An Immunohistochemical Study on Testicular Steroidogenesis in the Sunda
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Advance Publication The Journal of Veterinary Medical Science Accepted Date: 1 July 2019 J-STAGE Advance Published Date: 23 July 2019 ©2019 The Japanese Society of Veterinary Science Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Wildlife Science 2 Full paper 3 An immunohistochemical study on testicular steroidogenesis in the Sunda 4 porcupine (Hystrix javanica) 5 6 Anni NURLIANI1,2,3), Motoki SASAKI1,2)*, Teguh BUDIPITOJO4), Toshio 7 TSUBOTA5), Masatsugu SUZUKI2,6) and Nobuo KITAMURA1,2) 8 9 1)Department of Veterinary Medicine, Obihiro University of Agriculture and 10 Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan 11 2)United Graduate School of Veterinary Sciences, Gifu University, Gifu 12 501-1193, Japan 13 3)Department of Biology, Faculty of Mathematics and Natural Sciences, 14 Lambung Mangkurat University, South Kalimantan 70714, Indonesia 15 4)Department of Anatomy, Faculty of Veterinary Medicine, Gadjah Mada 16 University, Yogyakarta 55281, Indonesia 17 5)Laboratory of Wildlife Biology and Medicine, Department of Environmental 18 Veterinary Science, Graduate School of Veterinary Medicine, Hokkaido 19 University, Hokkaido 060-0818, Japan 20 6)Laboratory of Zoo and Wildlife Medicine, Faculty of Applied Biological 21 Science, Gifu University, Gifu 501-1193, Japan 22 23 24 25 *Correspondence to: SASAKI, M., Department of Veterinary Medicine, 26 Obihiro University of Agriculture and Veterinary Medicine, Obihiro, 27 Hokkaido 080-8555, Japan. e-mail: [email protected] 28 29 30 Running head: STEROIDOGENESIS IN PORCUPINE TESTES 31 32 1 33 ABSTRACT 34 In the testes of the Sunda porcupine (Hystrix javanica), the expression of the 35 steroidogenic acute regulatory protein (StAR) and steroidogenic enzymes, 36 such as cytochrome P450 side chain cleavage (P450scc), 3β-hydroxysteroid 37 dehydrogenase (3β-HSD), cytochrome P450 17α-hydroxylase (P450c17) and 38 cytochrome P450 aromatase (P450arom), was immunohistochemically 39 examined to clarify the location of steroidogenesis. In this study, complete 40 spermatogenesis (spermiogenesis) was observed in the testes of the 41 examined Sunda porcupine, and spermatozoa of the Sunda porcupine had a 42 spatulate sperm head unlike that of rats and mice which has an apical hook. 43 On immunostaining of StAR, P450scc, 3β-HSD, P450c17 and P450arom, 44 immunoreactivity for all proteins was only detected in the Leydig cells and 45 not observed within the seminiferous tubules, suggesting that the Leydig 46 cells can synthesize both androgen and estrogen from cholesterol in the 47 Sunda porcupine testes. 48 49 KEY WORDS: Leydig cell, porcupine, sperm head, steroidogenesis, testis 50 51 52 53 54 55 56 57 58 2 59 INTRODUCTION 60 The Sunda porcupine (Hystrix javanica), which is a species of rodent 61 in the family Hystricidae, is endemic to Indonesia and distributed in Java, 62 Bali, Sumbawa, Flores, Lombok, Madura, Tonahdjampea and South 63 Sulawesi [50]. H. javanica was previously considered to belong to same 64 species as H. brachyura [10], but Van Weers [47] examined the cranial and 65 external characters of H. javanica, and demonstrated it to be a separate 66 species from H. brachyura. Based on the Law of the Republic of Indonesia 67 number 7 of 1999 concerning the list of protected animals in Indonesia, only 68 H. brachyura is listed as a protected animal, whereas H. javanica is not 69 listed. However, from June 2018, H. javanica became a protected animal 70 based on the Law of Ministry of Environment and Forestry, Republic of 71 Indonesia (Number P.20/MENLHK/SETJEN/KUM.1/6/2018) related to 72 decline of their population. Although, on the IUCN Red List of Threatened 73 Species, this species is currently listed as being of least concern by 74 International Union for Conservation of Nature [27]. 75 Historically, people in Indonesia have hunted and consumed porcupine 76 meat because they believed it to have medicinal properties. The meat from 77 the porcupine tail was cooked and consumed as an aphrodisiac [3]. The 78 continuous hunting of the Sunda porcupine without considering conservation 79 principles may significantly reduce their population. Therefore, reproductive 80 strategies must be designed for conservation of this species, and knowledge 81 of aspects related to the reproduction of the Sunda porcupine is essential to 3 82 support its successful conservation. 83 The in vitro fertilization (IVF) is one of the assisted reproductive 84 technologies (ARTs) which could be applied as reproductive strategies to 85 increase the number of population of the Sunda porcupine. Regarding this 86 point, the knowledge about morphology of sperm head is important to predict 87 the outcome of IVF. The significance of morphology of sperm head as a 88 marker of successful fertilization has been reported previously [14]. 89 Information about the sperm head shape has not been available yet in the 90 Sunda porcupine and the findings in this study were provided as the leading 91 information. 92 Male reproductive function is regulated by steroid hormones such as 93 androgen and estrogen. In the Sunda porcupine, the testicular dynamics of 94 steroid production are not fully understood. Information on testicular 95 steroidogenesis is needed to improve our understanding of testicular systems 96 regulated by steroid hormones. 97 Gonadal steroidogenesis is controlled by changes in the steroidogenic 98 acute regulatory protein (StAR). StAR mediates the transfer of cholesterol 99 from the outer to the inner mitochondrial membrane. Cholesterol is then 100 converted to androgen via catalysis activity of steroidogenic enzymes such as 101 cytochrome P450 side chain cleavage (P450scc), 3β-hydroxysteroid 102 dehydrogenase (3β-HSD) and cytochrome P450 17α-hydroxylase (P450c17) 103 [28]. Then, another biosynthetic enzyme, cytochrome P450 aromatase 104 (P450arom), converts androgen into estrogen [20]. This study also 4 105 investigated the production site of steroid hormones in the testis of the 106 Sunda porcupine by examining the localization of these steroidogenic 107 enzymes immunohistochemically to clarify the reproductive process. 108 109 MATERIALS AND METHODS 110 Animals and Tissue Collection 111 Four wild male Sunda porcupines were obtained from Central Java, 112 Indonesia. The animals were euthanized by an overdose injection of ketamin 113 HCl (10-15 mg/kg) and xylazine HCl (0.10-0.15 mg/kg) administrated 114 intramuscularly before taking organ samples. The removed testes were 115 immediately fixed in Bouin’s fluid for 24 hr. After fixation, the tissue samples 116 were dehydrated in a graded series of ethanol, cleared in xylene and 117 embedded in paraffin. The samples were cut serially at 4 µm thickness and 118 mounted on aminopropyl-triethoxy-silane-coated slides (S9226, Matsunami 119 Glass, Osaka, Japan). After deparaffinization, the testicular tissue sections 120 were stained with hematoxylin and eosin (HE) to examine the general 121 structure. All experimental procedures were authorized by the Ethics 122 Committee for the Use of Animals at Gadjah Mada University (Number: 123 0014/EC-FKH/Eks/2017). Collection permit (number: 124 SK.56/KSDAE/SET/KSA.2/2/2018) was issued by the Directorate General of 125 Natural Resources and Ecosystem Conservation (Direktorat Jenderal 126 Konservasi Sumber Daya Alam dan Ekosistem, Jakarta, Indonesia) under 127 the Ministry of Environment and Forestry of the Republic of Indonesia. 5 128 129 Immunohistochemistry 130 To examine the localization of StAR and steroidogenic enzymes in 131 testes, the sections were stained immunohistochemically using an 132 avidin-biotin peroxidase complex (ABC) method. The primary antibodies 133 used in this study are summarized in Table 1. For the ABC method, the 134 sections were deparaffinized in xylene, rehydrated in graded alcohols and 135 washed with tap water. After microwaving in high pH target retrieval 136 solution (1:10, S3307; Dako Cytomation, Inc., Carpinteria, CA, U.S.A.) for 137 antigen retrieval, the endogenous peroxidase activity was blocked with 138 methanol containing 0.3% H2O2 for 10 min at room temperature (RT). Then, 139 the sections were incubated with normal goat serum for 30 min at RT to 140 prevent nonspecific staining. The samples were incubated with each primary 141 antibody overnight at 4°C. The biotinylated anti-rabbit IgG (1:200 dilution, 142 BA-1000, Vector Laboratories, Burlingame, CA, U.S.A.) as the secondary 143 antibody was applied for 30 min at RT, and the sections were then incubated 144 with the Vectastain Elite ABC Kit (1:2 dilution, PK-6100, Vector 145 Laboratories) for 30 min at RT. The immunoreactive sites were visualized 146 using Tris HCl buffer containing 0.02% 3,3’-diaminobenzidine 147 tetrahydrochloride (DAB) and 0.006% H2O2. The negative control sections 148 were prepared without the primary antibody. 149 150 RESULTS 6 151 On histological observation of testes of the Sunda porcupine, the 152 seminiferous tubules with several stages of spermatogenesis were observed, 153 although the stage number of the spermatogenetic (seminiferous epithelium) 154 cycle was not identified in this study (Fig. 1). Spermatozoa of the Sunda 155 porcupine have a spatulate sperm head (Fig. 1C, D). Among the seminiferous 156 tubules, the interstitial tissue, including the Leydig cells exhibiting a round 157 nucleus, was identified (Fig. 1A-C). 158 The localization of StAR and steroidogenic enzymes in the testes is 159 shown in Figure 2. Immunoreactivities for StAR,