Human epididymal protein 6 (HE6; also known as GPR64) is an orphan member of the LNB-7TM (B 2 ) subfamily of G-protein-coupled receptors. Family members are characterized by the dual presence of a secretin-like (type II) seven-transmembrane (7TM) domain and a long cell adhesion-like extracellular domain. HE6 is specifically expressed within the efferent ductules and the initial segment of the epididymis, ductal systems involved in spermatozoon maturation. Here, we report that targeted deletion of the 7TM domain of the murine HE6 gene results in male infertility. Mutant mice reveal a dysregulation of fluid reabsorbtion within the efferent ductules, leading to a backup of fluid accumulation in the testis and a subsequent stasis of spermatozoa within the efferent ducts. The fertility phenotype of HE6 knockout mice identifies this receptor as a potential nonsteroidal, nontesticular target for future male contraceptives and identifies an in vivo function for a member of this unusual gene family.In the last few years, molecular biology has made substantial advances in understanding how sperm are made and how fertilization occurs. However, disappointingly little of this new information has translated into relevance for the clinic, particularly in terms of novel contraceptive agents for men.Spermatozoa, produced in the seminiferous tubules of the testis, are passed via the rete testis through the efferent ductules to the epididymis. It is within this organ that the spermatozoa acquire their capabilities for forward motility and oocyte fertilization (19,33,44). Although the molecular basis of this maturation process is as yet poorly understood, analyses of gene expression within the epididymis have revealed the identities of many secretory proteins (18,22) that potentially interact with and modify the sperm surface, enabling the mature spermatozoa to develop. The proximal epididymis and efferent ductules have a further function in the reabsorption of testicular fluid, concentrating the spermatozoa and establishing an adequate milieu for maturation of the spermatozoa (37). The epididymis, therefore, represents an alternative target organ for male contraception (5) and circumvents the potentially disadvantageous testicular steroidal regulation which is currently a favored approach (21).A seven-transmembrane (7TM) domain protein, human epididymis-specific protein 6 (HE6/GPR64) has been found to be highly expressed within the proximal epididymis (34) and recently within the efferent ductules (32). The gene encoding HE6 has been localized to the X-chromosomal region XF4, and orthologous highly conserved genes have been identified in the rat, mouse (32), and puffer fish (3). Sequence homology suggests that HE6 is a member of the newly defined LNB-7TM (B 2 ) subfamily of G-protein-coupled receptors (GPCRs) (13,42). Family members are characterized by a conserved secretin-like (type II) 7TM domain and a long N-terminal extracellular domain with an array of protein motifs considered to be involved in cell adhesion and protein-pro...

In the rat, the secretory glycoprotein DE/AEG is one of the main constituents of the epididymal fluid. We have recently reported the cloning of the cDNA for the related cysteine-rich secretory protein-1 (CRISP-1) from murine epididymis (Haendler et al., 1993; Endocrinology 133:192-198). The protein has now been isolated from the same organ and its N-terminal amino acid sequence has been determined. CRISP-1 exhibited an isoelectric point of approximately 6.8. High levels of CRISP-1 antigen were detected in the corpus and cauda of the epididymis, vas deferens, seminal vesicle, prostate, and in the salivary gland by immunohistochemistry. A quantitative analysis of the cauda epididymal fluid by sandwich ELISA revealed that CRISP-1 represented approximately 15% of the total protein. For heterologous expression, the CRISP-1 coding sequence was introduced into the pMPSV/CMV vector before transfection of baby hamster kidney (BHK) cells and selection with puromycin and neomycin. Expression in insect cells was achieved by co-transfection of Sf9 cells with a transfer vector and baculovirus DNA. Recombinant CRISP-1 was isolated in quantities sufficient for structural analysis. Ethyl maleimide treatment showed that all 16 cysteines were engaged in disulfide bonds. Proteolytic digestion demonstrated that the six cysteines localized in the N-terminal moiety formed three bonds with each other, suggesting the existence of two discrete domains in the protein.

The androgen dependency of the genes coding for the cysteine-rich secretory proteins (CRISP) was analysed in their main sites of expression. Male mice were treated with the gonadotropin-releasing hormone antagonist Ac-DNapAla-DClPhAla-DPyrAla-Ser-Tyr-DCtl-Leu-Lys(Mor)-Pro-DAla-NH 2 [DNapAla, D-2-naphthyl-Ala ; DClPhAla, D-4-chlorphenyl-Ala ; DPyrAla, D-pyridyn-3-yl-Ala ; DCtl, D-citrulline ; Lys(Mor), L-2-amino-6-(morpholin-4-yl)-hexanoic acid], and CRISP RNA levels were assessed by northern blot and competitive reverse transcriptase-mediated (RT)-PCR. In the salivary gland, CRISP-1 and to a lesser extent CRISP-3 expression was markedly reduced, in spite of an up-regulation of androgen receptor transcript levels. A down-regulation of CRISP-1 expression was also observed in the epididymis. Conversely, the levels of the testicular CRISP-2 transcripts were hardly affected at all. Female mice were ovariectomised and treated with testosterone propionate, and their salivary gland RNAs analysed. CRISP-1 and CRISP-3 RNA levels were significantly increased, and these effects were prevented by a concomitant treatment with the antiandrogen flutamide. Androgen receptor transcript levels were not affected by androgen administration but increased following antiandrogen treatment. CRISP expression during postnatal development was monitored by northern blot analysis. CRISP-1 and CRISP-2 transcripts were detected as early as 22 days after birth in the epididymis and testis, respectively, whereas CRISP-3 mRNA was visible only from day 30 in the salivary gland. A sharp increase of all CRISP levels was noted on day 40, coincident with the onset of sexual maturity. Altogether these results indicate that despite their high similarity, the CRISP genes are differentially regulated by androgens.Keywords : androgen; cysteine-rich secretory protein; epididymis; salivary gland; testis.The cysteine-rich secretory protein (CRISP) family was orig-ily of plants, which are induced upon infection by pathogens as inally described in the mouse [1, 2] and comprises evolutionarily part of the hypersensitive defense reaction [16, 17]. conserved polypeptides with a potential involvement in innate CRISP-1 is the best characterized member of the family and immunity. The CRISP genes are located on mouse chromosome its gene is mainly expressed in the corpus and cauda regions of 17 and human chromosome 6, in close vicinity to the major the epididymis, and in the male submandibular gland [1, 2, 9]. histocompatibility complex region [3Ϫ6]. They are expressed in Specific binding of its rat counterpart named acidic epididymal the specific granules of human neutrophils [7], in murine B cell glycoprotein and DE to spermatozoa heads has been reported, precursors [8], by glands with an exocrine function and by mu-leading to speculation about a role in sperm maturation [18Ϫ cosal epithelial surfaces [1, 2, 9, 10], which suggests a role in 20], but this could not be confirmed in mouse or human [9, 10, non-specific defense reactions, similar to that of defensins [11]...

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Boar proacrosin (E.C. 3.4.21.10, Mw 53 kD) was isolated by a modified method and subjected to autoactivation. Previously described molecular intermediates of 49 and 43 kD and a stable form (beta-acrosin, 35 kD) were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoactivation was expedited in the presence of either zona pellucida glycoproteins, fucoidan, or DNA. The end point of this accelerated conversion was the complete degradation of otherwise stable beta-acrosin via the formation of a characteristic active intermediate protein of 30 kD. All intermediate molecular forms observed during proacrosin activation/conversion exhibited the N-terminal sequence of the boar acrosin heavy chain, indicating a C-terminal processing mechanism. Hence zona pellucida glycoproteins stimulate proacrosin activation as well as acrosin degradation. Such a mechanism of proenzyme activation and degradation is to our knowledge described here for the first time and points to a previously unrecognized role of zona pellucida during gamete interaction.