DOI: 10.2436/20.1501.02.6 Endocrinologia molecular (Jaume Reventós, ed.) Treballs de la SCB. Vol. 56 (2005) 59-74 AN OVERVIEW OF THE PROACROSIN/ACROSIN SYSTEM IN HUMAN SPERMATOZOA Mónica H. Vazquez-Levin,1 Laura I. Furlong,1,3 Carolina M. Veaute 1,4 and P. Daniel Ghiringhelli 2 1 Instituto de Biología y Medicina Experimental. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Universidad de Buenos Aires. 2 Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM), Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes. 3 Current address: Research Unit on Biomedical Informatics (GRIB) IMIM/UPF. 4 Current address: Cátedra de Inmunología Básica, Universidad Nacional del Litoral. Corresponding author: Mónica H. Vazquez-Levin. Instituto de Biología y Medicina Experimental. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Universidad de Buenos Aires. Vuelta de Obligado, 2490. 1428 Buenos Aires, Argentina. E-mail: [email protected]. RESUM Entre totes les proteïnases espermàtiques semblants a la tripsina estudiades, l’acrosina (EC 3.4.21.10) ha estat identificada en totes les espècies estudiades, i ha estat associada amb elpoten- cial fertilitzador de l’esperma. En aquesta breu revisió, volem presentar els principals aspectes cell. ulars, moleculars i bioquímics del sistema proacrosina/acrosina definits pel nostre grup de recerca i per molts altres equips de recerca del camp de la biologia de la reproducció. Com a resultat de tots aquests estudis, presentem el model putatiu de la interacció del sistema proa- crosina/acrosina amb les glicoproteïnes de la zona pell. úcida i la demostració de l’activació del proenzim i la seva activitat com a proteïnasa. Paraules clau: humans, fertilització, esperma, acrosina, zona pell. úcida. SUMMARY Among all of the sperm acrosomal trypsin-like proteinases studied, acrosin (EC 3.4.21.10) has been identified in all of the species evaluated and has been associated with humansperm fertility potential. In this report, a brief overview of cellular, biochemical, and molecular aspects related to the human proacrosin/acrosin system, has been compiled from studies performed by our research team, as well as contributions by numerous groups from the clinical and basic field of reproductive biology. As a result of these studies, a putative model for the interaction 60 M. H. VAZQUEZ-LEVIN, L. I. FURLONG, C. M. VEAUTE AND P. D. GHIRINGHELLI between the proacrosin/acrosin system and the zona pellucida glycoproteins, in association with proenzyme activation and proteinase activity, is presented. Keywords: human, fertilization, sperm, acrosin, zona pellucida. Abbreviations: Zona pellucida (ZP); hu- from the clinical and basic field of reproduc- man preproacrosin (h-preproacrosin); human tive biology. proacrosin (h-proacrosin); bp (base pair); mRNA (messenger ribonucleic acid); 50UTR (50 untranslated region); Domain I (DI); Do- MOLECULAR CLONING main II (DII); Domain III (DIII); amino termi- OF THE SEQUENCE ENCODING nus (N-terminus), carboxy terminus (C-ter- H-PROACROSIN minus), in vitro fertilization and early embryo transfer (IVF-ET), enzyme-linked immunoas- The cDNA encoding h-proacrosin (Baba et say (ELISA). al., 1989a; Adham et al., 1990), as well as the Sperm-egg interaction is a specialized cell human genomic sequence (Keime et al., 1990; adhesion process that leads to fertilization Vazquez-Levin et al., 1992), have been pre- and the activation of development. Once viously reported. The h-proacrosin structural spermatozoa reach the vicinity of the egg, gene pattern (exon/intron) is very similar to they interact with the glycoproteins of the that characterized in other members of the egg’s extracellular coat, called the zona pel- serine proteinases family: it is organized into lucida (ZP). Sperm-binding to the ZP trig- five exons (exon 1: 77 bp from the ATG start- gers the exocytosis of the acrosomal gran- ing codon, exon 2: 204 bp, exon 3: 284 bp, ule (acrosomal exocytosis), wherein fusion exon 4: 146 bp, and exon 5: 555 bp), which are of the sperm plasma membrane and the arranged in two clusters, separated by a long outer acrosomal membrane occurs, followed intron (Keime et al., 1990). The amino acids of by the release of the acrosomal content; the acrosin active site are localized on exon acrosome-reacted sperm penetrate the ZP and 2 (His69), 3 (Asp123) and 5 (Ser221), and the finally bind and fuse to the egg’s plasma substrate recognition site, Asp215, is located membrane (Yanagimachi, 1994; Wassarman on exon 4 (Keime et al., 1990; Vazquez-Levin et al., 2001). Evidence of the participation of et al., 1992) (The nucleotide sequence reported sperm’s trypsin-like proteinases in the acro- for human proacrosin at the EMBL/GenBank somal exocytosis and ZP penetration first can be found under the following accesion came from experiments showing a block- numbers: Y00970, Baba et al., 1989; X17349, ade on sperm penetration by the addition Keime et al., 1990; M77378-M77381, Vazquez- of trypsin inhibitors (Liu and Baker, 1993; Levin et al., 1992.) In addition, the amino acids Llanos et al., 1993). Among all of the acro- involved in proenzyme processing towards somal trypsin-like proteinases studied acrosin the activation to a mature enzyme (see be- (EC 3.4.21.10) has been associated with hu- low) are localized in exon 2 and 5. Moreover, man sperm fertility potential (see below). In exon 5 contains a Pro rich region, which is this report, a brief overview of cellular, bio- absent in other serine proteinases. The tran- chemical, and molecular aspects related to scription initiation site was localized to the the h-proacrosin/acrosin system, will be pre- C residue at position –74, upstream from the sented. Data has been compiled from stud- translation initiation codon ATG (Keime et ies performed by our research team, as well al., 1990); sequence analysis of over 1 kbp nu- as contributions made by numerous groups cleotides from the 50UTR showed an absence AN OVERVIEW OF THE PROACROSIN/ACROSIN SYSTEM IN HUMAN SPERMATOZOA 61 of an identifiable TATA and CCAAT boxes though the enzyme was first found in round and the presence of a highly GC-rich region, spermatids (Nayernia et al., 1992). Partial con- as well as several regulatory elements iden- trol of acrosin gene translation may result tified in other testis specific genes (Keime et from the association of the DNA/RNA bind- al., 1990; Vazquez-Levin et al., 1992; Vazquez- ing protein MSY2 to stored or translationally Levin et al., 1996; Schulten et al., 2001). delayed acrosin transcripts (Yang et al., 2005). Although the h-pre-proacrosin gene was initially described as a single copy gene mapped to chromosome 22 (q13-qter) (Ad- STRUCTURAL FEATURES ham et al., 1989), a report by Fan et al. (2002), OF H-PROACROSIN identified the truncated copy of exons 4and 5 of the h-proacrosin gene in chromosome 2, The primary structure of h-proacrosin has in a region called the 2qFUS region, which re- been deduced from its cDNA sequence (Baba sulted from the fusion of duplications in the et al., 1989a; Adham et al., 1990). The proen- sub-telomeric regions of chromosomes 9p and zyme N-terminus is preceded by a highly 22q, now located in the 2q13-2q14.1 region. hydrophobic, cleavable signal peptide of 19 residues. The protein sequence of proacrosin can be divided into three major domains: DI, EXPRESSION OF H-PROACROSIN which encodes the amino acids 1-23 of the DURING SPERMATOGENESIS light chain, DII or catalytic domain, which contains the residues of the heavy chain for Expression of the pre-proacrosin gene is the mature enzyme, and DIII or tail domain, specific to male germ cells, finding anmRNA which comprises the C-terminal region of the form of approximately 1.6 kbp in all of the heavy chain (see figure 1a). species studied; however, evidence regard- The amino acid sequence of h-proacrosin ing the cell stage where transcription and shows an overall high degree of similar- translation occur, is still controversial. The hu- ity to those reported in other mammals: man protein has been detected in pachytene DI is highly conserved within all of the spermatocytes (Escalier et al., 1991), although species (91-97% similarity), and the same is regulation of mRNA transcription and trans- observed in DII (75-92%). In contrast, DIII lation in humans has not been reported. shows a broad range of sequence conserva- The expression of mouse acrosin mRNA tion across the species (27% ascidian/mouse and its functional association with polysomes and 83% boar/human) and is not present was first detected in pachytene spermato- in other members of the serine proteases cytes, and increased transcription and trans- superfamily. In most mammals, except the lation was found throughout spermiogenesis mouse and rat, this region is highly hy- (Kashiwabara et al., 1990). In the rat, transcrip- drophilic and unique because of its high tion of the acrosin gene was found at day 19 of Pro content; on the other hand, the ascidian spermatogenesis, which does not contain hap- proacrosin C-terminus is not Pro rich and has loid cells (Nayernia et al., 1994), but proacrosin two CUB domains (Kodama et al., 2001). Dif- biosynthesis was first identified in early sper- ferent functions have been proposed for the matids (Phi-van et al., 1983); similarly, trans- proenzyme DIII (Klemm et al., 1991; Mori et genic mice, carrying a construct with 2.3 kbp al., 1995; Kodama et al., 2001; see below). of the proacrosin 50 flanking sequence and The molecular weight of h-proacrosin de- the CAT reporter, showed transcription of the duced from its amino acidic sequence is 43,860 CAT gene in pachytene spermatocytes, al- Daltons, although a higher Mr (55-60 KDa) 62 M. H. VAZQUEZ-LEVIN, L.