Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp. (C) 2010 Elsevier Inc. All rights reserved.

Effect of insulin-like growth factor-I during preantral follicular culture on steroidogenesis, in vitro oocyte maturation, and embryo development in mice

Insulin-like growth factor-I (IGF-I) is involved in the regulation of ovarian follicular development and has been shown to potentiate the FSH responsiveness of granulosa cells from preantral follicles. The aim of the present study was to investigate the effect of IGF-I during preantral follicular culture on steroidogenesis, subsequent oocyte maturation, fertilization, and embryo development in mice. Preantral follicles were isolated mechanically and cultured for 12 days in a simplified culture medium supplemented with 1% fetal calf serum, recombinant human FSH, transferrin, and selenium. In these conditions, follicles were able to grow and produce oocytes that could be matured and fertilized. The first experiment analyzed the effect of different concentrations of IGF-I (0, 10, 50, or 100 ng/ml) added to the culture medium on the follicular survival, steroidogenesis, and the oocyte maturation process. The presence of IGF-I during follicular growth increased the secretion of estradiol but had no effect on the subsequent oocyte survival and maturation rates. In the second experiment, IGF-I (0 or 50 ng/ml) was added to the culture medium during follicular growth, oocyte maturation, or both, and subsequent oocyte fertilization and embryo development rates were evaluated. Oocyte fertilization rates were comparable in the presence or absence of IGF-I. However, the blastocyst development rate was enhanced after follicular culture in the presence of IGF-I. Moreover, the total cell number of the blastocysts observed after differential labeling staining was also higher when follicles were cultured or matured in the presence of IGF-I.

Long-range Transcriptome Sequencing Reveals Cancer Cell Growth Regulatory Chimeric mRNA

mRNA chimeras from chromosomal translocations often play a role as transforming oncogenes. However, cancer transcriptomes also contain mRNA chimeras that may play a role in tumor development, which arise as transcriptional or post-transcriptional events. To identify such chimeras, we developed a deterministic screening strategy for long-range sequence analysis. High-throughput, long-read sequencing was then performed on cDNA libraries from major tumor histotypes and corresponding normal tissues. These analyses led to the identification of 378 chimeras, with an unexpectedly high frequency of expression (˜2 x 10(-5) of all mRNA). Functional assays in breast and ovarian cancer cell lines showed that a large fraction of mRNA chimeras regulates cell replication. Strikingly, chimeras were shown to include both positive and negative regulators of cell growth, which functioned as such in a cell-type-specific manner. Replication-controlling chimeras were found to be expressed by most cancers from breast, ovary, colon, uterus, kidney, lung, and stomach, suggesting a widespread role in tumor development.

Prokineticin ligands and receptors are expressed in the human fetal ovary and regulate germ cell expression of COX2

<p>CONTEXT: Fetal ovarian development and primordial follicle formation underpin future female fertility. Prokineticin (PROK) ligands regulate cell survival, proliferation and angiogenesis in adult reproductive tissues including the ovary. However, their expression and function during fetal ovarian development remains unclear.</p><p>OBJECTIVE: To investigate expression and localization of the PROK ligands, receptors and their downstream transcriptional targets in the human fetal ovary.</p><p>SETTING: This study was conducted at the University of Edinburgh.</p><p>PARTICIPANTS: Ovaries were collected from 37 morphologically normal human fetuses.</p><p>DESIGN AND MAIN OUTCOME MEASURES: mRNA and protein expression of PROK ligands and receptors was determined in human fetal ovaries using qRT-PCR, immunoblotting and immunohistochemistry. Functional studies were performed using a human germ tumour cell line (TCam-2) stably transfected with PROKR1.</p><p>RESULTS: Expression of PROK1 and PROKR1 was significantly higher in mid-gestation ovaries (17-20 weeks) than at earlier gestations (8-11 and 14-16 weeks). PROK2 significantly increased across the gestations examined. PROKR2 expression remained unchanged. PROK ligand and receptor proteins were predominantly localised to germ cells (including oocytes within primordial follicles) and endothelial cells, indicating these cell types to be the targets of PROK signalling in the human fetal ovary. PROK1 treatment of a germ cell line stably-expressing PROKR1 resulted in ERK phosphorylation, and elevated COX2 expression.</p><p>CONCLUSIONS: Developmental changes in expression and regulation of COX2 and pERK by PROK1 suggest that PROK ligands may be novel regulators of germ cell development in the human fetal ovary, interacting within a network of growth and survival factors prior to primordial follicle formation.</p>

Insulin and insulin-like growth factor signaling increases proliferation and hyperplasia of the ovarian surface epithelium and decreases follicular integrity through upregulation of the PI3-kinase pathway

<p>BACKGROUND: The ovarian surface epithelium responds to cytokines and hormonal cues to initiate proliferation and migration following ovulation. Although insulin and IGF are potent proliferative factors for the ovarian surface epithelium and IGF is required for follicle development, increased insulin and IGF activity are correlated with at least two gynecologic conditions: polycystic ovary syndrome and epithelial ovarian cancer. Although insulin and IGF are often components of in vitro culture media, little is known about the effects that these growth factors may have on the ovarian surface epithelium morphology or how signaling in the ovarian surface may affect follicular health and development.</p><p>METHODS: Ovaries from CD1 mice were cultured in alginate hydrogels in the presence or absence of 5 μg/ml insulin or IGF-I, as well as small molecule inhibitors of IR/IGF1R, PI 3-kinase signaling, or MAPK signaling. Tissues were analyzed by immunohistochemistry for expression of cytokeratin 8 to mark the ovarian surface epithelium, Müllerian inhibiting substance to mark secondary follicles, and BrdU incorporation to assess proliferation. Changes in gene expression in the ovarian surface epithelium in response to insulin or IGF-I were analyzed by transcription array. Extracellular matrix organization was evaluated by expression and localization of collagen IV.</p><p>RESULTS: Culture of ovarian organoids with insulin or IGF-I resulted in formation of hyperplastic OSE approximately 4-6 cell layers thick with a high rate of proliferation, as well as decreased MIS expression in secondary follicles. Inhibition of the MAPK pathway restored MIS expression reduced by insulin but only partially restored normal OSE growth and morphology. Inhibition of the PI 3-kinase pathway restored MIS expression reduced by IGF-I and restored OSE growth to a single cell layer. Insulin and IGF-I altered organization of collagen IV, which was restored by inhibition of PI 3-kinase signaling.</p><p>CONCLUSIONS: While insulin and IGF are often required for propagation of primary cells, these cytokines may act as potent mitogens to disrupt cell growth, resulting in formation of hyperplastic OSE and decreased follicular integrity as measured by MIS expression and collagen deposition. This may be due partly to altered collagen IV deposition and organization in the ovary in response to insulin and IGF signaling mediated by PI 3-kinase.</p>

The role of transforming growth factor-beta 1 in steroidogenesis, cell proliferation, and apoptosis in cultured bovine granulosa cells

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Studying the cytomechanic aspects of pollen tube growth behavior using Lab-On-Chip technology

L'élongation cellulaire de cellules cultivant bout comme hyphae fongueux, inculquez hairs, des tubes de pollen et des neurones, est limité au bout de la cellule, qui permet à ces cellules d'envahir l'encerclement substrate et atteindre une cible. Les cellules cultivant bout d'équipement sont entourées par le mur polysaccharide rigide qui régule la croissance et l'élongation de ces cellules, un mécanisme qui est radicalement différent des cellules non-walled. La compréhension du règlement du mur de cellule les propriétés mécaniques dans le contrôle de la croissance et du fonctionnement cellulaire du tube de pollen, une cellule rapidement grandissante d'équipement, est le but de ce projet. Le tube de pollen porte des spermatozoïdes du grain de pollen à l'ovule pour la fertilisation et sur sa voie du stigmate vers l'ovaire le tube de pollen envahit physiquement le stylar le tissu émettant de la fleur. Pour atteindre sa cible il doit aussi changer sa direction de croissance les temps multiples. Pour évaluer la conduite de tubes de pollen grandissants, un dans le système expérimental vitro basé sur la technologie de laboratoire-sur-fragment (LOC) et MEMS (les systèmes micro-électromécaniques) ont été conçus. En utilisant ces artifices nous avons mesuré une variété de propriétés physiques caractérisant le tube de pollen de Camélia, comme la croissance la croissance accélérée, envahissante et dilatant la force. Dans une des organisations expérimentales les tubes ont été exposés aux ouvertures en forme de fente faites de l'élastique PDMS (polydimethylsiloxane) la matière nous permettant de mesurer la force qu'un tube de pollen exerce pour dilater la croissance substrate. Cette capacité d'invasion est essentielle pour les tubes de pollen de leur permettre d'entrer dans les espaces intercellulaires étroits dans les tissus pistillar. Dans d'autres essais nous avons utilisé l'organisation microfluidic pour évaluer si les tubes de pollen peuvent s'allonger dans l'air et s'ils ont une mémoire directionnelle. Une des applications auxquelles le laboratoire s'intéresse est l'enquête de processus intracellulaires comme le mouvement d'organelles fluorescemment étiqueté ou les macromolécules pendant que les tubes de pollen grandissent dans les artifices LOC. Pour prouver que les artifices sont compatibles avec la microscopie optique à haute résolution et la microscopie de fluorescence, j'ai utilisé le colorant de styryl FM1-43 pour étiqueter le système endomembrane de tubes de pollen de cognassier du Japon de Camélia. L'observation du cône de vésicule, une agrégation d'endocytic et les vésicules exocytic dans le cytoplasme apical du bout de tube de pollen, n'a pas posé de problèmes des tubes de pollen trouvés dans le LOC. Pourtant, le colorant particulier en question a adhéré au sidewalls du LOC microfluidic le réseau, en faisant l'observation de tubes de pollen près du difficile sidewalls à cause du signal extrêmement fluorescent du mur. Cette propriété du colorant pourrait être utile de refléter la géométrie de réseau en faisant marcher dans le mode de fluorescence.

Differential expression of mRNAs encoding co-receptor (betaglycan) and activin type-I preovulatory and prehierarchical follicles of the putative inhibin and type-II receptors in the laying hen ovary

Ovarian follicle development is primarily regulated by an interplay between the pituitary gonadotrophins, LH and FSH, and ovary-derived steroids. Increasing evidence implicates regulatory roles of transforming growth factor-beta (TGF beta) superfamily members, including inhibins and activins. The aim of this study was to identify the expression of mRNAs encoding key receptors of the inhibin/activin system in ovarian follicles ranging from 4 mm in diameter to the dominant F1 follicle (similar to 40 turn). Ovaries were collected (n=16) from inid-sequence hens maintained on a long-day photoschedule (16h of light:8 h of darkness). All follicles removed were dissected into individual granulosa and thecal layers. RNA was extracted and cDNA synthesized. Real-time quantitative PCR was used to quantify the expression of niRNA encoding betaglycan, activin receptor (ActR) subtypes (type-I, -IIA and -IIB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); receptor expression data were normalized to GAPDH expression. Detectable levels of ActRI, -IIA and -IIB and the inhibin co-receptor (betaglycan) expression were found in all granulosa and thecal layers analysed. Granulosa ActRI mRNA peaked (P < 0(.)05) in 8-9(.)9 mm follicles, whereas ActRIIA rose significantly from 6-7(.)9 mm to 8-9(.)9 nun, before filling to F3/2; levels then rose sharply (3-fold) to F1 levels. Granulosa betaglycan niRNA expression rose 3-fold from 4-5(.)9 min to 8-9(.)9 mm, before falling 4-fold to F3/2; levels then rose sharply (4-fold) to F1 levels. ActRIIB levels did not vary significantly during follicular development. Thecal ActRI mRNA expression was similar from 4-7(.)9 mm then decreased significantly to a nadir at the F4 position, before increasing 2-fold to the F1 (P < 0(.)05). Although thecal ActRIIB and -IIA expression did not vary significantly from 4 nim to F3, ActRIIB expression increased significantly (2-fold) from F3 to F1 and ActIIA, increased 22-fold from F2 to F1 (P < 0(.)05). Thecal betaglycan fell to a nadir at F6 after follicle selection; levels then increased significantly to F2, before filling similar to 50% in the F I. In all follicles studied expression of betaglycan and ActRI (granulosa: 1-0(.)65, P < 0-001, n=144/group; theca: r=0(.)49, P < 0-001, n=144/group) was well correlated. No significant correlations were identified between betaglycan and ActRIIA or -IIB. Considering all follicles analysed, granulosa mRNA expression of betaglycan, ActRI ActRIIA and ActRIIB were all significantly lower than in corresponding thecal tissue (betaglycan, 11(.)4-fold; ActRIIB, 5(.)1-fold; ActR(.) 3-8-fold: ActRIIA, 2(.)8-fold). The co-localization of type-I and -II activin receptors and betaglycan on granulosa and thecal cells are consistent with a local auto/paracrine role of inhibins and activins in modulating ovarian follicle development, selection and progression in the domestic fowl.

Differential expression of mRNAs encoding co-receptor (betaglycan) and activin type-I preovulatory and prehierarchical follicles of the putative inhibin and type-II receptors in the laying hen ovary

Ovarian follicle development is primarily regulated by an interplay between the pituitary gonadotrophins, LH and FSH, and ovary-derived steroids. Increasing evidence implicates regulatory roles of transforming growth factor-beta (TGF beta) superfamily members, including inhibins and activins. The aim of this study was to identify the expression of mRNAs encoding key receptors of the inhibin/activin system in ovarian follicles ranging from 4 mm in diameter to the dominant F1 follicle (similar to 40 turn). Ovaries were collected (n=16) from inid-sequence hens maintained on a long-day photoschedule (16h of light:8 h of darkness). All follicles removed were dissected into individual granulosa and thecal layers. RNA was extracted and cDNA synthesized. Real-time quantitative PCR was used to quantify the expression of niRNA encoding betaglycan, activin receptor (ActR) subtypes (type-I, -IIA and -IIB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); receptor expression data were normalized to GAPDH expression. Detectable levels of ActRI, -IIA and -IIB and the inhibin co-receptor (betaglycan) expression were found in all granulosa and thecal layers analysed. Granulosa ActRI mRNA peaked (P < 0(.)05) in 8-9(.)9 mm follicles, whereas ActRIIA rose significantly from 6-7(.)9 mm to 8-9(.)9 nun, before filling to F3/2; levels then rose sharply (3-fold) to F1 levels. Granulosa betaglycan niRNA expression rose 3-fold from 4-5(.)9 min to 8-9(.)9 mm, before falling 4-fold to F3/2; levels then rose sharply (4-fold) to F1 levels. ActRIIB levels did not vary significantly during follicular development. Thecal ActRI mRNA expression was similar from 4-7(.)9 mm then decreased significantly to a nadir at the F4 position, before increasing 2-fold to the F1 (P < 0(.)05). Although thecal ActRIIB and -IIA expression did not vary significantly from 4 nim to F3, ActRIIB expression increased significantly (2-fold) from F3 to F1 and ActIIA, increased 22-fold from F2 to F1 (P < 0(.)05). Thecal betaglycan fell to a nadir at F6 after follicle selection; levels then increased significantly to F2, before filling similar to 50% in the F I. In all follicles studied expression of betaglycan and ActRI (granulosa: 1-0(.)65, P < 0-001, n=144/group; theca: r=0(.)49, P < 0-001, n=144/group) was well correlated. No significant correlations were identified between betaglycan and ActRIIA or -IIB. Considering all follicles analysed, granulosa mRNA expression of betaglycan, ActRI ActRIIA and ActRIIB were all significantly lower than in corresponding thecal tissue (betaglycan, 11(.)4-fold; ActRIIB, 5(.)1-fold; ActR(.) 3-8-fold: ActRIIA, 2(.)8-fold). The co-localization of type-I and -II activin receptors and betaglycan on granulosa and thecal cells are consistent with a local auto/paracrine role of inhibins and activins in modulating ovarian follicle development, selection and progression in the domestic fowl.

Regulation and action of fibroblast growth factor 17 in bovine follicles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expression of fibroblast growth factor-8 and its cognate receptors, fibroblast growth factor receptor (FGFR)-3c and-4, in fetal bovine preantral follicles

Paracrine cell signaling is thought to be important for ovarian follicle development, and a role for some members of the fibroblast growth factor (FGF) family have been suggested. In the present study, we tested the hypothesis that FGF-8 and its cognate receptors (FGFR-3c and FGFR-4) are expressed in bovine preantral follicles. Reverse transcription-polymerase chain reaction was used to amplify bovine FGF-8, FGFR-3c, and FGFR-4 from preantral follicle samples and a variety of fetal and adult tissues. All three genes were widely expressed in fetal tissues, with a restricted expression pattern in adult tissues. FGF-8 and FGFR-3c were expressed in secondary follicles in 70% of fetuses examined, whereas FGFR-4 expression was significantly less frequent (20%). FGFR-3c expression frequency was significantly lower in primordial compared to secondary follicles, and FGF-8 expression showed a similar trend. FGFR-4 was only observed when all follicle classes of an individual were expressing both FGF-8 and FGFR-3c. We conclude that FGF-8 and its receptors are expressed in preantral follicles in a developmentally regulated manner. (C) 2005 Wiley-Liss, Inc.

Fibroblast growth factor-10 maintains the survival and promotes the growth of cultured goat preantral follicles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The fibroblast growth factor family: involvement in the regulation of folliculogenesis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Testosterone stimulates growth and secretory activity of the female prostate in the adult gerbil (Meriones unguiculatus)

The prostate of the female gerbil (Meriones unguiculatus) is similar to the human female prostate (Skene gland) and, despite its reduced size, it is functional and shows secretory activity. However, virtually nothing is known about its physiological regulation. This study was thus undertaken to evaluate the behavior of the gerbil female prostate in a hyperandrogenic condition. Adult females received subcutaneous injections of testosterone cypionate (1 mg/kg body weight every 48 h) up to 21 days. Circulating levels of testosterone and estradiol were monitored, and the prostate and ovaries subjected to structural and immunocytochemical analyses. The treatment resulted in sustained high levels of circulating testosterone, and caused a transient increase in estradiol. There was an increase in epithelial cell proliferation accompanied by significant reorganization of the epithelium and an apparent reduction in secretory activity, followed by a progressive increase in luminal volume density and accumulation of secretory products. Immunocytochemistry identified the expression of androgen receptor and a prostate-specific antigen (PSA)-related antigen in prostatic epithelial cells. A circulating PSA-related antigen was also found, and its concentration showed strong negative correlation with circulating estrogen. Epithelial dysplasia was detected in the prostate of treated females. Analysis of the ovaries showed the occurrence of a polycystic condition and stromal cell hyperplasia. The results indicate that testosterone has a stimulatory effect on the female prostate, inducing epithelial cell proliferation, differentiation, secretory activity, and dysplasia. The results also suggest that prostatic growth and activity, polycystic ovaries, and ovarian stromal cell hyperplasia are related to a hyperandrogenic condition in females.

Expression and Function of Fibroblast Growth Factor 18 in the Ovarian Follicle in Cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)