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1.
Int J Mol Sci ; 22(19)2021 Sep 23.
Article in English | MEDLINE | ID: mdl-34638585

ABSTRACT

Mammalian sperm must undergo two post-testicular processes to become fertilization-competent: maturation in the male epididymis and capacitation in the female reproductive tract. While caput epididymal sperm are unable to move and have not yet acquired fertilization potential, sperm in the cauda epididymis have completed their maturation, can move actively, and have gained the ability to undergo capacitation in the female tract or in vitro. Due to the impossibility of mimicking sperm maturation in vitro, the molecular pathways underlying this process remain largely unknown. We aimed to investigate the use of caput epididymal ligation as a tool for the study of sperm maturation in mice. Our results indicate that after seven days of ligation, caput sperm gained motility and underwent molecular changes comparable with those observed for cauda mature sperm. Moreover, ligated caput sperm were able to activate pathways related to sperm capacitation. Despite these changes, ligated caput sperm were unable to fertilize in vitro. Our results suggest that transit through the epididymis is not required for the acquisition of motility and some capacitation-associated signaling but is essential for full epididymal maturation. Caput epididymal ligation is a useful tool for the study of the molecular pathways involved in the acquisition of sperm motility during maturation.


Subject(s)
Cyclic AMP/metabolism , Phosphorylation/physiology , Sperm Maturation/physiology , Sperm Motility/physiology , Spermatozoa/physiology , Animals , Epididymis/metabolism , Epididymis/physiology , Female , Fertilization/physiology , Ligation/methods , Male , Mice , Signal Transduction/physiology , Sperm Capacitation/physiology , Spermatozoa/metabolism
2.
Mol Hum Reprod ; 26(1): 14-29, 2020 01 01.
Article in English | MEDLINE | ID: mdl-31778536

ABSTRACT

In the epididymis, prevention of autoimmune responses against spermatozoa and simultaneous protection against pathogens is important for male fertility. We have previously shown that mononuclear phagocytes (MPs) are located either in the epididymal interstitium or in close proximity to the epithelium. In the initial segments (IS), these 'intraepithelial' MPs extend slender luminal-reaching projections between epithelial cells. In this study, we performed an in-depth characterisation of MPs isolated from IS, caput-corpus and cauda epididymis of CX3CR1EGFP+/- mice that express EGFP in these cells. Flow cytometry analysis revealed region-specific subsets of MPs that express combinations of markers traditionally described in 'dendritic cells' or 'macrophages'. RNA sequencing identified distinct transcriptomic signatures in MPs from each region and revealed specific genes involved in inflammatory and anti-inflammatory responses, phagosomal activity and antigen processing and presentation. Functional fluorescent in vivo labelling assays showed that higher percentages of CX3CR1+ MPs that captured and processed antigens were detected in the IS compared to other regions. Confocal microscopy showed that in the IS, caput and corpus, circulatory antigens were internalised and processed by interstitial and intraepithelial MPs. However, in the cauda only interstitial MPs internalised and processed antigens, while intraepithelial MPs did not take up antigens, indicating that all antigens have been captured before they reached the epithelial lining. Cauda MPs may thus confer a stronger protection against blood-borne pathogens compared to proximal regions. By identifying immunoregulatory mechanisms in the epididymis, our study may lead to new therapies for male infertility and epididymitis and identify potential targets for immunocontraception.


Subject(s)
CX3C Chemokine Receptor 1/immunology , Epididymis/immunology , Fertility/genetics , Phagocytes/immunology , Spermatozoa/immunology , Transcriptome/immunology , Animals , Antigen Presentation , Antigens, CD/genetics , Antigens, CD/immunology , Autoantigens/genetics , Autoantigens/immunology , CX3C Chemokine Receptor 1/deficiency , CX3C Chemokine Receptor 1/genetics , Cell Communication , Chemokines, CC/genetics , Chemokines, CC/immunology , Dendritic Cells/cytology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Epididymis/cytology , Epididymis/metabolism , Epithelial Cells/cytology , Epithelial Cells/immunology , Epithelial Cells/metabolism , Gene Expression Profiling , Gene Expression Regulation , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/immunology , Male , Mice , Mice, Knockout , Phagocytes/cytology , Phagocytes/metabolism , Protein Transport , Receptors, Interleukin/genetics , Receptors, Interleukin/immunology , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Spermatozoa/cytology , Spermatozoa/metabolism
3.
J Cell Sci ; 133(5)2019 11 18.
Article in English | MEDLINE | ID: mdl-31636115

ABSTRACT

Epithelial cells are immune sensors and mediators that constitute the first line of defense against infections. Using the epididymis, a model for studying tubular organs, we uncovered a novel and unexpected role for professional proton-secreting 'clear cells' in sperm maturation and immune defense. The epididymal epithelium participates in the maturation of spermatozoa via the establishment of an acidic milieu and transfer of proteins to sperm cells, a poorly characterized process. We show that proton-secreting clear cells express mRNA transcripts and proteins that are acquired by maturing sperm, and that they establish close interactions with luminal spermatozoa via newly described 'nanotubes'. Mechanistic studies show that injection of bacterial antigens in vivo induces chemokine expression in clear cells, followed by macrophage recruitment into the organ. Injection of an inflammatory intermediate mediator (IFN-γ) increased Cxcl10 expression in clear cells, revealing their participation as sensors and mediators of inflammation. The functional diversity adopted by clear cells might represent a generalized phenomenon by which similar epithelial cells decode signals, communicate with neighbors and mediate mucosal immunity, depending on their precise location within an organ.


Subject(s)
Epididymis/cytology , Epithelial Cells/physiology , Immunity, Mucosal , Protons , Sperm Maturation , Spermatozoa/cytology , Animals , Chemokine CXCL10/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protein Transport , Seminal Vesicles/cytology , Sperm Motility
4.
J Physiol ; 597(7): 1957-1973, 2019 04.
Article in English | MEDLINE | ID: mdl-30746715

ABSTRACT

KEY POINTS: In the epididymis, elaborate communication networks between epithelial cells are important with respect to establishing an optimal acidic luminal environment for the maturation and storage of spermatozoa, which is essential for male fertility. Proton secretion by epididymal clear cells is achieved via the proton pumping V-ATPase located in their apical membrane. In the present study, we dissect the molecular mechanisms by which clear cells respond to luminal ATP and adenosine to modulate their acidifying activity via the adenosine receptor ADORA2B and the pH-sensitive ATP receptor P2X4. We demonstrate that the hydrolysis of ATP to produce adenosine by ectonucleotidases plays a key role in V-ATPase-dependent proton secretion, and is part of a feedback loop that ensures acidification of the luminal compartment These results help us better understand how professional proton-secreting cells respond to extracellular cues to modulate their functions, and how they communicate with neighbouring cells. ABSTRACT: Cell-cell cross-talk is crucial for the dynamic function of epithelia, although how epithelial cells detect and respond to variations in extracellular stimuli to modulate their environment remains incompletely understood. In the present study, we used the epididymis as a model system to investigate epithelial cell regulation by luminal factors. In the epididymis, elaborate communication networks between the different epithelial cell types are important for establishing an optimal acidic luminal environment for the maturation and storage of spermatozoa. In particular, clear cells (CCs) secrete protons into the lumen via the proton pumping V-ATPase located in their apical membrane, a process that is activated by luminal alkalinization. However, how CCs detect luminal pH variations to modulate their function remains uncharacterized. Purinergic regulation of epithelial transport is modulated by extracellular pH in other tissues. In the present study, functional analysis of the mouse cauda epididymis perfused in vivo showed that luminal ATP and adenosine modulate the acidifying activity of CCs via the purinergic ADORA2B and P2X4 receptors, and that luminal adenosine content is itself regulated by luminal pH. Altogether, our observations illustrate mechanisms by which CCs are activated by pH sensitive P2X4 receptor and ectonucleotidases, providing a feedback mechanism for the maintenance of luminal pH. These novel mechanisms by which professional proton-secreting cells respond to extracellular cues to modulate their functions, as well as how they communicate with neighbouring cells, might be translatable to other acidifying epithelia.


Subject(s)
Adenosine Triphosphate/pharmacology , Adenosine/pharmacology , Epididymis/physiology , Purinergic Agents , Purinergic Agonists/pharmacology , Vacuolar Proton-Translocating ATPases/metabolism , Animals , Epididymis/drug effects , Gene Expression Regulation , Hydrogen-Ion Concentration , Male , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Purinergic Antagonists/pharmacology , Receptor, Adenosine A2B/genetics , Receptor, Adenosine A2B/metabolism , Receptors, Purinergic P2X4/genetics , Receptors, Purinergic P2X4/metabolism , Vacuolar Proton-Translocating ATPases/genetics
5.
Sci Rep ; 8(1): 17531, 2018 12 03.
Article in English | MEDLINE | ID: mdl-30510210

ABSTRACT

Epididymal Cysteine Rich Secretory Proteins 1 and 4 (CRISP1 and CRISP4) associate with sperm during maturation and play different roles in fertilization. However, males lacking each of these molecules individually are fertile, suggesting compensatory mechanisms between these homologous proteins. Based on this, in the present work, we generated double CRISP1/CRISP4 knockout (DKO) mice and examined their reproductive phenotype. Our data showed that the simultaneous lack of the two epididymal proteins results in clear fertility defects. Interestingly, whereas most of the animals exhibited specific sperm fertilizing ability defects supportive of the role of CRISP proteins in fertilization, one third of the males showed an unexpected epididymo-orchitis phenotype with altered levels of inflammatory molecules and non-viable sperm in the epididymis. Further analysis showed that DKO mice exhibited an immature epididymal epithelium and abnormal luminal pH, supporting these defects as likely responsible for the different phenotypes observed. These observations reveal that CRISP proteins are relevant for epididymal epithelium differentiation and male fertility, contributing to a better understanding of the fine-tuning mechanisms underlying sperm maturation and immunotolerance in the epididymis with clear implications for human epididymal physiology and pathology.


Subject(s)
Cell Differentiation , Epididymis/metabolism , Infertility, Male/genetics , Infertility, Male/metabolism , Membrane Glycoproteins/deficiency , Seminal Plasma Proteins/genetics , Animals , Epididymis/pathology , Epithelium/metabolism , Epithelium/pathology , Infertility, Male/pathology , Male , Mice , Mice, Knockout
6.
Biol Reprod ; 99(2): 373-383, 2018 08 01.
Article in English | MEDLINE | ID: mdl-29481619

ABSTRACT

Epididymal sperm protein CRISP1 has the ability to both regulate murine CatSper, a key sperm calcium channel, and interact with egg-binding sites during fertilization. In spite of its relevance for sperm function, Crisp1-/-mice are fertile. Considering that phenotypes can be influenced by the genetic background, in the present work mice from the original mixed Crisp1-/- colony (129/SvEv*C57BL/6) were backcrossed onto the C57BL/6 strain for subsequent analysis of their reproductive phenotype. Whereas fertility and fertilization rates of C57BL/6 Crisp1-/- males did not differ from those reported for mice from the mixed background, several sperm functional parameters were clearly affected by the genetic background. Crisp1-/- sperm from the homogeneous background exhibited defects in both the progesterone-induced acrosome reaction and motility not observed in the mixed background, and normal rather than reduced protein tyrosine phosphorylation. Additional studies revealed a significant decrease in sperm hyperactivation as well as in cAMP and protein kinase A (PKA) substrate phosphorylation levels in sperm from both colonies. The finding that exposure of mutant sperm to a cAMP analog and phosphodiesterase inhibitor overcame the sperm functional defects observed in each colony indicated that a common cAMP-PKA signaling defect led to different phenotypes depending on the genetic background. Altogether, our observations indicate that the phenotype of CRISP1 null males is modulated by the genetic context and reveal new roles for the protein in both the functional events and signaling pathways associated to capacitation.


Subject(s)
Fertility/genetics , Fertilization/genetics , Membrane Glycoproteins/genetics , Reproduction/genetics , Spermatozoa/metabolism , Acrosome Reaction/drug effects , Acrosome Reaction/genetics , Animals , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Female , Genetic Background , Male , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Progesterone/pharmacology , Sperm Motility/genetics , Spermatozoa/drug effects
8.
J Am Soc Nephrol ; 29(2): 545-556, 2018 02.
Article in English | MEDLINE | ID: mdl-29222395

ABSTRACT

Acidosis is an important complication of AKI and CKD. Renal intercalated cells (ICs) express the proton pumping vacuolar H+-ATPase (V-ATPase) and are extensively involved in acid-base homeostasis. H+ secretion in type A intercalated cells (A-ICs) is regulated by apical vesicle recycling and stimulated by cAMP. In other cell types, cAMP is increased by extracellular agonists, including adenosine, through purinergic receptors. Adenosine is a Food and Drug Administration-approved drug, but very little is known about the effect of adenosine on IC function. Therefore, we investigated the role of adenosine in the regulation of V-ATPase in ICs. Intravenous treatment of mice with adenosine or agonists of ADORA2A and ADORA2B purinergic P1 receptors induced V-ATPase apical membrane accumulation in medullary A-ICs but not in cortical A-ICs or other IC subtypes. Both receptors are located in A-IC apical membranes, and adenosine injection increased urine adenosine concentration and decreased urine pH. Cell fractionation showed that adenosine or an ADORA2A or ADORA2B agonist induced V-ATPase translocation from vesicles to the plasma membrane and increased protein kinase A (PKA)-dependent protein phosphorylation in purified medullary ICs that were isolated from mice. Either ADORA2A or ADORA2B antagonists or the PKA inhibitor mPKI blocked these effects. Finally, a fluorescence pH assay showed that adenosine activates V-ATPase in isolated medullary ICs. Our study shows that medullary A-ICs respond to luminal adenosine through ADORA2A and ADORA2B receptors in a cAMP/PKA pathway-dependent mechanism to induce V-ATPase-dependent H+ secretion.


Subject(s)
Adenosine A2 Receptor Agonists/pharmacology , Adenosine/metabolism , Adenosine/pharmacology , Epithelial Cells/enzymology , Vacuolar Proton-Translocating ATPases/metabolism , Acid-Base Equilibrium , Adenosine A2 Receptor Antagonists/pharmacology , Animals , Cell Membrane/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Homeostasis , Kidney/cytology , Male , Mice , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Transport/drug effects , Receptor, Adenosine A2A , Receptor, Adenosine A2B , Transport Vesicles , Urinalysis
9.
Proc Natl Acad Sci U S A ; 114(46): E9989-E9998, 2017 11 14.
Article in English | MEDLINE | ID: mdl-29089413

ABSTRACT

Prior RNA sequencing (RNA-seq) studies have identified complete transcriptomes for most renal epithelial cell types. The exceptions are the cell types that make up the renal collecting duct, namely intercalated cells (ICs) and principal cells (PCs), which account for only a small fraction of the kidney mass, but play critical physiological roles in the regulation of blood pressure, extracellular fluid volume, and extracellular fluid composition. To enrich these cell types, we used FACS that employed well-established lectin cell surface markers for PCs and type B ICs, as well as a newly identified cell surface marker for type A ICs, c-Kit. Single-cell RNA-seq using the IC- and PC-enriched populations as input enabled identification of complete transcriptomes of A-ICs, B-ICs, and PCs. The data were used to create a freely accessible online gene-expression database for collecting duct cells. This database allowed identification of genes that are selectively expressed in each cell type, including cell-surface receptors, transcription factors, transporters, and secreted proteins. The analysis also identified a small fraction of hybrid cells expressing aquaporin-2 and anion exchanger 1 or pendrin transcripts. In many cases, mRNAs for receptors and their ligands were identified in different cells (e.g., Notch2 chiefly in PCs vs. Jag1 chiefly in ICs), suggesting signaling cross-talk among the three cell types. The identified patterns of gene expression among the three types of collecting duct cells provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.


Subject(s)
Aquaporin 2/metabolism , Epithelial Cells/metabolism , Kidney Tubules, Collecting/metabolism , Kidney/metabolism , Sequence Analysis, RNA/methods , Transcriptome , Animals , Anion Exchange Protein 1, Erythrocyte/metabolism , Anion Transport Proteins/metabolism , Base Sequence , Biomarkers/metabolism , Gene Expression , Gene Expression Profiling , Jagged-1 Protein/metabolism , Male , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , RNA/metabolism , Receptor, Notch2/metabolism , Signal Transduction , Sulfate Transporters , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptome/genetics
10.
J Cell Biol ; 210(7): 1213-24, 2015 Sep 28.
Article in English | MEDLINE | ID: mdl-26416967

ABSTRACT

Ca(2+)-dependent mechanisms are critical for successful completion of fertilization. Here, we demonstrate that CRISP1, a sperm protein involved in mammalian fertilization, is also present in the female gamete and capable of modulating key sperm Ca(2+) channels. Specifically, we show that CRISP1 is expressed by the cumulus cells that surround the egg and that fertilization of cumulus-oocyte complexes from CRISP1 knockout females is impaired because of a failure of sperm to penetrate the cumulus. We provide evidence that CRISP1 stimulates sperm orientation by modulating sperm hyperactivation, a vigorous motility required for penetration of the egg vestments. Moreover, patch clamping of sperm revealed that CRISP1 has the ability to regulate CatSper, the principal sperm Ca(2+) channel involved in hyperactivation and essential for fertility. Given the critical role of Ca(2+) for sperm motility, we propose a novel CRISP1-mediated fine-tuning mechanism to regulate sperm hyperactivation and orientation for successful penetration of the cumulus during fertilization.


Subject(s)
Calcium Channels/metabolism , Calcium Signaling/physiology , Membrane Glycoproteins/metabolism , Oocytes/metabolism , Sperm Motility/physiology , Sperm-Ovum Interactions/physiology , Spermatozoa/metabolism , Animals , Calcium/metabolism , Calcium Channels/genetics , Female , Male , Membrane Glycoproteins/genetics , Mice , Mice, Knockout , Oocytes/cytology , Spermatozoa/cytology
11.
Dev Biol ; 405(2): 237-49, 2015 Sep 15.
Article in English | MEDLINE | ID: mdl-26169470

ABSTRACT

Mammalian sperm must acquire their fertilizing ability after a series of biochemical modifications in the female reproductive tract collectively called capacitation to undergo acrosomal exocytosis, a process that is essential for fertilization. Actin dynamics play a central role in controlling the process of exocytosis in somatic cells as well as in sperm from several mammalian species. In somatic cells, small GTPases of the Rho family are widely known as master regulators of actin dynamics. However, the role of these proteins in sperm has not been studied in detail. In the present work we characterized the participation of small GTPases of the Rho family in the signaling pathway that leads to actin polymerization during mouse sperm capacitation. We observed that most of the proteins of this signaling cascade and their effector proteins are expressed in mouse sperm. The activation of the signaling pathways of cAMP/PKA, RhoA/C and Rac1 is essential for LIMK1 activation by phosphorylation on Threonine 508. Serine 3 of Cofilin is phosphorylated by LIMK1 during capacitation in a transiently manner. Inhibition of LIMK1 by specific inhibitors (BMS-3) resulted in lower levels of actin polymerization during capacitation and a dramatic decrease in the percentage of sperm that undergo acrosomal exocytosis. Thus, we demonstrated for the first time that the master regulators of actin dynamics in somatic cells are present and active in mouse sperm. Combining the results of our present study with other results from the literature, we have proposed a working model regarding how LIMK1 and Cofilin control acrosomal exocytosis in mouse sperm.


Subject(s)
Acrosome Reaction/physiology , Cofilin 1/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Exocytosis , Lim Kinases/metabolism , Sperm Capacitation/physiology , Actins/metabolism , Animals , Crosses, Genetic , Gene Expression Regulation , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Fluorescence , Phosphorylation , Signal Transduction , Spermatozoa/metabolism
12.
Asian J Androl ; 17(5): 711-5, 2015.
Article in English | MEDLINE | ID: mdl-26112483

ABSTRACT

Mammalian fertilization is a complex process that involves different steps of interaction between the male and female gametes. In spite of its relevance, the molecular mechanisms underlying this process still remain to be elucidated. The present review describes the contribution of our laboratory to the understanding of mammalian fertilization using Cysteine-RIch Secretory Proteins (CRISP) as model molecules. Substantial evidence obtained from in vitro assays and knockout models shows that epididymal CRISP1 associates with the sperm surface with two different affinities during maturation, and participates in the regulation of signaling pathways during capacitation as well as in both sperm-zona pellucida interaction and gamete fusion. These observations can be extended to humans as judged by our findings showing that the human homolog of the rodent protein (hCRISP1) is also involved in both stages of fertilization. Evidence supports that other members of the CRISP family secreted in the testis (CRISP2), epididymis (CRISP3-4) or during ejaculation (CRISP3) are also involved in sperm-egg interaction, supporting the existence of a functional redundancy and cooperation between homolog proteins ensuring the success of fertilization. Together, our observations indicate that CRISP proteins accompany spermatozoa along their transit through both the male and female reproductive tracts. We believe these results not only contribute to a better mechanistic understanding of fertilization but also support CRISP proteins as excellent candidates for future research on infertility and contraception.


Subject(s)
Epididymis/metabolism , Fertilization/physiology , Membrane Glycoproteins/metabolism , Sperm-Ovum Interactions/physiology , Animals , Female , Humans , Male , Sperm Capacitation/physiology , Spermatozoa/physiology
13.
Dev Biol ; 369(1): 43-53, 2012 Sep 01.
Article in English | MEDLINE | ID: mdl-22750823

ABSTRACT

Changes that occur to mammalian sperm upon epididymal transit and maturation render these cells capable of moving progressively and capacitating. Signaling events leading to mammalian sperm capacitation depend on the modulation of proteins by phosphorylation and dephosphorylation cascades. Recent experiments have demonstrated that the Src family of kinases plays an important role in the regulation of these events. However, sperm from cSrc null mice display normal tyrosine phosphorylation associated with capacitation. We report here that, despite normal phosphorylation, sperm from cSrc null mice display a severe reduction in forward motility, and are unable to fertilize in vitro. Histological analysis of seminiferous tubules in the testes, caput and corpus epididymis do not reveal obvious defects. However, the cauda epididymis is significantly smaller, and expression of key transport proteins in the epithelial cells lining this region is reduced in cSrc null mice compared to wild type littermates. Although previously, we and others have shown the presence of cSrc in mature sperm from cauda epididymis, a closer evaluation indicates that this tyrosine kinase is not present in sperm from the caput epididymis, suggesting that this protein is acquired by sperm later during epididymal maturation. Consistent with this observation, cSrc is enriched in vesicles released by the epididymal epithelium known as epididymosomes. Altogether, these observations indicate that cSrc is essential for cauda epididymal development and suggest an essential role of this kinase in epididymal sperm maturation involving cSrc extracellular trafficking.


Subject(s)
Epididymis/growth & development , Epididymis/metabolism , Spermatozoa/metabolism , Animals , Epididymis/cytology , Gene Expression Regulation, Developmental , Image Processing, Computer-Assisted , Male , Mice , Mice, Knockout , Organ Size , Proto-Oncogene Proteins pp60(c-src)/genetics , Proto-Oncogene Proteins pp60(c-src)/metabolism , Sperm Capacitation/physiology , Sperm Motility/physiology , Spermatozoa/cytology
14.
J Androl ; 33(6): 1360-70, 2012.
Article in English | MEDLINE | ID: mdl-22653965

ABSTRACT

Cysteine-rich secretory protein 2 (CRISP2) is a testicular sperm protein proposed to be involved in fertilization. With the aim of examining the relevance of CRISP2 for fertility and its potential use as a target for contraception, in the present work, male and female rats were immunized with recombinant CRISP2 coupled to maltose-binding protein (MBP) and evaluated for their subsequent fertility. As controls, animals were injected with either MBP or recombinant CRISP1. Enzyme-linked immunosorbent assay of sera collected at different intervals after immunization indicated that CRISP2 immunization raised specific antibodies in both sexes, with levels that increased as a function of time. Western blot studies revealed that anti-CRISP2 sera were capable of recognizing CRISP2 in testicular, epididymal, and sperm extracts, whereas histological studies showed no evidence of autoimmune orchitis or epididymitis. Indirect immunofluorescence experiments revealed the ability of anti-CRISP2 sera to recognize the native sperm protein in fresh, capacitated, and ionophore-induced acrosome-reacted cells. Moreover, anti-CRISP2 sera significantly inhibited the sperm ability to penetrate zona-free eggs, confirming the role of CRISP2 in rat gamete fusion. In spite of the presence of circulating anti-CRISP2 antibodies capable of inhibiting the sperm fertilizing ability, mating studies revealed no effects of CRISP2 immunization on male or female fertility, in contrast to the significant inhibition observed in both sexes in animals injected with CRISP1. Together, these observations indicated the immunogenic properties of testicular CRISP2 but do not support CRISP2 as a target for immunocontraception or as a molecule responsible for generating autoimmune orchitis or immunoinfertility.


Subject(s)
Fertilization/physiology , Glycoproteins/immunology , Animals , Cell Adhesion Molecules , Contraception, Immunologic , Female , Male , Rats , Rats, Sprague-Dawley , Rats, Wistar , Recombinant Proteins/immunology , Sperm Capacitation
15.
J Androl ; 32(6): 672-8, 2011.
Article in English | MEDLINE | ID: mdl-21441424

ABSTRACT

Rat epididymal CRISP1, the first described member of the evolutionarily conserved Cysteine-RIch Secretory Protein (CRISP) family, is expressed in the proximal regions of the epididymis and associates with the sperm during epididymal transit. Evidence indicates the existence of 2 populations of CRISP1 in spermatozoa: a major one, loosely bound, which is released during capacitation and, therefore, proposed as a decapacitating factor; and a minor one, strongly associated with spermatozoa that remains on the cells after capacitation and is proposed to participate in gamete interaction. Originally localized to the dorsal region of capacitated sperm, CRISP1 migrates to the equatorial segment with capacitation and acrosome reaction. Consistent with these localizations, in vitro fertilization experiments support the involvement of CRISP1 in the first step of sperm-zona pellucida (ZP) interaction and subsequent gamete fusion through its interaction with egg-complementary sites. The potential roles of CRISP1 in capacitation and fertilization have been further supported by the finding that capacitated spermatozoa from CRISP1 "knockout" animals exhibit low levels of protein tyrosine phosphorylation and have an impaired ability to fertilize zona-intact and zona-free eggs in vitro. Moreover, recent evidence from mutant spermatozoa reveals that CRISP1 mediates the stage of sperm binding to the ZP. Altogether, these observations support the view that CRISP1 is a multifunctional protein playing different roles during fertilization through its different associations with and localizations on spermatozoa. We believe these results contribute to a better understanding of the molecular mechanisms involved in both the fertilization process and the acquisition of sperm-fertilizing ability that occurs during epididymal maturation.


Subject(s)
Epididymis/metabolism , Fertilization , Membrane Glycoproteins/metabolism , Animals , Humans , Male , Mice , Rats , Sperm Capacitation , Sperm-Ovum Interactions , Spermatozoa/metabolism , Zona Pellucida/metabolism
16.
Neurochem Res ; 34(5): 953-63, 2009 May.
Article in English | MEDLINE | ID: mdl-18850267

ABSTRACT

The olfactory system in rats is part of the limbic region with extensive afferent connections with brain areas involved in the regulation of behaviour and autonomic responses. The existence of the endothelin system and catecholaminergic neurons in the olfactory bulb suggests that endothelins may modulate noradrenergic transmission and diverse olfactory mediated processes. In the present work we studied the effect of endothelin-1 and -3 on neuronal norepinephrine release and the short-term regulation of tyrosine hydroxylase in the olfactory bulb. Results showed that both endothelins increased tyrosine hydroxylase activity through the activation of a non-conventional endothelin G-protein coupled receptor, coupled to the stimulation of protein kinase A and C, as well as Ca(2+)/calmodulin-dependent protein kinase II. On the other hand, neither endothelin-1 nor endothelin-3 modified tyrosine hydroxylase total protein levels, but both peptides increased the phosphorylation of serine residues of the enzyme at sites 19 and 40. Furthermore, endothelins enhanced norepinephrine release in olfactory neurons suggesting that this event may contribute to increased tyrosine hydroxylase activity by reducing the feedback inhibition. Taken together present findings show a clear interaction between the endothelin system, and the catecholaminergic transmission in the olfactory bulb. Additional studies are required to evaluate the physiological functions regulated by endothelins at this brain level.


Subject(s)
Endothelin-1/physiology , Endothelin-3/physiology , Olfactory Bulb/metabolism , Tyrosine 3-Monooxygenase/metabolism , Adenylyl Cyclases/metabolism , Animals , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Endothelin-1/pharmacology , Endothelin-3/pharmacology , Enzyme Activation , Feedback, Physiological , Male , Neurons/metabolism , Norepinephrine/metabolism , Phosphorylation , Protein Kinase C/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Endothelin/agonists , Signal Transduction , Type C Phospholipases/metabolism
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