RESUMEN
Exposure of the fetal testis to numerous individual environmental chemicals (ECs) is frequently associated with dysregulated development, leading to impaired adult reproductive competence. However, 'real-life' exposure involves complex mixtures of ECs. Here we test the consequences, for the male fetus, of exposing pregnant ewes to EC mixtures derived from pastures treated with biosolids fertiliser (processed human sewage). Fetal testes from continuously exposed ewes were either unaffected at day 80 or exhibited a reduced area of testis immunostained for CYP17A1 protein at day 140. Fetal testes from day 140 pregnant ewes that were exposed transiently for 80-day periods during early (0-80 days), mid (30-110 days), or late (60-140 days) pregnancy had fewer Sertoli cells and reduced testicular area stained for CYP17A1. Male fetuses from ewes exposed during late pregnancy also exhibited reduced fetal body, adrenal and testis mass, anogenital distance, and lowered testosterone; collectively indicative of an anti-androgenic effect. Exposure limited to early gestation induced more testis transcriptome changes than observed for continuously exposed day 140 fetuses. These data suggest that a short period of EC exposure does not allow sufficient time for the testis to adapt. Consequently, testicular transcriptomic changes induced during the first 80 days of gestation may equate with phenotypic effects observed at day 140. In contrast, relatively fewer changes in the testis transcriptome in fetuses exposed continuously to ECs throughout gestation are associated with less severe consequences. Unless corrected by or during puberty, these differential effects would predictably have adverse outcomes for adult testicular function and fertility.
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Oveja Doméstica , Testículo , Animales , Femenino , Feto , Humanos , Masculino , Embarazo , Aguas del Alcantarillado/efectos adversos , Ovinos , Testículo/metabolismo , Testosterona/metabolismoRESUMEN
Gene expression in meiotic cells in the testis is characterized by intense transcriptional activity and alternative splicing. These processes are mainly controlled by RNA-binding proteins expressed strongly in germ cells. Functional impairments in any of these proteins' functions can lead to defects in meiosis and thus severe male infertility. Here, we have identified a homozygous frameshift mutation (NM_014469.4:c.301dup; p.Ser101LysfsTer29) in the RNA-binding motif protein, X-linked like 2 (RBMXL2) gene in a man with an azoospermia due to meiotic arrest. As RBMXL2 is known to be crucial for safeguarding the meiotic transcriptome in mice testes, we hypothesized that this variant leads to cryptic splice site poisoning. To determine the variant's impact on spermatogenesis, we confirmed the absence of RBMXL2 protein in the patient's testis tissue and then evidenced abnormal expression of several spermatogenesis proteins (e.g. meiosis-specific with coiled-coil domain) known to be altered in rbmxl2 knock-out mice with meiotic arrest. Our results indicate that RBMXL2's function in spermatogenesis is conserved in mammals. We hypothesize that deleterious variant in the RBMXL2 gene can result in male infertility and complete meiotic arrest, due to the disruption of gene expression by cryptic splice site poisoning.
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Azoospermia , Infertilidad Masculina , Humanos , Ratones , Animales , Masculino , Sitios de Empalme de ARN/genética , Mutación del Sistema de Lectura , Azoospermia/inducido químicamente , Azoospermia/genética , Azoospermia/metabolismo , Meiosis/genética , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Espermatogénesis/genética , Testículo/metabolismo , Proteínas de Unión al ARN/genética , Mutación , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
In animals, research in the past two decades has demonstrated the strong involvement of the endocannabinoid system (ECS) in numerous steps of the reproductive process, including ovarian physiology. Reproductive lifespan is closely related to the number of nongrowing ovarian follicles, called ovarian reserve (OR), which is definitively established during foetal life. Thus, OR damage may lead to poor reproductive outcomes and a shortened reproductive lifespan. We investigated whether prenatal ECS modulation had an effect on the OR at different ages in the rat offspring. Four groups of gestating female rats (F0) were exposed to the CB1-/CB2-receptor agonist WIN55212 (0.5 mg/kg), the CB1R inverse agonist SR141716 (3 mg/kg) or Δ9THC (5 mg/kg) and were compared to negative control groups. OR was histologically assessed at different postnatal timepoints (F1 individuals): postnatal day (PND) 6, PND40 and PND90. At PND6, prenatal exposure had no effect on OR. In the young adult group (PND90) exposed during gestation to WIN55212, we observed a CB1R-mediated delayed OR decrease, which was reversed by prenatal CB1R blockade by SR141716. Conversely, after prenatal SR141716 exposure, we observed higher OR counts at PND90. RT-PCR experiments also showed that prenatal ECS modulation perturbed the mRNA levels of ECS enzymes and OR regulation genes. Our findings support the role of the ECS in OR regulation during the foetal life of rats and highlight the need for further studies to elucidate its precise role in OR physiology.
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Agonistas de Receptores de Cannabinoides/toxicidad , Dronabinol/toxicidad , Reserva Ovárica/efectos de los fármacos , Ovario/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal , Receptor Cannabinoide CB1/agonistas , Animales , Benzoxazinas/toxicidad , Antagonistas de Receptores de Cannabinoides/farmacología , Agonismo Inverso de Drogas , Endocannabinoides/genética , Endocannabinoides/metabolismo , Femenino , Regulación de la Expresión Génica , Edad Gestacional , Morfolinas/toxicidad , Naftalenos/toxicidad , Reserva Ovárica/genética , Ovario/metabolismo , Ovario/fisiopatología , Embarazo , Ratas Wistar , Receptor Cannabinoide CB1/metabolismo , Rimonabant/farmacologíaRESUMEN
Topaz1 (Testis and Ovary-specific PAZ domain gene 1) is a germ cell specific gene highly conserved in vertebrates. The putative protein TOPAZ1 contains a PAZ domain, specifically found in PIWI, Argonaute and Zwille proteins. Consequently, Topaz1 is supposed to have a role during gametogenesis and may be involved in the piRNA pathway and contribute to silencing of transposable elements and maintenance of genome integrity. Here we report Topaz1 inactivation in mouse. Female fertility was not affected, but male sterility appeared exclusively in homozygous mutants in accordance with the high expression of Topaz1 in male germ cells. Pachytene Topaz1--deficient spermatocytes progress through meiosis without either derepression of retrotransposons or MSCI dysfunction, but become arrested before the post-meiotic round spermatid stage with extensive apoptosis. Consequently, an absence of spermatids and spermatozoa was observed in Topaz1(-/-) testis. Histological analysis also revealed that disturbances of spermatogenesis take place between post natal days 15 and 20, during the first wave of male meiosis and before the generation of haploid germ cells. Transcriptomic analysis at these two stages showed that TOPAZ1 influences the expression of one hundred transcripts, most of which are up-regulated in mutant testis at post natal day 20. Our results also showed that 10% of these transcripts are long non-coding RNA. This suggests that a highly regulated balance of lncRNAs seems to be essential during spermatogenesis for induction of appropriate male gamete production.
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Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Infertilidad Masculina/genética , Meiosis/genética , Animales , Perfilación de la Expresión Génica , Técnicas Histológicas , Masculino , Meiosis/fisiología , Ratones , Ratones Noqueados , ARN Largo no Codificante/genética , Testículo/metabolismoRESUMEN
Azoospermia (the complete absence of spermatozoa in the semen) is a common cause of male infertility. The etiology of azoospermia is poorly understood. Whole-genome analysis of azoospermic men has identified a number of candidate genes, such as the X-linked testis-expressed 11 (TEX11) gene. Using a comparative genomic hybridization array, an exonic deletion (exons 10-12) of TEX11 had previously been identified in two non-apparent azoospermic patients. However, the putative impact of this genetic alteration on spermatogenesis and the azoospermia phenotype had not been validated functionally. We therefore used a CRISPR/Cas9 system to generate a mouse model (Tex11Ex9-11del/Y) with a partial TEX11 deletion that mimicked the human mutation. Surprisingly, the mutant male Tex11Ex9-11del/Y mice were fertile. The sperm concentration, motility, and morphology were normal. Similarly, the mutant mouse line's testis transcriptome was normal, and the expression of spermatogenesis genes was not altered. These results suggest that the mouse equivalent of the partial deletion observed in two infertile male with azoospermia has no impact on spermatogenesis or fertility in mice, at least of a FVB/N genetic background and until 10 months of age. Mimicking a human mutation does not necessarily lead to the same human phenotype in mice, highlighting significant differences species.
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Azoospermia , Meiosis , Espermatogénesis , Animales , Masculino , Ratones , Espermatogénesis/genética , Meiosis/genética , Azoospermia/genética , Azoospermia/patología , Infertilidad Masculina/genética , Eliminación de Secuencia , Humanos , Testículo/metabolismo , Testículo/patología , Sistemas CRISPR-CasRESUMEN
BACKGROUND: Successful early folliculogenesis is crucial for female reproductive function. It requires appropriate gene specific expression of the different types of ovarian cells at different developmental stages. To date, most gene expression studies on the ovary were conducted in rodents and did not distinguish the type of cell. In mono-ovulating species, few studies have addressed gene expression profiles and mainly concerned human oocytes. RESULTS: We used a laser capture microdissection method combined with RNA-seq technology to explore the transcriptome in oocytes and granulosa cells (GCs) during development of the sheep ovarian follicle. We first documented the expression profile of 15 349 genes, then focused on the 5 129 genes showing differential expression between oocytes and GCs. Enriched functional categories such as oocyte meiotic arrest and GC steroid synthesis reflect two distinct cell fates. We identified the implication of GC signal transduction pathways such as SHH, WNT and RHO GTPase. In addition, signaling pathways (VEGF, NOTCH, IGF1, etc.) and GC transzonal projections suggest the existence of complex cell-cell interactions. Finally, we highlighted several transcription regulators and specifically expressed genes that likely play an important role in early folliculogenesis. CONCLUSIONS: To our knowledge, this is the first comprehensive exploration of transcriptomes derived from in vivo oocytes and GCs at key stages in early follicular development in sheep. Collectively, our data advance our understanding of early folliculogenesis in mono-ovulating species and will be a valuable resource for unraveling human ovarian dysfunction such as premature ovarian failure (POF).
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Regulación de la Expresión Génica , Folículo Ovárico/fisiología , Transcriptoma , Animales , Comunicación Celular/genética , Análisis por Conglomerados , Biología Computacional , Femenino , Células de la Granulosa/metabolismo , Humanos , Anotación de Secuencia Molecular , Oocitos/metabolismo , Especificidad de Órganos/genética , Reproducibilidad de los Resultados , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
DMRT1 is the testis-determining factor in several species of vertebrates, but its involvement in mammalian testes differentiation, where SRY is the testis-determining gene, remains ambiguous. So far, DMRT1 loss-of-function has been described in two mammalian species and induces different phenotypes: Disorders of Sex Development (46, XY DSD) in men and male infertility in mice. We thus abolished DMRT1 expression by CRISPR/Cas9 in a third species of mammal, the rabbit. First, we observed that gonads from XY DMRT1-/- rabbit fetuses differentiated like ovaries, highlighting that DMRT1 is involved in testis determination. In addition to SRY, DMRT1 is required in the supporting cells to increase the expression of the SOX9 gene, which heads the testicular genetic cascade. Second, we highlighted another function of DMRT1 in the germline since XX and XY DMRT1-/- ovaries did not undergo meiosis and folliculogenesis. XX DMRT1-/- adult females were sterile, showing that DMRT1 is also crucial for female fertility. To conclude, these phenotypes indicate an evolutionary continuum between non-mammalian vertebrates such as birds and non-rodent mammals. Furthermore, our data support the potential involvement of DMRT1 mutations in different human pathologies, such as 46, XY DSD as well as male and female infertility.
Animals that reproduce sexually have organs called gonads, the ovaries and testes, which produce eggs and sperm. These organs, which are different in males and females, originate from the same cells during the development of the embryo. As a general rule, the chromosomal sex of an embryo, which gets determined at fertilization, leads to the activation and repression of specific genes. This in turn, controls whether the cells that will form the gonads will differentiate to develop testes or ovaries. Disruption of the key genes involved in the differentiation of the gonads can lead to fertility problems, and in some cases, it can cause the gonads to develop in the 'opposite' direction, resulting in a sex reversal. Identifying these genes is therefore essential to know how to maintain or restore fertility. DMRT1 is a gene that drives the differentiation of gonadal cells into the testicular pathway in several species of animals with backbones, including species of fish, frogs and birds. However, its role in mammals where testis differentiation is driven by a different gene called SRY is not well understood. Indeed, when DMRT1 is disrupted in male humans it leads to disorders of sex development, while disrupting this gene in male mice causes infertility. To obtain more information about the roles of DMRT1 in mammalian species, Dujardin et al. disrupted the gene in a third species of mammal: the rabbit. Dujardin et al. observed that chromosomally-male rabbits lacking DMRT1 developed ovaries instead of testes, showing that in rabbits, both SRY and DMRT1 are both required to produce testes. Additionally, this effect is similar to what is seen in humans, suggesting that rabbits may be a better model for human gonadal differentiation than mice are. Additionally, Dujardin et al. were also able to show that in female rabbits, lack of DMRT1 led to infertility, an effect that had not been previously described in other species. The results of Dujardin et al. may lead to better models for gonadal development in humans, involving DMRT1 in the differentiation of testes. Interestingly, they also suggest the possibility that mutations in this gene may be responsible for some cases of infertility in women. Overall, these findings indicate that DMRT1 is a key fertility gene.
Asunto(s)
Trastorno del Desarrollo Sexual 46,XY , Testículo , Animales , Femenino , Masculino , Conejos , Trastorno del Desarrollo Sexual 46,XY/genética , Trastorno del Desarrollo Sexual 46,XY/metabolismo , Fertilidad/genética , Regulación del Desarrollo de la Expresión Génica , Gónadas/metabolismo , Mamíferos/genética , Procesos de Determinación del Sexo/genética , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Testículo/metabolismoRESUMEN
BACKGROUND: Ovarian granulosa cells (GC) are essential for the development and maturation of a proper oocyte. GC are sensitive to endocrine disruptors, including bisphenol A (BPA) and its analogue bisphenol S (BPS), plasticisers present in everyday consumer products. BPA exhibits greater binding affinity for the membrane oestrogen receptor (GPER) than for the nuclear oestrogen receptors (ERα and ERß). Here, we analysed the effects of BPA and BPS on the steroidogenesis of ovine GC in vitro, as well as their early mechanisms of action, the ovine being a relevant model to study human reproductive impairment. Disruption of GC steroidogenesis might alter oocyte quality and consequently fertility rate. In addition, we compared the effects of a specific GPER agonist (G-1) and antagonist (G-15) to those of BPA and BPS. Ewe GC were cultured with BPA or BPS (10 or 50 µM) or G-1 (1 µM) and/or G-15 (10 µM) for 48 h to study steroidogenesis. RESULTS: Both BPA and BPS (10 µM) altered the secretion of progesterone, however, only BPS (10 µM) affected oestradiol secretion. RNA-seq was performed on GC after 1 h of culture with BPA or BPS (50 µM) or G-1 (10 µM), followed by real-time PCR analyses of differentially expressed genes after 12, 24 and 48 h of culture. The absence of induced GPER target genes showed that BPA and BPS did not activate GPER in GC after 1 h of treatment. These molecules exhibited mainly independent early mechanisms of action. Gene ontology analysis showed that after 1 h of treatment, BPA mainly disrupted the expression of the genes involved in metabolism and transcription, while BPS had a smaller effect and impaired cellular communications. BPA had a transient effect on the expression of CHAC1 (NOTCH signalling and oxidative balance), JUN (linked to MAPK pathway), NR4A1 (oestradiol secretion inhibition), ARRDC4 (endocytose of GPCR) and KLF10 (cell growth, differentiation and apoptosis), while expression changes were maintained over time for the genes LSMEM1 (linked to MAPK pathway), TXNIP (oxidative stress) and LIF (cell cycle regulation) after 12 and 48 h, respectively. CONCLUSION: In conclusion, although they exhibited similar effects, BPA and BPS impaired different molecular pathways in GC in vitro. New investigations will be necessary to follow the temporal changes of these genes over time, as well as the biological processes involved.
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Células de la Granulosa , Oocitos , Femenino , Ovinos , Animales , Humanos , Hormonas Esteroides Gonadales , EstradiolRESUMEN
The negative in utero effects of bisphenol A (BPA) on female reproduction are of concern since the ovarian reserve of primordial follicles is constituted during the fetal period. This time-window is difficult to access, particularly in humans. Animal models and explant culture systems are, therefore, vital tools for investigating EDC impacts on primordial germ cells (PGCs). Here, we investigated the effects of BPA on prophase I meiosis in the fetal sheep ovary. We established an in vitro model of early gametogenesis through retinoic acid (RA)-induced differentiation of sheep PGCs that progressed through meiosis. Using this system, we demonstrated that BPA (3 ×10-7 M & 3 ×10-5 M) exposure for 20 days disrupted meiotic initiation and completion in sheep oogonia and induced transcriptomic modifications of exposed explants. After exposure to the lowest concentrations of BPA (3 ×10-7 M), only 2 probes were significantly up-regulated corresponding to NR2F1 and TMEM167A transcripts. In contrast, after exposure to 3 × 10-5 M BPA, 446 probes were deregulated, 225 were down- and 221 were up-regulated following microarray analysis. Gene Ontology (GO) annotations of differentially expressed genes revealed that pathways mainly affected were involved in cell-cycle phase transition, meiosis and spindle assembly. Differences in key gene expression within each pathway were validated by qRT-PCR. This study provides a novel model for direct examination of the molecular pathways of environmental toxicants on early female gametogenesis and novel insights into the mechanisms by which BPA affects meiosis I. BPA exposure could thereby disrupt ovarian reserve formation by inhibiting meiotic progression of oocytes I and consequently by increasing atresia of primordial follicles containing defective oocytes.
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Compuestos de Bencidrilo , Oogonios , Animales , Compuestos de Bencidrilo/toxicidad , Femenino , Humanos , Meiosis , Oocitos , Fenoles/toxicidad , OvinosRESUMEN
AROMATASE is encoded by the CYP19A1 gene and is the cytochrome enzyme responsible for estrogen synthesis in vertebrates. In most mammals, a peak of CYP19A1 gene expression occurs in the fetal XX gonad when sexual differentiation is initiated. To elucidate the role of this peak, we produced 3 lines of TALEN genetically edited CYP19A1 knockout (KO) rabbits that were devoid of any estradiol production. All the KO XX rabbits developed as females with aberrantly small ovaries in adulthood, an almost empty reserve of primordial follicles, and very few large antrum follicles. Ovulation never occurred. Our histological, immunohistological, and transcriptomic analyses showed that the estradiol surge in the XX fetal rabbit gonad is not essential to its determination as an ovary, or for meiosis. However, it is mandatory for the high proliferation and differentiation of both somatic and germ cells, and consequently for establishment of the ovarian reserve.
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Estrógenos/metabolismo , Ovario/embriología , Ovario/fisiología , Procesos de Determinación del Sexo/fisiología , Animales , Hormona Antimülleriana/metabolismo , Diferenciación Celular , Proliferación Celular , Familia 19 del Citocromo P450/metabolismo , Estradiol/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Gónadas , Mutación INDEL , Folículo Ovárico/fisiología , Ovulación , Fenotipo , Conejos , Diferenciación Sexual/fisiología , Testosterona/metabolismoRESUMEN
BACKGROUND: Successful achievement of early folliculogenesis is crucial for female reproductive function. The process is finely regulated by cell-cell interactions and by the coordinated expression of genes in both the oocyte and in granulosa cells. Despite many studies, little is known about the cell-specific gene expression driving early folliculogenesis. The very small size of these follicles and the mixture of types of follicles within the developing ovary make the experimental study of isolated follicular components very difficult.The recently developed laser capture microdissection (LCM) technique coupled with microarray experiments is a promising way to address the molecular profile of pure cell populations. However, one main challenge was to preserve the RNA quality during the isolation of single cells or groups of cells and also to obtain sufficient amounts of RNA.Using a new LCM method, we describe here the separate expression profiles of oocytes and follicular cells during the first stages of sheep folliculogenesis. RESULTS: We developed a new tissue fixation protocol ensuring efficient single cell capture and RNA integrity during the microdissection procedure. Enrichment in specific cell types was controlled by qRT-PCR analysis of known genes: six oocyte-specific genes (SOHLH2, MAEL, MATER, VASA, GDF9, BMP15) and three granulosa cell-specific genes (KL, GATA4, AMH).A global gene expression profile for each follicular compartment during early developmental stages was identified here for the first time, using a bovine Affymetrix chip. Most notably, the granulosa cell dataset is unique to date. The comparison of oocyte vs. follicular cell transcriptomes revealed 1050 transcripts specific to the granulosa cell and 759 specific to the oocyte.Functional analyses allowed the characterization of the three main cellular events involved in early folliculogenesis and confirmed the relevance and potential of LCM-derived RNA. CONCLUSIONS: The ovary is a complex mixture of different cell types. Distinct cell populations need therefore to be analyzed for a better understanding of their potential interactions. LCM and microarray analysis allowed us to identify novel gene expression patterns in follicular cells at different stages and in oocyte populations.
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Perfilación de la Expresión Génica/métodos , Células de la Granulosa/citología , Células de la Granulosa/metabolismo , Captura por Microdisección con Láser/métodos , Oocitos/citología , Oocitos/metabolismo , Ovinos/genética , Animales , Animales Recién Nacidos , Bovinos , Separación Celular , Femenino , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Ovinos/crecimiento & desarrolloRESUMEN
Spermatogenesis is a complex process regulated by a multitude of genes. The identification and characterization of male-germ-cell-specific genes is crucial to understanding the mechanisms through which the cells develop. The term "TEX gene" was coined by Wang et al. (Nat Genet. 2001; 27: 422-6) after they used cDNA suppression subtractive hybridization (SSH) to identify new transcripts that were present only in purified mouse spermatogonia. TEX (Testis expressed) orthologues have been found in other vertebrates (mammals, birds, and reptiles), invertebrates, and yeasts. To date, 69 TEX genes have been described in different species and different tissues. To evaluate the expression of each TEX/tex gene, we compiled data from 7 different RNA-Seq mRNA databases in humans, and 4 in the mouse according to the expression atlas database.Various studies have highlighted a role for many of these genes in spermatogenesis. Here, we review current knowledge on the TEX genes and their roles in spermatogenesis and fertilization in humans and, comparatively, in other species (notably the mouse). As expected, TEX genes appear to have a major role in reproduction in general and in spermatogenesis in humans but also in all mammals such as the mouse. Most of them are expressed specifically or predominantly in the testis. As most of the TEX genes are highly conserved in mammals, defects in the male (gene mutations in humans and gene-null mice) lead to infertility. In the future, cumulative data on the human TEX genes' physiological functions and pathophysiological dysfunctions should become available and is likely to confirm the essential role of this family in the reproductive process. Thirteen TEX genes are now referenced in the OMIM database, and 3 have been linked to a specific phenotype. TEX11 (on Xq13.1) is currently the gene most frequently reported as being associated with azoospermia.
RéSUMé: La spermatogenèse est un processus complexe régulé par une multitude de gènes. L'identification et la caractérisation des gènes spécifiques des cellules germinales mâles sont essentielles pour comprendre les mécanismes par lesquels les cellules se développent. Le terme «gène TEX¼ a été inventé par Wang et al. (Nat Genet. 2001; 27: 4226) après avoir utilisé l'hybridation soustractive d'ADNc (SSH) pour identifier de nouveaux transcrits qui n'étaient présents que dans la spermatogonie de souris. Puis, des orthologues TEX ont été trouvés chez d'autres vertébrés (mammifères, oiseaux et reptiles), des invertébrés et des levures. À ce jour, 69 gènes TEX (Testis expressed) ont été décrits dans différentes espèces et différents tissus. Pour évaluer l'expression de chaque gène TEX/tex, nous avons compilé les données de 7 bases de données différentes d'ARNm RNA-Seq chez l'homme, et 4 chez la souris selon la base de données de l'atlas d'expression.Diverses études ont mis en évidence le rôle de plusieurs de ces gènes dans la spermatogenèse. Ici, nous passons en revue les connaissances actuelles sur les gènes TEX et leurs rôles dans la spermatogenèse et la fécondation chez l'humain et, comparativement, chez d'autres espèces (notamment la souris). Comme prévu, les gènes TEX semblent avoir un rôle majeur dans la reproduction en général et dans la spermatogenèse chez l'homme, mais aussi chez d'autres mammifères comme la souris. La plupart d'entre eux sont exprimés spécifiquement ou principalement dans les testicules. Comme la plupart des gènes TEX sont hautement conservés chez les mammifères, des défauts chez le mâle (mutations géniques chez l'homme et KO murin) conduisent à l'infertilité. À l'avenir, l'accumulation des données sur les fonctions physiologiques et les dysfonctionnements physiopathologiques des gènes TEX humains devraient devenir disponibles et confirmer le rôle essentiel de cette famille dans le processus de reproduction. Treize gènes TEX sont désormais référencés dans la base de données OMIM, et 3 ont été liés à un phénotype spécifique. TEX11 (sur Xq13.1) est. actuellement le gène le plus fréquemment rapporté comme étant associé à l'azoospermie.
RESUMEN
Whereas the initially strategy for the genetic analysis of male infertility was based on a candidate gene approach, the development of next-generation sequencing technologies (such as whole-exome sequencing (WES)) provides an opportunity to analyze many genes in a single procedure. In order to recommend WES or whole-genome sequencing (WGS) after genetic counselling, an objective evaluation of the current genetic screening strategy for male infertility is required, even if, at present, we have to take into consideration the complexity of such a procedure, not discussed in this commentary.
RéSUMé: Alors que la stratégie actuelle d'analyse de génétique moléculaire de l'infertilité masculine est basée sur une approche dite "gène candidat", le développement des technologies de séquençage de nouvelle génération, comme le séquençage complet de l'exome (WES), offre la possibilité d'analyser de nombreux gènes en une seule technique.Afin de recommander le WES ou le séquençage complet du génome, après un conseil génétique, une évaluation objective des différentes stratégies de dépistage génétique est nécessaire, tout en prenant en considération que la complexité d'une utilisation des nouvelles technologies n'est pas abordé dans ce commentaire.
RESUMEN
Spermatogenesis involves coordinated processes, including meiosis, to produce functional gametes. We previously reported Topaz1 as a germ cell-specific gene highly conserved in vertebrates. Topaz1 knockout males are sterile with testes that lack haploid germ cells because of meiotic arrest after prophase I. To better characterize Topaz1 -/- testes, we used RNA-sequencing analyses at two different developmental stages (P16 and P18). The absence of TOPAZ1 disturbed the expression of genes involved in microtubule and/or cilium mobility, biological processes required for spermatogenesis. Moreover, a quarter of P18 dysregulated genes are long non-coding RNAs (lncRNAs), and three of them are testis-specific and located in spermatocytes, their expression starting between P11 and P15. The suppression of one of them, 4939463O16Rik, did not alter fertility although sperm parameters were disturbed and sperm concentration fell. The transcriptome of P18-4939463O16Rik -/- testes was altered and the molecular pathways affected included microtubule-based processes, the regulation of cilium movement and spermatogenesis. The absence of TOPAZ1 protein or 4930463O16Rik produced the same enrichment clusters in mutant testes despite a contrasted phenotype on male fertility. In conclusion, although Topaz1 is essential for the meiosis in male germ cells and regulate the expression of numerous lncRNAs, these studies have identified a Topaz1 regulated lncRNA (4930463O16Rik) that is key for both sperm production and motility.
RESUMEN
BACKGROUND: Although chromosome rearrangements are responsible for spermatogenesis failure, their impact depends greatly on the chromosomes involved. At present, karyotyping and Y chromosome microdeletion screening are the first-line genetic tests for patients with non-obstructive azoospermia. Although it is generally acknowledged that X or Y chromosome rearrangements lead to meiotic arrest and thus rule out any chance of sperm retrieval after a testicular biopsy, we currently lack markers for the likelihood of testicular sperm extraction (TESE) in patients with other chromosome rearrangements. RESULTS: We investigated the use of a single nucleotide polymorphism comparative genome hybridization array (SNP-CGH) and whole-exome sequencing (WES) for two patients with non-obstructive azoospermia and testicular meiotic arrest, a reciprocal translocation: t(X;21) and t(20;22), and an unsuccessful TESE. No additional gene defects were identified for the t(X;21) carrier - suggesting that t(X;21) alone damages spermatogenesis. In contrast, the highly consanguineous t(20;22) carrier had two deleterious homozygous variants in the TMPRSS9 gene; these might have contributed to testicular meiotic arrest. Genetic defect was confirmed with Sanger sequencing and immunohistochemical assessments on testicular tissue sections. CONCLUSIONS: Firstly, TMPRSS9 gene defects might impact spermatogenesis. Secondly, as a function of the chromosome breakpoints for azoospermic patients with chromosome rearrangements, provision of the best possible genetic counselling means that genetic testing should not be limited to karyotyping. Given the risks associated with TESE, it is essential to perform WES - especially for consanguineous patients.
RéSUMé: CONTEXTE: Si les réarrangements chromosomiques sont connus pour être à l'origine d'une altération de la spermatogenèse, leur impact dépend fortement des chromosomes impliqués. À l'heure actuelle, la réalisation d'un caryotype et le dépistage des microdélétions du chromosome Y sont les tests génétiques réalisés en première intention chez les patients atteints d'azoospermie non obstructive. S'il est généralement admis que les réarrangements impliquant les chromosomes X ou Y entraînent un arrêt méiotique et réduisent fortement les chances de retrouver des spermatozoïdes après une biopsie testiculaire, nous manquons de marqueurs permettant de définir une probabilité d'extraction de spermatozoïdes testiculaires chez les patients présentant d'autres réarrangements chromosomiques. RéSULTATS: Nous avons utilisé l'hybridation génomique comparative sur puces (SNP-CGH) et le séquençage entier de l'exome (SEE) pour deux patients présentant une azoospermie non obstructive avec arrêt méiotique, une translocation réciproque: t(X;21) et t(20;22), et sans spermatozoïde retrouvé après biopsie testiculaire Aucune autre anomalie génétique n'a été identifiée chez le patient porteur de la t(X;21) - ce qui suggère que la translocation seule altére la spermatogenèse. En revanche, le patient porteur de la t(20;22), consanguin, présentait deux variants homozygotes délétères dans le gène TMPRSS9 qui pourraient contribuer à l'arrêt méiotique. Le variant génétique a été confirmé par séquençage Sanger et par immunohistochimie sur des coupes de tissu testiculaire. CONCLUSIONS: Premièrement, nous faisons l'hypothèse d'un impact du défaut du gène TMPRSS9 sur la spermatogenèse. De plus, en fonction des points de cassures chromosomiques pour les patients azoospermes ayant une translocation réciproque, nous suggérons de ne pas limiter les analyses génétiques à la réalisation d'un caryotype afin d'affiner le conseil génétique. Compte tenu des risques associés à la TESE, il est essentiel de réaliser un SEE en amont et en particulier pour les patients consanguins. MOTS CLéS: Arrêt méiotique, azoospermie non obstructive, translocation, séquençage de l'exome, TMPRSS9.
RESUMEN
Bone morphogenetic protein (BMP) 1 is a vertebrate metalloproteinase of the astacin family. BMP1 plays a key role in regulating the formation of the extracellular matrix (ECM), particularly by processing the C-propeptide of fibrillar procollagens. BMP1 also promotes BMP signaling by releasing BMP signaling molecules from complexes with the BMP-antagonist chordin. As a result of BMP1's dual role in both ECM formation and BMP signaling, we hypothesized that BMP1 could play a role in ovarian physiology. Using the sheep ovary as a model system, we showed that BMP1 was expressed in the ovary throughout early fetal stages to adulthood. Furthermore, in adult ovaries, BMP1 was expressed along with chordin, BMP4, and twisted gastrulation, which together form an extracellular regulatory complex for BMP signaling. Within ovine ovaries, immunohistochemical localization demonstrated that BMP1 was present in granulosa cells at all stages of follicular development, from primordial to large antral follicles, and that the levels of BMP1 were not affected by the final follicle selection mechanism. In cultured granulosa cells, BMP1 expression was not affected by gonadotropins, but BMP4 and activin A had opposing effects on the levels of BMP1 mRNA. BMP1 appeared to be secreted into the follicular fluid of antral follicles, where it is able to exert procollagen C-proteinase and chordinase activities. Interestingly, BMP1 activity in follicular fluid decreased with follicular growth.
Asunto(s)
Proteína Morfogenética Ósea 1/metabolismo , Folículo Ovárico/metabolismo , Ovinos/metabolismo , Animales , Colágeno Tipo I/metabolismo , Femenino , Glicoproteínas/metabolismo , Gonadotropinas/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Procolágeno/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The M200V polymorphism of the human DMC1 protein, which is an essential, meiosis-specific DNA recombinase, was found in an infertile patient, raising the question of whether this homozygous human DMC1-M200V polymorphism may cause infertility by affecting the function of the human DMC1 protein. In the present study, we determined the crystal structure of the human DMC1-M200V variant in the octameric-ring form. Biochemical analyses revealed that the human DMC1-M200V variant had reduced stability, and was moderately defective in catalyzing in vitro recombination reactions. The corresponding M194V mutation introduced in the Schizosaccharomyces pombe dmc1 gene caused a significant decrease in the meiotic homologous recombination frequency. Together, these structural, biochemical and genetic results provide extensive evidence that the human DMC1-M200V mutation impairs its function, supporting the previous interpretation that this single-nucleotide polymorphism is a source of human infertility.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Polimorfismo de Nucleótido Simple , Secuencia de Aminoácidos , Arginina/química , Proteínas de Ciclo Celular/metabolismo , Emparejamiento Cromosómico , Cristalografía por Rayos X , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Meiosis/genética , Datos de Secuencia Molecular , Recombinasas/genética , Recombinación Genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
As with many other diseases, genetic testing in human azoospermia was initially restricted to karyotype analyses (leading to diagnostic chromosome rearrangement tests for Klinefelter and other syndromes). With the advent of molecular biology in the 1980s, genetic screening was broadened to analyses of Y chromosome microdeletions and the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Decades later, the emergence of whole-genome techniques has led to the identification of other genetic defects associated with human azoospermia. Although TEX11 and ADGRG2 defects are frequently described in men with azoospermia, most of the causal gene defects found to date are private (i.e. identified in a small number of consanguineous families). Here, we provide an up-to-date overview of all the types of genetic defects known to be linked to human azoospermia and try to give clinical practice guidelines according to azoospermia phenotype. Along with homozygous mutations, polymorphisms and epigenetic defects are also briefly discussed. However, as these variations predispose to azoospermia, a specific review will be needed to compile data on all the particular genetic variations reported in the literature.
Comme pour beaucoup de maladies humaines, les analyses génétiques en cas d'azoospermie étaient initialement limitées à la réalisation d'un caryotype, conduisant au diagnostic de réarrangements chromosomiques comme pour le syndrome de Klinefelter ou autres syndromes. L'avènement de la biologie moléculaire, dans les années 1980, a permis l'élargissement du dépistage génétique à la recherche des microdélétions du chromosome Y et aux anomalies du gène CFTR (cystic fibrosis transmembrane conductance regulator). Il a fallu attendre plusieurs décennies et l'apparition des techniques d'analyses du génome entier pour que soit réalisée l'identification d'autres anomalies génétiques associés à l'azoospermie humaine. Si les anomalies des gènes TEX11 et ADGRG2 sont fréquemment décrites dans la littérature pour les hommes présentant une azoospermie, la plupart des altérations génétiques découvertes à ce jour sont privées, identifiées dans un petit nombre de familles souvent consanguines.L'objectif dans cette revue est de fournir un aperçu actualisé de toutes les anomalies génétiques décrites dans la littérature et associées à l'azoospermie humaine tout en essayant de fournir des guides de conduite diagnostique en fonction du phénotype de l'azoospermie. En plus des mutations homozygotes et délétères, les polymorphismes et les défauts épigénétiques sont également brièvement abordés. Néanmoins, comme ces variations ne sont que de potentiels facteurs de prédisposition à l'azoospermie, une étude spécifique sera nécessaire pour compiler l'ensemble des données de la littérature pour chaque variant génétique.
RESUMEN
BACKGROUND: The increased incidence of diseases, including metabolic syndrome and infertility, may be related to exposure to the mixture of chemicals, which are ubiquitous in the modern environment (environmental chemicals, ECs). Xeno-detoxification occurs within the liver which is also the source of many plasma proteins and growth factors and plays an important role in the regulation of homeostasis. OBJECTIVES: The objective of this study was to investigate the effects of ECs on aspects of liver function, in a well characterized ovine model of exposure to a real-life EC mixture. METHODS: Four groups of sheep (nâ¯=â¯10-12/sex/treatment) were maintained long-term on control or sewage sludge-fertilized pastures: from conception to culling at 19â¯months of age in females and from conception to 7â¯months of age and thereafter in control plots until culling at 19â¯months of age in males. Environmental chemicals were measured in sheep livers and RNA and protein extracts were assessed for exposure markers. Liver proteins were resolved using 2D differential in-gel electrophoresis and differentially expressed protein spots were identified by liquid chromatography/tandem mass spectroscopy. RESULTS: Higher levels of polycyclic aromatic hydrocarbons (PAHs) and lower levels of polychlorinated biphenyls (PCBs) in the livers of control males compared to control females indicated sexually dimorphic EC body burdens. Increased levels of the PAHs Benzo[a]anthracene and chrysene and reduced levels of PCB 153 and PCB 180 were observed in the livers of continuously exposed females. EC exposure affected xenobiotic and detoxification responses and the liver proteome in both sexes and included major plasma-secreted and blood proteins, and metabolic enzymes whose pathway analysis predicted dysregulation of cancer-related pathways and altered lipid dynamics. The latter were confirmed by a reduction in total lipids in female livers and up-regulation of cancer-related transcript markers in male livers respectively by sewage sludge exposure. CONCLUSIONS: Our results demonstrate that chronic exposure to ECs causes major physiological changes in the liver, likely to affect multiple systems in the body and which may predispose individuals to increased disease risks.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Exposición a Riesgos Ambientales , Contaminantes Ambientales/toxicidad , Fertilizantes , Hígado/efectos de los fármacos , Hidrocarburos Policíclicos Aromáticos/toxicidad , Aguas del Alcantarillado , Animales , Femenino , Metabolismo de los Lípidos , Hígado/química , Masculino , Bifenilos Policlorados/toxicidad , Hidrocarburos Policíclicos Aromáticos/análisis , Medición de Riesgo , Aguas del Alcantarillado/química , Factores Sexuales , OvinosRESUMEN
BACKGROUND: The key steps in germ cell survival during ovarian development are the entry into meiosis of oogonies and the formation of primordial follicles, which then determine the reproductive lifespan of the ovary. In sheep, these steps occur during fetal life, between 55 and 80 days of gestation, respectively. The aim of this study was to identify differentially expressed ovarian genes during prophase I meiosis and early folliculogenesis in sheep. RESULTS: In order to elucidate the molecular events associated with early ovarian differentiation, we generated two ovary stage-specific subtracted cDNA libraries using SSH. Large-scale sequencing of these SSH libraries identified 6,080 ESTs representing 2,535 contigs. Clustering and assembly of these ESTs resulted in a total of 2,101 unique sequences depicted in 1,305 singleton (62.11%) and 796 contigs (37.9%) ESTs (clusters). BLASTX evaluation indicated that 99% of the ESTs were homologous to various known genes/proteins in a broad range of organisms, especially ovine, bovine and human species. The remaining 1% which exhibited any homology to known gene sequences was considered as novel. Detailed study of the expression patterns of some of these genes using RT-PCR revealed new promising candidates for ovary differentiation genes in sheep. CONCLUSION: We showed that the SSH approach was relevant to determining new mammalian genes which might be involved in oogenesis and early follicle development, and enabled the discovery of new potential oocyte and granulosa cell markers for future studies. These genes may have significant implications regarding our understanding of ovarian function in molecular terms, and for the development of innovative strategies to both promote and control fertility.