In vitro metabolism of pregnenolone to 7α-hydroxypregnenolone by rainbow trout embryos

Petkam, Rakpong; Renaud, R; Freitas, Ana; Canário, Adelino V.M.; Raeside, J. I.; Kime, David E.; Leatherland, J. F.

doi:10.1016/s0016-6480(02)00641-x

Cited by 14 publications

(7 citation statements)

References 27 publications

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“…This 7α‐hydroxylase activity probably explains the significant loss of radioactivity from the Sep‐Pak columns during the preliminary chromatography of the incubation medium. Similar losses of counts have been found following in vitro incubation of rainbow trout Oncorhynchus mykiss (Walbaum) follicles (Petkam, 2003) and embryos (Petkam et al ., 2003), with the ‘flow through’ counts appearing in the form of [ 3 H]H 2 O. [ 3 H]P 5 is labelled only at position 7, whilst [ 3 H]17OHP 4 is labelled at positions 1, 2, 6 and 7.…”

Section: Discussionsupporting

confidence: 70%

“…7α‐Hydroxylated steroids have been reported previously as the products of ovarian steroidogenesis in other teleostean species (Kime et al ., 1994; Ponthier et al ., 1998), and, using HPLC and gas chromatogrophy‐mass spectroscopy, this metabolite has been recently identified to be the only product of P 5 metabolism by rainbow trout embryos (Petkam et al ., 2003). These findings suggest a fairly broad distribution of ovarian 7‐hydroxylase activity in fishes, and even more widely, as shown by the 7α‐hydroxylated steroid synthesis from progestogens by mammalian neural tissues (Rose et al ., 1997).…”

Section: Discussionmentioning

confidence: 99%

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In vitro steroidogenesis of the gonads of the protogynous Pacific wrasse, Haliochoeres trimaculatus

Leatherland

Ogasawara

Rahman

et al. 2003

Journal of Fish Biology

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Testicular and ovarian fragments of the protogynous Pacific wrasse Haliochoeres trimaculatus were incubated in vitro with [3H]pregnenolone ([3H]P5), [3H]17‐hydroxyprogesterone ([3H]17OHP4), non‐radioactive (nr) 17β‐oestradiol (nrE2) or nrP5 to identify the major gonadal steroidogenic pathways and steroid products in females and in the two male variants of this species, the terminal phase (TP) and initial phase (IP) males. Both testis and ovarian tissues exhibited 7 hydroxylase activity resulting in the formation of 7α‐hydroxypregnenolone (7OHP5) from [3H]P5, and many HPLC peaks were identified as products of testicular (c. 29) and ovarian (c. 23) steroidogenesis, and only c. 50% of these metabolites co‐eluted with authentic reference standards; only very small amounts of conjugated steroid were synthesized from any of the precursors. [3H]P5 was converted by testis mainly to 7αOHP5, and two unknown steroids, whereas [3H]17OHP4 metabolism gave rise to [3H]17,20β‐dihydroxy‐4‐pregnen‐3‐one (DHP), 11‐ketotestosterone (11KT), and two unknown steroids. For ovarian tissues, [3H]17OHP4 and [3H]P5 were metabolized to form E2, oestrone (E1), androstenedione (A4), 20α‐ and 20β‐dihydroprogesterone (20αDHP and 20βDHP), 7αOHP5 (from [3H]P5) and a major unknown. The HPLC steroid profiles for testis incubations for IP and TP males were similar, however, the steroidogenic response of the testis of TP males to human chorionic gonadotrophin, in vitro (determined by hormone assay), was significantly higher than that of IP males.

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Section: Discussionsupporting

confidence: 70%

Section: Discussionmentioning

confidence: 99%

In vitro steroidogenesis of the gonads of the protogynous Pacific wrasse, Haliochoeres trimaculatus

Leatherland

Ogasawara

Rahman

et al. 2003

Journal of Fish Biology

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“…'Eyed stage' (26 dpf), late pre-hatch (40 dpf), and late yolk absorption (58 dpf) stage embryos were used to examine the capacity of embryos to metabolize [ 3 H]cortisol. For each developmental stage, the body of each embryo tissues was separated from the yolk-sac and cut into pieces as described previously (Petkam et al 2003a(Petkam et al , 2003b to allow penetration of cortisol. Tissue from ten 26 dpf embryos, eight 40 dpf embryos and two 58 dpf embryos (!12 replicates) were co-incubated with [ 3 H]cortisol in 2 ml supplemented M199 (described earlier) for 18 h at 8 8C on a tilt shaker.…”

Section: Ovulated Oocytes and Embryosmentioning

confidence: 99%

“…Sep-Pak C 18 cartridges (Waters Corp., Milford, MA, USA) were used for the SPE procedure as described previously (Khan et al 1997a, 1997b, Petkam et al 2003a, Barkataki et al 2011. Each cartridge was primed by adding 5 ml methanol followed by 5 ml ddH 2 O wash; each sample was mixed with double-distilled water (ddH 2 O) to make 5 ml, and this was then added to a primed cartridge and allowed to elute.…”

Section: Solid-phase Extractionmentioning

confidence: 99%

“…A mixture of 21 authentic steroid reference standards (Khan et al 1997b, Petkam et al 2003a was included in all HPLC runs to compare the elution times of the 3 H-labelled steroids with the standards; the absorbance of the standards was monitored at a light wavelength of 254 nm (representative chromatographs of the reference standards using the two protocols are shown in Fig. 1).…”

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confidence: 99%

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The in vitro metabolism of cortisol by ovarian follicles of rainbow trout (Oncorhynchus mykiss): comparison with ovulated oocytes and pre-hatch embryos

Li¹,

Christie²,

Leatherland³

2012

Reproduction

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REPRODUCTION RESEARCHThe in vitro metabolism of cortisol by ovarian follicles of rainbow trout (Oncorhynchus mykiss): comparison with ovulated oocytes and pre-hatch embryos Mao Li, Heather L Christie and John F Leatherland Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1Correspondence should be addressed to M Li who is now at Department of Integrated Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1; Email: mao@uoguelph.ca AbstractMid-vitellogenic stage rainbow trout (Oncorhynchus mykiss) ovarian follicles (both intact and yolk free (YF)), ovulated oocytes and embryos were co-incubated with [2,4,6,7-3 H]cortisol for 18 h to determine the degree and nature of the metabolism and biotransformation of the glucocorticoid. There was evidence of the conversion of cortisol to the less biologically potent glucocorticoid, cortisone, and the formation of glucocorticoid sulphates (both cortisol and cortisone) for all cell and tissue samples, suggesting the presence of 11b-hydroxysteroid dehydrogenase (11b-HSD) and glucocorticoid sulphotransferase (GST) activity at all stages; however, GST activity was particularly marked in both intact and YF ovarian follicles, suggesting an important role of follicles in limiting the exposure of oocyte to maternal cortisol. As there was no evidence of 11b-HSD or GST activity in ovarian fluid, the findings affirm that ovarian follicles (probably the thecal and granulosa cells) provide a barrier against the transfer of cortisol to the oocytes by forming sulphated steroids, whereas ovulated oocytes and early embryos have a more limited capacity to either metabolize or conjugate cortisol and are therefore more vulnerable at the post-ovulatory and early embryonic stages to increases in exposure to the glucocorticoid. (2012)). The relationship between the maternal stress response, maternal hypercortisolism and reproductive and embryonic outcomes is complex and still the object of ongoing research; however, some of the outcomes are most likely caused by metabolic or immune system-related actions of cortisol that indirectly impact the maternal involvement in the reproductive process; however, other responses can be attributed to the direct actions of cortisol on the developing embryo or fetus. In mammals, the antral fluid of the ovarian follicle regulates the exposure of the pre-ovulatory oocyte to cortisol, and the placental tissues act as a partial barrier to the transfer of maternal cortisol to the embryo and fetus. Because these protective barriers are not present in oviparous vertebrate species, there is the potential for the transfer of maternal cortisol to the oocytes. Consequently, if the oocytes and early embryos are unable to metabolize cortisol, embryogenesis may be compromised (Leatherland et al. 2010. The antral fluid of the ovarian follicles in mammals contains relatively high concentrations of unconjugated cortisol, the levels of which are regulated within narrow limits by the actions of two forms of 1...

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Gene structure and comparative and phylogenetic analyses of Catla catla CYP1A full-length cDNA and its responsiveness to benzo(a)pyrene and copper sulphate at early developmental stages

Kumari

Gireesh‐Babu

Kumar

et al. 2017

Fish Physiol Biochem

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In the present study, full-length CYP1A cDNA from Catla catla (Catla) has been identified, and its real-time quantitative reverse transcription PCR (qRT-PCR) expression has been evaluated in different tissues, developmental stages (0, 3, 6, 12 and 24 h and 5, 7 and 9 days post-fertilization) and copper sulphate and benzo(a)pyrene (BaP)-treated 5-day post-fertilization (dpf) larvae (6 to 6.5 mm). Various structural, comparative and phylogenetic analyses of the deduced amino acid sequence revealed that the identified gene of Catla belongs to the CYP1A1 subfamily. Among different tissues of Catla, the highest CYP1A expression was observed in the kidney followed by the liver, muscle, gill, intestine and brain. CYP1A mRNA expression was detected during all the larval developmental stages, including the unfertilized egg with the highest expression on 9 dpf. BaP (3.5 ppb) and copper sulphate (sublethal dose 0.516 ppm) challenge test for 96 h to Catla larvae revealed the highest CYP1A1 expression at 48 h post-challenge. CYP1A1 transcript also showed a concentration-dependent increase in expression following exposure at 1.75 and 3.5 ppb of BaP for 48 h. Its expression profiling indicates that it is functional at early developmental stages. It can also be used to develop a specific biomarker tool for monitoring environmental pollution.

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In vitro metabolism of pregnenolone to 7α-hydroxypregnenolone by rainbow trout embryos

Cited by 14 publications

References 27 publications

In vitro steroidogenesis of the gonads of the protogynous Pacific wrasse, Haliochoeres trimaculatus

In vitro steroidogenesis of the gonads of the protogynous Pacific wrasse, Haliochoeres trimaculatus

The in vitro metabolism of cortisol by ovarian follicles of rainbow trout (Oncorhynchus mykiss): comparison with ovulated oocytes and pre-hatch embryos

Gene structure and comparative and phylogenetic analyses of Catla catla CYP1A full-length cDNA and its responsiveness to benzo(a)pyrene and copper sulphate at early developmental stages

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