Expression Pattern of the Mitochondrial Capsule Selenoprotein mRNA in the Mouse Testis after Puberty; in situ Hybridization Study

Sang-Yoon NAM*, Seishi MAEDA, Kenji OGAWA1), Masamichi KUROHMARU, and Yoshihiro HAYASHI Department of Veterinary Anatomy, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113 and 1)Laboratory Research Center, The Institute of Physical and Chemical Research (RIKEN), 2–1 Hirosawa, Wako, Saitama 351–01, Japan

(Received 25 April 1997/Accepted 27 June 1997)

ABSTRACT. Mitochondrial capsule selenoprotein (MCS) has been known as a structural of the mitochondrial sheath in spermatozoa. In this study, to determine the expression pattern of MCS mRNA in the mouse testis after puberty, in situ hybridization using digoxigenin- labeled RNA probes for MCS was performed in the testes of 8- and 20-week-old ICR mice. In the testes of both ages, MCS mRNA first appeared in step 3 round spermatids, gradually increased during early spermiogenesis, and persisted a high level until step 14 spermatids. After the step 14 spermatids, the signal began to decline and was weakly detected in steps 15–16 spermatids. On the other hand, compared with that in the testes of 8-week-old mice, MCS mRNA level in the testes of 20-week-old mice increased over 2-fold at stages VI-III, while it slightly increased at stages IV-V. These findings suggest that MCS gene transcription may be up-regulated after puberty in the mouse testis. — KEY WORDS: in situ hybridization, mitochondrial capsule selenoprotein, puberty, testis. J. Vet. Med. Sci. 59(11): 983–988, 1997

Selenium (Se) is an essential micronutrient for mammals. digoxigenin (DIG)-labeled RNA probes for MCS in the Since it was first demonstrated that Se is a structural testes of 8- and 20-week-old mice. component of the active center of the enzyme glutathione (GSH-Px; EC 1.11.1.9.) [19], other MATERIALS AND METHODS selenoproteins have been identified using radiolabeled Se [2]. Among these, only 5 have been characterized in Experimental : Male ICR mice (3 weeks old) were mammalian tissues as selenoproteins containing purchased from Charles River, Japan Inc.(Yokohama). All which is encoded by an in-frame UGA codon, animals were housed in the room where the temperature (22 formerly known as a [3, 8, 15, 20, 23]. ± 2°C), relative humidity (55 ± 10%) and light/dark cycles Se has been known to play a part in the male reproductive (14L:10D) were controlled. They received food pellets system. In studies of animals fed a Se-deficient diet, the Se (CLEA Ltd., Tokyo, Japan) and water ad libitum. At 8 and retention was highest in the male reproductive organs. 20 weeks of age, animals were sacrificed under pentobarbital Abnormal spermatozoa with impaired motility due to anesthesia. For in situ hybridization studies, the testes were characteristic midpiece damage were frequently observed in perfused with Bouin’s fixative, immersed in the same the animals [4, 24, 25]. Tracer studies using 75Se fixative for 12 to 24 hr and subsequently processed for demonstrated that Se is localized within the midpiece of sectioning. spermatozoa and that it is bound to a polypeptide of 15,000 In situ hybridization: RNA probes were synthesized by to 17,000 daltons confined to the mitochondrial sheath [5, transcription of a pCRII plasmid (Invitrogen, Tokyo, Japan) 6, 14]. This molecule has been shown in the bull and rat to containing the MCS cDNA (592 bp) [16] with Sp6 or T7 be a major component of the keratinous mitochondrial RNA polymerase after linearization with Hind III or Xho I. capsule [7, 18] and it has been characterized as a - Hybridization was performed on the 5 µm sections of testes. and proline-rich selenoprotein [6, 17]. Recently, it was Deparaffinized sections were incubated in 10 µg/ml of reported that the mitochondrial capsule selenoprotein (MCS) proteinase K in phosphate buffered saline (PBS) at 37°C for cloned from mouse testes is a structural protein of the 5 to 30 min and treated with 4% paraformaldehyde in PBS mitochondrial sheath of spermatozoa [11, 12, 22]. Although for 10 min, 0.2 M HCl for 10 min, and 0.25% acetic it has been suggested that MCS is related to the formation anhydride in 0.1 M triethanolamine (pH 8.0) for 15 min. of the mitochondrial sheath in spermatozoa, the exact role Sections were prehybridized in 50% formamide (FA) plus of MCS in testes is still controversial. 2-strength standard saline citrate (SSC) at 42°C for 30 min In our previous study using Northern blot analysis, we and hybridized at 42°C for 16 hr by adding DIG-labeled demonstrated that the MCS mRNA greatly increases after riboprobes in the following solution: 50% FA, 10 mM Tris- puberty in the mouse testis. The level in the testis of 80- HCl (pH 7.6), 200 µg/ml yeast tRNA, 1-strength Denhardt’s week-old mouse increased about 6-fold as compared with reagent (the following component diluted with 500 ml of that of 3-week-old mouse [16]. In this study, to determine H2O: 0.1 g of Ficoll [Type 400; Pharmacia, Tokyo, Japan], the expression pattern of MCS mRNA in the mouse testis 0.1 g of polyvinylpyrrolidone, and 0.1 g of bovine serum after puberty, we performed in situ hybridization using albumin), 10% dextran sulfate, 600 mM NaCl, 0.25% SDS, 984 S-Y. NAM, ET AL. and 1 mM EDTA (pH 8.0) in DEPC-treated water. These stages VI-III, while it increased slightly at stages IV-V (Fig. sections were rinsed in 50% FA/2-strength SSC at 42°C for 3). 20 min 3 times and treated with 10 µg/ml of RNase A in In addition, the testes of both ages hybridized with a buffer (10 mM Tris-HCl [pH 7.6], 500 mM NaCl, and 1 DIG-labeled sense riboprobe for MCS showed no signals mM EDTA) at 37°C for 30 min. Sections were then washed over haploid cells or specific stages of tubules (data not in 2-strength SSC at 42°C for 20 min, followed by washing shown). in 0.2-strength SSC at 42°C for 20 min twice, and processed with immunohistochemical procedures. Each was incubated DISCUSSION with an anti-DIG alkaline phosphatase-conjugated antibody (Boehringer Mannheim, Tokyo, Japan) and developed with Se is necessary for spermatogenesis in mammals and its 5-bromo-4-chloro-3-indolyl phosphate and nitro blue concentration considerably increases in the testes during tetrazolium (Boehringer Mannheim, Tokyo, Japan) in 0.1 M puberal maturation [1]. In tissues, Se has been known to be Tris buffer for 1 hr, respectively. incorporated into a selenocysteine which recognizes specific Signal intensity analysis: To determine the relative level UGA codons in selenoprotein [8]. Previously, we of MCS mRNA in the testes of 8- and 20-week-old mice, demonstrated that MCS mRNA greatly increases after the signal intensity was measured with a laser densitometer puberty in the mouse testis [16]. In the present study, the (Scanning Densitometer; BIO-RAD, Tokyo, Japan) from expression of MCS mRNA changed stage-dependently and black and white negative films photographed under the same increased in the seminiferous epithelium of 20 weeks old as conditions on both of in situ analysis slides. An average of compared with that of 8 weeks old. 6 seminiferous tubules in each stage grouping was randomly Previous studies showed that during spermatogenesis, Se selected from the testes of both ages. The signal intensity is present in the form of selenoprotein and is localized in in the seminiferous tubules was analyzed from the the outer membrane of spermatozoa mitochondria [5, 18]. spermatids over spermatocytes. The data (mean ± SEM) Calvin et al. [7] reported that in rats, incorporation of 75Se for MCS mRNA was determined by the relative level of the into a selenoprotein begins in late primary spermatocytes, mRNA in the spermatids to that in the primary increases steadily during step 1–6 spermatids, is highest in spermatocytes where the signal was not detected. steps 7–12, declines during steps 13–15, and is undetectable Comparisons between both ages were performed using the during steps 16–19. In the present study using a DIG- Student’s t-test, and statistical significance was considered labeled riboprobe, MCS mRNA was expressed in step 3–16 at p<0.01. spermatids of mouse testes, which was consistent with the results using a radiolabeled riboprobe [22]. These findings RESULTS indicate that Se and MCS are necessary for spermatogenesis. On the other hand, many studies [9, 10] have In the testes of 8- and 20-week-old mice, the in situ demonstrated that aging results from imperfect protection analysis using a DIG-labeled antisense riboprobe for MCS against tissue damages, particularly mitochondrial injuries, showed that the MCS mRNA was concentrated in the upper which were brought about by free radicals produced during region of the seminiferous epithelium (spermatids) and that normal aerobic metabolism. It has been also known that the amount of signal varied greatly depending on the stage mitochondrial DNA is more susceptible to oxidative damage of the tubules (Fig. 1). At stage I, the MCS mRNA was not than nuclear DNA due to the lack of histones protecting the detected in the cytoplasm of step 1 spermatids, but was DNA and DNA repair enzymes, and the proximity of intensely expressed in the cytoplasm of step 13 spermatids mitochondrial DNA to oxidants generated during oxidative (Fig. 2A). At stages II-III, the signal first appeared in the phosphorylation [21]. Lee et al. [13] found that unlike cytoplasm of step 3 spermatids and was intensely expressed muscle and liver, the deletion of mitochondrial DNA in the cytoplasm of step 14 spermatids. At stages IV-VIII, induced by aging in humans is undetectable in the testes the signal began to increase during early spermiogenesis until age 60. Furthermore, previous studies [1, 4, 25] have and greatly increased in the cytoplasm of step 8 spermatids shown that the Se content in testes is especially high in Se- (Figs. 2B and 2C). At stages IX-XII, the signal attained a deficient animals as compared with that in main storage high level in the cytoplasm of steps 9–12 spermatids (Fig. tissues such as muscle and liver, and considerably increases 2D). No signal was observed in spermatogonia, in maturing animals after the initiation of spermatogenesis. spermatocytes, Sertoli cells, peritubular myoid cells, and Previously, we demonstrated that MCS mRNA begins to interstitial cells. On the other hand, the signal in the testes increase in mouse testes according to aging and the level in of 20-week-old mice remarkably increased as compared with the testes of 80-week-old mouse increases about 6-fold as that in the testes of 8-week-old mice (Figs. 1 and 2). compared with that of 3-week-old mouse. Additionally, its To quantify the stage-dependent changes of MCS mRNA expression in the testes remarkably decreases by Se at both ages, the signal intensities were measured with a deficiency [16]. In this in situ analysis, the MCS mRNA laser densitometer (see Materials and Methods). Compared was expressed in the cytoplasm of steps 3–16 spermatids. with that in the testes of 8-week-old mice, the MCS mRNA During the period, remarkable changes of mitochondrial level in 20-week-old mouse testes increased over 2-fold at morphology occurs. Moreover, the expression level of MCS INCREASE OF MCS mRNA IN TESTIS AFTER PUBERTY 985

Fig. 1. In situ hybridization using a DIG-labeled antisense riboprobe for MCS in testes. The signals of MCS mRNA are concentrated in the upper region of the seminiferous epithelium (spermatids). Compared with that in the testes of 8-week- old mouse, MCS mRNA is highly expressed in the testes of 20-week-old mouse. A) 8 weeks old, B) 20 weeks old. × 80. 986 S-Y. NAM, ET AL.

Fig. 2. In situ analysis using a DIG-labeled MCS riboprobe in 8- and 20-week-old mice testes. Compared with that in 8-week-old mouse testes, the expression of MCS mRNA in 20-week-old mouse testes greatly increases at stages I, VIII, and XII, while it slightly increases at stage V. A) stage I, B) stage V, C) stage VIII, D) stage XII. × 640. INCREASE OF MCS mRNA IN TESTIS AFTER PUBERTY 987

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