Molecular Reproduction and Development, Jul 13, 2017
The corpus luteum (CL) is a temporary endocrine organ essential for the survival and implantation... more The corpus luteum (CL) is a temporary endocrine organ essential for the survival and implantation of the embryo. For this reason, the development, function and regression of the CL are accurately regulated by various signalling pathways. In this thesis, we studied the involvement of Notch and Wnt/β-catenin pathways in the development and function of the rat CL. We also analysed their interaction with the progesterone action. The first chapter of this thesis was designed to study the action of Notch pathway on rat CL function, and its interaction with progesterone. Immature Sprague-Dawley female rats (21-23 day-old) were subcutaneously (sc) injected with eCG followed by hCG 48 hours later. The animals were euthanized 4 days after hCG administration. CLs were isolated and cultured in the presence of Aminoglutethimide (AG, which is a progesterone synthesis inhibitor), DAPT (which is a Notch pathway inhibitor), progesterone, DAPT in the presence of progesterone, or vehicle solution (DMSO). CLs were collected and the conditioned media were frozen. One or 4 hours after incubation, CL function was studied by measuring progesterone concentration in the conditioned medium. Besides, steroidogenic regulators, apoptotic parameters, PCNA content and AKT and ERK phosphorylation levels were measured in CLs protein extracts. DAPT produced a decrease in progesterone production along with a decrease in P450scc protein levels. The presence of progesterone in the culture medium caused an increase in StAR protein levels, and this effect was blocked by the inhibition of Notch pathway. DAPT produced an increase in pro:anti-apoptotic protein ratio and an increase in the levels of cleaved caspase 3. Both effects were blocked by the presence of progesterone in the culture medium. To study the proliferation of the CL cells, PCNA
The purpose of the present study was to evaluate the in vivo effect of the GnRH analogue leuproli... more 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 eCGtreated 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.
Fisiopatología endócrina. Fascículo XI: Función reproductiva femenina:#R##N#Bases fisiológicas. Fisiopatología y su diagnóstico: Bioquímica y Métodos diagnósticos, 2007
Selección del folículo dominante El reclutamiento cíclico y selección de los folículos representa... more Selección del folículo dominante El reclutamiento cíclico y selección de los folículos representa un proceso continuo, que eventualmente llega a la formación de uno o más folículos preovulatorios, cuyo número varía en cada especie. En los primeros días del ciclo menstrual en la mujer, aumentan los niveles circulantes de FSH, como consecuencia, una cohorte de folículos antrales escapa de la apoptosis que los llevaría a la atresia folicular. Dentro de este grupo, alrededor de 10 folículos antrales crecen más rápido y producen altos niveles de estrógenos e inhibina, seleccionándose entre éstos el folículo dominante. A pesar de que no se conoce exactamente por qué un folículo emerge como dominante, se postula que éste posee una mayor sensibilidad a FSH, debido a una mayor expresión de receptores de FSH y/o LH. Asociado a este proceso, el estradiol y los factores de crecimiento locales ejercen un efecto permisivo, amplificando la acción de la FSH en los folículos que están madurando. Sin embargo, el aumento de estradiol e inhibina, también ejerce una retroalimentación negativa sobre la liberación de FSH a nivel hipotálamo-hipófisis, lo cual evita que otros folículos sigan desarrollándose [1;2]. Además, la disminución de FSH provocaría un descenso en la actividad de la aromatasa dependiente de FSH lo que limita, como consecuencia, la disponibilidad de estrógenos en los folículos menos maduros. Esto llevaría a una disminución en la proliferación de las células de la granulosa y un aumento en los andrógenos, provocando una atresia irreversible. El folículo dominante debe retener una sensibilidad única a la FSH y de esa manera aumentar la proliferación de sus células granulosas, logrando así tener una mayor cantidad de receptores para esta gonadotrofina. De esta manera, los folículos seleccionados tendrán un aumento en los estrógenos mayor que los folículos restantes. Además, estos folículos seleccionados tendrán un incremento en células de la granulosa y una mayor vasculatura tecal, permitiendo una entrada preferencial de la FSH. Por lo tanto, los folículos dominantes no sólo tienen la ventaja de tener mayor número de receptores para FSH, sino que además poseen una gran vasculatura que permite un fácil acceso para esta hormona. En la rata, bajo el estímulo con FSH y en presencia de estrógenos, las células expresan receptores para LH y prolactina, los cuales van aumentando hasta llegar a valores máximos antes de la ovulación [3;4]. El aumento en los estrógenos provoca, por un mecanismo de retroalimentación positiva, la estimulación del pico preovulatorio de LH. Estos procesos, actuando en forma sincronizada, permitirían la selección de los folículos dominantes hasta llegar a la ovulación [1]. Por otro lado, se ha postulado que el folículo dominante produce factores atrésicos que inhiben el desarrollo de los folículos vecinos o subordinados [1;5].
Although advances in the prediction and management of ovarian hyperstimulation syndrome (OHSS) ha... more Although advances in the prediction and management of ovarian hyperstimulation syndrome (OHSS) have been introduced, complete prevention is not yet possible. Previously, we and other authors have shown that vascular endothelial growth factor, angiopoietins (ANGPTs) and sphingosine-1-phosphate are involved in OHSS etiology. In addition, we have demonstrated that ovarian protein levels of platelet-derived growth factor (PDGF) ligands -B and -D decrease in an OHSS rat model, whilst PDGFR-β and ANGPT2 remain unchanged. In the present work, we investigated the role of PDGF-B in OHSS by evaluating ligand protein levels in follicular fluid (FF) from women at risk of developing OHSS and by using an immature rat model of OHSS. We demonstrated that PDGF-B and PDGF-D are lower in FF from women at risk of developing OHSS compared to control patients (P < 0.05). In the OHSS rat model, PDGF-B (0.5 µg/ovary) administration decreased ovarian weight (P < 0.05), reduced serum progesterone (P &l...
Is ceramide-1-phosphate (C1P) an ovarian protective agent during alkylating chemotherapy? SUMMARY... more Is ceramide-1-phosphate (C1P) an ovarian protective agent during alkylating chemotherapy? SUMMARY ANSWER: Local administration of C1P drastically reduces ovarian damage induced by cyclophosphamide (Cy) via protection of follicular reserve, restoration of hormone levels, inhibition of apoptosis and improvement of stromal vasculature, while protecting fertility, oocyte quality and uterine morphology. WHAT IS KNOWN ALREADY: Cancer-directed therapies cause accelerated loss of ovarian reserve and lead to premature ovarian failure (POF). Previous studies have demonstrated that C1P regulates different cellular processes including cell proliferation, cell migration, angiogenesis and apoptosis. This sphingolipid may be capable of modulating vascular development and apoptosis in ovaries affected by chemotherapy. STUDY DESIGN, SIZE, DURATION: The 6-8-week-old mice were weighed and administered either a single intraperitoneal injection of Cy (75 mg/kg) or an equal volume of saline solution only for control mice. Control and Cy mice underwent sham surgery and received an intrabursal injection of saline solution, while Cy + C1P animal groups received 5 μl C1P, either 0.5 or 1 mM, under the bursa of both ovaries 1 h prior to Cy administration.
Are follicular fluid (FF) sphingosine-1-phosphate (S1P) levels in patients at risk of developing ... more Are follicular fluid (FF) sphingosine-1-phosphate (S1P) levels in patients at risk of developing ovarian hyperstimulation syndrome (OHSS) altered and in part responsible for the high vascular permeability observed in these patients. FF S1P levels are lower in FF from patients at risk of OHSS and treatment with S1P may reduce vascular permeability in these patients. Although advances have been made in the diagnosis, and management of OHSS and in basic knowledge of its development, complete prevention has proven difficult. A total of 40 FF aspirates were collected from patients undergoing ART. The women (aged 25-39 years old) were classified into a control group (n = 20) or a group at risk of OHSS (n = 20). The EA.hy926 endothelial cell line was used to assess the efffects of FF from patients at risk of OHSS with or without the addition of S1P. An animal model that develops OHSS in immature Sprague-Dawley rats were also used. Migration assays, confocal microscopy analysis of actin fil...
In this study, we investigated the interaction between the Notch pathway and progesterone to main... more In this study, we investigated the interaction between the Notch pathway and progesterone to maintain the functionality of the corpus luteum (CL). When Notch signaling is activated, the γ-secretase complex releases the active intracellular domains (NICD) of their receptors, which exert survival effects. We designed studies to analyze whether thein vitroinhibition of Notch affects progesterone production, steroidogenic regulators, apoptotic parameters, and signaling transduction pathways in the cultures of CL isolated from pregnant and superovulated rats. We detected a decrease in progesterone production when corpora lutea (CL) were incubated withN-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT), a γ-secretase inhibitor. This effect could be in part due to the decrease detected in the CL protein levels of P450scc because STAR and 3β-hydroxysteroid dehydrogenase were not affected by Notch inhibition. Besides, the addition of aminoglutethimide to the CL cultu...
The Journal of steroid biochemistry and molecular biology, 2014
The relationship between human chorionic gonadotropin and ovarian hyperstimulation syndrome (OHSS... more The relationship between human chorionic gonadotropin and ovarian hyperstimulation syndrome (OHSS) is partially mediated by vascular endothelial growth factor A (VEGF). The aim of this study was to investigate the effects of VEGF inhibition on the development of corpora lutea (CL) and cystic structures, steroidogenesis, apoptosis, cell proliferation, endothelial cell area, VEGF receptors (KDR and Flt-1), claudin-5 and occludin levels in ovaries from an OHSS rat model. The VEGF inhibitor used (VEGF receptor-1 (FLT-1)/Fc chimera, TRAP) decreased the concentrations of progesterone and estradiol as well as the percentage of CL and cystic structures in OHSS rats, and increased apoptosis in CL. Endothelial cell area in CL and KDR expression and its phosphorylation were increased, whereas claudin-5 and occludin levels were decreased in the OHSS compared to the control TRAP reversed these parameters. Our findings indicate that VEGF inhibition prevents the early onset of OHSS and decreases i...
Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of rep... more Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of reproductive age, and is characterized by abnormalities in ovarian angiogenesis, among other features. Consistent with this association, follicular fluid (FF) concentration and ovarian expression of vascular endothelial growth factor (VEGF) are increased in PCOS patients. In this study, we examined the protein levels of platelet-derived growth factor (PDGF) BB and DD (PDGFBB and PDGFDD), angiopoietin 1 and 2 (ANGPT1 and ANGPT2), and their soluble receptor sTIE2 in FF from PCOS and control patients undergoing assisted reproductive techniques. We also analyzed the effect of FF from PCOS and control patients on tight and adherens junction protein expression in an endothelial cell line. PDGFBB and PDGFDD were significantly lower whereas ANGPT1 concentration was significantly higher in FF from PCOS patients than from control patients. No changes were found in the concentration of ANGPT2 or sTIE2. Expression of claudin-5 was significantly increased in endothelial cells incubated for 24 hr in the presence of FF from PCOS versus from control patients, while vascular-endothelial cadherin, b-catenin, and zonula occludens 1 expression were unchanged. The changes observed in the levels of PDGF isoforms and ANGPT1 may prevent VEGF-induced vascular permeability in the PCOS ovary by regulating endothelial-cell-junction protein levels. Restoring the levels of angiogenic factors may provide new insights into PCOS treatment and the prevention of ovarian hyperstimulation syndrome in affected women.
Apoptosis is associated with the regression of the corpus luteum (CL) in many species. Since casp... more Apoptosis is associated with the regression of the corpus luteum (CL) in many species. Since caspases play a central role in apoptosis, we studied several initiators (-2, -8, and -9) and the main effector (-3) caspase in the CL during the estrous cycle of the rat. Two different populations of CL (old and new) were identified on ovaries at estrus and diestrus II (DII). Diminished (P< 0.05) luteal progesterone content and P450scc levels suggested that functional luteolysis occurred between the new CL at DII and old CL at estrus, whereas the decline (P< 0.05) in luteal weight indicated that structural regression was occurring between old CL at estrus to DII. Immunostaining for caspase-2 in luteal and endothelial cells appeared to increase as the luteal phase progressed, peaking at DII in the old CL. However, caspase-8 and -9 immunostaining showed little change with a slight increase at estrus in the old population. Notably, caspase-3 staining appeared to peak at DII in the new CL...
Reproductive success stems from a finely regulated balance between follicular maturation and atre... more Reproductive success stems from a finely regulated balance between follicular maturation and atresia, in which the role of carbohydrate structure is poorly understood. Here, we describe for the first time a fraction of purified recombinant human FSH that is capable of bringing about the cell death of granulosa cells and preventing follicular maturation in a rat model. Further analysis by mass spectrometry revealed the presence of the lectin Concanavalin-A (Con-A) within this fraction of recombinant FSH. Using both the fractionated FSH and Con-A, the observed cell death was predominantly located to the granulosa cells. Ex vivo culture of rat follicles demonstrated that follicle degeneration occurred and resulted in the release of a denuded and deteriorated oocyte. Moreover, in vivo experiments confirmed an increase in atresia and a corresponding reduction confined to follicle in early antral stage. As a mechanism of action, Con-A reduces ovarian proliferation, Von Willebrand staining...
Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of rep... more Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of reproductive age. It is characterized by anovulation, oligo- or amenorrhea, hyperandrogenism, obesity, and insulin resistance. PCOS patients present with elevated levels of vascular endothelial growth factor (VEGF) in serum and follicular fluid. In this study, we examined the ovarian expression of angiopoietins (ANGPT) and their receptor tyrosine kinase receptor (TIE2), involved in the stabilization of blood vessels, in a rat model of dehydroepiandrosterone-induced PCOS. We also analyzed the effect of ovarian VEGF inhibition on ANGPT/TIE2, follicular development, and vascular stability. VEGF levels were increased in the PCOS ovaries, whereas the levels of its receptor fetal liver kinase-1 were decreased. In addition, the periendothelial cell area and the ANGPT1 to ANGPT2 ratio in the ovary were increased in the PCOS group. Percentage of primary follicles was increased and the percentage of ...
Polycystic ovary syndrome (PCOS) is a heterogeneous disease characterized by hyperandrogenemia, h... more Polycystic ovary syndrome (PCOS) is a heterogeneous disease characterized by hyperandrogenemia, hirsutism, oligo-or amenorrhea, insulin resistance and anovulation. The aim of the present study was to evaluate if the balance between the ovarian expression of Bax (proapoptotic protein) and Bcl-2 (antiapoptotic protein) is altered in a PCOS model developed in rats by DHEA administration. In addition, the ovarian morphology and the circulating progesterone levels were evaluated. Histological studies confirmed the presence of follicular cysts, atretic follicles and the absence of corpora lutea in the ovaries from the PCOS group and a significant decrease in circulating progesterone levels. Immunohistochemical studies showed that the expression of Bcl-2 and Bax were mainly localized in granulosa cells of antral follicles in both groups. Bax expression was greater in preantral and antral follicles from PCOS ovarian sections than in Controls. In contrast, intense Bcl-2 immunostaining was observed in Control antral follicles, while Bcl-2 protein was either absent in preantral follicles or weakly expressed in antral follicles from PCOS rats. These results were partially confirmed by western studies. Data revealed that the ovarian level of Bcl-2 protein was lower in PCOS than in Control, and that there were no differences in Bax ovarian levels between groups. However, Bax/ Bcl-2 ratio was significantly higher in PCOS group than in Control group. In conclusion, an increase in ovarian apoptosis through an imbalance among the Bcl-2 family members may be involved in the transformation of growing follicles in cystic follicles in the ovaries from DHEAinduced PCOS rats.
Experiments were designed to investigate the expression and regulation of vascular endothelial gr... more Experiments were designed to investigate the expression and regulation of vascular endothelial growth factor (VEGF) in the primate corpus luteum (CL) throughout the luteal life span in the natural menstrual cycle. Corpora lutea were collected during the early (ECL; Days 3-5 post-LH surge), mid (MCL; Day 6-8 post-LH surge), mid-late (MLCL; Days 10-12 post-LH surge), late (LCL; Days 14-16 post-LH surge), and very late (Days 17-18 post-LH surge) luteal phase. Specific primers were designed to amplify mRNAs encoding VEGF isoforms 206, 189, 183, 165, 145, and 121. Only two cDNA products were obtained by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends; cloning and sequencing confirmed their 98% homology to the corresponding human VEGF 165 and 121 sequences. Semiquantitative RT-PCR assays indicated that VEGF 165 mRNA levels increased (P Ͻ 0.05) from ECL to MLCL but then declined (P Ͻ 0.05) by LCL. Although VEGF 121 mRNA levels were limited in ECL, they increased significantly in MCL (P Ͻ 0.05). Levels of VEGF protein, as measured by Western blot analysis, were two-to fourfold higher for VEGF 165 versus VEGF 121. Also, VEGF 165 levels were higher (P Ͻ 0.05) in ECL and MCL compared to those at later stages. During 2-day culture, preparations of dispersed luteal cells secreted VEGF into the media; the highest levels were observed in ECL and declined (P Ͻ 0.05) by LCL. Regardless of luteal stage, hypoxic conditions increased (P Ͻ 0.05) VEGF levels, whereas LH exposure increased (P Ͻ 0.05) progesterone, but not VEGF, in the media. These results are consistent with a dynamic, local regulation of VEGF production during the life span of the primate CL that is not directly controlled by LH.
Analogs of GnRH, including agonists (GnRH-a) and antagonists (GnRH-ant), have been widely used to... more 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.
The purpose of this study was to evaluate the role of inhibin A in follicular development and apo... more The purpose of this study was to evaluate the role of inhibin A in follicular development and apoptosis-related mechanisms in preantral and early antral follicles from prepubertal diethylstilbestrol (DES)-treated rats. Granulosa cells isolated from the ovaries of 23-to 25-day-old rats were cultured in serum-free medium containing FSH (20 ng/ml), transforming growth factor  (5 ng/ml), and estradiol (50 ng/ml) in the presence or absence of different concentrations of recombinant human inhibin A. 3 H-Thymidine incorporation was decreased in the presence of Inh, but no significant changes were observed in progesterone and estradiol levels in culture medium. An increase in low molecular weight DNA fragmentation indicative of apoptosis and an increase in the levels of Bax protein with no changes in Bcl-2 protein levels were evident in early antral follicles incubated for 24 h with Inh. For each animal, Inh (0.5 g/ovary) was injected intrabursally in one ovary, and the contralateral ovary served as a control. Ovarian histology revealed an inhibitory effect of Inh treatment on the follicular development induced by DES. At 24 h after Inh injection, the number of preantral follicles was increased compared with controls, whereas the number of early antral follicles was decreased. In addition, in vivo Inh treatment caused an increase in the percentage of apoptotic cells in preantral and early antral follicles. These results suggest that inhibin produced by the dominant follicle may act as a paracrine factor inhibiting the growth of neighboring follicles, thus participating in the mechanism of follicular selection.
The aim of the present study was to examine the acute and chronic effects of the gonadotropin-rel... more The aim of the present study was to examine the acute and chronic effects of the gonadotropin-releasing hormone agonist (GnRH-a) leuprolide acetate (LA) on the expression of the steroidogenic acute regulatory protein (StAR), the cytochrome P450 side-chain cleavage enzyme (P450 scc), and steroid production in antral ovarian follicles obtained from prepubertal equine choriogonadotropin (eCG)-treated rats. Follicular contents of StAR and P450 scc proteins were measured by Western blotting following in vivo injection of eCG (control) and eCG؉LA (LA) to prepubertal rats. Treatment with eCG for 2 h resulted in no change in StAR protein content, but it was markedly increased at 4 and 8 h after hormone treatment. However, coadministration of eCG؉LA produced a significant increase (P Ͻ 0.05) in StAR protein levels at 2, 4, and 8 h when compared with eCG treatment. Acute and chronic treatment with either eCG or eCG؉LA did not alter the P450 scc protein levels in freshly isolated follicles. The increase in StAR protein expression following LA treatment was qualitatively similar to StAR mRNA expression, as determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Furthermore, administration of eCG demonstrated a time-dependent increase (2-8 h) in the levels of StAR mRNA, and these levels were markedly increased by eCG؉LA. However, the temporal response pattern of StAR mRNA was much greater at 2 h following LA administration when compared with controls. In addition, 48 h of LA treatment in eCG-treated rats resulted in a significant increase (P Ͻ 0.05) in follicular progesterone levels, whereas significant decreases in androgen (testosterone and androsterone) and estradiol levels were observed. Similar results were obtained when serum androgens and estradiol were measured, but serum progesterone levels were unchanged. Collectively, these findings demonstrate that the inhibitory effect of LA on ovarian androgen and estradiol levels is related to changes in the follicular levels of StAR protein and steroid production.
Studies were designed to examine the expression and activity of four caspases that contribute to ... more Studies were designed to examine the expression and activity of four caspases that contribute to the initial (caspases-2, -8, and -9) and final (caspase-3) events in apoptosis in the rat corpus luteum (CL) during pregnancy ( days 7, 17, 19, and 21 of gestation), postpartum ( days 1 and 4), and after injection (0, 8, 16, 24, and 36 h) of the physiological luteolysin PGF2α. In addition, the temporal relationship of caspase expression/activity relative to steroid production and luteal regression was evaluated. During pregnancy, the activity of all four caspases was significantly greater on day 19, before a decline in CL progesterone (P) and CYP11A1 levels at day 21 of gestation. The levels of the caspase-3 active fragment (p17, measured by Western blot) also increased at days 19 and 21 of pregnancy. Immunohistochemical analyses detected specific staining for the caspases in luteal cells (large and small) as well as in endothelial cells. However, the percentage of apoptotic cells did no...
Molecular Reproduction and Development, Jul 13, 2017
The corpus luteum (CL) is a temporary endocrine organ essential for the survival and implantation... more The corpus luteum (CL) is a temporary endocrine organ essential for the survival and implantation of the embryo. For this reason, the development, function and regression of the CL are accurately regulated by various signalling pathways. In this thesis, we studied the involvement of Notch and Wnt/β-catenin pathways in the development and function of the rat CL. We also analysed their interaction with the progesterone action. The first chapter of this thesis was designed to study the action of Notch pathway on rat CL function, and its interaction with progesterone. Immature Sprague-Dawley female rats (21-23 day-old) were subcutaneously (sc) injected with eCG followed by hCG 48 hours later. The animals were euthanized 4 days after hCG administration. CLs were isolated and cultured in the presence of Aminoglutethimide (AG, which is a progesterone synthesis inhibitor), DAPT (which is a Notch pathway inhibitor), progesterone, DAPT in the presence of progesterone, or vehicle solution (DMSO). CLs were collected and the conditioned media were frozen. One or 4 hours after incubation, CL function was studied by measuring progesterone concentration in the conditioned medium. Besides, steroidogenic regulators, apoptotic parameters, PCNA content and AKT and ERK phosphorylation levels were measured in CLs protein extracts. DAPT produced a decrease in progesterone production along with a decrease in P450scc protein levels. The presence of progesterone in the culture medium caused an increase in StAR protein levels, and this effect was blocked by the inhibition of Notch pathway. DAPT produced an increase in pro:anti-apoptotic protein ratio and an increase in the levels of cleaved caspase 3. Both effects were blocked by the presence of progesterone in the culture medium. To study the proliferation of the CL cells, PCNA
The purpose of the present study was to evaluate the in vivo effect of the GnRH analogue leuproli... more 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 eCGtreated 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.
Fisiopatología endócrina. Fascículo XI: Función reproductiva femenina:#R##N#Bases fisiológicas. Fisiopatología y su diagnóstico: Bioquímica y Métodos diagnósticos, 2007
Selección del folículo dominante El reclutamiento cíclico y selección de los folículos representa... more Selección del folículo dominante El reclutamiento cíclico y selección de los folículos representa un proceso continuo, que eventualmente llega a la formación de uno o más folículos preovulatorios, cuyo número varía en cada especie. En los primeros días del ciclo menstrual en la mujer, aumentan los niveles circulantes de FSH, como consecuencia, una cohorte de folículos antrales escapa de la apoptosis que los llevaría a la atresia folicular. Dentro de este grupo, alrededor de 10 folículos antrales crecen más rápido y producen altos niveles de estrógenos e inhibina, seleccionándose entre éstos el folículo dominante. A pesar de que no se conoce exactamente por qué un folículo emerge como dominante, se postula que éste posee una mayor sensibilidad a FSH, debido a una mayor expresión de receptores de FSH y/o LH. Asociado a este proceso, el estradiol y los factores de crecimiento locales ejercen un efecto permisivo, amplificando la acción de la FSH en los folículos que están madurando. Sin embargo, el aumento de estradiol e inhibina, también ejerce una retroalimentación negativa sobre la liberación de FSH a nivel hipotálamo-hipófisis, lo cual evita que otros folículos sigan desarrollándose [1;2]. Además, la disminución de FSH provocaría un descenso en la actividad de la aromatasa dependiente de FSH lo que limita, como consecuencia, la disponibilidad de estrógenos en los folículos menos maduros. Esto llevaría a una disminución en la proliferación de las células de la granulosa y un aumento en los andrógenos, provocando una atresia irreversible. El folículo dominante debe retener una sensibilidad única a la FSH y de esa manera aumentar la proliferación de sus células granulosas, logrando así tener una mayor cantidad de receptores para esta gonadotrofina. De esta manera, los folículos seleccionados tendrán un aumento en los estrógenos mayor que los folículos restantes. Además, estos folículos seleccionados tendrán un incremento en células de la granulosa y una mayor vasculatura tecal, permitiendo una entrada preferencial de la FSH. Por lo tanto, los folículos dominantes no sólo tienen la ventaja de tener mayor número de receptores para FSH, sino que además poseen una gran vasculatura que permite un fácil acceso para esta hormona. En la rata, bajo el estímulo con FSH y en presencia de estrógenos, las células expresan receptores para LH y prolactina, los cuales van aumentando hasta llegar a valores máximos antes de la ovulación [3;4]. El aumento en los estrógenos provoca, por un mecanismo de retroalimentación positiva, la estimulación del pico preovulatorio de LH. Estos procesos, actuando en forma sincronizada, permitirían la selección de los folículos dominantes hasta llegar a la ovulación [1]. Por otro lado, se ha postulado que el folículo dominante produce factores atrésicos que inhiben el desarrollo de los folículos vecinos o subordinados [1;5].
Although advances in the prediction and management of ovarian hyperstimulation syndrome (OHSS) ha... more Although advances in the prediction and management of ovarian hyperstimulation syndrome (OHSS) have been introduced, complete prevention is not yet possible. Previously, we and other authors have shown that vascular endothelial growth factor, angiopoietins (ANGPTs) and sphingosine-1-phosphate are involved in OHSS etiology. In addition, we have demonstrated that ovarian protein levels of platelet-derived growth factor (PDGF) ligands -B and -D decrease in an OHSS rat model, whilst PDGFR-β and ANGPT2 remain unchanged. In the present work, we investigated the role of PDGF-B in OHSS by evaluating ligand protein levels in follicular fluid (FF) from women at risk of developing OHSS and by using an immature rat model of OHSS. We demonstrated that PDGF-B and PDGF-D are lower in FF from women at risk of developing OHSS compared to control patients (P < 0.05). In the OHSS rat model, PDGF-B (0.5 µg/ovary) administration decreased ovarian weight (P < 0.05), reduced serum progesterone (P &l...
Is ceramide-1-phosphate (C1P) an ovarian protective agent during alkylating chemotherapy? SUMMARY... more Is ceramide-1-phosphate (C1P) an ovarian protective agent during alkylating chemotherapy? SUMMARY ANSWER: Local administration of C1P drastically reduces ovarian damage induced by cyclophosphamide (Cy) via protection of follicular reserve, restoration of hormone levels, inhibition of apoptosis and improvement of stromal vasculature, while protecting fertility, oocyte quality and uterine morphology. WHAT IS KNOWN ALREADY: Cancer-directed therapies cause accelerated loss of ovarian reserve and lead to premature ovarian failure (POF). Previous studies have demonstrated that C1P regulates different cellular processes including cell proliferation, cell migration, angiogenesis and apoptosis. This sphingolipid may be capable of modulating vascular development and apoptosis in ovaries affected by chemotherapy. STUDY DESIGN, SIZE, DURATION: The 6-8-week-old mice were weighed and administered either a single intraperitoneal injection of Cy (75 mg/kg) or an equal volume of saline solution only for control mice. Control and Cy mice underwent sham surgery and received an intrabursal injection of saline solution, while Cy + C1P animal groups received 5 μl C1P, either 0.5 or 1 mM, under the bursa of both ovaries 1 h prior to Cy administration.
Are follicular fluid (FF) sphingosine-1-phosphate (S1P) levels in patients at risk of developing ... more Are follicular fluid (FF) sphingosine-1-phosphate (S1P) levels in patients at risk of developing ovarian hyperstimulation syndrome (OHSS) altered and in part responsible for the high vascular permeability observed in these patients. FF S1P levels are lower in FF from patients at risk of OHSS and treatment with S1P may reduce vascular permeability in these patients. Although advances have been made in the diagnosis, and management of OHSS and in basic knowledge of its development, complete prevention has proven difficult. A total of 40 FF aspirates were collected from patients undergoing ART. The women (aged 25-39 years old) were classified into a control group (n = 20) or a group at risk of OHSS (n = 20). The EA.hy926 endothelial cell line was used to assess the efffects of FF from patients at risk of OHSS with or without the addition of S1P. An animal model that develops OHSS in immature Sprague-Dawley rats were also used. Migration assays, confocal microscopy analysis of actin fil...
In this study, we investigated the interaction between the Notch pathway and progesterone to main... more In this study, we investigated the interaction between the Notch pathway and progesterone to maintain the functionality of the corpus luteum (CL). When Notch signaling is activated, the γ-secretase complex releases the active intracellular domains (NICD) of their receptors, which exert survival effects. We designed studies to analyze whether thein vitroinhibition of Notch affects progesterone production, steroidogenic regulators, apoptotic parameters, and signaling transduction pathways in the cultures of CL isolated from pregnant and superovulated rats. We detected a decrease in progesterone production when corpora lutea (CL) were incubated withN-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT), a γ-secretase inhibitor. This effect could be in part due to the decrease detected in the CL protein levels of P450scc because STAR and 3β-hydroxysteroid dehydrogenase were not affected by Notch inhibition. Besides, the addition of aminoglutethimide to the CL cultu...
The Journal of steroid biochemistry and molecular biology, 2014
The relationship between human chorionic gonadotropin and ovarian hyperstimulation syndrome (OHSS... more The relationship between human chorionic gonadotropin and ovarian hyperstimulation syndrome (OHSS) is partially mediated by vascular endothelial growth factor A (VEGF). The aim of this study was to investigate the effects of VEGF inhibition on the development of corpora lutea (CL) and cystic structures, steroidogenesis, apoptosis, cell proliferation, endothelial cell area, VEGF receptors (KDR and Flt-1), claudin-5 and occludin levels in ovaries from an OHSS rat model. The VEGF inhibitor used (VEGF receptor-1 (FLT-1)/Fc chimera, TRAP) decreased the concentrations of progesterone and estradiol as well as the percentage of CL and cystic structures in OHSS rats, and increased apoptosis in CL. Endothelial cell area in CL and KDR expression and its phosphorylation were increased, whereas claudin-5 and occludin levels were decreased in the OHSS compared to the control TRAP reversed these parameters. Our findings indicate that VEGF inhibition prevents the early onset of OHSS and decreases i...
Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of rep... more Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of reproductive age, and is characterized by abnormalities in ovarian angiogenesis, among other features. Consistent with this association, follicular fluid (FF) concentration and ovarian expression of vascular endothelial growth factor (VEGF) are increased in PCOS patients. In this study, we examined the protein levels of platelet-derived growth factor (PDGF) BB and DD (PDGFBB and PDGFDD), angiopoietin 1 and 2 (ANGPT1 and ANGPT2), and their soluble receptor sTIE2 in FF from PCOS and control patients undergoing assisted reproductive techniques. We also analyzed the effect of FF from PCOS and control patients on tight and adherens junction protein expression in an endothelial cell line. PDGFBB and PDGFDD were significantly lower whereas ANGPT1 concentration was significantly higher in FF from PCOS patients than from control patients. No changes were found in the concentration of ANGPT2 or sTIE2. Expression of claudin-5 was significantly increased in endothelial cells incubated for 24 hr in the presence of FF from PCOS versus from control patients, while vascular-endothelial cadherin, b-catenin, and zonula occludens 1 expression were unchanged. The changes observed in the levels of PDGF isoforms and ANGPT1 may prevent VEGF-induced vascular permeability in the PCOS ovary by regulating endothelial-cell-junction protein levels. Restoring the levels of angiogenic factors may provide new insights into PCOS treatment and the prevention of ovarian hyperstimulation syndrome in affected women.
Apoptosis is associated with the regression of the corpus luteum (CL) in many species. Since casp... more Apoptosis is associated with the regression of the corpus luteum (CL) in many species. Since caspases play a central role in apoptosis, we studied several initiators (-2, -8, and -9) and the main effector (-3) caspase in the CL during the estrous cycle of the rat. Two different populations of CL (old and new) were identified on ovaries at estrus and diestrus II (DII). Diminished (P< 0.05) luteal progesterone content and P450scc levels suggested that functional luteolysis occurred between the new CL at DII and old CL at estrus, whereas the decline (P< 0.05) in luteal weight indicated that structural regression was occurring between old CL at estrus to DII. Immunostaining for caspase-2 in luteal and endothelial cells appeared to increase as the luteal phase progressed, peaking at DII in the old CL. However, caspase-8 and -9 immunostaining showed little change with a slight increase at estrus in the old population. Notably, caspase-3 staining appeared to peak at DII in the new CL...
Reproductive success stems from a finely regulated balance between follicular maturation and atre... more Reproductive success stems from a finely regulated balance between follicular maturation and atresia, in which the role of carbohydrate structure is poorly understood. Here, we describe for the first time a fraction of purified recombinant human FSH that is capable of bringing about the cell death of granulosa cells and preventing follicular maturation in a rat model. Further analysis by mass spectrometry revealed the presence of the lectin Concanavalin-A (Con-A) within this fraction of recombinant FSH. Using both the fractionated FSH and Con-A, the observed cell death was predominantly located to the granulosa cells. Ex vivo culture of rat follicles demonstrated that follicle degeneration occurred and resulted in the release of a denuded and deteriorated oocyte. Moreover, in vivo experiments confirmed an increase in atresia and a corresponding reduction confined to follicle in early antral stage. As a mechanism of action, Con-A reduces ovarian proliferation, Von Willebrand staining...
Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of rep... more Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of reproductive age. It is characterized by anovulation, oligo- or amenorrhea, hyperandrogenism, obesity, and insulin resistance. PCOS patients present with elevated levels of vascular endothelial growth factor (VEGF) in serum and follicular fluid. In this study, we examined the ovarian expression of angiopoietins (ANGPT) and their receptor tyrosine kinase receptor (TIE2), involved in the stabilization of blood vessels, in a rat model of dehydroepiandrosterone-induced PCOS. We also analyzed the effect of ovarian VEGF inhibition on ANGPT/TIE2, follicular development, and vascular stability. VEGF levels were increased in the PCOS ovaries, whereas the levels of its receptor fetal liver kinase-1 were decreased. In addition, the periendothelial cell area and the ANGPT1 to ANGPT2 ratio in the ovary were increased in the PCOS group. Percentage of primary follicles was increased and the percentage of ...
Polycystic ovary syndrome (PCOS) is a heterogeneous disease characterized by hyperandrogenemia, h... more Polycystic ovary syndrome (PCOS) is a heterogeneous disease characterized by hyperandrogenemia, hirsutism, oligo-or amenorrhea, insulin resistance and anovulation. The aim of the present study was to evaluate if the balance between the ovarian expression of Bax (proapoptotic protein) and Bcl-2 (antiapoptotic protein) is altered in a PCOS model developed in rats by DHEA administration. In addition, the ovarian morphology and the circulating progesterone levels were evaluated. Histological studies confirmed the presence of follicular cysts, atretic follicles and the absence of corpora lutea in the ovaries from the PCOS group and a significant decrease in circulating progesterone levels. Immunohistochemical studies showed that the expression of Bcl-2 and Bax were mainly localized in granulosa cells of antral follicles in both groups. Bax expression was greater in preantral and antral follicles from PCOS ovarian sections than in Controls. In contrast, intense Bcl-2 immunostaining was observed in Control antral follicles, while Bcl-2 protein was either absent in preantral follicles or weakly expressed in antral follicles from PCOS rats. These results were partially confirmed by western studies. Data revealed that the ovarian level of Bcl-2 protein was lower in PCOS than in Control, and that there were no differences in Bax ovarian levels between groups. However, Bax/ Bcl-2 ratio was significantly higher in PCOS group than in Control group. In conclusion, an increase in ovarian apoptosis through an imbalance among the Bcl-2 family members may be involved in the transformation of growing follicles in cystic follicles in the ovaries from DHEAinduced PCOS rats.
Experiments were designed to investigate the expression and regulation of vascular endothelial gr... more Experiments were designed to investigate the expression and regulation of vascular endothelial growth factor (VEGF) in the primate corpus luteum (CL) throughout the luteal life span in the natural menstrual cycle. Corpora lutea were collected during the early (ECL; Days 3-5 post-LH surge), mid (MCL; Day 6-8 post-LH surge), mid-late (MLCL; Days 10-12 post-LH surge), late (LCL; Days 14-16 post-LH surge), and very late (Days 17-18 post-LH surge) luteal phase. Specific primers were designed to amplify mRNAs encoding VEGF isoforms 206, 189, 183, 165, 145, and 121. Only two cDNA products were obtained by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends; cloning and sequencing confirmed their 98% homology to the corresponding human VEGF 165 and 121 sequences. Semiquantitative RT-PCR assays indicated that VEGF 165 mRNA levels increased (P Ͻ 0.05) from ECL to MLCL but then declined (P Ͻ 0.05) by LCL. Although VEGF 121 mRNA levels were limited in ECL, they increased significantly in MCL (P Ͻ 0.05). Levels of VEGF protein, as measured by Western blot analysis, were two-to fourfold higher for VEGF 165 versus VEGF 121. Also, VEGF 165 levels were higher (P Ͻ 0.05) in ECL and MCL compared to those at later stages. During 2-day culture, preparations of dispersed luteal cells secreted VEGF into the media; the highest levels were observed in ECL and declined (P Ͻ 0.05) by LCL. Regardless of luteal stage, hypoxic conditions increased (P Ͻ 0.05) VEGF levels, whereas LH exposure increased (P Ͻ 0.05) progesterone, but not VEGF, in the media. These results are consistent with a dynamic, local regulation of VEGF production during the life span of the primate CL that is not directly controlled by LH.
Analogs of GnRH, including agonists (GnRH-a) and antagonists (GnRH-ant), have been widely used to... more 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.
The purpose of this study was to evaluate the role of inhibin A in follicular development and apo... more The purpose of this study was to evaluate the role of inhibin A in follicular development and apoptosis-related mechanisms in preantral and early antral follicles from prepubertal diethylstilbestrol (DES)-treated rats. Granulosa cells isolated from the ovaries of 23-to 25-day-old rats were cultured in serum-free medium containing FSH (20 ng/ml), transforming growth factor  (5 ng/ml), and estradiol (50 ng/ml) in the presence or absence of different concentrations of recombinant human inhibin A. 3 H-Thymidine incorporation was decreased in the presence of Inh, but no significant changes were observed in progesterone and estradiol levels in culture medium. An increase in low molecular weight DNA fragmentation indicative of apoptosis and an increase in the levels of Bax protein with no changes in Bcl-2 protein levels were evident in early antral follicles incubated for 24 h with Inh. For each animal, Inh (0.5 g/ovary) was injected intrabursally in one ovary, and the contralateral ovary served as a control. Ovarian histology revealed an inhibitory effect of Inh treatment on the follicular development induced by DES. At 24 h after Inh injection, the number of preantral follicles was increased compared with controls, whereas the number of early antral follicles was decreased. In addition, in vivo Inh treatment caused an increase in the percentage of apoptotic cells in preantral and early antral follicles. These results suggest that inhibin produced by the dominant follicle may act as a paracrine factor inhibiting the growth of neighboring follicles, thus participating in the mechanism of follicular selection.
The aim of the present study was to examine the acute and chronic effects of the gonadotropin-rel... more The aim of the present study was to examine the acute and chronic effects of the gonadotropin-releasing hormone agonist (GnRH-a) leuprolide acetate (LA) on the expression of the steroidogenic acute regulatory protein (StAR), the cytochrome P450 side-chain cleavage enzyme (P450 scc), and steroid production in antral ovarian follicles obtained from prepubertal equine choriogonadotropin (eCG)-treated rats. Follicular contents of StAR and P450 scc proteins were measured by Western blotting following in vivo injection of eCG (control) and eCG؉LA (LA) to prepubertal rats. Treatment with eCG for 2 h resulted in no change in StAR protein content, but it was markedly increased at 4 and 8 h after hormone treatment. However, coadministration of eCG؉LA produced a significant increase (P Ͻ 0.05) in StAR protein levels at 2, 4, and 8 h when compared with eCG treatment. Acute and chronic treatment with either eCG or eCG؉LA did not alter the P450 scc protein levels in freshly isolated follicles. The increase in StAR protein expression following LA treatment was qualitatively similar to StAR mRNA expression, as determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Furthermore, administration of eCG demonstrated a time-dependent increase (2-8 h) in the levels of StAR mRNA, and these levels were markedly increased by eCG؉LA. However, the temporal response pattern of StAR mRNA was much greater at 2 h following LA administration when compared with controls. In addition, 48 h of LA treatment in eCG-treated rats resulted in a significant increase (P Ͻ 0.05) in follicular progesterone levels, whereas significant decreases in androgen (testosterone and androsterone) and estradiol levels were observed. Similar results were obtained when serum androgens and estradiol were measured, but serum progesterone levels were unchanged. Collectively, these findings demonstrate that the inhibitory effect of LA on ovarian androgen and estradiol levels is related to changes in the follicular levels of StAR protein and steroid production.
Studies were designed to examine the expression and activity of four caspases that contribute to ... more Studies were designed to examine the expression and activity of four caspases that contribute to the initial (caspases-2, -8, and -9) and final (caspase-3) events in apoptosis in the rat corpus luteum (CL) during pregnancy ( days 7, 17, 19, and 21 of gestation), postpartum ( days 1 and 4), and after injection (0, 8, 16, 24, and 36 h) of the physiological luteolysin PGF2α. In addition, the temporal relationship of caspase expression/activity relative to steroid production and luteal regression was evaluated. During pregnancy, the activity of all four caspases was significantly greater on day 19, before a decline in CL progesterone (P) and CYP11A1 levels at day 21 of gestation. The levels of the caspase-3 active fragment (p17, measured by Western blot) also increased at days 19 and 21 of pregnancy. Immunohistochemical analyses detected specific staining for the caspases in luteal cells (large and small) as well as in endothelial cells. However, the percentage of apoptotic cells did no...
Uploads
Papers by Marta Tesone