CRISP1 Human

What is human CRISP1 and where is it expressed in the reproductive system?

Human CRISP1 belongs to the Cysteine-Rich Secretory Protein (CRISP) family, a subgroup of the CRISP, Antigen 5, and PR-1 (CAP) superfamily. It is primarily synthesized in the epididymis in an androgen-dependent manner and associates with sperm during epididymal maturation . Interestingly, recent research has revealed that CRISP1 is also expressed in the female reproductive tract, specifically in the ovaries, oviducts, uteri, and most notably, in the cumulus cells surrounding the egg . This dual presence in both male and female reproductive tracts suggests a more complex role in fertilization than previously understood.

To study CRISP1 expression, researchers typically employ RT-PCR for mRNA detection and Western blotting with specific antibodies for protein detection. Immunofluorescence techniques are valuable for localizing CRISP1 in tissues and cells, as demonstrated in studies showing CRISP1 labeling in cumulus cells from control mice but not from CRISP1 knockout mice .

How does human CRISP1 structurally and functionally differ from rodent CRISP proteins?

Human CRISP1 differs from rodent CRISP proteins in several important ways:

Human CRISP1 appears to be the functional equivalent of both rodent CRISP1 and CRISP4, as supported by its involvement in both gamete fusion and sperm-zona pellucida binding . This suggests an evolutionary consolidation of functions in humans, where a single protein performs roles that are divided between two proteins in rodents . Despite structural differences in the metal-binding site, human CRISP1 still exhibits zinc-dependent oligomerization properties through the conserved His142 in the CAP domain .

What are the key structural domains of human CRISP1 and their functional significance?

Human CRISP1 contains several key structural domains with specific functions:

• CAP Domain: Located at the N-terminal region, this domain contains a metal-binding site important for zinc-dependent oligomerization. Despite human CRISP1 lacking one of the two conserved histidines typically found in this domain, the remaining His142 is essential for zinc binding and subsequent oligomerization .

• CRISP Domain: Located at the C-terminal region, containing two signature motifs known as Signature 1 (S1) and Signature 2 (S2). Structure-function studies have revealed that the egg-binding activity of CRISP1 resides specifically in the 12-amino acid region corresponding to S2 .

• Metal-Binding Site: Studies using maltose-binding protein (MBP)-tagged human CRISP1 have demonstrated that zinc specifically induces oligomerization of both full-length CRISP1 and its CAP domain. This oligomerization is reversible upon zinc removal by EDTA, suggesting a regulatory mechanism that may be important during sperm maturation in the epididymis .

The three-dimensional conformation of CRISP1 is critical for its function, as demonstrated by experiments showing that heat-denatured and DTT-treated native CRISP1, as well as bacterially expressed recombinant CRISP1, lacked specific activities exhibited by the properly folded native protein .

How does CRISP1 regulate sperm calcium channels and what are the implications for hyperactivation?

CRISP1 has been identified as a novel regulator of sperm calcium channels, particularly CatSper, which is essential for hyperactivated motility and fertility. Patch clamping experiments have revealed that CRISP1 can modulate CatSper and TRPM8 channels, thereby regulating calcium influx and sperm hyperactivation .

When sperm are exposed to a CRISP1 gradient, several effects on motility are observed:

• Significant decrease in the percentage of hyperactivated sperm

• Increase in the proportion of sperm with linear motility pattern

• Enhanced orientation of sperm movement

Computer-assisted sperm analysis (CASA) studies have shown that exposure of capacitated sperm to native CRISP1 produces a significant reduction in motility parameters associated with hyperactivation. This regulatory ability appears to be critical for guiding sperm through the cumulus mass to reach the egg .

This modulation of calcium channels and hyperactivation represents a fine-tuning mechanism that facilitates sperm navigation through the cumulus and successful fertilization .

What is the significance of zinc-dependent oligomerization of human CRISP1?

The zinc-dependent oligomerization of human CRISP1 represents a potential regulatory mechanism in sperm function. Research utilizing a maltose-binding protein (MBP)-tagging approach has demonstrated that:

• Zinc specifically induces oligomerization of both MBP-tagged human CRISP1 (MBP-CRISP1) and the MBP-tagged CAP domain of CRISP1 (MBP-CRISP1 ΔC) in vitro

• The conserved His142 in the CAP domain is essential for this zinc-dependent oligomerization

• CRISP1 oligomers dissociate into monomers upon zinc removal by EDTA, indicating a reversible process

This oligomerization property is particularly significant in the context of epididymal sperm maturation, where protein condensation is a characteristic process and is known to be zinc-dependent. The ability of CRISP1 to form and dissolve oligomers in response to zinc concentration may contribute to the formation of functional protein complexes involved in mammalian fertilization .

The methodological approach of using MBP-tagging combined with low expression levels in XL-1 Blue bacteria has been crucial for obtaining moderate yields of soluble recombinant human CRISP1, allowing the investigation of these zinc-dependent properties that might be difficult to study with other expression systems .

How does human CRISP1 participate in sperm-zona pellucida binding?

Human CRISP1 plays a crucial role in sperm-zona pellucida (ZP) binding, as evidenced by several experimental approaches:

• Hemizona binding assays: When human hemizona were inseminated with capacitated sperm in the presence of either anti-hCRISP1 antibody or bacterially-expressed recombinant hCRISP1 (rec-hCRISP1), a significant inhibition in the number of sperm bound per hemizona was observed compared to controls .

• Protein-protein interaction analysis: ELISA studies with human recombinant ZP proteins expressed in insect cells revealed that rec-hCRISP1 primarily interacts with ZP3 in a dose-dependent and saturable manner, indicating specificity of interaction .

• Immunofluorescence experiments: Studies using human ZP-intact eggs demonstrated the presence of complementary sites for hCRISP1 in the zona pellucida .

Importantly, controls showed that neither anti-hCRISP1 antibody nor rec-hCRISP1 affected capacitation-associated events such as sperm motility, protein tyrosine phosphorylation, or acrosome reaction, supporting that the observed inhibition was specific to the sperm-egg interaction level .

These findings establish human CRISP1 as a multifunctional protein involved not only in gamete fusion (a later stage of fertilization) but also in the preceding stage of sperm-ZP binding through its specific interaction with human ZP3 .

How does CRISP1 expressed by cumulus cells affect sperm orientation and fertilization?

A groundbreaking discovery in CRISP1 research is that it is expressed not only in the male reproductive tract but also by cumulus cells surrounding the egg . This cumulus-derived CRISP1 plays several important roles:

• Sperm orientation: Using a modified Zigmond chamber assay, research has shown that CRISP1 (at concentrations of 1-10 μM) produces a significant increase in the percentage of oriented sperm, comparable to established chemoattractants like progesterone .

• Modulation of hyperactivation: CRISP1 treatment leads to a significant decrease in the percentage of hyperactivated sperm, with the CRISP1-oriented population exhibiting significantly lower percentages of hyperactivated cells and higher levels of sperm with linear pattern compared to non-oriented cells .

• Facilitation of cumulus penetration: Fertilization experiments with cumulus-oocyte complexes (COCs) from CRISP1 knockout females showed impaired fertilization due to a failure of sperm to penetrate the cumulus, suggesting that cumulus CRISP1 is necessary for effective cumulus penetration .

The data suggest a model where CRISP1 expressed by cumulus cells creates a gradient that helps guide sperm through the cumulus mass by fine-tuning their hyperactivated motility into a more linear pattern when needed for directional movement. This represents a novel mechanism for successful mammalian fertilization that involves CRISP1 from both male and female gametes .

What experimental approaches are most effective for studying CRISP1's role in fertilization?

Several complementary experimental approaches have proven effective for studying CRISP1's multifaceted roles in fertilization:

• Recombinant protein production:

  • MBP-tagging approach with low expression levels in bacteria has been successful for obtaining soluble human CRISP1

  • Expression in insect cells for human ZP proteins to study interactions with CRISP1

• Binding and functional assays:

  • Hemizona binding assays to evaluate sperm-ZP binding

  • Gamete fusion assays using zona-free eggs to assess fusion capacity

  • ELISA-based protein-protein interaction studies

  • Modified Zigmond chamber for chemotactic/orientation assays

• Microscopy and imaging:

  • Immunofluorescence for localizing CRISP1 and binding sites

  • Computer-assisted sperm analysis (CASA) for motility parameters

• Electrophysiology:

  • Patch clamping of sperm to analyze ion channel regulation

• Genetic approaches:

  • Generation of knockout models (Crisp1-/-, Crisp1-/-Crisp4-/- double knockouts)

  • Fertilization studies comparing wild-type and knockout gametes

Each method addresses specific aspects of CRISP1 function, and combining multiple approaches has been key to understanding the protein's multifunctional nature in fertilization.

What are the challenges in developing and using recombinant human CRISP1 for experimental studies?

Developing functionally active recombinant human CRISP1 presents several challenges that researchers must address:

• Protein solubility: CRISP1 can be difficult to express in soluble form. Using a maltose-binding protein (MBP)-tagging approach combined with low expression levels in XL-1 Blue bacteria has proven effective in obtaining moderate yields of soluble recombinant protein .

• Protein conformation: The three-dimensional structure of CRISP1 is crucial for its function. Studies have shown that heat-denatured and DTT-treated native CRISP1, as well as some bacterially expressed recombinant CRISP1 preparations, lack specific activities of the properly folded native protein . This highlights the importance of verification methods to ensure proper folding.

• Functional verification: Researchers must confirm that recombinant CRISP1 retains the functional properties of the native protein. This can be assessed through:

  • Zinc-dependent oligomerization assays

  • Binding studies with ZP proteins and eggs

  • Sperm orientation and motility assays

• Expression system selection: Different experimental questions may require different expression systems:

  • Bacterial expression for structural studies and antibody production

  • Mammalian cell expression for functional studies requiring post-translational modifications

  • Insect cell expression for interaction partners like ZP proteins

The successful production of functional recombinant CRISP1 has been instrumental in delineating the specific regions of the protein responsible for different activities, such as the S2 region for egg binding and the CAP domain for zinc interaction .

How do knockout models help us understand CRISP1 function and what are their limitations?

Knockout mouse models have been invaluable tools for understanding CRISP1 function, though they come with specific limitations:

Benefits of CRISP1 knockout models:

• Functional redundancy insights: While Crisp1-/- mice were found to be fertile, they exhibited other reproductive deficiencies, revealing both the redundancy in the fertilization system and specific roles of CRISP1 .

• Combined deletion studies: The generation of Crisp1 and Crisp4 double knockout (DKO) mice, equivalent to removing all CRISP expression in humans, has provided insights into the combined functions of epididymal CRISPs .

• Stage-specific function analysis: Using gametes from knockout animals in controlled in vitro assays has allowed researchers to identify specific stages of fertilization affected by CRISP1 absence .

• Sex-specific contributions: By using knockout animals of both sexes, researchers discovered the unexpected role of CRISP1 expressed by cumulus cells in fertilization .

Limitations and considerations:

• Compensatory mechanisms: Knockout animals often develop compensatory mechanisms that mask phenotypes, especially for fertility-related genes .

• Strain-specific effects: Genetic background can influence the severity of phenotypes in knockout models, requiring careful control selection .

• In vivo vs. in vitro discrepancies: Some deficiencies observed in vitro (such as reduced fertilization rates) may not translate to reduced fertility in vivo due to beneficial and/or compensatory mechanisms operating in the natural environment .

• Translation to humans: Caution is needed when translating mouse findings to humans due to differences in epididymal histology and potentially different roles of CRISP proteins across species .

Researchers found that while single Crisp1-/- mice showed mild fertility defects, aging and combined deletion of Crisp1 and Crisp4 revealed more pronounced deficiencies, suggesting that CRISPs help maintain optimal sperm function and protect against age-related fertility decline .

What evidence supports the immunocontraceptive potential of human CRISP1?

Human CRISP1 shows promising potential as an immunocontraceptive target, supported by several lines of evidence:

• Nonhuman primate studies: Research in cynomolgus macaques (Macaca fascicularis) demonstrated that immunization with recombinant human CRISP1 (hCRISP1) or recombinant monkey CRISP1 (mkCRISP1) induced an immune response that increased over time and specifically recognized CRISP1 in sperm extracts .

• Antibody presence in reproductive tract: Both anti-hCRISP1 and anti-mkCRISP1 antibodies were detected in seminal plasma, indicating their ability to enter the male reproductive tract .

• In vivo binding to sperm: Fluorescent labeling observed in sperm exposed only to secondary antibody demonstrated that the anti-hCRISP1 antibodies bound to sperm cells in vivo .

• Sperm parameters unaffected: Importantly, sperm number, motility, and morphology were not affected by immunization, suggesting that the contraceptive effect would be specific to fertilization function rather than spermatogenesis or sperm health .

• Multiple roles in fertilization: CRISP1's involvement in multiple critical steps of fertilization (sperm-ZP binding, gamete fusion, and hyperactivation regulation) makes it an attractive target for contraception, as blocking these functions could effectively prevent fertilization .

These findings support both the potential involvement of anti-hCRISP1 antibodies in human immunoinfertility and the consideration of hCRISP1 as a candidate for immunocontraception development .

What contradictions exist in the literature regarding CRISP1's role in sperm capacitation between different species?

The literature reveals intriguing contradictions regarding CRISP1's role in sperm capacitation across different species:

• Contradictory effects on tyrosine phosphorylation:

  • In rats, the presence of CRISP1 during sperm capacitation was reported to inhibit protein tyrosine phosphorylation, suggesting a role as a decapacitation factor .

  • Surprisingly, capacitated sperm from Crisp1-/- mice exhibited lower levels of tyrosine phosphorylation than controls, indicating that in mice, CRISP1 may actually promote rather than inhibit this capacitation-associated event .

• Decapacitation factor hypothesis:

  • In rats, the loosely associated population of CRISP1 that is released during capacitation has been proposed to act as a decapacitation factor .

  • Evidence for this role in mice and humans is less clear, suggesting species-specific differences in function.

• Acrosome reaction relationship:

  • Despite lower levels of tyrosine phosphorylation in Crisp1-/- mouse sperm, these cells presented normal levels of both spontaneous and progesterone-induced acrosome reaction .

  • This challenges the conventional understanding of the relationship between tyrosine phosphorylation and acrosome reaction competence.

These contradictions highlight important species differences in CRISP1 function and suggest that:

• CRISP1 may play different regulatory roles during mouse and rat sperm capacitation

• Protein tyrosine phosphorylation may be required at lower levels than previously thought to achieve normal acrosome reaction and fertility

• Caution should be exercised when extrapolating findings across species, even between closely related rodents

These species-specific differences further complicate translation to human fertility, emphasizing the need for direct studies on human CRISP1 function.

What are the key unresolved questions about human CRISP1 that require further investigation?

Despite significant advances in understanding human CRISP1, several important questions remain:

• Molecular mechanisms of CatSper regulation: While CRISP1 has been identified as a regulator of CatSper channels, the precise molecular mechanism by which this regulation occurs remains unclear. Further structural and functional studies are needed to elucidate this interaction .

• Clinical relevance in human infertility: The contribution of CRISP1 dysfunction to human infertility cases requires systematic investigation. Correlations between CRISP1 variations/mutations and specific infertility phenotypes could provide valuable clinical insights .

• Sperm DNA integrity connection: Recent research suggests a potential link between CRISP proteins and sperm DNA integrity. This relationship and its impact on early embryogenesis deserves further exploration, especially given the incidence of sperm DNA fragmentation in male infertility .

• Interaction with immune system: The immunocontraceptive potential of CRISP1 raises questions about its natural interaction with the immune system in both male and female reproductive tracts. Understanding these interactions could shed light on immune privilege mechanisms and cases of immunoinfertility .

• Species-specific differences in function: Resolving the contradictions in CRISP1 function between species, particularly regarding its role in capacitation, requires further comparative studies that can inform human applications .

• Therapeutic applications: The potential for using recombinant CRISP1 or CRISP1-derived peptides in treating specific types of infertility remains largely unexplored but represents a promising avenue for translational research .

Addressing these questions will require interdisciplinary approaches combining structural biology, reproductive physiology, clinical studies, and advanced imaging techniques to fully elucidate the complex roles of CRISP1 in human reproduction.