Edda Töpfer-Petersen

Biochemical and Genetic Investigation of Round-headed Spermatozoa in Infertile Men including two Brothers and their Father

Summary: Acrösin and the outer acrosomal membrane (OAM) were studied in the spermatozoa of 9 infertile patients who differed in the number of round‐headed spermatozoa between 14 and 71% in their ejaculates. These sperm components were also investigated in two infertile brothers who exhibited exclusively round‐headed spermatozoa in their ejaculates, and in their fertile father. It turned out that round‐headed spermatozoa lack both acrosin and the OAM as studied by indirect immunofluorescent and immuno‐peroxidase staining technique, gelatinolysis tests and by acrosin activity measurements. The normally shaped spermatozoa of 6 of the 9 infertile patients were found to be positive for acrosin and the OAM as expected, but in the remaining three patients even these spermatozoa were abnormal; in one patient they were unstainable for acrosin and in two patients they were unstainable both for acrosin and the OAM. These results have been confirmed by studies with the gelatinolysis test. The father of the two brothers with exclusively acrosomeless spermatozoa had more than 94% of normally shaped spermatozoa. However, only 10% of these spermatozoa were acrosin positive and only 30% were positive for the OAM. On the basis of these results we postulate that the mode of inheritance of the round‐headed spermatozoa syndrome is polygenic rather than monogenic as suggested by previous authors.

Isolation and biochemical characterization of a zona pellucida-binding glycoprotein of boar spermatozoa

Lectin‐like molecules on the sperm surface are implicated in the process of gamete recognition and adhesion. We have isolated and biochemically characterized a 15 kDa glycoprotein from ejaculated boar sperm which possess zona pellucida‐binding‐ and haemagglutinating‐activity. The zona/ 15 kDa protein interaction is inhibited by fucoldan, suggesting that the glycoprotein is one of the sperm components which participate in the initial gamete interaction. N‐Terminal sequence analysis of the isolated 15 kDa glycoprotein showed that it may belong to the same sperm/egg recognition‐mediating protein family as the sea urchin sperm protein binding.

The complete primary structure of three isoforms of a boar sperm-associated acrosin inhibitor

Acrosin inhibitors of seminal vesicle origin, after binding to their acceptor molecules on the anterior part of ejaculated sperm, are thought to be important capacitation factors, protecting zona binding sites during sperm uterine passage, and then dissociating to allow sperm binding to the zona pellucida of the oocyte. Each species so far tested possess an heterogeneous population of isoinhibitors which may display overlapping but not identical biological functions. Here we report the complete primary structure of three isoforms of a boar sperm‐associated acrosin inhibitor, whose sequences are 90% identical to the seminal plasma counterpart. Despite this high analogy, the differences between the sperm‐associated and the seminal plasma inhibitors may confer to them different physico‐chemical properties which are postulated to be of functional importance.

Zona pellucida-binding of boar sperm acrosin is associated with the N-terminal peptide of the acrosin B-chain (heavy chain)

Recently, it has been shown that boar acrosin exhibits a carbohydrate‐binding activity with a specificity to fucose, by which it can bind to the oocyte zona pellucida. By limited autoproteolysis of a high‐molecular mass acrosin ( kDa), designated as α‐acrosin, a 15 kDa fragment was generated which interacts strongly with the porcine zona pellucida. Zona‐binding was demonstrated on protein blots and by the solid‐phase zonabinding assay utilizing biotinylated zona proteins. The zona‐binding peptide was isolated by reversed‐phase HPLC and analyzed for amino acid sequence. Its single N‐terminal sequence corresponded to that of the acrosin B‐chain (heavy chain). These data indicate that the zona‐binding properties of acrosin are associated with the N‐terminal peptide of the acrosin heavy chain.

Isolation and biochemical characterization of two isoforms of a boar sperm zona pellucida-binding protein

Protein-carbohydrate complementarity has been recognized as a general mechanism of gamete recognition and adhesion in the process of fertilization throughout the whole animal kingdom. It appears that carbohydrate-binding molecules on the anterior sperm head surface mediate the binding of the male gamete to certain glycoconjugates present on the eggs extracellular coat. Subtle differences in protein and carbohydrate conformation may confer to this interaction a species-specific character. The mechanism responsible for gamete recognition is, however, poorly understood. A step in its elucidation is the characterization of the complementary molecules on the egg and sperm surfaces. With this aim we report here the isolation and partial structural characterization of two isoforms of a zona pellucida-binding protein (which we call AWN-1 and AWN-2) from boar spermatozoa, including partial sequence determination, assignment of disulphide bonds and identification of an N-terminal blocking group. AWN-1 and AWN-2 were isolated from acid extracts of washed ejaculated sperm and were present in seminal vesicle secretions, but absent in samples of epididymal fluid, suggesting a seminal vesicle origin for these sperm proteins. No analogous protein sequence could be found in the MIPS data bank, indicating that the AWN proteins may belong to a novel mammalian protein family involved in fertilization.

Boar proacrosin is a single-chain molecule which has the N-terminus of the acrosin A-chain (light chain)

Boar proacrosin was isolated from spermatozoa by a novel procedure under conditions preventing proenzyme activation. The spermatozoal extract was fractionated by gel filtration and reversed‐phase FPLC, all in acidic solutions. Isolated proacrosin had a molecular mass of 55/53 kDa (doublet) and was devoid of amidolytic activity. Its single N‐terminal sequence corresponded to that of the 23‐residue acrosin A‐chain and continued with that of the acrosin B‐chain. Autoactivation at pH 7.8 did not influence the molecular mass. However, activated material contained two parallel N‐terminal sequences, those of the A‐ and B‐chain. Thus, activation of proacrosin is analogous to that of other serine proteinase proenzymes.

Acrosin and the acrosome in human spermatogenesis.

SummaryUsing the indirect immunofluorescent staining technique, the developmental patterns of (pro)acrosin and the outer acrosomal membrane were studied in human spermatogenesis. Specific antibodies against purified acrosin and outer acrosomal membranes from boar spermatozoa were raised in the rabbit and were found to crossreact with (pro)acrosin and outer acrosomal membrane from human spermatogenic cells. It was concluded that (pro)acrosin as well as the molecules building up the outer acrosomal membrane have been highly conserved during mammalian evolution. In the course of human spermatogenesis (pro)acrosin as well as the outer acrosomal membrane first appear in the haploid spermatids; the fluorescent areas of the individual cells steadily increase during spermiogenesis. Staining for acrosin and the outer acrosomal membrane, respectively, was found in identical compartments of the spermatogenic cells in juxtaposition to the nucleus. Round-headed spermatozoa from an infertile patient did not stain for (pro)acrosin or outer acrosomal membrane. The lack of the acrosin system was further substantiated by the gelatin substrate film technique demonstrating the absence of a gelatinolytic protease in round-headed spermatozoa. Hence, round-headed spermatozoa lack the acrosome with its constituent membrane proteins and the acrosin system housed by the acrosome of normal spermatozoa.

Autoradiographic localization and characterization of bradykinin receptors in human skin

High affinity [3H]bradykinin binding sites have been identified in human skin cryosections by in vitro autoradiography. Equilibrium binding studies were performed with increasing concentrations of [3H]bradykinin for 120 min in the presence of protease inhibitors at 4 degrees C. In saturation experiments a single class of high affinity binding sites was identified with a dissociation constant Kd of 1.2 +/- 0.8 nM (mean +/- S.E.M., n = 3) and a maximal binding capacity Bmax of 33 +/- 8 fmol [3H]bradykinin specifically bound/mg protein (mean +/- S.E.M., n = 3). Competition experiments revealed a rank order of potency with bradykinin being most effective (bradykinin = [Lys]bradykinin > [Met- Lys]bradykinin > [Tyr]bradykinin > [des-Arg9]bradykinin), whereas [des-Arg9]bradykinin was ineffective. This indicates a B2 subtype of bradykinin receptors in normal human skin. Morphological data: autoradiography revealed that bradykinin receptors were localized in the stratum basale of the epidermis. The data are consistent with the hypothesis, that these mitotic active keratinocytes express bradykinin binding sites, that fulfil the pharmacological criteria for true receptors. Diverse stimuli, including bradykinin, play a role in the mediation of cutaneous inflammatory responses (e.g. fluid extravasation, reactive cell proliferation, hyperalgesia). Our data indicate that specific kinin receptors of the stratum basale are likely to contribute to these effects.

A new Separation Method of Subcellular Fractions of Boar Spermatozoa

Eine neue Isolierungsmethode subcellulärer Fraktionen aus Eberspermatozoen

Complete localization of the disulfide bridges and glycosylation sites in boar sperm acrosin

Acrosin is a disulfide‐bonded two‐chain glycoprotein, which belongs to the serine proteinase family and which plays a central role in mammalian fertilization. The amino acid sequence of acrosin from different species has been recently derived by cDNA analysis. Boar sperm acrosin contains twelve cysteine residues forming two interchain and 4 intrachain disulfide bonds. Protein‐chemical and mass‐spectroscopic analyses of fragments and subfragments obtained by proteolytic and chemical degradation of the isolated protein allowed the unambiguous localization of all disulfide bridges and glycosylation points in boar acrosin. The 12 cysteines and the glycosylated asparagines in the porcine enzyme are absolutely conserved in number and position within all known acrosin sequences. Thus, the disulfide bond and glycosylation patterns outlined here are conserved during evolution and may be important for enzyme function.