Éditeur(s) : HAL CCSD Society for the Study of Reproduction
Résumé : International audience
Spermmotility notably depends on the structural integrity of the flagellum and the regulation ofmicrotubule dynamics. Although researchers have started to use " omics" techniques to characterize the human sperm's molecular landscape, the constituents responsible for the assembly, organization, and dynamics of the flagellum microtubule have yet to be fully defined. In this study, we defined a core set of 116 gene products associated with the human sperm microtubulome (including products potentially involved in abnormal ciliary phenotypes and male infertility disorders). To this end, we designed and applied an integrated genomics workflow and combined relevant proteomics, transcriptomics, and interactomics datasets to reconstruct a dynamic interactome map. By further integrating phenotypic information, we defined a disease-interaction network; this enabled us to highlight a number of novel factors potentially associated with altered sperm motility and male fertility. Lastly, we experimentally validated the expression pattern of two candidate genes (CUL3 and DCDC2C) that had never previously been associated with male germline differentiation. Our analysis suggested that CUL3 and DCDC2C's products have important roles in the sperm flagellum. Taken as a whole, our results demonstrate that an integrated genomics strategy can highlight relevant molecular factors in specific sperm components. This approach could be easily extended by including other " omics" data (from asthenozoospermic men, for example) and identifying other critical proteins from the human sperm microtubulome. Summary Sentence Although researchers have started to use "omics" techniques to characterize the human sperm's molecular landscape, the constituents responsible for the assembly, organization, and dynamics of the flagellum microtubule have yet to be fully defined.
ISSN: 0006-3363

hal-01515563
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01515563
DOI : 10.1095/biolreprod.116.143479
PUBMED : 28395323

Éditeur(s) : HAL CCSD
Résumé : International audience
STUDY QUESTION: Do freezing and in vitro culture procedures enhance the expression of proteins involved in apoptotic or autophagic pathways in murine pre-pubertal testicular tissue? SUMMARY ANSWER: IVM strongly modified apoptosis- and autophagy-related relative protein levels in mice testicular tissue whereas the impact of cryopreservation procedures was minimal at the end of the culture. WHAT IS KNOWN ALREADY: In vitro spermatogenesis remains a challenging technical issue as it imposes to find a very close balance between survival and death of germ cell natural precursors (i.e. gonocytes and spermatogonia), which will eventually undergo a complete spermatogenesis close to in vivo conditions. The establishment of efficient culture conditions coupled with suitable cryopreservation procedures (e.g. controlled slow freezing [CSF] and solid surface vitrification [SSV]) of pre-pubertal testicular tissue is a crucial step in the fields of fertility preservation and restoration to improve the spermatic yield obtained in vitro. STUDY DESIGN, SIZE, DURATION: Here, we study cryopreservation procedures (i.e. CSF or SSV) and the impact of culture media compositions. A first set of 66 mouse pre-pubertal testes were directly cultured during 30, 36, 38 and 60 days (D) from 2.5 to 6.5-day-old CD-1 mice to evaluate the impact of time-aspect of culture and to endorse the reverse phase protein microarrays (RPPM) technique as an adapted experimental tool for the field of in vitro spermatogenesis. Ninety others fresh, slow-frozen and vitrified pre-pubertal testes were cultured during 30 days for the principal study to evaluate the impact of cryopreservation procedures before and after culture. Thirty-four testes dissected from 2.5, 6.5, 36.5, 40.5, 42.5 and 62.5 days postpartum (dpp) mice, corresponding to the time frames of spermatogenesis orchestrated in vitro, were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: After in vitro culture, testicular tissue samples originated from 2.5 or 6.5-day-old CD-1 male mice were analyzed using RPPM. This targeted proteomic technique allowed us to assess the expression level of 29 apoptosis- and autophagy-related factors by normalizing blank-corrected signal values. In addition, morphological analyses (e.g. HES, PAS, TRA98 and CREM) and DNA fragmentation in intra-tubular cells (i.e. terminal deoxynucleotidyl transferase dUTP nick end labeling; TUNEL) were assessed for the distinct experimental conditions tested as well as for in vivo control mouse testes. MAIN RESULTS AND THE ROLE OF CHANCE: A validation of the RPPM procedure in the field of in vitro spermatogenesis was completed with assay and array robustness before a principal study concerning the evaluation of the impact of in vitro culture and cryopreservation procedures. The proportion of elongated spermatids and the total cell number per seminiferous tubule tended to be very different between the in vivo and in vitro conditions (P < 0.05), suggesting the presence of a beneficial regulation on the first spermatogenesis wave by intrinsic apoptosis (Caspase₉) and autophagy (Atg5) factors (P < 0.0003 and r2 = 0.74). Concerning the impact of culture media compositions, a basic medium (BM) composed of αMEM plus 10% KnockOut™ serum replacement and gentamicin supplemented with retinol (Rol) and vitamin E (Vit. E) was selected as the best culture medium for fresh 6.5 dpp tissue cultured during 30D with 27.7 ± 8.10% of seminiferous tubules containing elongated spermatids. Concerning the impact of cryopreservation procedures, SSV did not have any impact on the morphological parameters evaluated after culture in comparison to fresh tissue (FT) controls. The proportion of tubules with elongated spermatids on testicular explants cultured with BMRol+Vit. E was not different between SSV (6.6 ± 1.6%) and CSF (5.3 ± 1.9%); however, round spermatids were observed more frequently for SSV (19 ± 6.2%) than CSF (3.3 ± 1.9%, P = 0.0317). Even if the proportion of TUNEL-positive cells for BMRol+Vit. E was higher at D30 after SSV (4.12 ± 0.26%) than CSF (1.86 ± 0.12%, P = 0.0022) and FT (2.69 ± 0.33%, P = 0.0108), the DNA damages observed at the end of the culture (i.e. D30) were similar to respective 6.5 dpp controls. In addition, the relative protein level expression ratio of an apoptotic factor, the phosphorylated FADD on Fas, was reduced by 64-fold in vitrified testes cultured with BMRol+Vit. E. Furthermore, we found in this study that the StemPro®-34 SFM culture medium supplemented with growth factors (e.g. EGF, bFGF, GDNF and LIF) prevented the differentiation of spermatogonial stem cells in favor of a significant proliferation with a better architectural pattern than in vivo 6.5 dpp controls with an increase of seminiferous tubules area for FT (P = 0.0357) and CSF (P = 0.0317). LIMITATIONS REASONS FOR CAUTION: Despite our promising results, the evaluation of apoptotic- and autophagic-related proteins was studied for a limited amount of proteins and on global testicular tissue. WIDER IMPLICATIONS OF THE FINDINGS: The data presented herein will help to improve apoptotic and autophagic understanding during the first spermatogenic wave. Moreover, our findings illustrate for the first time that, using finely-tuned experimental conditions, a testicular in vitro culture combined with proteomic technologies may significantly facilitate the study of cryopreservation procedures and in vitro culture evaluations. This study may also contribute to improve work on testicular tissues from pre-pubertal and adolescent cancer survivors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a Ph.D. grant from the Rouen Normandie Université and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Institute for Research and Innovation in Biomedicine (IRIB), Agence de la Biomédecine, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Regional Development Fund (ERDF). The authors declare that there is no conflict of interest.
Molecular Human Reproduction

hal-01647081
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01647081
DOI : 10.1093/molehr/gax054
PUBMED : 29040674

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
BACKGROUND: Evidence of human sexual transmission during Zika virus emergence is a matter of concern, particularly in procreation, but to date, kinetics of seminal shedding and the effects of infection on human reproductive function have not been described. To investigate the effects of Zika virus infection on semen and clearance of Zika virus from semen and body fluids, we aimed to study a cohort of Zika virus-infected men. METHODS: This prospective observational study recruited men presenting with acute Zika virus infection at Pointe-à-Pitre University Hospital in Guadeloupe, French Caribbean, where a Zika virus outbreak occurred between April and November, 2016. Blood, urine, and semen were collected at days 7, 11, 20, 30, 60, 90, and 120 after symptom onset, and semen characteristics, such as total sperm count, sperm motility, vitality, and morphology, and reproductive hormone concentrations, such as testosterone, inhibin, follicle-stimulating hormone, and luteinising hormone, were assessed. At days 7, 11, and 20, semen was processed to isolate motile spermatozoa. Zika virus RNA was detected by RT-PCR using whole blood, serum, urine, seminal plasma, semen cells, and motile spermatozoa fractions. Zika virus was isolated from different sperm fractions on Vero E6 cultures. FINDINGS: 15 male volunteers (mean age 35 years [SD 5; range 25-44) with acute Zika virus infection and positive Zika virus RNA detection in blood or urine were enrolled. Total sperm count was decreased from median 119 × 10(6) spermatozoa (IQR 22-234) at day 7 to 45·2 × 10(6) (16·5-89·6) at day 30 and 70 × 10(6) (28·5-81·4) at day 60, respectively, after Zika virus infection. Inhibin values increased from 93·5 pg/mL (IQR 55-162) at day 7 to 150 pg/mL (78-209) at day 120 when total sperm count recovered. In motile spermatozoa obtained after density gradient separation, Zika virus RNA was found in three of 14 patients at day 7, four of 15 at day 11, and four of 15 at day 20, and replication-competent virus was found in the tested patient. Seminal shedding kinetics seemed heterogeneous among patients. Whole blood was the fluid most frequently positive for Zika virus RNA (62 of 92 samples) and three patients remained positive at day 120. INTERPRETATION: Semen alterations early after acute Zika virus infection might affect fertility and could be explained by virus effects on the testis and epididymis. Frequency of shedding and high viral load in semen, together with the presence of replicative virus in a motile spermatozoa fraction, can lead to Zika virus transmission during sexual contact and assisted reproduction procedures. Whole blood seems to be the best specimen for Zika virus RNA detection, diagnosis, and follow-up. FUNDING: Agence de la Biomédecine/Agence Régionale de Santé de la Guadeloupe/Inserm-REACTing.
ISSN: 0140-6736

hal-01647078
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01647078
DOI : 10.1016/S1473-3099(17)30444-9
PUBMED : 28838639

Éditeur(s) : HAL CCSD Elsevier Masson
Résumé : National audience
Sperm and embryos cryopreservation is a commonly applied technique for several years. Recently authorized in France, vitrification tends to replace gradually the conventional technique of slow freezing, so upsetting the practices in the management of patients. It allows from now on the cryopreservation of oocytes and opens new perspectives in egg donation either still in fertility preservation. This review thus attempted to examine the contribution of vitrification in the freezing of oocytes and human embryos at various stages of development. If obviously vitrification appears as the current method of choice for the cryopreservation of oocytes as well as blastocysts, the results are less cut as regards embryos to early stages. No increase in adverse obstetric and perinatal outcomes in children conceived from vitrified oocytes or embryos is noted in the literature
ISSN: 0368-2315

hal-01282445
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01282445
DOI : 10.1016/j.jgyn.2015.02.015
PUBMED : 25869444

Éditeur(s) : HAL CCSD American Chemical Society
Résumé : International audience
The present study is a contribution to the "neXt50 challenge", a coordinated effort across C-HPP teams to identify the SO most tractable missing proteins (MPs) on each chromosome. We report the targeted search of 38 theoretically detectable MPs from chromosomes 2 and 14 in Triton X-100 soluble and insoluble sperm fractions from a total of 15 healthy donors. A targeted mass spectrometry-based strategy consisting of the development of LC-PRM assays (with heavy labeled synthetic peptides) targeting 92 proteotypic peptides of the 38 selected MPs was used. Out of the 38 selected MPs, 12 were identified with two or more peptides and 3 with one peptide after extensive SDS-PAGE fractionation of the two samples and with overall low-intensity signals. The PRM data are available via ProteomeXchange in PASSEL (PASS01013). Further validation by immunohistochemistry on human testes sections and cytochemistry on sperm smears was performed for eight MPs with antibodies available from the Human Protein Atlas. Deep analysis of human sperm still allows the validation of MPs and therefore contributes to the C-HPP worldwide effort. We anticipate that our results will be of interest to the reproductive biology community because an in-depth analysis of these MPs may identify potential new candidates in the context of human idiopathic infertilities.
ISSN: 1535-3893

hal-01681656
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01681656
DOI : 10.1021/acs.jproteome.7b00374

Éditeur(s) : HAL CCSD
Résumé : International audience
BACKGROUND: There is increasing evidence that reproductive abnormalities are increasing in frequency in both human population and among wild fauna. This increase is probably related to exposure to toxic contaminants in the environment. The use of sentinel species to raise alarms relating to human reproductive health has been strongly recommended. However, no simultaneous studies at the same site have been carried out in recent decades to evaluate the utility of wild animals for monitoring human reproductive disorders. We carried out a joint study in Guadeloupe assessing the reproductive function of workers exposed to pesticides in banana plantations and of male wild rats living in these plantations. METHODS: A cross-sectional study was performed to assess semen quality and reproductive hormones in banana workers and in men working in non-agricultural sectors. These reproductive parameters were also assessed in wild rats captured in the plantations and were compared with those in rats from areas not directly polluted by humans. RESULTS: No significant difference in sperm characteristics and/or hormones was found between workers exposed and not exposed to pesticide. By contrast, rats captured in the banana plantations had lower testosterone levels and gonadosomatic indices than control rats. CONCLUSION: Wild rats seem to be more sensitive than humans to the effects of pesticide exposure on reproductive health. We conclude that the concept of sentinel species must be carefully validated as the actual nature of exposure may varies between human and wild species as well as the vulnerable time period of exposure and various ecological factors.
Environmental health : a global access science source [electronic resource]

hal-00669505
https://hal.archives-ouvertes.fr/hal-00669505
DOI : 10.1186/1476-069X-7-40
PUBMED : 18667078

Éditeur(s) : HAL CCSD
Résumé : International audience
Among infertile couples, approximately half have to face with male infertility. In men with non-obstructive azoospermia, surgical retrieval testicular sperm extraction (TESE) of spermatozoa can be attempted, but with low success rates. A specific biomarker that could predict residual spermatogenesis would obviously be of interest before performing TESE. Thus, our aim was to identify biomarkers of residual spermatogenesis in seminal plasma of patients with non-obstructive azoospermia (NOA) using an isotope-coded protein label (ICPL)-based proteomic strategy coupled with conventional protein assay. This retrospective study was conducted in 40 men with NOA at the University Hospital of Nantes and Proteomics Core facility Biogenouest - Inserm U1085 - IRSET, Rennes, France. Comparative ICPL proteomic screening of frozen seminal plasma and correlation with TESE outcome allowed the identification of some differentially expressed proteins. Among them, lectin galactoside-binding, soluble 3 binding protein (LGALS3BP) expression was further confirmed using conventional protein assay, and its interest as a predictor of TESE outcome was then evaluated and compared with conventional clinical and hormonal markers of residual spermatogenesis. Among the 12 differentially expressed proteins identified with comparative ICPL proteomic strategy, seminal LGALS3BP expression was found to be significantly higher in men with successful TESE. Finally, comparative ICPL proteomic screening of seminal plasma appears to be a promising approach for the identification of biomarkers of residual spermatogenesis. LGALS3BP, associated with clinical and hormonal markers, could potentially be used as a predictive marker of successful TESE outcome in patients with NOA.
Andrology

inserm-00818514
http://www.hal.inserm.fr/inserm-00818514
DOI : 10.1111/j.2047-2927.2012.00059.x
PUBMED : 23427166

Éditeur(s) : HAL CCSD
Résumé : International audience
The Chromosome-Centric Human Proteome Project (C-HPP) aims to identify "missing" proteins in the neXtProt knowledgebase. We present an in-depth proteomics analysis of the human sperm proteome to identify testis-enriched missing proteins. Using protein extraction procedures and LC-MS/MS analysis, we detected 235 proteins (PE2-PE4) for which no previous evidence of protein expression was annotated. Through LC-MS/MS and LC-PRM analysis, data mining, and immunohistochemistry, we confirmed the expression of 206 missing proteins (PE2-PE4) in line with current HPP guidelines (version 2.0). Parallel reaction monitoring acquisition and sythetic heavy labeled peptides targeted 36 ≪one-hit wonder≫ candidates selected based on prior peptide spectrum match assessment. 24 were validated with additional predicted and specifically targeted peptides. Evidence was found for 16 more missing proteins using immunohistochemistry on human testis sections. The expression pattern for some of these proteins was specific to the testis, and they could possibly be valuable markers with fertility assessment applications. Strong evidence was also found of four "uncertain" proteins (PE5); their status should be re-examined. We show how using a range of sample preparation techniques combined with MS-based analysis, expert knowledge, and complementary antibody-based techniques can produce data of interest to the community. All MS/MS data are available via ProteomeXchange under identifier PXD003947. In addition to contributing to the C-HPP, we hope these data will stimulate continued exploration of the sperm proteome. © 2016 American Chemical Society.
Journal of Proteome Research

hal-01406074
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01406074
DOI : 10.1021/acs.jproteome.6b00400
PUBMED : 27444420