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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 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."

"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."

"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. "

"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"

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Latar Belakang: Beberapa gen yang terekspresi spesfik di epididimis diduga

terlibat dalam proses pematangan sperma. Karakteristik gen yang terlibat dalampematangan sperma selain ekspresinya spesifik di epididimis juga dipengaruhioleh androgen, faktor testikuler, dan terekspresi pada saat masa pubertas. Salahsatu famili gen yang cukup banyak ditemukan terekspresi di epididimis adalahBeta Defensin. Gen Beta Defensin diketahui memiliki peran sebagai pertahananterhadap mikroba, namun diduga memiliki keterlibatan dalam proses pematangansperma karena ekspresinya banyak ditemukan di epididimis. Oleh karena itu,penelitian pada gen Beta Defensin terhadap perannya dalam proses pematangansperma perlu dilakukan. Berdasarkan studi sebelumnya diketahui bahwa salahsatu gen Beta Defensin yang terekspresi di epididimis yaitu Beta Defensin 2(Defb2), namun karakterisasi terhadap gen ini belum dilakukan. Dengandemikian, pada penelitian ini bertujuan untuk mengkarakterisasi gen Defb2 terkaitdengan perannya pada proses pematangan sperma.Metode: Analisis bioinformatika digunakan untuk mendapatkan informasi

mengenai struktur gen, signal peptide, dan domain fungsional pada gen Defb2.

Analisis qRT-PCR untuk mengetahui ekspresi relatif gen Defb2 pada berbagai

jaringan, regulasinya oleh androgen, pengaruh dari faktor testikular dan

ekspresinya pada perkembangan postnatal.Hasil: Defb2 merupakan protein sekretori karena memiliki signal peptide. Defb2

memiliki domain fungsional berupa N-myristoylation dan protein kinase-C. Gen

Defb2 terekspresi spesifik di epididimis khususnya pada bagian caput epididimis.

Defb2 ekspresinya dipengaruhi oleh androgen terbukti setelah perlakuan

gonadektomi, ekspresi Defb2 menjadi menurun dan kembali mengalami kenaikan

ketika diberikan testosteron eksogen. Defb2 juga ekspresinya dipengaruhi oleh

faktor testikuler terbukti setelah diberi perlakuan Efferent Duct Ligation (EDL)

maka ekspresi Defb2 langsung menurun bahkan terjadi apoptosis sel sehingga

pola ekspresi gen Defb2 sudah tidak bisa diamati. Begitu juga pada analisis

postnatal development terlihat ekspresi gen Defb2 mulai terdeteksi jelas pada hari

ke-15 yang merupakan masa pubertas mencit jantan.Kesimpulan: Defb2 merupakan gen yang terlibat dalam proses pematangan

sperma di epididimis yang dibuktikan dengan ekspresi spesifik di epididimis,

diregulasi oleh androgen dan faktor testikuler, serta mulai terekspresi pada masa

pubertas.

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Background: Some of the specific genes expression in the epididymis are

suspected to be involved in the process of sperm maturation. Characteristics of thegenes involved in sperm maturation in the epididymis-specific expression inaddition also influenced by androgens, testicular factors, and expressed at the timeof puberty. One of a family of genes that is pretty much found expressed in theepididymis is a Beta Defensins. Beta Defensin genes known to have a role as adefence against microbes, but suspected to have involvement in the process ofsperm maturation because the expression is found in the epididymis. Therefore,research on Beta Defensin genes against its role in sperm maturation processneeds to be done. Based on previous studies it is known that one of the BetaDefensin genes which expressed in the epididymis that is Beta Defensins 2(Defb2), but the characterization of this gene has not been made against. Thus,this research aims to characterize genes associated with the Defb2 role in theprocess of sperm maturation.Methods: Bioinformatics analysis was used to obtain information about the

structure of genes, signal peptides, and functional domains of the Defb2 gene.

qRT-PCR analysis to find out the relative gene expression of Defb2 on various

tissue, regulation by androgens, the effect of testicular factors and its expression

in postnatal development.Results: Defb2 is a secreted protein because it has signal peptides. Defb2 has a

functional domain in the form of N-myristoylation and kinase-C protein. Specific

genes expression of Defb2 in the epididymis is especially in the caput epididymis.

Defb2 expression influenced by androgens is proven after the gonadectomy, the

expression of Defb2 to be decreased and start increase again when exogenous

testosterone is given. Defb2 also its expression influenced by testicular factors

that proven after being given the treatment by Efferent Duct Ligation (EDL), then

the Defb2 expressions directly decreased and the cell apoptosis occurs even so

that the pattern of gene expression Defb2 already could not be observed. So also

on analysis of postnatal development seen gene expression Defb2 begins to be

detected clearly at day 15 which is a male mice puberty.Conclusions: Defb2 is a gene which is involved in the process of maturation of

sperm in the epididymis that is evidenced by specific expression in the

epididymis, be regulated by androgens and testicular factors, and as well as start

expressed at puberty.

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"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."

"Latar belakang: Beta defensin diekspresikan terutama oleh sel epitel pada permukaan mukosa berbagai organ seperti kulit, usus, mulut dan saluran genital. Studi sebelumnya menunjukkan bahwa beta defensin 30 (Defb30) terekspresi spesifik di epididimis. Defb30 merupakan peptida kationik berukuran kecil yang diduga berperan penting pada proses pematangan spermatozoa di epididimis dan juga berperan sebagai pertahanan host terhadap infeksi mikroba. Untuk mempelajari aktivitas antimikroba Defb30 ini diperlukan analisis pada tingkat protein dan hal tersebut memerlukan protein dalam jumlah yang cukup. Karena itu perlu dilakukan suatu rekayasa genetika untuk pembuatan protein rekombinan DEFB30.Metode: Gen sintetik penyandi protein DEFB30 yang telah dioptimasi kodonnya diklona ke dalam vektor pQE-80L, suatu plasmid yang mengandung sistem ekspresi untuk prokariota. Plasmid rekombinan yang mengandung sisipan gen target dikonfirmasi dengan analisis enzim restriksi dan sekuensing. Selanjutnya plasmid rekombinan di ekpresikan ke dalam E. coli BL21 dan diinduksi menggunakan IPTG (Isopropyl-1-Thio-d-Galactopyranoside) dengan berbagai waktu inkubasi. Deteksi protein rekombinan dilakukan dengan SDS-PAGE dan westernblotting. IMAC (Immobilized Metal Affinity Chromatography) digunakan untuk mempurifikasi protein rekombinan. Uji antimikroba protein rekombinan dilakukan dengan cara pengukuran nilai optical density (OD) dan dianalisis hasilnya menggunakan uji one way anova.Hasil: Gen sintetik penyandi protein rekombinan DEFB30 berhasil dikonstruksi pada plasmid pQE-80L. Ekspresi ke dalam E. coli BL21 menghasilkan suatu protein fusi setelah diinduksi menggunakan IPTG selama 4 jam. Hasil analisis protein rekombinan dengan westernblotting menggunakan antibodi Anti-His G-HRP menunjukkan terbentuk pita tebal yang berukuran diatas 10 kDa (±12 kDa). Uji antimikroba protein rekombinan DEFB30 menunjukkan bahwa DEFB30 dapat menghambat pertumbuhan bakteri Eschericia coli dan Bacillus subtilis.Kesimpulan: Gen sintetik penyandi beta defensin 30 berhasil diklona ke dalam plasmid pQE-80L. Ekspresikan protein rekombinan DEFB30 menghasilkan suatu protein fusi berukuran ±12kDa. Protein rekombinan DEFB30 terbukti memiliki sifat antimikroba terhadap Eschericia coli dan Bacillus subtilis.

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ackground: Beta defensins are primarily expressed by epithelial cells at mucosal surfaces, such as those in skin, gut, mouth and genital tracts. Previous studies have demonstrated that beta defensin 30 (Defb30) is exclusively expressed in the epididymis. Defb30 is known as a small cationic antimicrobial peptide which plays an important role in epididymal sperm maturation and also acts as a host defence against microbial infection. Study of Defb30 role in the antimicrobial activity requires generating DEFB30 protein for characterization. For the purpose of this study, genetic engineering was done for the manufacture of the DEFB30 recombinant protein.

Methods: In this study, according to the preferred codon in E. coli, the Defb30 gene was optimized and synthesized. The gene was cloned into pQE-80L vector and subsequently expressed in E. coli BL21; using IPTG (Isopropyl-1-Thio-d-Galactopyranoside) as an inducer. Detection of recombinant protein was carried out by using SDS-PAGE and westernblotting. IMAC (Immobilized Metal Affinity Chromatography) was used to purify recombinant protein. Optical density measurement was used to analyze antimicrobial property of the DEFB30 recombinant protein.

Results: The synthetic gene was successfully constructed into pQE-80L plasmid and expression of the recombinant protein in E. coli BL21 produced a fusion protein after being induced by IPTG for 4 hours. Westernblotting analysis using Anti-His G-HRP antibody showed band above 10kDa (±12kDa). Antimicrobial assay for DEFB30 recombinant protein showed inhibition towards growth rates of Eschericia coli and Bacillus subtilis.

Conclusion: Defb30 synthetic gene was succesfully cloned into pQE-80L plasmid. Expression of recombinant DEFB30 produced a fusion protein of ±12kDa. This recombinant protein has antimicrobial property towards Eschericia coli and Bacillus subtilis."

"[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. ]"