C. Barros

Human Sperm Penetration into Zona-Free Hamster Oocytes as a Test to Evaluate the Sperm Fertilizing Ability

Penetration menschlicher Spermatozoen in Zona pellucida‐freie Hamster‐Oozyten. Test zur überprüfung der Fertilisation von Spermatozoen

EARLY STEPS OF SPERM-EGG INTERACTIONS DURING MAMMALIAN FERTILIZATION

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95kDa tyrosine kinase‐protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, inin vitrofertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver‐enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea‐pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.

A Basic 18-Amino Acid Peptide Contains the Polysulfate-Binding Domain Responsible for Activation of the Boar Proacrosin/Acrosin System

Abstract Proacrosin is the zymogen of acrosin, a serine protease localized in the acrosomal matrix of mammalian sperm. Proacrosin/acrosin binds to solubilized zona pellucida glycoproteins (ZPGs) and various polysulfates in a non-enzymatic mechanism. In addition, both polysulfates and ZPGs induce proacrosin activation once they bind to the polysulfate-binding domain (PSBD) of the enzyme. We show here that the peptide 43IFMYHNNRRYHTCGGILL60 inhibited the proacrosin activation induced by either fucoidan or ZPGs. In addition, the peptide was recognized by the monoclonal antibody C5F10, which is directed against the PSBD region. Our data suggest that the PSBD is composed of many “subsites” that may or may not interact with each other.

Inhibition of mouse in vitro fertilization by an antibody against a unique 18 -amino acid domain in the polysulfate-binding domain of proacrosin/acrosin

OBJECTIVE To determine the contribution of the polysulfate-binding domain (PSBD) of acrosin during sperm penetration. DESIGN To inhibit the in vitro fertilization of mouse zona-intact oocytes by using a polyclonal antibody raised against an 18-amino acid peptide of proacrosin (anti-PSBD). SETTING Unit of Reproduction and Development, Faculty of Biological Sciences, Pontifical Catholic University of Chile. PATIENT(S) None. INTERVENTION(S) A polyclonal antibody against the 43IFMYHNNRRYHTCGGILL(60) peptide was raised in New Zealand female rabbits. The specificity of the antibody was evaluated by an ELISA. Zona-intact mouse oocytes were coincubated with capacitated spermatozoa for 3 hours in the presence of 0.63 mg/mL of the antibody or preimmune serum. As a control, we used zona-free mouse oocytes under the same experimental conditions. MAIN OUTCOME MEASURE(S) We evaluated the fertilization rate of zona-intact and zona-free mouse oocytes by phase-contrast microscopy. An oocyte was considered fertilized when at least one decondensed sperm head was found within the egg cytoplasm. We evaluated 50-60 mouse oocytes in each group in three independent experiments. RESULT(S) The anti-PSBD antibody inhibited the fertilization of zona-intact, but not zona-free, mouse oocytes, by capacitated spermatozoa. In addition, the binding of the anti-PSBD to proacrosin/acrosin in a solid-phase assay was inhibited in the presence of polysulfates (fucoidan). CONCLUSION(S) The anti-PSBD directed against the PSBD of proacrosin/acrosin inhibited the penetration of capacitated mouse spermatozoa through the zona pellucida. This antibody may be a useful tool to define the roles of the different domains of proacrosin/acrosin during gamete interaction.

Fertilizing characteristics of bovine sperm with flattened or indented acrosomes.

Frozen semen from a control bull (C: 89% morphologically normal sperm) and two bulls with acrosomal defects (K1: 92% flattened acrosomes; K2: 82% indented acrosomes) were used to investigate the fertilizing ability of bull sperm with flattened or indented acrosomes. In experiment 1, frozen-thawed sperm were evaluated for acrosomal integrity with fluorescent microscopy. In experiment 2, proteolytic activity of the acrosomal contents of sperm was evaluated through a gelatin digestion assay. In experiment 3, an IVF test system was used to determine the ability of sperm with flattened or indented acrosomes to bind to bovine oocytes and penetrate the zona pellucida. In experiment 4, IVM zona-free bovine oocytes (ZFO) were fertilized and examined to evaluate sperm chromatin decondensation. In experiment 1, bulls K1 and K2 had a lower proportion of sperm with intact acrosomes (0 and 13.6 +/- 4.5%, respectively) than bull C (30.2 +/- 5.6%) after 2h of incubation. In experiment 2, the proportion of sperm with proteolytic activity, as indicated by gelatin digestion around sperm heads, did not differ among bulls (C: 55%, n=410; K1: 43%, n=426; K2: 48%, n=324). In experiment 3, a lower proportion of sperm with flattened (K1) or indented acrosomes (K2) bound to oocytes than sperm from the control bull, C. The percentage of zona penetrated (55%, n=20; 13%, n=23; 4%, n=25) and the mean (+/- S.E.M.) number of sperm penetrating these zona pellucida (19.7 +/- 2.5; 6.9 +/- 1.0; and 2.6 +/- 0.5) was higher (P<0.05) for bull C than for bulls K1 or K2, respectively. In experiment 4, the percentage of ZFO penetrated (95%, n=20; 52%, n=30; 30%, n=33) and the mean (+/- S.E.M.) number of sperm with chromatin decondensation (7.8 +/- 1.6; 0.8 +/- 0.2; and 0.3 +/- 0.1) were also higher (P<0.05) for the control bull, C than for bulls K1 or K2, respectively. Results suggest that although sperm with the flattened or indented acrosomes had a tendency to undergo spontaneous acrosome reaction on incubation after thawing, the proteolytic activity of the acrosomal contents appeared to be normal. Sperm with the flattened or indented acrosomes also appeared to have a reduced ability to fuse with oolemma as demonstrated by confocal microscopy. This would impair the ability to penetrate ooplasm and undergo sperm chromatin decondensation.

Larval development and metamorphosis of cultured Tetrapygus niger (Echinodermata Echinoidea): an uncommon form of echinoplutei

Summary This study provides a qualitative and quantitative description of larval development of Tetrapygus niger to metamorphosis. After fertilization, the length of the planktonic interval (developmental time) for T. niger at room temperature (14–18°C) ranged from 94 to 120 days. Competent larvae were induced to metamorphose by transferring them into culture vessels containing bacterial films and diatoms. About 40% of the larvae reached the juvenile stage, with complete metamorphosis from the feeding larval stage to the feeding adult stage taking 4–6 days. Although the morphology of T. niger larvae is atypical compared with most regular extant echinoid species, it is similar to another arbacioid species, Arbacia punctulata. This suggests that larval morphology needs to be included in studies aimed at establishing phylogenetic relationships. The large larval size (>3.0 mm close to metamorphosis) and uniform pattern of ciliation observed in T. niger larvae have also been observed in non-feeding larvae. However, as T. niger larvae feed, we believe these characteristics reflect a functional solution to the swimming and feeding requirements of a long planktonic life. The maximum size (>4.5 mm) achieved by T. niger larvae in this in vitro study represents the largest recorded for echinoid larvae.

Purification and biochemical characterization of a trypsin-like enzyme present in the sperm of the rock shrimp, Rhynchocinetes typus

Summary The aim of the present work was to isolate, purify and characterize a trypsin-like enzyme from the sperm of the rock shrimp, Rhynchocinetes typus. Sperm proteins were extracted with 1 mM HCl in 10% glycerol at pH 3.0. Purification of the trypsin-like substance was effected by affinity chromatography using SBTI-agarose, yielding a specific activity on BAEE as substrate of 787 U/mg, with a recovery rate of 34%. Enzymatic activity was maximal at 27°C, pH of 8.0, 50 mM Ca2+ and 30 mM Mg2+. One hundred percent inhibition of enzymatic activity was obtained at 0.05 mM Zn2+. Kinetic analysis showed that the KM on BAEE as substrate at pH 8.0 was 2.5 x 10−5 M and the V MAX, reached was 198 U. It was also found that the enzyme had a substrate inhibition at concentrations higher than 0.06 mM of BAEE. These findings suggest that this enzyme has similar characteristics to other trypsin-like enzymes including acrosin.

Protease activity involvement in the passage of mammalian sperm through the zona pellucida

The interaction between acrosome-reacted sperm and zona pellucida proteins is not yet fully understood. Serine protease acrosin and its zymogen proacrosin have been proposed to fulfill this function due to their capacity to bind zona pellucida glycoproteins. However, the molecular mechanism underlying this interaction has been merely speculative. Here we show that fucoidan (a sulfated polysaccharide) and solubilized zona pellucida glycoproteins, but not soybean trypsin inhibitor, are able to detach bound spermatozoa, which suggests that live sperm binds to the zona pellucida in a non-enzymatical way. Interestingly, mild proteolytic digestion with acrosin or trypsin does not modify the structure of the zona pellucida, but rather results in fewer spermatozoa binding to the zona. These results agree with a model where the active site of acrosin digests the zona pellucida and binds through the polysulfate-binding domain through a three-dimensional zona structure rather than a single ligand.