Construction and Preliminary Characterization of a Series of Mouse and Rat Testis cDNA Libraries

We have constructed a series of 23 cDNA libraries from mouse and rat testicular cells. These include libraries made from whole, intact adult testes; seminiferous tubule cells from adult testes; combined populations of primary spermatocytes from 18‐day‐old mouse testes; and isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene spermatocytes, leptotene plus zygotene spermatocytes, juvenile pachytene spermatocytes, adult pachytene spermatocytes, round spermatids, Sertoli cells from 6‐, 8‐, 17‐, and 18–20‐day‐old mice, and peritubular cells from 18–20 day old mice, all recovered from outbred white Swiss (CD‐1) mice. We also constructed libraries from whole adult testes from five other lines of mice: C57 BI6/J, C3 HEB, BDF‐1, Balb/c, and 129 Sv. Finally, there are two libraries made from populations of Sertoli cells and peritubular cells isolated from testes of 20‐day‐old Sprague‐Dawley rats. Enzymatic dissociation, followed by gradient separation or plating/lysing techniques, was used to prepare populations of specific cell types in purities of 85–98%. cDNAs were synthesized from poly A+ mRNA primed with oligo dT and unidirectionally cloned into the lambda Uni‐Zap XR expression vector from Stratagene. Primary titers ranged from 2.1 ± 105 to 2.9 × 108 plaque‐forming units, and insert sizes averaged 1.0–1.2 kb. These libraries have been amplified once and submitted to the American Type Culture Collection (ATCC) for distribution to interested investigators. ATCC accession numbers are provided

Developmental origins of transgenerational sperm histone retention following ancestral exposures

Numerous environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of disease and phenotypic variation. Alterations in the germline epigenome are necessary to transmit transgenerational phenotypes. In previous studies, the pesticide DDT (dichlorodiphenyltrichloroethane) and the agricultural fungicide vinclozolin were shown to promote the transgenerational inheritance of sperm differential DNA methylation regions, non-coding RNAs and histone retention, which are termed epimutations. These epimutations are able to mediate this epigenetic inheritance of disease and phenotypic variation. The current study was designed to investigate the developmental origins of the transgenerational differential histone retention sites (called DHRs) during gametogenesis of the sperm. Vinclozolin and DDT were independently used to promote the epigenetic transgenerational inheritance of these DHRs. Male control lineage, DDT lineage and vinclozolin lineage F3 generation rats were used to isolate round spermatids, caput epididymal spermatozoa, and caudal sperm. The DHRs distinguishing the control versus DDT lineage or vinclozolin lineage samples were determined at these three developmental stages. DHRs and a reproducible core of histone H3 retention sites were observed using an H3 chromatin immunoprecipitation-sequencing (ChIP-Seq) analysis in each of the germ cell populations. The chromosomal locations and genomic features of the DHRs were analyzed. A cascade of epigenetic histone retention site alterations was found to be initiated in the round spermatids and then further modified during epididymal sperm maturation. Observations show that in addition to alterations in sperm DNA methylation and ncRNA expression previously identified, the induction of differential histone retention sites (DHRs) in the later stages of spermatogenesis also occurs. This novel component of epigenetic programming during spermatogenesis can be environmentally altered and transmitted to subsequent generations through epigenetic transgenerational inheritance.•Environmental induction of new transgenerational sperm histone retention sites.•Identification of a developmental cascade of histone retention.•Potential role of novel sperm histone retention in epigenetic inheritance

Systematic variation in mRNA 3′-processing signals during mouse spermatogenesis

Gene expression and processing during mouse male germ cell maturation (spermatogenesis) is highly specialized. Previous reports have suggested that there is a high incidence of alternative 3′-processing in male germ cell mRNAs, including reduced usage of the canonical polyadenylation signal, AAUAAA. We used EST libraries generated from mouse testicular cells to identify 3′-processing sites used at various stages of spermatogenesis (spermatogonia, spermatocytes and round spermatids) and testicular somatic Sertoli cells. We assessed differences in 3′-processing characteristics in the testicular samples, compared to control sets of widely used 3′-processing sites. Using a new method for comparison of degenerate regulatory elements between sequence samples, we identified significant changes in the use of putative 3′-processing regulatory sequence elements in all spermatogenic cell types. In addition, we observed a trend towards truncated 3′-untranslated regions (3′-UTRs), with the most significant differences apparent in round spermatids. In contrast, Sertoli cells displayed a much smaller trend towards 3′-UTR truncation and no significant difference in 3′-processing regulatory sequences. Finally, we identified a number of genes encoding mRNAs that were specifically subject to alternative 3′-processing during meiosis and postmeiotic development. Our results highlight developmental differences in polyadenylation site choice and in the elements that likely control them during spermatogenesis

Widespread Over-Expression of the X Chromosome in Sterile F1 Hybrid Mice

The X chromosome often plays a central role in hybrid male sterility between species, but it is unclear if this reflects underlying regulatory incompatibilities. Here we combine phenotypic data with genome-wide expression data to directly associate aberrant expression patterns with hybrid male sterility between two species of mice. We used a reciprocal cross in which F1 males are sterile in one direction and fertile in the other direction, allowing us to associate expression differences with sterility rather than with other hybrid phenotypes. We found evidence of extensive over-expression of the X chromosome during spermatogenesis in sterile but not in fertile F1 hybrid males. Over-expression was most pronounced in genes that are normally expressed after meiosis, consistent with an X chromosome-wide disruption of expression during the later stages of spermatogenesis. This pattern was not a simple consequence of faster evolutionary divergence on the X chromosome, because X-linked expression was highly conserved between the two species. Thus, transcriptional regulation of the X chromosome during spermatogenesis appears particularly sensitive to evolutionary divergence between species. Overall, these data provide evidence for an underlying regulatory basis to reproductive isolation in house mice and underscore the importance of transcriptional regulation of the X chromosome to the evolution of hybrid male sterility

HSP70-binding protein HSPBP1 regulates chaperone expression at a posttranslational level and is essential for spermatogenesis

Molecular chaperones play key roles during growth, development, and stress survival. The ability to induce chaperone expression enables cells to cope with the accumulation of nonnative proteins under stress and complete developmental processes with an increased requirement for chaperone assistance. Here we generate and analyze transgenic mice that lack the cochaperone HSPBP1, a nucleotide-exchange factor of HSP70 proteins and inhibitor of chaperone-assisted protein degradation. Male HSPBP1(−/−) mice are sterile because of impaired meiosis and massive apoptosis of spermatocytes. HSPBP1 deficiency in testes strongly reduces the expression of the inducible, antiapoptotic HSP70 family members HSPA1L and HSPA2, the latter of which is essential for synaptonemal complex disassembly during meiosis. We demonstrate that HSPBP1 affects chaperone expression at a posttranslational level by inhibiting the ubiquitylation and proteasomal degradation of inducible HSP70 proteins. We further provide evidence that the cochaperone BAG2 contributes to HSP70 stabilization in tissues other than testes. Our findings reveal that chaperone expression is determined not only by regulated transcription, but also by controlled degradation, with degradation-inhibiting cochaperones exerting essential prosurvival functions

The murine testicular transcriptome: Characterizing gene expression in the testis during the progression of spermatogenesis

One of the most promising applications of microarrays is the study of changes in gene expression associated with the growth and development of mammalian tissues. The testis provides an excellent model to determine the ability of microarrays to effectively characterize the changes in gene expression as an organ develops from birth to adulthood. To this end, a developmental testis gene expression time course profiling the expression patterns of �36 000 transcripts on the Affymetrix MGU74v2 GeneChip platform at 11 distinct time points was created to gain a greater understanding of the molecular changes necessary for and elicited by the development of the testis. Additionally, gene expression profiles of isolated testicular cell types were created that can aid in the further characterization of the specific functional actions of each cell type in the testis. Statistical analysis of the data revealed 11 252 transcripts (9846 unique) expressed differentially in a significant manner. Subsequent cluster analysis produced five distinct expressional patterns within the time course. These patterns of expression are present at distinct chronological periods during testis development and often share similarities with cell-specific expression profiles. Analysis of cell-specific expression patterns produced unique and characteristic groups of transcripts that provide greater insight into the activities, biological and chronological, of testicular cell types during the progression of spermatogenesis. Further analysis of this time course can provide a distinct and more definitive view into the genes implicated, known and unknown, in the maturation, maintenance, and function of the testis and the integrated process of spermatogenesis. developmental biology, gene regulation, spermatogenesis, testi

Impact of Treatment Adherence Intervention on a Social Skills Program Targeting Criticism Behaviours

The present study investigates the effect of a cognitive-behaviollral treatment adherence intervention in the course of a criticism skills group training program. Self-monitoring, goal setting, corrective feedback, behavioural contract, procedures to enhance commitment and reinforcement were the techniques used in this intervention. Eighty-six 23-year-Dld volunteer university participants, 62.2% females who reported they had difficulty giving/receiving criticism, and who had a high fear of negative evaluation, were randomly assigned to the treatment condition with or without the adherence intervention or to a waiting list control condition. We found treatment program attendance increased and percentage dropouts reduced by a factor of approximately one half. People who worked on treatment adherence reported differentially improved cognitive antecedents (reduced criticism concerns) and consequences (higher selfesteem/ self-efficacy) and demonstrated better skills (videotape ratings of trained judges) in giving criticism to others. On the other hand, people in the adherence condition reported no differentially improved affective antecedents (fear of negative evaluation) nor cognitive/ affective consequences (self-esteem/self-efficacy) nor did any demonstrate differentially better skills in the way they received criticism. Our results were interpreted in terms of an optimal arousal/avoidance learning paradigm whereupon people's adherence activities facilitated arousal optimal to greater program attendance and outcome gains in the proactive task of giving but not the more reactive task of receiving criticism.La présente étude enquête sur l'effet de l'intervention d'adhérence du traitement cognitif- behavioral à l'intérieur d'un programme d'entrainement de groupe sur les habiletés à critiquer. L'auto-observation, l'établissement de buts, le feed-back correctif, les contrats comportementaux, les procédures pour soutenir l'engagement et le renforcement étaient des techniques utilisées lors de l'intervention. Quatre-vingt-six participants volontaires âgés de 23 ans, étudiant à l'université, dont parmi ceux-ci 62,2% des femmes avaient signalés avoir de la difficulté à donner/à recevoir une critique et qui avaient une grande crainte de recevoir une évaluation négative, ont été assignées à la condition de traitement avec ou sans l'intervention d'adhérence ou bien aune liste d'attente de condition de contrôle. Nous avons trouvé que la participation au programme de traitement augmentait et que le pourcentage des décrocheurs diminuait par un facteur d'approximativement la moitié. Les participants qui ont travaillé avec le traitement d'adhérence soulignaient une amélioration différentielle des antécédents cognitifs (diminution de l'inquiétude face aux critiques) et des conséquences (estime de soi plus élevée/autoefficacité) et démontraient de meilleures habiletés (évaluations sur magnétoscope de juges entraînés) à offrir une critique aux autres. De l'autre côté, les participants du groupe de condition d'adhérence ont rapporté aucune amélioration différentielle des antécédents affectifs (peur d'une évaluation négative) de même que des conséquences cognitives/affectives (estime de soi/auto-efficacité) et n'ont montré aucune amélioration différentielle des habiletés concernant la façon dont ils reçoivent une critique. Nos résultats ont été interprétés en termes d'un paradigme d'apprentissage d'éveil/d'évitement optimal sur quoi les activités d'adhérence des participants facilitaient l'éveil optimal à une plus grande présence au programmée! les gains dans la tâche proactive de donner mais pas la tâche plus réactive de recevoir une critique

Developmental alterations in DNA methylation during gametogenesis from primordial germ cells to sperm

Because epigenetics is a critical component for gene expression, the hypothesis was tested that DNA methylation alterations are dynamic and continually change throughout gametogenesis to generate the mature sperm. Developmental alterations and stage-specific DNA methylation during gametogenesis from primordial germ cells (PGCs) to mature sperm are investigated. Individual developmental stage germ cells were isolated and analyzed for differential DNA methylation regions (DMRs). The number of DMRs was highest in the first three comparisons with mature PGCs, prospermatogonia, and spermatogonia. The most statistically significant DMRs were present at all stages of development and had variations involving both increases or decreases in DNA methylation. DMR-associated genes were identified and correlated with gene functional categories, pathways, and cellular processes. Observations identified a dynamic cascade of epigenetic changes during development that is dramatic during the early developmental stages. Complex epigenetic alterations are required to regulate genome biology and gene expression during gametogenesis.[Display omitted]•A dynamic cascade of epigenetic change throughout gametogenesis from PGC to sperm•Most dramatic epigenetic alterations in PGC and spermatogenic stem cell stages•Different DNA methylation regions between and within stages were identified•Complex epigenetic alterations required for gene expression during gametogenesisEpigenetics; Stem cells research; Developmental biolog

Environmentally induced transgenerational epigenetic reprogramming of primordial germ cells and the subsequent germ line

A number of environmental factors (e.g. toxicants) have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. Transgenerational inheritance requires the germline transmission of altered epigenetic information between generations in the absence of direct environmental exposures. The primary periods for epigenetic programming of the germ line are those associated with primordial germ cell development and subsequent fetal germline development. The current study examined the actions of an agricultural fungicide vinclozolin on gestating female (F0 generation) progeny in regards to the primordial germ cell (PGC) epigenetic reprogramming of the F3 generation (i.e. great-grandchildren). The F3 generation germline transcriptome and epigenome (DNA methylation) were altered transgenerationally. Interestingly, disruptions in DNA methylation patterns and altered transcriptomes were distinct between germ cells at the onset of gonadal sex determination at embryonic day 13 (E13) and after cord formation in the testis at embryonic day 16 (E16). A larger number of DNA methylation abnormalities (epimutations) and transcriptional alterations were observed in the E13 germ cells than in the E16 germ cells. These observations indicate that altered transgenerational epigenetic reprogramming and function of the male germline is a component of vinclozolin induced epigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigenetic inheritance are provided