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 BioScientifica
Résumé : International audience
The general rules established from mammalian species for the regulation of spermatogenesis by gonadotropins may not be fully relevant in fish. Particularly, Fsh is as potent as Lh to stimulate steroidogenesis and the Fsh receptor is expressed in Leydig cells. In seasonal breeders, Fsh is likely the major gonadotropin involved in spermatogenesis onset and Lh is required to support spermatogenesis progression and gamete release. However, the genes that relay the action of Fsh and Lh have been poorly investigated in fish. The present study was aimed at identifying gonadotropin-dependent genes expressed in the testis during fish puberty. We cultured pubertal trout testicular explants for 96 h, with or without gonadotropin, and analyzed transcriptome variations using microarrays. Fsh and Lh had similar effects on a large group of genes while other genes were preferentially regulated by one or the other gonadotropin. We showed that most of the responsive genes were expressed in somatic cells and exhibited relevant patterns during the seasonal reproductive cycle. Some genes preferentially modulated by Lh could be involved in testicular cell fate (pvrl1 and bty) or sperm maturation (ehmt2 and racgap1) and will deserve further examination. Besides Fsh's effects on the steroidogenic pathway, our study demonstrates that Fsh coordinates relevant stimulatory and inhibitory paracrine factors known to regulate early germ cell proliferation and differentiation. Some of these genes belong to major regulatory pathways including the Igf pathway (igf1b/igf3 and igfbp6), the Tgfb pathway (amh, inha, inhba, and fstl3), the Wnt pathway (wisp1), and pleiotrophin (mdka).
ISSN: 0952-5041

inserm-00812401
http://www.hal.inserm.fr/inserm-00812401
DOI : 10.1530/JME-12-0197
PUBMED : 23045716

Éditeur(s) : HAL CCSD John Wiley & Sons
Résumé : International audience
Kisspeptins are now considered key players in the neuroendocrine control of puberty and reproduction, at least in mammals. Most teleosts have two kiss genes, kiss1 and kiss2, but their sites of expression are still poorly documented. As a first step in investigating the role of kisspeptins in the European sea bass, a perciform fish, we studied the distribution of kiss1 and kiss2-expressing cells in the brain of males and females undergoing their first sexual maturation. Animals were examined at early and late in the reproductive season. We also examined the putative expression of estrogen receptors in kiss-expressing cells and, finally, we investigated whether kisspeptins are expressed in the pituitary gland. We show that kiss1-expressing cells were consistently detected in the habenula and, in mature males and females, in the rostral mediobasal hypothalamus. In both sexes, kiss2-expressing cells were consistently detected at the level of the preoptic area, but the main kiss2 mRNA-positive population was observed in the dorsal hypothalamus, above and under the lateral recess. No obvious sexual differences in kiss1 and kiss2 mRNA expression were detected. Additional studies based on confocal imaging clearly showed that most kiss1 mRNA-containing cells of the mediobasal hypothalamus strongly express ERα and slightly express ERβ2. At the pituitary level, both sexes exhibited kiss1 mRNA expression in most FSHβ-positive cells and never in LHβ-positive cells.
ISSN: 0092-7317

hal-00877326
https://hal.archives-ouvertes.fr/hal-00877326
PUBMED : 22886357
DOI : 10.1002/cne.23211

Éditeur(s) : HAL CCSD
Résumé : The ovary is not solely the unique source of oocytes necessary for fertilization but also a crucial conductor of endocrine regulations of the reproductive system. In the fetal ovary, oocytes grouped in clusters are surrounded by somatic pre-granulosa cells, altogether delineated by a basement membrane and constituting ovarian cords. The formation of the definitive ovarian functional units requires their fragmentation into follicles formed by a single oocyte surrounded by granulosa cells and delineated by a basement membrane. This partitioning occurs within a tiny period and is dependent on the correct timing of the meiotic process, on an apoptotic wave that specifically targets oocytes and the remodelling of the basement membrane. Follicle formation is a crucial event of ovarian development because the follicle stock formed at the end of this process is non-renewable. Therefore, any disturbance of this process can lead to reproductive abnormalities in adulthood. Recent data on endocrine disruptors displaying estrogenic activity have involved the fragile estrogenic homeostasis in the process. We aimed at better understanding how the endogen estrogen 17b-estradiol (E2) contributes to follicle formation. To assess it, Sprague-Dawley rats were treated with E2 at different doses during follicle formation, /i.e./ in the 3 days following birth. We show that although this treatment impairs the development of the hepatic and ovarian detoxification systems, the female neonate is able to increase its estrogen clearance capabilities. Nevertheless, E2 treatment within this critical period triggers a dose-dependent decrease of oocyte number per ovary. Regardless of the E2 dose, puberty is advanced. All treated females are at least transiently fertile, yet these reproductive capabilities rapidly decline. A high (10 µg/day) dose of E2 leads to a secondary infertility characterized by anovulation that probably result from a dysfunction of the whole reproductive tract. By contrast, a lower (0.1 µg/day) dose of E2, that does not significantly modify neonatal oocyte survival, leads to a progressive reproductive senescence likely due to ovarian failure. Long-term troubles may originate from precocious E2 impact on the ovary since it disturbs ovarian transcriptome and produces many lesions on ovarian cell DNA.
L'ovaire est au cœur de la physiologie de la reproduction féminine. Il est à la fois à l'origine des ovocytes nécessaires à la fécondation et acteur des régulations endocrines du système reproducteur. Les ovocytes de l'ovaire fœtal sont groupés en amas bordés de cellules somatiques et d'une membrane basale, formant les cordons ovariens. Ces cordons ovariens se fragmentent pour former les follicules ovariens: un ovocyte bordé de cellules somatiques et d'une membrane basale. Cette fragmentation se déroule sur une courte période (/in utero/ chez la femme et juste après la naissance chez les rongeurs) et fait intervenir une vague de mort cellulaire programmée touchant les ovocytes pendant leur méiose et un remodelage de la membrane basale. La formation des follicules est une période cruciale du développement ovarien puisque le stock de follicules formés à l'issue de ce processus est non renouvelable. Son altération peut donc être à l'origine de troubles de la fonction de reproduction chez le futur adulte. Il existe une fragile homéostasie œstrogénique en place au moment de la formation folliculaire: chez la femme, l'ovaire se développe /in utero/, sous l'influence des œstrogènes circulants maternels et la production ovarienne fœtale. Chez les rongeurs, les follicules se forment lors de la levée de l'imprégnation hormonale maternelle au moment de la naissance. Nous avons souhaité comprendre comment le 17b-œstradiol (E2), œstrogène endogène, pouvait contribuer à la mise en place des follicules. Pour cela, des rattes Sprague-Dawley ont été traitées pendant la période de formation des follicules avec de l'E2 à différentes doses entre 0.01 et 10 µg/jour. Nos travaux démontrent que le traitement de rattes par des œstrogènes entre leur naissance et 3 jours induit une réduction dose-dépendante de leur nombre d'ovocytes. Bien que le traitement entrave le développement normal du système de détoxication hépatique et ovarien, l'animal est capable d'accroître ses capacités d'élimination des œstrogènes. Ces capacités sont toutefois insuffisantes pour bloquer les effets du traitement. Quelle que soit la dose d'E2 utilisée (0.1 ou 10 µg/jour), la puberté est plus précoce chez les femelles exposées mais toutes sont fertiles, au moins transitoirement. Cependant le traitement induit une dégradation rapide des capacités reproductives. L'E2 modifie la dynamique folliculaire chez l'adulte, longtemps après l'arrêt du traitement. Si une forte dose d'E2 réduit la survie des ovocytes lors de la formation des follicules et conduit à une infertilité secondaire due à un dysfonctionnement général du tractus reproducteur, une dose faible n'affecte pas la survie des ovocytes à court terme mais conduit à une sénescence reproductive précoce vraisemblablement d'origine ovarienne. L'origine de ces troubles pourrait résider dans les effets précoces de l'exposition à E2 : cette molécule altère le transcriptome ovarien et induit de nombreuses lésions de l'ADN des cellules ovariennes.
https://tel.archives-ouvertes.fr/tel-00953657

NNT : 2013REN1S107
tel-00953657
https://tel.archives-ouvertes.fr/tel-00953657
https://tel.archives-ouvertes.fr/tel-00953657/document
https://tel.archives-ouvertes.fr/tel-00953657/file/CHALMEY_Clementine.pdf

Éditeur(s) : HAL CCSD Nature Publishing Group
Résumé : International audience
In cyclic females, FSH stimulates ovarian estradiol (E2) production and follicular growth up to the terminal stage. A transient elevation in circulating FSH and E2 levels occurs shortly after birth. But what could be the action of FSH on the ovary during this period, and in particular how it stimulates ovarian steroidogenesis without supporting terminal follicular maturation is intriguing. By experimentally manipulating FSH levels, we demonstrate in mice that the mid-infantile elevation in FSH is mandatory for E2 production by the immature ovary, but that it does not stimulate follicle growth. Importantly, FSH increases aromatase expression to stimulate E2 synthesis, however it becomes unable to induce cyclin D2, a major driver of granulosa cell proliferation. Besides, although FSH prematurely induces luteinizing hormone (LH) receptor expression in granulosa cells, LH pathway is not functional in these cells to induce their terminal differentiation. In line with these results, supplying infantile mice with a superovulation regimen exacerbates E2 production, but it does not stimulate the growth of follicles and it does not induce ovulation. Overall, our findings unveil a regulation whereby high postnatal FSH concentrations ensure the supply of E2 required for programming adult reproductive function without inducing follicular maturation before puberty.
ISSN: 2045-2322

hal-01516023
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01516023
DOI : 10.1038/srep46222
PUBMED : 28397811
PUBMEDCENTRAL : PMC5387682

Éditeur(s) : HAL CCSD
Résumé : International audience
In two swordtail species of the genus Xiphophorus, the onset of puberty has been shown to be modulated at the P locus by sequence polymorphism and gene copy-number variation affecting the type 4 melanocortin hormone receptor Mc4r. The system works through the interaction of two allelic types, one encoding wild type and the other dominant-negative receptors. We have analyzed the structure and evolution of the P locus in the platyfish Xiphophorus maculatus, where as many as nine alleles of P determining the onset of sexual maturity in males and females, fecundity in females, and adult size in males are located on both the X and Y chromosomes in a region linked to the master sex-determining locus. In this species, mc4r has been amplified to up to 10 copies on both the X and Y chromosomes through recent large serial duplications. Subsequently, mc4r paralogues have diverged considerably into many different subtypes. Certain copies have acquired new untranslated regions through genomic rearrangements, and transposable element insertions and other mutations have accumulated in promoter regions, possibly explaining observed deviations from the classical mc4r transcriptional pattern. In the mc4r-coding sequence, in-frame insertions and deletions as well as nonsense and missense mutations have generated a high diversity of Mc4r-predicted proteins. Most of these variants are expressed in embryos, adults, and/or tumors. Functional receptor characterization demonstrated major divergence in pharmacological behavior for Mc4r receptors encoded by different copies of platyfish mc4r, with differences in constitutive activity as well as binding and stimulation by hormones. The high degree of allelic and copy-number variation observed between individuals can explain the high level of polymorphism for sexual maturation, fecundity, and body size in the platyfish: multiple combinations of Mc4r variants with different biochemical properties might interact to modulate the melanocortin signaling that regulates the hypothalamus-pituitary-gonadal axis.
GENETICS

hal-01544912
https://hal.archives-ouvertes.fr/hal-01544912
DOI : 10.1534/genetics.113.155952

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
Growth charts are an essential clinical tool for evaluating a child's health and development. The current French reference curves, published in 1979, have recently been challenged by the 2006 World Health Organization (WHO) growth charts. To evaluate and compare the growth of French children who were born between 1981 and 2007, with the WHO growth charts and the French reference curves currently used. Anthropometric measurements from French children, who participated in 12 studies, were analyzed: 82,151 measurements were available for 27,257 children in different age groups, from birth to 18 years. We calculated and graphically compared mean z-scores based on the WHO and French curves, for height, weight and Body Mass Index (BMI) according to age and sex. The prevalence of overweight using the WHO, the French and International Obesity Task Force definitions were compared. Our population of children was on average 0.5 standard deviations taller than the French reference population, from the first month of life until puberty age. Mean z-scores for height, weight and BMI were closer to zero based on the WHO growth charts than on the French references from infancy until late adolescence, except during the first six months. These differences not related to breastfeeding rates. As expected, the prevalence of overweight depended on the reference used, and differences varied according to age. The WHO growth charts may be appropriate for monitoring growth of French children, as the growth patterns in our large population of French children were closer to the WHO growth charts than to the French reference curves, from 6 months onwards. However, there were some limitations in the use of these WHO growth charts, and further investigation is needed.
ISSN: 1932-6203

hal-01134193
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01134193
DOI : 10.1371/journal.pone.0120806
PUBMED : 25761138
PUBMEDCENTRAL : PMC4356547

Éditeur(s) : HAL CCSD
Résumé : International audience
Afro-­‐‑ Caribbean sprinters often reach high performance levels at an early age. Adolescence is a time of morphological and physiological changes. This study was designed to analyze the evolution in parameters of short sprint performance during adolescence in Afro-­‐‑ Caribbean boys, especially the stride number/body height ratio (SN/BH), which is at the interface of technical and morphological factors. Seventy-­‐‑ one 13-­‐‑ year-­‐‑ old boys performed vertical jumps and short sprint races. The races were filmed with a view to determine stride variables. Anthropometric parameters were also measured. The same tests were performed two years later. Body height and SN/BH were the main predictors of sprint performance. The delta of performance was principally explained by stride length and stride number. Although deterioration in technical parameters was expected, the parameters related to body size and stride length were the main sprint performance predictors rather than explosive force. These results could be useful in developing tests to detect sprint potential in youth.
Journal of Human Kinetics

hal-01163933
https://hal.univ-antilles.fr/hal-01163933

Éditeur(s) : HAL CCSD
Résumé : International audience
Afro-­ Caribbean sprinters often reach high performance levels at an early age. Adolescence is a time of morphological and physiological changes. This study was designed to analyze the evolution in parameters of short sprint performance during adolescence in Afro-­Caribbean boys, especially the stride number/body height ratio (SN/BH), which is at the interface of technical and morphological factors. Seventy-­‐‑ one 13-­‐‑ year-­‐‑ old boys performed vertical jumps and short sprint races. The races were filmed with a view to determine stride variables. Anthropometric parameters were also measured. The same tests were performed two years later. Body height and SN/BH were the main predictors of sprint performance. The delta of performance was principally explained by stride length and stride number. Although deterioration in technical parameters was expected, the parameters related to body size and stride length were the main sprint performance predictors rather than explosive force. These results could be useful in developing tests to detect sprint potential in youth.
Journal of Human Kinetics

hal-01143539
https://hal.univ-antilles.fr/hal-01143539

Éditeur(s) : HAL CCSD
Résumé : International audience
BACKGROUND: Worrying trends regarding human reproductive endpoints (e.g. semen quality, reproductive cancers) have been reported and there is growing circumstantial evidence for a possible causal link between these trends and exposure to endocrine disrupting chemicals (EDCs). However, there is a striking lack of human data to fill the current knowledge gaps. To answer the crucial questions raised on human reproductive health, there is an urgent need for a reproductive surveillance system to be shared across countries. METHODS: A multidisciplinary network named HUman Reproductive health and Global ENvironment Network (HURGENT) was created aiming at designing a European monitoring system for reproductive health indicators. Collaborative work allowed setting up the available knowledge to design such a system. Furthermore we conducted an overview of 23 potential indicators, based upon a weight of evidence (WoE) approach according to their potential relation with EDC exposure. RESULTS: The framework and purposes of the surveillance system are settled as well as the approach to select suitable reproductive indicators. The indicators found with the highest scores according to the WoE approach are prostate and breast cancer incidence, sex ratio, endometriosis and uterine fibroid incidence, indicators related to the testicular dysgenesis syndrome, precocious puberty incidence and reproductive hormone levels. CONCLUSION: Not only sentinel health endpoints, but also diseases with high burdens in public health are highlighted as prior indicators in the context of EDC exposure. Our work can serve as a basis to construct, as soon as possible, the first multi-country reproductive monitoring system
European Journal of Public Health

hal-01197348
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01197348
DOI : 10.1093/eurpub/ckv153
PUBMED : 26330492