Senescence Marker Protein-30/Gluconolactonase Expression in the Mouse Ovary during Gestation

Abstract

Senescence marker protein-30 (SMP30) was first described as a physiologic entity that decreases in the rat liver and kidney with aging. Previously, we established that SMP30 is the lactone-hydrolyzing enzyme gluconolactonase (GNL), which is involved in ascorbic acid (AA) biosynthesis. In the present study, we found SMP30/GNL mRNA expressed in the mouse ovary. To ascertain the reason for ovarian SMP30/GNL expression, we examined mice during gestation. SMP30/GNL mRNA expression was evident at the start of gestation, increased for the next eight days then decreased rapidly. Moreover, L-gulono-γ-lactone oxidase (Gulo) mRNA, which catalyzes the last step of AA, was found in the ovaries of these mice. The variations of these genes’ expression showed an inverse pattern to that of Cyp19a1 (aromatase) mRNA expression. Therefore, the SMP30/GNL and Gulo mRNA expression might be regulated by estrogen levels in the ovary. Since the presence of both SMP30/GNL and Gulo mRNAs could indicate that AA synthesis occurs in the ovary, we quantified AA levels in mouse ovaries during gestation. However, no correlation was found between changes of AA content and SMP30/GNL or Gulo mRNAs expression at this site. Moreover, we compared the changes of AA content during gestation between wild-type and SMP30/GNL knockout mice, which cannot synthesize AA, and found no significant differences between them. These results indicated that, although AA synthesis might occur in the ovaries, the amount of AA which is synthesized in ovaries must be quite low and insufficient to influence the AA content in ovary.

After the discovery of senescence marker protein-30 (SMP30) as a protein that decreases with aging in the liver, kidney, and lung,^1,2) SMP30 transcripts were detected in not only the liver, kidney, and lungs of mice but also in their brain and testes, as established by reverse transcription-polymerase chain reaction (RT-PCR) analysis.³⁾ In humans, SMP30 in parenchymal cells of the liver, proximal tubular cells of the kidney, acinal and ductal cells of the pancreas, and fasciculate cells of the adrenal cortex was seen by immunohistochemical staining.²⁾ To clarify the relationship between decreases of SMP30 and organ disorders associated with aging, we established SMP30 knockout mice.⁴⁾ These knockout mice are viable and fertile but lower in body weight and shorter in life span than their wild-type counterparts.⁵⁾ Throughout our experiments in vitro and in vivo, the livers from SMP30 knockout mice were far more susceptible to TNF-α- and Fas-mediated apoptosis than those from the wild-type.⁴⁾

Previously, we found that SMP30 is a gluconolactonase (GNL, EC 3.1.1.17) responsible for the conversion of L-gulonic acid to L-gulono-γ-lactone, an enzyme that participates in the penultimate step of the ascorbic acid (AA) biosynthetic pathway.⁶⁾ Therefore, SMP30/GNL knockout mice are unable to synthesize AA in vivo.⁶⁾ Humans, monkeys, and guinea pigs cannot synthesize AA in vivo because mutations in the gene encoding L-gulono-γ-lactone oxidase (Gulo) eliminate this essential enzyme.⁷⁾

Recently, gene expression profiling of differentially expressed genes in granulosa cells of bovine dominant follicles revealed that SMP30/GNL was one of the up-regulated genes located therein.⁸⁾ The ovary has long been recognized as a site of AA accumulation at high concentrations in the theca interna, granulosa, and luteal compartments.^9,10) However, AA synthesis was identified mainly in the livers of most mammalian species including mice and rats.¹¹⁾ AA is believed to undergo subsequent transport to the ovaries, where cellular uptake occurs through an energy-dependent process.¹²⁾ In this study, to ascertain the reason for ovarian SMP30/GNL expression, we examined SMP30/GNL, Cyp19a1 and Gulo expressions and AA levels in the mouse ovaries during gestation by using wild-type and SMP30/GNL knockout mice.

MATERIALS AND METHODS

Expression Analysis of SMP30/GNL and Gulo in Multiple Mouse Tissues

For the analyses of SMP30/GNL mRNA expression in multiple mouse tissues, commercially available RNAs of nine tissues (FirstChoice® Mouse Total RNA Survey Panel; Ambion, Austin, TX, U.S.A.) were used. Total RNA of 1.5 µg of each tissue was reverse transcribed using SuperScript II Reverse transcriptase (Invitrogen, Carlsbad, CA, U.S.A.) with random hexamer primers (TaKaRa Bio Inc., Otsu, Japan) following the supplier’s instructions. The PCR was performed in a thermal cycler GeneAmp PCR system 9700 (Applied Biosystems, Carlsbad, CA, U.S.A.) in 25 µL of a reaction mixture containing complementary DNA (cDNA) corresponding to 100 ng of the initial total RNA, 1×Blend Taq polymerase buffer, 0.2 mM of deoxynucleotide triphosphates, 0.2 µM of each primer and 0.5 U of Taq DNA polymerase (Blend Taq Plus; Toyobo, Osaka, Japan). Primers for mouse SMP30/GNL spanning the entire coding region were as follows: sense 5′-CAC TGT CCT TTT CCT GTG ACC-3′ and antisense 5′-TGG TCA CAG TCC CAT TTC AA-3′. Gulo: sense 5′-GTC ACA GTG GAA GCC GGT AT-3′ and antisense 5′-ACA CCA CCC GAA AGA GAC AC-3′. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh): sense 5′-TGG CAA AGT GGA GAT TGT TG-3′ and antisense 5′-TTA GTG GGG TCT CGC TCC T-3′ were used as a control. Primers were designed using Primer3 software.¹³⁾ The PCR parameters were: 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 m 45 s. PCR products were visualized by gel electrophoresis in 2.0% agarose with ethidium bromide staining.

Animals

Wild-type (C57BL/6Cr slc) mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). SMP30/GNL knockout mice were generated by the gene targeting technique described previously.⁴⁾ These mice cannot synthesize AA in vivo, because they lack SMP30/GNL, which is the penultimate enzyme in the AA biosynthetic pathway.⁶⁾ Wild-type and SMP30/GNL knockout mice were fed with an AA-deficient diet (CL-2, CLEA Japan, Tokyo, Japan) ad libitum. SMP30/GNL knockout mice had free access to water containing 1.5 g/L AA and 10 µM ethylenediaminetetraacetic acid to maintain AA levels in tissues that were similar to that of wild-type mice, and to eliminate any possible confounding influences of AA deficiency.¹⁴⁾ Wild-type mice had free access to water without AA. Throughout the experiments, animals were maintained on a 12-h light/dark cycle in a controlled environment. All experimental procedures using laboratory animals were approved by the Animal Care and Use Committee of the Tokyo Metropolitan Institute of Gerontology.

Preparation of Ovaries

Female mice at three- to five-months of age were mated with male mice overnight. The following morning, these females were checked for vaginal plugs to verify pregnancy (day 0). On 0, 2, 5, 8, 11, 14, 17, and 20 d of gestation, ovaries were removed and frozen with liquid nitrogen and then stored at −80°C until use.

Quantitative Real-Time Polymerase Chain Reaction (qPCR)

Total RNA was extracted from ovaries using Isogen reagent (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. The RNA samples were treated with TURBO DNase (TURBO DNA-free Kit; Applied Biosystems), and 1 µg of total RNAs was reverse transcribed using SuperScript II Reverse transcriptase with random hexamer primers. TaqMan® probes for qPCR were purchased from Applied Biosystems. Assay IDs are as follows, SMP30/GNL: Mm00485711_m1, Gulo: Mm00626646_m1, cytochrome P450, family 19, subfamily a, polypeptide 1 (Cyp19a1): Mm00484049_m1, and beta actin: Mm00607939_s1. The qPCR was performed in reaction mixture containing cDNA corresponding to 1 ng (for beta actin), 40 ng (for SMP30/GNL and Cyp19a1), and 80 ng (for Gulo) of the initial total RNA with Platinum qPCR SuperMix-UDG with ROX (Invitrogen) and TaqMan® probes. The RT-PCR equipment (Applied Biosystems 7300 Real Time PCR System) was used for detection.

AA Measurement

AA levels in ovaries were measured by using high-performance liquid chromatography (HPLC) and an electrochemical detector.^14,15) Tissues were homogenized in 14 volumes of 5.4% metaphosphate containing 1 mM ethylenediaminetetraacetic acid and centrifuged at 21000×g for 10 min at 4°C. Samples were analyzed by HPLC using an Atlantis dC18 column (4.6×150 mm; Nihon Waters, Tokyo, Japan). The mobile phase was 50 mM phosphate buffer (pH 2.8), 0.2 g/L of ethylenediaminetetraacetic acid, 2% methanol at a flow rate of 1.3 mL/min, and electrical signals were recorded by using an electrochemical detector (2465, Nihon Waters) with a glassy carbon electrode at +0.6 V.

Statistical Analysis

Results are expressed as means±S.E.M. The probability of statistical differences between wild-type and SMP30/GNL knockout mice was assessed by an independent two-sample t-test for means using PASW Statistics 18 software (SPSS, Inc., Chicago, IL, U.S.A.). Statistical differences were considered significant at p<0.05.

RESULTS AND DISCUSSION

SMP30/GNL mRNA Expression in the Mouse Ovary

In the first of these experiments, we estimated the expression of SMP30/GNL in mouse ovaries by RT-PCR. As Fig. 1 shows, amplicon derived from SMP30/GNL mRNA was clearly evident in ovarian tissues. SMP30/GNL mRNA expression was also detected in the liver, brain, thymus, heart, lung, testis, and kidney; however, the spleen contained no such distinguishable signals. The PCR products of these ovaries were sequenced and confirmed RT-PCR specificity.

Fig. 1. RT-PCR Analysis of SMP30/GNL in Multiple Mouse Tissues

The PCR products of SMP30/GNL, Gulo, and Gapdh were 1306, 1609, and 177 bps, respectively.

SMP30/GNL mRNA Expression in Ovaries during Gestation

We next quantified SMP30/GNL mRNA expression in the mouse ovary during gestation (Fig. 2A). The expression of SMP30/GNL mRNA increased from the day after plug detection up to day 8 and then decreased rapidly until day 11 of gestation. Afterward, the amount of SMP30/GNL mRNA remained nearly stable until day 20 of gestation. The SMP30/GNL mRNA expression levels at 5 and 8 d of gestation were 2.6- and 2.7-fold, respectively, higher than that at gestation day 20, which was virtually the lowest level throughout the gestational period.

Fig. 2. The qPCR Analysis of SMP30/GNL, Cyp19a1, and Gulo in Mouse Ovaries during Gestation

(A) SMP30/GNL mRNA expression levels. (B) Cyp19a1 mRNA expression levels. (C) Gulo mRNA expression levels. Black and gray columns represent wild-type and SMP30/GNL knockout mice, respectively. The mRNA expression levels were calculated as the ratio of mRNA expression of wild-type mice at 0 d of gestation. Values are expressed as means±S.E.M. of five animals.

Cyp19a1 mRNA Expression in Ovaries during Gestation

The likelihood that SMP30/GNL is involved in hormonal regulation was indicated by Grossman et al., who prevented the normal developmental increase of renal SMP30/GNL in male rats by the administration of 17β-estradiol.¹⁶⁾ Moreover, Maia et al. reported that estradiol down-regulated SMP30/GNL expression in the rat mammary gland and prostate.¹⁷⁾ Additionally, Cyp19a1 (aromatase) is known to catalyze the conversion of androgen to estrogen in the ovary.¹⁸⁾ Therefore, we quantified Cyp19a1 mRNA expression in mouse ovaries during gestation (Fig. 2B). Starting at day 5 of gestation, Cyp19a1 mRNA expression increased and maximized at day 14. Subsequently, the Cyp19a1 mRNA expression levels dropped rapidly until day 20 of gestation. Moreover, there was no significant difference of Cyp19a1 mRNA expression levels in ovaries during gestation between wild-type and SMP30/GNL knockout mice.

These results suggest that the reduced SMP30/GNL mRNA expression levels after 11 d of gestation resulted from the elevated estrogen levels; that is, SMP30/GNL mRNA expression might be regulated by estrogen content in the ovary.

Gulo mRNA Expression in the Ovary during Gestation

AA synthesis is known to occur mainly in the liver of most mammalian species including mice and rats.¹¹⁾ If SMP30/GNL participates in an AA synthetic process in the ovaries of mice, Gulo, which catalyzes the last step of AA production, must also be expressed in the ovary. Accordingly, mRNA expression of Gulo was examined by RT-PCR. Conclusive evidence of Gulo mRNA transcripts was detected in the ovarian samples from these mice (Fig. 1). Further, sequencing of these PCR products confirmed RT-PCR specificity. This is a first report to identify the presence of Gulo mRNA transcripts in the mouse ovary.

The next step was to examine Gulo mRNA expression levels in mouse ovaries during gestation (Fig. 2C). Gulo mRNA expression began to increase on the day of plug detection (day 0), and maximized on day 5, then continuously decreased until day 17, finally increasing slightly at day 20 of gestation. There was no significant difference of Gulo mRNA expression levels in ovaries during gestation between wild-type and SMP30/GNL knockout mice. Therefore, the SMP30/GNL defect had no influence on Gulo mRNA expression. Since the expression profile of Gulo was similar to that of SMP30/GNL, Gulo mRNA expression might also be regulated by estrogen content in the ovary.

AA Levels in the Mouse Ovary during Gestation

Since both SMP30/GNL and Gulo mRNAs were expressed and increased during the early gestational periods, we explored the possibility that AA might be synthesized in the mouse ovary. Quantification of AA levels in mouse ovaries during gestation revealed a gradual decline from the day of plug detection (day 0) until day 14 and then an increase until day 20 of gestation (Fig. 3).

Fig. 3. AA Levels in Mouse Ovary during Gestation

Black and gray columns represent wild-type and SMP30/GNL knockout mice, respectively. Values are expressed as means±S.E.M. of five animals.

However, the change of AA levels in mouse ovaries during gestation did not correlate with the changes of SMP30/GNL and Gulo mRNA expression, although the quantities of AA, SMP30/GNL, and Gulo mRNA expression after 11 d of gestation showed similar, relatively lower levels than at earlier gestational periods. Further, AA levels in ovaries were not significantly different between wild-type and SMP30/GNL knockout mouse during gestation. These results indicated that, although AA synthesis might occur in the ovaries, the amount must be too low to influence the AA content in ovary.

In conclusion, SMP30/GNL mRNA transcripts were detected in the ovaries of mice, and the expression of these transcripts increased from days 0 to 8 of gestation and decreased rapidly thereafter. In this study, we first detected Gulo mRNA transcripts and the expression profile of Gulo mRNA during gestation, which was similar to that of SMP30/GNL mRNA. Moreover, variations of these genes’ expression showed an inverse pattern to that of Cyp19a1 mRNA expression. Therefore, the SMP30/GNL and Gulo mRNA expression might be regulated by estrogen levels in the ovary. These results suggested the possibility that AA synthesis occurs in the mouse ovaries. However, the absence of AA synthesis caused by the SMP30/GNL defect had no apparent influence on the AA levels in ovaries during gestation. Therefore, AA incorporated from outside the ovarian cells must be the main source of ovarian AA. Further, since SMP30/GNL might have different functions in the mouse ovary, this newfound phenomenon warrants further study to determine the role of SMP30/GNL in ovaries.

Acknowledgment

This study is supported by JSPS KAKENHI Grant Numbers 24380073. The authors thank Ms. P. Minick for the excellent English editorial assistance. Vitamin C powder was kindly provided by DSM Nutrition Japan.

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