Expression and redistribution of cellular Bad, Bax, and Bcl-xL protein is associated with VCD-induced ovotoxicity in rats

X. Hu, P. Christian, I. G. Sipes, Patricia B Hoyer

Research output: Contribution to journalArticle

69 Citations (Scopus)

Abstract

Previous studies have shown that 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in rats is likely caused by acceleration of the normal rate of atresia (apoptosis). VCD-induced ovotoxicity is specific for small preantral follicles and is associated with increased activity of caspase cascades. The present study was designed to investigate the alteration of expression and distribution of several Bcl-2 family member proteins induced by dosing of VCD in rat small ovarian follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., 1 day; a time when ovotoxicity is not initiated), or dosed daily for 15 days (80 mg/kg, i.p., 15 days; a time when significant ovotoxicity is underway). Four hours following the final dose, livers and ovaries were collected. Ovarian small (25-100 μm) and large (100-250 μm) preantral follicles were isolated, and subcellular fractions (cytosolic and mitochondrial) were prepared. Compared with controls, levels of the proapoptotic protein, Bad, were greater in both cytosolic and mitochondrial fractions of small preantral follicles collected from 15-day VCD-treated rats (cytosol, 1.97 ± 0.16; mitochondria, 2.20 ± 0.24, VCD/control, P < 0.05). After 15 days of daily VCD dosing, total cellular antiapoptotic Bcl-xL protein levels were unaffected in small preantral follicles, but its distribution in mitochondrial and cytosolic components was altered (mitochondria, 0.635 ± 0.08; cytosol, 1.39 ± 0.14, VCD/control, P < 0.05). Likewise, VCD did not affect protein levels of proapoptotic Bax in small follicles on Day 15. However, consistent with a Bax-mediated mechanism of apoptosis, the relative ratio of Bax/Bcl-xL in the mitochondrial fraction of small preantral follicles was significantly increased by VCD dosing (1.62 ± 0.21, VCD/control, P < 0.05). Immunofluorescence staining intensity evaluated by confocal microscopy visualized cytochrome c protein in the cytosolic compartment in granulosa cells of preantral follicles in various stages of development. Relative to controls, within the population of small preantral follicles, staining intensity was less (P < 0.05) and presumably more diffuse, specifically in stage 1 primary follicles from VCD-treated animals (15 days). VCD caused none of these effects in large preantral follicles or liver (not targeted by VCD). These data provide evidence that the apoptosis induced by VCD in ovarian small preantral follicles of rats is associated with increased expression of Bad protein, re-distribution of Bcl-xL protein and cytochrome c from the mitochondria to the cytosolic compartment, and an increase in the Bax/ Bcl-xL ratio in the mitochondria. These observations are consistent with the involvement of Bcl-2 gene family members in VCD-induced acceleration of atresia.

Original languageEnglish (US)
Pages (from-to)1489-1495
Number of pages7
JournalBiology of Reproduction
Volume65
Issue number5
StatePublished - 2001

Fingerprint

Proteins
Mitochondria
bcl-Associated Death Protein
4-vinyl-1-cyclohexene dioxide
Apoptosis
Cytochromes c
Cytosol
Staining and Labeling
bcl-2 Genes
Ovarian Follicle
Subcellular Fractions
Granulosa Cells
Liver
Inbred F344 Rats
Caspases
Confocal Microscopy
Fluorescent Antibody Technique
Ovary
Population

Keywords

  • Apoptosis
  • Follicle
  • Ovary
  • Signal transduction
  • Toxicology

ASJC Scopus subject areas

  • Cell Biology
  • Developmental Biology
  • Embryology

Cite this

Expression and redistribution of cellular Bad, Bax, and Bcl-xL protein is associated with VCD-induced ovotoxicity in rats. / Hu, X.; Christian, P.; Sipes, I. G.; Hoyer, Patricia B.

In: Biology of Reproduction, Vol. 65, No. 5, 2001, p. 1489-1495.

Research output: Contribution to journalArticle

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AU - Christian, P.

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AU - Hoyer, Patricia B

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AB - Previous studies have shown that 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in rats is likely caused by acceleration of the normal rate of atresia (apoptosis). VCD-induced ovotoxicity is specific for small preantral follicles and is associated with increased activity of caspase cascades. The present study was designed to investigate the alteration of expression and distribution of several Bcl-2 family member proteins induced by dosing of VCD in rat small ovarian follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., 1 day; a time when ovotoxicity is not initiated), or dosed daily for 15 days (80 mg/kg, i.p., 15 days; a time when significant ovotoxicity is underway). Four hours following the final dose, livers and ovaries were collected. Ovarian small (25-100 μm) and large (100-250 μm) preantral follicles were isolated, and subcellular fractions (cytosolic and mitochondrial) were prepared. Compared with controls, levels of the proapoptotic protein, Bad, were greater in both cytosolic and mitochondrial fractions of small preantral follicles collected from 15-day VCD-treated rats (cytosol, 1.97 ± 0.16; mitochondria, 2.20 ± 0.24, VCD/control, P < 0.05). After 15 days of daily VCD dosing, total cellular antiapoptotic Bcl-xL protein levels were unaffected in small preantral follicles, but its distribution in mitochondrial and cytosolic components was altered (mitochondria, 0.635 ± 0.08; cytosol, 1.39 ± 0.14, VCD/control, P < 0.05). Likewise, VCD did not affect protein levels of proapoptotic Bax in small follicles on Day 15. However, consistent with a Bax-mediated mechanism of apoptosis, the relative ratio of Bax/Bcl-xL in the mitochondrial fraction of small preantral follicles was significantly increased by VCD dosing (1.62 ± 0.21, VCD/control, P < 0.05). Immunofluorescence staining intensity evaluated by confocal microscopy visualized cytochrome c protein in the cytosolic compartment in granulosa cells of preantral follicles in various stages of development. Relative to controls, within the population of small preantral follicles, staining intensity was less (P < 0.05) and presumably more diffuse, specifically in stage 1 primary follicles from VCD-treated animals (15 days). VCD caused none of these effects in large preantral follicles or liver (not targeted by VCD). These data provide evidence that the apoptosis induced by VCD in ovarian small preantral follicles of rats is associated with increased expression of Bad protein, re-distribution of Bcl-xL protein and cytochrome c from the mitochondria to the cytosolic compartment, and an increase in the Bax/ Bcl-xL ratio in the mitochondria. These observations are consistent with the involvement of Bcl-2 gene family members in VCD-induced acceleration of atresia.

KW - Apoptosis

KW - Follicle

KW - Ovary

KW - Signal transduction

KW - Toxicology

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