scholarly journals Zona pellucida protein ZP2 is expressed in the oocyte of Japanese quail (Coturnix japonica)

Reproduction ◽  
2010 ◽  
Vol 139 (2) ◽  
pp. 359-371 ◽  
Author(s):  
Mihoko Kinoshita ◽  
Daniela Rodler ◽  
Kenichi Sugiura ◽  
Kayoko Matsushima ◽  
Norio Kansaku ◽  
...  
Keyword(s):  
Western Blot ◽  
Zona Pellucida ◽  
Rnase Protection Assay ◽  
Follicular Development ◽  
Amorphous Structure ◽  
Rnase Protection ◽  
Zona Radiata ◽  
Oocyte Cytoplasm ◽  
First Time

The avian perivitelline layer (PL), a vestment homologous to the zona pellucida (ZP) of mammalian oocytes, is composed of at least three glycoproteins. Our previous studies have demonstrated that the matrix's components, ZP3 and ZPD, are synthesized in ovarian granulosa cells. Another component, ZP1, is synthesized in the liver and is transported to the ovary by blood circulation. In this study, we report the isolation of cDNA encoding quail ZP2 and its expression in the female bird. By RNase protection assay and in situ hybridization, we demonstrate that ZP2 transcripts are restricted to the oocytes of small white follicles (SWF). The expression level of ZP2 decreased dramatically during follicular development, and the highest expression was observed in the SWF. Western blot and immunohistochemical analyses using the specific antibody against ZP2 indicate that the 80 kDa protein is the authentic ZP2, and the immunoreactive ZP2 protein is also present in the oocytes. Moreover, ultrastructural analysis demonstrated that the immunoreactive ZP2 localizes to the zona radiata, the perivitelline space, and the oocyte cytoplasm in the SWF. By means of western blot analysis and immunofluorescence microscopy, we detected a possible interaction of the recombinant ZP2 with ZP3 and that this interaction might lead to the formation of amorphous structure on the cell surface. These results demonstrate for the first time that the avian ZP gene is expressed in the oocyte, and that the ZP2 protein in the oocyte might play a role for the PL formation in the immature follicles of the ovary.

Integrin alpha subunit mRNAs are differentially expressed in early Xenopus embryos

Development ◽  
1993 ◽  
Vol 117 (4) ◽  
pp. 1239-1249 ◽  
Author(s):  
C.A. Whittaker ◽  
D.W. DeSimone
Keyword(s):  
In Situ Hybridization ◽  
Rnase Protection Assay ◽  
Early Embryo ◽  
Mrna Levels ◽  
Rnase Protection ◽  
Dorsal Midline ◽  
Transmembrane Receptors ◽  
Early Embryos ◽  
Whole Mount

Adhesion of cells to extracellular matrix proteins is mediated, in large part, by transmembrane receptors of the integrin family. The identification of specific integrins expressed in early embryos is an important first step to understanding the roles of these receptors in developmental processes. We have used polymerase chain reaction methods and degenerate oligodeoxynucleotide primers to identify and clone Xenopus integrin alpha subunits from neurula-stage (stage 17) cDNA. Partial cDNAs encoding integrin subunits alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and an alpha IIb-related subunit were cloned and used to investigate integrin mRNA expression in early embryos by RNase protection assay and whole-mount in situ hybridization methods. Considerable integrin diversity is apparent early in development with integrins alpha 2, alpha 3, alpha 4, alpha 5 and alpha 6 each expressed by the end of gastrulation. Both alpha 3 and alpha 5 are expressed as maternal mRNAs. Zygotic expression of alpha 2, alpha 3, alpha 4 and alpha 6 transcripts begins during gastrulation. Integrin alpha 5 is expressed at relatively high levels during cleavage, blastula and gastrula stages suggesting that it may represent the major integrin expressed in the early embryo. We demonstrated previously that integrin beta 1 protein synthesis remains constant following induction of stage 8 animal cap cells with activin (Smith, J. C., Symes, K., Hynes, R. O. and DeSimone, D. W. (1990) Development 108, 289–298.). Here we report that integrin alpha 3, alpha 4 and alpha 6 mRNA levels increase following induction with 10 U/ml activin-A whereas alpha 5, beta 1 and beta 3 mRNA levels remain unchanged. Whole-mount in situ hybridization reveals that alpha 3 mRNAs are expressed by cells of the involuting mesoderm in the dorsal lip region of early gastrulae. As gastrulation proceeds, alpha 3 expression is localized to a stripe of presumptive notochordal cells along the dorsal midline. In neurulae, alpha 3 mRNA is highly expressed in the notochord but becomes progressively more restricted to the caudalmost portion of this tissue as development proceeds from tailbud to tadpole stages. In addition, alpha 3 is expressed in the forebrain region of later stage embryos. These data suggest that integrin-mediated adhesion may be involved in the process of mesoderm involution at gastrulation and the organization of tissues during embryogenesis.

scholarly journals C-kit gene is expressed by skin mast cells in embryos but not in puppies of Wsh/Wsh mice: age-dependent abolishment of c-kit gene expression

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3509-3516 ◽  
Author(s):  
M Yamazaki ◽  
T Tsujimura ◽  
E Morii ◽  
K Isozaki ◽  
H Onoue ◽  
...  
Keyword(s):  
Mast Cells ◽  
Mrna Expression ◽  
Messenger Rna ◽  
Rnase Protection Assay ◽  
Coding Region ◽  
Carboxypeptidase A ◽  
Rnase Protection ◽  
Kit Gene ◽  
Age Dependent

Abstract The Wsh is a mutant allele at the W (c-kit) locus of mice, but no significant abnormalities are found at the coding region of the Wsh allele. Since cultured mast cells derived from the spleen of Wsh/Wsh mice do not express messenger RNA (mRNA) of c-kit, we studied the interrelation between the number of mast cells and the magnitude of c- kit mRNA expression in the skin of Wsh/Wsh mice of various ages. The number of mast cells in the skin of Wsh/Wsh embryos of 18 days postcoitum (pc) was approximately 40% that of normal control (+/+) embryos, but the number of mast cells decreased exponentially after birth; the number dropped to 0.6% that of +/+ mice at day 150 after birth. A weak but apparent signal of c-kit mRNA was detectable in the skin of 18-day pc Wsh/Wsh embryos by RNase protection assay but not in the skin of 5-day-old Wsh/Wsh mice. The number of c-kit protein- containing cells was significantly greater in the skin of 18-day pc Wsh/Wsh embryos than in the skin of 5-day-old Wsh/Wsh mice. The abolishment of c-kit mRNA expression appeared to be specific, because the expression of mast cell carboxypeptidase A mRNA but not of c-kit mRNA was detectable by in situ hybridization in skin mast cells of 5- day-old Wsh/Wsh mice. Taken together, the expression of c-kit mRNA was abolished first, then the content of c-kit protein dropped to undetectable levels, and then the disappearance of Wsh/Wsh mast cells themselves followed.

scholarly journals Regional and maturation-associated expression of endothelin 2 in rat gastrointestinal tract.

1995 ◽  
Vol 43 (2) ◽  
pp. 203-209 ◽  
Author(s):  
S M de la Monte ◽  
T Quertermous ◽  
C C Hong ◽  
K D Bloch
Keyword(s):  
Gene Expression ◽  
Gastrointestinal Tract ◽  
Stromal Cells ◽  
Rnase Protection Assay ◽  
Secretory Activity ◽  
Intestinal Epithelial ◽  
Rnase Protection ◽  
Adult Rat ◽  
Vasoactive Peptides

Endothelin 2 (ET2), also referred to as vasoactive intestinal contractor peptide, is a member of a family of vasoactive peptides. ET2 is a potent constrictor of intestinal smooth muscle, and the mRNA that encodes it has been detected in murine intestinal extracts. To further investigate the potential physiological roles of ET2, we characterized the cellular distribution of ET2 gene expression in adult rat gastrointestinal tract. Using an RNAse protection assay, an overall proximal to distal gradient of increasing ET2 gene expression was observed from stomach to colon. In situ hybridization studies confirmed this finding and demonstrated ET2 mRNA localized in lamina propria stromal cells. Moreover, ET2 gene expression in stromal cells increased from crypt to villous tip. The results demonstrate that ET2 is produced by stromal cells in villi throughout the intestine. Increased ET2 gene expression at the villous tip is associated with more mature overlying epithelial cells, suggesting a possible role for this vasoactive peptide in intestinal epithelial differentiation or secretory activity.

Developmental expression of tissue inhibitor of metalloproteinase (TIMP) RNA

Development ◽  
1989 ◽  
Vol 105 (3) ◽  
pp. 575-583 ◽  
Author(s):  
S. Nomura ◽  
B.L. Hogan ◽  
A.J. Wills ◽  
J.K. Heath ◽  
D.R. Edwards
Keyword(s):  
Rnase Protection Assay ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Developmental Expression ◽  
Corpora Lutea ◽  
Tissue Inhibitor ◽  
Rnase Protection ◽  
Rna Probes ◽  
Order Of Magnitude

Single-stranded antisense RNA probes have been used to study the expression of the metalloproteinase inhibitor TIMP (tissue inhibitor of metalloproteinases), during mouse embryogenesis and in adult tissues. Using a sensitive RNase protection assay, low levels of transcript can be detected in a variety of tissues, including maternal deciduum, embryonic kidney, lung and amnion. Higher levels are seen in osteogenic tissues such as calvaria, while the highest level in any tissue is found in the ovary, though even here expression is an order of magnitude below that observed in growth factor-treated fibroblasts in vitro. Using the technique of in situ hybridization, TIMP transcripts can first be detected in osteogenic tissues in the head and limb at about 15.5 days post coitum, and increase in amount until birth. The high levels of TIMP RNA in the ovary are localized to cells of the corpora lutea.

scholarly journals Adrenomedullin expression in rat uterus is correlated with plasma estradiol

2002 ◽  
Vol 282 (1) ◽  
pp. E139-E146 ◽  
Author(s):  
Vicky A. Cameron ◽  
Dominic J. Autelitano ◽  
John J. Evans ◽  
Leigh J. Ellmers ◽  
Eric A. Espiner ◽  
...  
Keyword(s):  
Estrous Cycle ◽  
Rnase Protection Assay ◽  
Estrogen Replacement ◽  
Rat Uterus ◽  
Rnase Protection ◽  
Plasma Progesterone ◽  
Ovx Rats ◽  
Plasma Estradiol ◽  
Estradiol Levels

Levels of expression of adrenomedullin (AM) in the uterus have been reported to vary with the reproductive cycle. This study examines the relationships among uterine AM mRNA, the stage of the estrous cycle, and circulating estradiol and progesterone in cycling rats and in ovariectomized (OVX) rats without or with estrogen replacement (ER). Strong AM mRNA, AM immunoreactivity, and pro-AM NH2-terminal 20 peptide (PAMP) immunoreactivity were observed in endometrial stroma by use of in situ hybridization and immunocytochemistry. Endometrial expression was particularly intense at proestrus and estrus, with weaker expression in the myometrium. By RNase protection assay, significant differences in AM mRNA between the stages of the estrous cycle could not be established. However, levels of AM mRNA were positively correlated with plasma estradiol in cycling rats ( r = 0.56, P < 0.005) and in OVX and ER rats ( r = 0.92, P < 0.001) and were not correlated with plasma progesterone. Levels of AM mRNA were significantly reduced after OVX compared with cycling rats, and ER restored AM mRNA to levels equivalent to those seen at the peak of the cycle (proestrus). In conclusion, although AM expression in the uterus varies throughout the estrous cycle, it is more closely correlated with circulating estradiol levels than with the stage of the cycle itself.

scholarly journals C-kit gene is expressed by skin mast cells in embryos but not in puppies of Wsh/Wsh mice: age-dependent abolishment of c-kit gene expression

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3509-3516 ◽  
Author(s):  
M Yamazaki ◽  
T Tsujimura ◽  
E Morii ◽  
K Isozaki ◽  
H Onoue ◽  
...  
Keyword(s):  
Mast Cells ◽  
Mrna Expression ◽  
Messenger Rna ◽  
Rnase Protection Assay ◽  
Coding Region ◽  
Carboxypeptidase A ◽  
Rnase Protection ◽  
Kit Gene ◽  
Age Dependent

The Wsh is a mutant allele at the W (c-kit) locus of mice, but no significant abnormalities are found at the coding region of the Wsh allele. Since cultured mast cells derived from the spleen of Wsh/Wsh mice do not express messenger RNA (mRNA) of c-kit, we studied the interrelation between the number of mast cells and the magnitude of c- kit mRNA expression in the skin of Wsh/Wsh mice of various ages. The number of mast cells in the skin of Wsh/Wsh embryos of 18 days postcoitum (pc) was approximately 40% that of normal control (+/+) embryos, but the number of mast cells decreased exponentially after birth; the number dropped to 0.6% that of +/+ mice at day 150 after birth. A weak but apparent signal of c-kit mRNA was detectable in the skin of 18-day pc Wsh/Wsh embryos by RNase protection assay but not in the skin of 5-day-old Wsh/Wsh mice. The number of c-kit protein- containing cells was significantly greater in the skin of 18-day pc Wsh/Wsh embryos than in the skin of 5-day-old Wsh/Wsh mice. The abolishment of c-kit mRNA expression appeared to be specific, because the expression of mast cell carboxypeptidase A mRNA but not of c-kit mRNA was detectable by in situ hybridization in skin mast cells of 5- day-old Wsh/Wsh mice. Taken together, the expression of c-kit mRNA was abolished first, then the content of c-kit protein dropped to undetectable levels, and then the disappearance of Wsh/Wsh mast cells themselves followed.

Expression and localization of aquaporins in rat gastrointestinal tract

1999 ◽  
Vol 276 (3) ◽  
pp. C621-C627 ◽  
Author(s):  
Yu Koyama ◽  
Tadashi Yamamoto ◽  
Tatsuo Tani ◽  
Kouei Nihei ◽  
Daisuke Kondo ◽  
...  
Keyword(s):  
Small Intestine ◽  
In Situ Hybridization ◽  
Gastrointestinal Tract ◽  
Epithelial Cells ◽  
Cellular Localization ◽  
Basolateral Membrane ◽  
Rnase Protection Assay ◽  
Rnase Protection ◽  
Basolateral Membranes

A family of water-selective channels, aquaporins (AQP), has been demonstrated in various organs and tissues. However, the localization and expression of the AQP family members in the gastrointestinal tract have not been entirely elucidated. This study aimed to demonstrate the expression and distribution of several types of the AQP family and to speculate on their role in water transport in the rat gastrointestinal tract. By RNase protection assay, expression of AQP1–5 and AQP8 was examined in various portions through the gastrointestinal tract. AQP1 and AQP3 mRNAs were diffusely expressed from esophagus to colon, and their expression was relatively intense in the small intestine and colon. In contrast, AQP4 mRNA was selectively expressed in the stomach and small intestine and AQP8 mRNA in the jejunum and colon. Immunohistochemistry and in situ hybridization demonstrated cellular localization of these AQP in these portions. AQP1 was localized on endothelial cells of lymphatic vessels in the submucosa and lamina propria throughout the gastrointestinal tract. AQP3 was detected on the circumferential plasma membranes of stratified squamous epithelial cells in the esophagus and basolateral membranes of cardiac gland epithelia in the lower stomach and of surface columnar epithelia in the colon. However, AQP3 was not apparently detected in the small intestine. AQP4 was present on the basolateral membrane of the parietal cells in the lower stomach and selectively in the basolateral membranes of deep intestinal gland cells in the small intestine. AQP8 mRNA expression was demonstrated in the absorptive columnar epithelial cells of the jejunum and colon by in situ hybridization. These findings may indicate that water crosses the epithelial layer through these water channels, suggesting a possible role of the transcellular route for water intake or outlet in the gastrointestinal tract.

scholarly journals Temporal changes in inhibin subunit mRNAs during atresia of preovulatory follicles in the rat

1998 ◽  
Vol 159 (2) ◽  
pp. 331-340 ◽  
Author(s):  
JT Uilenbroek ◽  
AL Durlinger ◽  
M Tebar ◽  
P Kramer ◽  
RH van Schaik ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Gnrh Antagonist ◽  
Rnase Protection Assay ◽  
Protection Assay ◽  
Rnase Protection ◽  
Subunit Mrna ◽  
Preovulatory Follicles ◽  
Inhibin Subunits

This study aimed to investigate the time course of disappearance of the mRNAs of the various subunits of inhibin in follicles which become atretic. An animal model was used in which atresia of preovulatory follicles could be studied in a chronological order. Injection of gonadotrophin-releasing hormone (GnRH) antagonist (20 microg) at the morning of pro-oestrus (P) blocked ovulation and the 10-12 preovulatory follicles became gradually atretic. A second injection was given the next day to prevent delayed ovulation. The rate of atresia could be delayed by simultaneous administration of a subovulatory dose of human chorionic gonadotrophin (hCG) (0.5 IU) and could be advanced by administration of a fivefold larger amount of GnRH antagonist. Functional activity of follicles becoming atretic was studied by measuring oestradiol production after incubation of individual follicles for 4 h. Follicles isolated 24 h after the first injection of GnRH antagonist (P+24) already secreted significantly less oestradiol in vitro than follicles isolated at pro-oestrus, although they were morphologically not different from pro-oestrous follicles. Follicles isolated at P+24 from hCG-treated rats secreted more oestradiol compared with follicles from rats not treated with hCG. In contrast, follicles isolated at P+24 from rats that were given a fivefold larger amount of GnRH antagonist secreted less oestradiol. Once this model was validated, temporal changes in inhibin subunit mRNAs in follicles undergoing atresia were measured by in situ hybridization and RNase protection assay. In situ hybridization showed abundant alpha- and betaA-subunit mRNA in the whole granulosa layer of preovulatory follicles at P and P+24, while betaB-subunit mRNA was restricted to the antral layer and cumulus. At P+48 the amount of alpha- and betaA-subunit mRNA had declined and was restricted to the cumulus, whereas betaB-subunit mRNA was absent. In the atretic follicles present at P+72 and P+96, mRNAs of all three inhibin subunits were absent. Administration of 0.5 IU hCG delayed the decline in the amount of alpha, betaA and betaB mRNA in preovulatory follicles at P+48. RNase protection assay of inhibin subunits in isolated follicles revealed no changes between P and P+24. However, at P+48, the mRNAs of alpha- and betaA-subunits were decreased. Expression of the mRNA of betaB-subunit declined gradually from P to P+48. The present study demonstrates that in follicles which are becoming atretic, mRNAs of alpha- and betaA-subunits decline simultaneously with the appearance of pycnotic cells in the granulosa layer, while betaB-subunit mRNA declines earlier, simultaneously with the decrease in the ability to secrete oestradiol in vitro.

Developmental expression of human angiotensinogen in transgenic mice

1998 ◽  
Vol 274 (5) ◽  
pp. F932-F939 ◽  
Author(s):  
Gongyu Yang ◽  
Curt D. Sigmund
Keyword(s):  
In Situ Hybridization ◽  
Transgenic Mice ◽  
Developmental Regulation ◽  
Rnase Protection Assay ◽  
Late Gestation ◽  
Developmental Expression ◽  
Rnase Protection ◽  
Adult Mice ◽  
Specific Regulation

Transgenic mice containing the human angiotensinogen ( HAGT) gene were utilized to determine the developmental regulation of HAGT expression. RNase protection assay on total RNA obtained from whole transgenic fetuses revealed that HAGT expression was first detected at embryonic day 8.5( E8.5) and was abundant from E9.5 onward. The earliest expression of the HAGT transgene appeared to precede the earliest expression of the endogenous mouse AGT gene by 1–2 days. Northern blot analysis revealed moderate levels of HAGT mRNA in liver and kidney and low levels of HAGT mRNA in heart and brain from E16.5 ( day 16.5 of gestation) onward. HAGT mRNA in liver, although abundant during late gestation and in 2-wk-old and adult mice, decreased transiently around birth. In situ hybridization performed on sections from whole fetuses revealed that HAGTmRNA was restricted to the developing liver and heart between E9.5 and E11.5 but became more widespread to include the developing aorta, brain, subcutaneous tissues, and vertebra at E13.5. In situ hybridization analysis on fetal kidneys from late gestation, newborn, and 2-wk-old mice demonstrated a progressive restriction of HAGT mRNA to developing cortical proximal tubular cells. These data illustrate the developmental tissue-specific regulation of HAGTexpression and demonstrate that sequences present in the transgene can confer an appropriate developmental expression profile.

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