The cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily members are found in a remarkable range of organisms spanning each of the animal kingdoms. Within humans and mice, there are 31 and 33 individual family members, respectively, and although many are poorly characterized, the majority show a notable expression bias to the reproductive tract and immune tissues or are deregulated in cancers. CAP superfamily proteins are most often secreted and have an extracellular endocrine or paracrine function and are involved in processes including the regulation of extracellular matrix and branching morphogenesis, potentially as either proteases or protease inhibitors; in ion channel regulation in fertility; as tumor suppressor or prooncogenic genes in tissues including the prostate; and in cell-cell adhesion during fertilization. This review describes mammalian CAP superfamily gene expression profiles, phylogenetic relationships, protein structural properties, and biological functions, and it draws into focus their potential role in health and disease. The nine subfamilies of the mammalian CAP superfamily include: the human glioma pathogenesis-related 1 (GLIPR1), Golgi associated pathogenesis related-1 (GAPR1) proteins, peptidase inhibitor 15 (PI15), peptidase inhibitor 16 (PI16), cysteine-rich secretory proteins (CRISPs), CRISP LCCL domain containing 1 (CRISPLD1), CRISP LCCL domain containing 2 (CRISPLD2), mannose receptor like and the R3H domain containing like proteins. We conclude that overall protein structural conservation within the CAP superfamily results in fundamentally similar functions for the CAP domain in all members, yet the diversity outside of this core region dramatically alters target specificity and, therefore, the biological consequences. (Endocrine Reviews 29: 865-897, 2008)

The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function.

Changes in the concentration of intracellular Ca(2+) ([Ca(2+) ]i) trigger and/or regulate principal sperm functions during fertilization, such as motility, capacitation, and the acrosome reaction (AR). Members of the large TRP channel family participate in a variety of Ca(2+) -dependent cell signaling processes. The eight TRPM channel members constitute one of the seven groups belonging to this family. Here we document using RT-PCR experiments the presence of Trpm2, 4, 7, and 8 in mouse spermatogenic cells. Trpm8 transcription is up-regulated after day 30. The localization of TRPM8 protein in mouse sperm was confirmed by immunocytochemistry and Western blots. Patch clamp recordings in testicular mouse sperm revealed TRPM8 agonist (menthol and icilin) activated currents sensitive to TRPM8 inhibitors N-(4-t-Butylphenyl)-4-(3-Chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide (BCTC) and capsazepine. These findings are consistent with the presence of functional TRPM8 in mouse sperm. Furthermore, menthol induced a [Ca(2+) ]i increase and the AR in these cells, that were inhibited by capsazepine (20 µM) and BCTC (1.6 µM). Notably, the progesterone and zona pellucida-induced AR was significantly (>40%) inhibited by BCTC and capsazepine, suggesting the possible participation of TRPM8 channels in this reaction. TRPM family members present in sperm could be involved in other important signaling events, such as thermotaxis, chemotaxis, and mechanosensory transduction.

CRISPs are a group of 3 proteins found in mammals (4 in the mouse) which show a strong expression bias in the male reproductive organs. Whilst the function of most CRISPs are yet to be elucidated, mouse CRISP2 is a known regulator of the ion channel, ryanodine receptor. CRISP4 is most abundantly produced by the principal cells of the epididymis and are secreted into the lumen, where they adhere to sperm during epididymal transit. In this study we examined the role of CRISP4 ion channel regulation in mouse spermatozoa through cell assays and mouse models. The identification of the Transient Receptor Potential (TRP) ion channel, TRPM8 to interact with CRISP4 was confirmed using stably-transfected CHO cell lines. Production of CRISP4 KO mouse model, whilst males are fertile, they exhibit a subtle infertility phenotype characterized by a reduced ability to capacitate and undergo the acrosome reaction. This data is further emphasized by the ability of TRPM8 agonists, icillin and menthol, to inhibit the acrosome reaction in mouse spermatozoa that could be prevented by the addition of recombinant CRISP4 crisp domain. Corresponding to these data, CRISP4 is localized to the tail and head of mouse spermatozoa. In conclusion, we have demonstrated that CRISP4 is a regulator of TRPM8 in mouse spermatozoa, and due to its expression and localization pattern is an important protein in sperm epididymal maturation.

The glioma pathogenesis-related 1 (GLIPR1) family consists of three genes [GLIPR1, GLIPR1-like 1 (GLIPR1L1), and GLIPR1-like 2 (GLIPR1L2)] and forms a distinct subgroup within the cysteine-rich secretory protein (CRISP), antigen 5, and pathogenesis-related 1 (CAP) superfamily. CAP superfamily proteins are found in phyla ranging from plants to humans and, based largely on expression and limited functional studies, are hypothesized to have roles in carcinogenesis, immunity, cell adhesion, and male fertility. Specifically data from a number of systems suggests that sequences within the C-terminal CAP domain of CAP proteins have the ability to promote cell-cell adhesion. Herein we cloned mouse Glipr1l1 and have shown it has a testis-enriched expression profile. GLIPR1L1 is posttranslationally modified by N-linked glycosylation during spermatogenesis and ultimately becomes localized to the connecting piece of elongated spermatids and sperm. After sperm capacitation, however, GLIPR1L1 is also localized to the anterior regions of the sperm head. Zona pellucida binding assays indicate that GLIPR1L1 has a role in the binding of sperm to the zona pellucida surrounding the oocyte. These data suggest that, along with other members of the CAP superfamily and several other proteins, GLIPR1L1 is involved in the binding of sperm to the oocyte complex. Collectively these data further strengthen the role of CAP domain-containing proteins in cellular adhesion and propose a mechanism whereby CAP proteins show overlapping functional significance during fertilization.

Background information . CRISP2 (cysteine‐rich secretory protein 2) is a sperm acrosome and tail protein with the ability to regulate Ca 2+ flow through ryanodine receptors. Based on these properties, CRISP2 has a potential role in fertilization through the regulation of ion signalling in the acrosome reaction and sperm motility. The purpose of the present study was to determine the expression, subcellular localization and the role in spermatogenesis of a novel CRISP2‐binding partner, which we have designated SHTAP (sperm head and tail associated protein). Results . Using yeast two‐hybrid screens of an adult testis expression library, we identified SHTAP as a novel mouse CRISP2‐binding partner. Sequence analysis of all Shtap cDNA clones revealed that the mouse Shtap gene is embedded within a gene encoding the unrelated protein NSUN4 (NOL1/NOP2/Sun domain family member 4). Five orthologues of the Shtap gene have been annotated in public databases. SHTAP and its orthologues showed no significant sequence similarity to any known protein or functional motifs, including NSUN4. Using an SHTAP antiserum, multiple SHTAP isoforms (∼20–87 kDa) were detected in the testis, sperm, and various somatic tissues. Interestingly, only the ∼26 kDa isoform of SHTAP was able to interact with CRISP2. Furthermore, yeast two‐hybrid assays showed that both the CAP (CRISP/antigen 5/pathogenesis related‐1) and CRISP domains of CRISP2 were required for maximal binding to SHTAP. SHTAP protein was localized to the peri‐acrosomal region of round spermatids, and the head and tail of the elongated spermatids and sperm tail where it co‐localized with CRISP2. During sperm capacitation, SHTAP and the SHTAP—CRISP2 complex appeared to be redistributed within the head. Conclusions . The present study is the first report of the identification, annotation and expression analysis of the mouse Shtap gene. The redistribution observed during sperm capacitation raises the possibility that SHTAP and the SHTAP—CRISP2 complex play a role in the attainment of sperm functional competence.

BACKGROUND Functional male gametes are produced through complex processes that take place within the testis, epididymis and female reproductive tract. A breakdown at any of these phases can result in male infertility. The production of mutant mouse models often yields an unexpected male infertility phenotype. It is with this in mind that the current review has been written. The review aims to act as a guide to the ‘non-reproductive biologist’ to facilitate a systematic analysis of sterile or subfertile mice and to assist in extracting the maximum amount of information from each model. METHODS This is a review of the original literature on defects in the processes that take a mouse spermatogonial stem cell through to a fully functional spermatozoon, which result in male infertility. Based on literature searches and personal experience, we have outlined a step-by-step strategy for the analysis of an infertile male mouse line. RESULTS A wide range of methods can be used to define the phenotype of an infertile male mouse. These methods range from histological methods such as electron microscopy and immunohistochemistry, to hormone analyses and methods to assess sperm maturation status and functional competence. CONCLUSION With the increased rate of genetically modified mouse production, the generation of mouse models with unexpected male infertility is increasing. This manuscript will help to ensure that the maximum amount of information is obtained from each mouse model and, by extension, will facilitate the knowledge of both normal fertility processes and the causes of human infertility.

The Cysteine‐RIch Secretory Proteins (CRISPs) are abundantly produced in the male reproductive tract of mammals and within the venom of reptiles and have been shown to regulate ion channel activity. CRISPs, along with the Antigen‐5 proteins and the Pathogenesis related‐1 (Pr‐1) proteins, form the CAP superfamily of proteins. Analyses of EST expression databases are increasingly suggesting that mammalian CRISPs are expressed more widely than in the reproductive tract. We, therefore, conducted a reverse transcription PCR expression profile and immunohistochemical analyses of 16 mouse tissues to define the sites of production of each of the four murine CRISPs. These data showed that each of the CRISPs have distinct and sometimes overlapping expression profiles, typically associated with the male and female reproductive tract, the secretory epithelia of exocrine glands, and immune tissues including the spleen and thymus. These investigations raise the potential for a role for CRISPs in general mammalian physiology. Developmental Dynamics 237:3313–3323, 2008. © 2008 Wiley‐Liss, Inc.

BACKGROUND:Cysteine-rich secretory protein 2 (CRISP2) is localized to the human sperm acrosome and tail. It can regulate ryanodine receptors Ca(2+) gating and binds to mitogen-activated protein kinase kinase kinase 11 in the acrosome and gametogenetin 1 (GGN1) in the tail.METHODS AND RESULTS:In order to test the hypothesis that CRISP2 variations contribute to male infertility, we screened coding and flanking intronic regions in 92 infertile men with asthenozoo- and/or teratozoospermia and 176 control men using denaturing HPLC and sequencing. There were 21 polymorphisms identified, including 13 unreported variations. Three SNPs resulted in amino acid substitutions: L59V, M176I and C196R. All were only present in a heterozygous state and found in fertile men. However, the C196R polymorphism was of particular interest as it resulted in the loss of a strictly conserved cysteine involved in intramolecular disulphide bonding. Screening of an additional 637 infertile men identified 23 heterozygous C196R men to give an overall frequency of 3.6%, compared with 3.4% in control men. The functional significance of the C196R polymorphism was defined using a yeast two-hybrid assay. The C196R substitution resulted in the loss of CRISP2-GGN1 binding.CONCLUSIONS:Although none of the many polymorphisms identified herein showed a significant association with male infertility, functional studies suggested that the C196R polymorphism may compromise CRISP2 function.

Cysteine-rich secretory protein 2 (CRISP2) is a testis-enriched protein localized to the sperm acrosome and tail. CRISP2 has been proposed to play a critical role in spermatogenesis and male fertility, although the precise function(s) of CRISP2 remains to be determined. Recent data have shown that the CRISP domain of the mouse CRISP2 has the ability to regulate Ca(2+) flow through ryanodine receptors (RyR) and to bind to MAP kinase kinase kinase 11 (MAP3K11). To further define the biochemical pathways within which CRISP2 is involved, we screened an adult mouse testis cDNA library using a yeast two-hybrid assay to identify CRISP2 interacting partners. One of the most frequently identified CRISP2-binding proteins was gametogenetin 1 (GGN1). Interactions occur between the ion channel regulatory region within the CRISP2 CRISP domain and the carboxyl-most 158 amino acids of GGN1. CRISP2 does not bind to the GGN2 or GGN3 isoforms. Furthermore, we showed that Ggn1 is a testis-enriched mRNA and the protein first appeared in late pachytene spermatocytes and was up-regulated in round spermatids before being incorporated into the principal piece of the sperm tail where it co-localized with CRISP2. These data along with data on RyR and MAP3K11 binding define the CRISP2 CRISP domain as a protein interaction motif and suggest a role for the GGN1-CRISP2 complex in sperm tail development and/or motility.

The cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily members are found in a remarkable range of organisms spanning each of the animal kingdoms. Within humans and mice, there are 31 and 33 individual family members, respectively, and although many are poorly characterized, the majority show a notable expression bias to the reproductive tract and immune tissues or are deregulated in cancers. CAP superfamily proteins are most often secreted and have an extracellular endocrine or paracrine function and are involved in processes including the regulation of extracellular matrix and branching morphogenesis, potentially as either proteases or protease inhibitors; in ion channel regulation in fertility; as tumor suppressor or prooncogenic genes in tissues including the prostate; and in cell-cell adhesion during fertilization. This review describes mammalian CAP superfamily gene expression profiles, phylogenetic relationships, protein structural properties, and biological functions, and it draws into focus their potential role in health and disease. The nine subfamilies of the mammalian CAP superfamily include: the human glioma pathogenesis-related 1 (GLIPR1), Golgi associated pathogenesis related-1 (GAPR1) proteins, peptidase inhibitor 15 (PI15), peptidase inhibitor 16 (PI16), cysteine-rich secretory proteins (CRISPs), CRISP LCCL domain containing 1 (CRISPLD1), CRISP LCCL domain containing 2 (CRISPLD2), mannose receptor like and the R3H domain containing like proteins. We conclude that overall protein structural conservation within the CAP superfamily results in fundamentally similar functions for the CAP domain in all members, yet the diversity outside of this core region dramatically alters target specificity and, therefore, the biological consequences.

Mammalian cysteine rich secretory proteins are a family of four proteins exhibiting a high amino acid sequence similarity and belonging to the CAP (Cysteine rich secretory proteins, Antigen-5 proteins and the plant Pathogenesis related-1 proteins) superfamily of proteins. They are designated CRISP 1, 2, 3 and 4. Structurally, mammalian CRISP’s are characterised by 16 cysteine residues involved in intra-molecular di-sulfide bonds and the formation of 2 domains, ie., the CRISP domain (CD) and CAP domain. Whilst studies on mouse CRISP2 suggest that the CD is involved in ion channel regulation, studies on non-mammalian CAP superfamily members suggest that the CAP domain is involved in proteolytic activity.They are predominantly expressed and localised in the male reproductive tract, however, the EST expression databases suggest that mammalian CRISPs are expressed more widely than in the male reproductive tract. The objective of this study was therefore to conclusively define the expression and localisation of each CRISP protein in a mammalian system.A reverse transcription PCR expression profile and immunohistochemical analysis of 16 mouse tissue was conducted to establish the expression and localisation of each of the four CRISPs. These data showed that although the CRISPs have a strong expression and localisation bias to the male reproductive tract, they are widely distributed throughout the body in mice, including the ovary, uterus, and mammary gland. Whilst each CRISP has a clear expression profile, there was a striking localisation of androgen regulated CRISPs (1, 3, 4) in immune tissue including the spleen and thymus. Such a localisation raises the spectre of a role for CRISPs in the normal physiology and disease of several organs.

The cysteine-rich secretory proteins (CRISPs) were identified as a major androgen regulated protein in the epididymal lumen of the rat in 1975. CRISPs were subsequently identified in the male reproductive tract of all mammals investigated and to date they have been identified in the testis (CRISP2), the epididymis (CRISP1 and CRISP4) and the prostate (CRISP3). Their abundance in the reproductive tract has lead to the widely held view that CRISPs are reproductive tract-specific proteins. While a few reports described CRISP expression in a wider range of tissues, these have not been pursued in assessing the biological function of CRISPs. In particular, CRISP3 is expressed in several exocrine secretory glands including the salivary and mammary glands. We conducted a reverse transcription (rt) PCR expression profile analysis of CRISP1-4 in 16 mouse tissues to determine the expression profile. These data show CRISPs are NOT restricted to the reproductive tract. Our results show that CRISP1 is produced in skeletal muscle, the epididymis, spleen, thymus, eye, the lacrimal gland and the salivary gland; CRISP2 is produced in the lung, skeletal muscle, ovary, the lacrimal gland and the testis; CRISP3 is produced in the heart, uterus, the salivary gland, the mammary gland, skeletal muscle, spleen, thymus, blood and the lacrimal gland and that CRISP4 is produced in skeletal muscle, spleen, the epididymis and the thymus. Products from tissues not previously reported to produce CRISPs were sequenced for confirmation. This data represents the first uniform experimental approach to the characterization of the mouse CRISP expression profile. On each of those tissues where a transcript was detected, we conducted immunohistochemistry using a range of available CRISP antibodies to determine the cell types producing CRISP proteins. We have shown a wider expression profile of CRISPs and have summarized our data with the many literature reports to produce a comprehensive summary of CRISP expression in the mouse. These data highlight the general significance of this protein family which extends well beyond the historical notion of reproductive tract specific proteins.

The CAP superfamily is defined by two signature sequence motifs. Using these motifs 1281 CAP proteins are identified in 481 species. Within the human and mouse there are 31 and 33 CAP proteins respectively, contained within four distinct protein groups: (1) the human glioma pathogenesis-related 1 (GLIPR1) proteins; (2) the Golgi-associated pathogenesis-related 1 (GAPR-1) proteins; (3) the peptidase inhibitor 15 (Pi15) proteins and (4) the cysteine-rich secretory proteins (CRISPs). All four members of the CRISP sub-group are expressed in the male reproductive tract. While screening a testis expression library we identified a novel sequence with homology to the CAP protein superfamily. The sequence showed greatest similarity to the GLIPR1 family of proteins and was called GLIPR1 like-1 (GLIPR1L1). We showed by Northern blot and EST analysis that GLIPR1L11 expression was testis-specific. Following generation of a specific GLIPR1L1 antisera (T32) a developmental Western blot of testis proteins showed a T32 immunoreactive band at the expected size of 25 kDa at day 14 post-partum. From day 22, coinciding with the appearance of round spermatids, a T32 immunoreactive band at 37 kDa was observed which increased in intensity in the adult testis. The 25 kDa form was absent in epididymal sperm. Interestingly however, within the epididymis a proportion of the 37 kDa form was additionally modified to produce a 51 kDa immunoreactive band. Addition of ~12 kDa units is suggestive of post-translational modification by sumoylation. We confirmed successive sumoylation of GLIPR1L1 using dual fluorescence labeling Western blots and isoform-specific SUMO antibodies. GLIPR1L1 is the first protein shown to be directly sumoylated during within sperm and during epididymal maturation. Using immunofluorescence on testicular sections, epididymal mouse sperm and in vitro capacitated mouse sperm we showed that GLIPR1L1 is initially localized to the redundant nuclear envelop (RNE) where it is subsequently redistributed following capacitation to the apical aspect of the sperm head. This redistribution is consistent with a role for GLIPR1L1 in interaction with the oocyte and a role in fertilization and is also consistent with the reports of CAP function in sperm egg interaction. We have characterized a novel testis-specific sperm protein GLIPR1L1, have shown that sumoylation is an active mechanism during sperm maturation and that GLIPR1L1 undergoes an impressive redistribution to the peri-acrosomal region consistent with a role in fertilization.

The cysteine rich secretory proteins (Crisp) are predominantly found in the mammalian male reproductive tract and in the venom of reptiles. Crisps are two domain proteins with a structurally similar yet evolutionarily diverse N-terminal domain and a characteristic cysteine rich C-terminal domain which we refer to as the Crisp domain. Since their identification 30 years ago Crisp research in mammals has focused on the characterisation of their expression localization to infer function. While no doubt important observations, these have not substantially led to an understanding of the biochemical activity of the Crisps and their role in sperm function or fertilisation. Recently, we demonstrated that the Crisp-2 Crisp domain has a structure similar to ion channel toxins ShK and BgK and was itself able to regulate Ca2+ flux through ryanodine receptors. These data build upon the previous characterizations of reptile venom Crisps as regulators of several types of ion channels and permits for the first time a dissection of the biochemical activity of mammalian Crisps.

Cysteine-rich secretory protein (CRISP) 2 (previously TPX1) is a testis-enriched member of the CRISP family, and has been localized to both the sperm acrosome and tail. Like all members of the mammalian CRISP family, its expression pattern is strongly suggestive of a role in male fertility, but functional support for this hypothesis remains limited. In order to determine the biochemical pathways within which CRISP2 is a component, the putative mature form of CRISP2 was used as bait in a yeast two-hybrid screen of a mouse testis expression library. One of the most frequently identified interacting partners was mitogen-activated protein kinase kinase kinase 11 (MAP3K11). Sequencing and deletion experiments showed that the carboxyl-most 20 amino acids of MAP3K11 interacted with the CRISP domain of CRISP2. This interaction was confirmed using pull-down experiments and the cellular context was supported by the localization of CRISP2 and MAP3K11 to the acrosome of the developing spermatids and epididymal spermatozoa. Interestingly, mouse epididymal sperm contained an similar to 60-kDa variant of MAP3K11, which may have been a result of proteolytic cleavage of the longer 93-kDa form seen in many tissues. These data raise the possibility that CRISP2 is a MAP3K11-modifying protein or, alternatively, that MAP3K11 acts to phosphorylate CRISP2 during acrosome development.