RESUMO
Embryonic mortality in cattle is high, reaching 10-40 % in vivo and 60-70 % in vitro. Death of embryos involves reduced expression of genes related to embryonic viability, inhibition of DNA repair and increased DNA damage. In follicular granulosa cells, FGF18 from the theca layer increases apoptosis and DNA damage, so we hypothesized that FGF18 may also affect the oocyte and contribute to early embryonic death. The aims of this study were to identify the effects of FGF18 on cumulus expansion, oocyte maturation and embryo development from cleavage to blastocyst stage using a conventional bovine in vitro embryo production system using ovaries of abattoir origin. Addition of FGF18 during in-vitro maturation did not affect FSH-induced cumulus expansion or rates of nuclear maturation. When FGF18 was present in the culture system, rates of cleavage were not affected however, blastocyst and expanded blastocyst development was substantially inhibited (P < 0.05), indicating a delay of blastulation. The number of phosphorylated histone H2AFX foci per nucleus, a marker of DNA damage, was higher in cleavage-stage embryos cultured with FGF18 than in those from control group (P < 0.05). Furthermore, FGF18 decreased accumulation of PTGS2 and IFNT2 mRNA in blastocysts. In conclusion, these novel findings suggest that FGF18 plays a role in the regulation of embryonic death during the early stages of development by impairing DNA double-strand break repair and expression of genes associated with embryo viability and maternal recognition of pregnancy during the progression from oocyte to expanded blastocysts.
Assuntos
Blastocisto , Quebras de DNA de Cadeia Dupla , Fatores de Crescimento de Fibroblastos , Animais , Feminino , Bovinos , Blastocisto/efeitos dos fármacos , Blastocisto/fisiologia , Gravidez , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Cultura Embrionária/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacosRESUMO
Metabolic stress conditions caused by negative energy balance (NEB) have been associated with reduced fertility in cows. ß-hydroxybutyrate (BHBA) is the main circulating ketone body, which accumulates within follicular fluid. The aim of this study was to evaluate the effects of BHBA on follicle growth and on ovulatory mechanisms in cattle. At 72 h after intrafollicular injection, there was a decrease in follicular diameter in BHBA group compared to control (P = 0.02). Furthermore, follicle growth rate was reduced post-treatment with BHBA in comparison to the control group (P < 0.03). The BHBA intrafollicular injection in follicles ≥ 12 mm, however, did not affect E2 and P4 concentrations in the follicular fluid. In addition, the relative abundance of genes involved in the ovulatory cascade (ADAM 17, AREG, EREG, PTGS2), steroidogenesis (CYP19A1, 3BHSD, STAR), cellular stress (SOD1, CAT, GPX1, HSPA5, XBP1s, XBP1u, ATF4, ATF6), monocarboxylic acid transporters (SLC16A1, SLC16A7) and apoptosis (XIAP) was similar between groups. In conclusion, the results of this study indicate that the increase in intrafollicular concentrations of BHBA affects follicular growth, but it does not compromise the ovulatory cascade and cellular homeostasis in bovine granulosa cells.
Assuntos
Células da Granulosa , Folículo Ovariano , Ácido 3-Hidroxibutírico/metabolismo , Animais , Bovinos , Feminino , Fertilidade , Líquido Folicular , Células da Granulosa/metabolismoRESUMO
Failure to ovulate is a major cause of infertility. The critical pathway that induces ovulation involves the EGF and MAPK phosphorylation, but studies in rodents have suggested that the Hippo activator, YAP, is also involved. It is unknown whether YAP-dependent transcriptional activity is important for the LH- or EGF-induced ovulatory cascade in monovulatory species such as the cow. Using a well-defined preovulatory GC culture system, we employed pharmacological inhibitors to demonstrate that YAP signaling is critical for expression of EGFR and downstream target genes EREG, EGR1 and TNFAIP6. Most importantly, by using an ultrasound guided follicle injection system, we also showed that the classic Hippo signaling inhibitor Verteporfin inhibits GnRH-induced ovulation in vivo in cattle. In conclusion, YAP transcriptional activity is critical for EGF-like cascade induced by LH to promote ovulation in a monovulatory species.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Células da Granulosa/metabolismo , Ovulação/fisiologia , Proteínas de Sinalização YAP/fisiologia , Animais , Bovinos , Células Cultivadas , Feminino , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/fisiologia , Hormônio Luteinizante/farmacologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Ovulação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas de Sinalização YAP/genéticaRESUMO
Postpartum cows, mainly with metabolic diseases, such as ketosis, usually experience an increased number of services per conception. During ketosis, high concentrations of ß-hydroxybutyrate (BHBA) in follicular, uterine and oviductal fluid have been considered to cause subfertility in cows. However, the effect of sperm exposure to an environment with high BHBA concentration is not known. This study investigated the influence of high levels of BHBA on kinetics, oxidative status and morphology of bovine spermatozoa. To assess the effect of BHBA after sperm selection, bovine spermatozoa were incubated (180 min) with different BHBA concentrations: 0 (Control), 0.8, 2.4 or 5 mM. Sperm kinetics was evaluated after 30, 60, 120 and 180 min, and oxidative status and morphology were analysed at 180 min. Oxidative status was evaluated through the production of reactive oxidative species (ROS), total antioxidant capacity and lipid peroxidation. High concentrations of BHBA decreased the curvilinear velocity, straight line velocity, mean path velocity, linearity, straightness and hyperactivity of spermatozoa. However, there was no effect of BHBA on oxidative and antioxidant capacity as well as on sperm morphology. In conclusion, exposure of bovine spermatozoa to high levels of BHBA impairs sperm kinetics without altering oxidative and antioxidant mechanisms.
Assuntos
Doenças dos Bovinos , Cetose , Ácido 3-Hidroxibutírico , Animais , Bovinos , Feminino , Cinética , Masculino , EspermatozoidesRESUMO
Although prostaglandins are important in the ovulation process, a precise role for prostaglandin F2α (PGF) has not been elucidated. This study aimed to evaluate the regulation of PGF receptor mRNA (PTGFR) in granulosa cells and the local effect of PGF on ovulation and luteinization. In Experiment 1, using samples collected in vivo before (Day 2), during (Day 3) and after (Day 4) follicular deviation, expression of PTGFR in bovine granulosa cells was more abundant in the dominant follicle after deviation than in subordinates (P < 0.05). However, the expression of PTGFR was not regulated (P = 0.1) in preovulatory follicles at different time-points (0, 3, 6, 12 and 24 h) after ovulation induction with GnRH. In Experiment 2, to assess the role of systemic PGF treatment on luteinization and vascularization of preovulatory follicles, flunixin meglumine (FM), a nonsteroidal anti-inflammatory drug, was used to inhibit endogenous prostaglandin synthesis. Cows with preovulatory follicles were induced to ovulate with GnRH (0 h) and allocated to three groups: Control, with no further treatment; FM, treated with 2.2 mg/kg FM im 17 h after GnRH treatment; and FM + PGF, treated with FM 17 h after GnRH, followed by 25 mg dinoprost tromethamine (PGF) 23 h after GnRH treatment. FM injection was able to reduce the concentration of PGF in the follicular fluid (FF) (P < 0.001). However, contrary to our hypothesis, color Doppler ultrasound evaluations revealed decreased vascular flow in FM + PGF group (P < 0.05), and no effect of the treatments on intrafollicular P4 and E2 concentrations 24 h after GnRH. The prostaglandin metabolite (PGFM) concentrations in the FF were greater in cows receiving systemic PGF (P < 0.001), which prompted us to further check its role on ovulation. Therefore, in Experiment 3, in a final attempt to demonstrate the local effect of PGF on ovulation, cows with preovulatory follicles received an intrafollicular injection (IFI) of PBS (Control) or 100 ng/mL purified PGF (PGF group). PGF treatment did not affect the time of ovulation after IFI (66 ± 6.4 and 63 ± 8.5 h for control and PGF, respectively; P > 0.05), further suggesting that it has no direct effect in the ovulatory process. Based on our findings, we concluded that FM decreased PGF synthesis within the follicle, whereas PGF treatment decreased follicular vascularization. In addition, the in vivo model of intrafollicular injection evidenced that PGF alone is not able to locally induce ovulation.
Assuntos
Dinoprosta , Progesterona , Animais , Bovinos , Dinoprosta/farmacologia , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Luteinização , Folículo Ovariano , OvulaçãoRESUMO
In this study, a GnRH agonist, leuprolide acetate (LA), was given as a single depot injection before 48 h of life to Wistar female rats allotted to prenatal (E16-18) and postnatal androgenization (day 5 of life) by the use of testosterone propionate, looking for reproductive endpoints. Remarkably, a single injection of LA increased the estrus cycles in the postnatal group (PostN) from 0% to 25% of the estrus cycles in the postnatal LA treated group (PostN L). LA also reduced the serum testosterone levels and cysts and atretic follicles in PostN L in contrast with rats (>100 days) from the PostN group (p = 0.04). Prenatally androgenized rats (PreN) exhibited significant modifications in the hypothalamic genes, such as Gnrh. To the best of our knowledge, this is the first study to show that blockage of the GnRH axis with leuprolide acetate depot prevented the development of typical features (anovulation, cysts, atretic follicles) in a postnatal testosterone propionate rat model of PCOS.
Assuntos
Leuprolida/farmacologia , Síndrome do Ovário Policístico/tratamento farmacológico , Reprodução/efeitos dos fármacos , Animais , Anovulação/tratamento farmacológico , Anovulação/metabolismo , Ciclo Estral/efeitos dos fármacos , Feminino , Hormônio Liberador de Gonadotropina/metabolismo , Masculino , Folículo Ovariano/metabolismo , Síndrome do Ovário Policístico/metabolismo , Ratos , Ratos Wistar , Testosterona/metabolismo , Virilismo/tratamento farmacológico , Virilismo/metabolismoRESUMO
In the present work, two biochemical approaches were used to characterize PAGs isolated from Bos indicus fetal cotyledons removed at different gestational ages. The first procedure included acidic and ammonium sulfate precipitations, anion and cation exchange chromatographies and the second included pepstatin-agarose affinity chromatography. A bovine PAG radioimmunoassay was used to monitor the immunoreactivity throughout the isolation procedures. The most immunoreactive fractions issued from cation exchange and affinity chromatographies were analyzed by SDS-PAGE and Western blotting, before transfer to a polyvinylidene difluoride (PVDF) membrane for NH2-microsequence determination. Use SDS-PAGE and Western blotting, different isoforms of PAG with apparent molecular masses of 51 to 69 kDa and isoelectric points varying from 4.4 to 6.7 were identified in the placentas from different gestational ages. N-terminal microsequencing (10 to 25 aa long) indicates the expression of one single terminal amino acid sequence in the Bos indicus placenta, which is 100% identical to the bovine PAG-1.