High performance liquid chromatographic separation of steroids from ovarian follicles of fresh water perchAnabas testudineus: Identification and characterization of the maturation-inducing hormone
✍ Scribed by Bhattacharyya, Sanghamitra ;Sen, Utpal ;Bhattacharyya, Sachi Prasad ;Mukherjee, Dilip
- Book ID
- 102333822
- Publisher
- John Wiley and Sons
- Year
- 2002
- Tongue
- English
- Weight
- 181 KB
- Volume
- 292
- Category
- Article
- ISSN
- 0022-104X
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✦ Synopsis
Abstract
Accurate separation and identification of steroids from the postvitellogenic ovarian follicles of Indian climbing perch Anabas testudineus was performed by high‐performance liquid chromatography (HPLC) and specific radioimmunoassay (RIA). The steroids from such follicles, incubated in Cortland’s saline with or without homologous fish pituitary extract (FPE), were extracted with dichloromethane and separated on a μBondapak C~18~ column. Identification of the HPLC fractions was further confirmed by thin layer chromatography. As HPLC peaks for 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (DHP) and testosterone (T) were close, clear separation of these steroids and accurate measurement of their quantities was achieved by RIA of HPLC fractions using specific antibodies. Altogether, nine eluted fractions in the FPE‐untreated and ten in FPE‐treated samples were obtained. Of these, six were identified as: 5β‐pregnan‐3α,17α,20β‐triol (5β‐3α,17α,20β‐P); DHP; T; 17α‐hydroxyprogesterone (17α‐P~4~); progesterone (P~4~); and androstenedione (AD). Three fractions from untreated and four from FPE‐treated samples, however, remained unidentified. Of all the HPLC fractions examined for their relative maturational inducing (MI) potency on full grown (postvitellogenic) ovarian follicles of perch, the fraction identified as DHP was found to be the most effective inducer of germinal vesicle breakdown (GVBD) both at low and high concentrations. Fractions identified as 5β‐3α,17α,20β‐P and 17α‐P~4~ could induce only 32% and 20% GVBD at their highest concentration, while none of the unidentified fractions showed any MI activity. FPE caused increased production of DHP, testosterone, and 5β‐3α,17α,20β‐P. The qualitative differences between the fractions obtained from FPE‐treated samples and those from FPE‐untreated samples were only the appearance of a new polar metabolite of unknown function. The present study showed that, as a single steroid, DHP was the most potent MIH for A. testudineus. J. Exp. Zool. 292:565–572, 2002. © 2002 Wiley‐Liss, Inc.