Disertasi

Analisis In silico dan karakterisasi sel sperma setelah dikultur bersama dengan sel epitel kaput epididimis mencit yang ditransfeksi siRNA Defb20. = In silico analysis and sperm characterization after co-culture with siRNA-mediated knockdown of Defb20 in caput epididymal cell culture.

DEFB20 merupakan anggota keluarga protein beta defensin yang diduga memiliki peran penting dalam proses pematangan sperma di epididimis. Dugaan tersebut didasarkan pada ekspresi spesifik Defb20 pada bagian caput epididimis dan diregulasi oleh androgen dan faktor testicular. Namun mekanisme DEFB20 dalam proses pematangan sperma masih belum jelas. Beta defensin diduga memiliki peranan dalam regulasi influks kalsium melalui kanal ion CatSper yang terekspresi secara spesifik pada sel sperma Oleh sebab itu, penelitian ini bertujuan untuk menganalisis peranan Defb20 dalam pematangan sperma melalui pendekatan secara in silico dan in vitro. Studi in silico dilakukan dengan menggunakan teknik molecular docking untuk mengetahui interaksi antara DEFB20 dengan CatSper sub unit alfa (1-4). Studi in vitro dilakukan dengan memproduksi kultur primer sel kaput epididimis yang ditransfeksi dengan siRNA Defb20 untuk menghasilkan sel knockdown. Sel Sperma mencit dan manusia di ko-kultur pada medium kultur sel (Cell), medium kondisional (CM), sekretom (SC) dan medium kultur (MC) sebagai medium kontrol selama 3 jam pada suhu 37°C. Selanjutnya dilakukan karakterisasi sperma ditingkat molekuler. Uji statistika dilakukan dengan kemaknaan p < 0,05. Pendekatan in silico menggambarkan interaksi antara mDEFB20 dan mCatSper1-4 serta mDEFB20 dan hCatSper1-4 membutuhkan energi rendah, yang berikatan pada bagian situs aktif kanal ion mCatSper1 dan hCatSper3. Pada sperma mencit tidak ditemukan adanya perbedaan bermakna kadar kalsium total dan kualitas sperma antara kelompok yang diko-kultur pada medium yang ditransfeksi dengan siRNA Defb20 dan medium yang ditransfeksi dengan negatif siRNA. Kadar kalsium total sperma manusia mengalami peningkatan pada kelompok yang di kokultur bersama sel epitel kaput epididimis yang ditransfeksi siRNA Defb20. Peningkatan kadar kalsium total mempengaruhi secara bermakna terhadap penurunan kecepatan sperma yang diikuti dengan tren penurunan fosforilasi tirosin serta tren peningkatan reaksi akrosom dan caspase-3. Peningkatan kalsium total tidak berpengaruh pada viabilitas dan integritan membran sperma. Pembungkaman Defb20 tidak mempengaruhi kualitas sperma mencit, sedangkan pada sperma manusia berpengaruh terhadap konsentrasi total kalsium dan kecepatan sperma yang mengindikasikan mDEFB20 mencegah terjadinya reaksi akrosom spontan.
Kata Kunci: DEFB20, siRNA, epididimis, kalsium, kualitas sperma, fosforilasi tirosin.


DEFB20 belongs to the beta defensin protein family and is putatively involved in sperm maturation process. This notion is based on its specific expression in the epididymis, regulation by androgens and testicular factors, expression from puberty to adulthood, and classification as a secretory protein. However, the mechanism of DEFB20 in the sperm maturation process remains unclear. Beta defensins are believed to be involved in the regulation of calcium influx, while CatSper is a specific ion channel expressed in sperm. The aim of this research was to analyze the role of Defb20 in sperm maturation through both in silico and in vitro approaches. The study of in silico utilized molecular docking techniques to investigate the interactions between DEFB20 and CatSper subunits alpha (1-4). The study of in vitro involved the production of primary cultures of epididymal caput cells transfected with Defb20 siRNA to generate knockdown cells. Mouse and human sperm were co-cultured in cell culture medium (Cell), conditioned medium (CM), secretome (SC), and culture medium (MC) as a control for 3 hours at 37°C. Subsequently, both cellular and molecular sperm phenotype analyses were performed. Statistical tests were conducted with a significance level of p < 0.05. The in silico approach illustrated that the interaction between mDEFB20 and mCatSper1-4, as well as mDEFB20 and hCatSper1-4, it required low energy. These interactions were primarily observed at the active site of the mCatSper1 ion channel and hCatSper3. In mouse sperm, there were no significant differences in total calcium levels and sperm quality between those co-cultured with Defb20 siRNA-transfected medium and those co-cultured with negative siRNA-transfected medium. Conversely, total calcium levels significantly increased in human sperm after cultured with caput epididymal epithelial cells transfected with Defb20 siRNA. It significantly influenced a reduction in sperm velocity, accompanied by trends of decreased tyrosine phosphorylation, increased acrosome reactions, and caspase-3 levels. However, this increasing total calcium did not affect sperm viability or membrane integrity. Silencing Defb20 did not impact the quality of mouse sperm. In contrast, human sperm exhibited notable effects on total calcium concentration and sperm velocity, suggesting that mDEFB20 prevents spontaneous acrosome reactions. Keywords: siRNA, epididymis, calcium, sperm quality, tyrosine phosphorylation.