научная статья по теме EXPRESSION AND REGULATION OF ANG-2 IN MURINE OVARIES DURING SEXUAL MATURATION AND DEVELOPMENT OF CORPUS LUTEUM Биология

Текст научной статьи на тему «EXPRESSION AND REGULATION OF ANG-2 IN MURINE OVARIES DURING SEXUAL MATURATION AND DEVELOPMENT OF CORPUS LUTEUM»

МОЛЕКУЛЯРНАЯ БИОЛОГИЯ, 2012, том 46, № 6, с. 900-906

МОЛЕКУЛЯРНАЯ ^^^^^^^^^^^^^^ БИОЛОГИЯ КЛЕТКИ

UDC 577.21;577.17.171.52

EXPRESSION AND REGULATION OF Ang-2 IN MURINE OVARIES DURING SEXUAL MATURATION AND DEVELOPMENT OF CORPUS LUTEUM

© 2012 B. Guow, X.-M. Zhang1#, S.-J. Li2, X.-C. Tian1, S.-T. Wang1, D.-D. Li1, D.-F. Liu1*, Z.-P. Yue1*

1College of Animal Science and Veterinary Medicine, Jilin University, Changchun, P. R. China 2College of Life Science, Northeast Agricultural University, Harbin, P. R. China

Received March 14, 2012 Accepted for publication April 03, 2012

The aim of this study was to examine the expression and regulation of angiopoietin-2 (Ang-2) in murine ovaries during sexual maturation, gonadotropin treatment and luteal development by in situ hybridization and RT-PCR. By in situ hybridization Ang-2 mRNA was mainly localized in granulosa cells, thecal cells and corpus lu-teum, otherwise in oocytes. Moreover, Ang-2 mRNA was highly expressed in corpus luteum and granulosa cells of atretic follicles. According to RT-PCR data, Ang-2 mRNA was lowly expressed on day 10 after birth, then expression levels gradually increased and reached their highest values on day 25 after birth. In the superovulated model of immature mice, Ang-2 expression was strongly induced by equine chorionic gonadotropin (eCG) 48 h post the eCG injection, and was high from 0.5 to 13 h after hCG treatment. In situ hybridization showed that Ang-2 mRNA was highly expressed in corpus luteum from day 2 to 9 post the hCG injection, then the expression levels gradually declined on days 11 and 13 after hCG treatment. According to RT-PCR data, the levels of Ang-2 mRNA expression showed a decline after the hCG injection, with a nadir on day 3, followed by an increase, reaching the highest level on day 9 post-hCG injection. Then again Ang-2 expression gradually declined from day 11 to 15 after hCG injection. These results suggest that Ang-2 may be involved in follicular development, atresia, ovulation, and corpus luteum formation and regression.

Keywords: Ang-2, mouse, ovary, follicle, corpus luteum.

Angiogenesis, the development of new blood vessels from preexistent vasculature, in healthy adult animals is mainly limited to the reproductive system [1]. The development of new blood vessels in the ovaries is essential to guarantee the necessary supply of nutrients and hormones to promote follicular growth and corpus luteum formation. Accumulating data has shown that the vascular endothelial growth factor (VEGF) is present within both ovary follicles and corpora luteum and appears to play an integral role in endothelial cells and blood vessel regulation within the ovary [2, 3]. However, recent molecular analysis of the mechanism of angiogenesis has focused on the role of other growth factors, angiopoietins, which function in concert with VEGF on the formation, stabilization, and regression of blood vessels [4].

Angiopoietin-1 (Ang-1) affects vascular endothelial cells through an endothelial cell-specific tyrosine kinase receptor (Tie-2) and is involved in the maturation and stabilization of new blood vessels. On the other hand, Ang-2, an endogenous antagonist of Ang-1, plays an important role in loosening the supporting

The text was submitted by the author(s) in English.

# These authors contributed equally to this work.

* E-mail: zpyue@yahoo.com.cn; ccldf@163.com

cell matrix and destabilization of existing blood vessels together with VEGF. In the presence of VEGF, Ang-2 could stimulate endothelial cell proliferation and migration and promote further angiogenesis, whereas in the absence ofVEGF, Ang-2 could induce blood vessel destabilization and regression [4, 5]. At present, a number of studies found that Ang-2 is dynamically expressed in ovarian follicles and corpus luteum of different species, suggesting a regulating role of Ang-2 in ovarian angiogenesis. In rat ovaries, Ang-2 is strongly expressed in theca cells from early antral follicles and preovulatory follicles [6]. In ewe ovaries, Ang-2 was detected in granulosa cells and theca cells of follicles [7]. Intrafollicular injection ofAng-2 can prevent ovulation and development and function of the subsequent corpus luteum [8]. In addition, Ang-2 was also detected in corpus luteum of rats, cows, mares, monkeys and humans [9—13]. During prostaglandin F2a-in-duced luteolysis, Ang-2 expression increases [14—16]. These results indicate that Ang-2 is necessary for folli-cular growth and atresia, ovulation, and subsequent development and regression of corpus luteum. However, Ang-2 expression and regulation in murine ovaries during sexual maturation and luteal development are still not defined in detail. The aim of this study was to investigate Ang-2 expression in mouse ovaries dur-

ing sexual maturation, gonadotropin treatment, and luteal development by in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR).

EXPERIMENTAL

Sexual maturation. Immature female mice (Kunming White outbred strain, 21 days old) were caged in a controlled environment with a cycle of 14 L : 10 D. All animal procedures were approved by the Institutional Animal Care and Use Committee of Jilin University. Mouse ovaries were collected on days 10, 15, 20, 25, 30 and 40 after birth. There were at least three mice per group.

Gonadotropin treatment.The mice (21 days old) were superovulated with an i.p. injection of 5 IU equine chorionic gonadotropin (eCG). Ovaries were collected from the treated mice at 0.5, 1, 3, 6, 12, 24, 36 and 48 h after the eCG injection. Ovaries were collected directly from 21-day-old immature mice as controls. In addition, 21-day-old mice were superovulated with an i.p. injection of 5 IU eCG followed by administration of 5 IU of human chorionic gonadot-ropin (hCG) 48 h later. Ovaries were collected from these treated mice at 0.5, 1, 3, 5, 7, 9, 11 and 13 h post the hCG injection and on days 1, 2, 3, 4, 5, 7, 9, 11, 13 and 15 post the hCG injection respectively. Day 0 was designated as the day when hCG was injected. There were at least three mice per group

In situ hybridization. Total RNA extracted from the mouse uteri was reverse-transcribed and amplified with Ang-2 primers. Ang-2 forward primer 5'-GCATCTACACACTGACCTTC and reverse primer 5'-CTGGTTGGCTGATGCTAC were designed according to Mus musculus Ang-2 gene (Genbank accession number NM_007426). The amplification ofAng-2 cDNA was performed with 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. The amplified fragment (372 bp) of Ang-2 was recovered from the agarose gel and cloned into a pGEM-T plasmid (pGEM-T Vector System 1, "Promega", Madison, WI). The orientation of the Ang-2 fragment in the pGEM-T plas-mid was determined by PCR using a combination of primers for T7, SP6 and mouse Ang-2. The cloned Ang-2 fragment was further verified by sequencing. An Ang-2-containing plasmid was amplified with the primers for T7 and S6 to prepare templates for labeling. Digoxigenin (DIG)-labeled antisense and sense cRNA probes were transcribed in vitro using a DIG RNA labeling kit ("Roche Diagnostics GmbH", Mannheim, Germany).

Mouse ovaries were flash frozen in liquid nitrogen. Frozen sections (10 ^m) were mounted onto 3-ami-nopropyltriethoxy-silane ("Sigma")-coated slides and fixed in 4% paraformaldehyde solution in PBS. The sections were washed in PBS twice, treated with 1% Triton-100 for 20 min and washed again in PBS 3 times. After prehybridization in a solution of 50%

formamide and 5 x SSC (1 x SSC is 0.15 M sodium chloride, 0.015 M sodium citrate) at room temperature for 15 min, the sections were hybridized in the hybridization buffer (5 x SSC, 50% formamide, 0.02% BSA, 250 mg/mL yeast tRNA, 10% dextran sulfate, 1 mg/mL denatured DIG-labeled antisense or sense RNA probe for mouse Ang-2) at 55°C for 16 h. After hybridization, the sections were washed sequentially in 50% formamide/5 x SSC at 55°C for 15 min, 50% formamide/2 x SSC at 55°C for 30 min, 50% for-mamide/0.2 x SSC at 55°C twice for 30 min each, and 0.2 x SSC at room temperature for 5 min. After nonspecific binding was blocked in a 1% blocking reagent ("Roche") for 1 h, the sections were incubated with sheep anti-DIG antibodies, conjugated with alkaline phosphatase (1 : 5000, "Roche") in a 1% block reagent overnight at 4°C. The signal was visualized with 0.4 mM 5-bromo-4-chloro-3-indolyl phosphate and 0.4 mM nitrobluetetrazolium in a buffer containing 100 mM Tris-HCl, pH 9.5, 100 mM NaCl and 50 mM MgCl2. Endogenous alkaline phosphatase activity was inhibited with 2 mM levamisole ("Sigma"). All of the sections were counterstained with 1% methyl green in 0.12 M glacial acetic acid and 0.08 M sodium acetate for 30 min. The positive signal was visualized by a dark brown color.

RT-PCR.Total RNA was extracted from mouse ovaries with the TRIZOL reagent, digested with RQ1 DNase I and reverse-transcribed into cDNA with M-MLV Reverse Transcriptase ("Promega", Madison, WI). The amplified PCR fragment for Ang-2 was separated by 1.5% agarose gel electrophoresis, stained with ethidium bromide, and quantitated by optical density using the UVP laboratory imaging and analysis system ("UVP, Inc.", Upland, CA). The band densities for Ang-2 were normalized to GAPDH expression. To compare the intensities of RT-PCR products in a semi-quantitative way, we determined the exponential phase of amplification by performing 25—28—30—35 cycles for Ang-2 and GAPDH. The final amplification cycles were 25 for Ang-2 and GAPDH. As a negative control, each sample was run through PCR in the absence of reverse transcriptase or cDNA to rule out genomic DNA contamination. All the RT-PCR reactions were repeated at least three times with three batches of ovaries. Quantitative data is expressed as mean ± SEM and was analyzed by one-way ANOVA using the SPSS software program. The differences were considered significant at P < 0.05.

RESULTS

Ang-2 mRNA expression in mouse ovary during s

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