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"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."

"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."

"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."

"Latar Belakang : Proses pematangan sperma terjadi karena adanya interaksi antara sel sperma dan protein yang disekresikan oleh sel epitel yang melapisi saluran epididimis, sehingga menyebabkan perubahan morfologi dan biokimia pada membran spermatozoa. Sekresi protein pada saluran epididimis ditentukan oleh gen-gen yang diekspresikan secara spesifik di region tertentu misalnya pada region initial segmen, caput, corpus atau cauda sehingga pada masing-masing segmen terbentuk lingkungan spesifik (microenvironment) yang diperlukan untuk proses pematangan sperma. Gen yang menyandi protein yang terlibat dalam proses pematangan sperma diekspresikan secara spesifik pada epididimis dan ekspresinya diregulasi oleh androgen. Salah satu gen yang diduga berperan dalam proses pematangan sperma adalah gen Serpina1f. Berdasarkan urutan asam amino, Serpina1f dianggap sebagai anggota baru dari keluarga serpin dengan fungsi putatif sebagai serin protease inhibitor. Karakterisasi Serpina1f pada organ reproduksi pria khususnya epididimis dapat memberikan petunjuk mengenai perannya dalam proses pematangan sperma.Tujuan : Mengkarakterisasi gen Serpina1f di epididimis mencit.Desain : Penelitian ini menggunakan analisis bioinformatika dan eksperimentalMetode : Struktur gen, batas ekson-intron dan domain fungsional serta deteksi signal peptide pada Serpina1f dilakukan analisis bioinformatika. Untuk menganalisis ekspresi SERPINA1F tingkat protein dilakukan western imunoblotting, sedangkan untuk mengetahui lokasi protein SERPINA1F dilakukan imunohistokimia dan imunositokimia.Hasil : SERPINA1F merupakan anggota family SERPIN. Berdasarkan analisis Signal peptide, SERPINA1F merupakan protein sekretori. Hasil imunohistokimia pada jaringan epididimis mencit menunjukkan adanya reaksi antibodi dengan terwarnainya nukleus dan sitoplasma pada sel epitel initial segment dan caput. Sedangkan pada corpus dan cauda SERPINA1F terdeteksi hanya pada sitoplasma. Hasil analisis western imunoblotting dan imunositokimia pada protein sperma menunjukkan adanya asosiasi SERPINA1F dengan spermatozoa dan terdapat di seluruh bagian spermatozoa.Kesimpulan : SERPINA1F memiliki signal peptide pada asam amino 1 - 27 dan termasuk protein sekretori. Berdasarkan urutan asam aminonya, SERPINA1F yang termasuk famili SERPIN. Protein SERPINA1F diekspresikan di sel epitel initial segment, caput, corpus dan cauda. SERPINA1F berasosiasi dengan spermatozoa dan terdapat pada seluruh bagian spermatozoa.

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Background: Sperm maturation occur due to the interaction between the sperm cell and the proteins secreted by the epithelial cells lining the epididymis duct, causing morphological and biochemical changes in the sperm membrane. The secretion of proteins in epididymis duct is determined by genes that are expressed specifically in certain regions, for example in the region of the initial segment, caput, corpus or cauda that each segment forms microenvironment that is required for sperm maturation process. Genes that encode proteins involved in the process of sperm maturation in the epididymis specifically expressed and its expression is regulated by androgens. One of the genes thought to play a role in sperm maturation process is Serpina1f. Based on the amino acid sequence, Serpina1f considered as a new member of the serpin family with putative functions as a serine protease inhibitor. Characterization Serpina1f on male reproductive organs, especially the epididymis may provide a clue as to its role in sperm maturation process.Objective: To characterize Serpina1f in the epididymis of mice.Design: This study used a bioinformatics analysis and experimental.Methods: Bioinformatics analysis were used to know gene structure, exon-intron boundaries and functional domains as well as the detection of signal peptide on Serpina1f. To analyze protein expression levels SERPINA1F performed western immunoblotting, whereas for the location of the protein immunohistochemistry and immunocytochemistry SERPINA1F done.Results: SERPINA1F a member of the serpin family. Based on the analysis of signal peptide, a protein secretory SERPINA1F. The results of immunohistochemistry on epididymis tissue of mice showed an antibody reaction with colored nucleus and cytoplasm in epithelial cells of the initial segment and caput. While SERPINA1F detected only in the cytoplasm corpus and cauda. The results of western imunoblotting analysis and immunocytochemistry showed SERPINA1F associated with spermatozoa and found in all parts of the spermatozoa.Conclusion: SERPINA1F has a signal peptide at amino acids 1-27 and include secretory proteins. Based on their amino acid sequence, which includes SERPINA1F serpin family. The protein is expressed in epithelial cells SERPINA1F initial segment, caput, corpus and cauda. SERPINA1F associated with spermatozoa and found on all parts."

"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. "

"ABSTRAKLatar Belakang: Pematangan spermatozoa di epididimis terjadi melalui interaksi antara spermatozoa dengan protein yang disekresikan oleh sel epitel yang melapisi duktus epididimis. Sekresi protein tersebut menciptakan microenvironment yang diregulasi oleh gen-gen tertentu. Studi sebelumnya menunjukkan bahwa gen yang terlibat dalam pematangan spermatozoa pada umumnya terekspresi secara spesifik di epididimis dan dipengaruhi oleh androgen. Spink2 merupakan salah satu gen yang terekspresi di epididimis, namun regulasi ekspresinya masih belum diketahui. Tujuan penelitian ini adalah untuk mengkarakterisasi ekspresi dan regulasi gen Spink2 pada epididimis mencit jantan.Metode: Analisis secara in silico digunakan untuk mengetahui struktur gen dan prediksi sinyal peptida, serta domain fungsional gen Spink2. Quantitative real-time RT-PCR digunakan dalam mengukur ekspresi relatif gen Spink2 pada analisis spesifisitas jaringan, ketergantungan terhadap androgen dan faktor testikuler, serta post-natal development.Hasil: Spink2 termasuk dalam famili serine protease inhibitor yang ditandai dengan adanya domain Kazal type 2. Analisis signal peptide menunjukkan bahwa Spink2 merupakan protein sekretori. Spink2 terekspresi di testis dan epididimis, dengan ekspresi tertinggi berada di kaput epididimis. Ekspresi Spink2 pada mencit yang digonadektomi mengalami peningkatan setelah 6 jam, kemudian menurun mulai dari hari ke-1 hingga hari ke-5. Pemberian testosteron mampu mempertahankan ekspresi Spink2 pada 3 dan 5 hari setelah gonadektomi. Selain itu, pada analisis pengaruh faktor testikuler, ekspresi Spink2 menunjukkan adanya regulasi dari faktor testikuler pada semua kelompok setelah dilakukan efferent duct ligation EDL . Spink2 menujukkan regulasi post-natal yakni mulai terekspresi pada umur mendekati 22 hari.Kesimpulan: SPINK2 merupakan protein sekretori yang terekspresi pada kaput epididimis, serta diregulasi oleh androgen dan faktor testikuler. Spink2 tidak terekspresi secara konstitutif. Berdasarkan data tersebut Spink2 sangat berpotensi terlibat dalam proses pematangan spermatozoa di epididimis. Penelitian lebih lanjut diperlukan untuk mengkonfirmasi potensi tersebut.

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ABSTRACTBackground Sperm maturation in the epididymis occurs through interactions between sperm and proteins secreted by epithelium cells lining the epididymal duct. The secretion of these proteins creates a microenvironment that is regulated by certain genes. Previous studies showed that genes which are involved in sperm maturation process are expressed specifically in the epididymis and regulated by androgen. Spink2 is one of the epididymal genes, but the regulation of its expression is still unknown. Therefore, this study was aimed to characterize Spink2 expression and its regulation in the mouse epididymis.Method s In silico analysis was performed to determine the gene structure and identify the signal peptide, as well as the functional domain of Spink2. Quantitative real time RT PCR was performed to measure relative expression of Spink2 in the analyses of the tissue specificity, androgen dependency, testicular factor and post natal development.Result s Spink2 belongs to the serine protease inhibitor family which is characterized by the presence of Kazal type 2 domain. Signal peptide analysis showed that Spink2 amino acid sequence contains a signal peptide, indicating Spink2 is a secretory protein. Spink2 was expressed specifically in the testis and epididymis, with the highest level of its expression was in the epididymal caput. Spink2 expression increased after six hours and started to decrease on day 1 throughout day 5. Interestingly, administration of exogenous testosterone was able to maintain expression at the physiological level. In addition, Spink2 was slightly affected by testicular factors. During post natal development, Spink2 start to be expressed at day 22 before increased dramatically throughout day 60.Conclusion s Spink2 is a secretory protein that is expressed in caput region of the mouse epididymis and regulated by androgen. Spink2 is not constitutively expressed throughout development. Based on our data, may be involved in epididiymal sperm maturation process. Further studies are required to confirm its role. "

"Ekspresi gen antigen 85B perlu diketahui sebelum dijadikan kandidat vaksin tuberkulosis yang diharapkan bisa menggantikan vaksin BCG. Ekspresi tersebut bisa diketahui setelah gen antigen 85B diklona dan ditransformasikan ke dalam E. coli DH5α. Ekspresi gen diharapkan bisa lebih banyak dengan penggunaan signal peptide AQ1 endoxilanase. Gen antigen 85B yang berukuran 991 pb diklona ke dalam vektor pUC57 fragmen 2 yang berukuran sekitar 2500 pb, sehingga menghasilkan plasmid rekombinan yang berukuran sekitar 3500 pb. Hasil ekspresi dari plasmid rekombinan diuji dengan SDS-PAGE. Hasil penelitian menunjukkan gen antigen 85B berhasil diklona ke dalam vektor pUC57 fragmen 2, namun antigen tersebut belum berhasil diekspresikan.

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The expression of antigen 85B gene needed to be known before the antigen is used as tuberculosis vaccine candidate that is expected to replace the BCG vaccine. The expression of antigen 85B gene could be known after the gene was cloned and transformed into E. coli DH5α. The expression of the gene was expected to increase in number with the use of AQ1 endoxylanase signal peptide. Antigen 85B gene size 991 bp was cloned into a vector pUC57 fragmen 2 with size approximately 2500 bp, resulting in a recombinant plasmid with size approximately 3500 bp. The expression results of recombinant plasmid were tested with SDS-PAGE. The results showed antigen 85B gene successfully cloned into the vector pUC57 fragment 2, but these antigens have not been successfully expressed."

"ABSTRAKProses pematangan spermatozoa terjadi karena adanya interaksi antara protein dengan membran plasma spermatozoa. Walaupun proses pematangan spermatozoa ini sangat penting, namun gen yang berperan dalam sekresi protein di epididimis ini masih banyak yang belum dikarakterisasi. Gen-gen yang berperan dalam proses pematangan spermatozoa umumnya merupakan protein sekretorik, terekspresi pada segmen spesifik, diregulasi androgen, faktor testikular dan perkembangan postnatal. Pada penelitian sebelumnya diketahui bahwa b-defensin merupakan gen yang banyak terekspresi di organ reproduksi pria dan memiliki peran dalam pertahanan tubuh dan pematangan spermatozoa. Penelitian ini dilakukan untuk mengkarakterisasi ekspresi gen Defb20 untuk mengetahui perannya dalam proses pematangan spermatozoa. Studi in silico dilakukan untuk prediksi struktur gen, signal peptide dan domain fungsional. Quantitative real-time PCR digunakan untuk mengukur ekspresi gen Defb20 pada analisis sebaran jaringan, regulasi androgen dan faktor testikular serta postnatal developmen. Hasil penelitian mendapatkan bahwa sekuen Defb20 mengandung domain penting seperti N-myristoilation dan beberapa situs phosporilasi protein kinase yang mungkin berperan dalam mekanisme interaksi protein dengan membran plasma. Sekuen asam amino Defb20 mengandung signal peptides, mengindikasikan protein yang disekresikan dan terlibat dalam proses pematangan spermatozoa. -defensins 20 (Defb20) terekspresi spesifik di epididimis dengan ekspresi tertinggi terdapat pada kaput epididimis. Defb20 diregulasi oleh androgen yang ditunjukkan dengan adanya penurunan ekspresi Defb20 paska dilakukan gonadektomi dan kondisi ini dapat diperbaiki dengan pemberian hormon pengganti. Defb20 juga diregulasi oleh faktor testikular, yang dibuktikan dari menurunnya ekspresi Defb20 setelah ligasi pada duktus eferen (efferent duct ligation (EDL)). Defb20 mulai terekspresi pada hari ke-21 setelah lahir yang mengindikasikan gen Defb20 terekspresipada suatu periode perkembangan epididimis. Berdasarkan hasil penelitian disimpulkan bahwa Defb20 memiliki karakteristik ekspresi : mengandung signal peptide yang mengarahkan sintesis protein pada jalur sekretorik, spesifik terekspresi di epididimis, diregulasi androgen dan faktor testikular serta mulai terekspresi pada masa pubertas hingga dewasa

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ABSTRACTEpididymal sperm maturation occurs via interactions between sperm and proteins secreted by the epididymal epithelium. Although this is an important process, the genes that encode secreted proteins remain largely uncharacterized. The genes that play a role in sperm maturation process has character, among others; is a secretory protein, expressed specifically in the epididymis, regulated by androgen, testicular factor, and postnatal development. Previous studies showed that family of-defensins preferentially eaxpressed in male reproductive tracts and play an important role in both innate immunity and sperm fertility. This study aimed to characterize Defb20 to understand its role in sperm maturation. This study using in silico analyses and quantitative real-time PCR (qRT-PCR). In silico analyses were performed to predict gene structure, signal peptides and functional domains. Defb20 expression in various tissues, after gonadectomy, efferent duct ligation and postnatal development were measured using quantitative real-time RT-PCR. Defb20 sequence contains important domains such as N-myristoilation and kinase binding sites which are putatively involved in the protein activation and protein-plasma membrane interaction. The amino acid sequence of Defb20 contains signal peptides, indicating characteristic of secretory proteins involved in the sperm maturation. β-defensins 20 (Defb20) was expressed exclusively in the epididymis with the highest expression in the caput region. Defb20 was regulated by androgen showing down-regulation after gonadectomy and the expression was recovered after testosterone replacement. However, Defb20 was also regulated by testicular factors in which the expression was down-regulated after efferent duct ligation (EDL). The dependency on the androgen was further confirmed by postnatal expression analysis in which Defb20 begin to express at day21 postnatal indicating specific stage of expression after initial development of the epididymis. In conclusion, Defb20 have a potential to be involved in the epididymal sperm maturation process. Defb20 has characteristic expression; has a signal peptide sequence that directs synthesis in the secretory pathway, specifically expressed in the epididymis, androgen and testicular factors regulated, and expressed in puberty to adulthood"

"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."

"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."