A Key Enzyme in Placental Steroidogenesis: Detection of 3β-Hydroxysteroid Dehydrogenase/∆5-4 İsomerase (3β-HSD)’ın With Cloned Probes on Eukaryotic Vector

Öz

3β-hydroxysteroid dehydrogenase/∆5-4 isomerase (3β-HSD) is a key enzyme in placental steroidogenesis. To form steroid hormones, pregnenolone requires enzymes 3βhydroxysteroiddehydrogenase/∆5-4 isomerase (3β-HSD), which belongs to the ∆5 and ∆4 steroidogenic pathways. The created probes will help to identify the molecular basis of enzymes which play a role in steroidogenesis. Therefore, the objective of this study was to develop probes with cloning the target gene on a eukaryotic vector. Total RNA was isolated and RT-PCR was performed. DNA bands were extracted from agarose gel and purified. 3βHSD-DNAs were transformed on E.coli strain. Vector containing 3βHSD insert were digested with the restriction enzymes NcoI and NotI. In vitro transcription of the DIG-labelled cRNA was carried out by using DIG-RNA labelling mix. In situ hybridization was carried out with the probes. Specific staining was observed in uninucleated trophoblast cells where as negative control was negative. In conclusion, specific probes were generated with cloning the target gene on a eukaryote vector, whic are sitable for detection of steroidogenic enzymes with in situ hybridization.

Anahtar Kelimeler

• 3β-HSD
• placenta
• in situ hybridization

Plasental Steroidogeneziste Kilit Enzim: 3β-Hidroksisteroid Dehidrogenaz/∆5-4 İzomeraz (3β-HSD)’ın Ökaryon Vektörde Klonlanmış Prob’larla Belirlenmesi

Öz

3β-hidroksisteroid dehidrogenaz/∆5-4 izomeraz (3β-HSD) plasental steroidogenesiz’de kilit enzim olarak kabul edilir. Steroid hormonların oluşumu için, pregnenolon ∆5 ve ∆4 sentez yollarında kullanılan 3β- hidroksisteroid dehidrogenaz/∆5-4 izomeraz (3β-HSD)’a ihtiyaç duyar. Çalışmanın amacı, hedef genin, bir ökaryon vektor üzerinde klonlanmasıyla prob elde edilmesi ve in situ hibridizasyonda kullanılabilirliğinin gösterilmesidir. Bu amaçla total RNA izolasyonundan sonra RT-PCR yapıldı. DNA bantları agaroz jelden ayrılarak saflaştırıldı. 3βHSD-DNA’ların E.coli suşuna transformasyonu tamamlandıktan sonra 3βHSD insert içeren vektörden NcoI ve NotI restriksiyon enzimleriyle kesildi. DIG-işaretli cRNA’ların transkripsiyonu sonrasında in situ hibridizasyon yapıldı. Spesifik boyanmalar tek nukleuslu trofoblast hücrelerinde gözlenirken negatif kontrol grubunda boyanma gözlenmedi. Sonuç olarak bir ökaryon vector üzerinde, hedef gen klonlanarak steroidogenik enzimlerin belirlenmesinde kullanılmak üzere, in situ hibridizasyon tekniği için oldukça spesifik prob’lar elde edilmiştir.

Anahtar Kelimeler

• 3β-HSD
• plasenta
• in situ hibridizasyon

Kaynakça

1. Chaffin, C.L., Dissen, G.A., Stouffer, R.L., 2006. Hormonal regulation of steroidogenic enzyme expression in granulosa cells during the peri- ovulatory interval in monkeys. Mol. Hum. Re- prod., 6, 11–18.
2. Chih-Hsien, C., Hen-Wei, Wei., Leang-Shin, W., 2008. Generation and Utilization of P450 Cholesterol Side-Chain Cleavage Enzyme and 3b-Hydroxysteroid Dehydrogenase Antibodies for Universal Detection. Journal of Immunoassay & Immunochemistry. 29, 152–160.
3. Conley, A.J., Bird, I.M., 1997. The role of cytochrome P450 17 alphahydroxylose and 3 be- ta-hydroxysteroid integration of gonadal and adrenal steroidogene- sis via the delta 5 and delta 4 pathways of steri- dogenesis in mammals. Biol Reprod 56, 789–799.
4. Hall, P.F., 1984. Cellular organization for steroidogenesis. Int Rev Cytol. 86, 53-95.
5. Herrmann, M., Scholmerich, J., Straub, R.H., 2002. Influence of cytokines and growth factors on distinct steroidogenic enzymes in vitro: a short tabular data collection. Ann N Y Acad Sci. 966, 166-186.
6. Labrie, F., Simard, J., Luu-The, V., Pelletier, G., Belghmi, K., Belanger, A. 1994. Structure, regulation and role of 3 beta-hydroxysteroid dehydrogenase, dehydrogenase and aromatase enzymes in the formation of sex steroids in classical and peripheral intacrine tissues. Baillieres Clin Endocrinol Metab. 8, 451–474.
7. Mason, J.I., 1993. The 3b-hydroxysteroid dehydrogenase gene family of enzymes. Trends Endocrinol Metab. 4, 199–203.
8. Penning, T.M.,1997. Molecular endocrinology of hydroxysteroiddehydrogenases. Endocr Rev. 18, 281–305.

9. Rodway, M.R., Swan, C.L., Crellin, N.K., Gillio- Meina, C., Chedrese, P.J., 1999. Steroid regula- tion of progesterone synthesis in a stable porcine granulosa cell line: a role for progestins. J. Ster- oid. Biochem. Mol. Biol. 68, 173–180.
10. Sugawara, T., Fujimoto, S., 2004. The potential function of steroid sulphatase activity in steroid production and steroidogenic acute regulatory protein expression. Biochem J. 380, 153-160.
11. Tsumagari, S., Kamata, J., Takagi, K., Tanemura, K., Yosal, A., Takeishi, M., 1994. 3ß- Hydroxysteroiddehydrogenase gestagen concentrations in bovine cotyledons and caruncles during gestation and parturition. J Re- prod Fertil 102, 35-39. activity and