Role of apoptosis in functional luteolysis in the pregnant rabbit corpus luteum: evidence of a role for placental-derived factors in promoting luteal cell survival.

Corpora lutea (CL) were isolated from one rabbit ovary on days 4, 8, 16 (peak luteal function), 28 (functional regression) and 30 of pregnancy and processed for biochemical analysis of DNA integrity. Analysis of DNA integrity revealed the presence of oligonucleosomal fragments in day 28 and day 30 CL but not in day 16 CL. The extent of low molecular weight (<15 kb) DNA labeling was 6.6 +/- 0.84 fold higher in day 30 as compared to day 16 CL (mean +/- SEM; n = 4, P < 0.01). In a second series of experiments, healthy CL collected from day 16 pregnant rabbits were incubated for 2 h in the absence or presence of 250 microg/ml of placental extract (PE) obtained from day 16 and/or day 30 placentas. Analysis of DNA integrity revealed that extensive apoptosis occurred in CL incubated in medium alone and in medium containing day 30 PE. In contrast, day 16 PE significantly suppressed apoptosis vs control (70 +/- 4%). In a third series of experiments, expression of mRNA for bcl-x and bax was measured by Northern analysis of CL treated without and with day 16 PE using cRNA probes for bcl-x and bax developed in our laboratory by RT-PCR. Treatment with PE significantly reduced bax mRNA levels but did not change bcl-x mRNA levels. These studies provide evidence that functional luteolysis in the pregnant rabbit CL is correlated with the occurrence of apoptosis. The data suggest that a factor(s) derived from the placenta may be responsible for the prolongation of CL life span during pregnancy by its ability to alter the bax:bcl -x rheostat and suppress apoptosis.

Targeted expression of Bcl-2 in mouse oocytes inhibits ovarian follicle atresia and prevents spontaneous and chemotherapy-induced oocyte apoptosis in vitro.

Members of the Bcl-2 family serve as central checkpoints for cell death regulation, and overexpression of Bcl-2 is known to inhibit apoptosis in many cell types. To determine whether targeted expression of Bcl-2 could be used to protect female germ cells from apoptosis, we generated transgenic mice expressing fully functional human Bcl-2 protein only in oocytes. Transgenic mice were produced using a previously characterized 480-bp fragment of the mouse zona pellucida protein-3 (ZP3) gene 5'-flanking region to direct oocyte-specific expression of a human bcl-2 complementary DNA. Immunohistochemical analyses using a human Bcl-2-specific antibody showed that transgene expression was restricted to growing oocytes and was not observed in the surrounding ovarian somatic cells or in any other nonovarian tissues. Histomorphometric analyses revealed that ovaries collected from transgenic female mice possessed significantly fewer atretic small preantral follicles compared with wild-type sisters, resulting in a larger population of healthy maturing follicles per ovary. However, the number of oocytes ovulated in response to exogenous gonadotropin priming and the number of pups per litter were not significantly different among wild-type vs. transgenic female mice. Nonetheless, oocytes obtained from transgenic mice and cultured in vitro were found to be resistant to spontaneous and anticancer drug-induced apoptosis. We conclude that targeted expression of Bcl-2 only in oocytes can be achieved as a means to convey resistance of the female germ line to naturally occurring and chemotherapy-induced apoptosis.

Antioxidants mimic the ability of chorionic gonadotropin to suppress apoptosis in the rabbit corpus luteum in vitro: a novel role for superoxide dismutase in regulating bax expression.

We have recently reported that members of the bcl-2 gene family are expressed and estradiol regulated in rabbit luteal cells during corpus luteum (CL) regression, and that estradiol and hCG are effective inhibitors of apoptosis in the rabbit CL in vivo and in vitro. As Bcl-2 and related proteins are known to regulate levels of reactive oxygen species or their intermediates in cells as one possible mechanism to control apoptosis, the present studies were designed to examine if oxidative stress plays a role in luteal cell apoptosis during CL regression in the rabbit. In the first set of experiments, healthy CL obtained from day 11 pseudopregnant rabbits were incubated in serum-free medium for 2 h in the absence or presence of superoxide dismutase (SOD; 1.5-150 U/ml), ascorbic acid (1-100 mM), N-acetyl-L-cysteine (25 and 50 mM), or catalase (10-1000 U/ml). Cells within CL incubated in medium alone exhibited extensive apoptosis (examined by analysis of extracted DNA using 3'-end labeling), and this onset of apoptosis was blocked in a dose-dependent fashion by treatment with SOD, ascorbic acid, N-acetyl-L-cysteine, or catalase. In the second set of experiments, expression of bax and bcl-x in CL after in vitro treatment without and with 100 U/ml SOD was examined. Although SOD treatment did not alter the levels of bcl-x messenger RNA (mRNA) over the 2-h incubation period, this antioxidant enzyme significantly reduced the levels of bax mRNA in incubated CL. In the final set of experiments, we observed that expression of mitochondrial- or manganese-containing SOD was significantly increased by treatment of isolated CL with 1 microg/ml hCG in vitro, whereas bax mRNA levels were significantly reduced under the same culture conditions. Collectively, these data indicate that the gonadotropin-mediated inhibition of apoptosis in rabbit luteal cells involves enhanced expression of the oxidative stress response gene, manganese-containing SOD, whose protein product may then function to protect luteal cells directly from the damaging effect of reactive oxygen species and/or indirectly by acutely down-regulating expression of Bax, a prooxidant member of the Bcl-2 protein family.

Localization, regulation and possible consequences of apoptotic protease-activating factor-1 (Apaf-1) expression in granulosa cells of the mouse ovary.

The recent characterization of apoptotic protease-activating factor-1 (Apaf-1) in vertebrates as a putative homolog of the Caenorhabditis elegans gene, ced-4, indicates that the third major arm of the C. elegans programmed cell death machinery has also been conserved through evolution. Although apoptosis is now known to be important for ovarian follicular atresia in vertebrates, nothing is known of the role of Apaf-1 in ovarian function. Herein we show by immunohistochemical analysis that Apaf-1 is abundant in granulosa cells of early antral follicles whereas in vivo gonadotropin priming completely suppresses Apaf-1 expression and granulosa cell apoptosis. Western blot analysis of fractionated protein extracts prepared from granulosa cells before and after in vitro culture without hormonal support to induce apoptosis indicated that mitochondrial cytochrome c release, a biochemical step required for the activation of Apaf-1, occurs in granulosa cells cultured in vitro. Moreover, Western blot analysis of procaspase-3 processing, a principal downstream event set in motion by activated Apaf-1, indicated that healthy granulosa cells possess almost exclusively the inactive (pro-) form of the enzyme whereas granulosa cells deprived of hormonal support rapidly process procaspase-3 to the active enzyme. Lastly, we show that serum-starved granulosa cells activate caspase-3-like enzymes both prior to and after nuclear pyknosis, as revealed by a single-cell fluorescent caspase activity assay. These data, combined with previous observations regarding the role of homologs of the two other C. elegans cell death regulatory genes, ced-9 (Bcl-2 family members) and ced-3 (caspases), in atresia fully support the hypothesis that granulosa cell apoptosis is precisely coordinated by all three major arms of a cell death program conserved through evolution.

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.

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.

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.

Models to elucidate the regulation of adrenal cell death.

Hypophysectomy-induced apoptosis in the rat adrenal gland is slow (not apparent until 12-24 h) and in-situ, 3'-end labeling (ISEL) of DNA strand breaks is restricted to a subpopulation of zona reticularis cells. In addition, it is completely blocked by ACTH. By contrast, apoptosis in the intact rat adrenal gland, cultured in the absence of trophic support, is extensive and rapid. Culture-triggered apoptosis (as determined by oligonucleosome formation) is attenuated by ACTH and is largely restricted to the zonae fasciculata and reticularis even at 3 h (as determined by ISEL and DAPI cytochemistry). Thus, this organ culture system may help elucidate factors that can acutely regulate adrenocortical cell survival. Quartered glands have a nearly 2-fold increase in oligonucleosome formation compared to intact glands at 3 h and are resistant to the antiapoptotic action of ACTH. In contrast, ACTH-induced corticosterone secretion is not attenuated. Angiotensin II (Ang II) enhances culture-triggered apoptosis, and its apoptotic action is attenuated by ACTH. These observations suggest that 1) acute hormonal modulation of apoptosis may require some level of gross adrenal structural integrity, and 2) ACTH and Ang II act in an antagonistic fashion to regulate adrenocortical apoptosis. The apoptotic effect of Ang II may be mediated via the type 2 receptor.

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.

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.

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.

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.

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.

Activin inhibits basal and androgen-stimulated proliferation and induces apoptosis in the human prostatic cancer cell line, LNCaP.

LNCaP cells, derived from an androgen-sensitive cell line widely employed as an in vitro model of human prostate cancer, have been shown to express activin receptors. Activin is a local regulator of cellular growth, appears to play a key role in mesoderm induction and differentiation during development, and has been implicated in gonadal tumorigenesis. Follistatin, a monomeric glycoprotein that specifically binds and neutralizes activin, is often coexpressed with activin and, thus, modulates the autocrine/paracrine biological activity of this potent growth factor. We tested the hypothesis that LNCaP growth is modulated by the activin/follistatin system. Recombinant human activin A inhibited [3H]thymidine incorporation in a dose-dependent fashion with an ED50 of approximately 0.43 +/- 0.3 nM. Activin (0.1-3 nM) also inhibited dihydrotestosterone (DHT)-stimulated [3H]thymidine incorporation in LNCaP cells. Similarly, recombinant human inhibin A inhibited LNCaP proliferation, but was only 1/100th as potent as activin. Furthermore, activin (3 nM) induced a 3-fold increase in the extent of labeling of low mol wt DNA fragments typical of apoptosis. Activin-induced apoptosis was also indicated by an increase in the number of cells with reduced DNA content, as measured by flow cytometry of activin-treated cells. Both activin-mediated inhibition of cell proliferation and induction of apoptosis could be completely blocked by recombinant human follistatin. Based upon these results using an in vitro model, we speculate that activin functions locally to oppose androgen-driven cell proliferation and, thus, is a key factor controlling prostate growth. Reduced activin biosynthesis, increased follistatin secretion, or signaling defects in the activin receptor system should be further investigated in future studies as potential mechanisms underlying enhanced androgen-independent growth of human prostate cancer cells.

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.

Apoptotic cell death in the rat adrenal gland: an in vivo and in vitro investigation.

Adrenocortical cell apoptosis was studied by using an established in vivo model, the hypophysectomized rat, and an in vitro model, viz., rat adrenal glands in short-term organ culture. In vivo, apoptosis (biochemical autoradiographic analysis of internucleosomal DNA cleavage) was weak and not apparent until 12-24 h after hypophysectomy. In situ histochemical localization of 3'-end DNA strand breaks revealed that apoptosis in vivo occurred nearly exclusively in subpopulations of zona reticularis cells. Adrenocorticotropic hormone (ACTH) maintenance completely blocked these indices of apoptosis. By contrast, apoptosis (DNA fragmentation) in cultured rat adrenal glands without ACTH was extensive and relatively rapid, being apparent after 1 h and increasing with the duration of incubation. ACTH attenuated (by 44%) but did not completely block apoptosis in vitro. Thus, ACTH appears to be the sole pituitary hormone that forestalls apoptosis of terminally differentiated adrenocortical (zona reticularis) cells. However, the discrepancy between in vitro and in vivo models in terms of the magnitude and rate of DNA fragmentation suggests that, in vivo, other factors finely regulate the magnitude of adrenocortical apoptotic cell death.

Interleukin-1 beta-converting enzyme-related proteases (IRPs) and mammalian cell death: dissociation of IRP-induced oligonucleosomal endonuclease activity from morphological apoptosis in granulosa cells of the ovarian follicle.

The Caenorhabditis elegans death susceptibility gene, ced-3, has a number of homologs in vertebrate species, including interleukin-1 beta (IL-1 beta)-converting enzyme (ICE), Ich-1long, and CPP32. These genes, which encode a family of related proteases, have been shown to induce apoptosis when transfected into eukaryotic cells. However, it remains to be determined whether these proteases are involved in apoptotic cell death under physiological conditions. The purpose of these studies was to examine the role of ICE-related proteases (IRPs) in apoptosis using a physiologically relevant model system, the ovarian follicle. Somatic granulosa cells within ovarian follicles undergo apoptosis during follicular atresia, a process responsible for the depletion of greater than 95% of the follicles established in the postnatal ovary. To accomplish these studies, we cloned partial rat complementary DNAs encoding ICE, Ich-1, and CPP32 and used these complementary DNAs to examine the gonadotropin regulation of ICE, Ich-1, and CPP32 gene expression in the immature rat ovary. We also examined levels of ICE activity in healthy and atretic rat follicles by monitoring the conversion of exogenous pro-IL-1 beta to the active cytokine, and then evaluated the actions of recombinant IL-1 beta on apoptosis in follicles incubated in vitro. Finally, we tested the requirement for IRP activity in granulosa cell apoptosis and follicular atresia by incubating follicles without and with IRP inhibitors. Northern blot analysis of total RNA samples indicated that gonadotropin-promoted follicular survival was associated with reduced ovarian expression of messenger RNAs encoding Ich-1 and CPP32. In contrast, ICE messenger RNA levels were extremely low and were not affected by gonadotropin treatment. We were also unable to detect ICE activity in proteins extracted from either healthy or atretic rat follicles, collectively suggesting that ICE per se may not function in granulosa cell death. As another approach to determine whether ICE is involved in atresia, healthy antral follicles were isolated from ovaries of gonadotropin-primed immature rats and incubated for 24 h in the absence or presence of 100 ng/ml transforming growth factor-alpha (TGF alpha) without and with 100 ng/ml IL-1 beta. Granulosa cells within follicles incubated in medium alone exhibited extensive levels of apoptosis, and this onset of apoptosis was prevented by the inclusion of TGF alpha. Addition of IL-1 beta did not alter basal levels of apoptosis nor did the cytokine antagonize TGF-alpha-promoted follicle survival, providing additional evidence that ICE activity is not required for atresia to occur.(ABSTRACT TRUNCATED AT 400 WORDS)

Vasoactive intestinal peptide-mediated suppression of apoptosis in the ovary: potential mechanisms of action and evidence of a conserved antiatretogenic role through evolution.

Vasoactive intestinal peptide (VIP)-containing nerve fibers are present in ovarian follicles at all stages of development, and VIP, acting primarily via the cAMP pathway, has been reported to modulate many aspects of granulosa cell function. Herein we examined the effects of VIP and its potential mechanisms of action on apoptosis in antral follicles isolated from ovaries of gonadotropin-primed immature rats and incubated in vitro under serum-free conditions. Additionally, the effects of VIP on apoptosis in isolated avian granulosa cells incubated in vitro were used as a comparative model system to determine whether the ability of VIP to modulate apoptosis in the ovary has been conserved through evolution. Genomic DNA extracted from incubated rat antral follicles exhibited extensive levels of internucleosomal DNA cleavage characteristic of cell death via apoptosis. Treatment of follicles with VIP (1-1000 nM) caused a dose-dependent reduction in the extent of apoptotic DNA breakdown, with a maximal effect achieved with 100 nM VIP. Provision of the adenylyl cyclase activator, forskolin (10 microM), mimicked the inhibitory effect of VIP on apoptosis and concomitantly increased intrafollicular cAMP accumulation, suggesting a role for the cAMP pathway in mediating the immediate actions of VIP on follicular cell survival. Moreover, treatment of rat antral follicles with insulin-like growth factor-binding protein 3 (3 micrograms/ml) partially antagonized the ability of VIP (100 nM) to suppress apoptosis, suggesting involvement of endogenous insulin-like growth factor I in mediating the downstream actions of VIP in incubated rat antral follicles. To further confirm that VIP and activation of the cAMP pathway prevented atresia, individual rat antral follicles incubated for 24 h in the absence or presence of VIP (100 nM) or forskolin (10 microM) were fixed, embedded, and sectioned for morphological analysis. Follicles fixed immediately after isolation from equine CG-primed rat ovaries were classified as morphologically healthy, consistent with the absence of biochemical evidence for apoptosis (e.g. oligonucleosomes) in this pool of follicles. Follicles incubated for 24 h in the absence of tropic support displayed extensive granulosa cell pyknosis and disorganization characteristic of follicles at a moderate stage of atresia. Inclusion of VIP or forskolin maintained the morphological health status of incubated follicles at that resembling healthy follicles fixed immediately after isolation from ovaries of equine CG-primed rats. Lastly, extensive levels of internucleosomal DNA cleavage were also detected in avian granulosa cells incubated for 6 h under serum-free conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Lobe-specific apoptotic cell death in rat prostate after androgen ablation by castration.

It is well established that androgens are central to regulation of the growth of the mammalian prostate gland. Conversely, androgen deprivation by castration induces rapid cell death in the ventral prostate via an apoptotic mechanism. To date, most studies of cell death in the rodent prostate have focused on the ventral lobe, with little attention directed to the dorsal and lateral lobes. The results presented herein demonstrate that cell death in the rat prostate gland caused by castration is lobe specific. In particular, castration caused decreases in wet weights and protein contents of all three prostatic lobes, but these events were more rapid and profound in the ventral than in the dorsal and lateral lobes. Reduced epithelial cell size was apparent in the three lobes as well. However, castration resulted in loss of DNA content in the ventral lobe only. To confirm this finding, and to examine apoptosis of individual cells, we used in situ labeling of fragmented DNA, supported by biochemical analysis of DNA integrity in agarose gels. With both approaches, significant cell death in response to castration was seen in the ventral lobe but not the dorsal and lateral lobes. Taken together, these results clearly indicate that there are lobe-specific differences in the response of the rat prostate to androgen ablation by castration, with apoptotic cell death occurring in the ventral lobe of the prostate but to a far lesser extent, if at all, in the dorsal and lateral lobes. Moreover, castration caused apoptotic death of both epithelial and stromal cells of the ventral prostate, with these cells dying throughout the ductal network of the ventral prostate rather than being restricted to a particular region. We suggest that lobe-specific differences in androgen responsiveness in the rat prostate may provide an appropriate model for the study of androgen-independent prostatic cell survival during tumor progression.

Expression of the p53 and Wilms' tumor suppressor genes in the rat ovary: gonadotropin repression in vivo and immunohistochemical localization of nuclear p53 protein to apoptotic granulosa cells of atretic follicles.

We have previously demonstrated that the gonadotropin-mediated inhibition of apoptosis in ovarian granulosa cells is linked to changes in the expression of several cell death-related genes, including members of the bcl-2 gene family (bcl-2, bax, and bcl-x). Recently, the product of the p53 tumor suppressor gene, a protein reported to play a critical role in regulating cell proliferation and death, has been shown to directly modulate the transcriptional activity of the bcl-2 and bax genes. In addition, the actions of p53 may be amplified through a cooperative interaction with another tumor suppressor protein, the product of the Wilms' tumor suppressor gene (WT-1). Based on our identification of a potential role for bcl-2-related factors in regulating granulosa cell apoptosis and the reported function of p53 as a regulator of bcl-2 and bax gene transcription in extragonadal cells, the present studies were conducted to determine whether the p53 and WT-1 genes are expressed and gonadotropin regulated in the rat ovary and to investigate whether granulosa cell apoptosis is linked to elevated levels of tumor suppressor gene expression. Northern blot analysis of total RNA prepared from immature (27-day-old) rat ovaries revealed the presence of a single p53 messenger RNA (mRNA) transcript (2.0 kilobases) and multiple WT-1 messages (1.8, 3.5, and 7.5 kilobases). Subcutaneous injection of immature rats with 10 IU equine CG (eCG) reduced the levels of p53 and WT-1 mRNA to 71 +/- 9% (P < 0.05) and 46 +/- 3% (P < 0.05), respectively, of saline-treated control levels after 2 days. The inhibition of tumor suppressor gene expression by eCG treatment was associated with a marked reduction in the number of apoptotic granulosa cells and atretic follicles. Furthermore, immunohistochemical analysis revealed that p53 protein was localized exclusively to nuclei of apoptotic granulosa cells of atretic follicles, and that p53 immunostaining was reduced to undetectable levels after in vivo treatment with eCG. To further evaluate whether granulosa cell apoptosis is linked to increased expression of tumor suppressor genes, we analyzed levels of p53 and WT-1 mRNA in antral follicles induced to undergo atresia in vitro by serum-free culture.(ABSTRACT TRUNCATED AT 400 WORDS)