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"Epididimis adalah bagian dari alat reproduksi laki-laki yang berfungsi dalam pematangan sperma. Proses ini terjadi melalui interaksi antara sperma dan protein yang disekresikan oleh sel-sel epitel di epididimis. Protein-protein tersebut dikode oleh gen yang terekspresi secara spesifik di epididimis, salah satunya adalah Serpina1f. Akan tetapi, kebanyakan dari regulasi gen diatas belum dipahami dan perlu ditelilti, lebih lanjut. Serpina1f adalah salah satu gen yang menarik untuk di karakterisasikan. Gen ini diregulasi oleh androgen dan sudah dibuktikan oleg data yang diambil menggunakan mikroaray analisis. Penelitian ini bertujuan untuk menganalisa struktur gen dari Serpina1f sebagai dasar untuk mempelajari fungsi dari gen tersebut lebih lanjut, dan juga distribusi jaringan untuk menyeleksi apakah gen Serpina1f hanya terekspresi di epididymis yang pada akhirnya akan menentukan apakah gen ini cocok dikembangkan menjadi kontrasepsi non-hormonal bagi pria. Penelitian ini dilakukan sejak Mei 2012 sampai Februari 2013 di Laboratorium Departemen Biologi Universitas Indonesia dan dilaksanakan dengan menggunakan analisa bioinformatika dan analisa ekspresi gen. Hasil penelitian menunjukan bahwa gen Serpina1f sangat dominan pada daerah initial segment pada epididimis mencit sehingga cocok untuk dikembangkan menjadi kontrasepsi non-hormonal bagi pria.

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Epididymis is a part of male reproductive system which functions in the process of sperm maturation. This process is occurs by interaction between sperm and proteins that secreted by epithelial cells in the epididymis. Those proteins are encoded by epididymis specific genes, one of them is Serpina1f. However, many of those genes are not entirely well-characterized and need to be elaborated further. Serpina1f is one of the attractive genes to be characterized. It is regulated by androgen and has been proved by previous data obtained by microarray analysis.

This research was aimed to analyze gene structure of Serpina1f gene as a basis for more exploration regarding the function of this gene, and also to identify tissue distribution to determine whether Serpina1f gene is expressed only in epididymis, so that this gene is suitable to be developed as a target for non-hormonal male contraception.

This experiment was conducted from May 2012 to February 2013 in the Laboratory of Department of Biology Universitas Indonesia and it was performed by in-silico analysis and gene expression analysis. The result shows that Serpina1f gene is very dominant in initial segment in epididymis, thus suitable as a candidate for non-hormonal male contraception."

"Epididimis adalah bagian sistem reproduksi laki-laki yang berperan penting dalam proses pematangan sperma. Proses ini berlangsung karena adanya interaksi antara protein yang disekresikan oleh sel-sel epitel epididymis dengan sperma. Banyak gen yang mengatur protein-protein tersebut belum dipahami sepenuhnya, contohnya Serpina1f. Penelitian ini bertujuan untuk mengidentifikasi signal peptide sebagai dasar informasi untuk mempelajari fungsi gen tersebut lebih lanjut, dan ketergantungan androgen untuk secara dini menyeleksi apakah gen ini cocok dikembangkan menjadi kontrasepsi pria nonhormonal. Metode penelitian untuk memprediksi signal peptide adalah dengan menggunakan SignalP 4.0, sedangkan analisis ketergantungan androgen menggunakan real-time RT-PCR. Hasil penelitian ini memperlihatkan bahwa Serpina1f mengandung signal peptide pada 27 asam amino yang pertama, sedangkan ekspresi gen berubah sesuai level androgen. Hal ini menunjukkan bahwa Serpina1f menghasilkan secretory protein dan gen ini diregulasi androgen.

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Epididymis is a part of male reproductive system that plays important role in sperm maturation process. This process occurs by the interaction between proteins secreted by epididymal epithelial cells with sperm. Many of the genes that regulate the protein involved in this process have not been understood completely, such as Serpina1f. The objective of this research is to identify its signal peptide as basic information to further learn its function, and androgen dependency to early select whether this gene could be a target for a nonhormonal male contraception. SignalIP 4.0 was used to predict the presence of signal peptide in its amino acid sequence, while the androgen dependency analysis was performed by quantitative real-time RT-PCR using RNA samples from gonadectomized mice and gonadectomized mice plus androgen therapy. The result showed that Serpina1f contained signal peptide at the first 27 amino acid sequence, while its expression fluctuated according to androgen level. It can be concluded that Serpina1f encodes a secretory protein and it is an androgen dependent gene."

"Terdapatnya kasus infertilitas pria menimbulkan dugaan bahwa salah satu penyebabnya adalah tingkat integritas DNA sperma, sehingga pemeriksaan tingkat kerusakan DNA sperma dipandang perlu untuk dimasukan dalam analisa semen standar untuk menilai kesuburan pria. Namun demikian sampai saat ini masih terdapat ketidakseragaman laporan mengenai hubungan tingkat integritas DNA sperma dengan parameter standar kualitas spermatozoa seperti motilitas dan morfologi. Disamping itu terdapat berbagai metode pemeriksaan integritas DNA sperma dengan prinsip deteksi yang berbeda yang menyebabkan kesulitan dalam menginterpretasi hasil. Di antara metode tersebut adalah uji Sperm Chromatin Dispersion (SCD) assay dengan melihat pola penyebaran kromatin sperma dan Terminal Deoxynucleotidyl Transferase dUTP Nick-end Labelling (TUNEL) yang mampu mendeteksi patahan DNA. Berdasarkan masalah tersebut maka tujuan penelitian ini adalah untuk mengetahui hubungan antara tingkat integritas DNA sperma dengan parameter standar kualitas spermatozoa dan juga untuk mengetahui korelasi antara uji SCD dan TUNEL.Jenis penelitian ini menggunakan metode observasional analitik. Sampel yang diteliti berjumlah 36 sampel dengan rincian 23 sampel dari kelompok pria dengan parameter semen abnormal dibandingkan dengan 13 sampel kelompok pria dengan parameter semen normal. Masing-masing sampel dilakukan pemeriksaan integritas DNA dengan metode SCD dan TUNEL. Hasil pemeriksaan dari kedua metode ini kemudian dilakukan analisa korelasi.Hasil penelitian ini menunjukkan bahwa dengan metode SCD spermatozoa dengan parameter abnormal (n = 23) mempunyai kisaran indeks fragmentasi DNA (IFD) kriteria baik sebesar 34%, IFD sedang 26% dan IFD kurang 40%, sedangkan pada sperma dengan parameter normal (n = 13) dengan urutan kriteria yang sama menunjukkan kisaran IFD sebesar 46%, 46%, dan 8%. Pada pemeriksaan dengan menggunakan metode TUNEL, sperma dengan parameter abnormal diperoleh IFD baik, sedang dan kurang sebesar 35%, 35%, dan 30%, sedangkan sperma dengan parameter normal diperoleh IFD berkisar antara 31%, 61% dan 8%. Hal ini menunjukkan bahwa secara umum, baik pada uji SCD maupun TUNEL, terdapat kecenderungan tingginya IFD pada sampel abnormal walaupun hasil analisa statistik pada kedua metode tidak menunjukkan perbedaan yang signifikan. Dari analisa korelasi antara SCD dan TUNEL diperoleh hasil bahwa pemeriksaan dengan kedua metode menunjukkan korelasi yang kuat dan signifikan dengan nilai (r = 0.791). sehingga dapat disimpulkan bahwa pemeriksaan integritas DNA sperma dengan uji SCD maupun TUNEL memberikan hasil yang serupa.

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The incidence of man infertility leads to a notion that one of the possible cause is sperm DNA damage, therefore sperm DNA integrity test is thought to be necessary for a standard sperm analysis to assess male fertility. However, there is still lack of common reports regarding the relationship between sperm DNA integrity and its quality parameters such as motility and morphology. Besides, there are different methods of sperm DNA integrity test with different detection principles that lead to difficulties in interpreting the results. Among these methods are the Sperm Chromatin Dispersion test (SCD) that is based on detection of sperm chromatin spread pattern and Terminal Deoxynucleotidyl Transferase dUTP Nickend Labeling (TUNEL) capable of detecting sperm DNA strand break. Based on these problems, the purpose of this study was to determine the relationship between the levels of sperm DNA integrity and its quality parameters and also to determine the correlation between SCD and TUNEL test.

The observational analytic method was used in this study to analyze the relationship between sperm DNA integrity and its quality parameters. Thirty six samples consist of 23 samples from groups of men with abnormal semen parameters were compared with 13 samples from group with normal semen parameters. SCD and TUNEL test were performed on each sample from both groups. The relatioship between SCD and TUNEL was further analyzed using a correlation analysis.

The results on SCD method showed that spermatozoa with abnormal parameter (n = 23) had DNA fragmentation Index (DFI) ranged from good criteria 34%, average 26% and poor 40%, whereas sperm with normal parameters (n = 13) showed good, average and poor criteria of 46%, 46% and 8% respectively. The results on TUNEL method also showed DFI of abnormal sperm ranged from good 35%, average 35% and poor criteria of 30%, whereas sperm with normal parameters showed 31%, 61% and 8%, respectively. In general, this study showed that, in both methods, sperm with abnormal parameters showed a higher DFI compared to the normal samples, although the difference was not statistically significant. In addition, correlation analysis between SCD and TUNEL showed that both methods had a strong linear correlation (r = 0.791). Thus it can be concluded that sperm DNA integrity test using SCD and TUNEL gave similar results."

"Epididimis adalah saluran berbelit yang terletak antara testis dan vas deferens. Epididimis telah lama dikenal sebagai tempat proses pematangan sperma, yang merupakan interaksi antara sperma dan protein-protein yang disekresikan oleh sel-sel epitel epididimis. Salah satu protein yang diduga berperan penting dalam proses pematangan sperma adalah gen Defb42. Tujuan penelitian ini adalah untuk mengetahui struktur gen dan analisis sebaran jaringan dari gen Defb42. Data struktur gen Defb42 diperoleh dari database Unigene danUCSC Genome Bioinformatics, dimana cDNA, ekson, intron, dan sekuens asam amino diperoleh. Isolasi RNA dilakukan untuk jaringan dari 4 segmenepididimis (initial segment, caput, corpus, cauda) disertai dengan beberapa organ viseral lainnya. Real time RT-PCR dari RNA yang terelusi dilakukan untuk mengukur ekspresi relative Defb42 terhadap gen aktin beta. Hasil penelitian menunjukkan bahwa Defb42 termasuk dalam keluarga beta defensin karena memiliki 6 residu sistein. Ekspresi Defb42 relatif terhadap aktin ditemukan paling tinggi di initial segment dariepididimis, diikuti dengan caput, cauda, dan vas deferens. Disimpulkan bahwa gen Defb42 adalah gen beta defensin dan diekspresikan terutama di initial segment dari epididimis.

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Epididymis is a convoluted duct that is located between the testis and vas deferens. Epididymis has been known as the site of sperm maturation, which occur via interaction between the sperm and the proteins secreted by the epididymal epithelial cells. One of the proteins that is believed to play a major role in sperm maturation process is encoded by Defb42 gene. The objective of this research is to know the gene structure and tissue distribution analysis of Defb42 gene. Gene structure data was obtained from Unigene database and UCSC Genome Bioinformatics, in which cDNA, exons, introns, and amino acid sequence of Defb42 gene were received. RNA isolation was done for tissues of the four segments of epididymis (initial segment, caput, corpus, cauda) along with other visceral tissues. Real time RT-PCR of the isolated RNA was then performed in order to measure Defb42 relative expression to beta actin. The result showed that Defb42 gene belongs to the beta defensin family due to its conserved six cysteine residues. Defb42 expression relative to actin was highest in the initial segment of the epididymis, followed by caput, cauda, and vas deferens. In conclusion, Defb42 is a beta defensin gene and is mainly expressed in the epididymis and showed region-specific expression in the initial segment."

"[ABSTRAKLatar belakang: Proses pematangan sperma terjadi melalui interaksi spermatozoa dengan protein yang disekresikan ke lumen oleh sel epitel epididimis. Sekresi protein pada epididimis ditentukan oleh gen-gen yang terekspresi spesifik di epididimis. Ekspresi gen di epididimis dapat dipengaruhi oleh androgen atau faktor testikular. CD52 telah diketahui terekspresi di epididimis, namun regulasi yang mempengaruhi ekspresi gen CD52 di epididimis belum diketahui. Tujuan dari penelitian ini adalah untuk menganalisis ekspresi dan regulasi gen CD52 agar dapat memprediksi perannya di epididimis mencit. Metode: Analisis bioinformatika dilakukan untuk memprediksi sinyal peptida dan domain fungsional dari CD52. Quantitative real time RT-PCR digunakan untuk mengukur ekspresi relatif gen CD52 pada analisis spesifisitas jaringan, ketergantungan terhadap androgen dan faktor testikular, serta postnatal development. Hasil: CD52 memiliki sinyal peptida yang menunjukkan ciri protein sekretori dan terekspresi secara spesifik di epididimis. Ekspresi CD52 yang tertinggi terdapat di bagian cauda. Ekspresi CD52 pada mencit diregulasi oleh androgen yang ditandai dengan penurunan pada hari pertama dan ketiga setelah digonadektomi dan pemberian testosteron eksogen setelah gonadektomi dapat menjaga ekspresi CD52 50% dari kadar normalnya. Eksperimen dengan memberikan reseptor androgen antagonis (flutamide) juga mendukung bahwa ekspresi CD52 sangat tergantung terhadap androgen. Ekspresi CD52 menurun sangat bermakna hingga mencapai 93% dibandingkan dengan kontrol. Selain androgen, ekspresi CD52 juga dipengaruhi oleh faktor testikular. Ekspresi CD52 mengalami penurunan bermakna dari hari pertama hingga kelima setelah perlakuan efferent duct ligation (EDL) hingga mencapai 75% dari kontrol. Selain itu ekspresi CD52 juga dipengaruhi oleh perkembangan pasca lahir. Ekspresi CD52 meningkat di hari ke-15 hingga hari ke-60 pasca lahir. Kesimpulan: CD52 merupakan gen penyandi protein sekretori yang terekspresi spesifik di epididimis pada region cauda dan regulasinya dipengaruhi oleh androgen, faktor testikular, dan perkembangan pasca lahir.

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ABSTRACTBackground. Epididymal sperm maturation is occurs via interactions between sperm and proteins secreted by epididymal epithelium. These proteins are encoded by genes that are specifically expressed in a region-specific manner. Previous studies have demonstrated that epididymal genes are regulated by androgen and testicular factors. CD52 is an epididymal gene putatively involved in sperm maturation. However, the regulation of its expression in the epididymis has not been fully understood and little is known about its role during sperm maturation process. Therefore, this study was aimed to analyze the expression and regulation of CD52 in the mouse epididymis. Method. Bioinfomatic analyses were perfomed to predict signal peptides and functional domains of CD52. Quantitative real-time RT-PCR was used to analyze tissue distribution, androgen, testicular factors dependency and postnatal development. Results. CD52 amino acid sequence contains a signal peptide, indicating it is a secretory protein. CD52 exhibited region-spesific expression in the epididymis with the highest level was in cauda. Mice CD52 expression was regulated by androgen indicated by a decrease started at day 1 following a gonadectomy. Interestingly, testosterone replacement therapy was able to maintain the expression at 50% of normal level. Experiment by given androgen receptor antagonist, flutamide showed decrease of CD52 expression about 93% than control. It?s confirming that CD52 expression depend on androgen. Moreover, testicular factors also influenced CD52 expression. This was revealed by efferent duct ligation in which CD52 expression was reduced at day 1 to day 5 following the ligation. Finally, CD52 expression was developmentally regulated, this was indicated by increase in the level of expression start at day 15 postnatally. Conclusion: CD52 is a secretory protein and exhibited region-spesific expression in the cauda epididymis. It is regulated by androgen, testicular factors, and also affected by development stage. , Background. Epididymal sperm maturation is occurs via interactions between sperm and proteins secreted by epididymal epithelium. These proteins are encoded by genes that are specifically expressed in a region-specific manner. Previous studies have demonstrated that epididymal genes are regulated by androgen and testicular factors. CD52 is an epididymal gene putatively involved in sperm maturation. However, the regulation of its expression in the epididymis has not been fully understood and little is known about its role during sperm maturation process. Therefore, this study was aimed to analyze the expression and regulation of CD52 in the mouse epididymis. Method. Bioinfomatic analyses were perfomed to predict signal peptides and functional domains of CD52. Quantitative real-time RT-PCR was used to analyze tissue distribution, androgen, testicular factors dependency and postnatal development. Results. CD52 amino acid sequence contains a signal peptide, indicating it is a secretory protein. CD52 exhibited region-spesific expression in the epididymis with the highest level was in cauda. Mice CD52 expression was regulated by androgen indicated by a decrease started at day 1 following a gonadectomy. Interestingly, testosterone replacement therapy was able to maintain the expression at 50% of normal level. Experiment by given androgen receptor antagonist, flutamide showed decrease of CD52 expression about 93% than control. It’s confirming that CD52 expression depend on androgen. Moreover, testicular factors also influenced CD52 expression. This was revealed by efferent duct ligation in which CD52 expression was reduced at day 1 to day 5 following the ligation. Finally, CD52 expression was developmentally regulated, this was indicated by increase in the level of expression start at day 15 postnatally. Conclusion: CD52 is a secretory protein and exhibited region-spesific expression in the cauda epididymis. It is regulated by androgen, testicular factors, and also affected by development stage. ]"

"ABSTRAKLatar Belakang. Protein yang berperan penting dalam fungsi sperma berpotensi sebagai target molekul dalam upaya pengembangan bahan kontrasepsi pria. Salah satu protein yang terdapat pada sperma adalah protein kanal Voltage Dependent Anion Channel3 (VDAC3). VDAC3 berfungsi mengatur aliran ion dan metabolit termasuk ATP. Dari penelitian dengan menggunakan teknik knock-out mouse pada gen VDAC3 dilaporkan bahwa mencit jantan mutan VDAC3 homozigot mengalami penurunan yang signifikan dalam motilitas spermanya. Tujuan penelitian ini adalah memproduksi antibodi poliklonal VDAC3 melalui imunisasi protein rekombinan VDAC3 murni dan uji aktivitasnya terhadap motilitas dan viabilitas sperma manusia.Metode. Verifikasi keberhasilan pemotongan His fussion tag beserta 31 asam amino plasmid dari protein rekombinan dilakukan dengan teknik Western blott. ELISA digunakan untuk mengetahui titer IgG anti VDAC3sedangkan uji efektifitas antibodi VDAC3 terhadap fungsi sperma dilakukan dengan menghitung prosentase sperma yang tidak bergerak, waktu yang ditempuh sperma dalam jarak 0,1 mm. Analisa viabilitas sperma dilakukan dengan metode pewarnaan eosin.Hasil. Pada penelitian ini Western blotting dengan menggunakan antibodi Rabbit Anti VDAC Human menghasilkan pita tunggal dengan ukuran ~ 16 kDa, sedangkan penggunaan antibodi terhadap His (C-term) tidak menunjukan adanya pita. Hasil spektofotometri ELISA titer antibodi poliklonal VDAC3 yang berasal dari kelinci menunjukkan adanya peningkatan titer antibodi poliklonal VDAC3 setelah imunisasi dibandingkan dengan titer antibodi sebelum imunisasi (preimun serum). Hasil uji aktivitas antibodi poliklonal VDAC3 menunjukkan terjadi peningkatan jumlah sperma bergerak yang bermakna pada waktu 30 menit (p<0,05) dan 60 menit (p<0,05), juga terjadi peningkatan waktu tempuh sperma yang bermakna pada waktu 0-30 menit (p<0,05) setelah perlakuan. Penambahan antibodi poliklonal VDAC3 juga berpengaruh secara nyata terhadap persentase viabilitas spermatozoa yang hidup (p<0,05).Kesimpulan. VDAC3 poliklonal antibodi berhasil diproduksi melalui imunisasi dari VDAC3 rekombinan murni. Antibodi poliklonal anti-protein rekombinan Voltage Dependent Anion Channel-3 (VDAC3) dapat menurunkan motilitas dan viabilitas sperma manusia invitro secara bermakna.

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ABSTRACT

Background. Sperm-specific proteins that are important for sperm function can potentially be used as a target for developing a male contraceptive. One of the proteins found in the human sperm is Voltage Dependent Anion Channel3 (VDAC3). VDAC3 regulates the flow of ions and metabolites including ATP in the mitochondrial membrane and cell membrane of the eukaryotes. A previous study showed VDAC3 knockout mice had significant reduction in sperm motility. The purpose of this study was to produce polyclonal antibodies through immunization of pure VDAC3 recombinant protein and analyze its effect towards sperm motility and viability.

Methods. Removal of the His fussion tags plus 31 amino acids from the recombinant plasmid was verified using western immunoblotting. The titter of VDAC3 polyclonal antibody was determined by ELISA. The effect of VDAC3 antibodies against sperm qualities namely motility and viability was assessed using standard sperm analyses approved by the WHO.

Results. Western immunoblotting using Rabbit Anti Human VDAC3, produced a single band with size of ~ 16 kDa. No visible band was detected when anti-His (C-term) antibody was used in the analyses. Spectophotometric ELISA showed that the titer of VDAC3 polyclonal antibodies, derived from rabbits, polyclonal antibody increased better than the pre-immune. Analyses of VDAC3 polyclonal antibody against human sperm showed an increase in the number of sperm to move significant at 30 minutes (p < 0.05) and 60 minutes (p < 0.05), as well as an increase in sperm significant travel time at the time of 0-30 minutes (p < 0.05) after treatment. Polyclonal antibodies VDAC3 also significantly affect the percentage of sperm viability (p < 0.05).

Conclusion. Polyclonal antibody anti-VDAC3 was successfully produced via immunization of the pure recombinant VDAC3. Polyclonal antibody anti-recombinant protein Voltage Dependent Anion Channel-3 (VDAC3) may decrease human sperm motility and viability in vitro significantly."

"Latar Belakang. Voltage Dependent Anion Channel3 (VDAC3) merupakan salah satu protein yang terdapat pada sperma. Pada pengembangan imunokontrasepsi, protein tersebut dapat dijadikan antigen potensial dalam menurunkan fungsi sperma. Beberapa penelitian menunjukan bahwa anti-VDAC dapat mengganggu fungsi normal dan meningkatkan abnormalitas morfologi sperma. Imunisasi protein VDAC3 rekombinan terhadap kelinci diharapkan dapat memicu terbentuknya antibodi poliklonal anti-VDAC3. Antibodi tersebut kemudian dipurifikasi dan dievaluasi kemampuannya dalam mengikat antigen VDAC3 yang terdapat pada sperma manusia melalui pengamatan motilitas, viabilitas, integritas membran, dan integritas akrosom.Metode. Protein rekombinan VDAC3 diproduksi dengan cara mengkultur bakteri E.coli BL21 yang mengandung konstruksi vektor rekombinan. Protein rekombinan tersebut diimunisasikan pada kelinci kemudian diambil antiserumnya. Antibodi VDAC3 yang terkandung dalam antiserum dipurifikasi dengan metode kromatografi afinitas matriks sepharose protein A kemudian konsentrasinya diukur dengan metode ELISA dan dianalisis kemurniannya secara kualitatif dengan SDS-PAGE. Antibodi VDAC3 tersebut kemudian diuji kemampuannya terhadap sperma manusia normal dengan parameter: motilitas, kecepatan pergerakan, mortalitas (menggunakan metode pewarnaan Eosin Y), integritas membran ekor (menggunakan metode HOST), dan integritas akrosom (menggunakan metode FITC-PNA).Hasil. Kultur biakan E. coli dengan penambahan IPTG menunjukkan konsentrasi protein terlarut lebih tinggi yaitu 2,051 mg/ml dibandingkan dengan tanpa IPTG yaitu 1,528 mg/ml. Imunisasi protein VDAC3 rekombinan terhadap kelinci menghasilkan antiserum yang mengandung antibodi antiVDAC3 dengan titer tertinggi yaitu 3,504 pada kelinci B. Hasil SDS-PAGE antibodi yang dimurnikan menunjukan dua pita yang mendekati ukuran ~ 55 kDa dan ~25 kDa. Analisis statistik dari pengaruh antibodi VDAC3 murni terhadap motilitas, kecepatan gerakan, mortalitas, integritas membran ekor dan integritas akrosom sperma menunjukkan perbedaan yang signifikan (p <0,05) dengan kontrol preimun serum.

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ABSTRACTBackground. Voltage Dependent Anion Channel 3 (VDAC 3) is one of the proteins found in sperm. On the development of immunocontraception, this protein can be used as potential antigens to reduce normal function of sperm. Several studies have shown that anti-VDAC can reduce normal function of sperm and also increase the morphological abnormalities of sperm. VDAC3 recombinant protein immunization to rabbit is expected to produce of polyclonal antibodies anti-VDAC3. Then, the antibodies are purified and evaluated its ability to bind antigen VDAC3 contained in human sperma by observation of motility, sperm speed, viability, membrane integrity, and the integrity of the acrosome.Method. VDAC3 recombinant protein produced by culturing of E. coli BL21 that contains the construction of recombinant vector. This recombinant protein immunized to rabbits and then the antiserum taken from blood sample. VDAC3 antibodies that contained in antiserum purified by affinity chromatography matrix protein A Sepharose then the concentration was measured by ELISA and the purity analyzed by SDS-PAGE. The ability of VDAC3 antibodies were then tested to normal human sperma with parameters are: sperm motility, sperm speed, mortality (eosin Y staining method), membrane integrity (using HOST), and the integrity of the acrosome (using FITC-PNA).Results. Culture of E. coli with addition of IPTG showed a higher concentrations (2,051 mg/ml) than without IPTG (1,528 mg/ml). Immunization of protein VDAC3 recombinant to rabbit produce antiserum that contains antibodies with the highest titers 3,504 in rabbit B. SDS-PAGE analysis two protein fragmen with size of ~ 55 kDa and ~ 25 kDa. Statistical analysis of the effect of pure VDAC3 antibodies to motility, sperm speed, mortality, membrane integrity, and the integrity of the acrosome showed a significant difference (p < 0.05) with control preimun serum."

"ABSTRAKSalah satu kendala yang dihadapi manusia saat ini adalah pertambahan penduduk yang tidak terkendali yang menimbulkan banyak masalah baru di berbagai aspek kehidupan. Oleh sebab itu, pengendalian pertumbuhan penduduk harus dilakukan dengan berbagai metode kontrasepsi. Pengembangan kontrasepsi pria non-hormonal dengan menghambat proses pematangan sperma di epididimis menjadi hal yang menjanjikan. Sayangnya, gen yang berperan di dalam proses pematangan sperma di epididimis masih belum banyak dipelajari. Kami menganalisis beberapa kandidat gen yang diekspresikan di epididimis, salah satunya adalah Defensin Beta-42 (Defb42). Beberapa langkah metode yang kami lakukan adalah isolasi epididimis dan ekstraksi RNA, analisis bioinformatika, dan real-time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) untuk menganalisa ketergantungan ekspresi terhadap androgen dari gen Defb42 karena pematangan sperma bergantung pada androgen. Selain itu, proses pematangan sperma juga terjadi akibat interaksi antara sperma dan protein yang disekresikan oleh epitel epididimis. Oleh sebab itu, peptida sinyal harus dianalisis juga untuk mengecek apabila gen ini adalah protein sekretori. Hasilnya adalah gen ini diregulasi oleh androgen dan memiliki peptida sinyal. Hal ini membuat gen Defb42 menjadi kandidat gen yang menjanjikan untuk diteliti lebih lanjut dalam upaya pengembangan kontrasepsi non-hormonal pada pria.

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ABSTRACTOne of the problem nowadays is the uncontrolled population growth. This raises many other problems in every aspect of life. Therefore, the population growth must be controlled with many types of contraceptive agents. The development of male non-hormonal contraceptive agent by inhibiting the sperm maturation process in epididymis seems to be promising. We analyzed several candidates of gene which are expressed in epididymis; one of them is Defensin Beta-42 (Defb42) gene. Several method we conducted in this research are epididymis isolation and RNA extraction, bioinformatics analysis, and real time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) to analyze the expression dependency towards androgen of Defb42 gene because the sperm maturation is androgen-dependent process. Besides, the sperm maturation process occurs due to the interaction between sperm and protein secreted by epididymal epithelium. Therefore, the signal peptide has to be analyzed to confirm whether the candidate gene is a secretory protein. The results are this gene is regulated by androgen and has signal peptide properties. Therefore we can conclude that the gene is promising to be studied further in effort to develop male non-hormonal contraceptive agent."

"ABSTRAKLatar Belakang : Proses pematangan spermatozoa terjadi melalui interaksi spermatozoa dengan protein yang disekresikan ke lumen oleh sel-sel epitel epididimis. Sekresi protein akan menciptakan lingkungan yang mendukung proses pematangan spermatozoa. Namun gen penyandi protein yang terlibat dalam proses pematangan spermatozoa di epididmis masih belum banyak diketahui. Berdasarkan penelitian sebelumnya, gen-gen yang terlibat dalam proses pematangan spermatozoa ini memiliki kriteria antara lain protein sekretori, terekspresi spesifik di epididimis, dan menunjukkan eskpresi regional, diregulasi oleh faktor androgen dan faktor testikular. Defb30 merupakan salah satu gen yang perlu dilakukan karakterisasi lebih lanjut untuk mengetahui apakah gen tersebut memenuhi kriteria sebagai gen yang terlibat dalam proses pematangan spermatozoa. Tujuan dari penelitian ini adalah untuk melakukan karakterisasi gen Defb30 pada epididimis mencit.Desain : Penelitian ini menggunakan analisis bioinformatika dan Quantitative real-time PCR qRT-PCR .Metode : Analisis bioinformatika digunakan untuk memprediksi struktur gen, sinyal peptida dan domain fungsional. Analisis qRT-PCR digunakan untuk mengukur ekspresi relatif gen Defb30 terhadap analisis sebaran jaringan, regulasi terhadap androgen dan faktor testikular serta postnatal development.Hasil : Analisis sinyal peptida menggunakan signalP 4.1 menunjukkan bahwa Defb30 merupakan protein sekretori. Defb30 terekspresi secara spesifik di epididimis dan memiliki nilai spesifitas tinggi di bagian kaput epididimis. Ekspresi relatif gen Defb30 diregulasi oleh faktor endokrin berupa androgen, penurunan ekspresi relatif gen Defb30 terlihat pada hari pertama hingga hari ketiga gonadektomi dan testosteron diketahui mampu mencegah penurunan ekspresi Defb30 pada mencit yang telah digonadektomi. Analisis eksperimen efferent duct ligation menunjukkan gen Defb30 diregulasi oleh faktor testikular. Analisis postnatal development menunjukkan bahwa gen Defb30 mulai terekspresi pada hari ke-15 postnatal dan meningkat hingga usia dewasa.Kesimpulan : Defb30 merupakan protein sekretori yang terekspresi spesifik pada kaput epididimis dan diregulasi oleh androgen dan faktor testikluar.

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ABSTRACTBackground The process of sperm maturation occurs through interaction between sperm and proteins secreted by epididymal epithelial cells. The secretion of proteins will create micro environment suitable for spermmaturation. However, the role of protein encoding genes involved in the maturation process are not widely known. Based on previous studies the genes that are involved in spermmaturation process have characteristics such as secretory protein, specific expression in the epididymis and shows region specific expression, regulated by androgen and testicular factors. Defb30 is one of the genes that need further characterization to determine the putative function. Therefore, this study was aimed to characterize expression and regulation of Defb30 in the mouse epididymis.Methods Bioinformatics analysis was used to predict the structure of genes, peptide signals and functional domains. qRT PCR analysis was performed to measure the level of Defb30 expressionin the tissue distribution, regulation of andorgen and testicular factors and postnatal development.Result Peptide signal analysis using signalP 4.1 indicated that Defb30 was a secretory protein. Defb30 was expressed exclusively in the epididymis and had a high specificity in the caput. The expression of the Defb30 gene was regulated by androgen in which decreased of Defb30 expression was observed at the first day to the third day of gonadectomy and exogenous T was able to maintain Defb30 expression at 3d and 5d gonadectomized mice. efferent duct ligation showed that Defb30 was slightly regulated by testicular factors. Defb30 was developmentally regulated being expressed start at day 15 postnataly.Conclusions Defb30 is a secretory protein which is expressed specifically in the caput epididymis and it is regulated by androgen and testicular factors. "

"Spag11a diketahui terekspresi secara spesifik pada kaput epididimis sehingga dimungkinkan protein tersebut memiliki fungsi yang spesifik untuk maturasi spermatozoa. Studi peran SPAG11A dalam maturasi spermatozoa di epididimis memerlukan produksi protein SPAG11A untuk dikarakterisasi. Tujuan dari penelitian ini adalah untuk mengklon, mengekspreesikan, dan mengkarakterisasi sifat antimikroba dari protein rekombinan SPAG11A. Insert cDNA Spag11a yang dihasilkan melalui PCR diklon ke dalam vektor pET100/D-TOPO. Plasmid rekombinan kemudian diekspresikan ke Escherichia coli BL21 DE3 star. Deteksi dari fusi protein rekombinan dilakukan dengan SDS-PAGE dan Western Blotting. IMAC Immobilized Metal Affinity Chromatography digunakan untuk mempurifikasi protein rekombinan. Uji antimikroba protein rekombinan dianalisis melalui pengukuran Optical density.PCR amplifikasi dari cDNA kaput epididimis mencit menghasilkan insert Spag11a berukuran 210bp. Insert tersebut kemudian dikloning ke dalam pET100/D-TOPO menghasilkan 1 rekombinan plasmid dari 10 koloni yang diskrining. Ekspresi rekombinan klon ke dalam E.coli BL21 menghasilkan fusi protein setelah diinduksi IPTG selama 4 jam. Fusi protein dikonfirmasi menggunakan Western Blotting menggunakan antibodi yang mengenali N-terminal His-Tag 21kDa dan protein SPAG11A. Uji antimikroba protein rekombinan SPAG11A mununjukkan tidak ada inhibisi yang signifikan terhadap laju pertumbuhan E.coli dan Bacillus subtilis. Insert Spag11a yang berukuran 210bp berhasil diklon ke dalam vektor pET100/D-TOPO. Ekspresi rekombinan Spag11a menghasilkan fusi protein berukuran 21kDa. Protein rekombinan SPAG11A tidak membawa sifat antimikroba terhadap E.coli dan B. subtilis.

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Spag11a is known to be specifically expressed in the caput region of the epididymis suggesting a specific function for sperm maturation. Study of SPAG11A role in the epididymal sperm maturation requires generating SPAG11A protein for characterization. The objective of this study was to clone, express and characterize antimicrobial property of the recombinant SPAG11A. Spag11a cDNA insert was generated by PCR and cloned in TOPO vector. Recombinant DNA plasmid was subsequently expressed in E coli BL 21 star. Detection of recombinant fusion protein was carried out using SDS PAGE and western immunobloting. IMAC Immobilized Metal Affinity Chromatography was used to purify recombinant protein. Optical density measurement was used to analyse antimicrobial property of the recombinant protein. PCR amplification of mouse caput epididymis cDNA produced a 210 bp insert of Spag11a. Cloning of the insert into TOPO pET100 resulted in 2 recombinants out of 10 colonies that were screened. Expression of recombinant clones in the E coli BL21 produced a fusion protein after being induced IPTG for 4 hours. Fusion protein was confirmed by western immunobloting using two antibodies recognizing N terminal His Tag 21 kDa and SPAG11A protein. Antimicrobial assay for SPAG11A recombinant showed no significant inhibition towards growth rates of E coli and Bacillus subtilis. A 210 bp Spag11a insert was successfully cloned into TOPO pET100 vector. Expression of recombinant spag11a produced a fusion protein of 21 kDa. SPAG11A recombinant protein does not have antimicrobial property towards E coli and B subtilis."