Involvement of apoptosis in 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats

L. N. Springer, M. E. McAsey, J. A. Flaws, J. L. Tilly, I. G. Sipes, Patricia B Hoyer

Research output: Contribution to journalArticle

107 Citations (Scopus)

Abstract

Previous studies have determined that 4-vinylcyclohexene diepoxide (VCD) causes specific destruction of oocytes contained in small pre-antral (primordial and primary) ovarian follicles of Fischer 344 rats following 30 days of daily dosing with VCD. The purposes of this study were to identify the type of VCD-induced cell death occurring in small pre-antral follicles and to determine the earliest time following the onset of dosing when evidence of follicular destruction could first be detected. A significant decrease in the number of oocytes contained in small pre-antral follicles in ovaries of rats after 15 days of daily dosing (ip) with VCD (80 mg/kg) had been observed in preliminary experiments. Therefore, a study was conducted to determine the time of the onset of this follicular destruction by examination of follicular DNA integrity. Female Fischer 344 rats were dosed daily (80 mg/kg, ip) for 6, 8, 10, 12, or 14 days, and ovaries were removed 1, 4, or 24 hr after the final dose. Small pre-antral follicles (25-100 μm) were isolated by gentle dissociation of ovaries with collagenase, and follicles were sorted with micropipets. Genomic DNA was isolated from follicles and radiolabeled with [32P]dideoxy ATP, and the degree of fragmentation quantified by agarose gel electrophoresis and autoradiography. Degradation of DNA was evaluated by 32P content in low-molecular-weight fragments (<4 kilobase pairs). Degradation of DNA was not observed in follicles collected 24 hr after the final dose on any day. However, a random pattern of DNA degradation was observed, and was significantly greater (p < 0.05) compared with controls, when follicles were collected 4 hr following VCD administration on Days 10 and 12, but not on Days 6 or 8, of dosing. Although not significant, there was also evidence of DNA degradation in dosed animals on Day 14. Histological evaluation of small pre-antral follicles in ovarian sections during the early stages of VCD-induced DNA degradation (Day 10; 4 hr) demonstrated margination of chromatin along the nuclear membrane in oocytes and disruptions in focal contact between granulosa cells and oocytes, both features indicative of apoptosis. Furthermore, there was no sign of ruptured membranes in granulosa cells or oocytes or of an inflammatory response, characteristics of necrosis (pathological cell death). Whereas biochemical and morphological evidence of follicular destruction was seen 4 hr after dosing on Day 10, numbers of oocyte-containing primordial and primary follicles in VCD-treated animals were not different from controls at that time. These results demonstrate that the initial evidence of impending destruction of small pre-antral follicles is first consistently visualized following 10 days of daily dosing with VCD, although a measurable reduction in oocyte numbers has not yet occurred. Despite the fact that internucleosomal cleavage of genomic DNA was not observed, morphological evaluations support that granulosa cells and oocytes in primordial and primary follicles are destroyed via the induction of apoptosis.

Original languageEnglish (US)
Pages (from-to)394-401
Number of pages8
JournalToxicology and Applied Pharmacology
Volume139
Issue number2
DOIs
StatePublished - Aug 1996

Fingerprint

Oocytes
Rats
Apoptosis
DNA
Granulosa Cells
Degradation
Ovary
Ovarian Follicle
Inbred F344 Rats
Cell death
Animals
Cell Death
Membranes
DNA Cleavage
Focal Adhesions
4-vinyl-1-cyclohexene dioxide
Agar Gel Electrophoresis
Nuclear Envelope
Collagenases
Electrophoresis

ASJC Scopus subject areas

  • Pharmacology
  • Toxicology

Cite this

Involvement of apoptosis in 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats. / Springer, L. N.; McAsey, M. E.; Flaws, J. A.; Tilly, J. L.; Sipes, I. G.; Hoyer, Patricia B.

In: Toxicology and Applied Pharmacology, Vol. 139, No. 2, 08.1996, p. 394-401.

Research output: Contribution to journalArticle

Springer, L. N. ; McAsey, M. E. ; Flaws, J. A. ; Tilly, J. L. ; Sipes, I. G. ; Hoyer, Patricia B. / Involvement of apoptosis in 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats. In: Toxicology and Applied Pharmacology. 1996 ; Vol. 139, No. 2. pp. 394-401.
@article{85bc3437eb3442e389482dbabd04bd78,
title = "Involvement of apoptosis in 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats",
abstract = "Previous studies have determined that 4-vinylcyclohexene diepoxide (VCD) causes specific destruction of oocytes contained in small pre-antral (primordial and primary) ovarian follicles of Fischer 344 rats following 30 days of daily dosing with VCD. The purposes of this study were to identify the type of VCD-induced cell death occurring in small pre-antral follicles and to determine the earliest time following the onset of dosing when evidence of follicular destruction could first be detected. A significant decrease in the number of oocytes contained in small pre-antral follicles in ovaries of rats after 15 days of daily dosing (ip) with VCD (80 mg/kg) had been observed in preliminary experiments. Therefore, a study was conducted to determine the time of the onset of this follicular destruction by examination of follicular DNA integrity. Female Fischer 344 rats were dosed daily (80 mg/kg, ip) for 6, 8, 10, 12, or 14 days, and ovaries were removed 1, 4, or 24 hr after the final dose. Small pre-antral follicles (25-100 μm) were isolated by gentle dissociation of ovaries with collagenase, and follicles were sorted with micropipets. Genomic DNA was isolated from follicles and radiolabeled with [32P]dideoxy ATP, and the degree of fragmentation quantified by agarose gel electrophoresis and autoradiography. Degradation of DNA was evaluated by 32P content in low-molecular-weight fragments (<4 kilobase pairs). Degradation of DNA was not observed in follicles collected 24 hr after the final dose on any day. However, a random pattern of DNA degradation was observed, and was significantly greater (p < 0.05) compared with controls, when follicles were collected 4 hr following VCD administration on Days 10 and 12, but not on Days 6 or 8, of dosing. Although not significant, there was also evidence of DNA degradation in dosed animals on Day 14. Histological evaluation of small pre-antral follicles in ovarian sections during the early stages of VCD-induced DNA degradation (Day 10; 4 hr) demonstrated margination of chromatin along the nuclear membrane in oocytes and disruptions in focal contact between granulosa cells and oocytes, both features indicative of apoptosis. Furthermore, there was no sign of ruptured membranes in granulosa cells or oocytes or of an inflammatory response, characteristics of necrosis (pathological cell death). Whereas biochemical and morphological evidence of follicular destruction was seen 4 hr after dosing on Day 10, numbers of oocyte-containing primordial and primary follicles in VCD-treated animals were not different from controls at that time. These results demonstrate that the initial evidence of impending destruction of small pre-antral follicles is first consistently visualized following 10 days of daily dosing with VCD, although a measurable reduction in oocyte numbers has not yet occurred. Despite the fact that internucleosomal cleavage of genomic DNA was not observed, morphological evaluations support that granulosa cells and oocytes in primordial and primary follicles are destroyed via the induction of apoptosis.",
author = "Springer, {L. N.} and McAsey, {M. E.} and Flaws, {J. A.} and Tilly, {J. L.} and Sipes, {I. G.} and Hoyer, {Patricia B}",
year = "1996",
month = "8",
doi = "10.1006/taap.1996.0180",
language = "English (US)",
volume = "139",
pages = "394--401",
journal = "Toxicology and Applied Pharmacology",
issn = "0041-008X",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Involvement of apoptosis in 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats

AU - Springer, L. N.

AU - McAsey, M. E.

AU - Flaws, J. A.

AU - Tilly, J. L.

AU - Sipes, I. G.

AU - Hoyer, Patricia B

PY - 1996/8

Y1 - 1996/8

N2 - Previous studies have determined that 4-vinylcyclohexene diepoxide (VCD) causes specific destruction of oocytes contained in small pre-antral (primordial and primary) ovarian follicles of Fischer 344 rats following 30 days of daily dosing with VCD. The purposes of this study were to identify the type of VCD-induced cell death occurring in small pre-antral follicles and to determine the earliest time following the onset of dosing when evidence of follicular destruction could first be detected. A significant decrease in the number of oocytes contained in small pre-antral follicles in ovaries of rats after 15 days of daily dosing (ip) with VCD (80 mg/kg) had been observed in preliminary experiments. Therefore, a study was conducted to determine the time of the onset of this follicular destruction by examination of follicular DNA integrity. Female Fischer 344 rats were dosed daily (80 mg/kg, ip) for 6, 8, 10, 12, or 14 days, and ovaries were removed 1, 4, or 24 hr after the final dose. Small pre-antral follicles (25-100 μm) were isolated by gentle dissociation of ovaries with collagenase, and follicles were sorted with micropipets. Genomic DNA was isolated from follicles and radiolabeled with [32P]dideoxy ATP, and the degree of fragmentation quantified by agarose gel electrophoresis and autoradiography. Degradation of DNA was evaluated by 32P content in low-molecular-weight fragments (<4 kilobase pairs). Degradation of DNA was not observed in follicles collected 24 hr after the final dose on any day. However, a random pattern of DNA degradation was observed, and was significantly greater (p < 0.05) compared with controls, when follicles were collected 4 hr following VCD administration on Days 10 and 12, but not on Days 6 or 8, of dosing. Although not significant, there was also evidence of DNA degradation in dosed animals on Day 14. Histological evaluation of small pre-antral follicles in ovarian sections during the early stages of VCD-induced DNA degradation (Day 10; 4 hr) demonstrated margination of chromatin along the nuclear membrane in oocytes and disruptions in focal contact between granulosa cells and oocytes, both features indicative of apoptosis. Furthermore, there was no sign of ruptured membranes in granulosa cells or oocytes or of an inflammatory response, characteristics of necrosis (pathological cell death). Whereas biochemical and morphological evidence of follicular destruction was seen 4 hr after dosing on Day 10, numbers of oocyte-containing primordial and primary follicles in VCD-treated animals were not different from controls at that time. These results demonstrate that the initial evidence of impending destruction of small pre-antral follicles is first consistently visualized following 10 days of daily dosing with VCD, although a measurable reduction in oocyte numbers has not yet occurred. Despite the fact that internucleosomal cleavage of genomic DNA was not observed, morphological evaluations support that granulosa cells and oocytes in primordial and primary follicles are destroyed via the induction of apoptosis.

AB - Previous studies have determined that 4-vinylcyclohexene diepoxide (VCD) causes specific destruction of oocytes contained in small pre-antral (primordial and primary) ovarian follicles of Fischer 344 rats following 30 days of daily dosing with VCD. The purposes of this study were to identify the type of VCD-induced cell death occurring in small pre-antral follicles and to determine the earliest time following the onset of dosing when evidence of follicular destruction could first be detected. A significant decrease in the number of oocytes contained in small pre-antral follicles in ovaries of rats after 15 days of daily dosing (ip) with VCD (80 mg/kg) had been observed in preliminary experiments. Therefore, a study was conducted to determine the time of the onset of this follicular destruction by examination of follicular DNA integrity. Female Fischer 344 rats were dosed daily (80 mg/kg, ip) for 6, 8, 10, 12, or 14 days, and ovaries were removed 1, 4, or 24 hr after the final dose. Small pre-antral follicles (25-100 μm) were isolated by gentle dissociation of ovaries with collagenase, and follicles were sorted with micropipets. Genomic DNA was isolated from follicles and radiolabeled with [32P]dideoxy ATP, and the degree of fragmentation quantified by agarose gel electrophoresis and autoradiography. Degradation of DNA was evaluated by 32P content in low-molecular-weight fragments (<4 kilobase pairs). Degradation of DNA was not observed in follicles collected 24 hr after the final dose on any day. However, a random pattern of DNA degradation was observed, and was significantly greater (p < 0.05) compared with controls, when follicles were collected 4 hr following VCD administration on Days 10 and 12, but not on Days 6 or 8, of dosing. Although not significant, there was also evidence of DNA degradation in dosed animals on Day 14. Histological evaluation of small pre-antral follicles in ovarian sections during the early stages of VCD-induced DNA degradation (Day 10; 4 hr) demonstrated margination of chromatin along the nuclear membrane in oocytes and disruptions in focal contact between granulosa cells and oocytes, both features indicative of apoptosis. Furthermore, there was no sign of ruptured membranes in granulosa cells or oocytes or of an inflammatory response, characteristics of necrosis (pathological cell death). Whereas biochemical and morphological evidence of follicular destruction was seen 4 hr after dosing on Day 10, numbers of oocyte-containing primordial and primary follicles in VCD-treated animals were not different from controls at that time. These results demonstrate that the initial evidence of impending destruction of small pre-antral follicles is first consistently visualized following 10 days of daily dosing with VCD, although a measurable reduction in oocyte numbers has not yet occurred. Despite the fact that internucleosomal cleavage of genomic DNA was not observed, morphological evaluations support that granulosa cells and oocytes in primordial and primary follicles are destroyed via the induction of apoptosis.

UR - http://www.scopus.com/inward/record.url?scp=0030220009&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030220009&partnerID=8YFLogxK

U2 - 10.1006/taap.1996.0180

DO - 10.1006/taap.1996.0180

M3 - Article

C2 - 8806857

AN - SCOPUS:0030220009

VL - 139

SP - 394

EP - 401

JO - Toxicology and Applied Pharmacology

JF - Toxicology and Applied Pharmacology

SN - 0041-008X

IS - 2

ER -