Luteal Cell Apoptosis Research Articles

Endoplasmic reticulum stress-mediated apoptotic pathway is involved in corpus luteum regression in rats.

Endoplasmic reticulum stress (ERS), which is a novel pathway of regulating cellular apoptosis and the function of ERS during corpus luteum (CL) regression, is explored. Early-luteal stage (day 2), mid-luteal stage (day 7), and late-luteal stage (day 14 and 20) were induced, and the apoptosis of luteal cells was detected by a terminal 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL) assay. The apoptotic cells were increased with the regression of CL, especially during the late-luteal stage. The ERS markers glucose-regulated protein 78 (Grp78), CCAAT/enhancer-binding protein homologous protein (CHOP), X-box binding protein 1 (XBP1), activating transcription factor 6α (ATF6α), eukaryotic initiation factor 2α (eIF2α), inositol-requiring protein 1α (IRE1α), caspase 12, and apoptosis marker caspase 3 were analyzed by real-time polymerase chain reaction (PCR) and immunohistochemistry, in agreement with the results of the TUNEL assay; the expression levels of CHOP, caspase 12, and caspase 3 were increased during the process of CL regression. Luteal cells were isolated and cultured in vitro, and the apoptosis of luteal cells was induced by prostaglandin F2α. The ERS was attenuated by the ERS inhibitor tauroursodeoxycholic acid, and the apoptotic rate was analyzed by flow cytometry. The ERS markers Grp78, CHOP, XBP1s, ATF6α, eIF2α, IRE1α, caspase 12, and apoptotic execute marker caspase 3 were analyzed by real-time PCR and immunofluorescence, and the results suggested that the expression of CHOP, caspase 12, and caspase 3 were increased, and there was increased apoptosis of luteal cells. But the expression of IRE1α/XBP1s and eIF2α was not detected. Taken together, the ERS is involved in the CL regression of rats through the CHOP and caspase 12 pathway.

Swainsonine induces caprine luteal cells apoptosis via mitochondrial-mediated caspase-dependent pathway.

Swainsonine (SW) is an indolizidine alkaloid isolated from a number of poisonous plants. We have previously reported that SW inhibited luteal cell progesterone production by inducing caprine luteal cell apoptosis in vitro; however, the molecular mechanism of this phenomenon remains unclear. In this study, SW-treated luteal cells showed apoptosis characteristics, including nuclear fragmentation, DNA ladder formation, and phosphatidylserine externalization. Further studies showed that SW activated caspase-9 and caspase-3, which subsequently cleaved poly(ADP-ribose) polymerase. SW also increased in Bax/BcL-2 ratios, promoted Bax translocation from the cytosol to mitochondria, and triggered the release of cytochrome c from mitochondria into the cytoplasm. However, Fas and Fas ligand induction or caspase-8 activity did not appear any significant changes. Additional analysis also showed that pan-caspase inhibitor, caspase-9 inhibitor, or caspase-3 inhibitor almost completely protected the cells from SW-induced apoptosis, but not caspase-8 inhibitor. Overall, these data demonstrated that SW induced luteal cells apoptosis through a mitochondrial-mediated caspase-dependent pathway.

Expression of Aldo-keto Reductase 1C23 in the Equine Corpus Luteum in Different Luteal Phases

Regression of the corpus luteum (CL) is characterized by a decay in progesterone (P4) production (functional luteolysis) and disappearance of luteal tissues (structural luteolysis). In mares, structural luteolysis is thought to be caused by apoptosis of luteal cells, but functional luteolysis is poorly understood. 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes P4 into its biologically inactive form, 20α-hydroxyprogesterone (20α-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20α-HSD activity. To clarify whether AKR1C23 is associated with functional luteolysis in mares, we investigated the expression of AKR1C23 in the CL in different luteal phases. The luteal P4 concentration and levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA were higher in the mid luteal phase than in the late and regressed luteal phases (P<0.05), but the level of 3β-HSD protein was higher in the late luteal phase than in the regressed luteal phase (P<0.05). The luteal 20α-OHP concentration and the level of AKR1C23 mRNA were higher in the late luteal phase than in the early and mid luteal phases (P<0.05), and the level of AKR1C23 protein was also highest in the late luteal phase. Taken together, these findings suggest that metabolism of P4 by AKR1C23 is one of the processes contributing to functional luteolysis in mares.

Effects of Hypo‐ and Hyperthyroidism on Proliferation, Angiogenesis, Apoptosis and Expression of COX‐2 in the Corpus Luteum of Female Rats

Although thyroid dysfunction occurs frequently in humans and some animal species, the mechanisms by which hypo- and hyperthyroidism affect the corpus luteum have not been thoroughly elucidated. This study evaluated the levels of proliferative activity, angiogenesis, apoptosis and expression of cyclooxygenase-2 in the corpus luteum of female rats with thyroid dysfunction. These processes may be important in understanding the reproductive changes caused by thyroid dysfunction. A total of 18 adult female rats were divided into three groups (control, hypothyroid and hyperthyroid) with six animals per group. Three months after treatment to induce thyroid dysfunction, the rats were euthanized in the dioestrus phase. The ovaries were collected and immunohistochemically analysed for expression of the cell proliferation marker CDC-47, vascular endothelial growth factor (VEGF), VEGF receptor Flk-1 and cyclooxygenase-2 (COX-2). Apoptosis was evaluated using the TUNEL assay. Hypothyroidism reduced the intensity and area of COX-2 expression in the corpus luteum (p<0.05), while hyperthyroidism did not alter COX-2 expression in the dioestrus phase. Hypothyroidism significantly reduced the expression of CDC-47 in endothelial cells and pericytes in the corpus luteum, whereas hyperthyroidism did not induce a detectable change in CDC-47 expression (p>0.05). Hypothyroidism reduced the level of apoptosis in luteal cells (p<0.05) and increased VEGF expression in the corpus luteum. In contrast, hyperthyroidism increased the level of apoptosis in the corpus luteum (p<0.05). In conclusion, thyroid dysfunction differentially affects the levels of proliferative activity, angiogenesis and apoptosis and COX-2 expression in the corpus luteum of female rats.

Transforming Growth Factor Beta 1 Stimulates Profibrotic Activities of Luteal Fibroblasts in Cows1

Luteolysis is characterized by angioregression, luteal cell apoptosis, and remodeling of the extracellular matrix characterized by deposition of collagen 1. Transforming growth factor beta 1 (TGFB1) is a potent mediator of wound healing and fibrotic processes through stimulation of the synthesis of extracellular matrix components. We hypothesized that TGFB1 stimulates profibrotic activities of luteal fibroblasts. We examined the actions of TGFB1 on luteal fibroblast proliferation, extracellular matrix production, floating gel contraction, and chemotaxis. Fibroblasts were isolated from the bovine corpus luteum. Western blot analysis showed that luteal fibroblasts expressed collagen 1 and prolyl 4-hydroxylase but did not express markers of endothelial or steroidogenic cells. Treatment of fibroblasts with TGFB1 stimulated the phosphorylation of SMAD2 and SMAD3. [3H]thymidine incorporation studies showed that TGFB1 caused concentration-dependent reductions in DNA synthesis in luteal fibroblasts and significantly (P < 0.05) reduced the proliferative effect of FGF2 and fetal calf serum. However, TGFB1 did not reduce the viability of luteal fibroblasts. Treatment of luteal fibroblasts with TGFB1 induced the expression of laminin, collagen 1, and matrix metalloproteinase 1 as determined by Western blot analysis and gelatin zymography of conditioned medium. TGFB1 increased the chemotaxis of luteal fibroblasts toward fibronectin in a transwell system. Furthermore, TGFB1 increased the fibroblast-mediated contraction of floating bovine collagen 1 gels. These results suggest that TGFB1 contributes to the structural regression of the corpus luteum by stimulating luteal fibroblasts to remodel and contract the extracellular matrix.

Alterations of Circadian Clockworks During Differentiation and Apoptosis of Rat Ovarian Cells

Ovarian development is related to cell proliferation, differentiation, and apoptosis of granulosa cells and luteal cells under the control of various modulators, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and growth factors. In the present study, the expression of clock genes and the related regulation mechanism were analyzed in different ovarian cell types during differentiation and apoptosis. The authors focused on the circadian expression of Per2 as a core clock gene for the maintenance of circadian rhythms. By using a real-time monitoring system of the Per2 promoter activity, the circadian oscillation was analyzed in the granulosa and luteal cells from preantral follicles, antral follicles, and corpora lutea of immature Per2 promoter–destabilized luciferase transgenic rats that were primed with diethylstilbestrol, equine chorionic gonadotropin (eCG), and/or human CG. In addition, transcript levels of Per2, Bmal1, Clock, and Nampt were quantified by quantitative polymerase chain reaction (qPCR). Immunohistochemical studies revealed strong circadian rhythmicity of PER2 protein in the luteal cells, but apparently little rhythmicity in granulosa cells of both preantral and antral follicles. In vitro monitoring of promoter activity showed generation of several oscillations in luteal cells after exposure to dexamethasone (DXM), whereas oscillatory amplitudes of immature and mature granulosa cells were rapidly attenuating. The circadian rhythm of the Bmal1 transcript levels, but not the Per2 transcript, was very weak in the granulosa cells, as compared with that in luteal cells. Granulosa cells gained a strong circadian rhythm ability of the Per2 promoter activity after stimulation with FSH for 3 days. In contrast, LH had little effect on the circadian rhythm before stimulation of granulosa cells with FSH, probably owing to lack of LH receptor. In luteal cells, induction of apoptosis by inhibiting progesterone synthesis resulted in deregulation of Per2 circadian oscillation. Transcript levels of Bmal1 and Clock, but not Per2 and Nampt, were significantly decreased in apoptotic luteal cells. The Bmal1 transcript level was particularly reduced. Consequently, these results strongly suggest the circadian clockwork alters in ovarian cells during follicular development, luteinization, and apoptosis, and expression of Bmal1 may be related to the switch-on and switch-off of the circadian oscillation. (Author correspondence: mhattori@agr.kyushu-u.ac.jp)

Microarray analysis of differentially expressed microRNAs in non-regressed and regressed bovine corpus luteum tissue; microRNA-378 may suppress luteal cell apoptosis by targeting the interferon gamma receptor 1 gene

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules that down-regulate the expression of target genes in a sequence-dependent manner. Recent studies indicated that miRNAs are mechanistically involved in the regulation of the mammalian corpus luteum (CL). However, few studies have profiled the different miRNA expression patterns in bovine non-regressed and regressed CL. In this study, miRNA microarray was employed to investigate the different miRNA expression patterns in bovine CL. Among the 13 differentially expressed miRNAs, seven were preferentially expressed in non-regressed CL, while six miRNAs were more highly expressed in regressed CL. Real-time RT-PCR was used to validate the microarray results. Mir-378 miRNA, known to be associated with apoptosis, was 8.54-fold (P < 0.01) up-regulated in non-regressed CL, and the interferon gamma receptor 1 (IFNGR1) gene, which potentially plays a role in apoptosis of the luteal cell, was predicted to be the target of mir-378. The results of real-time RT-PCR of mir-378 and western blot analysis of the IFNGR1 protein at different stages of CL development showed that mir-378 decreased the expression of IFNGR1 protein but not IFNGR1 mRNA. Taken together, our data support a direct role for miRNA in apoptosis of bovine CL.

The Role of Autophagy in Corpus Luteum Regression in the Rat

Autophagy is associated with luteal cells death during regression of the corpus luteum (CL) in some species. However, the involvement of autophagy or the association between autophagy and apoptosis in CL regression are largely unknown. Therefore, we investigated the role of autophagy in CL regression and its association with apoptosis. Ovaries were obtained from pseudopregnant rats at Days 2 (early), 7 (mid-), and 14 and 20 (late-luteal stage) of the pseudopregnancy; autophagy-associated protein (microtuble-associated protein light chain 3 [LC3]) was immunolocalized and its expression level was measured. Luteal cell apoptosis was evaluated by measuring cleaved caspase 3 expression. LC3 expression increased slightly from early to mid-luteal stage, with maximal levels detected at the late-luteal stage in steroidogenic luteal cells. The expression level of the membrane form of LC3 (LC3-II) also increased during luteal stage progression, and reached a maximum at the end point of late-luteal stage (Day 20). This pattern coincided with cleaved caspase 3 expression. Furthermore, LC3-II expression increased, as did levels of cleaved caspase 3 in luteal cells cultured with prostaglandin F(2alpha) known to induce CL regression. These findings suggest that luteal cell autophagy is directly involved in CL regression, and is correlated with increased apoptosis. In addition, autophagic processes were inhibited using 3-methyladenine or bafilomycin A1 to evaluate the role of autophagy in apoptosis induction. Inhibition of autophagosome degradation by fusion with lysosomes (bafilomycin A1) increased apoptosis and cell death. Furthermore, inhibition of autophagosome formation (3-methyladenine) decreased apoptosis and cell death, suggesting that the accumulation of autophagosomes induces luteal cell apoptosis. In conclusion, these results indicate that autophagy is involved in rat luteal cell death through apoptosis, and is most prominent during CL regression.

Nitric Oxide and Luteal Blood Flow in the Luteolytic Cascade in the Cow

The corpus luteum (CL) of the estrous cycle in the cow is a dynamic organ which has a lifespan of approximately 17-18 days. The main function of the CL is to produce progesterone (P) that requires for achievement and maintenance of pregnancy. If pregnancy does not occur successfully, the CL must regress within a few days to induce the next chance of ovulation. As the CL matures, the steroidogenic cells establish contact with many capillary vessels, consequently the CL is composed of a large number of vascular endothelial cells that can account for up to 50% of all cells. Therefore, blood vessels and blood flow within the CL have an essential role in luteal function. Nitric oxide (NO), a strong vasorelaxant, is now known to play key roles in a variety of physiological process. Indeed, NO has established itself as a polyvalent molecule which plays a decisive role in regulating multiple functions within female reproductive system. In the CL, NO is produced and regulates luteal blood flow, P secretion and apoptosis of luteal cells as well as endothelial cells. This review describes the current investigation for possible roles of NO in the luteolytic cascade within the bovine CL.

Effect of Cytokines and Steroid Hormones on Nitric Oxide Generating System in Bovine Luteal Endothelial Cells.

Injection of a luteolytic dose of prostaglandin (PG) F2α induces an acute increase in plasma concentration of nitric oxide (NO) metabolites (nitrite/nitrate). Furthermore, NO suppresses progesterone (P4) production and induces apoptosis in bovine luteal cells. These findings suggest that NO plays important roles in luteolysis. However, the regulation of NO generating system in bovine CL is not fully understood. Although tumor necrosis factor α (TNF) and interferon γ (IFNG) produced by immune cells have been demonstrated stimulate NO production in extragonadal cells, the roles of TNF and IFNG in the regulation of NO production, NO synthase (NOS) expression and NOS activity in the bovine corpus luteum (CL) remain unclear. To elucidate whether NO generating system in luteal endothelial cells (LECs) is regulated by cytokines, the effects of TNF or IFNG on the expression of NOS (inducible NO synthase; iNOS and endothelial NO synthase; eNOS) mRNA, protein and NO production were examined. In addition, the direct effects of estrogen (E2) and P4 on NOS proteins and NOS activity were examined. LECs were obtained from mid CL (Days 8-12 of the estrous cycle) using enzymatic digestion and magnetic beads coated with lectin BS-1. Cultured bovine LECs were treated for 24 h with TNF (2.9 nM), IFNG (2.5 nM), E2 (10 nM) or P4 (1 μM). Both TNF and IFNG stimulated iNOS mRNA, protein expression and NO production. Furthermore, E2 stimulatediNOS protein expression, whereas P4 inhibited iNOS protein expression. In contrast to iNOS, eNOS protein expression was not affected by treatments with TNF, IFNG, E2 or P4. By observing the conversion of [3C]L-arginine to [3C]L-citrulline, we found that TNF, IFNG and E2 stimulated the NOS activity in cultured LECs (P<0.05). The overall results demonstrate that TNF, IFNG and E2 accelerate NO production by stimulating iNOS protein expression as well as NOS activity. On the other hand, P4 was able to suppress iNOS expression in LECs. This research was supported by JSPS Grants (No. 19580326 and No. 18380166), and by a research fund from the Livestock Technology Association (LTA), Japan. (poster)

Vascular dynamics in relation to immunolocalisation of VEGF-A, VEGFR-2 and Ang-2 in the bovine corpus luteum

Vascular dynamics during development and regression of the bovine corpus luteum were investigated morphometrically in relation to immunolocalisation of vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor receptor 2 (VEGFR-2) and angiopoietin 2 (Ang-2) in the different cell types. Angiogenesis and remodelling of the capillary network between day 8 and 17 of the oestrous cycle was driven by luteal cells, which were highly immunopositive for VEGF-A. Thereafter, immunoreactivity for VEGF-A and VEGFR-2 was mainly found in vascular smooth muscle cells, the predominant cell type in late luteolysis. During early regression, apoptosis of luteal and endothelial cells was closely correlated, resulting in a significant decrease of capillarity. In late regression, an increase in capillary density was found, suggesting that regression and transformation into the corpus albicans requires adequate perfusion. In the phases of vascular remodelling, i.e. mature and late regressing corpus luteum, high scores of Ang-2-immunopositive endothelial and smooth muscle cells were found. Therefore, it may be hypothesised that Ang-2 supports the angiogenic effects of VEGF-A in these luteal stages. Results emphasise that precise staging and a differentiated view on the cellular populations is important in evaluating the controlled regression and transformation of the corpus luteum.

Anti-Apoptotic Roles of Prostaglandin E2 and F2alpha in Bovine Luteal Steroidogenic Cells1

Production of prostaglandins (PGs) and expression of their receptors have been demonstrated in bovine corpus luteum (CL). The aim of the present study was to determine whether PGE2 and PGF2alpha have roles in bovine luteal steroidogenic cell (LSC) apoptosis. Cultured bovine LSCs obtained at the midluteal stage (Days 8-12 of the cycle) were treated for 24 h with PGE2 (0.001-1 microM) and PGF2alpha (0.001-1 microM). Prostaglandin E2 (1 microM) and PGF2alpha (1 microM) significantly stimulated progesterone (P4) production and reduced the levels of cell death in the cells cultured with or without tumor necrosis factor alpha (TNF)/interferon gamma (IFNG), in the presence and absence of FAS ligand (P < 0.05). Furthermore, DNA fragmentation induced by TNF/IFNG was observed to be suppressed by PGE2 and PGF2alpha. Prostaglandin E2 and PGF2alpha also attenuated mRNA expression of caspase 3 and caspase 8, as well as caspase 3 activity (P < 0.05) in TNF/IFNG-treated cells. FAS mRNA and protein expression were decreased only by PGF2alpha (P < 0.05). A specific P4 receptor antagonist (onapristone) attenuated the apoptosis-inhibitory effects of PGE2 and PGF2alpha in the absence of TNF/IFNG (P < 0.05). A PG synthesis inhibitor (indomethacin) reduced cell viability in PGE2- and PGF2alpha-treated cells (P < 0.05). A specific inhibitor of cyclooxygenase (PTGS), PTGS2 (NS-398), also reduced cell viability, whereas an inhibitor of PTGS1 (FR122047) did not affect it. The overall results suggest that PGE2 and PGF2alpha locally play luteoprotective roles in bovine CL by suppressing apoptosis of LSCs.

Cortisol Is a Suppressor of Apoptosis in Bovine Corpus Luteum1

Glucocorticoid (GC) acts as a modulator of physiological functions in several organs. In the present study, we examined whether GC suppresses luteolysis in bovine corpus luteum (CL). Cortisol (an active GC) reduced the mRNA expression of caspase 8 (CASP8) and caspase 3 (CASP3) and reduced the enzymatic activity of CASP3 and cell death induced by tumor necrosis factor (TNF) and interferon gamma (IFNG) in cultured bovine luteal cells. mRNAs and proteins of GC receptor (NR3C1), 11beta-hydroxysteroid dehydrogenase type 1 (HSD11B1), and HSD11B2 were expressed in CL throughout the estrous cycle. Moreover, the protein expression and the enzymatic activity of HSD11B1 were high at the early and the midluteal stages compared to the regressed luteal stage. These results suggest that cortisol suppresses TNF-IFNG-induced apoptosis in vitro by reducing apoptosis signals via CASP8 and CASP3 in bovine CL and that the local increase in cortisol production resulting from increased HSD11B1 at the early and midluteal stages helps to maintain CL function by suppressing apoptosis of luteal cells.

Apoptosis en la atresia folicular y la regresión del cuerpo lúteo. Revisión

This review analyzes the primary molecules involved in survival and in apoptotic death of antral follicle and corpus luteum (CL) cells. Gonadotropins, estrogens and growth factors such as IGF-I and VEGF, must be present in the follicular environment for follicles to remain healthy. While progesterone, LH and the growth factors mentioned above are vital for CL cells and for ovarian cell apoptosis. A large number of molecules participate in these processes, particularly: Fas/FasL and other members of the tumor necrosis factor (TNF) family and their ligands, pro-apoptotic members of the Bcl-2 protein family (Bax, Bok, Bim, Bad and Bid), cytochrome c, apoptosis-inducing factor (AIF), apoptotic protease activating factor 1 (Apaf-1), p53, initiator caspases (1, 8 and 9), effector caspases (3, 6 and 7), Smac/Diablo and the inhibitor of apoptosis proteins (IAPs). A follicular environment with low survival factor levels increases Fas expression and its interaction with ligand FasL in follicular cells, which results in apoptotic death, however, this inadequate environment can also cause the release of pro-apoptotic proteins from the mitochondrion. Apoptosis in luteal cells is induced by PGF2-a, which increases the expression of pro-apoptotic members such as Bax and death receptors (RTNF-alpha,,,).

Hypoxia Promotes Luteal Cell Death in Bovine Corpus Luteum1

Low oxygen caused by a decreasing blood supply is known to induce various responses of cells, including apoptosis. The present study was conducted to examine whether low-oxygen conditions (hypoxia) induce luteal cell apoptosis in cattle. Bovine midluteal cells incubated under hypoxia (3% O(2)) showed significantly more cell death than did those incubated under normoxia (20% O(2)) at 24 and 48 h of culture, and had significantly lower progesterone (P4) levels starting at 8 h. Characteristic features of apoptosis, such as shrunken nuclei and DNA fragmentation, were observed in cells cultured under hypoxia for 48 h. Hypoxia increased the mRNA expressions of BNIP3 and caspase 3 at 24 and 48 h of culture. Hypoxia had no significant effect on the expressions of BCL2 and BAX mRNA. Hypoxia also increased BNIP3 protein, and activated caspase-3. Treatment of P4 attenuated cell death, caspase-3 mRNA expression, and caspase-3 activity under hypoxia. Overall results of the present study indicate that hypoxia induces luteal cell apoptosis by enhancing the expression of proapoptotic protein, BNIP3, and by activating caspase-3, and that the induction of apoptosis by hypoxia is partially caused by a decrease in P4 production. Because hypoxia suppresses P4 synthesis in bovine luteal cells, we suggest that oxygen deficiency caused by a decreasing blood supply in bovine corpus luteum is one of the major factors contributing to both functional and structural luteolysis.

Forkhead transcription factor FOXO subfamily is essential for reactive oxygen species-induced apoptosis

Intracellular accumulation of reactive oxygen species is implicated in the pathogenesis of cancer and other diseases by disturbing proper cell cycle control or cell survival. Here, we show that the expression and phosphorylation of FOXO is drastically affected by H 2O 2 treatment, resulting in drastic induction of luteal cell apoptosis. Western blot analysis revealed that FOXO1a accumulated preferentially in the nucleus upon ROS stimuli, resulting in the transactivation of IRS promoter activity driven by H 2O 2-activated FOXO1a. Because ROS-induced cell death was suppressed by co-transfection of a FOXO3a mutant that lacks the activation-domain of transcription, transactivation of pro-apoptotic genes by FOXO was necessary to cause ROS-induced apoptosis. In fact, expression of several pro-apoptotic genes, such as Bim and BCL-6 was induced in H 2O 2-stimulated cells, and was blocked by co-transfection of dominant-negative type FOXO3a mutant. These findings indicate that FOXO is a key regulator of ROS-induced apoptosis in mammalian cells.

Expression and localisation of Bcl-2 and Bax proteins in developing rat ovary

Bcl-2 and Bax proteins localised mainly in granulosa cells. Primordial and primary follicles of new born rat ovary showed an intensive nuclear staining for Bax but faint staining for Bcl-2. In terms of staining intensity, no remarkable difference was observed within the same stage of developing follicle. Compared to new born rats, granulosa cells of adult and one month old rat ovary showed an increased staining both for Bcl-2 and Bax proteins. No staining was observed in primordial follicles of one month old and adult rats. However, granulosa cells of primary follicles, granulosa cells and theca cells in tertiary follicles of adult rat ovary also showed a strong staining for Bcl-2 and Bax proteins. Oocytes of follicles from different developmental stages revealed an apparent staining both for Bcl-2 and Bax proteins. However, in the more mature follicles oocytes stained more intensively. In developing corpus luteum a remarkable staining was observed for Bax. However, the staining was more prominent in regressing corpus luteum. Contrary to this, Bcl-2 stained the luteal cells in developing corpus luteum strongly, while in the fully developed corpus luteum no staining for Bcl-2 was observed. In conclusion, there was an apparent relation between the expression of the apoptosis regulating protein Bcl-2 and Bax and follicular development. Thus, during the follicular development Bcl-2 and Bax may be involved in granulosa cell demise in rat ovary. Furthermore, increased levels of Bax and decreased levels of Bcl-2 in the fully developed corpus luteum suggest that Bax plays a role in apoptosis of luteal cells in rat ovary.

Species-Related Differences in the Mechanism of Apoptosis During Structural Luteolysis

Luteolysis is defined as the loss of function and subsequent involution of the luteal structure. The luteolytic process is usually subdivided, whereby the decline in progesterone is described as functional luteolysis and the structural involution is described as structural luteolysis. After the corpus luteum ceases to produce progesterone, it decreases in size, experiences a loss of cellular integrity, and then disappears from the ovary as a result of apoptosis of luteal cells. However, the control mechanisms responsible for initiating and mediating apoptosis during structural luteolysis seem more complex than originally envisioned. Furthermore, efforts to elucidate the apoptotic mechanisms have been complicated by the fact that different mammalian species have different mechanisms for controlling luteal function. Therefore, it is of interest to know whether different mammalian species have different apoptotic mechanisms. The goal of this review was to focus on species-related differences in the mechanism of apoptosis during structural luteolysis in rodents, cattle and humans, the species that are used most for luteolysis research.

Endotoxin induces luteal cell apoptosis through the mitochondrial pathway

The effect of endotoxin (lipopolysacharide, LPS) exposure on luteal cells was studied using an in vitro cell culture system. Buffalo luteal cells were isolated from corpora lutea of the late luteal phase (days 14–16 post estrus) and exposed to various LPS doses (5, 10 and 100 μg/ml) for different time periods (6, 12, 18 or 24 h). The cultured cells were subsequently evaluated for oxidative stress (super oxide, nitric oxide, inducible nitric oxide synthase activity, reduced glutathione depletion and lipid peroxidation) and apoptotic markers (mitochondrial membrane potential, DNA fragmentation, apoptotic cells and cell viability). LPS exposure significantly increased the production of super oxide ( P < 0.05) and nitric oxide ( P < 0.01) and increased inducible nitric oxide synthase activity ( P < 0.01). LPS exposure further depleted reduced glutathione ( P < 0.05) levels and induced lipid peroxidation ( P < 0.05). LPS exposure also induced the loss of mitochondrial membrane potential ( P < 0.05), increased DNA fragmentation ( P < 0.01) and apoptosis ( P < 0.01) and decreased cell viability ( P < 0.01). LPS mediated apoptotic pathway in luteal cells was further characterized using a selected LPS dose (10 μg/ml). It was observed that LPS exposure induced mitochondrial translocation of proapoptotic protein Bax, increased the total Bad expression and down regulated the expression of antiapoptotic proteins Bcl2 and BclXL. LPS exposure further induced cytochrome c release and increased Caspase-9 ( P < 0.01) and Caspase-3 ( P < 0.01) activities. LPS exposure also inhibited luteal progesterone secretion ( P < 0.01). It was evident that the LPS mediated apoptotic effects could be prevented by the coincubation of luteal cells with mitochondrial permeability transition pore blocker Cyclosporine A, inducible nitric oxide synthase inhibitor N-[3-(aminomethyl)benzyl]acetamidine and oxidative stress scavenger N-acetyl cysteine. Our study clearly indicates that LPS induces oxidative stress mediated apoptosis in luteal cells through the mitochondrial pathway.

O-204: Angiotensin II (Ang II) regulates apoptosis in human granulosa-lutein cells (GL)

The human ovary has a complete and functional gonadotrophin-sensitive renin-angiotensin system and both types of AngII receptors, AT1 and AT2, are present in ovarian cells. We have shown that AngII is present in high concentrations in granulosa cells and follicular fluid of human preovulatory follicles and in luteal cells. AngII regulates ovulation and modulates steroidogenesis in rat luteal cells and human GL cells. Other investigators have shown that AngII regulates luteal cell apoptosis. Recently, we have shown that apoptosis of human GL cells correlates with several indicators of IVF outcome, such as embryo fragmentation and pregnancy rate, and is regulated by intraovarian growth factors. The aim of the present study was to test whether AngII could induce in vitro apoptosis of gonadotrophin-exposed human IVF GL cells. Following AngII exposure, apoptosis was assessed by caspace expression. Controls included the non-specific angiotensin receptor blocker Saralasin and the specific AT2 receptor (AT2-R) blocker CGP-42112A. GL cells from 87 IVF-intra-cytoplasmic sperm injection (ICSI) cycles were analyzed. Ovarian stimulation was performed using recombinant FSH and LH or HMG. The pregnancy rate for these cycles was 56.32%. Following oocyte recovery GL cells were collected from pooled follicular fluid (FF), washed and purified using a 50% percoll gradient and anti-CD45-coated magnetic beads. Cells were cultured without serum for 48 hours in the presence or absence of 10-11, 10-9, 10-7M AngII, with and without Saralasin10-5 or CGP-42112A 10-5M. The percentage of apoptotic cells was determined under a fluorescence microscope by staining with 10μm caspace FITV-VAD-FMK (a fluorescent marker for activated caspaces) and 0.5μg/ml propidium iodide. Statistical analysis was carried out using the student-t test. AngII in concentrations present in human follicle fluid caused increased apoptosis of GL cells compared to untreated control cells (10-11M p=0.312; 10-9M p<0,05; 10-7M p<0,05, respectively). This effect was partially inhibited by both Saralasin and CGP-42112A (18.15% and 33.01%, respectively, p<0,05). In these preliminary studies on human GL cells Ang II at concentrations that are present in FF from human IVF cycles (J Soc Gynecol Investig. 1994 1:118-27) is proapoptotic. The partial blockade by angiotensin receptor antagonists is consistent with a receptor-mediated action. Ovarian AII has been linked to fertility, steroidogenesis and polycystic ovarian syndrome. Further studies are underway to determine the relevance of these results to the outcome of IVF cycles and other clinical scenarios.