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Impaired 17,20-Lyase Activity in Male Mice Lacking Cytochrome b 5 in Leydig Cells

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ABSTRACT

Androgen and estrogen biosynthesis in mammals requires the 17,20-lyase activity of cytochrome P450 17A1 (steroid 17-hydroxylase/17,20-lyase). Maximal 17,20-lyase activity in vitro requires the presence of cytochrome b5 (b5), and rare cases of b5 deficiency in human beings causes isolated 17,20-lyase deficiency. To study the consequences of conditional b5 removal from testicular Leydig cells in an animal model, we generated Cyb5flox/flox:Sf1-Cre (LeyKO) mice. The LeyKO male mice had normal body weights, testis and sex organ weights, and fertility compared with littermates. Basal serum and urine steroid profiles of LeyKO males were not significantly different than littermates. In contrast, marked 17-hydroxyprogesterone accumulation (100-fold basal) and reduced testosterone synthesis (27% of littermates) were observed after human chorionic gonadotropin stimulation in LeyKO animals. Testis homogenates from LeyKO mice showed reduced 17,20-lyase activity and a 3-fold increased 17-hydroxylase to 17,20-lyase activity ratio, which were restored to normal upon addition of recombinant b5. We conclude that Leydig cell b5 is required for maximal androgen synthesis and to prevent 17-hydroxyprogesterone accumulation in the mouse testis; however, the b5-independent 17,20-lyase activity of mouse steroid 17-hydroxylase/17,20-lyase is sufficient for normal male genital development and fertility. LeyKO male mice are a good model for the biochemistry but not the physiology of isolated 17,20-lyase deficiency in human beings.

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Steroidogenesis stimulation with hCG leads to accumulation of Prog and 17OHP in LeyKO mice. The Cyb5flox/flox (WT) and LeyKO male mice 2–4 months of age (n = 5–7) were injected with 10 mIU of hCG or saline, killed 2 hours later, and exsanguinated. A, Plasma levels of Prog, 17OHP, T, AD, 11-deoxycortisol, corticosterone, and 11-deoxycorticosterone (11-DOC) determined by LC-MS/MS. B, Ratio of plasma concentrations of 21-carbon steroids (Prog + 17OHP) to 19-carbon steroids (AD + T) as a functional measure of in vivo 17,20-lyase activity. C, P450 17A1 activities, expressed as hydroxylase to lyase ratio in testicular homogenates from LeyKO and WT mice (n = 13–23). Testicular homogenates were incubated with either [3H]-Prog or [3H]-17OHP, and total products were measured by HPLC with radiochemical detection to assay 17-hydroxylase and 17,20-lyase activities, respectively (see Materials and Methods). Addition of recombinant b5 to the testicular homogenates (+b5) rescued the decreased 17,20-lyase activity in LeyKO homogenates but did not change activities in WT homogenates. Values are mean ± SD, and statistics were determined with 2-tailed t test (*, P < .05; **, P < .005; ***, P < .0005).
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Figure 2: Steroidogenesis stimulation with hCG leads to accumulation of Prog and 17OHP in LeyKO mice. The Cyb5flox/flox (WT) and LeyKO male mice 2–4 months of age (n = 5–7) were injected with 10 mIU of hCG or saline, killed 2 hours later, and exsanguinated. A, Plasma levels of Prog, 17OHP, T, AD, 11-deoxycortisol, corticosterone, and 11-deoxycorticosterone (11-DOC) determined by LC-MS/MS. B, Ratio of plasma concentrations of 21-carbon steroids (Prog + 17OHP) to 19-carbon steroids (AD + T) as a functional measure of in vivo 17,20-lyase activity. C, P450 17A1 activities, expressed as hydroxylase to lyase ratio in testicular homogenates from LeyKO and WT mice (n = 13–23). Testicular homogenates were incubated with either [3H]-Prog or [3H]-17OHP, and total products were measured by HPLC with radiochemical detection to assay 17-hydroxylase and 17,20-lyase activities, respectively (see Materials and Methods). Addition of recombinant b5 to the testicular homogenates (+b5) rescued the decreased 17,20-lyase activity in LeyKO homogenates but did not change activities in WT homogenates. Values are mean ± SD, and statistics were determined with 2-tailed t test (*, P < .05; **, P < .005; ***, P < .0005).

Mentions: Unlike human beings, exposure of male mice to a receptive female or soiled bedding from a receptive female activates the vomeronasal reflex and stimulates LH release and T synthesis, with circulating T increasing to 6 ng/mL within 20–60 minutes of exposure (33, 34). Thus, basal T production rates in mice are low and a poor test for a partial blockade in 17,20-lyase activity. To simulate a transient robust burst of androgen synthesis and P450 17A1-catalyzed steroidogenesis, LeyKO mice and floxed littermates were injected with 10 mIU of hCG and killed 2 hours later, when plasma T peaks after hCG stimulation (35). Plasma T increased approximately 100-fold (to 37 ± 12 ng/mL) after hCG stimulation in floxed animals relative to saline controls, whereas AD rose only 5-fold with negligible accumulation of 21-carbon precursors 17OHP or Prog (Figure 2A). In contrast, plasma T rose only 18-fold (to 11 ± 5 ng/mL) and AD 2-fold after hCG stimulation in LeyKO mice; however, a marked accumulation of plasma 17OHP (to 36 ± 12 ng/mL) and lesser accumulation of Prog was observed, relative to a slight increase in floxed controls. Thus, hCG stimulation elicited a similar augmentation of steroidogenesis in WT and LeyKO animals, but the lack of b5 in Leydig cells of LeyKO animals impaired 17,20-lyase activity sufficiently to detain the flux of steroids above the partial block and to afford marked 17OHP accumulation. Plasma dehydroepiandrosterone was less than 0.02 ng/mL in all 23 basal or hCG-stimulated samples tested from LeyKO mice and floxed littermates (data not shown).


Impaired 17,20-Lyase Activity in Male Mice Lacking Cytochrome b 5 in Leydig Cells
Steroidogenesis stimulation with hCG leads to accumulation of Prog and 17OHP in LeyKO mice. The Cyb5flox/flox (WT) and LeyKO male mice 2–4 months of age (n = 5–7) were injected with 10 mIU of hCG or saline, killed 2 hours later, and exsanguinated. A, Plasma levels of Prog, 17OHP, T, AD, 11-deoxycortisol, corticosterone, and 11-deoxycorticosterone (11-DOC) determined by LC-MS/MS. B, Ratio of plasma concentrations of 21-carbon steroids (Prog + 17OHP) to 19-carbon steroids (AD + T) as a functional measure of in vivo 17,20-lyase activity. C, P450 17A1 activities, expressed as hydroxylase to lyase ratio in testicular homogenates from LeyKO and WT mice (n = 13–23). Testicular homogenates were incubated with either [3H]-Prog or [3H]-17OHP, and total products were measured by HPLC with radiochemical detection to assay 17-hydroxylase and 17,20-lyase activities, respectively (see Materials and Methods). Addition of recombinant b5 to the testicular homogenates (+b5) rescued the decreased 17,20-lyase activity in LeyKO homogenates but did not change activities in WT homogenates. Values are mean ± SD, and statistics were determined with 2-tailed t test (*, P < .05; **, P < .005; ***, P < .0005).
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Figure 2: Steroidogenesis stimulation with hCG leads to accumulation of Prog and 17OHP in LeyKO mice. The Cyb5flox/flox (WT) and LeyKO male mice 2–4 months of age (n = 5–7) were injected with 10 mIU of hCG or saline, killed 2 hours later, and exsanguinated. A, Plasma levels of Prog, 17OHP, T, AD, 11-deoxycortisol, corticosterone, and 11-deoxycorticosterone (11-DOC) determined by LC-MS/MS. B, Ratio of plasma concentrations of 21-carbon steroids (Prog + 17OHP) to 19-carbon steroids (AD + T) as a functional measure of in vivo 17,20-lyase activity. C, P450 17A1 activities, expressed as hydroxylase to lyase ratio in testicular homogenates from LeyKO and WT mice (n = 13–23). Testicular homogenates were incubated with either [3H]-Prog or [3H]-17OHP, and total products were measured by HPLC with radiochemical detection to assay 17-hydroxylase and 17,20-lyase activities, respectively (see Materials and Methods). Addition of recombinant b5 to the testicular homogenates (+b5) rescued the decreased 17,20-lyase activity in LeyKO homogenates but did not change activities in WT homogenates. Values are mean ± SD, and statistics were determined with 2-tailed t test (*, P < .05; **, P < .005; ***, P < .0005).
Mentions: Unlike human beings, exposure of male mice to a receptive female or soiled bedding from a receptive female activates the vomeronasal reflex and stimulates LH release and T synthesis, with circulating T increasing to 6 ng/mL within 20–60 minutes of exposure (33, 34). Thus, basal T production rates in mice are low and a poor test for a partial blockade in 17,20-lyase activity. To simulate a transient robust burst of androgen synthesis and P450 17A1-catalyzed steroidogenesis, LeyKO mice and floxed littermates were injected with 10 mIU of hCG and killed 2 hours later, when plasma T peaks after hCG stimulation (35). Plasma T increased approximately 100-fold (to 37 ± 12 ng/mL) after hCG stimulation in floxed animals relative to saline controls, whereas AD rose only 5-fold with negligible accumulation of 21-carbon precursors 17OHP or Prog (Figure 2A). In contrast, plasma T rose only 18-fold (to 11 ± 5 ng/mL) and AD 2-fold after hCG stimulation in LeyKO mice; however, a marked accumulation of plasma 17OHP (to 36 ± 12 ng/mL) and lesser accumulation of Prog was observed, relative to a slight increase in floxed controls. Thus, hCG stimulation elicited a similar augmentation of steroidogenesis in WT and LeyKO animals, but the lack of b5 in Leydig cells of LeyKO animals impaired 17,20-lyase activity sufficiently to detain the flux of steroids above the partial block and to afford marked 17OHP accumulation. Plasma dehydroepiandrosterone was less than 0.02 ng/mL in all 23 basal or hCG-stimulated samples tested from LeyKO mice and floxed littermates (data not shown).

View Article: PubMed Central - PubMed

ABSTRACT

Androgen and estrogen biosynthesis in mammals requires the 17,20-lyase activity of cytochrome P450 17A1 (steroid 17-hydroxylase/17,20-lyase). Maximal 17,20-lyase activity in vitro requires the presence of cytochrome b5 (b5), and rare cases of b5 deficiency in human beings causes isolated 17,20-lyase deficiency. To study the consequences of conditional b5 removal from testicular Leydig cells in an animal model, we generated Cyb5flox/flox:Sf1-Cre (LeyKO) mice. The LeyKO male mice had normal body weights, testis and sex organ weights, and fertility compared with littermates. Basal serum and urine steroid profiles of LeyKO males were not significantly different than littermates. In contrast, marked 17-hydroxyprogesterone accumulation (100-fold basal) and reduced testosterone synthesis (27% of littermates) were observed after human chorionic gonadotropin stimulation in LeyKO animals. Testis homogenates from LeyKO mice showed reduced 17,20-lyase activity and a 3-fold increased 17-hydroxylase to 17,20-lyase activity ratio, which were restored to normal upon addition of recombinant b5. We conclude that Leydig cell b5 is required for maximal androgen synthesis and to prevent 17-hydroxyprogesterone accumulation in the mouse testis; however, the b5-independent 17,20-lyase activity of mouse steroid 17-hydroxylase/17,20-lyase is sufficient for normal male genital development and fertility. LeyKO male mice are a good model for the biochemistry but not the physiology of isolated 17,20-lyase deficiency in human beings.

No MeSH data available.


Related in: MedlinePlus