Deliverables Identification of a genetic association of the KIT, KITLG and PTEN genes with male infertility We have carried out genetic association studies in order to identify a possible role of the signalling pathway KIT/KITLG in male infertility. We selected the KIT, KITLG, PIK3R1, AKT1 and PTEN human genes involved in this specific pathway. The analyses of this genes revealed that the KIT, KITLG and PTEN genes could present genetic susceptibility factors affecting the male reproductive function in humans. We are currently analysing the joint effects of these genes under a multilocus approach. Positional cloning strategies for the identification of susceptibility factors within these genes have also been designed. Protocol to assess testicular damage by toxic agents - Average numbers of apoptotic germ cells per 100 tubule cross-sections. Types of germ cell studied: spermatogonia to assess effects on proliferating cells, spermatocytes to study effects on the meiotic process which is sensitive to changes in Sertoli cell function and spermatids which are particularly sensitive towards changes in temperature and hormone levels. - Seminiferous tubule diameter. The tubule diameter decreases when many cells enter apoptosis and when cells disappear by being sloughed off through the tubule lumen. - Abnormal epithelial morphology. Sometimes, germ cells proceed more slowly through their development or there are generations of germ cells missing. Also a diminished Sertoli cell function can cause the formation of vauoles in the epithelium. These abnormalities were assessed. - Diploid spermatids. Problems in spermatocyte development can lead to skipping of the first meiotic division and the formation of diploid spermatids easily recognizable by their large size. These 4 parameters in conjunction with testis weight gave a good impression of the effects of endocrine disrupters on testis development and spermatogenesis but will also be useful to assess effects of other possibly toxic compounds. Development of a DNA biobank of patients affected by male infertility and testicular cancer, and its corresponding population controls Neocodex is leading a multicentric project devoted to create a DNA bank from common diseases patients and samples from unrelated healthy controls representative from the Spanish general population. Once GENDISRUPT started, Neocodex began the recruitment of biologic samples from infertile patients and testicular tumours patients. All the samples were collected in anonymised fashion in private or public collaborating clinical entities. These referral centres are: - Centro Gutenberg (Málaga). - Hospital Universitario Virgen del Rocío (Sevilla) - Hospital General Universitario de Alicante (Alicante) - Fundació Puigvert (Barcelona) - Hospital La Paz (Madrid) All the infertile patients fulfil the following inclusion criteria: primary infertility, absence of any known cause of infertility, absence of radiotherapy or chemotherapy, azoospermia or severe oligozoospermia (sperm counts < 5 million spz/ml), absence of Y chromosome microdeletions according to Simoni et al., (1999). Written informed consent was obtained from all the patients and controls included in the present project. The Institutional Review Boards of referral centres and Neocodex S.L. approved this protocol. Deoxyribonucleic acid banking and genetic tests included in this study have complied with the international regulations regarding the collection, treatment, storage and use of genetic data (International Bioethics Committee, UNESCO SHS-503/2001/CIB-8/3). Sample type: Available number Germinal DNA from severe oligozoospermic or azoospermic males: 167 DNA from control population: 1000 DNA from testicular cancer patients: 101 Fresh tissues from sterile males (testes): 7 Tissue from males affected of testicular carcinoma: 100. Generation of targeted microarrays for ED assays Our experiments provided a system by which small ologonucleotides from specific genes could be bound to glass surfaces containing a three dimensional matrix. These gene sequences could be used to investigate the effect of endocrine disruption. It is viewed that one of the effects of ED may ell be a change in gene expression, by measuring the effect in different tissues in response to a range of potential EDs than a diagnostic could be made. Such slides, as well as the technology to produce similar arrays and to read arrays could have wide ranging uses. Instead of studying single genes multiple gene families can give an indication of exposure and by accumulating many samples, can also give an indication of mechanism. For Genetix the work is important as it can validate the use of array and scanner technology and maintain the R and D in this highly competitive area as well as sustaining and providing new jobs. Effect of endocrine disruptors on primordial germ cell and fetal testis development The present results represent the first indication that exposure to estrogens during embryonic life may have profound effect on PGC growth and differentiation through they action on gonadal somatic cells. Evidence is provided that in association with genetic background and conditions (i.e. KL/c-Kit mutations, PTEN inactivation) favouring increased and/or prolonged PGC proliferation and the availability of factors affecting PGC differentiation, embryo exposure to high levels of estrogens or EDs constitutes a risk for PGC transformation in tumorigenic cells. This process can be at the origin of germ cell tumour formation in the testis and in other extragonadal sites where PGCs can misallocated. This information can be used as basis for genetic studies in humans focused on the expression of polymorphism for KIT/KIL and Akt/PTEN in male infertility and testicular tumours. The in vitro assay devised on fetal testis cells represents a novel test useful for rapid estrogenic and anti-estrogenic activity screening and to investigate the molecular pathways underlying the effect of these compounds on testis development. Focused microarray analyses started in the present project offer precious clues for the identification of crucial molecular pathways of germ and testis somatic cell development that can be deregulated by exposure to environmental EDs. Definition of gene expression signatures of effects of Endocrine Disruptors in testis development. Molecular markers. Designed of specific DNA arrays An Endocrine Disruptor (ED) is an exogenous substance that causes adverse health effect in an intact organism, or its progeny, secondary to changes in endocrine functions. The reproductive systems have been considered a crucial target of EDs generating gene deregulation. Consequently, the analysis of the genetic effects of EDs on developing gonads has repercussions on the effects upon the individuals and the progeny. By gene-expression microarrays, the effects of four different EDs: lindane, bisphenol-A, monoester of ethylhexyl phthalate and zearalenone and estradiol, upon testis development were investigated. Three different dosages for each compound were supplied with the drinking water to mice during three precise periods of development: - To the mothers before mating; - Same dosages maintained during pregnancy; and - Maintained four weeks after birth. The mouse transcriptome were analysed. Comparative analysis of all data defined gene expression signatures. Most of the EDs analysed in this study have a distinctive pattern of gene deregulation from the Estradiol. Phthalate and zearalenone showed defined pattern. After validation of data obtained from microarray analysis, selection of specific genes can be processed to build a specific GENDISRUPT microarray that could be patented and used in the analysis in animal models of suspected EDs. Identification of genetic interactions between genes involved in the synthesis or actions of estrogens in human male infertility We have analysed five genes (ESR1, ESR2, FSHR, CYP19A1 and NRIP1) in human male infertility under a multilocus approach. We selected a Single Nucleotide Polymorphism (SNP marker) for each candidate gene. The statistical analyses were carried out by using three different software: SUMSTAT, Permutation and Model Free analyses (PM) and Estimating haplotypes (EH) developed by Dr. Ott. The results of the analyses revealed the polygenic nature of human male infertility, and the genetic interaction of these five genes in this phenotype. A manual search for genotype combinations revealed two protective and a risk pattern for male infertility in our population. This result has been recently published (galan et al., 2005). Molecular analysis of the human ESR1 gene in male infertility. Implication of the ESR1 gene in male infertility In a previous genetic analysis, we detected a genetic association of a genetic variant within the 5´ region of the ESR1 gene with male infertility. These results indicate the putative presence of a mutation or a genomic rearrangement within the ESR1 locus. To identify the possible mutation, we designed and analysed a high density panel of markers within the ESR1 gene. This panel is comprised of 14 SNP markers along the genomic region of the ESR1 gene. The results derived from the marker panel analyses firmly support the existence of this putative mutation. We are currently performing the positional cloning of this mutation by increasing the density of the marker panel. Once we have identified the mutation, a testing protocol to detect its presence in human samples will be developed, and with this protocols different series of patients affected by estrogen related diseases will be analysed. Morphological effects of intra-uterine exposure to endocrine disrupters on testis development Female mice were given various endocrine disrupters or estradiol in the drinking water from 14 days before conception up till birth of the young and subsequently mother and young were given the compounds up till 4 weeks of age of the youing. Three different treatment periods were employed, up till conception, up till birth and up till 28 days after birth. MEHP (100 _M, 500 _M, 1 mM) and Zearalenone (4 mg/l, 12 mg/l, 20 mg/l) did not cause consistent, dose dependent effects. However, Bisphenol A (0,4 _g/l, 500 _g/l, 50mg/l, 200mg/l), Lindane (50mg/l, 100mg/l, 200mg/l) and estradiol (20 _g/l, 40 _g/l, 0,16mg/l) all caused increased numbers of apoptotic cells after the longest treatment but also when the treatment was stopped at birth indicating longterm effects. After treatment with Lindane and estradiol germ cell apoptosis caused a significant shrinkage of the tubule diameter after the longest exposure period. Finally, bisphenol A and lindane both caused a doubling of the number of diploid spermatids after the longest treatment period and the highest dose and estradiol even after all three doses. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
