RESUMO
The objective of the study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A), inducible nitric oxide synthase (iNOS) and endothelial (eNOS) isoforms in time-defined follicle classes before and after GnRH application in the cow. Ovaries containing pre-ovulatory follicles or corpora lutea were collected by transvaginal ovariectomy (n = 5 cows/group) as follow: (I) before GnRH administration; (II) 4h after GnRH; (III) 10h after GnRH; (IV) 20h after GnRH; (V) 25h after GnRH; and (VI) 60h after GnRH (early corpus luteum). The mRNA abundance of HIF1A in the follicle group before GnRH was high, followed by a significant down regulation afterwards with a minimum level 25h after GnRH (close to ovulation) and significant increase only after ovulation. The mRNA abundance of iNOS before GnRH was high, decreased significantly during LH surge, with minimum levels afterwards. In contrast, the mRNA of eNOS decreased in the follicle group 20h after GnRH, followed by a rapid and significant upregulation just after ovulation. Immunohistochemically, the granulosa cells of antral follicles and the eosinophils of the theca tissue as well of the early corpus luteum showed a strong staining for HIF1A. The location of the eosinophils could be clearly demonstrated by immunostaining with an eosinophil-specific antibody (EMBP) and transmission electron microscopy. In conclusion, the parallel and acute regulated expression patterns of HIF1A and NOS isoforms, specifically during the interval between the LH surge and ovulation, indicate that these paracrine factors are involved in the local mechanisms, regulating final follicle maturation, ovulation and early luteal angiogenesis.
Assuntos
Bovinos/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Folículo Ovariano/enzimologia , Ovulação/metabolismo , Animais , Corpo Lúteo/irrigação sanguínea , Feminino , Hormônio Liberador de Gonadotropina/administração & dosagem , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Óxido Nítrico Sintase/metabolismo , Folículo Ovariano/metabolismo , RNA Mensageiro/metabolismoRESUMO
The aim of this study was to characterize certain prostaglandin family members in the bovine corpus luteum (CL) during the estrous cycle and pregnancy. The CL tissue was assigned to the stages 1-2, 3-4, 5-7, 8-12, 13-16 and >18 days (after regression) of the estrous cycle and 1-2, 3-4, 6-7, and >8 months of pregnancy. In these samples, we investigated prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE), their receptors (PTGFR, PTGER2, and PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS), and PTGE synthase (PTGES). The expression of messenger RNA (mRNA) was measured by reverse transcription quantitative polymerase chain reaction, hormones by enzyme immunoassay, and localization by immunohistochemistry. The mRNA expression of COX-2, PTGFS, and PTGES in CL during the early-luteal phase was high followed by a continuous and significant downregulation afterward, as well as during all phases of pregnancy. The concentration of PTGF in CL tissue was high during the early-luteal phase, decreased significantly in the mid-luteal phase, and increased again afterward. In contrast, the concentration of PTGE increased significantly during the late-luteal phase followed by a decrease during regression. The PTGE level increased again during late pregnancy. Immunohistochemically, the large granulose-luteal cells show strong staining for COX-2 and PTGES during the early-luteal stage followed by lower activity afterward. During pregnancy, most of the luteal cells were only weakly positive or negative. In conclusion, our results indicate that the examined prostaglandin family members are involved in the local mechanisms that regulate luteal function, specifically during CL formation, function, and regression and during pregnancy in the cow.
Assuntos
Corpo Lúteo/metabolismo , Ciclo-Oxigenase 2/biossíntese , Dinoprosta/biossíntese , Dinoprostona/biossíntese , Ciclo Estral/fisiologia , Hidroxiprostaglandina Desidrogenases/biossíntese , Prostaglandina-E Sintases/biossíntese , Animais , Bovinos , Ciclo-Oxigenase 2/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Hidroxiprostaglandina Desidrogenases/genética , Fase Luteal/metabolismo , Gravidez , Prostaglandina-E Sintases/genética , RNA Mensageiro/genética , Receptores de Prostaglandina/biossínteseRESUMO
The aim of this study was to characterize the expression patterns and localization of the thrombospondin family members (THBS1, THBS2) and their receptors (CD36 and CD47) in bovine ovaries. First, the antral follicles were classified into 5 groups based on the follicle size and estradiol-17beta (E2) concentration in the follicular fluid (< 0.5, 0.5-5, 5-40, 40-180 and >180 E2 ng/ml). Second, the corpus luteum (CL) was assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16 and >18 of the estrous cycle and of pregnancy (month 1-2, 3-4, 6-7 and > 8). Third, the corpora lutea were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after inducing luteolysis by injecting a prostaglandin F2alpha analog. The mRNA expression of examined factors was measured by RT-qPCR, steroid hormone concentration by EIA, and localization by immunohistochemistry. The mRNA expression of THBS1, THBS2, CD36, and CD47 in the granulosa cells and theca interna was high in the small follicles and reduced in the preovulatory follicles. The mRNA expression of THBS1, THBS2, and CD47 in the CL during the estrous cycle was high, but decreased significantly during pregnancy. After induced luteolysis, thrombospondins increased significantly to reach the maximum level at 12 h for THBS1, 24 h for THBS2, and 48 h for CD36. The temporal expression and localization pattern of the thrombospondins and their specific receptors in the antral follicles and corpora lutea during the different physiological phases of the estrous cycle and induced luteolysis appear to be compatible with their inhibitory role in the control of ovarian angiogenesis.
Assuntos
Corpo Lúteo/fisiologia , Regulação da Expressão Gênica , Folículo Ovariano/fisiologia , Ovário/fisiologia , Trombospondinas/metabolismo , Animais , Antígenos CD36/metabolismo , Antígeno CD47/metabolismo , Bovinos , Estradiol/metabolismo , Ciclo Estral , Feminino , Perfilação da Expressão Gênica , Células da Granulosa/metabolismo , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Luteólise , Gravidez , Prenhez , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Prostaglandins are synthesized from arachidonic acid through the catalytic activities of cyclooxygenase, while the production of different prostaglandin types, prostaglandin F2 alpha (PGF) and prostaglandin E2 (PGE), are regulated by specific prostaglandin synthases (PGFS and PGES). Prostaglandin ligands (PGF and PGE) bind to specific high-affinity receptors and initiate biologically distinct signalling pathways. In the ovaries, prostaglandins are known to be important endocrine regulators of female reproduction, in addition to maintaining local function through autocrine and/or paracrine effect. Many research groups in different animal species have already identified a variety of factors and molecular mechanisms that are responsible for the regulation of prostaglandin functions. In addition, prostaglandins stimulate their intrafollicular and intraluteal production via the pathway of prostaglandin self-regulation in the ovary. Therefore, the objective of the review article is to discuss recent findings about local regulation patterns of prostaglandin ligands PGF and PGE during different physiological stages of ovarian function in domestic ruminants, especially in bovine. In conclusion, the discussed local regulation mechanisms of prostaglandins in the ovary may stimulate further research activities in different methodological approaches, especially during final follicle maturation and ovulation, as well as corpus luteum formation and function.
Assuntos
Ovário , Prostaglandinas , Feminino , Bovinos , Animais , Prostaglandinas/metabolismo , Ovário/fisiologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Ruminantes/metabolismo , Folículo Ovariano/fisiologia , Corpo Lúteo/metabolismoRESUMO
The objective of the study was to characterize the mRNA expression patterns of specific steroid hormone receptors namely, estrogen receptors (ESRRA-estrogen related receptor alpha and ESRRB-estrogen related receptor beta) and progesterone receptors (PGR) in superovulation-induced bovine follicles during the periovulation and subsequent corpus luteum (CL) formation. The bovine ovaries (n = 5 cow / group), containing preovulatory follicles or early CL, were collected relative to injection of the gonadotropin-releasing hormone (GnRH) at (I) 0 h, (II) 4 h, (III) 10 h, (IV) 20 h, (V) 25 h (preovulatory follicles) and (VI) 60 h (CL, 2-3 days after induced ovulation). In this experiment, we analyzed the steroid receptor mRNA expression and their localization in the follicle and CL tissue. The high mRNA expression of ESRRA, ESRRB, and PGR analyzed in the follicles before ovulation is significantly reduced in the group of follicles during ovulation (25 h after GnRH), rising again significantly after ovulation in newly formed CL, only for ESRRA and PGR (P < 0.05). Immunohistochemically, the nuclei of antral follicles' granulosa cells showed a positive staining for ESRRA, followed by higher activity in the large luteal cells just after ovulation (early CL). In contrast, the lower PGR immunopresence in preovulatory follicles increased in both small and large luteal cell nuclei after follicle ovulation. Our results of steroid receptor mRNA expression in this experimentally induced gonadotropin surge provide insight into the molecular mechanisms of the effects of steroid hormones on follicular-luteal tissue in the period close to the ovulation and subsequent CL formation in the cow.
RESUMO
The objective of the study was to evaluate the expression patterns of prostaglandin F2alpha (PGF), prostaglandin E2 (PGE), PGF receptor (FP), PGE receptors (EP2 and EP4), prostaglandin-endoperoxide synthase 2 (PTGS2) and prostaglandin synthases (PGFS and PGES) in corpora lutea (CL) during experimentally induced luteolysis in cow. The Fleckvieh cows in the mid-luteal phase (days 8-12, control group) were injected with cloprostenol (PGF analogue), and CL were collected by transvaginal ovariectomy before (days 8-12, control group) and at 0.5, 2, 4, 12, 24, 48 and 64 h after PGF application (n = 5 per group). The mRNA expression was determined by RT-qPCR, the hormone concentrations by enzyme immunoassay and localization by immunohistochemistry. PTGS2 gene expression increased significantly 2 h after PGF application, followed by continuous and significant downregulation afterwards. The PGF tissue concentration increased significantly just after PGF injection and again during structural luteolysis (after 12 h), whereas PGE concentration significantly decreased during structural luteolysis. The FP receptor mRNA decreased significantly at 2 h and again at 12 h after PGF. In contrast, EP4 receptor mRNA increased significantly just after the PGF application (0.5 h). The immunostaining of PGES and PTGS2 on day 15-17 shows numerous positive luteal cells, followed by lower activity afterwards on day 18 (luteolysis). In conclusion, the changes of examined prostaglandin family members in CL tissue after PGF application may be key components of the local mechanisms regulating the cascade of actions leading to functional and subsequent structural luteolysis in the bovine ovary.
Assuntos
Células Lúteas , Luteólise , Animais , Bovinos , Corpo Lúteo/metabolismo , Dinoprosta/metabolismo , Dinoprosta/farmacologia , Feminino , Células Lúteas/metabolismo , Luteólise/genética , Luteólise/metabolismo , Progesterona/metabolismo , Prostaglandinas/metabolismoRESUMO
The essential role of endometrial prostaglandin F2 alpha (PTGF) for induction of the corpus luteum (CL) regression is well documented in the cow. However, the acute effects of PTGF on known local luteotropic factors (oxytocin [OXT] and its receptor, insulin-like growth factor [IGF] 1, and progesterone and its receptor), the principal angiogenic factor vascular endothelial growth factor (VEGF) A and the capillary destabilization factor angiopoietin (ANGPT) 2 were not thoroughly studied in detail. The aim of this study was therefore to evaluate the tissue concentration of these factors during PTGF induced luteolysis. In addition the mRNA expression of progesterone receptor (PGR), OXT receptor (OXTR), IGF1, IGFBP1, ANGPT1, and ANGPT2 was determined at different times after PTGF treatment. Cows (n = 5 per group) in the mid-luteal phase (Days 8-12, control group) were injected with the PTGF analog (cloprostenol), and CL were collected by transvaginal ovariectomy at 0.5, 2, 4, 12, 24, 48, and 64 h after injection. The mRNA expression was analyzed by quantitative real-time PCR, and the protein concentration was evaluated by enzyme immunoassay or radioimmunoassay. Progesterone concentrations, as well as mRNA expression of PGR, in CL tissue were significantly down-regulated by 12 h after PTGF. Tissue OXT peptide and OXTR mRNA decreased significantly after 2 h, followed by a continuous decrease of OXT mRNA. IGF1 and VEGFA protein already decreased after 0.5 h. By contrast, the IGFBP1 mRNA was up-regulated significantly after 2 h to a high plateau. ANGPT2 protein and mRNA significantly increased during the first 2 h, followed by a steep decrease after 4 h. The acute decrease of local luteotropic activity and acute changes of ANGPT2 and VEGFA suggest that modulation of vascular stability may be a key component in the cascade of events leading to functional luteolysis.
Assuntos
Corpo Lúteo/metabolismo , Dinoprosta/fisiologia , Fase Luteal/metabolismo , Luteólise/fisiologia , Angiopoietina-2/genética , Angiopoietina-2/metabolismo , Animais , Bovinos , Feminino , Regulação da Expressão Gênica , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Neovascularização Fisiológica/fisiologia , Ocitocina/genética , Ocitocina/metabolismo , Progesterona/genética , Progesterona/metabolismo , RNA Mensageiro/análise , Receptores de Progesterona/metabolismo , Transdução de Sinais/fisiologia , Fatores de Tempo , Distribuição Tecidual , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Leptin, the hormonal product of the obese (ob) gene, circulates in the blood at levels paralleling those of fat reserves and regulates satiety. In cattle, leptin has also been implicated in the control of ovarian function, but its local production in the ovary and role in the control of ovarian function in autocrine/paracrine manner is unknown. The aims of this study were to document the expression of leptin and its receptor (Ob-R) in detail in bovine corpus luteum (CL) obtained from different stages of the oestrous cycle and during pregnancy-and to determine if the leptin/Ob-R system is expressed clearly in bovine follicles during final growth to preovulatory follicles. Real-time RT-PCR (qPCR) was applied to investigate mRNA expression of examined factors. In general, we demonstrated leptin and its receptor transcripts and leptin protein are consistent with in vivo luteinisation of bovine CL and decline coincidental with luteal regression. The highest co-expression of leptin/Ob-R system was observed in TI and GC of the smallest follicles with E2 concentration <0.5 ng/ml followed by significant down regulation in growing follicles with the increase of follicular size and E2 content in the follicular fluid. Furthermore, expression of the leptin/Ob-R system does not show any significant variation in the CL throughout pregnancy. To conclude, our results are the first to demonstrate the possible involvement of locally produced leptin/Ob-R system in the bovine ovary, suggesting roles in the function and/or development of the CL and growth of small follicles in an autocrine/paracrine fashion.
Assuntos
Corpo Lúteo/metabolismo , Regulação da Expressão Gênica/fisiologia , Leptina/biossíntese , Folículo Ovariano/metabolismo , Comunicação Parácrina/fisiologia , Receptores para Leptina/biossíntese , Animais , Comunicação Autócrina/fisiologia , Bovinos , Corpo Lúteo/citologia , Ciclo Estral/fisiologia , Feminino , Folículo Ovariano/citologia , Gravidez/metabolismoRESUMO
Follicular development, follicular rupture, and corpus luteum (CL) formation are accompanied by extensive tissue remodeling. We examined whether heparanase (HPSE), which cleaves heparan sulfate glycosaminoglycans, is induced during these processes. Prostaglandin F2alpha injection, which initiated luteolysis and the development of a preovulatory follicle, moderately increased HPSE mRNA in bovine granulosa cells (GCs). GnRH, used to induce gonadotropin surge, markedly augmented HPSE mRNA levels 12 h after its injection. The temporal pattern of HPSE gene expression in follicular-luteal transition was further examined in follicles collected before, and 4, 10, 20, 25, and 60 h after GnRH injection. HPSE mRNA increased transiently 10-20 h after GnRH injection to levels 10-fold higher than in untreated heifers. HPSE protein levels were similarly elevated 20 h after GnRH injection in GCs, but not in the theca layer. Cyclooxygenase-2 (PTGS2) mRNA peaked before ovulation when HPSE levels returned to baseline levels. HPSE mRNA abundance also remained low in the CLs. The antiprogesterone, RU-486, elevated HPSE levels in GC culture, suggesting that progesterone secreted by CLs may inhibit HPSE. HPSE immunostaining was more abundant in GCs than thecae. In cultured GCs, LH induced a transient increase in HPSE mRNA 3-6 h after its addition, but not at 24 h. However, PTGS2 mRNA was clearly induced at this time. These findings suggest that: 1) HPSE may play a role in ovulation but much less so during CL development, and 2) GC-derived HSPE may be a novel member of the LH-induced extracellular matrix-degrading enzyme family and may contribute to follicular rupture.
Assuntos
Glucuronidase/genética , Células da Granulosa/enzimologia , Hormônio Luteinizante/farmacologia , Animais , Aromatase/genética , Bovinos , Feminino , Regulação da Expressão Gênica , Glucuronidase/biossíntese , Hormônio Liberador de Gonadotropina/fisiologia , Células da Granulosa/efeitos dos fármacos , Lactação/fisiologia , Hormônio Luteinizante/fisiologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Ovulação , Reação em Cadeia da Polimerase , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genéticaRESUMO
The aim of this study was to investigate the possible participation of fibroblast growth factor (FGF) family members (FGF1, FGF2 and FGF7 and their receptors) in porcine follicles (polyovulatory species) under special consideration for FGF2 during final growth. A classification of follicles was done by size and follicular fluid content of oestradiol-17beta, progesterone and prostaglandin F2alpha. The mRNA expression of examined factors was analysed by real-time PCR. The hormone concentration was estimated by enzyme immunoassay, protein characterisation by western blotting and localisation by immunohistochemistry. Follicle tissue separated in theca interna and granulosa cells was extracted and tested for mRNA of FGF1, FGF2, FGF7 and receptors (FGFR1IIIc, FGFRIIIb and FGFR2IIIc). Additionally, the mRNA expression of FSHR, LHR and aromatase cytochrome P450 for further characterisation of follicles was analysed. Significantly, higher FGF2 protein levels were measured in stroma when compared with total follicle or corpus luteum tissue. This result was confirmed by western blot with two strong bands. Immunological localisation of FGF2 only in stroma (fibroblasts) confirms the protein measurements. The results show a clear difference for FGF2 protein expression during final growth of follicles if monovulatory (bovine) and polyovulatory (porcine) species are compared. FGF2 protein in porcine ovary may be (due to localisation and concentration in stroma) important for support of angiogenesis of more follicles (polyovulatory species) and not of a single follicle like in cows.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Folículo Ovariano/metabolismo , Suínos/metabolismo , Animais , Aromatase/genética , Western Blotting , Dinoprosta/metabolismo , Ensaio de Imunoadsorção Enzimática , Estradiol/metabolismo , Feminino , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 7 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Imuno-Histoquímica , Progesterona/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores do FSH/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores do LH/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos/genéticaRESUMO
The aim of this study was to characterize the regulation pattern of prostaglandin family members namely prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE), their receptors (PTGFR, PTGER2, PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS), and PTGE synthase (PTGES) in the bovine follicles during preovulatory period and early corpus luteum (CL). Ovaries containing preovulatory follicles or CL were collected by transvaginal ovariectomy (n = 5 cows/group), and the follicles were classified: (I) before GnRH treatment; (II) 4 h after GnRH; (III) 10 h after GnRH; (IV) 20 h after GnRH; (V) 25 h after GnRH, and (VI) 60 h after GnRH (early CL). In these samples, the concentrations of progesterone (P4), estradiol (E2), PTGF and PTGE were investigated in the follicular fluid (FF) by validated EIA. Relative mRNA abundance of genes encoding for prostaglandin receptors (PTGFR, PTGER2, PTGER4), COX-2, PTGFS and PTGES were quantified by RT-qPCR. The localization of COX-2 and PTGES were investigated by established immunohistochemistry in fixed follicular and CL tissue samples. The high E2 concentration in the FF of the follicle group before GnRH treatment (495.8 ng/ml) and during luteinizing hormone (LH) surge (4 h after GnRH, 574.36 ng/ml), is followed by a significant (P<0.05) downregulation afterwards with the lowest level during ovulation (25 h after GnRH, 53.11 ng/ml). In contrast the concentration of P4 was very low before LH surge (50.64 mg/ml) followed by a significant upregulation (P < 0.05) during ovulation (537.18 ng/ml). The mRNA expression of COX-2 increased significantely (P < 0.05) 4 h after GnRH and again 20 h after GnRH, followed by a significant decrease (P < 0.05) after ovulation (early CL). The mRNA of PTGFS in follicles before GnRH was high followed by a continuous and significant downregulation (P < 0.05) afterwards. In contrast, PTGES mRNA abundance increased significantely (P < 0.05) in follicles 20 h after GnRH treatment and remained high afterwards. The mRNA abundance of PTGFR, PTGER2, and PTGER4 in follicles before GnRH was high, followed by a continuous and significant down regulation afterwards and significant increase (P < 0.05) only after ovulation (early CL). The low concentration of PTGF (0.04 ng/ml) and PTGE (0.15 ng/ml) in FF before GnRH, increased continuously in follicle groups before ovulation and displayed a further significant and dramatic increase (P < 0.05) around ovulation (101.01 ng/ml, respectively, 484.21 ng/ml). Immunohistochemically, the granulosa cells showed an intensive signal for COX-2 and PTGES in follicles during preovulation and in granulosa-luteal cells of the early CL. In conclusion, our results indicate that the examined bovine prostaglandin family members are involved in the local mechanisms regulating final follicle maturation and ovulation during the folliculo-luteal transition and CL formation.
RESUMO
Several factors participate in regulation of growth and development as well as angiogenesis of the uterus during pregnancy, and hence little is known about the role of hormonal regulation of vascular endothelial growth factor (VEGF)-receptor system expression. This study has examined the effect of insulin-like growth factor-I (IGF-I), relaxin (RLX), oxytocin (OT) and prostaglandin (PG) E(2), on VEGF secretion and VEGF-receptor system mRNA expression in the porcine endometrial stromal cells. IGF-I and RLX were identified as the most effective inducers of VEGF secretion and mRNA expression. Although PGE(2) stimulated VEGF secretion and VEGF164 mRNA expression, OT inhibited both secretion and mRNA expression of VEGF. When tested for VEGF receptors (R), all factors failed to affect their mRNA expression. Media conditioned by stromal cells collected after IGF-I and RLX treatment significantly increased endothelial cell proliferation and this effect was blocked by soluble VEGFR-1. These data suggest that during early pregnancy IGF-I, RLX and PGE(2) can affect VEGF expression in the endometrium and therefore may support uterine and embryo development, implantation and pregnancy.
Assuntos
Dinoprostona/metabolismo , Endométrio/citologia , Fator de Crescimento Insulin-Like I/metabolismo , Ocitocina/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Relaxina/metabolismo , Células Estromais/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Gravidez , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Transdução de Sinais/fisiologia , Células Estromais/citologia , Suínos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Angiogenesis, changes in blood flow, and extracellular matrix remodeling are the processes associated with the development and demise of the bovine corpus luteum (CL) during the estrous cycle. APJ (putative receptor protein related to angiotensin type 1 receptor) is a G-protein-coupled receptor, and its ligand, apelin, has been identified as a novel regulator of blood pressure and as an angiogenic factor. We hypothesized that the apelin-APJ system is involved in luteal function. This study investigated whether apelin-APJ exists in bovine CL and determined their expression profiles and localization during luteal phase and prostaglandin F(2)(alpha) (PGF(2)(alpha))-induced luteolysis. During the luteal phase, apelin mRNA expression increased from early to late CL and decreased in regressed CL. APJ mRNA expression increased from early to mid-CL and remained elevated in late and regressed CL. Apelin and APJ proteins were immunohistochemically detected only in the smooth muscle cells of intraluteal arterioles during the luteal phase. PGF(2)(alpha) stimulated apelin and APJ mRNA expression at 0.5-2 and 2 h respectively, and then the mRNA expression of apelin-APJ was inhibited from 4 h during PGF(2)(alpha)-induced luteolysis. Additionally, apelin mRNA and protein were stimulated at 1 h after PGF(2)(alpha) injection only in the periphery of mid- but not early CL. The present study indicated that the apelin-APJ was localized in the smooth muscle cells of intraluteal arterioles, and responded to PGF(2)(alpha) at the periphery of mid-CL in the cow. Thus, the apelin-APJ system may be involved in the maturation of CL and the luteolytic cascade as a regulator of intraluteal arterioles in cow.
Assuntos
Quimiocinas/genética , Corpo Lúteo/metabolismo , Ciclo Estral/fisiologia , Receptores Acoplados a Proteínas G/genética , Animais , Arteríolas , Bovinos , Quimiocinas/análise , Corpo Lúteo/irrigação sanguínea , Corpo Lúteo/química , Dinoprosta/farmacologia , Feminino , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Luteólise , Músculo Liso Vascular/química , Músculo Liso Vascular/metabolismo , RNA Mensageiro/análise , Receptores Acoplados a Proteínas G/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Estimulação QuímicaRESUMO
We previously established a bovine experimental model showing that the corpus luteum (CL) does not appear following aspiration of the preovulatory follicle before the onset of LH surge. Using this model, the present study aimed to determine the profile of follicular development and the endocrinological environment in the absence of CL with variable nadir circulating progesterone (P(4)) concentrations during the oestrous cycle in cattle. Luteolysis was induced in heifers and cows and they were assigned either to have the dominant follicle aspirated (CL-absent) or ovulation induced (CL-present). Ultrasound scanning to observe the diameter of each follicle and blood collection was performed from the day of follicular aspiration or ovulation and continued for 6 days. The CL-absent cattle maintained nadir circulating P(4) throughout the experimental period and showed a similar diameter between the largest and second largest follicle, resulting in co-dominant follicles. Oestradiol (E(2)) concentrations were greater in the CL-absent cows than in the CL-present cows at day -1, day 1 and day 2 from follicular deviation. The CL-absent cows had a higher basal concentration, area under the curve (AUC), pulse amplitude and pulse frequency of LH than the CL-present cows. After follicular deviation, the CL-absent cows showed a greater basal concentration, AUC and pulse amplitude of growth hormone (GH) than the CL-present cows. These results suggest that the absence of CL accompanying nadir circulating P(4) induces an enhancement of LH pulses, which involves the growth of the co-dominant follicles. Our results also suggest that circulating levels of P(4) and E(2) affect pulsatile GH secretion in cattle.
Assuntos
Bovinos/fisiologia , Hormônios do Corpo Lúteo/fisiologia , Corpo Lúteo/fisiologia , Folículo Ovariano/crescimento & desenvolvimento , Animais , Corpo Lúteo/cirurgia , Estradiol/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Fase Folicular/fisiologia , Hormônio do Crescimento/sangue , Fator de Crescimento Insulin-Like I/análise , Hormônio Luteinizante/sangue , Modelos Animais , Folículo Ovariano/diagnóstico por imagem , Ovulação/fisiologia , Progesterona/sangue , UltrassonografiaRESUMO
The purpose of the present study was to investigate the effects of prostaglandin (PG) E(2) and tumor necrosis factor (TNF) alpha on expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in cultured porcine luteal cells. Real-time PCR was used for quantification of VEGF and its receptors mRNA, whereas VEGF release by luteal cells was determined by radioimmunoassay (RIA). Only the highest dose of PGE(2) (1 microM) after 6 hr of incubation stimulated VEGF release by luteal cells collected in the mid-luteal phase (P < 0.05). Moreover, PGE(2) (100 nM, 1 microM) significantly stimulated VEGF secretion by luteal cells in the late phase and during pregnancy on Days 10-12 (P < 0.05). Elevated mRNA expression of both VEGF 120 and VEGF 164 isoforms was found in luteal cells cultured with PGE(2). The lack of an effect of PGE(2) on VEGF receptors mRNA expression was observed. TNFalpha was able to significantly stimulate VEGF release from cells obtained in the mid- and late luteal phase or during early pregnancy. All tested doses enhanced mRNA levels of VEGF 120 isoform, but not VEGF 164. Additionally, TNFalpha was able to decrease Flk-1/KDR mRNA expression, whereas Flt-1 mRNA levels were not affected. These results indicated that PGE(2) and TNFalpha influenced VEGF ligand-receptor system expression in porcine luteal cells and may therefore play an important role in regulation of luteal functions during the estrous cycle and pregnancy in pigs.
Assuntos
Dinoprostona/farmacologia , Células Lúteas/fisiologia , Prenhez/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Células Cultivadas , Dinoprostona/fisiologia , Ciclo Estral , Feminino , Células Lúteas/efeitos dos fármacos , Gravidez , Sus scrofa , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Active angiogenesis and progesterone (P) synthesis occur in parallel during development of the corpus luteum (CL). Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are known to stimulate angiogenesis and P synthesis in vitro. The aim of the present study was to investigate the impact of bFGF or VEGF on the CL development in the cow by using a specific antibody against bFGF or VEGF. bFGF antibody, VEGF antibody, or saline as a control (n = 4 cows/treatment) were injected directly into the CL immediately after ovulation (Day 1), and the treatment was continued for 3 times/day over 7 days. Luteal biopsies were applied on Day 8 of the estrous cycle to determine the expression of genes associated with P synthesis and angiogenesis. Intraluteal injections with the bFGF antibody or the VEGF antibody markedly decreased the CL volume, plasma P concentration and StAR mRNA expression. bFGF antibody treatment decreased the mRNA expression of bFGF, FGF receptor-1, VEGF120, and angiopoietin (ANPT)-1, and increased ANPT-2/ANPT-1 ratio. However, VEGF antibody treatment decreased ANPT-2 mRNA expression and ANPT-2/ANPT-1 ratio. These results indicate that local neutralization of bFGF or VEGF changes genes regulating angiogenesis and P synthesis, and remarkably suppresses the CL size and P secretion during the development of CL in the cow, supporting the concept that bFGF and VEGF control the CL formation and function.
Assuntos
Anticorpos/farmacologia , Bovinos/fisiologia , Corpo Lúteo/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Angiopoietina-1/genética , Angiopoietina-1/metabolismo , Angiopoietina-2/genética , Angiopoietina-2/metabolismo , Animais , Corpo Lúteo/crescimento & desenvolvimento , Corpo Lúteo/metabolismo , Corpo Lúteo/fisiologia , Ciclo Estral/sangue , Ciclo Estral/efeitos dos fármacos , Ciclo Estral/genética , Feminino , Fator 2 de Crescimento de Fibroblastos/imunologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Progesterona/sangue , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
The aim of the present study was to evaluate the pattern of regulation of vascular endothelial growth factor (VEGF)-A (isoforms 121, 165, 189), VEGF receptor tyrosine kinases (VEGF-R1 and VEGF-R2), matrix metalloproteinase (MMP)-1, MMP-2, MMP-14, MMP-19, tissue-specific inhibitor of metalloproteinases (TIMP)-1, TIMP-2, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) in time-defined follicle classes before (0 h) and after the application of gonadotrophin-releasing hormone (GnRH). Bovine ovaries containing periovulatory follicles or new corpora lutea (CL; Days 1-2) were collected 0, 4, 10, 20 and 25 h (follicles) or 60 h (CL) after the injection of GnRH. Transcripts of VEGF isoforms (VEGF(121), VEGF(165), VEGF(189)) were upregulated 4 h after GnRH injection (during the luteinising hormone (LH) surge) and decreased thereafter to lowest levels around ovulation. All VEGF isoforms and their receptors were upregulated again after ovulation. The VEGF peptide concentration in follicular fluid decreased 20 h after GnRH injection, followed by an increase in follicles 25 h after GnRH. Expression of MMP-1 mRNA increased rapidly 4 h after GnRH injection and remained high during the entire experimental period. In contrast, MMP-19 mRNA increased significantly only after ovulation. Expression of TIMP-1 mRNA increased 4 h after GnRH and again after ovulation. Expression of tPA mRNA increased 4 h after GnRH and remained high during the entire experimental period, whereas expression of uPA transcripts increased significantly only after ovulation. Both uPAR and PAI-1 mRNA levels increased in follicles 4 h after GnRH and again after ovulation. The amount of MMP-1 protein (immunolocalisation) increased in follicles 10 h after GnRH: additional staining was observed in the granulosa cell layer. In conclusion, the temporal and spatial pattern of regulation of VEGF and extracellular matrix-degrading proteinases during periovulation suggests they are important mediators of the LH-dependent rupture of bovine follicles and for early CL formation (angiogenesis).
Assuntos
Bovinos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/farmacologia , Metaloproteinases da Matriz/metabolismo , Folículo Ovariano/efeitos dos fármacos , Ativadores de Plasminogênio/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Análise de Variância , Animais , Primers do DNA/genética , Feminino , Imuno-Histoquímica , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
The effect of insulin-like growth factor-I (IGF-I), relaxin (RLX) and luteinizing hormone (LH) on vascular endothelial growth factor (VEGF) in vitro secretion by endometrial stromal cells in pigs was investigated on days 10-12 and 20-22 of gestation. LH-stimulated stromal cell secretion of VEGF did not differ among tested days of early pregnancy. However, IGF-I- and RLX-mediated release of VEGF depended on the day of pregnancy. It seems that IGF-I and RLX may be considered as potent activators of VEGF-mediated angiogenesis in porcine endometrium, and their action may be more pronounced during maternal recognition of pregnancy.
Assuntos
Endométrio/citologia , Fator de Crescimento Insulin-Like I/farmacologia , Hormônio Luteinizante/farmacologia , Prenhez/fisiologia , Relaxina/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Células Cultivadas , Feminino , Neovascularização Fisiológica/efeitos dos fármacos , Gravidez , Células Estromais/metabolismo , Suínos , Fatores de TempoRESUMO
The aim of the present study was, to investigate the effects of oxytocin administration on Day 7 post-ovulation on progesterone secretion, pregnancy rate and embryonic growth in mares. Endogenous stimulation of oxytocin release was compared to the administration of native oxytocin or the long-acting oxytocin analogue carbetocin. At Day 7 after ovulation, mares had to undergo four treatments in a crossover design: (a) control, (b) oxytocin (10 IU i.v.), (c) carbetocin (280 microg i.m.) and (d) cervical dilation. On Day 13, all mares (8 of 8 mares) were pregnant on groups control, oxytocin and carbetocin and only 6 of 8 mares on group dilation. In one mare uterine fluid accumulation and uterine edema from Day 6 to 13 and early embryonic death by Day 11 occurred during dilation treatment. Another mare, which did not become pregnant during dilation treatment, developed uterine fluid accumulation and uterine edema from Day 10 to 14. Mean growth rates of the conceptuses did not differ among treatment groups and individual growth rates varied in a wide range from -0.1 to 0.8 cm per day. At Day 13, mean diameters of conceptuses yielded 1.4+/-0.1 cm in control group, 1.5+/-0.1 in oxytocin and carbetocin group and 1.3+/-0.2 cm in dilation group. Secretion of progesterone was not affected by treatments. Administration of oxytocin and carbetocin caused similar maximum plasma concentrations of oxytocin, but onset and duration of peaks differed. Maximum concentrations after intramuscular application of carbetocin were obtained almost 20 min later when compared to intravenous administration of oxytocin. Duration of peaks after injection of the long-acting oxytocin analogue was more than three-fold longer than after administration of native oxytocin. In conclusion, the present study showed that single administration of oxytocin or its long-acting analogue carbetocin at Day 7 after ovulation did not affect progesterone secretion, pregnancy rate and embryonic growth. Two possible scenarios concerning the effects of cervical dilation were observed: In the majority of mares, dilation of the caudal half to two-third of the cervical lumen up to a diameter of 4.5 cm had no negative consequences on progesterone secretion and pregnancy outcome. However, cervical dilation caused uterine inflammation and subsequent luteolysis in two mares and early embryonic death in one of them. Thus, manipulation of the cervix itself seems not to have negative impact on success rates of transcervical transfer of embryos in the mare.
Assuntos
Colo do Útero/fisiologia , Desenvolvimento Embrionário , Cavalos/embriologia , Cavalos/fisiologia , Ocitocina/farmacologia , Progesterona/metabolismo , Animais , Colo do Útero/efeitos dos fármacos , Estudos Cross-Over , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Feminino , Ocitócicos/farmacologia , Ocitocina/análogos & derivados , Ocitocina/fisiologia , Gravidez , Taxa de Gravidez , Distribuição Aleatória , Fatores de TempoRESUMO
In the female reproductive system, as in a few adult tissues, angiogenesis occurs as a normal process and is essential for normal tissue growth and development. In the ovary, new blood vessel formation facilitates oxygen, nutrients, and hormone substrate delivery, and also secures transfer of different hormones to targeted cells. Ovarian follicle and the corpus luteum (CL) have been shown to produce several angiogenic factors, however, vascular endothelial growth factor (VEGF) is thought to play a paramount role in the regulation of normal and abnormal angiogenesis in the ovary. Expression of VEGF in ovarian follicles depends on follicular size. Inhibition of VEGF expression results in decreased follicle angiogenesis and the lack of the development of mature antral follicles. The permeabilizing activity of VEGF is thought to be involved in follicle antrum formation and in the ovulatory process. In the CL, VEGF expression corresponds to different patterns of angiogenesis during its lifespan. In most the species, higher VEGF expression in the early luteal phase is essential for the development of a high-density capillary network in the CL. However, high VEGF expression may be still maintained in the mid-luteal phase to increase vascular permeability that results in enhancement of luteal function. During gestation, VEGF is thought to be important for the persistence of the CL function for a longer than in the nonfertile cycle period of time. Further elucidation of specific roles of VEGF in ovarian physiology may help to understand the phenomenon of luteal insufficiency and reveal novel strategies of ovarian angiogenesis manipulation to alleviate infertility or to control fertility.