Kim I. Tilly

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.

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.

Expression of superoxide dismutase, catalase and glutathione peroxidase in the bovine corpus luteum: evidence supporting a role for oxidative stress in luteolysis.

Apoptosis, a type of physiological or active cell death, has been implicated as a mechanism underlying regression of the corpus luteum (CL) in the rat, bovine, rabbit and ovine ovary. Previousin vitro studies of cultured luteal cells have also provided evidence which suggests that reactive oxygen species play an important role in luteolysis in the rodent ovary. To further evaluate the potential role of oxidative stress in luteal cell demise, changes in the expression of several enzymes known to protect cells from oxidative stress were investigated using bovine CL collected from ovaries of non-pregnant (day 21 of the estrous cycle; regressed CL) and pregnant (day 21 of pregnancy; functional CL) animals. Biochemical analysis of genomic DNA extracted from these two pools of CL demonstrated the presence of extensive levels of internucleosomal DNA cleavage characteristic of cell death via apoptosis in regressed, but not in functional, CL. Northern blot analysis of total RNA indicated that functional CL expressed significantly higher levels of mRNA encoding secreted superoxide dismutase (SEC-SOD, 1.9 kb) and manganese-containing or mitochondrial SOD (Mn-SOD, multiple transcripts) as compared to regressed CL. Similarly, levels of mRNA encoding catalase (2.1 kb), an enzyme responsible for detoxification of peroxides to water, were significantly higher in functional versus regressed CL. From these data, we conclude that a decline in expression of specific oxidative response genes occurs during luteolysis, and that maintained expression of these genes in the CL during pregnancy may prevent oxidative damage and delay regression.

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 pr...

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

Abstract.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.

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 μM), 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.

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 μg/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.

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-1β-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.

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, adreno-corticotropin (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.

Dissecting the Functions, Mechanisms, and Genes of Physiological Cell Death in Reproductive Tissues: An Overview

Physiological cell death, a process referred to most often as apoptosis or programmed cell death, is fundamental to almost every aspect of normal tissue development, function, and homeostasis in multicellular organisms (1–3). In reproductive physiology, cell death plays a fundamental role in phenotypic sex determination during embryogenesis, and maintains important functions throughout postnatal life in the gonads, reproductive tract, and accessory reproductive tissues of both males and females. For instance, controlled cellular deletion has been characterized during Mullerian duct regression in males (Chapter 6) (4) and Wolffian duct regression in females (5), ovarian germ cell endowment and depletion (Chapters 2, 3, and 8–11) (6–8), spermatogenesis (Chapter 16) (9), early blastocyst development (Chapter 4) and embryo implantation (Chapter 5), parturition (Chapter 7), and luteolysis (Chapters 13 and 14). Furthermore, physiological cell death serves to balance cellular replication and maintain general homeostasis or cyclicity of nongonadal reproductive tissues such as the prostate (Chapter 17) (10), uterus (11, 12), and breast (Chapters 12 and 17) (13).