RESUMEN

Novel male contraceptives will promote gender equality in sharing contraceptive responsibility. The sperm-associated protein epididymal protease inhibitor (EPPIN) is a promising target for non-hormonal male contraception. EPPIN interacts with the semen coagulum protein semenogelin-1 (SEMG1) on the sperm surface, leading to transient inhibition of sperm motility after ejaculation. Small organic molecules targeting EPPIN's SEMG1-binding are under development as male contraceptives. Here, we combined computational approaches to uncover key aspects underlying EPPIN binding to SEMG1 and small organic ligands. We generated a human EPPIN model showing a typical arrangement of the WFDC (Whey-acid four disulfide core)-type and Kunitz-type domains, connected by a hinge region. Determining the EPPIN model's intrinsic motion by molecular dynamics simulations and normal mode analysis revealed a conformation, presenting a binding pocket that accommodates SEMG1Glu229-Gln247, EP055, and EP012. EPPIN's residues Phe63 and Lys68 (WFDC domain), Asp71 (hinge region), and Asn113, Asn114, and Asn115 (Kunitz domain) were identified as hot spots for SEMG1, EP055, and EP012 binding. Moreover, hydrophobic and hydrophilic residues in the WFDC and Kunitz domains allow plasma membrane anchoring, orienting the EPPIN binding pocket to the solvent. Targeting EPPIN's essential residues for its biomolecular interactions may improve the rational design of EPPIN ligands as spermiostatic compounds.

Asunto(s)

Anticonceptivos Masculinos , Humanos , Masculino , Anticonceptivos Masculinos/farmacología , Ligandos , Semen , Motilidad Espermática , Anticonceptivos

RESUMEN

Men have two practical choices for contraception; the condom which has a high typical use failure rate or vasectomy. New male hormonal and non-hormonal contraceptives are under development that target either the production of sperm (spermatogenesis) or the delivery of sperm. One particular target is the sperm protein EPPIN, which is present on the surface of human spermatozoa. EP055 is a small organic compound that targets EPPIN on the surface of sperm and inhibits motility. EP055 was tested in cynomolgus (Macaca fascicularis) males to determine its plasma half-life after intravenous (i.v.) infusion of a single dose and for binding to its target tissues. Our initial study demonstrated a plasma half-life for EP055 of 10.6 minutes. In a second study examination of macaque testis, epididymis, and plasma after i.v. infusion of a single dose of compound EP055 (63.25 mg/kg) demonstrated that EP055 was detected in testis and epididymis two hours and six hours post-infusion. We initiated a trial in rhesus (Macaca mulatta) males to assess the availability of EP055 in semen and its effect on sperm motility as a measure of the drug's efficacy. Four macaques were infused with a low dose (75-80 mg/kg) followed by a recovery period and a subsequent high dose (125-130 mg/kg) of EP055. After high dose administration, sperm motility fell to approximately 20% of pretreatment levels within 6 hours post-infusion; no normal motility was observed at 30 hours post-infusion. Recovery of sperm motility was obvious by 78 hours post-infusion; with full recovery in all animals by 18 days post-infusion. EP055 has the potential to be a male contraceptive that would provide a reversible, short-lived pharmacological alternative.

Asunto(s)

Anticonceptivos Masculinos/farmacología , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Animales , Anticonceptivos Masculinos/farmacocinética , Macaca fascicularis , Masculino

RESUMEN

Developing a non-hormonal male contraceptive requires identifying and characterizing an appropriate target and demonstrating its essential role in reproduction. Here we review the development of male contraceptive targets and the current therapeutic agents under consideration. In addition, the development of EPPIN as a target for contraception is reviewed. EPPIN is a well characterized surface protein on human spermatozoa that has an essential function in primate reproduction. EPPIN is discussed as an example of target development, testing in non-human primates, and the search for small organic compounds that mimic contraceptive antibodies; binding EPPIN and blocking sperm motility. Although many hurdles remain before the success of a non-hormonal male contraceptive, continued persistence should yield a marketable product.

Asunto(s)

Anticoncepción , Anticonceptivos Masculinos/farmacología , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Animales , Anticonceptivos Masculinos/uso terapéutico , Humanos , Proteínas Inhibidoras de Proteinasas Secretoras/genética

RESUMEN

Semenogelin I (SEMG1) is found in human semen coagulum and on the surface of spermatozoa bound to EPPIN. The physiological significance of the SEMG1/EPPIN interaction on the surface of spermatozoa is its capacity to modulate sperm progressive motility. The present study investigates the hypothesis that the interacting surface of SEMG1 and EPPIN co-evolved within the Hominoidea time scale, as a result of adaptive pressures applied by their roles in sperm protection and reproductive fitness. Our results indicate that some amino acid residues of SEMG1 and EPPIN possess a remarkable deficiency of variation among hominoid primates. We observe a distinct residue change unique to humans within the EPPIN sequence containing a SEMG1 interacting surface, namely His92. In addition, Bayes Empirical Bayes analysis for positive selection indicates that the SEMG1 Cys239 residue underwent positive selection in humans, probably as a consequence of its role in increasing the binding affinity of these interacting proteins. We confirm the critical role of Cys239 residue for SEMG1 binding to EPPIN and inhibition of sperm motility by showing that recombinant SEMG1 mutants in which Cys239 residue was changed to glycine, aspartic acid, histidine, serine or arginine have reduced capacity to interact to EPPIN and to inhibit human sperm motility in vitro. In conclusion, our results indicate that EPPIN and SEMG1 rapidly co-evolved in primates due to their critical role in the modulation of sperm motility in the semen coagulum, providing unique insights into the molecular co-evolution of sperm surface interacting proteins.

Asunto(s)

Adaptación Fisiológica , Evolución Molecular , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Proteínas de Secreción de la Vesícula Seminal/metabolismo , Espermatozoides/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Filogenia , Selección Genética , Proteínas de Secreción de la Vesícula Seminal/química , Proteínas de Secreción de la Vesícula Seminal/genética , Motilidad Espermática , Espermatozoides/citología

RESUMEN

EPPIN (epididymal protease inhibitor; SPINLW1), an antimicrobial cysteine-rich protein containing both Kunitz and whey acidic protein (WAP)-type four disulfide core protease inhibitor consensus sequences, is a target for male contraception because of its critical role in sperm motility. Here, we characterized EPPIN's expression and cellular distribution in rat tissues and its in vivo regulation by androgens in the epididymis. EPPIN (mRNA and protein) was abundantly expressed in the rat testis and epididymis; we also found that the vas deferens, seminal vesicles, and brain were novel sites of EPPIN expression. PCR studies demonstrated that in addition to Sertoli cells, spermatogenic cells expressed Eppin mRNA. EPPIN was immunolocalized in Sertoli cells and spermatogenic cells (pachytene spermatocytes and round and elongated spermatids) and in epithelial cells and spermatozoa from efferent ductules and epididymis. EPPIN staining was observed on the middle and principal pieces of the flagellum of testicular spermatozoa. Epididymal spermatozoa had more intense EPPIN staining on the flagellum, and the EPPIN staining became apparent on the head and neck regions. This suggested that the EPPIN found on maturing spermatozoa was secreted primarily by the epithelial cells of the epididymis. Surgical castration down-regulated EPPIN expression levels (mRNA and protein) in the caput and cauda epididymis, an effect reversed by testosterone replacement. Altogether, our data suggested that EPPIN expression in rats is more widespread than in humans and mice, and is androgen-dependent in the epididymis. This species could be used as an experimental model to further study EPPIN's role in male fertility.

Asunto(s)

Genitales Masculinos/metabolismo , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Maduración del Esperma , Espermatozoides/metabolismo , Andrógenos/metabolismo , Animales , Encéfalo/metabolismo , Castración , Células Cultivadas , Epidídimo/metabolismo , Masculino , Proteínas Inhibidoras de Proteinasas Secretoras/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Proteínas de Secreción de la Vesícula Seminal/genética , Proteínas de Secreción de la Vesícula Seminal/metabolismo , Vesículas Seminales/metabolismo , Motilidad Espermática , Cola del Espermatozoide/metabolismo , Conducto Deferente/metabolismo

RESUMEN

Epididymal protease inhibitor (EPPIN) is found on the surface of spermatozoa and works as a central hub for a sperm surface protein complex (EPPIN protein complex [EPC]) that inhibits sperm motility on the binding of semenogelin I (SEMG1) during ejaculation. Here, we identify EPPIN's amino acids involved in the interactions within the EPC and demonstrate that EPPIN's sequence C102-P133 contains the major binding site for SEMG1. Within the same region, the sequence F117-P133 binds the EPC-associated protein lactotransferrin (LTF). We show that residues Cys102, Tyr107, and Phe117 in the EPPIN C-terminus are required for SEMG1 binding. Additionally, residues Tyr107 and Phe117 are critically involved in the interaction between EPPIN and LTF. Our findings demonstrate that EPPIN is a key player in the protein-protein interactions within the EPC. Target identification is an important step toward the development of a novel male contraceptive, and the functionality of EPPIN's residues Cys102, Tyr107, and Phe117 offers novel opportunities for contraceptive compounds that inhibit sperm motility by targeting this region of the molecule.

Asunto(s)

Anticonceptivos Masculinos , Terapia Molecular Dirigida , Dominios y Motivos de Interacción de Proteínas , Proteínas Inhibidoras de Proteinasas Secretoras/química , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Proteínas de Secreción de la Vesícula Seminal/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Anticonceptivos Masculinos/análisis , Anticonceptivos Masculinos/química , Anticonceptivos Masculinos/aislamiento & purificación , Anticonceptivos Masculinos/metabolismo , Descubrimiento de Drogas , Humanos , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Terapia Molecular Dirigida/métodos , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Puntual/fisiología , Unión Proteica/genética , Dominios y Motivos de Interacción de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas/fisiología , Proteínas Inhibidoras de Proteinasas Secretoras/antagonistas & inhibidores , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Proteínas de Secreción de la Vesícula Seminal/química , Proteínas de Secreción de la Vesícula Seminal/genética

RESUMEN

BACKGROUND: Studies published in the 1970s by Mostafa S. Fahim and colleagues showed that a short treatment with ultrasound caused the depletion of germ cells and infertility. The goal of the current study was to determine if a commercially available therapeutic ultrasound generator and transducer could be used as the basis for a male contraceptive. METHODS: Sprague-Dawley rats were anesthetized and their testes were treated with 1 MHz or 3 MHz ultrasound while varying power, duration and temperature of treatment. RESULTS: We found that 3 MHz ultrasound delivered with 2.2 Watt per square cm power for fifteen minutes was necessary to deplete spermatocytes and spermatids from the testis and that this treatment significantly reduced epididymal sperm reserves. 3 MHz ultrasound treatment reduced total epididymal sperm count 10-fold lower than the wet-heat control and decreased motile sperm counts 1,000-fold lower than wet-heat alone. The current treatment regimen provided nominally more energy to the treatment chamber than Fahim's originally reported conditions of 1 MHz ultrasound delivered at 1 Watt per square cm for ten minutes. However, the true spatial average intensity, effective radiating area and power output of the transducers used by Fahim were not reported, making a direct comparison impossible. We found that germ cell depletion was most uniform and effective when we rotated the therapeutic transducer to mitigate non-uniformity of the beam field. The lowest sperm count was achieved when the coupling medium (3% saline) was held at 37 degrees C and two consecutive 15-minute treatments of 3 MHz ultrasound at 2.2 Watt per square cm were separated by 2 days. CONCLUSIONS: The non-invasive nature of ultrasound and its efficacy in reducing sperm count make therapeutic ultrasound a promising candidate for a male contraceptive. However, further studies must be conducted to confirm its efficacy in providing a contraceptive effect, to test the result of repeated use, to verify that the contraceptive effect is reversible and to demonstrate that there are no detrimental, long-term effects from using ultrasound as a method of male contraception.

Asunto(s)

Anticoncepción/métodos , Epidídimo/citología , Recuento de Espermatozoides , Testículo/citología , Terapia por Ultrasonido/métodos , Animales , Calor , Masculino , Meiosis , Ratas , Ratas Sprague-Dawley , Terapia por Ultrasonido/instrumentación

RESUMEN

The development of a new male contraceptive requires a transition from animal model to human and an understanding of the mechanisms involved in the target's inhibition of human spermatozoan fertility. We now report that semenogelin (SEMG1) and anti-EPPIN antibodies to a defined target site of 21 amino acids on the C terminal of EPPIN cause the loss of intracellular calcium, as measured by Fluo-4. The loss of intracellular calcium explains our previous observations of an initial loss of progressive motility and eventually the complete loss of motility when spermatozoa are treated with SEMG1 or anti-EPPIN antibodies. Thimerosal can rescue the effects of SEMG1 on motility, implying that internal stores of calcium are not depleted. Additionally, SEMG1 treatment of spermatozoa decreases the intracellular pH, and motility can be rescued by ammonium chloride. The results of this study demonstrate that EPPIN controls sperm motility in the ejaculate by binding SEMG1, resulting in the loss of calcium, most likely through a disturbance of internal pH and an inhibition of uptake mechanisms. However, the exact steps through which the EPPIN-SEMG1 complex exerts its effect on internal calcium levels are unknown. Anti-EPPIN antibodies can substitute for SEMG1, and, therefore, small-molecular weight compounds that mimic anti-EPPIN binding should be able to substitute for SEMG1, providing the basis for a nonantibody, nonhormonal male contraceptive.

Asunto(s)

Calcio/metabolismo , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Proteínas de Secreción de la Vesícula Seminal/metabolismo , Espermatozoides/metabolismo , Anticuerpos Antiidiotipos/farmacología , Anticoncepción , Humanos , Concentración de Iones de Hidrógeno , Masculino , Proteínas Inhibidoras de Proteinasas Secretoras/inmunología , Proteínas de Secreción de la Vesícula Seminal/farmacología , Motilidad Espermática/efectos de los fármacos , Motilidad Espermática/fisiología , Espermatozoides/efectos de los fármacos

RESUMEN

Our laboratory has characterized EPPIN [epididymal protease inhibitor; SPINLW1] as a novel gene on human chromosome 20q12-13.2, which encodes a cysteine-rich protein of 133 amino acids with a calculated molecular mass of 15.283 kDa, containing both Kunitz-type and WAP (whey acidic protein)-type four-disulfide core consensus sequences. Eppin is secreted by Sertoli cells in the testis and epididymal epithelial cells; it is predominantly a dimer, although multimers often exist, and in its native form eppin is found on the human sperm surface complexed with LTF (lactotransferrin) and clusterin. During ejaculation SEMG (semenogelin) from the seminal vesicles binds to the eppin protein complex, initiating a series of events that define eppin's function. Eppin's functions include (i) modulating PSA (prostate-specific antigen) enzyme activity, (ii) providing antimicrobial protection and (iii) binding SEMG thereby inhibiting sperm motility. As PSA hydrolyses SEMG in the ejaculate coagulum, spermatozoa gain progressive motility. We have demonstrated that eppin is essential for fertility because immunization of male monkeys with recombinant eppin results in complete, but reversible, contraception. To exploit our understanding of eppin's function, we are developing compounds that inhibit eppin-SEMG interaction and mimic anti-eppin, inhibiting sperm motility. These compounds should have potential as a male contraceptive.

Asunto(s)

Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Secuencia de Aminoácidos , Animales , Anticonceptivos Masculinos/metabolismo , Epidídimo/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Antígeno Prostático Específico/metabolismo , Proteínas Inhibidoras de Proteinasas Secretoras/química , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Proteínas de Secreción de la Vesícula Seminal/química , Proteínas de Secreción de la Vesícula Seminal/genética , Proteínas de Secreción de la Vesícula Seminal/metabolismo , Espermatozoides/citología , Espermatozoides/metabolismo , Testículo/metabolismo

RESUMEN

BACKGROUND: NASP (Nuclear Autoantigenic Sperm Protein) is a histone chaperone that is present in all dividing cells. NASP has two splice variants: tNASP and sNASP. Only cancer, germ, transformed, and embryonic cells have a high level of expression of the tNASP splice variant. We examined the consequences of tNASP depletion for prostate cancer PC-3 cells. METHODS: tNASP was depleted from prostate cancer PC-3 cells, cervical cancer HeLa cells, and prostate epithelial PWR-1E cells using lentivirus expression of tNASP shRNA. Cell cycle changes were studied by proliferation assay with CFSE labeling and double thymidine synchronization. Gene expression profiles were detected using RT(2)Profiler PCR Array, Western and Northern blotting. RESULTS: PC-3 and HeLa cells showed inhibited proliferation, increased levels of cyclin-dependant kinase inhibitor p21 protein and apoptosis, whereas non-tumorigenic PWR-1E cells did not. All three cell types showed decreased levels of HSPA2. Supporting in vitro experiments demonstrated that tNASP, but not sNASP is required for activation of HSPA2. CONCLUSIONS: Our results demonstrate that PC-3 and HeLa cancer cells require tNASP to maintain high levels of HSPA2 activity and therefore viability, while PWR-1E cells are unaffected by tNASP depletion. These different cellular responses most likely arise from changes in the interaction between tNASP and HSPA2 and disturbed tNASP chaperoning of linker histones. This study has demonstrated that tNASP is critical for the survival of prostate cancer cells and suggests that targeting tNASP expression can lead to a new approach for prostate cancer treatment.

Asunto(s)

Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Autoantígenos/genética , Proliferación Celular/efectos de los fármacos , Proteínas Nucleares/genética , Neoplasias de la Próstata/patología , ARN Interferente Pequeño/farmacología , Adenocarcinoma/genética , Apoptosis/genética , Autoantígenos/fisiología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Chaperonas de Histonas/antagonistas & inhibidores , Chaperonas de Histonas/genética , Chaperonas de Histonas/fisiología , Humanos , Masculino , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/fisiología , Neoplasias de la Próstata/genética

RESUMEN

The Laboratories for Reproductive Biology at the University of North Carolina at Chapel Hill began collaboration with Human Genome Sciences (Rockville, Maryland) to sequence a human epididymal library and identify epididymal-specific genes. Among the first clones obtained from Human Genome Sciences was a clone for EPPIN (official symbol, SPINLW1). Our laboratory has described EPPIN (epididymal protease inhibitor) as a novel gene on human chromosome 20q12-13.2 that encodes a cysteine-rich protein containing both Kunitz-type and WAP-type 4-disulfide core consensus sequences that characterize it as a protease inhibitor. EPPIN expresses 3 mRNA splice variants that encode 2 protein isoforms found in the testis and epididymis. Of the 2 isoforms, 1 is secreted and 1 lacks a secretory signal piece. EPPIN is predominantly a dimer, although multiples often exist, and in its native form, EPPIN is found on the sperm surface complexed with lactotransferrin and clusterin. During ejaculation, semenogelin from the seminal vesicles is bound to the EPPIN protein complex, initiating a series of events that define EPPIN's function: modulating prostate-specific antigen (PSA) activity, providing antimicrobial protection, and binding semenogelin, thereby inhibiting sperm motility. As PSA hydrolyzes semenogelin in the ejaculate coagulum, spermatozoa gain progressive motility. Using immunization as a tool to study antigen function, we demonstrated that EPPIN is essential for fertility because immunization of male monkeys with recombinant EPPIN results in complete, but reversible, contraception. To exploit our understanding of EPPIN's function, we have developed a high-throughput screen to look for compounds that inhibit EPPIN-semenogelin interaction and mimic anti-EPPIN, inhibiting sperm motility. These compounds are now being developed into a nonhormonal male contraceptive.

Asunto(s)

Anticonceptivos Masculinos/farmacología , Epidídimo/efectos de los fármacos , Epidídimo/enzimología , Ensayos Analíticos de Alto Rendimiento , Inhibidores de Proteasas/farmacología , Proteínas Inhibidoras de Proteinasas Secretoras/antagonistas & inhibidores , Proteínas de Secreción de la Vesícula Seminal/antagonistas & inhibidores , Animales , Eyaculación , Fertilización , Humanos , Masculino , Ratones , Antígeno Prostático Específico/metabolismo , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Proteínas de Secreción de la Vesícula Seminal/metabolismo , Motilidad Espermática , Testículo/metabolismo

RESUMEN

Biomarkers for early detection of cancer have great clinical diagnostic potential. Numerous reports have documented the generation of humoral immune responses that are triggered in response to changes in protein expression patterns in tumor tissues and these biomarkers are referred to as tumor associated antigens (TAAs). Using a high-throughput technology, we previously identified 65 proteins as diagnostically useful TAAs by profiling the humoral immune responses in ovarian cancer (OVCA) patients. Here we determined the expression status of some of those TAAs in tissues from OVCA patients. The protein expression patterns of 4 of those 65 antigens, namely NASP, RCAS1, Nijmegen breakage syndrome1 (NBS1) and eIF5A, along with p53 and Her2 (known molecular prognosticators) and two proteins that interact with NBS1, MRE11 and RAD50, were assessed by immunohistochemistry (IHC). NASP and RCAS1 proteins were more frequently expressed in ovarian cancer tissues than with normal ovarian tissue and serous cystadenomas and MRE11 was less frequently expressed. When evaluated simultaneously, only NASP and MRE11 remained statistically significant with sensitivity of 66% and specificity of 89%. None of these proteins' expression levels were prognostic for survival. Together, our results indicate that occurrence of humoral immune responses against some of these TAAs in OVCA patients is triggered by antigen protein overexpression.

Asunto(s)

Antígenos de Neoplasias/administración & dosificación , Biomarcadores de Tumor/análisis , Neoplasias Ováricas/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/biosíntesis , Autoantígenos/biosíntesis , Cistadenoma Seroso/diagnóstico , Cistadenoma Seroso/metabolismo , Proteínas de Unión al ADN/biosíntesis , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Proteína Homóloga de MRE11 , Persona de Mediana Edad , Estadificación de Neoplasias , Proteínas Nucleares/biosíntesis , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Análisis de Matrices Tisulares , Adulto Joven

RESUMEN

Eppin (epididymal protease inhibitor [SPINLW1]) is present in a protein complex on the human sperm surface that contains lactotransferrin, clusterin, and semenogelin (SEMG1). During ejaculation the presence of semenogelin inhibits sperm progressive motility until semenogelin is hydrolyzed by prostate-specific antigen (PSA). Although eppin binds all three components in its protein complex, the binding of semenogelin to eppin appears to be critical for the inhibition of progressive motility. The effect of the originally identified seminal plasma motility inhibitor fragment has not been clearly defined on live spermatozoa. Therefore, we have used recombinant semenogelin (rSEMG1) and its fragments, including a semenogelin mutant in which cysteine 239 was changed to glycine, coupled with a computer assisted sperm analysis assay to study the motility inhibitory properties of semenogelin. Each fragment and the mutant were tested for their effects on motility. Recombinant semenogelin significantly inhibited sperm progressive motility in a dose- and time-dependent manner. The C-terminal semenogelin fragment (amino acids 164-283) containing cysteine 239 significantly inhibited sperm progressive motility, whereas the N-terminal fragment (amino acids 24-163), a short C-terminal fragment (amino acids 172-215) without cysteine 239, and the mutant fragment (amino acids 24-283 with glycine 239) did not inhibit motility. After treatment with recombinant semenogelin, spermatozoa could be washed and treated with PSA, partially reversing the inhibition of progressive motility. Cysteine 239 of rSEMG1 appears to be the critical amino acid for both binding to eppin and inhibiting sperm motility.

Asunto(s)

Proteínas de Secreción de la Vesícula Seminal/farmacología , Motilidad Espermática/efectos de los fármacos , Sitios de Unión/genética , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Humanos , Masculino , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Unión Proteica/fisiología , Estructura Terciaria de Proteína/genética , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Proteínas de Secreción de la Vesícula Seminal/química , Proteínas de Secreción de la Vesícula Seminal/genética , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Espermatozoides/fisiología , Factores de Tiempo

RESUMEN

In mammalian spermatocytes, cell division cycle protein 2 (CDC2)/cyclin B1 and the chaperone heat shock protein A2 (HSPA2) are required for the G2-->M transition in prophase I. Here, we demonstrate that in primary spermatocytes, linker histone chaperone testis/embryo form of nuclear autoantigenic sperm protein (tNASP) binds the heat shock protein HSPA2, which localizes on the synaptonemal complex of spermatocytes. Significantly, the tNASP-HSPA2 complex binds linker histones and CDC2, forming a larger complex. We demonstrate that increasing amounts of tNASP favor tNASP-HSPA2-CDC2 complex formation. Binding of linker histones to tNASP significantly increases HSPA2 ATPase activity and the capacity of tNASP to bind HSPA2 and CDC2, precluding CDC2/cyclin B1 complex formation and, consequently, decreasing CDC2/cyclin B1 kinase activity. Linker histone binding to NASP controls the ability of HSPA2 to activate CDC2 for CDC2/cyclin B1 complex formation; therefore, tNASP's role is to provide the functional link between linker histones and cell cycle progression during meiosis.

Asunto(s)

Autoantígenos/metabolismo , Proteína Quinasa CDC2/metabolismo , Ciclina B1/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas Nucleares/metabolismo , Espermatocitos/metabolismo , Complejo Sinaptonémico/metabolismo , Animales , Proteínas de Ciclo Celular , Histonas/metabolismo , Masculino , Meiosis , Ratones

RESUMEN

BACKGROUND: NASP (Nuclear Autoantigenic Sperm Protein) is a linker histone chaperone required for normal cell division. Changes in NASP expression significantly affect cell growth and development; loss of gene function results in embryonic lethality. However, the mechanism by which NASP exerts its effects in the cell cycle is not understood. To understand the pathways and networks that may involve NASP function, we evaluated gene expression in HeLa cells in which NASP was either overexpressed or depleted by siRNA. METHODS: Total RNA from HeLa cells overexpressing NASP or depleted of NASP by siRNA treatment was converted to cRNA with incorporation of Cy5-CTP (experimental samples), or Cy3-CTP (control samples). The labeled cRNA samples were hybridized to whole human genome microarrays (Agilent Technologies, Wilmington, Delaware, USA). Various gene expression analysis techniques were employed: Significance Analysis of Microarrays (SAM), Expression Analysis Systematic Explorer (EASE), and Ingenuity Pathways Analysis (IPA). RESULTS: From approximately 36 thousand genes present in a total human genome microarray, we identified a set of 47 up-regulated and 7 down-regulated genes as a result of NASP overexpression. Similarly we identified a set of 56 up-regulated and 71 down-regulated genes as a result of NASP siRNA treatment. Gene ontology, molecular network and canonical pathway analysis of NASP overexpression demonstrated that the most significant changes were in proteins participating in organismal injury, immune response, and cellular growth and cancer pathways (major "hubs": TNF, FOS, EGR1, NFkappaB, IRF7, STAT1, IL6). Depletion of NASP elicited the changed expression of proteins involved in DNA replication, repair and development, followed by reproductive system disease, and cancer and cell cycle pathways (major "hubs": E2F8, TP53, FGF, FSH, FST, hCG, NFkappaB, TRAF6). CONCLUSION: This study has demonstrated that NASP belongs to a network of genes and gene functions that are critical for cell survival. We have confirmed the previously reported interactions between NASP and HSP90, HSP70, histone H1, histone H3, and TRAF6. Overexpression and depletion of NASP identified overlapping networks that included TNF as a core protein, confirming that both high and low levels of NASP are detrimental to cell cycle progression. Networks with cancer-related functions had the highest significance, however reproductive networks containing follistatin and FSH were also significantly affected, which confirmed NASP's important role in reproductive tissues. This study revealed that, despite some overlap, each response was associated with a unique gene signature and placed NASP in important cell regulatory networks.

Asunto(s)

Autoantígenos/genética , Ciclo Celular/fisiología , Cuello del Útero/citología , Cuello del Útero/fisiología , Perfilación de la Expresión Génica , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , División Celular/fisiología , Supervivencia Celular/fisiología , Femenino , Células HeLa , Humanos , ARN Interferente Pequeño

RESUMEN

Epididymal protease inhibitor (eppin [official symbol, SPINLW1]) is of interest as a male contraceptive target because of its specificity and location on the human sperm surface. We have examined the effect of anti-eppin antibodies from infertile male monkeys and the effect of recombinant human semenogelin on human sperm motility. Anti-eppin antibodies significantly decreased the progressive motility of human spermatozoa as measured by decreased total distance traveled, decreased straight-line distance, and decreased velocity. Anti-eppin treatment of spermatozoa significantly increased the amount of cAMP present in nonprogressive spermatozoa; however, approximately 25% of antibody-treated spermatozoa could be rescued by the addition of cAMP-acetoxymethyl ester, indicating that anti-eppin-treated spermatozoa have a compromised ability to utilize cAMP. Addition of recombinant human semenogelin has a concentration-dependent inhibitory effect on progressive motility (increased tortuosity and decreased velocity). We tested the hypothesis that anti-eppin antibodies bound to eppin would subsequently block semenogelin binding to eppin. Anti-eppin antibodies from infertile monkeys inhibited eppin from binding to semenogelin. Addition of affinity-purified antibodies made to the dominant C-terminal epitope of eppin had an inhibitory effect on progressive motility (increased tortuosity, decreased velocity, and straight distance). Our results suggest that the eppin-semenogelin binding site is critical for the removal of semenogelin in vivo during semen liquefaction and for the initiation of progressive motility. We conclude that the eppin-semenogelin binding site on the surface of human spermatozoa is an ideal target for a nonsteroidal male contraceptive.

Asunto(s)

Anticuerpos/farmacología , Anticonceptivos Masculinos/farmacología , AMP Cíclico/farmacología , Infertilidad Masculina/inmunología , Proteínas Inhibidoras de Proteinasas Secretoras/inmunología , Motilidad Espermática/efectos de los fármacos , Animales , Anticuerpos/aislamiento & purificación , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Haplorrinos/inmunología , Humanos , Masculino , Unión Proteica/efectos de los fármacos , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Proteínas de Secreción de la Vesícula Seminal/metabolismo , Motilidad Espermática/inmunología

RESUMEN

Eppin (SPINLW1; serine peptidase inhibitor-like with Kunitz and WAP domains 1 (eppin); epididymal protease inhibitor) coats the surface of human ejaculate spermatozoa and originates from Sertoli and epididymal epithelial cells. In this study, we have isolated native eppin from ejaculate supernatants (seminal plasma) and washed ejaculate spermatozoa using column chromatography and two-dimensional SDS-PAGE, and identified by mass spectrometry and Western blots an eppin protein complex (EPC) containing lactotransferrin (LTF; also known as lactoferrin), clusterin (CLU), and semenogelin (SEMG1). To confirm the association of eppin with LTF, CLU, and SEMG1, antibodies to CLU and LTF were used to immunoprecipitate CLU and LTF from human sperm lysates. In both cases identical results were obtained, namely, the immunoprecipitate of the EPC. Additionally, we localized eppin, LTF, and CLU in human Sertoli cells and on human testicular and ejaculate spermatozoa, implying that the EPC is present on spermatozoa from the time they leave the seminiferous tubule. On ejaculate spermatozoa eppin, LTF, and CLU colocalize on the tail. The identification of the EPC components suggests that LTF, CLU, and/or eppin receptors may function as sperm plasma membrane receptors for the EPC, implicating the complex as a central player in a network of protein-protein interactions on the human sperm surface. The EPC may provide a surface network with microbicidal properties that protects spermatozoa as well as regulates the sperm's transition to a motile, capacitated sperm.

Asunto(s)

Proteínas Inhibidoras de Proteinasas Secretoras/aislamiento & purificación , Semen/química , Espermatozoides/química , Western Blotting , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Clusterina/química , Clusterina/aislamiento & purificación , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunohistoquímica , Lactoferrina/química , Lactoferrina/aislamiento & purificación , Masculino , Proteínas Inhibidoras de Proteinasas Secretoras/química , Proteínas de Secreción de la Vesícula Seminal/química , Proteínas de Secreción de la Vesícula Seminal/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

A multichaperone nucleosome-remodeling complex that contains the H1 linker histone chaperone nuclear autoantigenic sperm protein (NASP) has recently been described. Linker histones (H1) are required for the proper completion of normal development, and NASP transports H1 histones into nuclei and exchanges H1 histones with DNA. Consequently, we investigated whether NASP is required for normal cell cycle progression and development. We now report that without sufficient NASP, HeLa cells and U2OS cells are unable to replicate their DNA and progress through the cell cycle and that the NASP(-/-) null mutation causes embryonic lethality. Although the null mutation NASP(-/-) caused embryonic lethality, null embryos survive until the blastocyst stage, which may be explained by the presence of stored NASP protein in the cytoplasm of oocytes. We conclude from this study that NASP and therefore the linker histones are key players in the assembly of chromatin after DNA replication.

Asunto(s)

Autoantígenos/genética , Autoantígenos/fisiología , Histonas/química , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Animales , Autoantígenos/metabolismo , Línea Celular Tumoral , Proliferación Celular , ADN/química , Femenino , Vectores Genéticos , Genotipo , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Proteínas Nucleares/metabolismo

RESUMEN

NASP (nuclear autoantigenic sperm protein) is a histone H1 binding protein expressed in all cells undergoing division. We have previously reported the sequence for the mouse NASP gene and analyzed its proximal promoter region in silico to determine putative regulatory regions. In this report we describe various factors regulating the transcription of NASP. Luciferase assays using 3T3 fibroblasts show that the region +9 to -135 nt (PR1C) provides the core transcriptional activity for NASP and that extending this region out to -976 nucleotides partially represses activity. However, when luciferase reporter assays were done in transfected pachytene spermatocytes, the cells that exhibit the highest NASP expression, a different gene regulation picture was revealed. In spermatogenic cells, PR1C is still a relatively strong core promoter, but unlike 3T3 cells, if the construct is extended to -3002 nucleotides there is marked enhancement of transcription. Electrophoretic mobility shift assays with 3T3 nuclear extracts were used to study the PR1C core promoter in greater detail. In the region immediately upstream of the transcription initiation site we identified two closely associated Sp1 binding sites and a binding site for an Ets family member. Supershift assays further confirmed the presence of Sp1 bound to their respective sites suggesting that Sp1 and Ets are the primary activators of the NASP promoter.

Asunto(s)

Autoantígenos/biosíntesis , Regulación de la Expresión Génica/fisiología , Proteínas Nucleares/biosíntesis , Regiones Promotoras Genéticas/fisiología , Espermatocitos/fisiología , Células 3T3 , Animales , Autoantígenos/genética , Proteínas de Ciclo Celular , Masculino , Ratones , Proteínas Nucleares/genética , Unión Proteica/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Espermatocitos/citología

RESUMEN

Nuclear autoantigenic sperm protein (NASP) is a linker histone binding protein that is cell-cycle regulated. Synchronized HeLa cells are delayed in progression through the G1/S border when transiently transfected to overexpress full-length NASP, but not the histone-binding site (HBS) deletion mutant (NASP-DeltaHBS). The purpose of the current study was to identify possible NASP-associated proteins in HeLa cell nuclei that could elucidate NASP's influence on the cell cycle and chromatin remodeling. For this purpose, we employed a new approach: mass spectrometry identification of initially cross-linked proteins after their separation in a second dimension by reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Of the twelve proteins identified, three appear to be relevant to NASP's function: heat shock protein 90 (HSP90), DNA-activated protein kinase, and ATP-dependent DNA helicase II (70-kDa subunit). Individual protein-protein interactions were tested by immunoprecipitation techniques. This new method can be used for expedited identification of binding partners of different proteins in enriched fractions and as a complementary or alternative strategy to the yeast two-hybrid system and immunoprecipitation methods.

Asunto(s)

Autoantígenos/química , Autoantígenos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Western Blotting , Reactivos de Enlaces Cruzados , Electroforesis en Gel Bidimensional , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Masculino , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Succinimidas