Fertilization and early embryology: Human follicular fluid inhibits the binding of human spermatozoa to zona pellucidain vitro

The effect of human follicular fluid on human zona pellucidabinding of spermatozoa was investigated using the hemizona bindingassay (HZA). This effect was compared to that of progesterone,a known component of human follicular fluid. Exposure of spermatozoato 25% pooled human follicular fluid for 1 h significantly reducedthe number of spermatozoa bound to zona pellucida when comparedto those without human follicular fluid treatment (149.1 ±30.7 versus 177.1 ± 33.8, P 0.01). The same phenomenonwas observed after 3 h of treatment The corresponding numbersof bound spermatozoa were 140.4 ± 19.1 and 200.2 ±23.4 (P 0.0001). Progesterone (1.0µg/ml) stimulated thezona pellucida-binding capacity of spermatozoa significantlyunder the same conditions (P 0.01). The numbers of bound spermatozoaafter 1 and 3 h progesterone treatment were 235.5 ± 44.7(control, 168.1 ± 32.9) and 204.3 ± 27.4 (control,162.3 ± 20.1) respectively. HZA comparing the effectsof human follicular fluid and progesterone at concentrationsequivalent to those found in human follicular fluid using matchinghemizonae confirmed the inhibitory effect of human follicularfluid on sperm binding to zona pellucida (80.4 ± 28.4versus 149.8 ± 35.2, P 0.05). This inhibitory effectwas also found in another eight individual human follicularfluid samples. Both human follicular fluid and progesteronedid not affect the motility and viability of the treated spermatozoawhen compared to the controls with the same incubation period.Although more spermatozoa underwent the acrosome reaction after1 and 3 h of human follicular fluid treatment than in the control,the extent was comparable to those after progesterone treatmentThese results suggested that human follicular fluid inhibitedthe zona pellucida-binding capacity of spermatozoa in vitro.This inhibitory effect of human follicular fluid was not mediatedby progesterone, and did not result from the effects of humanfollicular fluid on sperm motility, viability and acrosome reaction.     

The effects of follicular fluid from patients with different indications for IVF treatment on the binding of human spermatozoa to the zona pellucida

This study compared the effects of human follicular fluid (hFF) from women with endometriosis, tubal factor and male factor on the zona binding capacity of human spermatozoa. Samples of hFF were collected from 30 patients, 10 patients for each of the indications of infertility, at the time of oocyte retrieval in an in-vitro fertilization/embryo transfer programme. The hemizona binding assay (HZA) was used to assess the effect of these hFF on the zona binding potential of human spermatozoa. The mean numbers of spermatozoa bound to the zona pellucida after treating the spermatozoa with hFF from endometriosis, tubal factor and male factor were 90.5 +/- 20.9, 108.9 +/- 22.3 and 101.2 +/- 13.4 respectively. These were significantly lower than their corresponding controls, the spermatozoa of which were incubated with Earle's balanced salt solution (endometriosis 238.7 +/- 34.7; tubal factor 210.8 +/- 41.6; male factor 205.4 +/- 26.3; P <0.002). The hemizona binding index (HZI) was similar between male factor samples (52.0 +/- 6.7) and tubal factor samples (53.8 +/- 4.2). Spermatozoa incubated with hFF from endometriosis patients (36.0 +/- 4.1) had an HZI that was significantly lower than those treated with hFF from tubal factor patients (P <0.01). Probably due to small sample size, the differences in HZI between endometriosis samples and male factor samples did not reach statistical significance (P = 0.076). These data suggest that there was a stronger sperm-zona binding inhibitory effect of hFF from patients with endometriosis than from those without the disease.      

An IgG-Fc Binding Protein in Seminal Fluid*

ABSTRACT: Human seminal fluid, at low dilutions, prevented the binding of aggregated human IgG (AHG) to bull spermatozoa. Seminal fluids from vasectomized men were also inhibitory. Preincubation of the seminal fluid with the spermatozoa prior to washing and addition to AHG had no inhibitory effect, indicating that the fluid component was reacting directly with AHG. Human seminal fluid was fractionated by gel exclusion chromatography on Ultrogel AcA-34, and AHG inhibitory activity was found in fractions corresponding to a molecular weight of 94,000. The activity in this fraction was stable to boiling for 10 min. It was sensitive to pronase but resistant to glycosidase, phospholipase C, neuraminidase, ribonuclease, and deoxyribonuclease, indicating that it was a protein. The gel filtration fraction readily bound recrystallized Fc and AHG; IgG was bound to a lesser extent, and no reactivity was observed with F(ab')₂, IgA, or IgM. Thus, the seminal fluid fraction appeared to specifically react with the Fc portion of IgG. The seminal fluid Fc-binding protein was isolated by affinity chromatography on Fc coupled to CNBr-activated Sepharose 4B. Scatchard analysis revealed that the binding of the seminal fluid Fc-binding protein to recrystallized Fc is reversible and had a Kd of approximately 3 × 10^?6 M.     

The influence of solubilized porcine zona pellucida protein on the binding capacity of human spermatozoa.

The acrosome reaction, sperm-zona pellucida binding, sperm-oolemma binding/fusion and subsequent fertilization are known to be influenced by homologous as well as heterologous follicular fluid and zona pellucida protein. In this study, the effect was investigated of different concentrations of solubilized porcine zona pellucida protein on the zona binding potential of human spermatozoa under hemizona assay conditions. Human spermatozoa incubated with 617 and 142 micrograms/ml porcine zona pellucida protein showed a statistically significant increase in zona binding when compared with control spermatozoa (106.5 +/- 18.0 versus 60.9 +/- 29.0, P less than 0.02 and 111.0 +/- 26.6 versus 63.0 +/- 25.5, P less than 0.0001, respectively). Concentrations of 67 micrograms/ml porcine zona pellucida protein did not show a significant increase in zona binding (78.7 +/- 21.7 versus 66.7 +/- 25.4, P greater than 0.05). Control zona binding values for different experiments did not differ significantly (60.9 +/- 29.0; 63.0 +/- 25.5; and 66.7 +/- 25.4, P greater than 0.6). In conclusion, it seems likely that a factor(s) present in the porcine zona pellucida might play a beneficial role during human sperm-oocyte binding. The results of the study might be used in future investigations to manipulate gamete interaction to such an extent that improved fertilization rates can be accomplished.     

Fertilization and early embryology: The factors affecting sperm binding to the zona pellucida in the hemizona binding assay

Sperm binding to the zona pellucida is a prerequisite for fertilization.The hemizona binding assay (HZA) is commonly used to evaluatethe zona-binding capacity of spermatozoa. The present studyreports three factors that affect HZA. They were the base mediumused, the protein source and the size of pipette used for removingloosely bound spermatozoa during HZA. The number of spermatozoabound on the hemizona was compared between (1) Earle's balancedsalt solution (EBSS) and Ham's F-10, and (2) between human serumand bovine serum albumin (BSA). Results indicated that EBSSand human serum both significantly increase the number of boundspermatozoa when compared to Ham's F-10 (P < 0.0001, pairedt-test) and BSA (P < 0.05, paired t-test) respectively. Pipettesof different diameters were used to study the effect of sizein removing loosely bound spermatozoa on hemizona. Data showedthat the diameter of the pipette should be 200 mm, in ordernot to remove bound spermatozoa excessively. These results emphasizethe importance of standardization of the protocol of the hemizonaassay worldwide to be able to compare results between differentlaboratories.     

Characterization and isolation of SOB2, a human sperm protein with a potential role in oocyte membrane binding

G12 monoclonal antibody (mAb), one of a library of constructed mAb directed against human sperm proteins, was found by immunoperoxidase staining to label the post-acrosomal and neck regions of fixed human cauda epididymal and ejaculated spermatozoa. Epithelium and fluid of caput epididymis were strongly labelled while there was no staining on testis and efferent ducts. Western lot analysis revealed that G12 antibody reacted with proteins of 17.5, 18 and 19 kDa in human spermatozoa. This pattern seems to be specific for mature human spermatozoa, as it has not been observed either in other human tissues tested, or in spermatozoa from different animals. SOB2, the corresponding protein, was isolated from NP40-extracted human spermatozoa by using preparative electrophoresis, followed by isoelectrofocusing according to its isoelectric point of 6.4 G12 Fab fragments strongly inhibited binding of human spermatozoa to zona-free hamster oocytes (up to 86% inhibition at 200 micrograms/ml). Impairment of binding was dependent on the concentration of purified G12 immunoglobulin (Ig)G1, and significant even at 10 micrograms/ml. There was no inhibitory effect of G12 antibody on sperm motility parameters or triggering of the acrosome reaction and it did not inhibit binding to human zona pellucida. These results indicate that SOB2 is likely to participate in membrane oocyte binding, and my be potential candidate for the development of a contraceptive vaccine.      

Purification and characterization of a sperm-binding glycoprotein from human endometrium

A sialic-acid-binding protein (SABP) was purified to apparenthomogeneity from human endometrial scrapings taken at variousstages of the menstrual cycle from normal cycling females. The54 kDa monomer was found to be an O-linked glycoprotein witha total carbohydrate content of 34%. This protein agglutinatedwashed 2% v/v rabbit red blood cells (RBC) in the presence ofcalcium. Amongst sialic acids and sialoglycoproteins testedfor haemagglutination inhibitory activities, N-glycolyI neuraminicacids and human 1-acid glycoprotein were found to be the mostpotent, the agglutination activity being totally abolished ondesialylation of the RBC in the presence of neuraminidase. Westernblot studies showed it to be present in the uterine fluid butabsent in normal female serum and in full-term placenta. Itwas also absent in endometrial homogenates of some cases ofunexplained primary infertility. Specific binding studies andScatchard analysis revealed that 125I-labelled human SABP ligandcan bind to human spermatozoa with a Ka= 2.6 x 109 M-1, theirreceptors probably being glycoconjugates having a terminal sialicacid moiety, since the sperm–protein interaction couldalso be abolished when spermatozoa were desialylated with neuraminidase.The binding occurred specifically on the sperm head plasma membraneand decreased markedly when spermatozoa were previously capacitatedin vitro using human serum albumin, implicating the possibleloss of a sialoglycoprotein receptor to which the ligand bindsduring capacitation. The biological importance of this sperm-bindingsecretory glycoprotein and its functional significance in humanreproduction have been discussed.     

Isolation and biological characterization of boar sperm capacitation-related antigen

PROBLEM: Monoclonal anti-capacitated sperm antibody has been used as a probe to identify, isolate, and characterize specific, fertilization-related antigen with some characteristic features that point to its possible significance in immunocontraception. METHOD OF STUDY: Fast protein liquid chromatography (FPLC), enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and isoelectrofocusing were used for isolation, immunochemical and physicochemical characterization of a monoclonal antibody (mAb) 1F10 cognate antigen. Sperm-zona pellucida binding and hemizona assay were used for testing the biological roles of mAb 1F10 and Ag 1F10 in boar and human fertilization processes. RESULTS: The Ag 1F10 was found to be eluted in the eighth protein peak of FPLC-fractionated Nonidet P40 (NP40) extracts of capacitated boar spermatozoa. The SDS-PAGE and immunoelectrofocusing experiments showed that Ag 1F10 is a protein composed of a single peptide chain with a relative molecular mass of 68/70 kDa and an isoelectric point of 3.5. It was demonstrated that the zona binding activity of spermatozoa preincubated in the presence of mAb 1F10 was significantly inhibited both in porcine and human in vitro fertilization (IVF) systems. A dose-dependent manner of inhibition of the sperm/ligand activities of porcine and human zona pellucida was observed when the effect of purified Ag 1F10 was investigated by its preincubation with zona pellucida. CONCLUSIONS: It is assumed that the protein bearing the epitope recognized by mAb 1F10 may be accepted as one of the molecules with receptor function in sperm-zona pellucida interaction during fertilization.     

Tyrosine phosphorylation of a 95 kDa protein and induction of the acrosome reaction in human spermatozoa by recombinant human zona pellucida glycoprotein 3

Protein tyrosine phosphorylation and induction of the acrosome reaction (AR) in non-capacitated and capacitated human spermatozoa was investigated in response to recombinant human zona pellucida glycoprotein (rhZP3) produced by Chinese hamster ovary cells transfected with a plasmid containing human ZP3 cDNA. rhZP3-containing medium promoted the AR in a high proportion of capacitated spermatozoa (48.6 +/- 3.2%; P < 0.01) compared with control (no rhZP3) samples (14.8 +/- 2.1%). However, rhZP3-containing medium did not cause increased acrosomal exocytosis in non-capacitated spermatozoa (16.8 +/- 3.0%). Induction of the AR was associated with increased tyrosine phosphorylation of a 95 +/- 5 kDa epitope only in capacitated spermatozoa. A dose-dependent increase in the protein phosphorylation of a 95 kDa epitope in response to rhZP3 was detected by [gamma-32P]- ATP labelling of detergent-solubilized sperm proteins. When spermatozoa were co-incubated with monoclonal antibody 97.25 (mAb 97.25) recognizing a 95 kDa tyrosine kinase epitope, there was no rhZP3 induction of tyrosine phosphorylation of the 95 kDa protein. Such co- incubation also markedly inhibited the AR (23.9 +/- 3.1%). These results support the model that initial interaction of the fertilizing spermatozoon with ZP3 involves the tyrosine phosphorylation of a 95 kDa tyrosine kinase protein and that this requires capacitation.      

Purification and characterization of cell wall proteins of Listeria monocytogenes

Two proteins were purified from Listeria monocytogenes cell wall using detergent extraction, Superose 6 gel chromatography. Mono Q cation-exchange chromatography and Superose 12 gel chromatography. Proteins were shown to form complexes of ca. 300 kDa and pI 4.7. These complexes could not be dissociated in 6 M urea, however, during SDS-PAGE 79 and 39 kDa monomers were formed. Immunoblot analysis showed that the proteins under investigation were common to all listerial strains tested, but were absent in strains of other bacteria. We propose that the proteins investigated here could serve as immunoserological markers for Listeria.     

Clinical application of fast protein liquid chromatography (FPLC) system

FPLC system is a high-performance liquid chromatography system, designed specifically for separation of proteins, peptides and polynucleotides. Many kinds of column have been prepared, including Mono Q, Mono S, Mono P, Superose, etc. for ion exchange chromatography, chromatofocussing, gel filtration, hydrophobic chromatography, reversed phase chromatography and affinity chromatography. It is a fast and simple system for separation of proteins in various body fluids, particularly plasma, urine and cerebrospinal fluid.     

Purification and characterization of Plasmodium falciparum succinate dehydrogenase

Succinate dehydrogenase (SDH), a Krebs cycle enzyme and complex II of the mitochondrial electron transport system was purified to near homogeneity from the human malarial parasite Plasmodium falciparum cultivated in vitro by FPLC on Mono Q, Mono S and Superose 6 gel filtration columns. The malarial SDH activity was found to be extremely labile. Based on Superose 6 FPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing-PAGE analyses, it was demonstrated that the malarial enzyme had an apparent native molecular mass of 90 +/- 8 kDa and contained two major subunits with molecular masses of 55 +/- 6 and 35 +/- 4 kDa (n = 8). The enzymatic reaction required both succinate and coenzyme Q (CoQ) for its maximal catalysis with Km values of 3 and 0.2 microM, and k(cat) values of 0.11 and 0.06 min(-1), respectively. Catalytic efficiency of the malarial SDH for both substrates were found to be relatively low (approximately 600-5000 M(-1) s(-1)). Fumarate, malonate and oxaloacetate were found to inhibit the malarial enzyme with Ki values of 81, 13 and 12 microM, respectively. The malarial enzyme activity was also inhibited by substrate analog of CoQ, 5-hydroxy-2-methyl-1,4-naphthoquinone, with a 50% inhibitory concentration of 5 microM. The quinone had antimalarial activity against the in vitro growth of P. falciparum with a 50% inhibitory concentration of 0.27 microM and was found to completely inhibit oxygen uptake of the parasite at a concentration of 0.88 microM. A known inhibitor of mammalian mitochondrial SDH, 2-thenoyltrifluoroacetone. had no inhibitory effect on both the malarial SDH activity and the oxygen uptake of the parasite at a concentration of 50 microM. Many properties observed in the malarial SDH were found to be different from the host mammalian enzyme.     

Molecular basis of human sperm-zona pellucida interaction

The recognition of carbohydrate sequences by complimentary receptors has been shown to be a critical factor in gamete interaction in many different animal species. We have proposed the hypothesis that, in the human, sperm binding to the zona pellucida requires a 'selectin-like' interaction. We have used the hemizona assay (a unique internally controlled bioassay that evaluates tight binding of human sperm to the homologous zona pellucida) and advanced methods of carbohydrate analysis to test this hypothesis. We provide compelling evidence that demonstrates that oligosaccharide recognition is also required for specific, tight human gamete binding. Hapten inhibition tests, zona pellucida lectin-binding studies and removal/modification of functional carbohydrates by chemical and enzymatic methods provide evidence for the involvement of defined carbohydrate moieties in initial binding. Our studies suggest the existence of distinct zona-binding proteins on human sperm that can bind to selectin ligands. Additionally, results suggest a possible convergence in the types of carbohydrate sequences recognized during initial human gamete binding and immune/inflammatory cell interactions. Full characterization of the glycoconjugates that manifest selectin-ligand activity on the human zona pellucida will allow for a better understanding of human gamete interaction in physiological and pathological situations.     

Fertilization and early embryology: Female age does not affect the capacity of human zona pellucida to bind spermatozoa

The purpose of this study was to evaluate the effect of femaleage on the capacity of the zona pellucida to bind spermatozoa.A total of 1008 unfertilized oocytes obtained from 210 women(aged 21–43 years) participating in the in-vitro fertilizationprogramme were tested using a hemizona assay. Spermatozoa takenfrom a cryopreserved pool of fertile donor specimens servedas a control in the hemizona assay, and were used to assessthe ability of the zona pellucida to bind spermatozoa. The mean± SD number of spermatozoa attached to the hemizona was107 ± 42. The binding capacity of different oocytes fromthe same cohort varied substantially (coefficient of variation= 28%). Age was not found to be correlated with the number ofspermatozoa bound to the zona pellucida (r = -0.02; P > 0.1).It was concluded that female age has no role in the abilityof the human zona pellucida to bind spermatozoa. Key words:female age/spermatozoa/zona binding.     

Further characterization and partial amino acid sequence of a cysteine proteinase from Trypanosoma cruzi

A cysteine proteinase from epimastigotes of Trypanosoma cruzi, Tul 2 stock, has been purified to homogeneity from cell-free extracts obtained by freezing and thawing, by a procedure involving ammonium sulfate fractionation, DEAE-Sephacel chromatography, and gel filtration on Sephadex G-200; when necessary, further purification was attained by fast protein liquid chromatography on Mono Q and Superose 6 columns. The purified enzyme was strongly inhibited by leupeptin, antipain and chymostatin (I50 values of 0.25, 0.75 and 1 microM, respectively), little inhibited by elastatinal, and unaffected by pepstatin A. The enzyme is a glycoprotein, as shown by binding to ConA-Sepharose and elution with alpha-methyl-D-mannopyranoside and alpha-methyl-D-glucopyranoside. Partial amino acid sequences were obtained from the N-terminal end (32 amino acids) of the carbamidomethylated enzyme, and from a tryptic peptide (14 amino acids) of the pyridylethylated enzyme. Both regions show considerable homology with papain and some cathepsins, such as cathepsin L, thus showing that the enzyme belongs to the cysteine proteinase family.     

Factors influencing sperm-zona pellucida binding in vitro using the intact zona model

The zona pellucida binding assay assesses the ability of spermatozoa to bind to the zona pellucida. The present study investigated the influence of: (i) prior oocyte exposure to spermatozoa on subsequent sperm-zona pellucida binding in vitro; and (ii) cryopreservation of oocytes. Only oocytes obtained from fertile donors were used and the binding capacity of non-inseminated, cryopreserved oocytes was compared with both inseminated/unfertilized, cryopreserved oocytes and inseminated/unfertilized, non-cryopreserved oocytes recovered from in- vitro fertilization cultures on sperm-zona pellucida binding using an intact zona model. There was no statistically significant difference in sperm-zona binding between non-inseminated, cryopreserved oocytes (range 9.6-23.2), inseminated/unfertilized, cryopreserved oocytes (range 15.0-16.0) and inseminated/ unfertilized, non-cryopreserved oocytes (range 3.3-23.0). The coefficient of variation for sperm binding to all oocyte groups was very large (range 37-121%). We conclude that neither prior exposure of human oocytes to human spermatozoa nor cryopreservation of human oocytes influences the subsequent binding of a different population of spermatozoa to the zona pellucida. However, large oocyte-to-oocyte variation of sperm-zona binding may diminish the usefulness of this assay in clinical practice and as a research tool.      

Cell surface binding sites for progesterone on human spermatozoa

This study demonstrates the presence of [3H]-progesterone binding protein on the cell surface of human spermatozoa. The binding protein is masked and is exposed after treatment of spermatozoa with surfactants such as digitonin. Specific binding of [3H]-progesterone is observed after the washed spermatozoa or the crude cell membrane fractions are treated with 0.1% digitonin at 0-4 degrees C for 30 min R5020, 17alpha-hydroxyprogesterone and testosterone only partially compete with [3H]-progesterone for binding whereas the synthetic steroids STS557, RU486, ZK98.299 and ZK98.734 do not competitively inhibit the binding of [3H]-progesterone to spermatozoa. Progesterone binding sites are located in the acrosomal region of spermatozoa, as indicated by the use of fluorescein isothiocyanate-labelled progesterone-bovine serum albumin. The binding protein cross-reacts with the monoclonal antibodies to uterine progesterone receptor and has a molecular weight of approximately 85 kDa. This is the first study which demonstrates the presence of masked progesterone binding sites on the cell surface of human spermatozoa that are exposed after treatment with surfactants. These binding sites seem to have structural homology with the intracellular progesterone receptor but differ in terms of ligand specificity and location. The study further demonstrates that follicular fluid facilitates progesterone binding to spermatozoa.      

Anti-SLIP1-reactive proteins exist on human spermatozoa and are involved in zona pellucida binding.

Sulpholipid immobilizing protein 1 (SLIP1) is an evolutionarily conserved 68 kDa plasma membrane protein, present selectively in germ cells. We have previously shown that mouse sperm SLIP1 is involved in sperm-zona pellucida (ZP) binding. In this report, we extended our study to the human system. Immunoblotting demonstrated that anti-SLIP1-reactive proteins (mol. wt 68 and 48 kDa) could be extracted from human spermatozoa by an ATP-containing solution, a result that is consistent with observations in other species. Direct immunofluorescence, using Cy3-conjugated anti-SLIP1 IgG, revealed SLIP1 staining over the acrosomal region, with higher intensity at the posterior area. Using the human sperm-ZP binding assay, we demonstrated that pretreatment of human spermatozoa from three donors with anti-SLIP1 IgG revealed lower numbers of zona-bound spermatozoa, as compared to the corresponding control spermatozoa treated with normal rabbit serum IgG. This decrease in zona pellucida binding was not from an antibody-induced decline in sperm motility or an increase in the premature acrosome reaction. The results strongly suggest that anti-SLIP-reactive proteins on human spermatozoa play an important role in ZP binding.     

Progesterone promotes the acrosome reaction in capacitated human spermatozoa as judged by flow cytometry and CD46 staining.

The acrosome reaction is a necessary prerequisite for spermatozoa to acquire fertilizing ability. Several different moieties appear to promote the acrosome reaction through different pathways, including solubilized zona pellucidae, recombinant zona protein ZP3, follicular fluid, calcium ionophores, and mannosylated bovine serum albumin (BSA). Although many investigators have presented evidence that progesterone also promotes the acrosome reaction through the mediation of a non-genomic cell membrane receptor, this concept has been challenged. Other workers have suggested that progesterone does not promote an acrosome reaction in human spermatozoa, as judged by the detection of CD46, a complement regulatory protein present on the inner acrosome membrane, through flow cytometric analysis of large numbers of spermatozoa. Prior investigations were criticized by the limited numbers of spermatozoa enumerated visually, the use of non-specific staining techniques, and the failure to eliminate dead spermatozoa during the scoring of the acrosome reaction. We have repeated these experiments, using both a supravital dye to eliminate dead spermatozoa from flow cytometric analysis, and anti-CD46 monoclonal antibody to score acrosome-reacted spermatozoa. Care was taken to validate the adequacy of capacitation conditions, which were proven by the ability of spermatozoa to acrosome react in response to mannosylated BSA and to penetrate zona-free hamster eggs. Confocal microscopy was used to confirm that CD46 immunostaining was limited to the acrosomal region of the spermatozoon head. Our results indicate that progesterone does promote an acrosome reaction within capacitated spermatozoa.     

The trypanocidal Cape buffalo serum protein is xanthine oxidase.

Plasma and serum from Cape buffalo (Syncerus caffer) kill bloodstream stages of all species of African trypanosomes in vitro. The trypanocidal serum component was isolated by sequential chromatography on hydroxylapatite, protein A-G, Mono Q, and Superose 12. The purified trypanocidal protein had a molecular mass of 150 kDa, and activity correlated with the presence of a 146-kDa polypeptide detected upon reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid sequences of three peptide fragments of the 146-kDa reduced polypeptide, ligand affinity and immunoaffinity chromatography of the native protein, and sensitivity to pharmacological inhibitors, identified the trypanocidal material as xanthine oxidase (EC 1.1.3.22). Trypanocidal activity resulted in the inhibition of trypanosome glycolysis and was due to H2O2 produced during catabolism of extracellular xanthine and hypoxanthine by the purine catabolic enzyme.