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Biomedical subjects

J L Tilly

Publications and source records attributed to J L Tilly.

At least 55 records · Page 3Linked to original sources
PubMed1998

High and low molecular weight DNA cleavage in ovarian granulosa cells: characterization and protease modulation in intact cells and in cell-free nuclear autodigestion assays.

To continue elucidation of the biochemical and molecular pathways involved in the induction of apoptosis in granulosa cells (GC) of ovarian follicles destined for atresia, we characterized the occurrence and protease modulation of high and low molecular weight (MW) DNA fragmentation during rat GC death. Atresia of ovarian follicles, occurring either spontaneously in vivo or induced in vitro, was associated with both high MW and internucleosomal (low MW) DNA cleavage. Incubation of follicles in the presence of a putative irreversible and non-competitive inhibitor of caspase-1 (interleukin-1beta-converting enzyme or ICE), sodium aurothiomalate (SAM), completely prevented internucleosomal, but not high MW, DNA cleavage. As reported previously, morphological features of apoptosis (pyknosis, cellular condensation) and atresia (granulosa cell disorganization, oocyte pseudomaturation) remained detectable in SAM-treated follicles. The potential involvement of proteases in endonuclease activation was further analyzed in cell-free assays using nuclei from both GC (which autodigest their DNA) and HeLa cells (HC, which do not autodigest their DNA unless incubated with extracts prepared from other cell types). Crude cytoplasmic extracts prepared from GC induced both high MW and internucleosomal DNA cleavage in HC nuclei. The induction of low, but not high, MW DNA cleavage in HC nuclei by GC extracts was suppressed by pretreatment of the extracts with SAM or with any one of the serine protease inhibitors, dichloroisocoumarin (DCI), N-tosyl-L-leucylchloromethylketone (TLCK) or N-tosyl-L-phenylchloromethylketone (TPCK). Interestingly, SAM and DCI also prevented cation-induced low MW DNA fragmentation in GC nuclei; however, TLCK and TPCK were without effect. Our results support a role for cytoplasmic and nuclear serine proteases in the activation of the endonuclease(s) responsible for internucleosomal DNA cleavage during apoptosis.

Animals↗
PubMed1998

Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary.

Recent data support a role for apoptosis, under tight regulatory control by bcl-2, oxidative stress response, tumor suppressor, and CASP gene family members, in mediating granulosa cell demise during follicular atresia in the rodent and avian ovary. Herein we evaluated the occurrence of apoptosis in the human and baboon ovary relative to follicular health status, and analyzed expression of several cell death genes in these tissues. In situlocalization of DNA strand breaks in fixed human and baboon ovarian tissue sections indicated that apoptosis was essentially restricted to granulosa cells of atretic antral follicles. Biochemical analysis of DNA oligonucleosomes in individual follicles isolated from baboon ovaries during the ovulatory phase revealed the presence of apoptotic DNA fragments in subordinate but not dominant follicles, thus substantiating the in situ labeling studies. Messenger RNA transcripts encoded by the bax death susceptibility gene, the bcl-xlong survival gene, the bcl-xshort pro-apoptosis gene, the p53 tumor suppressor gene, and two members of the CASP gene family (CASP-2/Ich-1, CASP-3/CPP32), were detected by Northern blot analysis of total RNA prepared either from human ovaries or from Percoll-purified granulosa-lutein cells obtained from patients undergoing assisted reproductive technologies. Lastly, immunohistochemical localization of the BAX death-susceptibility protein in the human ovary revealed abundant expression in granulosa cells of early atretic follicles, whereas BAX protein was extremely low or non-detectable in healthy or grossly-atretic follicles. We conclude that apoptosis occurs during, and is probably responsible for, folicular atresia in the human and baboon ovary. Moreover, apoptosis in the human ovary is likely controlled by altered expression of the same cohort of cell death regulatory factors recently implicated as primary determinants of apoptosis induction or suppression in the rodent ovary.

Adult↗
PubMed1998

Estradiol-mediated suppression of apoptosis in the rabbit corpus luteum is associated with a shift in expression of bcl-2 family members favoring cellular survival.

In the rabbit, estradiol is the primary luteotropic hormone. Estradiol withdrawal results in a rapid decline in serum progesterone and eventually in corpus luteum (CL) regression. The objective of this study was to determine whether estradiol modulates luteal cell apoptosis. In the first experiment, rabbits were randomly assigned to one of five experimental groups. An empty capsule (control) or estradiol-filled Silastic capsule was inserted s.c. on Day 0 of pseudopregnancy (day of hCG administration). On Day 11 of pseudopregnancy, some of the group I (control) and group II (estradiol capsule) rabbits were subjected to laparotomy, and one ovary from each rabbit was perfused in vitro to determine progesterone secretion rates. The CL from the contralateral ovary were dissected, snap-frozen, and stored at -70 degrees C until analyzed for internucleosomal DNA cleavage (apoptosis). Estradiol-containing capsules were removed from some of the remaining rabbits on Days 8, 9, and 10 to initiate estradiol deprivation. Rabbits were then subjected to laparotomy 24, 48, or 72 h after capsule removal (groups III, IV, and V, respectively), and ovaries or CL were processed as described above. Deprivation of estradiol for 24 (group III), 48 (group IV), or 72 (group V) h in vivo reduced in vitro progesterone secretion rates by more than 90% as compared to that in ovaries collected from estradiol capsule-intact animals. After in vivo endogenous estradiol suppression, withdrawal of exogenous estradiol resulted in luteal cell apoptosis, which increased in a time-dependent manner. Northern blot analysis revealed an increase in bax mRNA levels and a decrease in bcl-x mRNA levels coincident with luteal cell apoptosis induced by estradiol withdrawal. These data demonstrate that changes in progesterone production caused by estradiol exposure and deprivation are in part related to luteal cell apoptosis, and alterations in the expression of bcl-2 gene family members may be one of the mechanisms by which estradiol exerts its luteotropic effect in the rabbit CL.

Animals↗
PubMed1997

Germ cell apoptosis after treatment of cryptorchidism with human chorionic gonadotropin is associated with impaired reproductive function in the adult.

Cryptorchidism results in impaired fertility. Reduced numbers of testicular germ cells can be shown histologically during the first years of life. The process causing germ cell loss in cryptorchid prepubertal boys is unknown, but it could be the result of a form of programmed cell death known as apoptosis. 25 adult men with a history of surgically treated cryptorchidism were studied, 15 of whom had received an unsuccessful human chorionic gonadotropin (hCG) therapy before orchidopexy. Apoptotic DNA fragmentation was assayed in testis biopsies taken during orchidopexy by end-labeling, both in extracted DNA and histochemically in situ. Only a few scattered apoptotic spermatogonias were seen by end-labeling of biopsies from patients not treated with hCG, whereas more extensive labeling of spermatogonia was seen after hCG treatment. As estimated by gel electrophoresis, the amount of low molecular weight DNA was 4.3-fold higher in the hCG-treated group when compared with the level in scrotal testis of non-hCG-treated patients (P < 0.001). About 20 yr after the biopsy, the low molecular weight DNA fragmentation correlated negatively with the testis volume (r = -0.84; P < 0.001) and positively with serum FSH levels (r = 0.73; P < 0.001). Findings in the semen analysis were similar between the groups. Apoptotic loss of spermatogonia after hCG treatment of cryptorchidism warrants reevaluation of the safety of this treatment.

Adult↗
PubMed1997

Induction of PreB cell apoptosis by 7,12-dimethylbenz[a]anthracene in long-term primary murine bone marrow cultures.

Numerous studies demonstrate that polycyclic aromatic hydrocarbons (PAH) suppress immunity by modifying the function of both B and T cells. Relatively few studies have assessed the effects of these common environmental chemicals on immature lymphocytes. In the present study, long-term primary bone marrow cultures were employed to investigate the effects of a prototypic PAH and aryl hydrocarbon receptor (AhR) agonist, 7,12-dimethylbenz[a]anthracene (DMBA), on immature B lymphocytes. In this system, immature preB cells are maintained in a supportive microenvironment provided by bone marrow stromal cells. Results presented here demonstrate that (1) exposure of primary bone marrow cultures to DMBA results in preB cell death by apoptosis; (2) notably low doses of DMBA (> or = 10(-8) M) induce preB cell apoptosis; (3) in long-term cultures, bone marrow stromal cells, but not preB cells, express AhR mRNA and protein as determined by in situ hybridization, RT-PCR, and immunoblotting; (4) freshly isolated unfractionated bone marrow cells, but not purified bone marrow B cells, express AhR protein as assessed by immunohistochemistry; (5) alpha-naphthoflavone, a competitive AhR inhibitor and cytochrome P450 antagonist, completely blocks DMBA-induced preB cell apoptosis in primary bone marrow cultures; and (6) DMBA or benzo[a]pyrene injection in vivo results in bone marrow cell apoptosis consistent with the death of hematopoietic cells clustered around stromal elements. The results implicate programmed cell death as a mechanism underlying DMBA-mediated immunosuppression and suggest that preB cell death is influenced by local interactions with AhR+ bone marrow stromal cells.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1997

Hormonal modulation of apoptosis in the rat adrenal gland in vitro is dependent on structural integrity.

The intact rat adrenal gland in short-term (3-h) organ culture may be amenable for the identification of factors involved in regulating adrenal cell apoptosis under defined conditions. In this model, culturing in the absence of trophic support (basal; control) triggered apoptosis in the intact rat adrenal gland; oligonucleosome formation, a measure of apoptosis, was 56.4-fold greater than that of glands snap-frozen at the start of incubation. Angiotensin II (Ang II) (100 nM) enhanced apoptosis by 67% over control. By contrast, adrenocorticotropin (ACTH) (100 nM) attenuated basal apoptosis by 59% and antagonized the enhanced apoptosis induced by Ang II back to the control level. Quartering of the glands enhanced basal oligonucleosome formation 182.2% greater than that of intact glands. Interestingly, quartering of the glands abolished the influences of Ang II and ACTH on apoptotic DNA fragmentation, but did not alter ACTH-induced corticosterone secretion. These data suggest that some level of gross adrenal structural information or compartmentalization, sufficiently disrupted by quartering, is required for the hormonal modulation of adrenal cell survival.

Adrenal Glands↗
PubMed1997

Apoptosis-associated signaling pathways are required for chemotherapy-mediated female germ cell destruction.

Female sterility resulting from oocyte destruction is an unfortunate, and in many cases inevitable, consequence of chemotherapy. We show that unfertilized mouse oocytes exposed to therapeutic levels of the antitumor drug, doxorubicin (DXR), undergo apoptosis; however, fertilized oocytes do not initiate apoptosis, but enter cell-cycle arrest, when treated with DXR. Apoptosis induced by DXR in oocytes is blocked by sphingosine-1-phosphate, an inhibitor of ceramide-promoted cell death. Oocytes from Bax-deficient, but not p53-null, female mice display complete resistance to DXR-induced apoptosis in vivo and in vitro. Pretreatment of oocytes with a specific peptide inhibitor of caspases also abrogates the apoptotic response to DXR. These findings indicate that oocyte destruction caused by chemotherapy can be prevented by manipulation of apoptosis-associated signaling pathways.

Amino Acid Chloromethyl Ketones↗
PubMed1997

The genes of cell death and cellular susceptibility to apoptosis in the ovary: a hypothesis.

Recent advances in the field of cell death, primarily derived from gene-transfer experiments and manipulation of tumor cell lines in vitro, have identified key genes responsible for determining whether or not a given cell will initiate apoptosis. However, comparatively less is known of the role that the products of these genes play in physiological settings of cell death. In the ovary, a tremendous level of normal cell death takes place in the germline throughout the later stages of fetal development. This process is responsible for setting the absolute number of oocytes ('eggs') available for subsequent development and ovulation during adult life. Interestingly, death remains the fate of the vast majority of oocytes that survive the waves of attrition during fetal life and are endowed in the post-natal ovary as primordial follicles. This pool of oocytes is lost indirectly as a consequence of the death of the somatic (granulosa) cells that, in the case of a small percentage of the total follicles, support and nourish the oocyte until its release at ovulation. Due to the magnitude of cell death that occurs normally within the female gonad during both fetal development and post-natal life, the ovary has proven to be an excellent model to study the role of cell death genes in a physiological setting of endocrine-regulated apoptosis. It is now known that a diverse spectrum of pro- and anti-apoptosis susceptibility genes, including members of the bcl-2 and CASP (ced-3/Ice) gene families, are expressed in germ cells and/or somatic cells of the ovary. Many, but not all, of these genes are regulated by specific survival factors, such as gonadotropins and growth factors, and changes in the temporal patterns of cell death gene expression suggest an intimate association exists between the products of these genes and activation of cellular suicide. Moreover, pathological oocyte destruction, such as that triggered by exposure of female germ cells to chemotherapeutic compounds or environmental toxicants, may also be dependent upon gene-driven apoptosis. As such, this review will discuss data supporting the hypothesis that the susceptibility of ovarian cells to death induction is dependent upon the pattern of cell death gene expression occurring within those cells prior to and/or concomitant with receipt of the stimulus for apoptosis. Elucidation of the relationship between germ cell loss and cell death genes may allow future intervention into the process of oocyte depletion associated with normal and pathophysiological reproductive senescence.

Journal Article↗
PubMed1997

Cleavage of cytoskeletal proteins by caspases during ovarian cell death: evidence that cell-free systems do not always mimic apoptotic events in intact cells.

Several lines of evidence support a role for protease activation during apoptosis. Herein, we investigated the involvement of several members of the CASP (cysteine aspartic acid-specific protease; CED-3- or ICE-like protease) gene family in fodrin and actin cleavage using mouse ovarian cells and HeLa cells combined with immunoblot analysis. Hormone deprivation-induced apo-ptosis in granulosa cells of mouse antral follicles incubated for 24 h was attenuated by two specific peptide inhibitors of caspases, zVAD-FMK and zDEVD-FMK (50-500 microM), confirming that these enzymes are involved in this paradigm of cell death. Proteolysis of actin was not observed in follicles incubated in vitro while fodrin was cleaved to the 120 kDa fragment that accompanies apoptosis. Fodrin, but not actin, cleavage was also detected in HeLa cells treated with various apoptotic stimuli. These findings suggest that, in contrast to recent data, proteolysis of cytoplasmic actin may not be a component of the cell death cascade. To confirm and extend these data, total cell proteins collected from mouse ovaries or non-apoptotic HeLa cells were incubated without and with recombinant caspase-1 (ICE), caspase-2 (ICH-1) or caspase-3 (CPP32). Immunoblot analysis revealed that caspase-3, but not caspase-1 nor caspase-2, cleaved fodrin to a 120 kDa fragment, wheres both caspases-1 and -3 (but not caspase-2) cleaved actin. We conclude that CASP gene family members participate in granulosa cell apoptosis during ovarian follicular atresia, and that collapse of the granulosa cell cytoskeleton may result from caspase-3-catalyzed fodrin proteolysis. However, the discrepancy in the data obtained using intact cells (actin not cleaved) versus the cell-free extract assays (actin cleaved) raises concern over previous conclusions drawn related to the role of actin cleavage in apoptosis.

Journal Article↗
PubMed1997

Cumulus cells are required for the increased apoptotic potential in oocytes of aged mice.

Recent studies with female ICR mice have suggested that oocyte DNA fragmentation is one reason for poor oocyte quality and lower fertility associated with ageing. Since it was not determined if this increased 'apoptotic' potential in aged oocytes is due to changes within the oocyte itself or within the microenvironment of cumulus cells (CC) surrounding the germ cell, we sought to clarify if CC were required to affect the rate of apoptosis in oocytes maintained in vitro. Intact cumulus-oocyte complexes (COC) were retrieved by superovulation of virgin female ICR mice at 7 weeks ('young') or 34-35 weeks ('aged') of age. One-half of the COC in each group were incubated at 37 degrees C in human tubal fluid medium under paraffin oil for 24 h. The other half of the COC in each group were denuded of CC and incubated under the same conditions (denuded oocytes; DO). Following incubation, COC were stripped of adherent CC by gentle pipetting. All DO were then fixed and checked by light microscopy for morphological changes characteristic of apoptosis. In young mice, the presence of CC had no significant effect on oocyte death rate (18 +/- 9% and 14 +/- 6% apoptotic oocytes in COC and DO, respectively; P > 0.05). However, in aged mice the percentage of CC-enclosed oocytes that underwent apoptosis was significantly greater as compared to the death rate in DO (48 +/- 3% versus 19 +/- 8% apoptotic oocytes, respectively; P < 0.05). This increased death potential was due to the presence of CC since the occurrence of apoptosis in DO of aged versus young mice was not significantly different (19 +/- 8% versus 14 +/- 6% apoptotic oocytes, respectively; P > 0.05). These results demonstrate that the age-dependent acceleration of apoptosis in oocytes maintained in vitro requires the CC.

Aging↗
PubMed1997

Increased bax and interleukin-1beta-converting enzyme messenger ribonucleic acid levels coincide with apoptosis in the bovine corpus luteum during structural regression.

Recent reports have demonstrated that prostaglandin F2alpha-induced generation of reactive oxygen species or their intermediates inhibits progesterone synthesis and may also serve as a trigger for apoptosis in the corpus luteum (CL). BCL-2, an inhibitor of apoptosis in a wide variety of cell types, has been reported to prevent oxidative stress-induced cell death. Thus, the present studies were conducted to determine whether levels of mRNA encoding BCL-2 and related members of this gene family (BAX and BCL-Xshort, which induce apoptosis; BCL-Xlong, a BCL-2 homologue that prevents apoptosis) differed in functional (Day 21 of pregnancy) versus regressed (Day 21 of the estrous cycle) CL in the bovine ovary. Levels of mRNAs encoding p53, a transcriptional regulator of the bcl-2 and bax genes, and interleukin-1beta-converting enzyme (ICE), a protein recently implicated in the induction of apoptosis whose expression may be enhanced by oxidative stress, were also assessed. Partial cDNA clones encoding bovine bax, bcl-x, p53, and Ice were isolated using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique with total RNA prepared from functional or regressed CL. A bovine bcl-2 cDNA could not be isolated from luteal tissue RNA despite the use of several primer pairs for amplification. Total RNA was then extracted from functional or regressed CL and analyzed by Northern blot analysis. The occurrence of apoptosis in regressed CL, as evidenced by the presence of internucleosomal DNA cleavage, was associated with a significant increase in both bax and Ice mRNA levels as compared with levels of bax and Ice expression in functional CL (p < 0.05, n = 3). There were no significant differences in bcl-x or p53 mRNA levels in functional versus regressed CL. Analysis of bcl-x mRNA by RT-PCR revealed that the long form was the primary, if not only, mRNA expressed in functional and regressed bovine luteal tissue. On the basis of data that increased expression of bax is associated with, and may be required for, apoptosis in ovarian granulosa cells and germ cells, we propose that BAX may play a similar role in apoptosis induction during luteal regression. Moreover, the increased Ice mRNA levels in regressed CL provides the first evidence that the ICE family of death proteases may be involved in luteolysis.

Animals↗
PubMed1997

Expression of bcl-2 and nr-13 in hen ovarian follicles during development.

Follicle atresia is initiated within the granulosa cell layer of ovarian follicles and is mediated via the process of apoptosis. In the hen, at least two populations of granulosa cells can be distinguished during follicle development on the basis of their inherent susceptibility or resistance to apoptosis, in vitro. Given the previously established correlation between expression of bcl-xLong and hen granulosa cell resistance to apoptosis, the present studies were conducted to characterize expression of bcl-2 and an avian bcl-2 homologue, nr-13, in follicles at various stages of development. Levels of nr-13 mRNA were significantly higher only in granulosa cells from the largest (F1) preovulatory follicle compared to 3- to 5-mm prehierarchal follicles. By comparison, bcl-2 mRNA levels were 5- to 9-fold higher in granulosa cells from the three largest preovulatory follicles compared to those from follicles 9 to 12 mm in diameter and prehierarchal follicles. The increase in neither nr-13 nor bcl-2 was correlated with the stage of follicle development associated with the acquisition of resistance to apoptosis in granulosa cells (e.g., at the 9- to 12-mm stage). Results from the present studies do not support a close correlation between constitutive expression of nr-13 or bcl-2 mRNA and the transition from a state of apoptosis susceptibility to apoptosis resistance in hen granulosa cells. Thus, it is proposed that nr-13 and bcl-2 play more of a supportive role in regulating additional aspects of ovarian cell function such as cell proliferation and/or differentiation.

Animals↗
PubMed1997

Differential expression of members of the bcl-2 gene family in proliferative and secretory human endometrium: glandular epithelial cell apoptosis is associated with increased expression of bax.

Glandular epithelial cells of the human endometrium initiate apoptosis in the secretory phase of the cycle. To better understand the regulation of apoptosis in this paradigm of endocrine-regulated cell turnover, we studied the expression of the cell death regulatory genes, bax, bcl-2, and bcl-x, in human proliferative and secretory endometria relative to the absence or presence of apoptosis. As assessed by immunohistochemistry, levels of BAX protein were modest in proliferative endometrium and increased dramatically in the secretory phase when apoptosis was most prevalent. Expression of BAX was predominantly localized to epithelial cells of the functionalis layer of the secretory endometrium. In contrast, BCL-2 immunoreactivity was maximal during the proliferative phase and decreased in the secretory phase. Moreover, BCL-2 was topographically concentrated in the basalis layer. Immunoreactive BCL-X protein was observed mostly in glandular epithelial cells of the human endometrium. Compared with proliferative endometrium, secretory endometrium showed stronger BCL-X staining, especially in the functionalis layer. By Western blotting we confirmed that both proliferative and secretory endometrium expressed the long or antiapoptosis form as well as the short or proapoptosis form of the BCL-X protein. Taken together, our results demonstrate a coordinated pattern of expression of bcl-2 gene family members in human endometrium during the menstrual cycle, with a shift toward greater levels of the proapoptosis protein, BAX, occurring in glandular epithelial cells during the secretory phase of the cycle. Therefore, we conclude that cyclic changes in endometrial growth and regression may be precisely regulated by shifts in the ratio or balance of BCL-2 and related proteins in glandular epithelial cells.

Apoptosis↗
PubMed1996

A program of cell death and extracellular matrix degradation is activated in the amnion before the onset of labor.

Fetal membranes usually rupture during the process of labor. Premature fetal membrane rupture occurs not infrequently and is associated with significant fetal and maternal morbidity. The mechanisms of normal and pathologic fetal membrane rupture are not well understood. We have examined structural and biochemical changes in the rat amnion as labor approaches in order to characterize this process in normal pregnancy. Here we report that before the onset of active labor the amnion epithelial cells undergo apoptotic cell death which encompasses degradation of 28S ribosomal subunit RNA and associated P proteins and fragmentation of nuclear DNA. Concurrent with these cellular changes, the amnion type I collagen matrix is degraded with the accumulation of three-quarter length type I collagen fragments in extraembryonic fluid, characteristic of the cleavage of fibrillar collagen by interstitial collagenase. Western blot and immunohistochemical analyses confirmed that interstitial collagenase protein appears in association with the loss of amnion type I collagen. We conclude that amnion epithelial cells undergo a process of programmed cell death associated with orchestrated extracellular matrix degradation which begins before the onset of active labor. Thus, fetal membrane rupture is likely to be the result of biochemical changes as well as physical forces.

Amnion↗
PubMed1996

The molecular basis of ovarian cell death during germ cell attrition, follicular atresia, and luteolysis.

Physiological cell death mechanisms (termed apoptosis, programmed cell death, active cell death, and biological cell death) play a fundamental role in the cyclic function of the ovary as it strives in each cycle to ovulate a viable egg for fertilization. Healthy ovarian follicles and corpora lutea are also required for the secretion of steroids to prepare the female reproductive tract for embryo implantation and gestation. Using molecular biological approaches combined with classic histological examinations, several recent studies have confirmed the occurrence of apoptosis in female germ cells (oogonia and oocytes) during fetal ovarian development, granulosa cells during follicular atresia, and cells of the corpus luteum during luteolysis. Additionally, new light has been shed on the potential gene products which function to precisely coordinate the balance of life and death in the ovary. It is the purpose of this review to discuss the concepts of physiological cell death as they relate to ovarian function, and to offer testable hypotheses concerning the intracellular effector pathways responsible for directing ovarian cell fate in response to changes in hormonal stimuli.

Apoptosis↗
PubMed1996

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

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, i.p.) 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 microns) 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.

Animals↗