Identification of putative drug targets for human sperm-egg interaction defect using protein network approach

Background: Sperm-egg interaction defect is a significant cause of in-vitro fertilization failure for infertile cases. Numerous molecular interactions in the form of protein-protein interactions mediate the sperm-egg membrane interaction process. Recent studies have demonstrated that in addition to experimental techniques, computational methods, namely protein interaction network approach, can address protein-protein interactions between human sperm and egg. Up to now, no drugs have been detected to treat sperm-egg interaction disorder, and the initial step in drug discovery research is finding out essential proteins or drug targets for a biological process. The main purpose of this study is to identify putative drug targets for human sperm-egg interaction deficiency and consider if the detected essential proteins are targets for any known drugs using protein-protein interaction network and ingenuity pathway analysis. Results: We have created human sperm-egg protein interaction networks with high confidence, including 106 nodes and 415 interactions. Through topological analysis of the network with calculation of some metrics, such as connectivity and betweenness centrality, we have identified 13 essential proteins as putative drug targets. The potential drug targets are from integrins, fibronectins, epidermal growth factor receptors, collagens and tetraspanins protein families. We evaluated these targets by ingenuity pathway analysis, and the known drugs for the targets have been detected, and the possible effective role of the drugs on sperm-egg interaction defect has been considered. These results showed that the drugs ocriplasmin (Jetrea (c)), gefitinib (Iressa (c)), erlotinib hydrochloride (Tarceva (c)), clingitide, cetuximab (Erbitux (c)) and panitumumab (Vectibix (c)) are possible candidates for efficacy testing for the treatment of sperm-egg interaction deficiency. Further experimental validation can be carried out to confirm these results. Conclusion: We have identified the first potential list of drug targets for human sperm-egg interaction defect using the protein interaction network approach. The essential proteins or potential drug targets were found using topological analysis of the protein network. These putative targets are promising for further experimental validation. These study results, if validated, may develop drug discovery techniques for sperm-egg interaction defect and also improve assisted reproductive technologies to avoid in-vitro fertilization failure

Systematic analysis of protein interaction network associated with azoospermia

Non-obstructive azoospermia is a severe infertility factor. Currently, the etiology of this condition remains elusive with several possible molecular pathway disruptions identified in the post-meiotic spermatozoa. In the presented study, in order to identify all possible candidate genes associated with azoospermia and to map their relationship, we present the first protein-protein interaction network related to azoospermia and analyze the complex effects of the related genes systematically. Using Online Mendelian Inheritance in Man, the Human Protein Reference Database and Cytoscape, we created a novel network consisting of 209 protein nodes and 737 interactions. Mathematical analysis identified three proteins, ar, dazap2, and esr1, as hub nodes and a bottleneck protein within the network. We also identified new candidate genes, CREBBP and BCAR1, which may play a role in azoospermia. The gene ontology analysis suggests a genetic link between azoospermia and liver disease. The KEGG analysis also showed 45 statistically important pathways with 31 proteins associated with colorectal, pancreatic, chronic myeloid leukemia and prostate cancer. Two new genes and associated diseases are promising for further experimental validation

Genetic control of cell fate specification in Caenorhabditis elegans germline.

The precise regulation of germ cell fates (sperm or oocyte) lies at the heart of reproduction and fertility. The nematode Caenorhabditis elegans hermaphrodites produce a discrete number of sperm during larval development and then switch to produce oocyte during adulthood. A number of positive (e.g., fbf genes) and negative (e.g., gld-3) regulators are important for this switch. Here, we found that aberrant activation of MPK-1 (an ERK homolog) by removal of both fbf-1 and lip-1 partially inhibits sperm-oocyte switch, resulting in Mog (masculinization of germline) sterility. The fbf-1 gene encodes a conserved PUF (Pumilio and FBF) RNA-binding protein and the lip-1 gene encodes an MPK-1/ERK phosphatase. Notably, inhibition of MPK-1/ERK signaling by either genetic mutation or chemical inhibition reprograms the germ cell fate and thus helps in regaining the fertility. We also found that fbf-1; lip-1 Mog sterility was enhanced by the depletion of G2/M cell cycle regulators, including CYB-3/Cyclin B, CDK-1/CDK1, and CDC-25.1/CDC25. Markedly, cdc-25.1 mRNA is a direct target of FBF-1. These results suggest that FBF-1 and LIP-1 may promote sperm-oocyte switch by activating MPK-1/ERK signaling and G2/M cell cycle progression.M.S

Xenopus DPPA2 is a Direct Inhibitor of Microtubule Polymerization Required for Nuclear Assembly

The eukaryotic nucleus mediates the genomic functions of information storage and gene expression, but must be completely rebuilt after every open mitosis as well as during fertilization. Nuclear abnormalities are observed in many tissue malignancies and congenital disorders, but the causes and effects of such pathologies remain poorly understood. Here we use cell-free Xenopus egg extracts to investigate the contribution of the DNA-binding protein Dppa2 to nuclear assembly. In Dppa2-depleted extracts, nuclei are small and deformed, assemble incomplete nuclear envelopes and fail DNA replication. We show that Dppa2 directly depolymerizes microtubules, and must be localized on DNA to tune local microtubule dynamics for proper nuclear assembly. In the absence of Dppa2, excess microtubules compromise nuclear shape, while ectopically abolishing microtubules retards nuclear expansion. Dppa2 can be bypassed with an appropriate dose oDppa2 nocodazole, but only during a narrow, defined time window. This reveals strict spatial and temporal demands for regulated microtubule dynamics during nuclear formation. We further study the effect of Dppa2 on dynamic microtubule growth reconstituted with purified tubulin, and find that Dppa2 stimulates microtubule catastrophe. This effect is dependent on GTP hydrolysis, and we explore the possibility that Dppa2 activates the intrinsic GTPase activity of microtubule polymers

GEMC1, a novel factor required for chromosomal DNA replication

In eukaryotic cells DNA replication begins from multiple origins. During the process of initiation, the DNA replication fork is established at each origin. In lower eukaryotes many factors required for chromosomal DNA replication have been identified. However, the regulation of DNA replication in complex multi-cellular organisms is still poorly understood. In this thesis I report the identification of GEMC1 (GEMinin Coiled-coil containing protein 1), a novel vertebrate factor belonging to a new protein family required to initiate chromosomal DNA replication. GEMC1 contains a domain similar to Geminin, a fundamental regulator of DNA replication (McGarry and Kirschner, 1998). GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. I show that Xenopus GEMC1 (XlGEMC1) binds the checkpoint and replication factor TopBP1, which promotes XlGEMC1 binding to chromatin during pre-replication complex (pre-RC) assembly. Moreover, I demonstrate that XlGEMC1 directly interacts with the replication factors Cdc45 and Cdk2/CyclinE by which it is heavily phosphorylated. Phosphorylated XlGEMC1 stimulates initiation of DNA replication. Inhibition of XlGEMC1 function with XlGEMC1 neutralizing antibodies prevents DNA replication onset by blocking Cdc45 loading onto chromatin. Inhibition of XlGEMC1 expression by morpholino antisense oligos is lethal for embryonic development. Furthermore, down-regulation of mouse GEMC1 (mGEMC1) expression by siRNA (small interfering RNA) oligos prevents initiation of DNA replication in somatic vertebrate cells. Data presented in this thesis suggest that GEMC1 promotes initiation of chromosomal DNA replication in higher eukaryotes by mediating TopBP1 and Cdk2 dependent Cdc45 recruitment onto replication origins

Molecular mechanisms of centromere inheritance in Xenopus egg extract

Tesis doctoral inédita, leída en Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 29/10/2013The centromere is a specialized chromosomal region that provides the foundation for the assembly of the kinetochore, a macromolecular assembly that orchestrates chromosome segregation. In most organisms, centromeres are defined epigenetically by the presence of a histone H3 variant called Centromere Protein A (CENP-A). A group of sixteen proteins that co-immunoprecipitate with CENP-A and localize to centromeres, the Constitutive Centromere Associated Network or CCAN, participate in kinetochore assembly and function. Among them, CENP-C, CENP-T and CENP-W appear to be closest to CENP-A. The propagation and maintenance of this epigenetic mark from one generation to the next is essential for cell division. In this thesis we have used the Xenopus laevis egg cell-free system to explore these processes in molecular detail. Chromosomes assembled in vitro in these extracts build functional kinetochores and recapitulate most aspects of the CENP-A deposition process described for human cells. Using a combination of biochemistry and fluorescence microscopy we have dissected the contributions of CCAN proteins CENP-C, CENP-T and CENP-W to CENP-A deposition and kinetochore assembly. We found that CENP-C, CENP-T and CENP-W are recruited to centromeres at different times although the three of them are essential for full kinetochore assembly in mitosis. We also established that CENP-C is required for CENP-A loading whereas CENP-T and CENP-W are not. Biochemical characterization of these proteins in the soluble egg extract revealed known and novel interactions among the three proteins and with additional factors. In particular, CENP-C associates with CENP-W and at least three other proteins, HJURP, the CENP-A chaperone; M18BP1, a member of the Mis18 complex, and the chromatin remodeler FACT. While HJURP and M18BP1 had been previously shown to be essential for CENP-A deposition, we demonstrate here that FACT can be detected at centromeres in mitosis and is required for the loading of new CENP-A. We speculate that CENP-C is stored in the egg extract forming a macromolecular complex with many of the proteins implicated in CENP-A assembly in order to facilitate their recruitment to centromeres in the rapid embryonic cycles that follow fertilization.El centrómero es el locus cromosómico en el que se construye el cinetocoro, una estructura multiproteica que media la interacción entre los cromosomas y los microtúbulos del huso y que orquesta la segregación cromosómica. En la mayor parte de los organismos, los centrómeros están definidos epigenéticamente por la presencia de una variante de la histona H3 que se denomina CENP-A (de Centromere Protein A). Se ha descrito un grupo de dieciséis proteínas centroméricas que interaccionan con CENP-A, que reciben el nombre de CCAN (por Constitutive Centromere Associated Network), que son importantes para el correcto ensamblaje y funcionamiento del cinetocoro. Dentro de este grupo, CENP-C, CENP-T y CENP-W parecen estar más próximas a CENP-A. El mantenimiento de la marca epigenética del centrómero de una generación a la siguiente es crucial para una correcta segregación cromosómica. Con el objetivo de entender los mecanismos que regulan la incorporación de CENP-A a la cromatina centromérica y la formación del cinetocoro hemos empleado un sistema in vitro basado en extractos obtenidos de huevos de Xenopus laevis en los que es posible ensamblar cromosomas con cinetocoros funcionales. Mediante técnicas bioquímicas y microscopía de fluorescencia, en este trabajo nos hemos preguntado acerca de la interacción entre CENP-C, CENP-T y CENPW, su dinámica de asociación a cromatina, su contribución a la formación del cinetocoro y a la incorporación de nuevos nucleosomas CENP-A que ocurre en cada ciclo celular. Hemos observado que estas tres proteínas se localizan en el centrómero de los cromosomas obtenidos in vitro en distintos momentos del ciclo celular si bien las tres son esenciales para la formación del cinetocoro en mitosis. En tanto que CENP-C se requiere para la incorporación de CENPA, no es ese el caso de CENP-T y CENP-W. Curiosamente, CENP-C aparece en el extracto soluble asociada a HJURP, M18BP1 y el complejo remodelador de cromatina FACT. Estudios anteriores habían descrito el papel de HJURP y M18BP1 en la incorporación de CENP-A. Aquí demostramos por primera vez que FACT juega un papel fundamental en dicha incorporación, al menos en nuestro sistema experimental. Así pues, es posible que CENP-C se almacene en el citoplasma de los oocitos asociado a los principales factores encargados del ensamblaje de cromatina centromérica para guiarlos hacia el centrómero y facilitar este proceso en los primeros ciclos celulares que tienen lugar tras la fertilización

Role of ubiquitination in <em>Caenorhabditis elegans</em> development and transcription regulation during spermatogenesis

Regulation of gene function can be achieved through a variety of mechanisms. In this dissertation, I present the genetic and molecular characterization of two genes involved in two distinct mechanisms of control. Each gene was initially identified by its functional role in sperm development in the model organism Caenorhabditis elegans. The first gene, uba-1, is an essential enzyme involved in protein turnover through ubiquitin-mediated proteolysis. A temperature-sensitive allele, (uba-1)it129, was isolated in a classical genetic screen for mutations that cause sperm-specific sterility. The second gene, spe-44, encodes a putative transcription factor. Its identification by microarray screening for sperm-enriched genes led to the cytological analysis of the deletion allele spe-44(ok1400), by reverse genetics approach. it129 encodes a conditional allele of uba-1, the sole E1 ubiquitin-activating enzyme in C. elegans. E1 functions at the apex of the ubiquitin-mediated conjugation pathway, and its activity is necessary for all subsequent steps in the reaction. Ubiquitin is covalently conjugated to various target proteins. Poly-ubiquitination typically results in target protein degradation, which provides an essential mechanism for the dynamic control of protein levels. Homozygous mutants of uba-1(it129) manifest pleiotropic phenotypes, and include novel roles for ubiquitination in sperm fertility, control of body size, and sex-specific development. We propose a model whereby proteins normally targeted for proteasomal degradation instead persist in uba-1(it129ts) and impair critical cellular processes. The second gene, spe-44, was identified as a putative sperm gene regulator in C. elegans based on its up-regulated expression during spermatogenesis and its significant sequence homology to the DNA-binding SAND domain. Genetic analysis of a deletion allele of spe-44(1400) has revealed its functional role during sperm development. Cytological analysis of spe-44(ok1400) showed developmental arrest of spermatocytes prior to spermatid differentiation. spe-44 mRNA is expressed in a narrow spatial and temporal window, just prior to spermatocyte differentiation, consistent with its functional role during spermatogenesis. Future study will be directed to find putative targets of spe-44 and the mechanisms that regulate gene expression using microarray analysis and yeast-one hybrid screens. These studies will help to understand transcriptional regulatory aspects of spermatogenesis in C. elegans

Characterization of germ cell development in free-living and parasitic flatworms

Sexually reproducing organisms must ensure correct specification and maintenance of germ cells for species survival. Germ cells must be carefully protected from inappropriate differentiation while simultaneously maintaining their identity as highly specialized totipotent cells. Several germ cell-intrinsic mechanisms, including transcriptional and post-transcriptional regulation, are key to executing germ cell-specific processes. Study of molecules necessary for the proper specification, maintenance, and differentiation of germ cells can lead to a deeper understanding of mechanisms underlying totipotency, infertility, tumorigenesis, evolutionary development, and even pathogenesis. Here we study germ cells in the context of both free-living and parasitic flatworms. We use the freshwater planarian Schmidtea mediterranea to understand the functions of germ cell-specific genes crucial for the development and proliferation of early male germ cells. Additionally, we also characterize genes essential for the tremendous reproductive output of the parasite Schisosoma mansoni, a causative agent of the a major neglected tropical disease, schistosomiasis. It was recently shown that in S. mediterranea, a male germ cell-specific component of the ubiquitous Nuclear Factor-Y family of transcription factors, NF-YB, is necessary for testis maintenance. In Chapter 2, we characterize the cellular mechanism behind NF-YB-mediated maintenance of the male germline stem cells, known as spermatogonial stem cells, or SSCs. We demonstrate that NF-YB is not necessary for the specification of germ cells, but plays a role in the self-renewal and proliferation of SSCs. Furthermore, we show that other components of the NF-Y complex in S. mansoni are similarly necessary for the proliferation of male germ cells, indicating molecular and functional conservation of this family of transcription factors. Next, we characterize the planarian homologs of boule, which belongs to a family of RNA-binding proteins known to play important roles in human infertility. In Chapter 3, we adopt an evolutionary-developmental approach and show that unlike other invertebrate boule genes, one of the planarian boule paralogs is required for SSC maintenance. Intriguingly, this early germ cell function, thought to be specific to vertebrate representatives of this gene family, appears to have evolved independently and earlier than previously thought. Our results demonstrate that the planarian can be a tractable invertebrate model system for understanding the germ cell functions of genes that are conserved between planarians and vertebrates. Finally, in Chapter 4, we perform an unbiased transcriptomic screen to identify genes necessary for germ cell development in the parasite S. mansoni. The prodigious egg laying capacity of these parasites is the cause behindunderlies the morbidity of the disease schistosomiasis. Using a large-scale RNA interference-based functional approach, we identify genes functioning in different stages of schistosome male gametogenesis, as well as genes necessary for egg production in the mammalian host. Together, our studies uncover novel roles for numerous genes critical for germ cell development in free-living and parasitic flatworms

Proteínas do espermatozoide como alvos para contraceção e biomarcadores de fertilidade

Doutoramento em BiomedicinaO elevado número de gravidezes indesejadas a nível mundial (~41%) reflete a necessidade premente de novos métodos contracetivos. Para já, os contracetivos masculinos estão limitados ao preservativo, ao coito interrompido e à vasectomia dado que ainda não existem métodos farmacológicos disponíveis. Por outro lado, nos países desenvolvidos, 15% dos casais são inférteis e, em metade dos casos, as causas estão relacionadas com fatores masculinos, sendo a infertilidade idiopática o tipo mais comum de infertilidade masculina. Os principais objetivos deste trabalho consistiram em (1) identificar, caracterizar e modular alvos não-hormonais para a contraceção masculina; e (2) estabelecer um conjunto de biomarcadores para avaliar a fertilidade masculina. No que toca à identificação de alvos para a contraceção masculina, foi dada especial atenção a duas proteínas e aos seus interactors: a fosfoproteína fosfatase 1 (PPP1) e a proteína percursora amilóide (APP). Caracterizámos o interactoma da subunidade catalítica gama 2 da PPP1 (PPP1CC2), uma isoforma específica do testículo e espermatozoide. Demonstrando, pela primeira vez, a interação entre a PPP1CC2 e a A-kinase anchor protein 4 (AKAP4), uma proteína expressa especificamente no testículo essencial para a motilidade do espermatozoide, e o potencial desse complexo como alvo contracetivo. A motilidade do espermatozoide foi, então, modulada eficazmente recorrendo a cell penetrating peptides (CPPs) como sistemas de transporte intracelular de sequências peptídicas direcionadas para padrões únicos de interações proteicas. Um outro interactor da PPP1CC2, a several ankyrin repeat protein variant 2 (SARP2), foi também caracterizado em testículo e espermatozoide. Descrevemos ainda, o interactoma da APP em testículo e espermatozoide humano sendo que a nossa abordagem permitiu a identificação de novos interactores e o reconhecimento de interactores-chave na fertilidade masculina, particularmente na interação espermatozoide-oócito, o que representa um potencial mecanismo alvo para a contraceção masculina. Identificámos diversas proteínas sinalizadoras cuja atividade se correlaciona com parâmetros seminais distintos, tendo o potencial para integrar uma plataforma de diagnóstico que poderá ter várias aplicações: explicar situações de infertilidade idiopática; explicar o insucesso de técnicas de procriação medicamente assistida (PMA) ou abortos de repetição; auxiliar a escolha da técnica de PMA mais apropriada; avaliar a eficácia de intervenções médicas; e clarificar os mecanismos responsáveis pela deterioração da qualidade dos espermatozoides associada com a idade. Para além do potencial para integrar um painel de biomarcadores, essas proteínas permitiram o reconhecimento de vias de sinalização responsáveis por regular funções específicas do espermatozoide que podem ser utilizadas com fins terapêuticos.The large number of unintended pregnancies worldwide (~41%) highlights the need for new contraceptive methods. Still, male contraceptive methods are limited to condoms, withdrawal or vasectomy as, currently, no pharmaceutical contraceptive agents exist for men. In contrast, in developed countries, infertility affects 15% of couples attempting to conceive and in half of these cases the cause is related to male reproductive issues. Moreover, idiopathic infertility remains the most common type of male infertility. The main goals of this work were to (1) identify, characterize and modulate non-hormonal targets for male contraception; and (2) establish a biomarker “fingerprint” to assess male fertility. Concerning the identification of targets for male contraception, particular attention was given to two distinct proteins and its interacting partners: phosphoprotein phosphatase 1 (PPP1) and amyloid precursor protein (APP). We characterized the interactome of PPP1 catalytic subunit gamma 2 (PPP1CC2), a testis-enriched/sperm-specific PPP1 isoform, in human testis/spermatozoa. We demonstrated for the first time the interaction between PPP1CC2 and A-kinase anchor protein 4 (AKAP4), a testis-specific protein essential for sperm motility, in human spermatozoa and the potential of the complex as a contraceptive target. Sperm motility was then successfully modulated by specific protein complex disruption using cell-penetrating peptides (CPPs) as a drug intracellular delivery system. Herein, we demonstrated for the first time the potential of CPPs to deliver peptide sequences that target unique protein-protein interactions in spermatozoa. Another PPP1CC2 interacting protein – several ankyrin repeat protein variant 2 (SARP2) – was also characterized in testis and spermatozoa. Moreover, we provided the first report on APP interactome in human testis/spermatozoa and our approach allowed the identification of novel interactions and the recognition of key APP interacting proteins for male reproduction, particularly in spermoocyte interaction, which represent a potential mechanism for male contraception modulation. We identified several signaling proteins that showed a high degree of differential activity in spermatozoa samples with distinct seminal parameters and have the potential to integrate a diagnostic array, which may have several applications: explain idiopathic infertility, failure in assisted reproductive techniques (ART) or repeated abortion; choice of the appropriate ART; assess the efficacy of medical interventions; and clarify the mechanisms responsible for age-dependent declines in spermatozoa quality. Besides the potential to integrate a biomarker "fingerprint" to assess sperm quality, those proteins allowed the recognition of the signaling pathways accountable for regulating specific spermatozoa functions that by future modulation could serve for therapeutic proposes