ARTICLE |
Correspondence to: Thomas Walther, University Hospital Benjamin Franklin, Dept. of Cardiology and Pneumology, Free University of Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany. E-mail: thomas.walther@ukbf.fu-berlin.de
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Summary |
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The Mas proto-oncogene encodes a G-protein-coupled receptor with the common seven transmembrane domains and may be involved in the actions of angiotensins. Because Mas is highly expressed in testis, we investigated the cell type-specificity and the onset of expression of the gene in this organ. Using an RNase protection assay, it could be shown that neither whole testes nor cultured Sertoli and Leydig cells of 12-day-old mice express Mas mRNA. Mas expression is first detected in 18-day-old mice and thereafter increases continuously until 6 months of age. By in situ hybridization, the expression could be localized to Leydig cells and Sertoli cells, the signals being much more pronounced in the former. A weak signal was detected in primary spermatocytes. The strong ontogenetically controlled and cell type-specific expression of this membrane-bound receptor in testis implicates a role for the Mas proto-oncogene in testis maturation and function.
(J Histochem Cytochem 50:691695, 2002)
Key Words: Mas proto-oncogene, in situ hybridization, Leydig cells, Sertoli cells, RNase protection assay
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Introduction |
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THE Mas proto-oncogene was first detected through its tumorigenic activity in in vivo tumor assays (
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Materials and Methods |
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Animals
Inbred mice of different ages were used for the experiments. The animals were maintained under standardized conditions with an artificial 12-hr darklight cycle, with free access to food and water. Animals were sacrificed by cervical dislocation. After preparation, all tissue samples were immediately snap-frozen in liquid nitrogen. This research was in compliance with the Guide for the Care and Use of Laboratory Animals published by the OPRR (Office for Protection against Research Risks) of the National Institutes of Health (Washington, DC) (NIH Publication No. 85-23, revised 1985).
Cell Culture
Commercially available cell lines TM3 (testis, Leydig cell, mouse BALB/c, isolated from 1113-day-old animals) and TM4 (testis, Sertoli cell, mouse BALB/c, isolated from 1113-day-old mice) from ATCC (American Type Culture Collection; Manassas, VA) were used for cell culture experiments. For at least 3 days, cells were grown in a monolayer (94/16-mm Petri dish) in medium as recommended by ATCC.
RNA Isolation
RNA from cultured cells was isolated using TRIzol reagent (Life Technologies; Gaithersburg, MD). Cells were lysed by adding 1.5 ml of TRIzol reagent to the culture dish. Each cell lysate was transferred to a 2-ml Eppendorf tube and centrifuged (12,000 x g, 15 min, 4C) after adding 0.3 ml of chloroform. The upper aqueous phase was transferred to a fresh tube and RNA was precipitated by mixing with 1 ml of isopropanol. Probes were centrifuged (12,000 x g, 10 min, 4C). After an additional washing step, the RNA pellet was air-dried and dissolved in DEPC-treated water. RNA of mouse organs was also isolated using TRIzol reagent according to the manufacturer's protocol, as described previously (
RNase Protection Assay
Mas expression was analyzed by RPA using commercially available Ambion RPA II kits (AMS Biotechnology; Witney, UK), according to the protocol of the manufacturer. Thirty µg total RNA of testis, brain, and cultured cells and 50 µg of yeast as a control were used for RPA. Via PCR, a 175-bp fragment was amplified from mouse Mas cDNA using the 5' primer CAAGCCTCTAGCCCTCTGTCC and the 3' primer GGAGGCATTTCTGCTGGAGG and subcloned in a T-vector (Promega; Mannheim, Germany). A T7-polymerase reaction transcribed a 225-bp radioactive probe complementary to the 175 nucleotides of the Mas mRNA and a 144-bp radioactive probe complementary to 127 nucleotides of the mRNA encoding the L32 housekeeping gene (commercially available L32 probe template; PharMingen International, San Diego, CA). RNA samples were hybridized with approximately 20,000 cpm of the radiolabeled Mas antisense probe and 10,000 cpm of the L32 antisense probe. The hybridized fragments, once protected from RNase A + T1 digestion, were separated by electrophoresis on a denaturing gel (5% polyacrylamid, 8 M urea) and analyzed using a FUJIX BAS 2000 Phospho-Imager system (Raytest GmbH; Straubenhardt, Germany).
Tissue Collection, Fixation, and Processing
Adult (4-month-old) male mice were used for experiments. Mas knockout mice of relevant age were used as a negative control. The animals were sacrificed by cervical dislocation, testes were quickly removed and fixed at 4C in 4% paraformalde/1 x PBS for 12 hr, dehydrated in ethanol, and embedded in paraffin for histology and in situ hybridization according to standard techniques. Serial cross-sections of testis (58-µm thickness) were mounted on Superfrost plus slides (Menzel Glass; Braunschweig, Germany), deparaffinized, and kept at -70C. On the day of the experiment, the testis sections were quickly brought to room temperature and used for in situ hybridization. For histological examination, sections were counterstained with hematoxylin and eosin.
DNARNA In Situ Hybridization
Control slides were digested with RNase A (400 µg/ml for overnight), and washed three times in fresh 2 x SSC (pH 7.0). All slides were rinsed briefly in 1 x PBS and incubated for 12 min in pepsin solution (10 mg/100 ml 0.01 M HCl). After 5-min rinsing in 1 x PBS, the sections were postfixed in 3% paraformaldehyde, rinsed in 1 x PBS, dehydrated in graded ethanol up to 95%, and air-dried. For in situ hybridization, a MasDNA probe, spanning positions from -165 to +150 in relation to the translation start site of Mas (
After in situ hybridization, sections were examined under a Leica fluorescent microscope equipped with an HBO100 lamp, a x63 or x40 (NA 1.6) oil immersion objective, and an appropriate set of optical filters. Images of metaphase and interphase preparations were recorded using a CCD camera connected to a Q-FISH image analysis system. In addition, the following controls were prepared: slides were processed as described above but in the absence of labeled probe, or slides were processed as described above but with biotin-labeled pBR 322 without insert.
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Results |
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To localize the expression of the Mas proto-oncogene to a cell type in testis, commercially available Leydig (TM3) and Sertoli cell lines (TM4), originally isolated from 1113-day-old mice, were cultured. RNA from these cells was isolated and used in RPA to detect Mas gene expression (Fig 1). Neither Leydig nor Sertoli cell lines from young mice showed any detectable Mas expression. Furthermore, Mas mRNA was not found in the testis of a 2-week-old mouse. In contrast, forebrain and testis RNA of an adult (3-month-old) mouse gave signals, which were strongest in the adult testes. All RNA used showed a similar expression of the housekeeping gene L32, confirming the integrity of the RNA.
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To examine the ontogenetic profile of Mas expression in testis, animals at the age of 16, 18, 20, 22, 25, 30 days and 1.5, 3, and 6 months were analyzed. For each time point, RNA of six testes isolated from mice of independent litters was used for RPA (Fig 2A). Mas expression was undetectable in testis of 16-day-old mice and started to be visible in 18-day-old animals. Then it continued to increase from 20 days to 6 months of age (Fig 3). To exclude a first peak of Mas expression after birth and to clarify whether there is a further increase in Mas mRNA in old animals, we also characterized the Mas expression in neonatal testes and testes of 1-year-old mice. Although no Mas mRNA could be detected after birth, there was also no further elevation in Mas expression in 12-month-old animals compared to 6-month-old mice (Fig 2B and Fig 3).
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To identify the cell type expressing the Mas in the adult testis in vivo, four testes of 3-month-old mice were isolated and prepared for fluorescent in situ hybridization (FISH). Using this technique, a distinct expression of Mas gene was detectable in both Leydig and Sertoli cells (Fig 4), the signal being much stronger in Leydig cells. In addition, weak expression could be detected in some round cells in the presumptive position of primary spermatocytes. The result was reproducible in all testes. Mas mRNA was not observed in later stages of the germ-cell lineage. No signal was observed in testes of Mas-deficient mice used as controls (data not shown).
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Discussion |
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Because it was shown that expression of the Mas proto-oncogene is highest in testis of mice, it was of great interest to analyze the ontogenetic profile and the cell type specifity of the Mas expression in this organ. Using RPA, we could demonstrate strong expression of the Mas gene in adult mouse testis, whereas the whole organ or lines of Leydig and Sertoli cells isolated from young mice did not show any Mas mRNA expression. In these cell lines it was not possible to clarify which cell type is responsible for the strong Mas expression in an adult murine testis. Therefore, the results show that TM3 and TM4 may be good models for young testicular cells but may not be representative for adult Leydig and Sertoli cells, respectively. Furthermore, it should be noted that TM3 and TM4 are permanent cell lines and may have lost some characteristics of the cell type of origin.
As described for rats (
Because we could not detect any Mas expression in newborn mice, the expression of Mas in fetal Leydig cells seems unlikely. The onset of Mas expression between Days 35 and 56 in the rat (
Mas is not expressed in tissues of the female genital tract (data not shown) and therefore cannot interact with the components of the reninangiotensin system (RAS) detected in ovary (for review see
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Acknowledgments |
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Supported in part (EP and TB) by RFBR grant no. 98-04-50002 and by an MDC fellowship (NA).
Received for publication July 26, 2001; accepted December 5, 2001.
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