RESUMO
PURPOSE: We hypothesized that the chemokine SDF1/CXCR4 system was present in feline cumulus-oocyte complexes (COCs) and that COCs cultured with SDF1 would directly upregulate gene expression in the ovulatory cascade. METHODS: Ovaries (n = 50) were obtained from adult domestic cats during the breeding season and COCs were recovered from antral follicles. Because IVM media triggers cumulus-oocyte expansion, culture conditions needed to be optimized to study periovulatory genes. After optimization, the effects of 25 ng/ml SDF1 and the CXCR4 inhibitor were examined in a COC culture for 3, 12, and 24 h. RESULTS: MEM-hepes with 1% of charcoal stripped-FBS was the optimized culture medium, assessed by the expansion of COCs at 24 h in the gonadotropin (GNT) group but not in the media with serum alone. The mRNA expression of HAS2, TNFAIP6, PTX3, and AREG peaked at 3 h in GNT group as compared to all other groups (p < 0.05). COCs cultured with SDF1 showed increased HAS2 and TNFAIP6 mRNA expression at 3 h compared to negative controls and to the CXCR4 inhibitor group. CXCR4 and SDF1 immunostaining was present in both cumulus cells and the oocyte. CONCLUSIONS: These results demonstrate that GNT stimulation upregulates key periovulatory genes and expansion in feline COCs from antral follicles, and support the use of this culture system to examine molecular processes within the COC. In addition, SDF1 directly promotes key periovulatory genes in feline COCs, suggesting that the SDF1-CXCR4 pathway may extend its function beyond a chemoattractant, and may play a direct role within the COC.
Assuntos
Quimiocina CXCL12/metabolismo , Células do Cúmulo/fisiologia , Regulação da Expressão Gênica , Oócitos/fisiologia , Ovulação/genética , Animais , Gatos , Técnicas de Cultura de Células/métodos , Quimiocina CXCL12/farmacologia , Meios de Cultura/química , Meios de Cultura/farmacologia , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Gonadotropinas/farmacologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Receptores CXCR4/metabolismoRESUMO
PURPOSE: The current study was designed to evaluate the response of individual intact antral follicles from adult female domestic cats to a luteinizing hormone (LH) stimulus in vitro by assessing cumulus-oocyte expansion (C-OE) and steroid production. METHODS: C-OE and steroid levels (estradiol [E2] and progesterone [P4]) obtained from individual antral feline follicles (n = 366 follicles; n = 56 cats) were analyzed after 12 or 24 h of culture in the presence or absence of LH (low [3.4 ng/ml] or high [100 ng/ml]). RESULTS: At the end of the culture, the highest percentage of expanded cumulus-oocyte complexes (COCs) was observed in the LH groups at 12 or 24 h in comparison to their controls (p < 0.001). There was a significant increase in expanded COCs when comparing LH concentrations (high vs. low) at 12 or 24 h. Higher levels of both E2 and P4 were observed in the media from antral follicles after 12 and 24 h of culture in the presence of LH (both concentration, p < 0.05). There was no association between hormone levels and follicle diameter; high variability was observed in the steroid levels produced by antral follicles within all treatment groups. CONCLUSIONS: These data indicate, for the first time, that feline antral follicles (0.5-2 mm) from different stages of the natural estrous cycle can be cultured and will respond to an LH stimulus, based on an increase in steroid levels as well as C-OE after 12 or 24 h in culture.
Assuntos
Folículo Ovariano/fisiologia , Ovário/fisiologia , Animais , Gatos , Células Cultivadas , Estradiol/metabolismo , Feminino , Hormônio Luteinizante/farmacologia , Oócitos/citologia , Oócitos/fisiologia , Folículo Ovariano/efeitos dos fármacos , Ovário/citologia , Progesterona/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
PURPOSE: The small antral follicles (SAFs) from the ovarian medulla can be a potential source of oocytes for infertility patients, but little is known about their ability to yield mature oocytes. This study evaluated the response of these SAFs to a stimulatory bolus of human corionic gonadotropin (hCG) in vitro. METHODS: Oocyte nuclear maturation and hormone production (estradiol [E2], progesterone [P4]), antimullerian hormone [AMH]) by individual intact SAFs (n = 91; >0.5 mm; n = 5 monkeys) was evaluated after 34 h of culture in the absence (control) or presence of hCG. RESULTS: Of the total cohort (n = 91), 49 % of SAFs contained degenerating oocytes. The percentage of healthy oocytes able to reinitiate meiosis to the metaphase I (MI) and MII was greater (p < 0.05) after hCG compared to controls. E2, P4 and AMH levels were higher (p < 0.05) in SAF cultures containing germinal vesicle (GV) oocytes compared to those with MII oocytes regardless of hCG exposure. SAF with MI oocytes produced more E2, but less (p < 0.05) P4 and AMH compared to SAFs containing GV oocytes (p < 0.05). Follicles ≥1 mm produced more (p < 0.05) E2, whereas follicle diameter did not correlate with P4 or AMH levels. Only P4 increased (p < 0.05) in response to hCG, regardless of follicle size or oocyte maturity. SAFs containing degenerating oocytes produced similar levels of E2, P4 and AMH compared to SAFs containing healthy oocytes. CONCLUSIONS: These data indicate, for the first time, that oocytes within primate SAFs can reinitiate meiosis in vitro in the absence of hCG, but nuclear maturation is enhanced in SAFs cultured with hCG. Oocyte nuclear maturation within SAFs in is associated with decreased E2, P4 and AMH levels. Furthermore, hormone content within the culture media does not necessarily reflect oocyte quality.
Assuntos
Técnicas de Maturação in Vitro de Oócitos , Macaca mulatta/crescimento & desenvolvimento , Oócitos/citologia , Folículo Ovariano/citologia , Animais , Hormônio Antimülleriano/metabolismo , Estradiol/metabolismo , Feminino , Fertilização in vitro , Gonadotropinas/metabolismo , Humanos , Meiose , Oócitos/crescimento & desenvolvimento , Oogênese/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Gravidez , Progesterona/metabolismoRESUMO
BACKGROUND: In non-primates, the epidermal growth factor (EGF) and EGF-related ligands such as amphiregulin (AREG) serve as critical intermediates between the theca/mural cells and the cumulus-oocyte-complex (COC) following the mid-cycle LH surge. Studies were designed in primates (1) to analyze AREG levels in follicular fluid (follicular fluid) obtained from pre-ovulatory follicles, as well as (2) to assess dose-dependent effects of AREG on oocytes from small antral follicles (SAFs) during culture, including meiotic and cytoplasmic maturation. METHODS: Controlled ovulation protocols were performed on rhesus monkeys (n=12) to determine AREG content within the single, naturally selected dominant follicle after an ovulatory stimulus. Using healthy COCs (n=271) obtained from SAFs during spontaneous cycles (n=27), in vitro maturation (IVM) was performed in the absence or presence of physiological concentrations of AREG (10 or 100 ng/ml) with or without gonadotrophins (FSH, 75 mIU/ml; LH, 75 mIU/ml). At the end of the culture period, oocyte meiotic maturation was evaluated and ICSI was performed (n=111), from which fertilization and early embryo development was followed in vitro. RESULTS: AREG levels in follicular fluid from pre-ovulatory follicles increased (P<0.05) following an ovulatory bolus of hCG at 12, 24 and 36 h post-treatment. At 12 h post-hCG, AREG levels in follicular fluid ranged from 4.8 to 121.4 ng/ml. Rhesus macaque COCs incubated with 10 ng/ml AREG in the presence of gonadotrophins displayed an increased percentage of oocytes that progressed to the metaphase II (MII) stage of meiosis (82 versus 56%, P<0.05) and a decreased percentage of metaphase I (MI) oocytes (2 versus 23%, P<0.05) relative to controls, respectively. The percentage of either MI or MII oocytes at the end of the culture period was not different between oocytes cultured with 100 ng/ml AREG or in media alone. Fertilization and first cleavage rates obtained by ICSI of all IVM MII oocytes were 93 and 98%, respectively, and did not vary among treatment groups. Of the MII oocytes that fertilized (n=103), 37 were randomly selected and maintained in culture to assess developmental potential. A total of 13 early blastocysts were obtained, with four embryos developing to expanded blastocysts. CONCLUSIONS: These data indicate that AREG levels increase in rhesus macaque pre-ovulatory follicles after an ovulatory stimulus, and a specific concentration of AREG (10 ng/ml) enhances rhesus macaque oocyte nuclear maturation but not cytoplasmic maturation from SAFs obtained during the natural menstrual cycle. However, owing to the small number of samples in some treatment groups, further studies are now required.
Assuntos
Glicoproteínas/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Oócitos/citologia , Anfirregulina , Animais , Blastocisto/citologia , Células Cultivadas , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Líquido Folicular/metabolismo , Ligantes , Hormônio Luteinizante/metabolismo , Macaca mulatta , Meiose , Oogênese , Folículo Ovariano/citologia , Ovário/fisiologia , Fatores de TempoRESUMO
We investigated the expression and cell localization of NOTCH1, NOTCH4, and the delta-like ligand DLL4 in corpus luteum (CL) from pregnant rats during prostaglandin F2alpha (PGF2alpha)-induced luteolysis. We also examined serum progesterone (P(4)) and CL proteins related to apoptosis after local administration of the notch inhibitor N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT). Specific staining for NOTCH1 and NOTCH4 receptors was detected predominantly in large and small luteal cells. Furthermore, in line with the fact that the notch intracellular domain is translocated to the nucleus, where it regulates gene expression, staining was evident in the nuclei of luteal cells. In addition, we detected diffuse cytoplasmic immunostaining for DLL4 in small and large luteal cells, in accordance with the fact that DLL4 undergoes proteolytic degradation after receptor binding. The mRNA expression of Notch1, Notch4, and Dll4 in CL isolated on Day 19 of pregnancy decreased significantly after administration of PGF2alpha. Consistent with the mRNA results, administration of PGF2alpha to pregnant rats on Day 19 of pregnancy decreased the protein fragment corresponding to the cleaved forms of NOTCH1/4 CL receptors. In contrast, no significant changes were detected in protein levels for the ligand DLL4. The local intrabursal administration of DAPT decreased serum P(4) levels and increased luteal levels of active caspase 3 and the BAX:BCL2 ratio 24 h after the treatment. These results support a luteotropic role for notch signaling to promote luteal cell viability and steroidogenesis, and they suggest that the luteolytic hormone PGF2alpha may act in part by reducing the expression of some notch system members.
Assuntos
Corpo Lúteo/metabolismo , Dinoprosta/metabolismo , Luteólise/metabolismo , Proteínas de Membrana/metabolismo , Proteínas da Gravidez/metabolismo , Receptor Notch1/metabolismo , Receptores Notch/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Núcleo Celular/metabolismo , Corpo Lúteo/efeitos dos fármacos , Corpo Lúteo/ultraestrutura , Feminino , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Luteólise/sangue , Proteínas de Membrana/genética , Fragmentos de Peptídeos/metabolismo , Gravidez , Proteínas da Gravidez/antagonistas & inibidores , Proteínas da Gravidez/genética , Progesterona/sangue , Inibidores de Proteases/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Receptor Notch4 , Receptores Notch/antagonistas & inibidores , Receptores Notch/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
The stage at which follicle-enclosed cumulus-oocyte complexes achieve developmental competence in primates is unknown. Therefore, studies were designed to characterize the ability of oocytes in small antral follicles present during the menstrual cycle to spontaneously resume meiosis, fertilize, and support early embryo development. Ovaries were removed from adult rhesus monkeys (n = 12) during the early follicular phase (Days 3-4) of spontaneous cycles. Small antral follicles were divided into five groups according to their diameter; group I: <0.5 mm; group II: 0.5-0.99 mm; group III: 1.0-1.49 mm; group IV: 1.5-1.99 mm; and group V: 2.0-2.5 mm. The cumulus-oocyte complex from healthy small antral follicles (devoid of dark oocytes or granulosa cells) were extracted (n = 199) and cultured for 48 h under different conditions: in TALP (tyrode, albumin, lactate, pyruvate) medium alone, SAGE medium alone, or plus gonadotropins. At 48 h, oocyte meiotic status and diameter were measured after treatment of cumulus-oocyte complexes with hyaluronidase. Cumulus-oocyte complexes derived from follicles of 0.5- to 2-mm diameter contain oocytes that typically reinitiate meiosis in the absence or presence of gonadotropins and fertilize via in vitro fertilization or intracytoplasmic sperm injection. Moreover, the inseminated oocytes can reach the morula stage but arrest. Thus, the ability of these oocytes to complete maturation, as monitored from subsequent embryonic development after fertilization, is suboptimal. Further studies on primate IVM of oocytes from SAFs are warranted in order for them to be considered as an additional, novel source of gametes for fertility preservation in cancer patients.
Assuntos
Fertilização in vitro , Fase Folicular/fisiologia , Meiose/fisiologia , Oócitos/fisiologia , Folículo Ovariano/fisiologia , Animais , Células do Cúmulo/fisiologia , Desenvolvimento Embrionário , Feminino , Macaca mulatta , Masculino , Microscopia Confocal , Oócitos/ultraestrutura , GravidezRESUMO
Since the regression of the corpus luteum (CL) occurs via a tightly controlled apoptotic process, studies were designed to determine if local administration of the antiapoptotic agent sphingosine 1-phosphate (S1P) effectively blocks the luteolytic action of prostaglandin F-2alpha (PGF-2alpha). On day 19 of pregnancy, 2 hr before systemic PGF-2alpha administration, rats were injected intrabursa with either S1P or vehicle (control). The activity of four caspases, which contribute to the initial (caspase-2, -8, and -9) and final (caspase-3) events in apoptosis was measured in pooled CL from four individual ovaries at 0 and 4 hr after PGF-2alpha injection. The expression of the phosphorylated form of AKT (pAKT) and tumor necrosis factor-alpha (TNF-alpha) was analyzed by ELISA. In addition, cell death was evaluated by electronic microscopy (EM) in CL 4 and 36 hr after PGF-2alpha injection. The activity of caspase-2, -3, and -8 was significantly greater by 4 hr after PGF-2alpha, but not caspase-9 activity. In contrast, expression of pAKT and TNF-alpha decreased significantly. Administration of S1P suppressed (P < 0.05) these effects, decreasing caspase activities and increasing pAKT and TNF-alpha expression. The administration of S1P also significantly decreased the percentage of luteal apoptotic cells induced by PGF-2alpha. PGF-2alpha treatment increased the prevalence of luteal cells with advanced signs of apoptosis (i.e., multiple nuclear fragments, chromatin condensation, or apoptotic bodies). S1P treatment suppressed these changes and increased the blood vessel density. These results suggest that S1P blocks the luteolytic effect of the PGF-2alpha by decreasing caspase-2, -3, and -8 activities and increasing AKT phosphorylation and TNF-alpha expression.
Assuntos
Dinoprosta/metabolismo , Luteólise , Lisofosfolipídeos/farmacologia , Esfingosina/análogos & derivados , Animais , Caspase 2/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Corpo Lúteo/metabolismo , Corpo Lúteo/ultraestrutura , Feminino , Luteólise/efeitos dos fármacos , Luteólise/fisiologia , Lisofosfolipídeos/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Gravidez , Progesterona/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Esfingosina/metabolismo , Esfingosina/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Members of the tumor necrosis factor (TNF)-receptor (R) family may be involved in the tissue remodeling that occurs in the primate corpus luteum (CL) during development and regression. As a first step towards addressing this issue, studies assessed TNF ligand-R expression and regulation in CL collected from monkeys during the early (ECL, Days 3-5), mid (MCL, Days 7-8), mid-late (MLCL, Days 10-11), late (LCL, Days 14-16), and very late (VLCL, menses) luteal phase of the menstrual cycle. CL were also collected after gonadotropin and/or steroid ablation and replacement (with hLH and the progestin R5020) for 3 days at mid-late luteal phase. TNF-alpha, -beta, FAS ligand (FASL), and TNF-R1 mRNA levels were two- to sixfold greater (P < 0.05) at the MLCL or LCL phase as compared to earlier (ECL, MCL). In contrast, TNF-R2 and FAS mRNA levels did not change during the luteal phase. Immunohistochemical staining for TNF-beta, TNF-R1, TNF-R2, FAS, and FASL was observed in luteal cells, whereas only TNF-beta staining was observed in endothelial cells. Several TNF-R components were influenced by LH and/or steroid ablation; notably, steroid ablation reduced (P < 0.05) luteal TNF-alpha, but not TNF-beta, mRNA levels, which was prevented by progestin treatment. In contrast, steroid ablation increased (P < 0.05) luteal cell immunostaining for FAS and FASL, which was reduced by progestin treatment. Thus, several members of the TNF R-ligand family are expressed in the primate CL in an LH- and/or progestin-dependent manner. Peak expression in the late luteal phase may signify a role for the TNF-R system in death receptor-mediated apoptosis during luteolysis.
Assuntos
Corpo Lúteo/metabolismo , Ciclo Menstrual/fisiologia , Receptores do Fator de Necrose Tumoral/metabolismo , Fatores de Necrose Tumoral/metabolismo , Abortivos Esteroides/farmacologia , Animais , Corpo Lúteo/citologia , Corpo Lúteo/efeitos dos fármacos , Di-Hidrotestosterona/análogos & derivados , Di-Hidrotestosterona/farmacologia , Feminino , Humanos , Macaca , Gravidez , Promegestona/farmacologia , RNA Mensageiro/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Fatores de Necrose Tumoral/genéticaRESUMO
Studies were designed to determine whether: 1) changes in caspase expression or activity occur in the macaque corpus luteum (CL) during its lifespan in the menstrual cycle, and 2) LH acting directly or via ovarian steroids regulates luteal caspases. Caspase-2, -3, -8, and -9 mRNAs were detectable by semiquantitative RT- or real time-PCR in CL, but levels did not differ between the early, mid, mid-late, late, and very-late luteal phases. Immunostaining for caspase-2 and -3 proteins was observed in luteal cells and appeared to peak by mid to mid-late stage. Enzyme activity for caspase-2, -3, -8, and -9 increased (P < 0.05) by mid-late stage, and then declined by the very-late stage. Treatment with GnRH antagonist + LH at the mid-late stage increased caspase-2, -8, and -9, but not -3, activity, compared with controls. Coadministration of a steroid synthesis inhibitor (trilostane) with GnRH antagonist + LH reduced (P < 0.05) caspase-2, -8, and -9 activity. Progestin (R5020) replacement during trilostane treatment did not restore caspase activity. Thus, initiator and effector caspases are present during CL development and regression in the menstrual cycle. The increased caspase activity at mid-late stage suggests that apoptosis is involved in early luteolysis in primates. Gonadotropin, perhaps via local steroids, modulates initiator caspases in the primate CL.
Assuntos
Caspases/metabolismo , Corpo Lúteo/enzimologia , Di-Hidrotestosterona/análogos & derivados , Ciclo Menstrual/metabolismo , Animais , Caspase 2 , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/genética , Di-Hidrotestosterona/farmacologia , Feminino , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Hormônio Luteinizante/farmacologia , Macaca mulatta , Promegestona/farmacologia , RNA Mensageiro/análiseRESUMO
Protease genes were identified that exhibited increased mRNA levels before and immediately after rupture of the naturally selected, dominant follicle of rhesus macaques at specific intervals after an ovulatory stimulus. Quantitative real-time PCR validation revealed increased mRNA levels for matrix metalloproteinase (MMP1, MMP9, MMP10, and MMP19) and a disintegrin and metalloproteinase with thrombospondin-like repeats (ADAMTS1, ADAMTS4, ADAMTS9, and ADAMTS15) family members, the cysteine protease cathepsin L (CTSL), the serine protease urokinase-type plasminogen activator (PLAU), and the aspartic acid protease pepsinogen 5 (PGA5). With the exception of MMP9, ADAMTS1, and PGA5, mRNA levels for all other up-regulated proteases increased significantly (P < 0.05) 12 h after an ovulatory human chorionic gonadotropin (hCG) bolus. MMP1, -10, and -19; ADAMTS1, -4, and -9; CTSL; PLAU; and PGA5 also exhibited a secondary increase in mRNA levels in 36-h postovulatory follicles. To further determine metalloproteinase involvement in ovulation, vehicle (n = 4) or metalloproteinase inhibitor (GM6001, 0.5 µg/follicle, n = 8) was injected into the preovulatory follicle at the time of hCG administration. Of the eight GM6001-injected follicles, none displayed typical stigmata indicative of ovulation at 72 h after hCG; whereas all four vehicle-injected follicles ovulated. No significant differences in mean luteal progesterone levels or luteal phase length occurred between the two groups. Subsequent histological analysis revealed that vehicle-injected follicles ruptured, whereas GM6001-injected follicles did not, as evidenced by an intact stroma and trapped oocytes (n = 3). These findings demonstrate metalloproteinases are critical for follicle rupture in primates, and blocking their activity would serve as a novel, nonhormonal means to achieve contraception.
Assuntos
Metaloproteases/genética , Metaloproteases/fisiologia , Folículo Ovariano/enzimologia , Animais , Gonadotropina Coriônica/farmacologia , Estrogênios/biossíntese , Feminino , Macaca mulatta , Análise de Sequência com Séries de Oligonucleotídeos , Folículo Ovariano/fisiologia , Progesterona/biossíntese , RNA Mensageiro/análiseRESUMO
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 PGF2alpha. 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 not increase in the CL until postpartum. Following PGF2alpha injection, there was a significant decrease in CL P by 24 h, although the activity of all four caspases did not increase until 36 h posttreatment. The active p17 fragment of caspase-3 also significantly increased at 36 h post-PGF2alpha. These results suggest that an increase in the activity of caspases-2, -8, -9, and -3 is associated with the early events of natural luteolysis at the end of pregnancy. Also, the exogenous administration of the luteolysin PGF2alpha may regulate members of the caspase family.
Assuntos
Caspases/metabolismo , Corpo Lúteo/enzimologia , Prenhez/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Caspases/biossíntese , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Dinoprosta/farmacologia , Ativação Enzimática , Feminino , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Isoenzimas/biossíntese , Isoenzimas/metabolismo , Luteólise , Masculino , Período Pós-Parto/metabolismo , Gravidez , Progesterona/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
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. Enzyme activity of caspase-9 increased (P < 0.05) in the new CL at DII, followed by that of caspase-2 and -3 in old CL at estrus. Caspase-8 activity did not change at any stage. The number of apoptotic cells increased at DII in the old CL. These results suggest an important role for this protease family during early events of luteolysis in the rat estrous cycle.
Assuntos
Caspases/metabolismo , Corpo Lúteo/enzimologia , Ciclo Estral/fisiologia , Animais , Apoptose , Western Blotting/métodos , Caspase 2/análise , Caspase 3/análise , Caspase 8/análise , Caspase 9/análise , Caspases/análise , Enzima de Clivagem da Cadeia Lateral do Colesterol/análise , Feminino , Imuno-Histoquímica/métodos , Marcação In Situ das Extremidades Cortadas , Progesterona/análise , Ratos , Ratos Sprague-DawleyRESUMO
The GnRH agonist leuprolide acetate (LA) inhibited DNA synthesis in epidermal growth factor-stimulated human granulosa luteinized cell cultures. This effect was blocked by the prior addition of a GnRH antagonist antide (ANT), and this compound per se was able to produce a stimulatory effect of DNA synthesis on basal conditions. Leuprolide acetate produced an increase in the percentage of apoptotic cells, and when these two factors were co-incubated, ANT blocked the apoptotic effect produced by LA.