enteritidis (SE) inoculated on the eggshell was examined upon storage for up to 20 d at real-life conditions (15 to 25°C and 45 to 75% relative humidity (RH)). 2. Penetration was assessed by emptying the egg contents and filling the eggs with a selective medium that allowed visualising Salmonella growth on the inside of the shell and membrane complex. 3. The study of survival on the eggshells was based on viable counts and showed that numbers of surviving organisms decreased over time. Survival was inversely related to storage temperature and RH. Although the average counts decreased over time, a limited proportion of shells carried high numbers of SE at all storage conditions. 4. Penetration spots were observed earlier using an increased storage temperature due to increased growth rates of SE on the agar. After 20 d of storage a similar percentage (c. 44·7%) of eggshells became penetrated, irrespective of the storage conditions tested in this study. 5. The higher the Salmonella shell contamination at the end of storage, the higher the probability that the eggshell was penetrated. Salmonella shell counts exceeding 4 log cfu yielded more than a 90% probability of eggshell penetration occurring.

The inhibition of the human sperm phosphatidylinosytol 3‐kinase by LY294002 does not interfere with sperm/oocyte interaction

It has recently been reported that the selective inhibition of phosphatidylinosytol 3-kinase (PI3K) enhances human sperm motility. However, little information exists on a possible role of PI3K in other sperm functions involved in the fertilization process. In this study, we investigated whether LY294002 could affect human sperm ability to fuse with oocytes, by means of the hamster egg penetration test (HEPT). The effect on acrosome reactions (AR) and on sperm/zona pellucida (ZP) binding was also evaluated. The pre-incubation with scalar doses of LY294002 (0.1, 1 and 10 microm) did not interfere with sperm ability to fuse with oocytes either in the conventional version of the HEPT or in the version enhanced with progesterone (P). No interference with the stimulatory effect on AR exerted by P or mannose-bovine serum albumin (mannose-BSA) was revealed. Finally, LY294002 had no effect on sperm/ZP binding. These results indicate that the inhibition of PI3K by LY294002 does not interfere with sperm interaction with oocytes. This is noteworthy in the view of a possible clinical use of LY294002 as an in vitro stimulator of the sperm motility of asthenozoospermic patients for assisted reproduction techniques.

Immunogenicity and contraceptive potential of recombinant human sperm associated antigen (SPAG9)

Human sperm-associated antigen 9 (hSPAG9) is a potential target for sperm-based contraceptive vaccine in lieu of its location on the sperm acrosomal compartment and its implication in sperm–egg interaction. SPAG9 is an acrosomal molecule which is not only restricted to a specific region (domain) of the acrosome but also undergoes relocation to the equatorial region in a stage-specific manner during acrosome reaction, demonstrating its potential role in sperm–egg binding. Human SPAG9 nucleotide sequence revealed 94% identity with macaque SPAG9 and 96.8% with baboon SPAG9 over the entire sequence. The amino acid sequence comparison of human SPAG9 with macaque and baboon revealed an overall homology of 84.9% and 90.6%, respectively. The presence of a high level of homology at the amino acid and nucleotide levels indicates that SPAG9 is conserved in macaque, baboon and human sharing common function and common origin in the biological past. Immunogenicity studies were carried in rats, which demonstrated that recombinant hSPAG9 protein adsorbed on alum is highly immunogenic. Antibodies thus generated after immunization reacted with recombinant human SPAG9 (rhSPAG9) and native SPAG9 protein from human sperm in Western blot analysis. In an in vitro assay, anti-rhSPAG9 antibodies inhibited sperm adherence to or penetration in zona-free hamster egg penetration test. Further, anti-SPAG9 antibodies inhibited the binding of human sperm to intact human oocyte as well as to matched hemi-zonae, indicating that the recombinant protein is a suitable contraceptive vaccinogen. Together these results demonstrate that the rhSPAG9 adsorbed on alum is immunogenic in nature, which is a permissible adjuvant for immunogenicity and fertility trials in non-human primates.

Characterization of immune response in mice to plasmid DNA encoding human sperm associated antigen 9 (SPAG9)

To examine the immunogenicity of the plasmid DNA encoding human sperm associated antigen 9 (hSPAG9), the cDNA corresponding to hSPAG9 was cloned in mammalian expression vector pcDNA 3.1 down stream of cytomegalovirus promoter. Immunization of female BALB/cJ mice with pcDNA–hSPAG9 plasmid DNA in saline by intramuscular (i.m.), by adsorbing onto gold microcarriers (delivered by gene gun) and by recombinant hSPAG9 (r-hSPAG9) protein generated antibody response against Escherichia coli expressed r-hSPAG9 protein and native SPAG9 in human sperm. Although mice immunized with r-hSPAG9 protein exhibited highest antibodies titres ( P < 0.001), the difference in the antibody titres seen by the two modes of plasmid DNA delivery were not significant ( P > 0.05). A dominant IgG1 isotype response was observed in mice immunized with pcDNA–hSPAG9 plasmid DNA delivered by gene gun as compared to a mixed IgG1–IgG2a isotype response in mice immunized with r-hSPAG9 protein and pcDNA–hSPAG9 plasmid DNA delivered by i.m. Further, antibodies generated by pcDNA–hSPAG9 plasmid DNA localized acrosomal compartment of human sperm and inhibited sperm adherence to or penetration in zona-free hamster egg penetration test. These studies for the first time, demonstrate the feasibility of generating an immune response to sperm specific hSPAG9 protein by DNA vaccine and that antibodies thus generated recognize native SPAG9 in human sperm.

Monoclonal antibody c262 counteracts the stimulatory effect of progesterone on sperm–oocyte fusion

Some evidence suggests that the non-genomic effects exerted by progesterone (P) on human spermatozoa are mediated by membrane receptor(s) displaying the C-terminal domain, but not the N-terminal domain of the genomic P receptor (PR). This study aimed at determining whether the monoclonal antibody (mAb) c-262, directed against the C-terminal domain of the genomic PR, counteracts the stimulatory effect of P on the human sperm ability to fuse with oocytes. Sperm/oocyte fusion was evaluated by means of the hamster egg penetration test. The brief exposure of capacitated spermatozoa to P produced a stimulatory effect on sperm/oocyte fusion. mAb c262 counteracted this stimulatory effect in a dose-dependent manner. No counteraction was observed when capacitated spermatozoa were pre-exposed to PGR-312, a mAb directed against the N-terminal domain of the genomic PR. These results reinforce the hypothesis that the non-genomic effects exerted by P on human spermatozoa are mediated by membrane receptor(s) displaying the C-terminal domain, but not the N-terminal domain of the genomic PR.

The Spermatozoa Protein, SLLP1, Is a Novel Cancer-Testis Antigen in Hematologic Malignancies.

SLLP1 is a unique non-bacteriolytic c-lysozyme-like protein isolated from human spermatozoa. Antisera to SLLP1 blocks binding in the hamster egg penetration assay, suggesting that SLLP1 may be involved in sperm/egg adhesion. A recent study by dot blot analysis on RNA showed that SLLP1 was expressed only in the testis and in Burkitt lymphoma Raji cell line, suggesting that further studies are warranted to determine and characterize SLLP1 expression in tumor cells, in particular, fresh tumor specimens.Using a pair of sequence-specific primers in RT-PCR, we found that SLLP1 transcripts could be detected in 5/8 myeloma cell lines, suggesting that SLLP1 may be expressed in tumor cells from some hematologic malignancies. When we applied the investigations to 52 primary hematologic malignant specimens, SLLP1 transcripts were detected in 6/17 myeloma, 4/14 CML, 3/11 CLL, 2/9 AML and 0/1 hairy cell leukemia. In contrast, SLLP1 transcripts were not detected in the peripheral blood (n=12) or bone marrow (n=3) from any healthy donors. The specificity of the PCR products was confirmed by either sequence analysis or restriction digest with Pvu II.SLLP1 transcripts were translated into its corresponding protein in these tumor cells. Using tumor cell lysate in Western blot analysis, we detected SLLP1 protein in the myeloma cell lines and also in fresh malignant specimens, although positivities were only observed in specimens with high RT-PCR signals. All PCR-negative specimens were also negative in Western blot analysis. The specificity of the Western blot signals were confirmed in all cases by blocking assays with a high concentration of recombinant SLLP1 protein.We next investigated the expression of SLLP1 in a large panel of normal tissues using RT-PCR and real time quantitative PCR. Both approaches showed that SLLP1 is a novel Cancer-Testis antigen in hematologic malignancies. SLLP1 was detected, at a level of 8206 copies/0.25 mcg total RNA, only in normal testis. We also found that the SLLP1 mRNA copy numbers in fresh hematologic tumor specimens were up to 2316 copies/0.25 mcg total RNA, i.e. more than 25% of the level found in normal testis. We cloned and generated SLLP1 recombinant protein from E coli and used the purified recombinant SLLP1 in an ELISA system to detect anti-SLLP1 antibodies. Using sera from 24 healthy donors and the mean + 2SD as the cut-off signal intensities, we found that high titer IgG antibodies directed at SLLP1 could be detected in the sera from 2/9 AML, 5/23 CLL, 6/27 CML and 14/51 myeloma patients. The specificity of the antibodies was confirmed in Western blot analysis. Probably due to the decreased sensitivity of the detection system in Western blot analysis, only 1/2 AML, 3/5 CLL, 4/6 CML and 7/14 myeloma SLLP1 antibody+ sera produced a signal in the Western blot analysis. Interesting, IgG2 was by far the commonest SLLP1 antibodies in these patients. There was a good correlation between SLLP1 gene expression and immune responses.In summary, SLLP1 is a novel CT antigen in hematologic malignancies and is capable of eliciting B-cell immune responses in vivo in cancer-bearing patients. Our results support SLLP1 as a protein target appropriate for further in vitro study to define its suitability for immunotherapy.

A simplified technique for visualizing decondensed sperm heads in the Hamster Egg Penetration Test (HEPT)

The Hamster Egg Penetration Test (HEPT) is a valuable tool in determining the ability of a patient’s sperm to penetrate an oocyte. One of the challenges of the test is the difficulty in mounting the oocytes to read the test. Oocytes must be flattened to the proper depth to visualize decondensed sperm heads in order to obtain an accurate result without causing the oocytes to lyse. The objective of this study was to evaluate the use of the Makler® chamber and compare it to other methods currently in use for reading the HEPT.

Molecular cloning, expression of testicular transcript abundant in germ cells and immunobiological effects of the recombinant protein.

It has been well documented that antisperm antibodies can be causative factors for infertility. In this report we have identified a protein on human sperm referred as human sperm-associated protein (HSAP) using serum of an immunoinfertile woman; it is thus a sperm-specific protein--a candidate molecule for control of fertility. An immunoinfertile woman serum showing head-head sperm agglutination and acrosomal localization, reacted with human sperm protein of apparent molecular weight of 48 kDa on Western blot. Anti-48 kDa antiserum was raised in rabbit by eluting 48 kDa protein and was used to screen the human testis cDNA expression library. A putative positive hsap cDNA clone was obtained, sequenced and subjected to tissue specificities studies by Northern blotting. The cell type-specific expression was done using in situ RNA hybridization studies. To obtain recombinant HSAP (r-HSAP), hsap cDNA was cloned in pET 22b(+) expression vector. r-HSAP was expressed as polyhistidine fusion protein in Escherichia coli and purified. Rabbits were immunized with the purified r-HSAP, which led to generation of antibodies. In order to evaluate in vitro immunocontraceptive potential, the anti-r-HSAP antibodies were characterized by agglutination assay, zona-free hamster egg penetration assay, indirect immunofluorescence (IIF) assay, and by flow cytometry analysis. We have cloned a human testis gene encoding a protein (HSAP) of 328 amino acids. Antibodies against the purified recombinant protein specifically recognized approximately 40 kDa r-HSAP, and a cognate 48 kDa protein band in human sperm extract in Western blot procedure. The anti-r-HSAP antibodies localized acrosomal compartment, inhibited sperm binding/attachment in zona-free hamster penetration assay and revealed surface binding with human live sperm by flow cytometry. The cDNA sequence has been submitted to EMBL and has been given the accession number Y16676. This study has put in evidence that novel sperm-specific r-HSAP has role in sperm function and may have application in the development of a contraceptive vaccine. The availability of the recombinant protein will facilitate studies on the assessment of its potential as a contraceptive immunogen.

Annexin V binding to plasma membrane predicts the quality of human cryopreserved spermatozoa.

Cryopreservation-induced stress may result in membrane injury with consequent decrease of sperm motility and fertilizing capacity. This study has investigated the relationship between human spermatozoa tolerance to cryopreservation and the loss of plasma membrane asymmetry, especially translocation of phosphatidylserine (PS) from the inner to the outer leaflet. The prospective study was performed on semen samples from 31 men. Conventional characteristics of 20 semen were analysed, before and after cryopreservation as well as externalization of PS assessed by annexin V-staining in combination with the propidium iodide which stains dead cells. The fertilizing capacity was evaluated by a zona free hamster egg penetration test in 11 freeze/thaw spermatozoa samples. The percentages of vital annexin V-negative and annexin V-positive spermatozoa in post-thaw samples were significantly lower than in pre-freeze ones (10 +/- 3 vs. 25 +/- 5% and 22 +/- 3 vs. 34 +/- 4% respectively), while the percentages of dead spermatozoa annexin V-negative and annexin V-positive had increased (42 +/- 4 vs. 23 +/- 4% and 23 +/- 4 vs. 16 +/- 2% respectively). The percentages of progressive and total motile spermatozoa were significantly correlated with the percentage of vital annexin V-negative spermatozoa before as well as after cryopreservation. Furthermore, recovery of motile spermatozoa after freeze/thaw was strongly correlated (p < 0.002) with the proportion of vital annexin V-negative spermatozoa in fresh semen. The percentage of penetration of zona-free hamster eggs was correlated (p < 0.02) with the percentage of live annexin V-negative cryopreserved spermatozoa capacitated for 3 h. These findings provide evidence that annexinV-binding staining in combination with PI brings additional information to predict the outcome of cryopreserved ejaculated sperm and may be used as a novel and reliable marker to study the freeze/thaw process.

Genes and male infertility: what can go wrong?

Genes and male infertility: what can go wrong?

Pre-penetration events in fungal parasitism of nematode eggs

The present investigation deals with the main factors involved in early infection of nematode eggs by fungal parasites. We studied the effect of hydrophobicity on appressorium formation by germlings of Pochonia rubescens (syn. Verticillium suchlasporium), P. chlamydosporia (syn. V. chlamydosporium) and Lecanicillium lecanii (syn. V. lecanii). Appressoria were frequently formed on hydrophobic surfaces such as polyvinyl chloride or polystyrene and were infrequently formed on hydrophilic materials such as glass or aluminium. Infected eggs probed with the FITC-labelled lectin Concanavalin-A showed intense labelling corresponding to appressoria formed by fungal parasites on the eggshell surface. Proteolytic activity was found in extracts from conidia and germlings of fungal parasites (especially P. chlamydosporia) in the absence of nematode eggs. Addition of the serine proteinase inhibitors phenylmetylsulphonyl fluoride (PMSF) or diisopropyl fluorophosphonate (DFP) to the extracts reduced their proteolytic activity. PMSF was the most effective inhibitor. Zymography also revealed proteolytic activity in extracts from the three fungi tested. This activity mostly corresponded to bands of Rf's of substrate degradation similar to that of purified main protease (P32) from P. rubescens. Other bands with molecular weight higher than P32 (low Rf) were found especially for P. chlamydosporia extracts. For L. lecanii only bands of low Rf were found. Serum anti-P32 partially inhibited proteolytic activity of extracts from conidia and germlings. Application of PMSF and DFP to the inoculum, reduced egg penetration for the three species studied. PMSF caused the highest reduction in eggs infected by L. lecanii, while DFP significantly reduced egg infection by both P. chlamydosporia and L. lecanii. Our results therefore show hydrophobicity, appresorium formation and protease production as factors involved in early parasitism of nematode eggs.

Effect of human sperm exposure to progesterone on sperm–oocyte fusion and sperm–zona pellucida binding under various experimental conditions

In this study, the effect of human sperm exposure to progesterone on sperm/oocyte fusion, using the hamster egg penetration test, and on sperm/zona pellucida (ZP) binding, using the hemizona assay, was investigated under various experimental conditions. A brief exposure of human spermatozoa to progesterone exerted a stimulatory effect on sperm/oocyte fusion which was dose-dependent, capacitation-dependent, influenced by the source of serum albumin in capacitating medium, and was higher than that produced by the exposure to progesterone from the onset of capacitation. The exposure of capacitated spermatozoa to progesterone during 20 min-spermatozoa/ZP-coincubation produced an enhancement of ZP-binding, which was not significantly influenced by the source of serum albumin in capacitating medium. A significantly lower ZP-binding was exhibited by spermatozoa exposed to progesterone from the beginning of capacitation. These results indicate that progesterone exerts a stimulatory effect on human sperm's fertilizing ability, which occurs mainly in post-capacitation events directly involved in sperm/oocyte fusion and in ZP-binding. Conditions optimizing these effects are provided. They should be taken into account in the standardization of experimental and clinical studies designed to evaluate the response of human spermatozoa to progesterone.

Use of Luminescent Campylobacter jejuni ATCC 33291 To Assess Eggshell Colonization and Penetration in Fresh and Retail Eggs

A luminescent phenotype in Campylobacter jejuni ATCC 33291, generated by a transcriptional fusion between the C. jejuniflaA σ28 promoter and the luxCDABE genes of Xenorhabdus luminescens on plasmid pRYluxCDABE, was used to examine colonization and penetration of fresh and retail eggs. C. jejuni colonized both fresh and retail eggs at 37, 40, and 42°C under microaerophilic conditions. Fresh eggs were more heavily colonized than retail eggs. Under aerobic conditions, fresh eggs were colonized at similar levels for all three temperatures. C. jejuni was found to penetrate the eggshell in 2 of 48 (4.2%) fresh eggs assessed. Although the lux+ phenotype did not provide an effective means of predicting penetration sites, it was effective at visualizing eggshell colonization. Also, it effectively demonstrated the organism's opportunistic nature, as eggshell surfaces with flaws and slight cracks were extensively colonized and easily detected by a photon counting charge-coupled device camera. Using scanning electron microscopy, C. jejuni ATCC 33291 was visualized on both the exterior and interior surfaces of the egg membranes indicating penetration of these barriers.

Every CatSper is Sacred

Sperm undergo a process called capacitance, part of which involves increasing their motility in the female reproductive tract. Ren et al. have found and characterized a new Ca 2+ channel expressed in sperm tails, termed CatSper, that is required for sperm motility and male fertility. CatSper –/– mice, although infertile, produced normal amounts of sperm and exhibited normal sexual behavior. However, CatSper-deficient mice had immotile sperm that could only fertilize eggs without the zona pellucida (but not intact eggs), suggesting that CatSper may be necessary for egg penetration. CatSper was activated by cyclic AMP and resulted in Ca 2+ influx, suggesting that tail movement depended on increased intracellular Ca 2+ concentrations. These results indicate that CatSper might be a useful target for contraception with minimal side effects, as CatSper is only expressed in the testis. Garbers provides a helpful News &amp; Views commentary. D. Ren, B. Navarro, G. Perez, A. C. Jackson, S. Hsu, Q. Shi, J. L. Tilly, D. E. Clapham, A sperm ion channel required for sperm motility and male fertility. Nature 413 , 603-609 (2001). [Online Journal] D. L. Garbers, Swimming with sperm. Nature 413 , 579-580 (2001). [Online Journal]

Pokeweed antiviral protein: a potential nonspermicidal prophylactic antiviral agent

Objective: To investigate the effects of pokeweed antiviral protein (PAP), a 29-kDa anti-human immunodeficiency virus (HIV) protein purified from the leaves of Phytolacca americana, on human sperm function. Design: Prospective, controlled study. Setting: Reproductive biology department. Patient(s): Seven sperm donors. Intervention(s): Human sperm and female genital tract epithelial cells were exposed to PAP ranging in concentration from 1 to 1,000 μg/mL. Main Outcome Measures: Effect of PAP on sperm motility, kinematics, and sperm penetration through bovine mucus, as well as binding, penetration, and fusion of zona-free hamster eggs. Results: Exposing human sperm to PAP (IC 50 p24 = 14 ± 2 nM) did not affect sperm motility and kinematics over a dose range of 1 to 1,000 μg/mL. Treating sperm with either 100 or 1,000 μg/mL of PAP had no effect on cervical mucus penetrability, nor did it affect sperm binding, penetration, and fusion of zona-free hamster eggs. PAP was noncytotoxic to genital-tract epithelial cells. Conclusions: The broad-spectrum antiviral agent PAP was nontoxic to human sperm and female genital tract epithelial cells even at a concentration 2,000 times higher than its IC 50 value against HIV-1. PAP has particular clinical usefulness both as a nonspermicidal intravaginal microbicide and as a prophylactic antiviral agent that can inactivate infective viruses and virus-infected cells in semen before assisted reproductive technology procedures are undertaken.