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"ABSTRAKPendahuluan. VDAC merupakan protein kanal ion yang bertanggung jawab atas aliran ion Ca2+ dan ATP dalam flagela spermatozoa. Defisiensi gen VDAC3 pada mencit dan mutasi gen VDAC3 pada manusia menyebabkan penurunan motilitas spermatozoa, sehingga VDAC3 dapat dijadikan antigen potensial untuk pengembangan vaksin kontrasepsi laki-laki. Tujuan penelitian ini adalah memproduksi protein rekombinan hVDAC3 dari gen hVDAC3 ekson 5-8 spesifik spermatozoa, dan digunakan sebagai antigen untuk produksi antibodi poliklonal pada kelinci.Metode. Gen hVDAC3 ekson 5-8 spermatozoa diperoleh melalui RT PCR, gen disisipkan ke plasmid pET100/D-TOPO dan diklona dalam E coli TOP 10. Analisis gen sisipan dengan PCR, enzim restriksi dan sekuensing DNA. Protein rekombinan hVDAC3 diekspresikan dalam E coli BL21 StarTM (DE3). Karakterisasi protein dilakukan dengan uji Bradford, SDS PAGE, western blot dan purifikasi protein dengan resin Ni-NTA. Antibodi poliklonal diperoleh dengan cara imunisasi protein rekombinan hVDAC3 ke kelinci dan diukur dengan indirect ELISA. Determinasi lokasi hVDAC3 di spermatozoa dengan metode immunoflurosence.Hasil. Amplifikasi PCR gen hVDAC3 ekson 5-8 berukuran 435 pb dan analisis BLAST menunjukkan 100% identik dengan gen VDAC3 manusia dari bank gen. Vektor rekombinan berukuran 6195 pb mengekspresikan protein rekombinan hVDAC3 berukuran 20 kDa. Antibodi poliklonal telah diproduksi kelinci secara bermakna (p<0.05) dengan titer 2.817, dan antibodi dapat berikatan dengan protein hVDAC3 di kepala dan flagela spermatozoa. Selanjutnya, antibodi poliklonal ini akan digunakan dalam pengembangan vaksin kontrasepsi pada laki-laki.

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ABSTRACT

Introduction. Voltage dependent anion channels (VDAC), also known as mitochondrial porins, are group of proteins in mitochondrial outer membrane that allow the passage of metabolites across the mitochondrial outer membrane, and are involved in ions and ATP transport in sperm flagella. Deficiency and mutation of VDAC3 may cause abnormality in structure and motility of human spermatozoa. VDAC3 could be a potential target to develop non hormonal male contraceptive vaccine. The objective of the study was to produce hVDAC3 recombinant proteins from exon 5 to 8 of human sperm VDAC3 spesific gene.

This recombinant protein was subsequenly used as an antigen to produce polyclonal antibodies in rabbits. Methods. hVDAC3 sperm gene obtained by RT PCR, this gene was inserted into plasmid pET 100/D-TOPO and cloned in E coli TOP 10. The gene was analyzed by PCR method, restriction enzymes and DNA sequencing. The proteins expressed in E coli BL21 StarTM (DE3). Characterization of proteins was evaluated by Bradford method, SDS PAGE and western blot. The recombinant protein was purified with NI-NTA resin. Polyclonal antibodies were obtained by immunization of hVDAC3 recombinant protein into rabbits. Indirect ELISA was done to analyze the antibody. Localization of the VDAC3 recombinant protein in human spermatozoa was evaluated by immunofluorescence method.

Result. By doing PCR amplification and BLAST analysis, the study showed that the hVDAC3 gene had 100% identical to hVDAC3 genes in data bank. E coli BL21 StarTM (DE3) containing recombinant vector (6195 bp) expressed the recombinant protein of hVDAC3 in 20 kDa. This protein produced polyclonal antibodies that bound VDAC3 protein on the head and flagella of human spermatozoa."

"Pendahuluan: Sindroma ovarium polikistik (SOPK) merupakan masalah reproduksi yang sering terjadi pada perempuan usia reproduksi, namun hingga saat ini etiopatogenesis SOPK masih belum jelas. Penelitian ini bertujuan menganalisis peran sel granulosa folikel ovarium dalam etiologi SOPK, keterkaitan genotip FSHR Asn680Ser dengan patogenesis SOPK dan peran gen CYP19A1(aromatase) dalam patogenesis SOPK. Metode: Penelitian ini menggunakan desain penelitian analitik observasional berbentuk studi seran lintang (cross sectional study) dan dilakukan di Departemen Biologi FKUI, Klinik Yasmin-RSCM Kencana dan Laboratorium terpadu FKUI pada tahun 2011-2014. Sebanyak 142 subyek penelitian (66 pasien SOPK dan 76 pasien bukan SOPK) terlibat dalam penelitian ini. Sampel penelitian berupa darah tepi dan cairan folikel ovarium yang diaspirasi ketika proses ovum pick up sebagai sumber sel granulosa. Dilakukan isolasi DNA untuk analisis RFLP polimorfisme FSHR Asn680Ser, dilakukan kultur sel granulosa untuk mengetahui kemampuan proliferasi sel granulosa dan analisis ekspresi mRNA aromatase sel granulosa dengan metode RT-qPCR. Hasil: Indeks proliferasi sel dan ekspresi mRNA aromatase sel granulosa pada kelompok SOPK lebih rendah secara bermakna dibandingkan bukan SOPK (p<0,05). Tidak ditemukan perbedaan bermakna distribusi genotip FSHR Asn680Ser antara kelompok SOPK dan bukan SOPK (p>0,05), tidak ditemukan perbedaan bermakna antara kadar hormon FSH basal berdasarkan variasi genotip FSHR Asn680Ser pada kelompok SOPK dan bukan SOPK (p>0,05). Tidak ditemukan perbedaan yang bermakna indeks proliferasi sel granulosa berdasarkan variasi genotip FSHR Asn680Ser baik pada kelompok SOPK maupun bukan SOPK (p>0,05). Tidak terdapat korelasi antara indeks proliferasi sel granulosa dengan ekspresi mRNA aromatase (p>0,05). Kesimpulan: Indeks proliferasi sel dan tingkat ekspresi mRNA aromatase sel granulosa kelompok SOPK menurun dibandingkan kelompok bukan SOPK. Genotip FSHR Asn680Ser tidak menentukan kerentanan individu untuk menderita SOPK. Kadar hormon FSH basal dan indeks proliferasi sel granulosa tidak berbeda antara kelompok SOPK dan bukan SOPK berdasarkan variasi genotip FSHR Asn680Ser. Tidak ada korelasi antara indeks proliferasi sel dengan ekspresi mRNA aromatase sel granulosa pada penelitian ini.

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Introduction: Polycystic ovary syndrome (PCOS) is a common reproductive problem in women at reproductive age, but until now aetiopathogenesis of PCOS has not been fully understood. The objective of this study was to analyse interrelationship between proliferation of ovarian follicular granulosa cells, CYP19A1 expression and polymorphism at codon 680 of FSHR towards the etiology of PCOS.

Methods: Observational analytic in the form of cross-sectional study was used in this research. The study was carried out between 2011-2014 at the Department of Biology, Integrated laboratory of Faculty of Medicine University of Indonesia and Yasmin clinic at the Cipto Mangunkusumo Hospital. A total of 142 subjects (66 patients with PCOS and 76 patients without PCOS) were involved in this study. Granulosa cells for culture were obtained from ovarian follicular fluid and total RNA was isolated from the cells. DNA samples were extracted from peripheral blood. Granulosa cell proliferation index was determined by counting under a phase-contrast microscope. CYP19A1 expression was measured by qRT-PCR, whereas polymorphism at Asn680Ser FSHR was performed by RFLP.

Result: Cell proliferation index and CYP19A1 mRNA expression levels in the granulosa cells of the PCOS group was significantly lower than non-PCOS (p < 0.05). There was no significant difference found in Asn680Ser FSHR genotype distribution between PCOS and non-PCOS group (p > 0.05). Based on Asn680Ser FSHR genotype variation, no significant difference was found between basal FSH hormone levels in the PCOS and non- PCOS group (p > 0.05) and FSHR genotype variation did not correlate with granulosa cell proliferation index between PCOS and non-PCOS group (p > 0.05). Moreover, there was no correlation between the granulosa cell proliferation index with aromatase mRNA expression levels (p > 0.05).

Conclusion: Cell proliferation index and CYPA1 expression of granulosa cells in PCOS group were lower compared to the non PCOS group although no correlation was found between the two parameters. Asn680Ser FSHR genotype did not correlate with individual susceptibility to PCOS. FSHR genotype variation did not correlate with basal FSH levels and granulosa cell proliferation index between PCOS and non-PCOS."

"Astenozoospermia merupakan penyebab umum terjadinya infertilitas pria. Motilitas spermatozoa didukung oleh homeostasis sel dan energi yang dihasilkan dari hidrolisis ATP. Na+,K+-ATPase dan Ca2+-ATPase bekerja pada transpor aktif ion di membran plasma untuk pertahanan homeostasis melalui regulasi proses metabolisme. Motilitas spermatozoa berawal pada proses morfogenesis di testis dan maturasi di epididimis serta memerlukan protein-protein fungsional seperti outer dense fiber (ODF) 1 dan 2. Aktivitas motorik terlaksana oleh protein kompleks dinein dengan ATPase dinein yang membebaskan energi dari ATP.Dalam penelitian ini dilakukan analisis ekspresi protein Outer Dense Fiber (ODF) 1 dan 2 serta aktivitas enzim Na+,K+-ATPase, Ca2+-ATPase dan dinein ATPase spermatozoa pada pria infertil astenozoospermia. Analisis semen dilakukan secara mikroskopik disertai uji viabilitas dan HOS. Aktivitas enzim diukur berdasarkan kemampuan ATPase melepaskan fosfat anorganik dari ATP dan ditentukan sebagai aktivitas spesifik. Sebagai kontrol digunakan spermatozoa normozoospermia.Didapati bahwa motilitas spermatozoa astenozoospermia (AG) cenderung lebih rendah dibanding dengan normozoospermia (NG). Hampir seluruh parameter, baik motilitas (VAP, VSL dan VCL), ekpresi dan kekompakan protein ODF1 dan ODF2 serta aktivitas spesifik Na+,K+-ATPase dan dinein ATPase, mengalami kecenderungan penurunan pada AG dibandingkan NG, kecuali aktivitas spesifik Ca2+-ATPase yang mengalami peningkatan secara bermakna pada AG dibandingkan NG. ODF berkorelasi positif dengan motilitas, Na+,K+-ATPase, morfologi, viabilitas dan integritas membran pada kelompok NG.

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Asthenozoospermia is a common cause in male infertility. Sperm motility and cell homeostasis are supported by energy generated from the hydrolysis of ATP in the cells, mediated by ATPases such as Na+, K+-ATPase, Ca2+-ATPase and dynein ATPase. In addition, sperm motility is initiated by the process of morphogenesis in the testis and maturation process in the epididymis. The morphogenesis of spermatozoa tail requires proteins such as outer dense fiber proteins (ODF) 1 and 2.

This study aims to evaluate the expression of Outer Dense Fiber (ODF) 1 and 2 protein, as well as the activity of the Na+,K+-ATPase, Ca2+-ATPase and dynein ATPase in asthenozoospermia infertile men. Microscopic semen analysis was carried out by CASA, equipped with the viability and HOS test. ATPase activity was determined based on its ability to release inorganic phosphate (Pi) from ATP and Pi concentration was measured as the intensity of the blue color of phosphomolibdate with a spectrophotometer.

In the AG group, almost all parameters, both motility (VAP, VSL and VCL), expression and density of protein ODF1 and ODF2 and the enzyme specific activities of Na+,K+-ATPase and dynein ATPase, experienced a downward tendency compared to the NG group. However, the specific activity of Ca2+-ATPase exhibited significant increase in the AG compared to the NG group. ODF correlates positively with motility, Na+,K+-ATPase, morphology, viability and membrane integrity in the NG group."

"Pendahuluan : Infertilitas merupakan masalah yang dialami pasangan suami istri, dimana faktor laki-laki berkontribusi sebesar 40%. Salah satu penyebab infertilitas dari faktor laki-laki adalah gangguan remodelling kromatin yang terjadi selama proses spermiogenesis. Pada proses ini histon akan digantikan oleh protein protamin yang menyebabkan DNA lebih padat dan kompak. Regulasi protamin dipengaruhi oleh kerja protein CREM yang merupakan faktor transkripsi pada gen protamin. Pada tahapan ini dibutuhkan peran androgen yang akan aktif setelah berikatan dengan reseptor androgen (AR), sehingga aktivitas AR sangat menentukan keberhasilan remodeling kromatin. Penelitian ini bertujuan menganalisis ekspresi protein CREM, Protamin 1 dan Protamin 2 pada spermatozoa laki-laki infertil dan kaitannya dengan variasi pengulangan CAG gen reseptor androgen. Metode: Desain penelitian cross sectional. Sampel spermatozoa berasal dari 30 pasien infertil OA/OAT dan 10 pria fertil sebagai kontrol. Protein dan DNA spermatozoa diekstraksi dari tiap-tiap individu. Analisis ekspresi protein CREM, protamin 1 dan protamin 2 dilakukan dengan teknik western immunoblotting. Distribusi protein CREM, protamin 1 dan protamin 2 dianalisis dengan teknik imunositokimia. Pemeriksaan jumlah pengulangan (CAG) dilakukan dengan sekuensing DNA spermatozoa. Hasil: Analisis protein CREM, protamin 1 dan 2 pada laki-laki infertil menunjukan ekspresi yang menurun dibandingkan dengan pria fertil. Penurunan ekspresi protein terlihat pada frekuensi keberadaan pita lebih rendah pada laki-laki infertil secara signifikan. Ekspresi protein CREM berhubungan dengan Protamin 1. Tidak terdapat hubungan ekspresi protein CREM dengan protamin 2, dan ekspresi protamine 1 dengan protamin 2. Analisis imunositokimia menunjukkan bahwa Protein CREM, Protamin 1 dan 2 diekspresikan pada daerah kepala spermatozoa laki-laki fertil dan infertil. Rerata jumlah pengulangan CAG pada laki-laki infertil 29,6 sedangkan pada laki-laki fertil 28,9. Hasil analisis statistik menunjukan tidak ada hubungan signifikan antara jumlah pengulangan CAG gen reseptor androgen dengan tingkat ekspresi CREM, Protamin 1 dan Protamin 2. Kesimpulan: Protein CREM, Protamine 1 dan protamin 2 diekspresikan lebih rendah pada spermatozoa laki-laki infertil dan tidak ada hubungan dengan pengulangan jumlah CAG gen reseptor androgen. Ekspresi protein CREM berhubungan dengan protein protamin 1.

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Introduction : Infertility is a problem experienced by married couples, and causes originated from male factors contribute around 40% of total cases. One of those factor is disturbance in chromatin remodeling during spermiogenesis. During this process, an important event in which histone protein is replaced by protamine takes place. As a result, DNA becomes more compact in size, bond by protamines protein. The expression of protamines is influenced by CREM which is a transcription factor regulating protamine genes. Protamine is transcripted in round spermatid, and translated in elongated spermatid, a process which is dependent on Androgen action. The aims of this study was to analyze CREM and protamine expression in spermatozoa from infertile patients and its correlation with CAG repeats variation of androgen receptor gene.

Method: This cross sectional study was conducted from December 2012 through March 2015. Protein and DNA sperm were extracted from spermatozoa of infertile men. CREM, and protamines expressions were analyzed by using western immunobloting. Localization and distribution of CREM and protamines expression were analyzed by immunocytochemistry. Examination of CAG repeat was performed by DNA sequencing.

Result: CREM and protamines were found to be down-regulated in infertile men compared to fertile individuals. A significant association was found between CREM and protamine 1 expression, but not with protamine 2. No significant association was found between protamine 1 and protamine 2. Analyses using immunocytochemistry showed reduced expression in CREM and Protamine from infertile patient compared to normal individuals. The average CAG repeat of infertile men was 29.6 compared to 28,9 of fertile donors. Statistical analysis showed no significant association between expression level of CREM and Protamine towards the number of CAG repeats variation androgen receptor gene.

Conclusion: CREM, protamine 1 and protamine 2 expression are lower in spermatozoa of infertile male and no association with CAG repeats variation of androgen receptor gene. CREM expression is associated with protamine 1."

"Latar Belakang: Hiperglikemia akut maupun kronis dapat menyebabkanperubahan patalogis pada lapisan serabut saraf retina RNFL . Pada tahap dinilapisan sel ganglion GCs mengalami apoptosis, yang diregulasi oleh proteinBrn3b. Proses ini diketahui terjadi lebih awal sebelum perubahan histopatologispada RNFL terjadi pada diabetes. Proses apoptosis ditandai dengan, peningkatanstres oksidatif, peningkatan NO, NF-??, TNF-?, dan Caspase 3 pada patogenesisglaukoma primer sudut terbuka GPSTa . Asumsi klinis saat ini bahwa penderitadiabetes melitus dalam jangka waktu lama lebih dari 5 tahun kemungkinan besarberisiko terjadinya glaukoma. Namun kenyataannya dalam pengalaman klinistidak semua pasien diabetes melitus dapat terjadi GPSTa. Berdasarkan temuanklinis tersebut diduga ada peran faktor genetik yang mendasari etiologi dariGPSTa pada pasien diabetes melitus. Tujuan: Mengetahui peran gen Brn3b pada apoptosis di RGCs dan kaitannyadengan perubahan kuantitas NO, Caspase 3, NF-?B, dan TNF-? sebagai prediksiglaukoma dini pada tikus diabetes. Metode: Penelitian ekperimental in vivo menggunakan tikus jantan SpraqueDawleyusia ge; 2 bulan dengan berat 150-200 gram diambil secara random sejakNopember 2015 hingga Juli 2016. Hewan coba dibagi menjadi 2 kelompok yaitu: kelompok perlakuan diinjeksi intraperitoneal STZ 50 mg/kg dalam 0.01M buffersitrat dalam ph 4.5 dan kelompok kontrol tidak ada perlakuan. Glukosa darahpuasa diperiksa 3 hari setelah injeksi STZ, dan dikonfirmasi ge; 250 mg/dL.Jaringan retina dibagi menjadi dua bagian pada kelompok perlakuan maupunkontrol yakni retina kanan untuk IHK Caspase 3 dan TNF-? dan retina kiridibagi dua bagian yaitu untuk pemeriksaan quantitative Real-time PCR RNAdiekstraksi untuk analisis ekspresi gen Brn3b dan pemeriksaan ELISA NO danNF-?B. Hasil: Terjadi penurunan ekpresi gen Brn3b pada tikus perlakuandibandingkan ekspresi gen Brn3b kontrol sebesar 1.2677 kali bulan kedua, 1.1348kali bulan keempat, dan 2.4600 kali bulan keenam. Disisi lain terjadi penurunankuantitas NO dan peningkatan kuantitas Caspase 3, NF-?B, dan TNF-?. Kesimpulan: Ekspresi mRNA Brn3b berbanding terbalik dengan apoptosis padaRGCs. Kuantitas NO, Caspase 3, NF-?B, dan TNF-? dipengaruhi oleh ekspresiBrn3b pada RGCs akibat hiperglikemia pada tikus diabetes.

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Background: Acute and chronic hyperglycemia may pathologically changeretinal nerve fiber layer RNFL . At early stage, ganglion cells GCs undergoapoptosis, which is regulated by a Brn3b protein. This change is known to occurat earlier time before retinal histological changes can be detected in diabeticpatients. The apoptosis process is marked by an increase in oxidative stress, a risein NO, NF-??, caspase 3, and TNF-? levels in pathogenesis of primary open angleglaucoma POAG . Current clinical assumption proposes that individualssuffering from diabetes mellitus for more than 5 years have greater risk ofglaucoma. Nonetheless, clinical experience shows that not all diabetic patientsdevelop POAG. Based on clinical examinations it is suspected that there is agenetic factor that caused the etiology of POAG in diabetes mellitus patients.

Purpose: To learn the role of Brn3b gene in apoptosis of RGCs and its effect onthe changing of quantity of NO, Caspase 3, NF-?B, and TNF-? as early predictorof glaucoma in diabetic rats.

Methods: Experimental in-vivo study was carried out using male SpraqueDawleyrats with age ge; 2 months, weighing 150-200 grams. The rats wererandomly selected from November 2015 to July 2016. The animals were dividedinto two groups. Group receiving intraperitoneal injection of STZ 50 mg/kg in0.01M citric buffer and pH 4,5; 2 and control group with no treatment. Fastingblood glucose was checked 3 days after injection of STZ and hyperglycemia wasdetermined as fasting blood glucose ge; 250 mg/dL. Retinal tissue was divided intotwo parts both experimental and control groups respectively : a right retina forIHC Caspase 3 and TNF-? ; b left retina was divided into two parts for thepurpose of quantitative Real-Time PCR test RNA extraction for Brn3b geneexpression analysis and ELISA test NO and NF-?B.

Results: Experimental group showed a decrease in Brn3b expression compared tocontrol group 1.2677-fold lower on 2nd month; 1.1348-fold lower on 4th monthand 2.4600-fold lower on 6th month . On the other hand, there was a decrease ofNO and there was increased of Caspase 3, NF-?B and TNF-? quantity.

Conclusion: The expression of mRNA Brn3b is inversely proportional toapoptosis in RGCs. The quantity of NO, Caspase 3, NF-?B and TNF-? isinfluenced by expression of Brn3b in RGCs caused by hyperglycemia in diabeticrats."