Kajian kualitas dan protein membran Spermatozoa tikus (Rattus norvegicus) akibat pemaparan 2-Methoxyethanol
HAYATI, Alfiah, Promotor Prof.drh. Soesanto Mangkoewidjojo, M.Sc.,Ph.D
2007 | Disertasi | S3 BiologiSenyawa 2-methoxyethanol (2-ME) adalah salah satu bahan toksik yang berpotensi merusak organ reproduksi jantan. Senyawa ini dapat masuk ke dalam tubuh melalui berbagai cara yaitu kontak langsung melalui kulit, pernafasan dan pencernaan. Informasi tentang kerusakan sel spermatogenik karena pemaparan 2-ME sudah banyak diketahui, namun bagaimana kerusakan spermatozoa yang mencerminkan kemampuannya dalam fertilisasi, khususnya pada ultrastruktur, biokimiawi, dan protein membran belum diketahui. Penelitian ini bertujuan untuk mengkaji pengaruh frekuensi dan lama waktu pemaparan 2-ME terhadap kualitas dan protein membran spermatozoa tikus serta tingkat kerusakan spermatozoa setelah pemaparan 2-ME. Sebanyak 140 ekor tikus strain Wistar digunakan dalam penelitian ini. Empat puluh ekor untuk pemeriksaan kualitas spermatozoa (motilitas, morfologi, integritas membran, jumlah spermatozoa dan leukosit, kadar reactive oxigen species (ROS) dan malondialdehyde (MDA)) dan uji apoptosis epididimis. Tikus-tikus tersebut dibagi menjadi 8 kelompok, masing-masing 5 ekor. Empat kelompok pertama, sebagai kelompok kontrol. Kelompok 1, 2 dan 3 disuntik dengan larutan NaCl fisiologis setiap hari secara subkutan sebanyak 1, 3 dan 6 kali/minggu selama 1 minggu, dan kelompok 4 disuntik 12 kali selama 2 minggu. Kelompok perlakuan (4 kelompok berikutnya) diberi suntikan 0,2 ml dari 200 mg/kg.bb 2-ME dengan frekuensi dan lama waktu yang sama dengan kelompok kontrol. Seratus ekor tikus digunakan untuk mengetahui profil protein membran spermatozoa dan uji imunositokimia. Tikus tersebut dibagi menjadi 5 kelompok, masing-masing 20 ekor. Satu kelompok sebagai kontrol disuntik larutan NaCl fisiologis dan 4 kelompok lainnya disuntik 0,2 ml dari 200 mg/kg.bb 2-ME sebanyak 1, 3 dan 6 kali/minggu selama 1 minggu dan 12 kali selama 2 minggu. Motilitas spermatozoa diperiksa di bawah mikroskop cahaya. Gerak spermatozoa yang diukur adalah gerak progresif ke depan. Morfologi spermatozoa diamati dengan pewarnaan eosin-nigrosin. Integritas membran spermatozoa ditentukan dengan uji hypoosmotic swelling. Jumlah spermatozoa dan leukosit dihitung dengan hemositometer. Kadar ROS dalam suspensi spermatozoa diukur menggunakan metode khemiluminisensi, dan kadar MDA spermatozoa ditetapkan dengan analisis thiobarbituric acid (TBA). Profil protein membran spermatozoa diukur dengan teknik sodium dodecyl sulphate-polyacrilamide gel electrophoresis (SDS-PAGE). Ketebalan pita protein diukur dengan densitometer, sedangkan distribusi protein membran spermatozoa diperiksa dengan imunositokimia dengan menggunakan antibodi monoklonal 2D6. Apoptosis detection kit S7100-S7101 digunakan untuk mengamati apoptosis sel epitel luminal epididimis. Analisis varian digunakan untuk mengetahui perbedaan antar perlakuan, dilanjutkan dengan uji beda nyata terkecil (BNT) dan uji Student-t dengan taraf signifikansi 5% (P < 0,05). Data kualitatif dianalisis secara deskriptif. Hasil pengukuran motilitas, morfologi dan integritas membran spermatozoa menunjukkan ada penurunan yang nyata setelah pemaparan 2-ME sebanyak 3 kali/minggu atau lebih (P<0,05). Sebagian besar morfologi spermatozoa tidak normal ditandai dengan cytoplasmic droplet pada kepala dan ekor, ekor bengkok/melingkar. Ultrastruktur nampak membran sel tidak utuh, mitokondria mengecil dan serabut padat menjadi tidak beraturan di bagian ekor. Jumlah spermatozoa kelompok perlakuan sampai pemaparan 6 kali/minggu tidak berbeda nyata (P>0,05) dibandingkan dengan kontrol, tetapi lama pemaparan 12 kali selama 2 minggu jumlah spermatozoa turun secara nyata (P<0,05). Jumlah leukosit dalam suspensi spermatozoa, ROS dan MDA semua kelompok perlakuan mengalami peningkatan secara nyata (P<0,05) dibandingkan kontrol. Hasil elektroforesis menunjukkan adanya perbedaan profil protein antar kelompok. Profil protein membran dengan berat molekul (BM) 60 kDa tampak pada kelompok kontrol dan perlakuan 1 dan 2 (1 dan 3 kali/minggu), tetapi tidak terekspresi setelah pemaparan 6 kali/minggu atau lebih. Pengamatan imunositokimia spermatozoa menunjukkan bahwa intensitas warna fluoresen protein 24 kDa menurun dengan bertambahnya waktu pemaparan 2-ME, intensitas warna terendah terdapat pada kelompok setelah pemaparan 12 kali dalam 2 minggu. Pewarnaan H dan E pada kelompok perlakuan menunjukkan bahwa inti sel berwarna ungu gelap dan lebih kecil daripada inti sel yang normal pada kontrol. Apoptosis sel epitel luminal epididimis tampak pada semua kelompok perlakuan, beberapa sel epitel luminal epididimis mempunyai inti kecil dan berwarna kecoklatan (gelap) sedangkan sitoplasma berwarna hijau. Kesimpulan penelitian ini adalah 1) Kualitas spermatozoa tikus turun setelah pemaparan 2-ME dosis 200 mg/kg.bb/hari 3 kali/minggu atau lebih. 2) Pemaparan 2-ME 6 kali/minggu atau lebih, menyebabkan protein membran spermatozoa dengan BM 60 kDa tidak terekspresi. 3) Kerusakan spermatozoa setelah pemaparan 2-ME meliputi perubahan pada struktur membran sel, serabut tebal dan mitokondria; motilitas, morfologi normal dan integritas membran spermatozoa. Efek ini terkait dengan peningkatan jumlah leukosit, kadar ROS dan MDA, serta apoptosis sel epitel luminal epididimis.
A 2-methoxyethanol (2-ME) compound is one of the toxic agents potentially affect male reproduction. This toxicant may enter in the body through skin, by inhalation or oral. The 2-ME is used primarily for industrial solvent to produce resins, lacquers, and paints. In mammals, the 2-ME may affect on testicular structures and functions. Many information of testis damages caused by 2-ME have been documented, but lack of reports of its effect in rat spermatozoa structure, biochemistry, and spermatozoa membrane protein. The main aim of this study was to determines the effect of the duration and frequency of 2-ME exposures on spermatozoa quality, protein membrane of spermatozoa, and to have better understandings in the severity of spermatozoa degeneration in rats. One hundred and forty male mature Wistar rats were used in this research. Forty rats were used for analyzing spermatozoa qualities (motility, morphology, membrane integrity, spermatozoa and leukocyte counts, reactive oxygen species (ROS), malondialdehyde (MDA)), and apoptosis as well. These 40 rats were divided further into 8 groups of 5 each. The first 4 groups were used as controls, given 0.2 ml of physiological saline subcutaneously. The first, second and third groups were given 1, 3 and 6 times/week for 1 week, respectively and the 4th group was given 12 times for 2 weeks. The second 4 groups, instead of being given physiological saline, they were injected subcutaneously with 0.2 ml of 200 mg/kg/d 2-ME with the same frequency and duration that of the first 4 groups. One hundred rats were used in the second experiment to analysis protein membrane of spermatozoa and immunocytochemistry. The rats were divided into 5 groups of 20, each. One group was the control, it was injected with physiological saline. Four treatment groups (groups 1, 2, 3, and 4) were given 0.2 ml of 200 mg/kg/day 2-ME in 1, 3 and 6 times/week for one week, respectively, and group 4 was given 12 times for two weeks. Spermatozoa motility was evaluated using spermatozoa tracks that were visually progressive under a light microscope. Spermatozoa morphology slides were examined using eosin-nigrosin stain. Membrane integrity was evaluated using hypoosmotic swelling test. The spermatozoa and leukocyte counts in spermatozoa suspension were done by haemocytometer. Generation of ROS concentration was quantified using chemiluminescence technique, and MDA concentration was determined by the thiobarbituric acid assay. The protein profiles of spermatozoa membrane were analyzed by sodium dodecyl sulphate-polyacrilamide gel electrophoresis (SDS-PAGE) techniques, and estimated by dencytometer. Immunocytochemical analysis of spermatozoa was observed using 2D6 monoclonal antibody, and the apoptotic epididymis epithelial cells were detected by In Situ Apoptosis Detection Kit (S7100-S7101). The different among treatments were analyzed using Anova, followed by LSD and student T test to obtain the significance the difference (P < 0.05). Qualitative data analyzed describtively. The results of investigation revealed hyperactive motility of the control rat spermatozoa. Mean values of motility, morphology and membrane integrity decreased after the exposure to 2-ME for 3 times/week (P<0.05). The major morphological abnormalities observed were cytoplasmic droplets on head-tail, curving and bent tails and damages in spermatozoa membrane included membrane lysis with discontinued features, the shrunken mitochondria, and disarrangement of dense fibers of tail membrane. The spermatozoa counts of treated rats (up to 6 times/week for one week) showed no significant difference (P>0.05), but in the highest dose treatment (12 times/week for two week) differed significantly (P<0.05) compared with those of the control. The leukocytes counts, ROS and MDA concentration in all of the treated rats were significant increased (P<0.05) compared with those of the control. The protein membrane of spermatozoa compositions in the treatmentgroups had different profiles. Protein of 60 kDa appeared in the control and treated rats (1 and 3 times/week groups), but it was not expressed in the treated rats of 6 times/week exposure or more. Immonocytochemical analysis of the spermatozoa showed spermatozoa membrane protein especially of 24 kDa. This protein concentration decreased in the treated rats (after 3 times/week) as shown by decreasing fluorescent intensity of yellow color. A number of epididymis epithelial cells of the treated rats stained with H and E showed purple (dark) color and had smaller nuclei than the normal nuclei. The apoptosis was detected in all of the treated rats those of in epididymis epithelial cells showing chocolate (dark) and shrunken nuclei with green color of cytoplasm. The conclusions of the research were 1) exposure to 200 mg/kg/day 2-ME after 3 times/week or more decreased spermatozoa quality. 2) the time of exposure to the 2-ME affected on the profiles of protein membrane. Protein 60 kDa was susceptible to exposure for 6 times/week or more, 3) Exposure to 2-ME caused damages in rat spermatozoa involving membrane structures, dense fibers, mitochondria; alterations of protein membrane profiles; and eventually decreasing in motility, morphology and membrane integrity. These effects were accompanied by increasing in the leuko
Kata Kunci : Sistem Reproduksi Jantan,Senyawa 2 ME, Spermatozoa, 2-methoxyethanol, ROS, MDA, protein membrane, immunocytochemistry, apoptosis
