Epigallocatechin-3-gallate added after thawing to frozen dog semen: Effect on sperm parameters and ability to bind to oocytes' zona pellucida.

Dog sperm cryopreservation is gaining importance both in breeding dogs for commercial purposes and for pet animals. Anyway, cryopreservation of mammalian spermatozoa, including dog ones, induces some negative effect on sperm fertility, leading to a lower use of this technique and limiting its widespread use. Therefore, studies to improve the quality of canine semen after cryopreservation could have a relevant impact on both the scientific advancement and the clinical practice. The aim of the present work was to investigate the putative ameliorative effect of Epigallochatechin-3-gallate (EGCG) addition to post thawing medium on dog sperm motility, mitochondrial activity, acrosome integrity and on zona-binding ability (zona binding assay). Spermatozoa were thawed in Tris-fructose-citrate medium supplemented with EGCG (0, 25 and 50 µM) and sperm motility, mitochondrial activity and acrosome integrity were assayed at 0.5, 1.5, 3 and 6 h after post thawing incubation at 37 °C. An aliquot of semen from each treatment group after 1.5 h post thawing incubation was washed and used to perform heterologous (using porcine oocytes) or homologous zona binding assay. The results obtained showed that no significant effect is exerted by EGCG on sperm parameters analysed neither at 0.5, 1.5, 3 or 6 h after thawing excepting for the reduction of the percentage of live cells with active mitochondria at the higher dose at 6 h; furthermore, both homologous or heterologous zona binding ability, was not influenced by EGCG. In conclusion, EGCG supplementation to thawing medium does not improve dog sperm quality or zona binding capacity.

Lipid Regulation of Acrosome Exocytosis.

Lipids are critical regulators of mammalian sperm function, first helping prevent premature acrosome exocytosis, then enabling sperm to become competent to fertilize at the right place/time through the process of capacitation, and ultimately triggering acrosome exocytosis. Yet because they do not fit neatly into the "DNA--RNA-protein" synthetic pathway, they are understudied and poorly understood. Here, we focus on three lipids or lipid classes-cholesterol, phospholipids, and the ganglioside G(M1)--in context of the modern paradigm of acrosome exocytosis. We describe how these various- species are precisely segregated into membrane macrodomains and microdomains, simultaneously preventing premature exocytosis while acting as foci for organizing regulatory and effector molecules that will enable exocytosis. Although the mechanisms responsible for these domains are poorly defined, there is substantial evidence for their composition and functions. We present diverse ways that lipids and lipid modifications regulate capacitation and acrosome exocytosis, describing in more detail how removal of cholesterol plays a master regulatory role in enabling exocytosis through at least two complementary pathways. First, cholesterol efflux leads to proteolytic activation of phospholipase B, which cleaves both phospholipid tails. The resultant changes in membrane curvature provide a mechanism for the point fusions now known to occur far before a sperm physically interacts with the zona pellucida. Cholesterol efflux also enables G(M1) to regulate the voltage-dependent cation channel, Ca(V)2.3, triggering focal calcium transients required for acrosome exocytosis in response to subsequent whole-cell calcium rises. We close with a model integrating functions for lipids in regulating acrosome exocytosis.

Methyl-ß-Cyclodextrin Improves Sperm Capacitation Status Assessed by Flow Cytometry Analysis and Zona Pellucida-Binding Ability of Frozen/Thawed Bovine Spermatozoa.

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. Cyclodextrins added to the sperm culture medium have been described to induce in vitro sperm capacitation, enabling its use in protein-free media. However, the additive capacitating effect of methyl-ß-cyclodextrin (MßCD) in the medium containing bovine serum albumin (BSA) is unknown in the bovine species. In this study, we evaluated the effects of incubating frozen-thawed bovine spermatozoa in a BSA-containing medium supplemented with MßCD on different sperm quality and functional parameters. Sperm viability decreased with the addition of MßCD in a dose-dependent manner (p < 0.05), and DNA damage could be observed but only with the highest concentration of MßCD. However, pre-incubation of spermatozoa in MßCD-supplemented medium improved the capacitation status as assessed by the increase in plasma membrane fluidity, intracellular calcium concentration, induced acrosome reactivity and zona pellucida (ZP)-binding ability (p < 0.05). Thus, we conclude that MßCD supplementation is able to enhance the capacitation status of frozen-thawed bovine spermatozoa cultured in capacitation medium containing BSA and could result in a valid strategy for its application on artificial reproductive technologies such as in vitro fertilization or intracytoplasmic sperm injection.

Effects of a lecithin and catalase containing semen extender and a second dilution with different enhancing buffers on the quality of cold-stored canine spermatozoa.

In the present study, a diluent containing 0.8% lecithin (Minitube®, Tiefenbach, G) for the cold storage of canine semen was compared to a Tris-egg yolk extender (TRIS-EY) containing 20% egg yolk. For this purpose, aliquots of 10 mixed ejaculates (main fractions) were either diluted with TRIS-EY or with three lecithin extenders containing 0.8% lecithin with or without catalase and tyrosine. All samples were examined by computer assisted sperm analysis (CASA), chlortetracycline assay (CTC) and flow cytometry, sperm chromatin structure assay (SCSA) and zona pellucida binding assay (ZBA). Samples were then cold stored for 8 d and examinations repeated at days 3 and 8. Measurement in the CASA were repeated daily and prior to measurement, each sample was diluted with each of 4 enhancers with or without acetylcarnitine. The use of an enhancer proved to be essential for all extenders and after 8 d of cooling, progressive motility (P) and viability (V) still averaged > 70% and > 80% with the lecithin extenders containing additives, whereas with TRIS-EY and without additives it was significantly lower (P < 0.05). The percentage of capacitated spermatozoa did not differ between extender groups and there was no significant increase in acrosome reactions (AR) within 3 d. The chromatin status of cells was not changed by cooling within 8 d. The ZBA showed that with additives, significantly more spermatozoa bound to oocytes when a lecithin extender with additives was used (P < 0.05). In conclusion, the 0.8% lecithin extender containing catalase, conserved P and V during 8 d of cold storage better than the TRIS-EY extender, however, only when an enhancer was used; addition of acetylcarnitine to the enhancer did not further improve semen quality. The here introduced lecithin extender / enhancer combination is a useful tool for prolonged storage of cooled semen with excellent longevity and binding ability; addition of tyrosine to the extender did not improve semen quality.

Effect of N-acetyl-D-glucosamine on bovine sperm-oocyte interactions.

N-Acetyl-D-glucosamine (GlcNAc) is a major component of glycosaminoglycan, which is involved in sperm-oocyte interactions. We examined the effect of adding GlcNAc and other monosaccharides, D-mannose and D-fucose, to the in vitro fertilization (IVF) medium on bovine sperm-oocyte interactions. In medium in which sperm and a zona pellucida (ZP) were co-incubated with monosaccharides for 5 min, addition of GlcNAc (5 or 25 mM) significantly reduced the number of sperm that attached to the ZP. Pretreatment of gametes with GlcNAc (5 mM) prior to co-incubation also suppressed sperm-ZP attachment. Addition of GlcNAc (5 or 25 mM) to the medium in which sperm and a ZP were co-incubated for 5 h, however, significantly increased the number of sperm binding to and penetrating the ZP in a concentration-related manner. The other monosaccharides, D-fucose and D-mannose, did not have this effect. Supplementation of the sperm-oocyte co-incubation medium with 5 mM GlcNAc also enhanced the rate of polyspermic fertilization. When the ZPs were removed from the oocytes, GlcNAc did not affect the fertilization rate. Furthermore, incubation of sperm with 5 mM GlcNAc induced sperm membrane destabilization and an acrosome reaction, as evidenced by the hypo-osmotic swelling test and fluorescein isothiocyanate-labeled peanut agglutinin/propidium iodide (FITC-PNA/PI) staining. Finally, GlcNAc suppressed ZP hardening following fertilization, as determined by measuring the time required for pronase to dissolve the ZP. In conclusion, supplementation of IVF medium with GlcNAc has various effects on sperm-oocyte interactions including suppression of initial attachment, induction of sperm membrane destabilization and acrosome reaction, increase in the number of sperm secondarily bound to and penetrating the ZP, suppression of ZP hardening following sperm-oocyte co-incubation and increase in the rate of polyspermic fertilization.

Oviduct-specific glycoprotein modulates sperm-zona binding and improves efficiency of porcine fertilization in vitro.

Oviduct-specific glycoprotein (OGP) displays estrus-associated regional and temporal differences in expression and localizes to the zona pellucida, perivitelline space, and plasma membrane of oviductal oocytes and embryos, suggesting that it may have a role in regulation of fertilization and/or early embryonic development. The aims of this study were to evaluate the effect of exogenous OGP on in vitro fertilization (IVF) and embryo development in the pig using a defined serum-free culture system. In vitro-matured porcine oocytes were incubated with homologous OGP (0, 1, 10, 20, and 40 microg/ml) for 3 h and then washed prior to IVF. Exposure of oocytes to 10 or 20 microg/ml porcine OGP (pOGP) significantly reduced the incidence of polyspermy compared with the control (P < 0.01) while maintaining high penetration rates. When oocytes, spermatozoa, or both were preincubated with 10 microg/ml pOGP prior to IVF, the incidence of polyspermy was similarly reduced (P < 0.01) by all three treatments without affecting penetration rates. The ability of spermatozoa to undergo calcium ionophore-induced acrosome reaction was similar with or without exposure to pOGP. However, significantly fewer spermatozoa (P < 0.01) bound to the zona pellucida when oocytes were preincubated with pOGP. To evaluate the effect of pOGP on embryo development, embryos were cultured in pOGP-supplemented medium for 48 h or 144 h. Both transient and continuous exposure to pOGP significantly enhanced cleavage and blastocyst formation rate compared with the control (P < 0.01). These data demonstrate that exposure of either in vitro-matured oocytes or spermatozoa to pOGP decreased polyspermy and spermatozoa binding while maintaining high penetration rates of pig oocytes fertilized in vitro. Furthermore, pOGP exerted an embryotrophic effect independent of effects demonstrated on spermatozoa and oocytes at fertilization.

Effect of azorellanone, a diterpene from Azorella yareta Hauman, on human sperm physiology.

Previous studies have shown that cyclic terpenes extracted from plants decrease sperm motility and concentration in rats. In this work, we studied the effect 13-alpha-hydroxy-7-oxoazorellano (azorellanone), a cyclic diterpene extracted from Azorella yareta Hauman, on in vitro human sperm physiology. Sperm aliquots, capacitated for 4.5 or 20 hours, were incubated for 15 minutes with different concentrations of azorellanone. Then, the effects of azorellanone on sperm motility, viability, binding to the human zona pellucida, progesterone-induced acrosome reactions and increase in intracellular Ca(2)(+) concentration, and trypsin and chymotrypsin-like protease activities were evaluated. Sperm motility was evaluated according to World Health Organization (WHO) guidelines; sperm viability with the supravital dye Hoescht 33258; sperm-zona binding with the hemizona assay; progesterone-induced acrosome reaction with fluorescent lectin; intracellular Ca(2)(+) level with fura 2; and protease activity with the synthetic substrates N-t-Boc-Gln-Ala-Arg-Amido-4-methylcoumaryn and Succinyl-Leu-Leu-Val-Tyr-Amido-4-methylcoumaryn. The results obtained indicate that azorellanone inhibited sperm motility in a concentration-dependent manner at 0.15, 1.5, and 3 mM, while sperm viability was only inhibited at 3 mM. Treatment with azorellanone significantly inhibited sperm-zona binding, progesterone-induced acrosome reactions, and intracellular Ca(2)(+) concentration. Treatment of free-swimming sperm with azorellanone did not affect protease activity; however, the incubation of sperm extracts with azorellanone significantly inhibited both trypsin-like and chymotrypsin-like protease activities. In conclusion, azorellanone has a significant effect on the different parameters that characterize human sperm physiology.

Ionic calcium levels in oviduct explant-conditioned media from an Australian marsupial, the brushtail possum (Trichosurus vulpecula) and its relevance to in vitro fertilization.

Gametes from the brushtail possum (Trichosurus vulpecula), an Australian marsupial, require exposure to oviductal cells and/or their secretions before sperm binding and penetration of the zona pellucida can occur. Sperm-egg fusion, the next critical step in fertilization has not previously been reported in vitro. Here we describe the refinement of an oviduct epithelial cell (OEC) explant culture system using two different media to obtain in vitro sperm-egg fusion in the brushtail possum for the first time. Conditioned media from OEC explant cultures were supplemented with either 1% fetal calf serum (FCS) or 1 mg/ml polyvinyl alcohol and used for co-culture of epididymal sperm and superovulated eggs. Under these conditions zona penetration rates varied from 0 to 46% and sperm-egg fusion from 0 to 20%. Analysis of explant conditioned media indicated that qualitative and quantitative differences between batches could account, at least partially, for the large variability in zona penetration rates. Conditioned media that contained approximately 1 mM of ionic calcium were most effective for achieving sperm capacitation, zona binding, and penetration and sperm-egg fusion. The reorientation of the sperm head to T-shape, an indicator of capacitation in the brushtail possum, was closely linked with the concentration of calcium present in vitro.

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 2. The timing of in vitro nuclear oocyte maturation.

The time course of in vitro red deer nuclear oocyte maturation was determined. Ovaries were obtained at slaughter and oocytes were aspirated from follicles greater than 2mm in diameter. Oocytes with compact cumulus cells were matured in 50 microl microdrops (10 per drop) under mineral oil containing TCM 199 supplemented with 0.33 mM pyruvate, 10 microg LH and FSH, 1 microg oestradiol and 10% foetal bovine serum. Oocytes were matured at 39 degrees C and 5% CO(2) in air. At 3h intervals (0-27 h) oocytes were removed from incubation, cumulus expansion scored and removed, and fixed oocytes in ethanol:acetic acid (3:1) for 48 h. Oocytes were stained with lacmoid (1%) and nuclear maturation assessed. Oocytes were arrested in the germinal vesicle (GV) stage at aspiration and up to 6h of incubation. The nuclear membrane began to disperse after 6h and by 10.6+/-0.6h of incubation 75% of the oocytes exhibited germinal vesicle breakdown (GVBD). The mean time for 50% of the oocytes to reach metaphase one (MI) and metaphase two (MII) was 11.7+/-0.4 and 24.8+/-0.9h, respectively. Cumulus oophorus were tightly compacted at aspiration and did not begin expansion until 12h of culture. Full expansion was complete by 18 h of culture. Corona radiata cells did not begin expansion until 15 h and were fully expanded by 24h. Results indicate that in vitro red deer oocyte maturation follows a similar time course of nuclear maturation as reported for bovine and ovine oocytes.

Successful development in vitro of hamster 8-cell embryos to 'zona-escaped' and attached blastocysts: assessment of quality and trophoblast outgrowth.

The peri-implantation development involves zona escape (hatching) of blastocysts and their attachment and proliferation. These events are difficult to study in vivo, so in this study hamster 8-cell embryos were cultured through the hatched and attached blastocyst stages using different formulations of hamster embryo culture medium (HECM)-2. Supplementation of succinate, amino acids, vitamins (inositol, pantothenate, choline chloride) and bovine serum albumin (BSA) to HECM-2 supported 100% development of 'zona-escaped' blastocysts. In this medium (designated as hatching, i.e. HECM-2h) all blastocysts invariably deflated and escaped from focally lysed zonae, which underwent complete dissolution. In their presence, pre-morula stage embryos also escaped from zonae. Omission of BSA from HECM-2h failed to support zona escape whereas that of vitamins reduced zona escape (34.0% +/- 7.0). Blastocysts with the potential to undergo zona escape in HECM-2h were of high quality as they had a higher mean cell number (MCN) than the MCN of those developing in BSA-free HECM-2h (35.2 +/- 1.6 v. 24.3 +/- 1.1). Cell allocation (i.e. trophectoderm to inner cell mass ratio) in blastocysts remained unaltered in both media (2.6 +/- 0.2 v. 2.7 +/- 0.2). Supplementation of 10% bovine fetal serum (BFS) to HECM-2h was detrimental to the development of blastocysts (22.3% +/- 7.4) and none of them underwent zona escape. Interestingly, BFS was required either as a supplement to the medium or as a coating on dishes for azonal blastocysts to attach (> or = 70%) and exhibit trophoblast (TB) outgrowth (30.3 x 10(3) +/- 2.9 x 10(3) micron 2 at 48 h). These results show that HECM-2h supports maximal development of zona-escaped blastocysts with the potential to attach and exhibit TB outgrowth, and there is a developmental stage-specific requirement for serum during peri-attachment in hamster development.

Cryopreservation of oocytes and embryos: use of a mouse model to investigate effects upon zona hardness and formulate treatment strategies in an in-vitro fertilization programme.

Mouse oocytes and embryos were obtained following ovulation induction of (C57B16 x CBA) F1 animals. Zonae pellucidae were exposed to alpha-chymotrypsin in phosphate-buffered medium (PB1) supplemented with 3 mg/ml bovine serum albumin upon a heated stage (37 degrees C) and were observed constantly through an inverted microscope. The endpoint of the bioassay was the limits of the zona no longer being seen clearly at x 200 magnification, and the time taken for each zona to dissolve was recorded. A dose-dependent response in dissolution time was clearly seen, with 1% alpha-chymotrypsin being chosen as the routine working solution. Cryopreservation of 2-cell mouse embryos using propanediol did not cause zona hardening but induced a small and significant softening, as gauged by the time taken for zona dissolution (2181 +/- 167 versus 1864 +/- 82 s). Zona hardening was not suspected to occur after the freezing of human embryos as there was no difference in implantation rates per embryo for in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles between fresh [IVF: 63/644 (9.7%); ICSI: 51/330 (15.5%)] and frozen embryos [IVF: 36/458 (7.9%); ICSI: 18/112 (16.1%)]. Conversely, significant hardening of the zonae of mature oocytes was seen following cryopreservation (747 +/- 393 s) compared with freshly ovulated oocytes (151 +/- 68 s). It is concluded that (i) the freezing of murine oocytes with propanediol results in zona hardening, implying a possible benefit of ICSI after the cryopreservation of human oocytes, and (ii) the cryopreservation of embryos is not associated with zona hardening or reduced implantation, making microdissection of the zona in such cases generally unwarranted.

Protein kinase C plays an important role in the human zona pellucida-induced acrosome reaction.

To investigate the involvement of protein kinases in signal transduction in the human zona pellucida (ZP)-induced acrosome reaction (AR), the effects of protein kinase (PK) activators, dibutyryl cAMP (PKA) and cGMP (PKG), phorbol 12-myristate 13-acetate (PMA, PKC), and the PKC inhibitor, staurosporine were studied. Sperm samples were obtained from normozoospermic men with normal sperm-ZP binding. Oocytes were obtained from other patients with failure of fertilization in vitro. Motile spermatozoa selected by a swim-up technique were pre-incubated with 2.5-20 microM PMA, 1 mM dibutyryl cAMP or cGMP, 3 mM pentoxifylline or 0.125-2.0 microM staurosporine for 30 min and then incubated with four oocytes for 2 h in human tubal fluid supplemented with bovine serum albumin. The spermatozoa bound to the ZP were dislodged by repeatedly aspirating the oocytes with a small bore pipette and the state of the AR was determined by fluorescein-labelled Pisum sativum agglutinin. Motility and movement characteristics were assessed by computer-assisted sperm analysis (CASA) after incubation of spermatozoa with PMA for 30 min and 2 h. The dibutyryl cAMP and cGMP analogues had a small positive effect (P < 0.05) but pentoxifylline had no effect on stimulating the ZP-induced AR (P > 0.05). In contrast, PMA stimulated ZP-induced AR in a marked dose-dependent manner. Only the highest concentrations (15-20 microM) of PMA significantly decreased percentage motility (P < 0.001). Doses of 2.5-15 microM of PMA significantly stimulated ZP-induced AR without decreasing motility (P < 0.001). The PKC inhibitor, staurosporine (0.125-0.25 microM) significantly inhibited ZP-induced AR without affecting motility (P < 0.001). Sperm samples from 33 normozoospermic men were used for studies of the ZP-induced AR augmented with 15 microM PMA. One sample did not show a response to PMA stimulation. Among the 14 men with low ZP-induced AR, half had normal responses to PMA and other half had low responses to PMA. In conclusion, activation or inhibition of PKC significantly increases or decreases human ZP-induced AR suggesting that PKC plays a important role in the signal transduction pathway for the physiological AR.

Analysis of polysulfate-binding domains in porcine proacrosin, a putative zona adhesion protein from mammalian spermatozoa.

Proacrosin is one of the major proteins found within the acrosomal vesicle of mammalian spermatozoa. Previous work has shown that it binds non-enzymatically and with high affinity to polysulfate groups on zona pellucida glycoproteins (ZPGPs) thereby leading to the hypothesis that at fertilization it functions as a secondary ligand molecule to retain acrosome-reacted spermatozoa on the surface of the egg. In the present work we have investigated the nature and extent of the polysulfate binding domain on boar sperm proacrosin using a combination of group-specific modifying reagents, fragmentation analysis, peptide synthesis and expression of deletion recombinants in E. coli bacteria. Taken overall, our results show that arginine, lysine and histidine residues located between Gly 93 and Ala 275, together with the participation of His 47 and Arg 50, are necessary for maximum polysulfate binding activity. The secondary and tertiary structure of this central peptide domain is also important to ensure correct alignment of basic residues with complementary sulfate groups on ZPGPs. Proacrosin, therefore, has many properties in common with other polysulfate binding proteins, such as antithrombin III and sea urchin sperm binding, in having a conformation-dependent domain containing basic amino acids that mediates specific protein-protein interactions. These observations strengthen the hypothesis that proacrosin is a multifunctional protein with a major role as a ligand molecule at fertilization.

Participation of glycosylated residues in the human sperm acrosome reaction: possible role of N-acetylglucosaminidase.

Sperm binding to the egg zona pellucida is mediated by complementary protein-carbohydrate interaction. This binding results in the exocytosis of the sperm acrosome, or acrosome reaction (AR). We report the effect of different neoglycoproteins (sugar residues covalently bound to bovine serum albumin) on the human sperm AR. p-Aminophenyl-N-acetyl-beta-D-glucosaminide-BSA (BSA-GlcNAc) and p-aminophenyl-alpha-D-mannopyranoside-BSA (BSA-Man) at 1 micrograms/ml were capable of inducing the greatest percentages of AR (3-fold stimulation with respect to controls), while other NeoGPs had only a weak effect on this process. The BSA-GlcNAc-induced acrosome reaction was inhibited by N-acetylglucosamine (GlcNAc), p-nitrophenyl-GlcNAc, and purified soluble beta-N-acetylglucosaminidase (beta NAG). The induction of the AR with BSA-Man could be inhibited by mannose, while soluble alpha-mannosidase was only partially effective. These data suggest that binding sites for GlcNAc and mannose may be involved in the induction of the AR in human sperm. The characteristics of the BSA-GlcNAc induction suggest that the beta NAG molecule may be the mediator of this effect.

Induction of physiological acrosome reactions in caput epididymal spermatozoa of mice.

Mouse caput spermatozoa are considered immature and thus unable to fertilize oocytes. In this study, we determined whether washing mouse caput spermatozoa increased their ability to acrosome react in response to a physiological stimulus. The results obtained showed that mouse caput spermatozoa incubated in Earles' modified medium containing calcium chloride and supplemented with BSA and pyruvate for 1 h at 37 degrees C and then washed acrosome reacted in response to both solubilized zonae and immunoaggregation of a zona binding site. In addition, the material removed from caput spermatozoa by washing blocked induced acrosome reactions of cauda spermatozoa. The data indicate that mouse caput epididymal spermatozoa, if incubated and washed, can undergo physiological acrosome reactions.