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1.
Methods Mol Biol ; 1965: 107-127, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31069671

RESUMEN

Major limitations in understanding the direct effects of endocrine-disrupting chemicals (EDCs) and cell signaling events in ovarian cellular dynamics in mammals include a lack of proper and simple tools/techniques as well as gaps in knowledge regarding the critical window(s) of vulnerability. Identifying and validating such tools and evaluating the effects of EDCs on molecular dynamics and cellular events during the critical windows of ovarian development are very important to improve the fertility in women and preserve the future health of the developing fetuses. Therefore, we developed a fetal whole ovarian ex vivo culture model. Ex vivo ovary culture models allow varying culture parameters in a highly controlled manner and thus have the potential to allow a more thorough evaluation for reproductive toxicity studies and drug response. This chapter describes clear and thorough details for setting up and maintaining an ex vivo culture system from the rat ovaries and further analyses of mRNA and protein expressions and estimating follicle numbers.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Disruptores Endocrinos/toxicidad , Ovario/citología , Ovario/embriología , Animales , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Modelos Biológicos , Folículo Ovárico/citología , Folículo Ovárico/metabolismo , Ovario/efectos de los fármacos , Ovario/metabolismo , Ratas , Reproducción
2.
Reprod Domest Anim ; 54(7): 964-971, 2019 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-31006155

RESUMEN

During the sex differentiation, the primordial germ cells (PGCs) pass through a differentiation, becoming spermatogonial cells in males and oocytes in females. In this phase, there is difference in gene expression and differentiation potency between males and females. Specific cell markers have been essential in the PGC meiosis beginning and become oocyte cells. However, there are few studies about germline in domestic animals. The domestic dog (Canis lupus familiaris) is an interesting animal model to be used in the investigation about the mammal development because it has several biochemical and physiological similarities to humans. In addition, some additional investigations about dogs may contribute to a better understanding of the biology and genetic components, improving clinical veterinary and zoological sciences. Here, we elucidated by immunofluorescence and quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), the dynamics of the expression of pluripotent (POU5F1 and NANOG) and germline (DDX4, DAZL and DPPA3) markers that are very important in the development of female canine germ cells during 35-50 days post-fertilization (dpf). The female canine germ cells were positive for pluripotent markers during middle developmental period. The number of DDX4, DAZL and DPPA3 cells increased along the germ cell maturation from 45 to 50 dpf. We provided an expression analysis of the pluripotent and germline markers in paraffin sections using the middle and later periods in female canine germ cells. The results can contribute the understanding about the timeline of each marker along the maturation of female canine germ cells. These results have a great significance to demonstrate the germ cell profile changes because it may allow the development of protocols about in vitro germ cell derivation.


Asunto(s)
Perros/embriología , Regulación del Desarrollo de la Expresión Génica , Oocitos/metabolismo , Animales , Diferenciación Celular/genética , ARN Helicasas DEAD-box/genética , Células Germinales Embrionarias/citología , Células Germinales Embrionarias/metabolismo , Femenino , Proteína Homeótica Nanog/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Oocitos/citología , Ovario/citología , Ovario/embriología , Proteínas de Unión al ARN/genética
3.
Int J Mol Sci ; 20(7)2019 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-30965605

RESUMEN

The doublesex and mab-3 related transcription factor (DMRT) gene family involvement in sex development is widely conserved from invertebrates to humans. In this study, we identified a DM (Doublesex/Mab-3)-domain gene in Macrobrachium nipponense, which we named MniDMRT11E because it has many similarities to and phylogenetically close relationships with the arthropod DMRT11E. Amino acid alignments and structural prediction uncovered conservation and putative active sites of the DM domain. Real-time PCR analysis showed that the MniDMRT11E was highly expressed in the ovary and testis in both males and females. Cellular localization analysis showed that DMRT11E was mainly located in the oocytes of the ovary and the spermatocyte of the testis. During embryogenesis, the expression level of MniDMRT11E was higher at the cleavage stage than at other stages. During the different stages of ovarian development, MniDMRT11E expression gradually increased from OI to OIII and decreased to the lowest level at the end of OIV. The results indicated that MniDMRT11E probably played important roles in embryonic development and sex maturity in M. nipponense. MniDMRT11E dsRNA injection also significantly reduced vitellogenin (VG) expression and significantly increased insulin-like androgenic gland factor (IAG) expression, indicating a close relationship in gonad development.


Asunto(s)
Proteínas de Artrópodos/metabolismo , Palaemonidae/embriología , Palaemonidae/metabolismo , Animales , Proteínas de Artrópodos/genética , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Masculino , Ovario/embriología , Ovario/metabolismo , Palaemonidae/genética , Testículo/embriología , Testículo/metabolismo
4.
Int J Mol Sci ; 20(7)2019 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-30965614

RESUMEN

Luteinizing hormone (LH), a pituitary gonadotropin, coupled with LH receptor (LHR) is essential for the regulation of the gonadal maturation in vertebrates. Although LH homolog has been detected by immunocytochemical analysis, and its possible role in ovarian maturation was revealed in decapod crustacean, so far there is no molecular evidence for the existence of LHR. In this study, we cloned a novel LHR homolog (named EsLHR) from the Chinese mitten crab Eriocheir sinensis. The complete sequence of the EsLHR cDNA was 2775bp, encoding a protein of 924 amino acids, sharing 71% amino acids identity with the ant Zootermopsis nevadensis LHR. EsLHR expression was found to be high in the ovary, while low in testis, gill, brain, and heart, and no expression in the thoracic ganglion, eye stalk, muscle, and hepatopancreas. Quantitative PCR revealed that the expression level of EsLHR mRNA was significantly higher in the ovaries in previtellogenic (Pvt), late vitellogenic (Lvt), and germinal vesicle breakdown (GVBD) stages than that in the vitellogenic (Mvt) and early vitellogenic (Evt) stages (P < 0.05), and, the highest and the lowest expression were in Lvt, and Evt, respectively. The strong signal was mainly localized in the ooplasm of Pvt oocyte as detected by in situ hybridization. The crab GnRH homolog can significantly induce the expression of EsLHR mRNA at 36 hours post injection in vivo (P < 0.01), suggesting that EsLHR may be involved in regulating ovarian development through GnRH signaling pathway in the mitten crab.


Asunto(s)
Braquiuros/metabolismo , Receptores de HL/metabolismo , Animales , Braquiuros/embriología , ADN Complementario/metabolismo , Femenino , Masculino , Ovario/embriología , Ovario/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Receptores de HL/genética , Testículo/embriología , Testículo/metabolismo
5.
Gen Comp Endocrinol ; 277: 130-140, 2019 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-30951722

RESUMEN

Expression of adamts9 (A disintegrin and metalloprotease with thrombospondin type-1 motif, member 9) increases dramatically in the somatic cells surrounding oocytes during ovulation in vertebrates from zebrafish to human. However, the function of Adamts9 during ovulation has not been determined due to the embryonic lethality of knockouts in mice and Drosophila. To identify the role of Adamts9 during ovulation we generated knockout (adamts9-/-) zebrafish using CRISPR/Cas9 and characterized the effects of the mutation. From 1047 fish generated by crossing adamts9+/- pairs, we found significantly fewer adult adamts9-/- fish (4%) than predicted by Mendelian ratios (25%). Of the mutants found, there was a significant male bias (82%). Only 3 female mutants were identified (7%), and they had small ovaries with few stage III and IV oocytes compared to wildtype (wt) counterparts of comparable size and age. Astoundingly, the remaining mutants (11%) did not appear to have normal testis or ovaries. Instead there was a pair of transparent, ovarian-like membranous shells that filled the abdominal cavity. Histological examination confirmed that shells were largely empty with no internal structure. Surprisingly, seminiferous tubules and various spermatocytes including mature spermatozoa were observed on the periphery of these transparent shells. No female or female like knockouts were observed to release eggs, and no ovulated oocytes were observed in histological sections. To our knowledge, this is the first report of an adamts9 global knockout model in any adult vertebrates and the first description of how gonadal sex and structure are affected- highlighting the importance of Adamts9 during gonadal development and the value of zebrafish as a model organism.


Asunto(s)
Proteína ADAMTS9/metabolismo , Ovario/embriología , Ovario/metabolismo , Pez Cebra/metabolismo , Proteína ADAMTS9/deficiencia , Proteína ADAMTS9/genética , Animales , Secuencia de Bases , Femenino , Fertilización , Técnicas de Inactivación de Genes , Homocigoto , Infertilidad Femenina/genética , Masculino , Mutación/genética , Ovario/anomalías , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Razón de Masculinidad , Análisis de Supervivencia
6.
Gen Comp Endocrinol ; 277: 122-129, 2019 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-30951723

RESUMEN

Gonadal soma-derived factor (Gsdf) is a unique TGF-ß factor essential for both ovarian and testicular development in Hd-rR medaka (Oryzias latipes). However, the downstream genes regulated by Gsdf signaling remain unknown. Using a high-throughput proteomic approach, we identified a significant increase in the expression of the RNA-binding protein Igf2bp3 in gsdf-deficient ovaries. We verified this difference in transcription and protein expression against normal gonads using real-time PCR quantification and Western blotting. The genomic structure of igf2bp3 and the syntenic flanking segments are highly conserved across fish and mammals. igf2bp3 expression was correlated with oocyte development, which is consistent with the expression of the igf2bp3 ortholog Vg1-RBP/Vera in Xenopus. In contrast to the normal ovary, cysts of H3K27me3- and Igf2bp3-positive germ cells were dramatically increased in the one-month-old gsdf-deficient ovary, indicating that the gsdf depletion led to a dysregulation of Igf2bp3-mediated oocyte development. Our results provide novel insights into the Gsdf-Igf2bp3 signaling mechanisms that underlie the fundamental process of gametogenesis; these mechanisms may be well conserved across phyla.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Oocitos/metabolismo , Oryzias/genética , Proteínas de Unión al ARN/genética , Factor de Crecimiento Transformador beta/deficiencia , Secuencia de Aminoácidos , Animales , Proliferación Celular , Secuencia Conservada , Evolución Molecular , Femenino , Perfilación de la Expresión Génica , Histonas/metabolismo , Lisina/metabolismo , Masculino , Oogénesis/genética , Ovario/embriología , Ovario/metabolismo , Filogenia , Proteómica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/genética , Factor de Crecimiento Transformador beta/metabolismo
7.
PLoS One ; 14(3): e0214130, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30901367

RESUMEN

Cells on the surface of the mesonephros give rise to replicating Gonadal Ridge Epithelial-Like (GREL) cells, the first somatic cells of the gonadal ridge. Later germ cells associate with the GREL cells in the ovigerous cords, and the GREL cells subsequently give rise to the granulosa cells in follicles. To examine these events further, 27 bovine fetal ovaries of different gestational ages were collected and prepared for immunohistochemical localisation of collagen type I and Ki67 to identify regions of the ovary and cell proliferation, respectively. The non-stromal cortical areas (collagen-negative) containing GREL cells and germ cells and later in development, the follicles with oocytes and granulosa cells, were analysed morphometrically. Another set of ovaries (n = 17) were collected and the expression of genes associated with germ cell lineages and GREL/granulosa cells were quantitated by RT-PCR. The total volume of non-stromal areas in the cortex increased significantly and progressively with ovarian development, plateauing at the time the surface epithelium developed. However, the proportion of non-stromal areas in the cortex declined significantly and progressively throughout gestation, largely due to a cessation in growth of the non-stroma cells and the continued growth of stroma. The proliferation index in the non-stromal area was very high initially and then declined substantially at the time follicles formed. Thereafter, it remained low. The numerical density of the non-stromal cells was relatively constant throughout ovarian development. The expression levels of a number of genes across gestation either increased (AMH, FSHR, ESR1, INHBA), declined (CYP19A1, ESR2, ALDH1A1, DSG2, OCT4, LGR5) or showed no particular pattern (CCND2, CTNNB1, DAZL, FOXL2, GATA4, IGFBP3, KRT19, NR5A1, RARRES1, VASA, WNT2B). Many of the genes whose expression changed across gestation, were positively or negatively correlated with each other. The relationships between these genes may reflect their roles in the important events such as the transition of ovigerous cords to follicles, oogonia to oocytes or GREL cells to granulosa cells.


Asunto(s)
Bovinos/embriología , Regulación del Desarrollo de la Expresión Génica , Ovario/embriología , Animales , Bovinos/genética , Femenino , Células Germinativas/citología , Células Germinativas/metabolismo , Células de la Granulosa/citología , Células de la Granulosa/metabolismo , Mesonefro/citología , Mesonefro/embriología , Mesonefro/metabolismo , Ovario/citología , Ovario/metabolismo
8.
Aquat Toxicol ; 210: 215-226, 2019 May.
Artículo en Inglés | MEDLINE | ID: mdl-30875550

RESUMEN

Roundup and other glyphosate-based herbicides are the most commonly used herbicides in the world, yet their effects on developing fish embryos are not clearly understood. The present study, therefore, examined developmental teratogenic effects and adult-onset reproductive effects of exposure to environmentally relevant concentrations of glyphosate and Roundup in Japanese medaka fish (Oryzias latipes). Hd-rR strain medaka embryos were exposed to 0.5 mg/L glyphosate, 0.5 mg/L and 5 mg/L Roundup (glyphosate acid equivalent) for the first 15 days of their embryonic life and then allowed to sexually mature without further exposure. Whole body tissue samples were collected at 15 days post fertilization (dpf) and brain and gonad samples were collected in mature adults. Hatching success and phenotypic abnormalities were recorded up until 15 dpf. Roundup (0.5 mg/L) and glyphosate decreased cumulative hatching success, while glyphosate exposure increased developmental abnormalities in medaka fry. Expression of the maintenance DNA methyltransferase gene Dnmt1 decreased, whereas expression of methylcytosine dioxygenase genes (Tet1, Tet2 and Tet3) increased in fry at 15 dpf suggesting that epigenetic alterations increased global DNA demethylation in the developing fry. Fecundity and fertilization efficiency were not altered due to exposure. Among the reproduction-related genes in the brain, kisspeptin receptor (Gpr54-1) expression was significantly reduced in females exposed to 0.5 mg/L and 5 mg/L Roundup, and Gpr54-2 was reduced in the 0.5 mg/L Roundup treatment group. No change in expression of these genes was observed in the male brain. In the testes, expression of Fshr and Arα was significantly reduced in medaka exposed to 0.5 mg/L Roundup and glyphosate, while the expression of Dmrt1 and Dnmt1 was reduced in medaka exposed to 0.5 mg/L glyphosate. No change in expression of these genes was observed in the ovaries. The present study demonstrates that Roundup and its active ingredient glyphosate can induce developmental, reproductive, and epigenetic effects in fish; suggesting that ecological species, mainly fish, could be at risk for endocrine disruption in glyphosate and Roundup-contaminated water bodies.


Asunto(s)
Embrión no Mamífero/efectos de los fármacos , Disruptores Endocrinos/toxicidad , Epigénesis Genética/efectos de los fármacos , Glicina/análogos & derivados , Oryzias/crecimiento & desarrollo , Contaminantes Químicos del Agua/toxicidad , Animales , Relación Dosis-Respuesta a Droga , Desarrollo Embrionario/efectos de los fármacos , Femenino , Glicina/toxicidad , Masculino , Oryzias/embriología , Oryzias/genética , Ovario/efectos de los fármacos , Ovario/embriología , Reproducción/efectos de los fármacos
9.
PLoS One ; 14(3): e0213575, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30856218

RESUMEN

Movement and expansion of mesonephric-derived stroma appears to be very important in the development of the ovary. Here, we examined the expression of 24 genes associated with stroma in fetal ovaries during gestation (n = 17; days 58-274) from Bos taurus cattle. RNA was isolated from ovaries for quantitative RT-PCR. Expression of the majority of genes in TGFß signalling, stromal transcription factors (NR2F2, AR), and some stromal matrix genes (COL1A1, COL3A1 and FBN1, but not FBN3) showed a positive linear increase with gestational age. Expression of genes associated with follicles (INSL3, CYP17A1, CYP11A1 and HSD3B1), was low until mid-gestation and then increased with gestational age. LHCGR showed an unusual bimodal pattern; high levels in the first and last trimesters. RARRES1 and IGFBP3 also increased with gestational age. To relate changes in gene expression in stromal cells with that in non stromal cells during development of the ovary we combined the data on the stromal genes with another 20 genes from non stromal cells published previously and then performed hierarchical clustering analysis. Three major clusters were identified. Cluster 1 genes (GATA4, FBN3, LHCGR, CYP19A1, ESR2, OCT4, DSG2, TGFB1, CCND2, LGR5, NR5A1) were characterised by high expression only in the first trimester. Cluster 2 genes (FSHR, INSL3, HSD3B1, CYP11A1, CYP17A1, AMH, IGFBP3, INHBA) were highly expressed in the third trimester and largely associated with follicle function. Cluster 3 (COL1A1, COL3A1, FBN1, TGFB2 TGFB3, TGFBR2, TGFBR3, LTBP2, LTBP3, LTBP4, TGFB1I1, ALDH1A1, AR, ESR1, NR2F2) had much low expression in the first trimester rising in the second trimester and remaining at that level during the third trimester. Cluster 3 contained members of two pathways, androgen and TGFß signalling, including a common member of both pathways namely the androgen receptor cofactor TGFß1 induced transcript 1 protein (TGFB1I1; hic5). GATA4, FBN3 and LHCGR, were highly correlated with each other and were expressed highly in the first trimester during stromal expansion before follicle formation, suggesting that this could be a critical phase in the development of the ovarian stroma.


Asunto(s)
Bovinos/embriología , Regulación del Desarrollo de la Expresión Génica , Ovario/embriología , Animales , Bovinos/genética , Bovinos/metabolismo , Femenino , Redes Reguladoras de Genes , Edad Gestacional , Familia de Multigenes , Ovario/citología , Ovario/metabolismo , Embarazo , Transducción de Señal , Células Tecales/citología , Células Tecales/metabolismo , Transcriptoma , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo
10.
Gen Comp Endocrinol ; 277: 17-19, 2019 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-30769011

RESUMEN

Gonadotropins (GtHs) and their receptors (follicle-stimulating hormone receptor, FSHR; luteinizing hormone receptor, LHR) are involved in the regulation of gametogenesis and play important roles during the reproductive cycles in vertebrate species, including fish. This minireview focuses on the molecular characterization and quantification of GtHs (common glycoprotein α subunit CGα, FSHß, and LHß) and their receptors (FSHR and LHR) throughout the reproductive cycle of female turbot Scophthalmus maximus. Information about GtHs, FSHR, LHR as well as other ligand-receptors interaction from different teleosts are also included in this review for the implications they may have on the functions of GtHs, FSHR and LHR in the reproductive development of turbot. These findings may enhance our understanding of the physiological roles of the GtHs, FSHR and LHR in controlling of flatfish ovarian development during the reproductive cycle and contributing to the improvement of management strategies for turbots in captivity.


Asunto(s)
Peces Planos/genética , Gonadotropinas/metabolismo , Ovario/embriología , Ovario/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Femenino
11.
Anat Histol Embryol ; 48(2): 102-109, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30450614

RESUMEN

Wilms' tumour 1 gene (WT1) is essential for the development of mammalian urogenital system. However, the expression pattern of WT1 in the development of porcine urogenital organs is still unclear. Here, we examined the expression of WT1 mRNA and protein in porcine kidneys, ovaries and testes from embryonic days 35 and 60 (E35d, E60d, n = 3) to the newborn (0d, n = 4) and adult (210d, n = 3) stages, using real-time PCR and immunofluorescent staining. Real-time PCR analysis showed that porcine kidneys, ovaries and testes all expressed high level of WT1 mRNAs, especially in adult testes (p < 0.05 or 0.01 vs. kidney and ovary, respectively). Morphologically, characteristic microstructures of the kidneys, ovaries and testes were observed and discerned at all four stages. Immunofluorescently, WT1 expression was detected in a dynamic and context-specific pattern during the development of these organs. Taken together, porcine urogenital organs express relatively high levels of WT1 mRNA. Dynamical and context-specific expression profile of WT1 in these organs occurs during their development, implying its close association with the development and function of porcine kidney, ovary and testis.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Riñón/metabolismo , Ovario/metabolismo , Porcinos/metabolismo , Testículo/metabolismo , Proteínas WT1/metabolismo , Animales , Femenino , Riñón/embriología , Masculino , Ovario/embriología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Porcinos/crecimiento & desarrollo , Testículo/embriología , Proteínas WT1/genética
12.
Environ Pollut ; 244: 747-756, 2019 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30384080

RESUMEN

The correlation between endocrine active contaminants in the environment and alterations in reproductive development of Sarotherodon melanotheron from Lagos lagoon has been investigated. Sediment and a total of 155 fish (74 males and 81 females) were collected between November 2014-March 2015 from selected contaminated sites (Ikorodu, Oworonshoki, Makoko and Idumota) and a putative control site (Igbore) along the lagoon. Sediment contaminant analysis revealed, significantly higher concentration of lindane, dieldrin, 4-iso-nonylphenol, 4-t-octylphenol and monobutyltin cation at the contaminated sites. Examination of gross morphological and histological changes of fish gonads showed a 27.4% prevalence of intersex in the sampled fish, of which 78% were males (testes-ova) and 22% were females (ovo-testis). Quantitative PCR (qPCR) of liver transcripts revealed the presence of vitellogenin (vtg) levels in male fish from contaminated sites. Zona radiata proteins (zrp) mRNA levels were significantly higher in females, compared to male fish. In general, significantly lower vtg and zrp transcripts levels were recorded at Igbore (control site), compared with contaminated sites. Principal component analysis (PCA) showed site and sex relationship in biological responses and contaminants, including trace metals, demonstrating that measured endocrine responses in fish were associated with contaminant burden in sediment. In addition, positive relationships were observed in male fish from Idumota, Oworonshoki and Ikorodu with vtg and dieldrin/4-iso-nonyphenol, with higher levels in male fish, compared to females. Further, contaminants from the Makoko, Oworonshoki and Ikorodu sites were positively associated with higher GSI and zrp in females. More importantly, the severity of intersex and changes in vtg transcripts imply a progressive feminization of male fish with concomitant alteration in the reproductive health of fish inhabiting the Lagos lagoon.


Asunto(s)
Cíclidos/embriología , Cíclidos/fisiología , Trastornos del Desarrollo Sexual/inducido químicamente , Disruptores Endocrinos/análisis , Disruptores Endocrinos/toxicidad , Sedimentos Geológicos/química , Contaminantes Químicos del Agua/análisis , Contaminantes Químicos del Agua/toxicidad , Animales , Dieldrín/análisis , Dieldrín/toxicidad , Femenino , Hexaclorociclohexano/análisis , Hexaclorociclohexano/toxicidad , Hígado/metabolismo , Masculino , Nigeria , Ovario/efectos de los fármacos , Ovario/embriología , Fenoles/análisis , Fenoles/toxicidad , Testículo/efectos de los fármacos , Testículo/embriología , Vitelogeninas/metabolismo
13.
Fundam Clin Pharmacol ; 33(2): 199-207, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30216532

RESUMEN

Dexamethasone (Dx) is often used in obstetric practice to promote fetal lung maturation and to prevent respiratory distress syndrome when the risk of preterm delivery persists. This therapy enables survival of the newborn, but also is associated with deleterious effects on the offspring, such as reproductive disorders. The aim of this study was to determine specifically whether prenatal exposure to Dx disturbs the physiological balance between proliferation and apoptosis of germinative cells (GC) in the ovary of 19- and 21-day-old fetuses and thus induces developmental programming of the female reproductive system. Pregnant Wistar rats (n = 10/group), separated into control (vehicle) and Dx-treated (0.5 mg/kg body mass) groups, received injections on gestational days 16, 17, and 18. Exposure to Dx lowered the volume of the fetal ovary by 30% (P < 0.05) in 21-day-old fetuses, as well as the total number of GC in the ovary by 21% (P < 0.05). When compared to the controls, in Dx-exposed fetuses, the total number of PCNA-positive GC was 27% lower at 19 days and 71% lower at 21 days old (P < 0.05), while total numbers of caspase-3-positive GC were 2.3-fold and 34% higher, respectively (P < 0.05). Our results demonstrate that prenatal exposure to Dx diminished proliferation but increased the rate of germinative cell apoptosis, with consequently reduced total germinative cell number and ovary volume. Impairment of fetal oogenesis and fewer GC in the fetal ovary compromise the oogonial stock and thus may constitute a risk of female fertility.


Asunto(s)
Dexametasona/toxicidad , Células Germinales Embrionarias/efectos de los fármacos , Oogénesis/efectos de los fármacos , Ovario/efectos de los fármacos , Óvulo/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Caspasa 3/metabolismo , Proliferación Celular/efectos de los fármacos , Células Germinales Embrionarias/metabolismo , Femenino , Edad Gestacional , Ovario/embriología , Ovario/metabolismo , Óvulo/metabolismo , Embarazo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas Wistar
14.
Reprod Fertil Dev ; 31(5): 847-854, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30554591

RESUMEN

DEAD-box helicase 4 (DDX4; also known as vasa) is essential for the proper formation and maintenance of germ cells. Although DDX4 is conserved in a variety of vertebrates and invertebrates, its roles differ between species. This study investigated the function of DDX4 in chicken embryos by knocking down its expression using retroviral vectors that encoded DDX4-targeting microRNAs. DDX4 was effectively depleted invitro and invivo via this approach. Male and female gonads of DDX4-knockdown embryos contained a decreased number of primordial germ cells, indicating that DDX4 is essential to maintain a normal level of these cells in chicken embryos of both sexes. Expression of doublesex and mab-3 related transcription factor 1 (DMRT1) and sex determining region Y-box 9 (SOX9), which are involved in testis determination and differentiation, was normal in male gonads of DDX4-knockdown embryos. In contrast, expression of cytochrome P450 family 19 subfamily A member 1 (CYP19A1), which encodes aromatase and is essential for ovary development, was significantly decreased in female gonads of DDX4-knockdown embryos. Expression of forkhead box L2 (FOXL2), which plays an important role in ovary differentiation, was also slightly reduced in DDX4-knockdown embryos, but not significantly. Based on several pieces of evidence FOXL2 was hypothesised to regulate aromatase expression. The results of this study indicate that aromatase expression is also regulated by several additional pathways.


Asunto(s)
ARN Helicasas DEAD-box/genética , Células Germinativas/citología , Ovario/metabolismo , Diferenciación Sexual/fisiología , Testículo/metabolismo , Animales , Aromatasa/genética , Aromatasa/metabolismo , Pollos , ARN Helicasas DEAD-box/metabolismo , Femenino , Proteína Forkhead Box L2/genética , Proteína Forkhead Box L2/metabolismo , Técnicas de Silenciamiento del Gen , Células Germinativas/metabolismo , Masculino , Ovario/embriología , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Testículo/embriología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
15.
Aging (Albany NY) ; 10(12): 4175-4187, 2018 12 26.
Artículo en Inglés | MEDLINE | ID: mdl-30591620

RESUMEN

Di (2-ethylhexyl) phthalate (DEHP), an estrogen-like compound that is a ubiquitous environmental contaminant, has been reported to adversely affect human and mammalian reproduction. Many studies have found that exposure to DEHP during pregnancy perturbs female germ cell meiosis and is detrimental to oogenesis. Previous studies have demonstrated that melatonin (MLT) is beneficial to reproductive endocrinology, oogenesis, and embryonic development as the ability to antioxidative and antiapoptotic. However, whether the meiotic defect of germ cells exposed to DEHP could be rescued by MLT is not clear. Here, we cultured 12.5 days post coitum (dpc) fetal mouse ovaries for 6 days, exposed them to 100 µM DEHP with or without 1 µM MLT in vitro.. The results showed that DEHP exposure induced the abnormal formation of DNA double-strand breaks (DSBs), and inhibited the repair of DSBs during meiotic recombination. In addition, we found defective oocytes were prone to undergo apoptosis. Notably, this defect could be remarkably ameliorated by the addition of MLT via a reduction of the levels of reactive oxygen species and an inhibition of apoptosis. In conclusion, our data revealed that MLT had a protective action against the meiotic deterioration of fetal oocytes induced by DEHP in the mouse in vitro.


Asunto(s)
Dietilhexil Ftalato/toxicidad , Meiosis/efectos de los fármacos , Melatonina/farmacología , Oocitos/efectos de los fármacos , Animales , Células Cultivadas , Femenino , Melatonina/administración & dosificación , Ratones , Ovario/embriología , Especies Reactivas de Oxígeno
16.
J Morphol ; 279(11): 1603-1614, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30397936

RESUMEN

Light microscopy studies of the female American lobster Homarus americanus reproductive system are essentially nonexistent or outdated. Based on samples taken in the spring, summer, and autumn from the southern Gulf of St. Lawrence between 1994 and 2014, and using a combination of histological and scanning electron microscope techniques, we propose an ovarian cycle with 10 stages, identifying for the first time a recovery stage. Also, an atypical resorption stage, characterized by massive reabsorption of mature oocytes, is occasionally observed during summer months. The oviducts are composed of connective tissue (elastic and collagen fibers) with no muscle or secretory activities. Their epithelium shows a cyclic pattern and phagocytosis activities linked to spawning. Although the role of the seminal receptacle is to store and protect semen, free spermatozoa (i.e., without the spermatophoric wall and the acellular gelatinous substance that constitute the semen) were also observed in its posteriolateral grooves immediately prior to spawning, which is consistent with an external fertilization mechanism at the seminal receptacle. Unexpectedly, free spermatozoa were observed externally near two pore-like structures located on the gonopore's operculum, not at the seminal receptacle, after spawning; hence, more work is needed to fully understand the fertilization mechanism for the American lobster.


Asunto(s)
Genitales Femeninos/anatomía & histología , Genitales Femeninos/fisiología , Nephropidae/anatomía & histología , Nephropidae/fisiología , Animales , Femenino , Genitales Femeninos/ultraestructura , Nephropidae/ultraestructura , Oogénesis , Ovario/citología , Ovario/embriología
17.
Endocrinology ; 159(10): 3515-3523, 2018 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-30169775

RESUMEN

Estrogens regulate vertebrate development and function through binding to nuclear estrogen receptors α and ß (ERα and ERß) and the G protein-coupled estrogen receptor (GPER). Studies in mutant animal models demonstrated that ERα and ERß are required for normal ovary development and function. However, the degree to which GPER signaling contributes to ovary development and function is less well understood. Previous studies using cultured fish oocytes found that estradiol inhibits oocyte maturation in a GPER-dependent manner, but whether GPER regulates oocyte maturation in vivo is not known. To test the hypothesis that GPER regulates oocyte maturation in vivo, we assayed ovary development and function in gper mutant zebrafish. We found that homozygous mutant gper embryos developed into male and female adults with normal sex ratios. Adult mutant fish exhibited normal secondary sex characteristics and fertility. Additionally, mutant ovaries were histologically normal. We observed no differences in the number of immature versus mature oocytes in mutant versus wild-type ovaries from both young and aged adults. Furthermore, expression of genes associated with sex determination and ovary function was normal in gper mutant ovaries compared with wild type. Our findings suggest that GPER is not required for sex determination, ovary development, or fertility in zebrafish.


Asunto(s)
Ovario/metabolismo , Receptores Acoplados a Proteínas G/genética , Análisis para Determinación del Sexo/métodos , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Genotipo , Masculino , Mutación , Oocitos/citología , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Ovario/embriología , Ovario/crecimiento & desarrollo , Receptores Acoplados a Proteínas G/metabolismo , Razón de Masculinidad , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo
18.
Differentiation ; 103: 5-13, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30245193

RESUMEN

We present a detailed review of fetal development of the male and female human urogenital tract from 8 to 22 weeks gestation at the macroscopic and morphometric levels. Human fetal specimens were sexed based on macroscopic identification of fetal testes or ovaries, Wolffian or Müllerian structures and the presence of the SRY gene in the specimens at or near the indifferent stage (8-9 weeks). Specimens were photographed using a dissecting microscope with transmitted and reflected light. Morphometric measurements were taken of each urogenital organ. During this time period, development of the male and female urogenital tracts proceeded from the indifferent stage to differentiated organs. The kidneys, ureters, and bladder developed identically, irrespective of sex with the same physical dimensions and morphologic appearance. The penis, prostate and testis developed in males and the clitoris, uterus and ovary in females. Androgen-dependent growth certainly influenced size and morphology of the penile urethra and prostate, however, androgen-independent growth also accounted for substantial growth in the fetal urogenital tract including the clitoris.


Asunto(s)
Diferenciación Celular/genética , Ovario/ultraestructura , Testículo/ultraestructura , Sistema Urogenital/ultraestructura , Femenino , Desarrollo Fetal , Feto , Genitales/embriología , Genitales/crecimiento & desarrollo , Genitales/ultraestructura , Humanos , Masculino , Ovario/embriología , Ovario/crecimiento & desarrollo , Testículo/embriología , Testículo/crecimiento & desarrollo , Sistema Urogenital/crecimiento & desarrollo
19.
Sci Rep ; 8(1): 12791, 2018 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-30143724

RESUMEN

Oocytes, including from mammals, lack centrioles, but neither the mechanism by which mature eggs lose their centrioles nor the exact stage at which centrioles are destroyed during oogenesis is known. To answer questions raised by centriole disappearance during oogenesis, using a transgenic mouse expressing GFP-centrin-2 (GFP CETN2), we traced their presence from e11.5 primordial germ cells (PGCs) through oogenesis and their ultimate dissolution in mature oocytes. We show tightly coupled CETN2 doublets in PGCs, oogonia, and pre-pubertal oocytes. Beginning with follicular recruitment of incompetent germinal vesicle (GV) oocytes, through full oocyte maturation, the CETN2 doublets separate within the pericentriolar material (PCM) and a rise in single CETN2 pairs is identified, mostly at meiotic metaphase-I and -II spindle poles. Partial CETN2 foci dissolution occurs even as other centriole markers, like Cep135, a protein necessary for centriole duplication, are maintained at the PCM. Furthermore, live imaging demonstrates that the link between the two centrioles breaks as meiosis resumes and that centriole association with the PCM is progressively lost. Microtubule inhibition shows that centriole dissolution is uncoupled from microtubule dynamics. Thus, centriole doublets, present in early G2-arrested meiotic prophase oocytes, begin partial reduction during follicular recruitment and meiotic resumption, later than previously thought.


Asunto(s)
Centriolos/metabolismo , Células Germinativas/metabolismo , Oocitos/metabolismo , Animales , Proteínas de Unión al Calcio/metabolismo , Centriolos/efectos de los fármacos , Centrosoma/efectos de los fármacos , Centrosoma/metabolismo , Femenino , Células Germinativas/citología , Células Germinativas/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Metafase/efectos de los fármacos , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Nocodazol/farmacología , Oocitos/citología , Oocitos/efectos de los fármacos , Oogonios/citología , Oogonios/efectos de los fármacos , Oogonios/metabolismo , Ovario/embriología , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Polos del Huso/efectos de los fármacos , Polos del Huso/metabolismo , Tubulina (Proteína)/metabolismo
20.
J Hazard Mater ; 358: 101-112, 2018 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-29990796

RESUMEN

The female reproductive lifespan is largely determined by the size of the primordial follicle pool, which is established early in life. We previously reported that Di (2-ethylhexyl) phthalate (DEHP), an environmental endocrine disruptor and a widely-spreading plasticizer, impairs primordial folliculogenesis. In the present study, we found DEHP significantly altered the number and sex ratio of the offspring of neonatal-exposed mice. Furthermore, by a neonatal exposure model and an ovary culture model, it showed that DEHP activated autophagy in the ovary, with increased autophagy-related gene expression and recognizable autophagosomes, while inhibition of autophagy by 3-MA attenuated the adverse impact of DEHP on primordial folliculogenesis. Moreover, key components of AMPK-SKP2-CARM1 signalling were up-regulated by DEHP in the ovary, and AMPK inhibitor Compound C reduced autophagy-related gene expression and partially recovered primordial follicle assembly. Collectively, this study demonstrates that DEHP induces autophagy by activating AMPK-SKP2-CARM1 signalling in mice perinatal ovaries, which results in disrupted primordial folliculogenesis and reduced female fertility.


Asunto(s)
Autofagia/efectos de los fármacos , Dietilhexil Ftalato/toxicidad , Disruptores Endocrinos/toxicidad , Ovario/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Animales , Animales Recién Nacidos , Femenino , Fertilidad/efectos de los fármacos , Ratones Endogámicos BALB C , Ovario/embriología , Ovario/crecimiento & desarrollo , Ovario/ultraestructura , Embarazo , Efectos Tardíos de la Exposición Prenatal/patología
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