Christiane Kirchhoff

Male-specific Modification of Human CD52

CD52 is an unusually short, bipolar glycopeptide bearing a highly charged N-linked carbohydrate moiety and a glycosylphosphatidylinositol membrane anchor. It is exclusively expressed on lymphocytes and in the male genital tract where it is shed into the seminal plasma and inserts into the sperm membrane. The sperm surface molecule has potential significance as a target for antibodies that inhibit sperm function and gamete interaction. Western blot analyses suggested cell type-specific modifications of the antigen. It was purified from seminal plasma and a detailed structural analysis performed. The majority of anchor structures in male genital tract CD52 showed 2-inositol palmitoylation, rendering molecules insensitive toward phospholipase C, and asn-1-alkyl-2-lyso-glycerol structure in place of the diacylated anchor described by Treumann et al.(Treumann, A., Lifely, M. R., Schneider, P., and Ferguson, M. A. (1995) J. Biol. Chem. 270, 6088–6099).N-Glycans of the male genital tract product were based on bi-, tri-, and tetraantennary structures of highly charged (up to -7), terminally sialylated complex-type sugars. A substantial proportion carried varying numbers of lactosamine repeats of which nearly 30% were branched. Different from lymphocytes, 10–15% of allN-glycans of the male genital tract antigen also contained peripheral fucose. These data confirm that male genital tract CD52 is distinct from the lymphocyte form by both N-linked glycans and COOH-terminal attached lipid anchor.

International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G Protein–Coupled Receptors

The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein–coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential.

Targeted Deletion of the Epididymal Receptor HE6 Results in Fluid Dysregulation and Male Infertility

ABSTRACT Human epididymal protein 6 (HE6; also known as GPR64) is an orphan member of the LNB-7TM (B2) 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.

Function of human epididymal proteins in sperm maturation.

Summary Human post‐testicular proteins were cloned by subtractive screening of epididymal cDNA libraries, employing testis as the primary negative control. This method identified six human epididymal cDNAs, named HE1‐HE6, which are derived from abundant epididymal mRNAs. With the exception of HE5, which turned out to be identical to the lymphocyte surface antigen CD52, they represented completely novel human gene products. To date, there is little information on their function and the mechanism of their deposition on the sperm surface. Unlike the sperm coating antigens, CD52 binds firmly to the sperm membrane via its GPI anchor during epididymal passage. Its synthesis is carefully regulated by the epididymal epithelium. From the results of both in vivo and in vitro studies it was concluded that androgen and temperature are principal factors synergistically modulating epididymal CD52 expression. The human counterparts of two well‐known major rodent epididymal proteins, secretory epididymal glutathione peroxidase (sGPX) and acidic epididymal glycoprotein (AEG = Protein DE), were not cloned by the subtractive screening approach, but by RT‐PCR amplification.

Screening for biomarkers of spermatogonia within the human testis: a whole genome approach

BACKGROUND A key step in studying the biology of spermatogonia is to determine their global gene expression profile. However, disassociation of these cells from the testis may alter their profile to a considerable degree. To characterize the molecular phenotype of human spermatogonia, including spermatogonial stem cells (SSCs), within their cognate microenvironment, a rare subtype of human defective spermatogenesis was exploited in which spermatogonia were the only germ cell type. METHODS The global expression profile of these samples was assessed on the Affymetrix microarray platform and compared with tissues showing homogeneous Sertoli-cell-only appearance; selected genes were validated by quantitative real-time PCR and immunohistochemistry on disparate sample sets. RESULTS Highly significant differences in gene expression levels correlated with the appearance of spermatogonia, including 239 best candidates of human spermatogonially expressed genes. Specifically, fibroblast growth factor receptor 3 (FGFR3), desmoglein 2 (DSG2), E3 ubiquitin ligase c-CBL (casitas B-cell lymphoma), cancer/testis antigen NY-ESO-1 (CTAG1A/B), undifferentiated embryonic cell transcription factor 1 (UTF1) and synaptosomal-associated protein, 91 kDa homolog (SNAP91) were shown to represent specific biomarkers of human spermatogonia. CONCLUSIONS These biomarkers, specifically the surface markers FGFR3 and DSG2, may facilitate the isolation and enrichment of human stem and/or progenitor spermatogonia and thus lay a foundation for studies of long-term maintenance of human SSCs/progenitor cells, spermatogonial self-renewal, clonal expansion and differentiation.

Novel Antimicrobial Peptide of Human Epididymal Duct Origin

Abstract HE2, a gene expressed specifically in human epididymis, gives rise to multiple mRNAs that encode a group of small cationic secretory peptides. Localization of HE2 within the defensin gene cluster and prediction that β-defensin-like modules exist suggest that these peptides have antimicrobial activity and represent components of the innate epithelial defense system of the epididymal duct. Reverse transcription-polymerase chain reaction analysis confirmed the occurrence of eight human HE2-derived transcripts, including minor mRNA variants, that had previously been shown only in animal species. Employing isoform-specific antibodies against the predicted HE2 products, multiple 4- to 8-kDa peptides were detected in human epididymal epithelium, epididymal fluid, and ejaculate. N-terminal microsequencing has suggested a proteolytic processing of these peptides by a furin-like proprotein convertase, which cleaves a propiece from the longer precursor peptides. HE2α and HE2β1, representing major peptide isoforms in the human epididymis, were recombinantly expressed, and their susceptibility to furin cleavage was demonstrated in vitro and in vivo. Processed recombinant peptides and chemosynthetic fragments were included in antimicrobial tests. In addition to the β-defensin-like HE2β1 with its expected antibacterial function, HE2α C-terminal fragments showed antibacterial activity against Escherichia coli, although it showed no significant similarity to β-defensins nor to any other known protein family.

Interaction of the human epididymal protein CD52 (HE5) with epididymal spermatozoa from men and cynomolgus monkeys

A monoclonal antibody (CAMPATH‐1G) against the human lymphocyte surface protein CD52, which is similar to the epididymal secretion HE5, was used to ascertain the presence of this protein on maturing primate spermatozoa by flow cytometry. The percentage of human viable spermatozoa stained specifically with this antibody increased from sperm in spermatocoeles (0.5%), to the efferent ducts (3.8%), corpus (47.2%), and cauda (85.7%) epididymidis. Positive cells revealed staining mainly over the whole tail and postacrosomal region of the sperm head. Spermatozoa (∼10%) from both the efferent ducts and corpus epididymidis took up additional antigen when incubated with human distal cauda epididymidal plasma as a source of CD52, and 12–22% of human testicular sperm (from spermatocoeles) took up CD52 from human seminal plasma. In the cynomolgus monkey, nonspecific binding of control IgG was greater than that in human males and net CD52 staining was measurable only on ∼30% of corpus sperm where it was mainly on the principal piece. Neither caput nor cauda sperm took up human CD52 upon incubation with human seminal plasma, but an additional 27% of corpus sperm expressed CD52. Such uptake of CD52 was drastically reduced, or did not occur, when seminal plasma had been fractionated by filtration through 0.1 μm filters (filtrate II) or 300,000 Da cutoff filters (filtrate III), respectively. Western blots revealed that CD52 contents were much reduced in filtrate II and nondetectable in filtrate III of seminal plasma. Similar reduction of CD52 in the filtrate of cauda epididymidal plasma indicates the association of this epididymal secretion with large molecular factors and suggests their involvement as carriers in the in vivo transfer of the secretion onto the epididymal sperm surface. The in vitro uptake of CD52 by some but not all immature sperm and the detection by Western blotting of much less CD52 in the corpus than the cauda luminal plasma suggest that the acquisition of this epididymal secretion by spermatozoa depends on their maturation status as well as the availability of the protein in the epididymal lumen. Mol. Reprod. Dev. 48:267–275, 1997.

New Insights into the Origin, Structure and Role of CD52: A Major Component of the Mammalian Sperm Glycocalyx

The sperm glycocalyx represents the primary interface between the male gamete and its environment, and gamete interaction inevitably involves interaction with this structure. Thus, it has potential significance as a target for antibodies that inhibit sperm function. Still, little is known about the components and biological role of the sperm glycocalyx. Despite the apparent complexity of the sperm membrane, surface carbohydrate labelling experiments show a high selectivity suggesting that carbohydrate side chains of CD52, an unusually short, bipolar glycopeptide of epididymal origin, form major components of the sperm glycocalyx in all mammalian species investigated. Acquisition of the highly sialylated, lipid-anchored CD52 antigen is one of the few well-defined modifications that occur to the sperm membrane during epididymal passage. It would explain changes in lectin-binding patterns and also the remarkable surface charge differences occurring during epididymal transit, most probably attributable to its terminal sialic acid residues. CD52 seems to be immunodominant on human spermatozoa, and antibodies directed against it can agglutinate and completely immobilize human sperm in the presence of complement. Expression of the same peptide backbone in lymphocytes had largely discounted its consideration as a candidate for contraceptive development. However, the recent proof of male-specific modifications indicates the feasibility of this approach.

Global human tissue profiling and protein network analysis reveals distinct levels of transcriptional germline-specificity and identifies target genes for male infertility

BACKGROUND Mammalian spermatogenesis is a process that involves a complex expression program in both somatic and germ cells present in the male gonad. A number of studies have attempted to define the transcriptome of male meiosis and gametogenesis in rodents and primates. Few human transcripts, however, have been associated with testicular somatic cells and germ cells at different post-natal developmental stages and little is known about their level of germline-specificity compared with non-testicular tissues. METHODS We quantified human transcripts using GeneChips and a total of 47 biopsies from prepubertal children diagnosed with undescended testis, infertile adult patients whose spermatogenesis is arrested at consecutive stages and fertile control individuals. These results were integrated with data from enriched normal germ cells, non-testicular expression data, phenotype information, predicted regulatory DNA-binding motifs and interactome data. RESULTS Among 3580 genes for which we found differential transcript concentrations in somatic and germ cells present in human testis, 933 were undetectable in 45 embryonic and adult non-testicular tissues, including many that were corroborated at protein level by published gene annotation data and histological high-throughput protein immunodetection assays. Using motif enrichment analyses, we identified regulatory promoter elements likely involved in germline development. Finally, we constructed a regulatory disease network for human fertility by integrating expression signals, interactome information, phenotypes and functional annotation data. CONCLUSIONS Our results provide broad insight into the post-natal human testicular transcriptome at the level of cell populations and in a global somatic tissular context. Furthermore, they yield clues for genetic causes of male infertility and will facilitate the identification of novel cancer/testis genes as targets for cancer immunotherapies.

Novel sperm-binding proteins of epididymal origin contain four fibronectin type II-modules

Novel fibronectin type II (Fn2)‐module proteins were cloned from human and canine epididymal cDNA libraries. cDNA sequences predicted a highly conserved protein family, related but not homologous to ungulate seminal plasma proteins (approximately 50% sequence identity), and the first known examples of proteins with four tandemly arranged Fn2‐domains. By Northern blot and in situ hybridization analyses the encoding mRNAs were shown to be abundant products of the epididymal duct epithelium, but not detectable in other tissues. Homologous mRNAs were identified in the epididymides of various mammals, representing members of this novel protein family of epididymal origin. Within the Fn2‐module‐encoding stretches, species homologues displayed > 85% sequence identity, but showed high variability at their predicted N‐termini. An antipeptide antiserum in Western blot analyses detected 30–35 kDa immunoreactive protein bands in epididymal tissue, cauda epididymidal fluid, and sperm membrane protein preparations. The tandem arrangement of increasing numbers of Fn2‐modules might functionally correspond to the tendency to form oligomers that has been described for lipid‐binding proteins. Mol. Reprod. Dev. 58:88–100, 2001.