Gonadotropin-releasing hormone antagonist antide inhibits apoptosis of preovulatory follicle cells in rat ovary

Analogs of GnRH, including agonists (GnRH-a) and antagonists (GnRH-ant), have been widely used to inhibit gonadotropin pituitary release. Aside from the effect of GnRH analogs on the pituitary-gonadal axis, studies have shown that GnRH has extrapituitary effects, particularly on rat and human ovaries. In the present study, we evaluated the direct in vivo effects of the GnRH-a, leuprolide acetate (LA), or the GnRH-ant, Antide (Ant), either singly or together, on ovarian follicular development in prepubertal eCG-treated rats. LA significantly decreased ovarian weight, whereas Ant increased ovarian weight compared with controls; however, coinjection of both compounds had no effect. In addition, LA increased the number of preantral follicles (PFs) and atretic follicles, and decreased the number of early antral follicles (EAFs) and preovulatory follicles (POFs). Coinjection of Ant interfered with this LA effect. Ant alone increased the number of POFs compared with that of controls. Analysis of apoptosis has shown that LA increases the percentage of apoptotic cells in PFs, EAFs, and POFs; however, Ant prevented this effect. In addition, Ant alone decreased the percentage of apoptotic cells in EAFs and POFs. Data have shown that Ant per se inhibited BAX translocation from cytosol to mitochondria and retained cytochrome C in the mitochondria, whereas LA induced cytochrome C release. We conclude that Ant inhibits apoptosis in preovulatory follicles through a decrease of BAX translocation to mitochondria, suggesting that GnRH may act as a physiological intraovarian modulator factor that is able to interfere with follicular development through an increase in apoptotic events mediated by an imbalance among the BCL-2 family members.     

Inhibition of cytochrome P-450 C17 enzyme by a GnRH agonist in ovarian follicles from gonadotropin-stimulated rats

Our objective was to study the direct action of a GnRH-I agonist, leuprolide acetate (LA), on ovarian steroidogenesis in preovulatory follicles obtained from equine chorionic gonadotropin (eCG)-treated rats. Previously, we have demonstrated an inhibitory effect of LA on steroidogenesis and follicular development. In this study, we tested the hypothesis that gonadotropin-releasing hormone (GnRH) exerts its negative effect on follicular development by inhibiting thecal cytochrome P-450 C17 (P450C17) alpha-hydroxylase expression and, consequently, androgen synthesis. Studies were carried out in prepubertal female rats injected with either eCG (control) or eCG plus LA (LA) and killed at different time points. Immunohistochemical studies indicated that LA induced steroidogenic acute regulatory protein (StAR) expression mainly in theca cells of preantral and antral follicles. In addition, serum progesterone levels increased significantly (P < 0.05), whereas those of androsterone decreased (P < 0.05) after 8 h of LA treatment. This inhibition caused by LA seemed to be a consequence of the decreased expression of follicular P450C17 alpha-hydroxylase, as demonstrated by Western blot and RT-PCR techniques. In vitro studies using follicles isolated from 48-h-eCG-treated rats and cultured with LA showed a significant (P < 0.05) inhibition of FSH-induced androsterone follicular content as well as P450C17 alpha-hydroxylase protein levels, as determined by Western analysis. However, LA increased StAR protein expression in these follicles without significant changes in P450scc enzyme levels. Taking all these findings into account, we suggest that GnRH-I exerts a direct inhibitory action on gonadotropin-induced follicular development by decreasing the temporal expression of the P450C17 enzyme and, consequently, androgen production, thus reducing the supply of estrogens available to developing follicles.     

Gonadotropin-releasing hormone agonist affects rat ovarian follicle development by interfering with FSH and growth factors on the prevention of apoptosis

Apoptosis is the biological process by which follicular cells are eliminated in atretic follicles. The aim of the present study was to examine the in vitro effect of a GnRH-a (leuprolide acetate, LA) and its interactions with FSH, dibutyryl cAMP, and growth factors (IGF-I, EGF, and FGF) on follicular apoptosis in early antral ovarian follicles obtained from prepubertal DES- treated rats. Follicles cultured 24 hr in the absence of hormones showed spontaneous onset of apoptotic DNA fragmentation. The presence of FSH suppressed the spontaneous onset of apoptotic DNA fragmentation (75-85%). Quantitative estimation of DNA cleavage from ovarian follicles revealed no significant changes in DNA fragmentation after in vitro LA treatment (1-100 ng/ml). However, coincubation with LA interfered partially with the effects of FSH on apoptosis suppression. This apoptosis suppression was also obtained by treatment with dibutyryl cAMP (80%), and was partially prevented by the presence of LA in the cultures. Follicles were cultured 24 hr with FGF, EGF, or IGF-I, and these factors suppressed DNA fragmentation (70, 60, and 70% respectively), while the presence of LA (100 ng/ml) in the culture medium prevented this effect. In conclusion, we show that the rescue from apoptotic DNA fragmentation produced in early antral follicles by FSH, cAMP, and growth factors, is prevented by coincubation with LA. This GnRH analog would thus interfere in the pathway of FSH, cAMP and/or growth factors by an as yet unknown mechanism.     

Expression of VEGF-A,Flt-1, and Flk-1 in the arterial endothelial cells of the uterine broad ligament throughout the estrous cycle

The aim of the present study was to determine the immunoreactivity of vascular endothelial growth factor (VEGF-A) and its two receptors, viz., Flt-1 (fms-like tyrosine kinase) and Flk-1 (fetal liver kinase), on the surface of endothelial cells of the uterine artery and its branches and of the arcuate arteries in the area of the uterine broad ligament during various phases of the estrous cycle in the pig. We also investigated their expression to determine whether this was phase-related. The highest immunoreactivity for VEGF-A was observed in the uterine artery and arcuate arteries at the early luteal phase and in the branches of the uterine artery during the follicular phase of the estrous cycle. The strongest immunostaining intensity of Flt-1 was found in the uterine artery and its branches at the follicular phase and in arcuate arteries at the mid-luteal phase, whereas Flk-1 immunostaining was at its highest in the uterine artery at the mid-luteal phase and in the branches of the uterine artery and arcuate arteries at the follicular phase. Additionally, VEGF-A expression was assessed by semi-quantitative Western blot analysis, which revealed significantly higher levels of VEGF-A protein during the early luteal and the follicular phase of the estrous cycle (P < 0.001). The phase-related differences in the immunoreactivity and expression of VEGF-A and VEGF receptors suggest that these factors are hormone-dependent during the estrous cycle in the pig.     

Effect of staurosporine-induced apoptosis on endothelial nitric oxide synthase in transfected COS-7 cells and primary endothelial cells

Nitric oxide (NO) may block apoptosis by inhibiting caspases via S-nitrosylation of cysteines. Here, we investigated whether effector caspases might cleave and thereby inhibit endothelial nitric oxide synthase (eNOS). Exposure of eNOS-transfected COS-7 cells and bovine aortic endothelial cells to staurosporine resulted in significant loss of 135-kDa eNOS protein and activity, and appearance of a 60-kDa eNOS fragment; effects were inhibited by the general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp[OMe]-fluoromethyl ketone (zVAD-fmk). In eNOS-transfected COS-7 cells, staurosporine-induced activation of caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage coincided with increased eNOS degradation and decreased activity. Loss of eNOS activity was greater than the degree of proteolysis. Incubation of immunoprecipitated eNOS with caspase-3, caspase-6 or caspase-7 resulted in eNOS cleavage. Staurosporine, a general protein kinase inhibitor, also reduced phosphorylation and decreased calmodulin binding, an effect that may explain the reduction in activity. eNOS, therefore, is both an inhibitor of apoptosis and a target of apoptosis-associated proteolysis.     

Enhancement of neoangiogenesis and follicle survival by sphingosine-1-phosphate in human ovarian tissue xenotransplants

Ovarian transplantation is one of the key approaches to restoring fertility in women who became menopausal as a result of cancer treatments. A major limitation of human ovarian transplants is massive follicular loss during revascularization. Here we investigated whether sphingosine-1-phosphate or its receptor agonists could enhance neoangiogenesis and follicle survival in ovarian transplants in a xenograft model. Human ovarian tissue xenografts in severe-combined-immunodeficient mice were treated with sphingosine-1-phosphate, its analogs, or vehicle for 1-10 days. We found that sphingosine-1-phosphate treatment increased vascular density in ovarian transplants significantly whereas FTY720 and SEW2871 had the opposite effect. In addition, sphingosine-1-phosphate accelerated the angiogenic process compared to vehicle-treated controls. Furthermore, sphingosine-1-phosphate treatment was associated with a significant proliferation of ovarian stromal cell as well as reduced necrosis and tissue hypoxia compared to the vehicle-treated controls. This resulted in a significantly lower percentage of apoptotic follicles in sphingosine-1-phosphate-treated transplants. We conclude that while sphingosine-1-phosphate promotes neoangiogenesis in ovarian transplants and reduces ischemic reperfusion injury, sphingosine-1-phosphate receptor agonists appear to functionally antagonize this process. Sphingosine-1-phosphate holds great promise to clinically enhance the survival and longevity of human autologous ovarian transplants.     

Effects of a gonadotropin-releasing hormone agonist on rat ovarian follicle apoptosis: regulation by epidermal growth factor and the expression of Bcl-2-related genes

The purpose of the present study was to evaluate the in vivo effect of the GnRH analogue leuprolide acetate (LA) on follicular development and apoptosis-related mechanisms in preovulatory ovarian follicles (POF) obtained from prepubertal eCG-treated rats. Serum progesterone and estradiol levels were measured, and a significant decrease in circulating estradiol levels was observed in the LA group, whereas serum progesterone levels remained unchanged. Ovarian histology revealed an inhibitory effect of LA treatment on the follicular development induced by eCG. After 48 h of LA treatment, the numbers of atretic and preantral follicles were increased as compared with controls, whereas the number of antral follicles had decreased. Cells undergoing DNA fragmentation were quantified by performing in situ 3' end labeling of DNA with digoxygenin-dUTP on ovarian sections. LA treatment caused an increase in the percentage of apoptotic cells in preantral and antral follicles. DNA isolated from these POF incubated 24 h in serum-free medium exhibited the typical apoptotic DNA degradation pattern. Treatment of follicles with epidermal growth factor (EGF) suppressed the spontaneous onset of DNA fragmentation, and a similar effect was observed in LA follicles. POF obtained from LA-treated rats showed no changes in Bcl-2 or Bax protein levels. However, a reduction in the Bcl-xL:Bcl-xS ratio was observed, with a greater decrease in Bcl-xL compared with Bcl-xS during the incubation, suggesting a lower stability of the Bcl-xL isoform in the LA group. These results indicate that in vivo GnRH agonist treatment produces an increase in the apoptosis process in POF from eCG-treated rats, and this effect is reversed in vitro by EGF. This GnRH analogue also reduced the stability of the Bcl-xL protein, thus interfering with follicular development by an as yet unknown mechanism.     

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.     

Carotene cleavage activity in bovine ovarian follicles

Carotene cleavage activity has been demonstrated in bovine ovarian follicles by incubating cell-free homogenates from granulosa cells with 15, 15'-(14)C-beta-carotene. Enzyme activity was highly dependent on the quality of follicles studied. Highest cleavage activity was found in large, preovulatory follicles. The correlation between follicular fluid concentration of vitamin A and the conversion rate obtained with the corresponding enzyme preparations was significantly positive. No correlation, however, could be shown between cleavage activity and beta-carotene, alpha-tocopherol or cholesterol. The only form of vitamin A in follicular fluid of cattle was found to be retinol. The results support the hypothesis that a local conversion of beta-carotene into vitamin A in follicular structures is responsible for the increase of the intrafollicular concentration of vitamin A during follicular development.     

Caspase-2 involvement during ionizing radiation-induced oocyte death in the mouse ovary

In mammals, the pool of primordial follicles at birth is determinant for female fertility. Exposure to IR during oogonia proliferation and the diplotene stages of ovarian development induced the virtual disappearance of primordial follicles in the postnatal ovary, while half the follicular reserve remained present after irradiation during the zygotene/pachytene stages. This sensitivity difference was correlated with the level of caspase-2 expression evaluated by immunohistochemistry. At the diplotene stage, Western blot and caspase activity analysis revealed that caspase-2 was activated 2 h after irradiation and a significant increase in the number of oocytes expressing cleaved caspase-9 and -3 occurred 6 h after treatment. Inhibition of caspase-2 activity prevented the cleavage of caspase-9 and partially prevented the loss of oocytes in response to irradiation. Taken together, our results show that caspase-2-dependent activation of the mitochondrial apoptotic pathway is one of the mechanisms involved in the genotoxic stress-induced depletion of the primordial follicle pool.     

In vivo and in vitro activation of caspase-8 and -3 associated with Helicobacter pylori infection

In vivo and in vitro studies have shown an increase in apoptosis in gastric epithelial cells in persons infected with Helicobacter pylori. H. pylori-induced activation of caspase-8 and -3 was evaluated using a human gastric adenocarcinoma cell line (AGS) and gastric tissue from humans and monkeys colonized with H. pylori. The enzymatic activity of caspase-8 was detected only in AGS cells exposed to H. pylori up to 24 h. The active form of caspase-8 was present by Western blot after exposure to H. pylori for 3 h and persisted through 24 h. Caspase-3 activity was present in AGS cells exposed to H. pylori for 3 h, reaching a maximum after 24 h (a sevenfold increase in activity). Caspase-8-mediated cleavage of procaspase-3 generated a 20-kDa band (indicative of the presence of active caspase-3) present only in AGS cells exposed to H. pylori. Active caspase-3 staining was markedly increased in gastric mucosa from infected persons and animals, compared to uninfected controls by immunohistochemistry. Stimulation of downstream events leading to apoptosis, such as the cleavage of PARP (poly adenosine-diphosphate-ribose polymerase) and DFF45 (DNA fragmentation factor 45) as a result of activation of caspase-3, was evaluated. PARP was cleaved, resulting in the presence of both an 89- and a 24-kDa band along with DFF45, resulting in the presence of 10- and 12-kDa bands only in gastric cells exposed to H. pylori. Our data show that H. pylori stimulates the activation of caspases and downstream mediators of caspase-induced apoptosis. This suggests that H. pylori-induced apoptosis is mediated through caspase pathways, which include the activation of caspase-8 and subsequent cleavage and activation of caspase-3. This is consistent with caspase-3 activation that was found in the gastric mucosa of humans and monkeys infected with H. pylori.     

Regulation of follicular luteinization by a gonadotropin-releasing hormone agonist: Relationship between steroidogenesis and apoptosis

The purpose of this study was to evaluate the effects of GnRH-analog (Leuprolide acetate, LA) administration on follicular luteinization in equine chorionic gonadotropin plus human chorionic gonadotropin (eCG + hCG)-superovulated prepubertal treated rats. Results indicate that LA treatment decreases circulating levels of progesterone (P) and P accumulation in collagenase-dispersed ovarian cell cultures, though estradiol(E₂) production is increased. These data suggest that cells from the LA group may be less luteinized following gonadotropin treatment. Studies performed on histological ovarian sections after different times of eCG administration showed that LA injections produce lower amounts of corpora lutea and antral follicles, and a greater number of atretic and preantral follicles. The basal and LH-stimulated P and progestagen accumulations are decreased in incubations of corpora lutea isolated from the LA group. In addition, the mitochondrial cholesterol side-chain cleavage (P450_SCC) levels in corpora lutea from LA-treated rats are reduced, indicating that the decrease in P production observed is due in part to an alteration in the steroidogenic luteal capability. Immunocytochemical localization of nuclei exhibiting DNA fragmentation by the technique of terminal deoxynucleotidyl transferase end-labeling showed that LA treatment causes an increase in the number of apoptotic cells in preantral and antral follicles at all times studied (1, 2, 4, or 7 days of LA administration). A similar effect, though less pronounced, was observed in corpora lutea. It is concluded that LA treatment produces a failure in the steroidogenic luteal capability and an increase of apoptotic mechanisms in the ovary, producing as a consequence an interference in the follicular recruitment, growth, and luteinization induced by gonadotropins. Mol. Reprod. Dev. 51:287–294, 1998. © 1998 Wiley-Liss, Inc.     

Combined application of camptothecin and the guanylate cyclase activator YC-1: Impact on cell death and apoptosis-related proteins in ovarian carcinoma cell lines

Camptothecin analogs and guanylate cyclase activator YC-1 [3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole] have been shown to induce apoptosis in cancer cells. However, the combined effect of camptothecin analogs and YC-1 on the viability of epithelial ovarian cancer cells remains uncertain. We assessed the combined effect of YC-1 on the camptothecin toxicity in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. Camptothecin and YC-1 induced apoptosis in OVCAR-3 and SK-OV-3 cells in a dose- and time-dependent manner. Both compounds induced nuclear damage, decreased Bid and Bcl-2 protein levels, enhanced cytochrome c release, activated caspase-3 and upregulated tumor suppressor p53. Camptothecin decreased Bax protein levels, whereas YC-1 increased Bax levels. YC-1 enhanced the camptothecin-induced changes in the apoptotic protein levels and increased apoptotic effect of camptothecin on ovarian carcinoma cell lines. The results suggested that YC-1 may enhance a camptothecin toxicity against ovarian carcinoma cell lines by increasing activation of the caspase-8 and Bid pathway as well as activation of the mitochondria-mediated apoptotic pathway, leading to cytochrome c release and subsequent caspase-3 activation. Combination of camptothecin analogs and YC-1 may provide a therapeutic benefit against ovarian adenocarcinoma.     

MicroRNA-205 affects mouse granulosa cell apoptosis and estradiol synthesis by targeting CREB1

MicroRNA-205 (miR-205) is involved in various physiological and pathological processes, but its biological function in follicular atresia remains unclear. In this study, we investigated miR-205 expression in mouse granulosa cells (mGCs) and analyzed its functions in primary mGCs by performing a series of in vitro experiments. Quantitative real-time polymerase chain reaction showed that miR-205 expression was significantly higher in early atretic follicles and progressively atretic follicles than in healthy follicles. miR-205 overexpression in mGCs significantly promoted apoptosis and caspase-3/9 activities, as well as inhibited estrogen (E2) release and cytochrome P450 family 19 subfamily A polypeptide 1 (CYP19A1, a key gene in E2 production) expression. Bioinformatics and luciferase reporter assays revealed that the gene encoding cyclic AMP response element (CRE)-binding protein 1 (CREB1) was a direct target of miR-205 in mGCs. CREB1 upregulation partially rescued the effects of miR-205 on apoptosis, caspase-3/9 activities, E2 production, and CYP19A1 expression on mGCs. These results indicate that miR-205 might play an important role in ovarian follicular development and provide new insights into follicular atresia     

Poly (ADP-ribose) polymerase cleavage monitored in situ in apoptotic cells

During apoptosis, the activation of a family of cysteine proteases, or caspases, results in proteolytic cleavage of numerous substrates. Antibody probes specific for neoepitopes on protein fragments generated by caspase cleavage provide a means to monitor caspase activity at the level of the individual cell. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a well-known substrate for caspase-3 cleavage during apoptosis. Its cleavage is considered to be a hallmark of apoptosis. Here, we demonstrate that an affinity-purified polyclonal antibody to the p85 fragment of PARP is specific for apoptotic cells. Western blots show that the antibody recognizes the 85-kDa (p85) fragment of PARP but not full-length PARP. We demonstrate a time course of PARP cleavage and DNA fragmentation in situ using the PARP p85 fragment antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in Jurkat cells treated with anti-Fas. Furthermore, our results indicate that the p85 fragment of PARP resulting from caspase cleavage during apoptosis is rapidly localized outside the condensed chromatin but not in the cytoplasm.     

SIRT6 deficiency causes ovarian hypoplasia by affecting Plod1-related collagen formation

SIRT6 is a key member of the mammalian sirtuin family of conserved nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylases. Previous studies have shown that SIRT6 can regulate metabolism, DNA damage repair and aging. Ovarian aging process usually share similar mechanisms with general aging, which is characterized by decreases in both numbers of ovarian follicles and the quality of oocytes. It is reported that the expression level of SIRT6 was significantly decreased in the ovaries of aged mice, and the level of SIRT6 was positively correlated with ovarian reserve, indicating that SIRT6 may be potential markers of ovarian aging. However, its biological roles in follicular development are still unclear. Here, we explored the effect of SIRT6 on follicular development and found that ovarian development was interrupted in SIRT6 knockout (KO) mice, leading to disruptions of puberty and the estrus cycle, significant decreases in numbers of secondary and antral follicles, and decreased collagen in the ovarian stroma. Plod1, a lysyl hydroxylase that is vital for collagen crosslinking and deposition, was decreased at both the mRNA and protein levels in SIRT6-deficient ovaries and granulosa cells (GCs). Additionally, we found abnormal estrogen levels in both SIRT6 KO mice and SIRT6 KD GCs, accompanied by decreases in the levels of the estrogen biosynthesis genes Cyp11a1, Cyp19a1, Mgarp, and increases in the levels of TNF-α and NF-κB. These results confirmed the effect of SIRT6 on follicular development and revealed a possible molecular mechanism for SIRT6 involvement in follicular development via effects on estrogen biosynthesis and collagen formation.     

Heteromeric complex formation of ASK2 and ASK1 regulates stress-induced signaling

Apoptosis signal-regulating kinase 2 (ASK2) is an interaction partner of the highly related ASK1. Here, we describe a regulatory function of ASK2 in stress signaling-induced cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Increased cleavage of caspase-3 and PARP was demonstrated by overexpression as well as knockdown of ASK2 after stress-induction by serum-starvation. We show that ectopically expressed ASK2 homo-oligomerized while endogenous ASK2 and ASK1 formed hetero-oligomers, which decreased upon serum-starvation. Co-expression of ASK2 and ASK1 stabilized these two proteins and reduced starvation-induced caspase-3 activation and degradation of PARP. Analysis of the intracellular localization of ASK2 exhibited a similar localization compared with ASK1 in the nucleus, cytoplasm, and in mitochondria. We propose that ASK2 regulates stress-induced caspase-3 and PARP cleavage in a dose-dependent manner by heteromeric complex formation with ASK1.