Icariin impacts molecular changes in estrogen receptors but not other reproductive parameters in female mice

2018 ◽  
Author(s):  
Caroline W Muneri ◽  
Seth Y Afedo ◽  
Yaping Xu ◽  
Guochao Ni ◽  
Qingxia Shen ◽  
...  
Keyword(s):  
Estrogen Receptors ◽  
In Vitro Maturation ◽  
Ovarian Development ◽  
Regulatory Protein ◽  
Reproductive Parameters ◽  
Rt Pcr ◽  
Steroidogenic Acute Regulatory ◽  
Estrogen Receptor Pathway ◽  
Cytochrome P450 Family

This study was conducted to assess the effects of icariin (ICA) an ingredient of Epimedium species, on reproductive parameters in mice. Serum, uterus and ovarian samples were collected after treatment. We determined the reproductive index (RI) and in vitro maturation (IVM) rates of oocytes. The serum was tested for 17b-estradiol (17b-E2) and progesterone (P4) using radioimmunoassay (RIA) and ovaries were analyzed for mRNA expression of Steroidogenic acute regulatory protein (Star), Cytochrome p450 family 19 subfamilies a polypeptide 1 (Cyp19a1) and 17b-hydroxysteroid dehydrogenase (Hsd17b1), genes using Real-time Quantitative RT-PCR (RT-qPCR). Protein expression of estrogen receptors (ER) a, b, and progesterone receptor A (PRA) were detected using western blot (WB). ERa from treatment group was increased (p less then 0.05) while ERb and PRA were decreased (p less then0.05). ICA regulated ovarian development through the estrogen receptor pathway. ERa expression was evidently increased and this may be the pathway that is utilized for its mode of action.

224 LOCALIZATION OF NITRIC OXIDE SYNTHASE ACTIVITY IN BUFFALO (BUBALUS BUBALIS) OOCYTES AND EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 211
Author(s):  
K. R. Babu ◽  
R. Sharma ◽  
K. P. Singh ◽  
A. George ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Epifluorescence Microscopy ◽  
Rt Pcr ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Nos Isoforms

Ovarian nitric oxide (NO) and that produced within the oocytes and embryos have been reported to play important roles in oocyte meiotic maturation and embryo development. Production of NO is catalyzed by NO synthase (NOS), which exists in 3 isoforms, the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms and the inducible (iNOS) isoform. We have previously shown that low concentrations of NO stimulate and high concentrations inhibit embryo development, and that endogenous NO produced by iNOS is necessary for optimal embryo development in the buffalo. The present study was aimed at localizing different isoforms of NOS and examining their relative mRNA abundance in buffalo oocytes and embryos. Oocytes from slaughterhouse ovaries were subjected to in vitro maturation in 100-μL droplets (10 to 15 oocytes/droplet) of in vitro maturation medium (TCM-199 + 10% FBS + 5 μg mL–1 of pFSH + 1 μg mL–1 of oestradiol-17β + 0.81 mM sodium pyruvate + 10% buffalo follicular fluid + 50 μg mL–1 of gentamicin) for 24 h in a CO2 incubator (5% CO2 in air) at 38.5°C. In vitro fertilization was carried out by incubating in vitro-matured oocytes with 2 to 4 million spermatozoa mL–1 for 18 h. The presumed zygotes were cultured on original beds of cumulus cells in in vitro culture medium (mCR2aa + 0.6% BSA + 10% FBS) for up to 8 days post-insemination. Immature and in vitro-matured oocytes and embryos at the 2-cell, 4-cell, 8- to 16-cell, morula, and blastocyst stages were examined for the presence of NOS isoforms by indirect immunofluorescence staining using epifluorescence microscopy and RT-PCR. Each experiment was repeated in triplicate, and data were analysed using one-way ANOVA, after arcsine transformation of percentage values. Expression of all 3 NOS isoforms was detected inside the cytoplasm, in all the stages of oocytes and embryos examined, by both immunofluorescence and RT-PCR. Abundance of the iNOS transcript was significantly higher (P ≤ 0.01) in the morula and blastocyst stages compared with that in immature and in vitro-matured oocytes and in embryos at the 2-cell, 4-cell, and 8- to 16-cell stages, indicating that its expression was up-regulated at the 8- to 16-cell stage. The expression of eNOS was significantly higher (P ≤ 0.05) in the immature and mature oocytes and in 8- to 16-cell stage embryos, morulae, and blastocysts than in the early-cleavage embryos at the 2- and 4-cell stages, indicating that it was down-regulated after fertilization and was up-regulated again at the 8- to 16-cell stage. Abundance of the nNOS transcript was not significantly different among all the stages of oocytes and embryos examined. These results demonstrate that different NOS isoforms are expressed in a dynamic manner during embryonic development in the buffalo. The role of an increase in expression of iNOS and eNOS at the 8- to 16-cell stage, at which a developmental block occurs in this species, needs to be examined.

scholarly journals Development of an experimental ovarian tumor: immunocytochemical analysis

2002 ◽  
pp. 387-395 ◽  
Author(s):  
E Sorianello ◽  
S Fritz ◽  
C Beyer ◽  
DB Hales ◽  
A Mayerhofer ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Regulatory Protein ◽  
Tumor Development ◽  
Female Rats ◽  
Nuclear Antigen ◽  
Normal Ovary ◽  
Rt Pcr ◽  
Thecal Cells ◽  
Junction Protein ◽  
Steroidogenic Acute Regulatory

OBJECTIVE: The aim of the present work was to study whether immunocytochemical parameters present in the normal ovary were altered after tumor development under high gonadotropin levels. METHODS: Ovarian tumors (luteoma): castrated female rats had an ovary grafted into the spleen; tumors were left to develop for 1, 2, 3 or 7 months. The presence of apoptotic cells (TUNEL method) and the expression of proliferating cell nuclear antigen (PCNA), gap junction protein (Cx43), steroidogenic acute regulatory protein (StAR), aromatase and synaptosome-associated protein of 25 kDa (SNAP-25) were determined by immunocytochemistry. Some of these findings were confirmed by RT-PCR (Cx43, StAR, SNAP-25). Inhibin subunit mRNAs were investigated by Northern blot. RESULTS: PCNA staining of tumors was mainly found in granulosa cells of transforming follicles and was absent from luteinized follicles. A nearly complete absence of apoptosis was observed. Cx43 was mainly found in follicles, while it was very weakly expressed or absent in luteinized follicles. StAR protein expression, indicating active steroidogenesis, was demonstrated only in luteinized follicles and in thecal cells, but was absent from granulosa cells. Aromatase immunoreactivity was very intense in granulosa and also present in luteal cells. Membrane-associated and cytoplasmic SNAP-25 immunostaining was determined in patches of endocrine cells in the follicles, as well as in the luteinized follicles. The expression of mRNAs for Cx43, StAR and SNAP-25 (RT-PCR) and inhibin subunits (Northern blots) were confirmed in 1-, 3- and 7-month-old tumors. CONCLUSIONS: These results indicated that luteoma most likely develop from unruptured follicles by hypertrophy and proliferation of follicular cells. Circulating gonadotropins seem to play a fundamental role in maintaining the expression of proteins typically expressed in normal ovary, while avoiding apoptosis in this tissue.

scholarly journals Peripheral-Type Benzodiazepine Receptor-Mediated Action of Steroidogenic Acute Regulatory Protein on Cholesterol Entry into Leydig Cell Mitochondria

2005 ◽  
Vol 19 (2) ◽  
pp. 540-554 ◽  
Author(s):  
Thierry Hauet ◽  
Zhi-Xing Yao ◽  
Himangshu S. Bose ◽  
Christopher T. Wall ◽  
Zeqiu Han ◽  
...  
Keyword(s):  
Regulatory Protein ◽  
Benzodiazepine Receptor ◽  
Steroidogenic Acute Regulatory Protein ◽  
Rate Determining Step ◽  
Human Choriogonadotropin ◽  
Leydig Tumor Cells ◽  
Peripheral Type ◽  
Steroidogenic Acute Regulatory ◽  
And Function

Abstract Hormone-induced steroid biosynthesis begins with the transfer of cholesterol from intracellular stores into mitochondria. Steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) have been implicated in this rate-determining step of steroidogenesis. MA-10 mouse Leydig tumor cells were treated with and without oligodeoxynucleotides (ODNs) antisense to PBR and StAR followed by treatment with saturating concentrations of human choriogonadotropin. Treatment with ODNs antisense but not missense for both proteins inhibited the respective protein expression and the ability of the cells to synthesize steroids in response to human choriogonadotropin. Treatment of the cells with either ODNs antisense to PBR or a transducible peptide antagonist to PBR resulted in inhibition of the accumulation of the mature mitochondrial 30-kDa StAR protein, suggesting that the presence of PBR is required for StAR import into mitochondria. Addition of in vitro transcribed/translated 37-kDa StAR or a fusion protein of Tom20 (translocase of outer membrane) and StAR (Tom/StAR) to mitochondria isolated from control cells increased pregnenolone formation. Mitochondria isolated from cells treated with ODNs antisense, but not missense, to PBR failed to form pregnenolone and respond to either StAR or Tom/StAR proteins. Reincorporation of in vitro transcribed/translated PBR, but not PBR missing the cholesterol-binding domain, into MA-10 mitochondria rescued the ability of the mitochondria to form steroids and the ability of the mitochondria to respond to StAR and Tom/StAR proteins. These data suggest that both StAR and PBR proteins are indispensable elements of the steroidogenic machinery and function in a coordinated manner to transfer cholesterol into mitochondria.

130 ANTI-APOPTOTIC pTEGT SHOWS A DIFFERENTIAL mRNA TRANSCRIPT LEVEL DURING IN VITRO MATURATION AND EARLY DEVELOPMENT OF PORCINE IVP EMBRYO

2006 ◽  
Vol 18 (2) ◽  
pp. 173
Author(s):  
J.-Y. Kim ◽  
J.-H. Kim ◽  
H.-Y. Choi ◽  
B.-W. Kim ◽  
S. J. Uhm ◽  
...  
Keyword(s):  
Early Development ◽  
In Vitro Maturation ◽  
Transcript Level ◽  
Early Embryo ◽  
Mrna Transcript ◽  
Rt Pcr ◽  
Mrna Transcript Level ◽  
Embryonic Genome ◽  
Apoptotic Effect

It is well known that very early development of the mammalian pre-implantation embryo is regulated by gene transcripts and proteins stored in the oocyte and that the embryonic genome gains control of development following 1 to 3 cleavage divisions. An active transcription and translation is required for chromatin condensation and germinal vesicle breakdown in pig oocyte. The transition from maternal to embryonic control of development is a gradual event, and following this transition, the maternally derived transcripts and proteins are gradually degraded. Successful embryonic development is dependent on the temporal and stage-specific expression of proper genes, but information on specific gene expression during early stages before zygotic gene activation (ZGA) is limited. Before activation of the embryonic genome, mRNA and proteins synthesized during oocyte growth and maturation contribute to early development. In this study, we compared the mRNA transcripts level among porcine immature, in vitro-matured and cleaved 2- to 4-cell stage embryos after in vitro fertilization to identify genes that show differential mRNA transcript levels during maturation and very early embryonic development. For the first strand cDNA synthesis, oligo (dT) primers were added to the total RNA isolated from each sample. Using annealing control primer (ACP)-based GeneFishing PCR, we detected tens of different genes showing differential mRNA transcript level (DRTL) and nine DRTL genes were identified to be KCRF, CAMSAP1, SMP1, FLJ20647, LOC132321, NADH1, NADH6, HERC3, and TEGT. Of 9 DRTL genes, TEGT showed higher mRNA transcript level at the immaturation stage, and mRNA transcript levels of the other 8 genes were increased after in vitro maturation. Therefore, we focused on TEGT (testis enhanced gene transcript), which is highly expressed in testis and also in oocytes before in vitro maturation. Differential mRNA transcripts pattern of CAMSAP1 and TEGT were confirmed using RT-PCR and real-time RT-PCR. Porcine TEGT (pTEGT) was cloned and sequenced to have an ORF of 714 bp nucleotides and to encode an integral membrane protein. When overexpressed in HEK293 cells, pTEGT suppressed apoptosis induced by etoposide. We found that pTEGT, but not TEGT-C (C-terminal deletion mutant), inhibited etoposide- and staurosporine-induced cell death. Next, we found that introduction of TEGT siRNA suppressed the anti-apoptotic effect of TEGT. Interestingly, expression of TEGT suppressed etoposide-induced ERK activation, suggesting that ERK phosphorylation is involved in the anti-apoptotic function of the gene. Several reports showed that apoptosis and MAP kinase signaling pathways play important roles in oocyte maturation and early embryo development. Therefore, the anti-apoptotic effect of TEGT was suggested to play a key role in the normal ooctye maturation and early embryo development. This work was supported by the Research Project on the Production of Bio-organs, Ministry of Agriculture and Forestry, Republic of Korea.

The Vent-Like Homeobox Gene Ventx2 Is a Novel Candidate for a Human Hematopoietic Regulatory Protein.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2278-2278
Author(s):  
Natalia Arseni ◽  
Farid Ahmed ◽  
Timothy J. Sadlon ◽  
Richard J. d’Andrea ◽  
Wolfgang Hiddemann ◽  
...  
Keyword(s):  
Regulatory Protein ◽  
Myeloid Cells ◽  
Homeobox Gene ◽  
Rt Pcr ◽  
Granulocytic Differentiation ◽  
Hematopoietic Stem ◽  
Hematopoietic Development ◽  
Functional Relevance ◽  
And Function

Abstract Identification of the genes which are critically involved in normal and leukemic hematopoiesis is a major goal for experimental and clinical hematology. Recent data indicate that a variety of regulatory molecules active in early development may also play a role in the maintenance of hematopoietic stem cells with repopulating activity. Since it was shown, that the Xvent-2 homeobox gene is part of the BMP-4 signalling pathway in Xenopus it is of particular interest to examine the expression profile and function of the human homologue Ventx2 in hematopoietic development. We first analysed Ventx2 expression by RT-PCR in CD3, CD19 and CD33 cells highly purified by FACS sort from peripheral blood of healthy donors. Expression of Ventx2 was detected in T- and B- as well as differentiated myeloid cells indicating that Ventx2 is expressed in multiple hematopoietic lineages. Furthermore, VENTX2 expression was recurrently detected in bone marrow samples from AML patients at diagnosis as determined by RT-PCR (n=6). In an attempt to characterize the functional relevance of Ventx2 expression for hematopoietic development we retrovirally engineered murine hematopoietic progenitor cells to constitutively express the gene using a MSCV-based retroviral construct with an IRES-EGFP cassette. Successfully transduced cells were injected into lethally irradiated mice or used for in vitro experiments. At the level of the clonogenic progenitor VENTX2 induced a 3fold increase in the number of CFU-G (n=5; p<0.001) compared to the GFP control (62 versus 25 CFU-G, respectively, per 1000 initially plated cells) without increasing the total number of colonies, indicating that VENTX2 promoted granulocytic differentiation in vitro. Re-plating assays confirmed the effect of the homeobox gene with an over 9fold increase in the number of secondary CFU-G (511 vs. 54, respectively, per 1000 initially plated cells). When the effect of VENTX-2 on the frequency of LTC-IC was determined by limiting dilution assay (n=2), no major differences were detected between the homeobox gene and the control arm (453 LTC-IC vs. 801 LTC-IC per 1x106 cells, respectively, p = n.s.). Furthermore, the number of colonies generated per LTC-IC did not significantly differ between the two arms (17 colonies for VENTX2 and 26 colonies for the control). In NOD/SCID mice VENTX2 induced a 2.9fold increase in the proportion of CD15+ mature myeloid cells within the GFP-positive compartment (n=7) compared to the control (n=9)(6.4 % vs. 2.2 %, respectively; p<0.02), translating into 4 x 106 (± 1 x 106) human CD15+ /GFP+ cells per mouse in the VENTX2 group compared to 2x106 cells (± 6 x 105) in the control. These findings characterize VENTX2 as a novel regulatory protein in human hematopoiesis and add information about the role of non-clustered homeobox genes in early blood development.

scholarly journals Gonadotropin Stimulation of Ovarian Fractalkine Expression and Fractalkine Augmentation of Progesterone Biosynthesis by Luteinizing Granulosa Cells

Endocrinology ◽  
2008 ◽  
Vol 149 (6) ◽  
pp. 2782-2789 ◽  
Author(s):  
Ping Zhao ◽  
Ananya De ◽  
Zeng Hu ◽  
Jing Li ◽  
Sabine M. Mulders ◽  
...  
Keyword(s):  
Real Time ◽  
Granulosa Cells ◽  
Regulatory Protein ◽  
Rt Pcr ◽  
Paracrine Factors ◽  
Transcript Levels ◽  
Preovulatory Follicles ◽  
Major Increase ◽  
Steroidogenic Acute Regulatory ◽  
Stimulation Of

Recent studies indicated that ovarian functions are regulated by diverse paracrine factors induced by the preovulatory increases in circulating LH. Based on DNA microarray analyses and real-time RT-PCR, we found a major increase in the transcript levels of a chemokine fractalkine after human chorionic gonadotropin (hCG) treatment during the preovulatory period in gonadotropin-primed immature mice and rats. Although CX3CR1, the seven-transmembrane receptor for fractalkine, was also found in murine ovaries, its transcripts displayed minimal changes. Using tandem RT-PCR and immunohistochemistry, fractalkine transcripts and proteins were localized in cumulus, mural granulosa, and theca cells as well as the oocytes, whereas CX3CR1 was found in the same cells except the oocyte. Real-time RT-PCR further indicated the hCG induction of fractalkine transcripts in different ovarian compartments, with the highest increases found in granulosa cells. In cultured granulosa cells, treatment with fractalkine augmented hCG stimulation of progesterone but not estradiol and cAMP biosynthesis with concomitant increases in transcript levels for key steroidogenic enzymes (steroidogenic acute regulatory protein, CYP11A, and 3β-hydroxysteroid dehydrogenase). In cultured preovulatory follicles, treatment with fractalkine also augmented progesterone production stimulated by hCG. Furthermore, treatment with fractalkine augmented the phosphorylation of P38 MAPK in cultured granulosa cells. The present data demonstrated that increases in preovulatory LH/hCG induce the expression of fractalkine to augment the luteinization of preovulatory granulosa cells and suggest the fractalkine/CX3CR1 signaling system plays a potential paracrine/autocrine role in preovulatory follicles.

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