RESUMO
A 60% pregnancy success for inseminations is targeted to optimize production efficiency for dairy cows within a seasonal, pasture-grazed system. Routine measures of pregnancy success are widely available but are limited, in practice, to a gestation stage beyond the first 28 d. Although some historical data exist on embryonic mortality before this stage, productivity of dairy systems and genetics of the cows have advanced significantly in recent decades. Accordingly, the aim was to construct an updated estimate of pregnancy success at key developmental stages during the first 70 d after insemination. Blood samples were collected for progesterone concentrations on d 0 and 7. A temporal series of 4 groups spanning fertilization through d 70 were conducted on 4 seasonal, pasture-grazed dairy farms (n = 1,467 cows) during the first 21 d of the seasonal breeding period. Morphological examination was undertaken on embryos collected on d 7 (group E7) and 15 (group E15), and pregnancy was diagnosed via ultrasonography on approximately d 28 and 35 (group E35) as well as d 70 (group E70). Fertilization, embryo, and fetal evaluation for viability established a pregnancy success pattern. Additionally, cow and on-farm risk factor variables associated with pregnancy success were evaluated. We estimated pregnancy success rates of 70.9%, 59.1%, 63.8%, 62.3%, and 56.7% at d 7, 15, 28, 35, and 70, respectively. Fertilization failure (15.8%) and embryonic arrest before the morula stage (10.3%) were the major developmental events contributing to first-week pregnancy failures. Embryo elongation failure of 7% contributed to pregnancy failure during the second week. The risk factors for pregnancy success that were related to the cows included interval between calving and insemination, and d-7 plasma progesterone concentrations, whereas insemination sire was associated with pregnancy outcome. Most pregnancy failure occurs during the first week among seasonal-calving pasture-grazed dairy cows.
Assuntos
Lactação , Progesterona , Feminino , Bovinos , Gravidez , Animais , Leite , Resultado da Gravidez/veterinária , Inseminação , Inseminação Artificial/veterinária , ReproduçãoRESUMO
Fertility of the dairy cow relies on complex interactions between genetics, physiology, and management. Mathematical modeling can combine a range of information sources to facilitate informed predictions of cow fertility in scenarios that are difficult to evaluate empirically. We have developed a stochastic model that incorporates genetic and physiological data from more than 70 published reports on a wide range of fertility-related traits in dairy cattle. The model simulates pedigree, random mating, genetically correlated traits (in the form of breeding values for traits such as hours in estrus, estrous cycle length, age at puberty, milk yield, and so on), and interacting environmental variables. This model was used to generate a large simulated data set (200,000 cows replicated 100 times) of herd records within a seasonal dairy production system (based on an average New Zealand system). Using these simulated data, we investigated the genetic component of lifetime reproductive success (LRS), which, in reality, would be impractical to assess empirically. We defined LRS as the total number of times, during her lifetime, a cow calved within the first 42 d of the calving season. Sire estimated breeding values for LRS and other traits were calculated using simulated daughter records. Daughter pregnancy rate in the first lactation (PD_1) was the strongest single predictor of a sire's genetic merit for LRS (R2 = 0.81). A simple predictive model containing PD_1, calving date for the second season and calving rate in the first season provided a good estimate of sire LRS (R2 = 0.97). Daughters from sires with extremely high (n = 99,995 daughters, sire LRS = +0.70) or low (n = 99,635 daughters, sire LRS = -0.73) LRS estimated breeding values were compared over a single generation. Of the 14 underlying component traits of fertility, 12 were divergent between the 2 lines. This suggests that genetic variation in female fertility has a complex and multifactorial genetic basis. When simulated phenotypes were compared, daughters of the high LRS sires (HiFERT) reached puberty 44.5 d younger and calved â¼14 d younger at each parity than daughters from low LRS sires (LoFERT). Despite having a much lower genetic potential for milk production (-400 L/lactation) than LoFERT cows, HiFERT cows produced 33% more milk over their lifetime due to additional lactations before culling. In summary, this simulation model suggests that LRS contributes substantially to cow productivity, and novel selection criteria would facilitate a more accurate prediction at a younger age.
Assuntos
Cruzamento , Bovinos/fisiologia , Fertilidade/genética , Reprodução/genética , Animais , Bovinos/genética , Feminino , Variação Genética , Masculino , Modelos Genéticos , Nova Zelândia , Seleção GenéticaRESUMO
Bovine adrenal chromaffin cells have nicotinic acetylcholine receptors (AChRs) that are activated by the splanchnic nerve, resulting in release of catecholamines from the cells. Examination of the AChRs can provide information about the regulation and turnover of synaptic components on neurons and endocrine cells. Previous studies have shown that mAb 35 recognizes the AChR on the cells. Here we show that mAb 35 can remove AChRs from the surface of the cells by antigenic modulation, and that the modulation can be used together with other methods to examine the stability and turnover of the receptors in the plasma membrane. Unexpectedly, the results indicate a disparity between the rate at which AChRs reappear on the cells and the rate at which the ACh response recovers after preexisting AChRs have been removed. Exposure of bovine adrenal chromaffin cultures to mAb 35 results in a parallel decrease in the magnitude of the nicotinic response and the number of AChRs on the cells. The decrease depends on the concentration and divalence of mAb 35, and on the time and temperature of the incubation. The antibody induces receptor aggregation in the plasma membrane under conditions where receptor loss subsequently occurs. After binding to receptor, mAb 35 appears to be internalized, degraded, and released from the cells through a temperature sensitive pathway that requires lysosomal function. These features are characteristic of antigenic modulation. Appearance of new AChRs on the cells either after antigenic modulation or after blockade of existing AChRs with monovalent antibody fragments occurs at a rate equivalent to 3% of the receptors present on control cells per hour. The rate of receptor loss from the cells was measured in the presence of either tunicamycin or puromycin to block appearance of new receptors. Both conditions indicated a receptor half-life of approximately 24 h and a rate of loss of approximately 3%/h. The finding that the rate of receptor loss equaled the rate of receptor appearance was consistent with the observation that the total number of AChRs on untreated cells did not increase with time. In the presence of tunicamycin, loss of receptor-mediated response to nicotine also occurred with a half-time of 24 h. Paradoxically, the rate of recovery of the nicotinic response, determined using two procedures, was more than twice as great as the rate at which new AChRs appeared on the cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Glândulas Suprarrenais/citologia , Grânulos Cromafim/metabolismo , Sistema Cromafim/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Variação Antigênica , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Centrifugação com Gradiente de Concentração , Grânulos Cromafim/ultraestrutura , Cromatografia em Gel , Meia-Vida , Receptores Nicotínicos/imunologiaRESUMO
Bovine adrenal chromaffin cells have nicotinic acetylcholine receptors (AChRs) that mediate release of catecholamines from the cells in response to synaptic input, and resemble neuronal AChRs in pharmacology and antigenic profile. Results presented here show that a cAMP-dependent process enhances the function of adrenal chromaffin AChRs as a population in the plasma membrane. This was demonstrated by showing that cAMP analogues cause specific increases both in the level of nicotine-induced catecholamine release from the cells and in the level of the nicotine-induced conductance change occurring in the cells. Neither de novo synthesis of receptors nor transport of preexisting intracellular receptors to the plasma membrane is necessary for the enhancement. The responsiveness of AChRs to regulation by the cAMP-dependent process appears to depend on the length of time the receptors have been on the cell surface. AChRs newly inserted into the plasma membrane generate a greater nicotinic response than do older AChRs and, unlike older AChRs, their response to agonist is not enhanced after treatment of the cells with cAMP analogues. The findings indicate that the AChRs and/or associated components undergo a maturation in the plasma membrane that alters their function and their regulation by secondary messenger systems.
Assuntos
Grânulos Cromafim/metabolismo , Sistema Cromafim/metabolismo , AMP Cíclico/farmacologia , Receptores Nicotínicos/metabolismo , Glândulas Suprarrenais/citologia , Animais , Catecolaminas/metabolismo , Bovinos , Membrana Celular/metabolismo , AMP Cíclico/análogos & derivados , Eletrofisiologia , Nicotina/farmacologia , Receptores Nicotínicos/efeitos dos fármacosRESUMO
The regulation of nicotinic acetylcholine receptors (AChRs) in chick ciliary ganglia was examined by using a radiolabeled anti-AChR mAb to quantitate the amount of receptor in ganglion detergent extracts after preganglionic denervation or postganglionic axotomy. Surgical transection of the preganglionic input to the ciliary ganglion in newly hatched chicks caused a threefold reduction in the total number of AChRs within 10 d compared with that present in unoperated contralateral control ganglia. Surgical transection of both the choroid and ciliary nerves emerging from the ciliary ganglion in newly hatched chicks to establish postganglionic axotomy led to a nearly 10-fold reduction in AChRs within 5 d compared with unoperated contralateral ganglia. The declines were specific since they could not be accounted for by changes in ganglionic protein or by decreases in neuronal survival or size. Light microscopy revealed no gross morphological differences between neurons in operated and control ganglia. A second membrane component of cholinergic relevance on chick ciliary ganglion neurons is the alpha-bungarotoxin (alpha-Bgt)-binding component. The alpha-Bgt-binding component also declined in number after either postganglionic axotomy or preganglionic denervation, but appeared to do so with a more rapid time course than did ganglionic AChRs. The results imply that cell-cell interactions in vivo specifically regulate both the number of AChRs and the number of alpha-Bgt-binding components in the ganglion. Regulation of these neuronal cholinergic membrane components clearly differs from that previously described for muscle AChRs.
Assuntos
Gânglios Parassimpáticos/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Bungarotoxinas/metabolismo , Comunicação Celular , Sobrevivência Celular , Galinhas , Denervação , Gânglios Parassimpáticos/citologia , Proteínas do Tecido Nervoso/metabolismo , Fatores de TempoRESUMO
Spinal cord cell cultures contain several types of neurons. Two methods are described for enriching such cultures with motoneurons (defined here simply as cholinergic cells that are capable of innervating muscle). In the first method, 7-day embryonic chick spinal cord neurons were separated according to size by 1 g velocity sedimentation. It is assumed that cholinergic motoneurons are among the largest cells present at this stage. The spinal cords were dissociated vigorously so that 95-98% of the cells in the initial suspension were isolated from one another. Cells in leading fractions (large cell fractions: LCFs) contain about seven times as much choline acetyltransferase (CAT) activity per unit cytoplasm as do cells in trailing fractions (small cell fractions: SCFs). Muscle cultures seeded with LCFs develop 10-70 times as much CAT as cultures seeded with SCFs and six times as much CAT as cultures seeded with control (unfractionated) spinal cord cells. More than 20% of the large neurons in LCF-muscle cultures innervate nearby myotubes. In the second method, neurons were gently dissociated from 4-day embryonic spinal cords and maintained in vitro. This approach is based on earlier observations that cholinergic neurons are among the first cells to withdraw form the mitotic cycle in the developing chick embryo (Hamburger, V. 1948. J. Comp. Neurol. 88:221-283; and Levi-Montalcini, R. 1950. J. Morphol. 86:253-283). 4-Day spinal cord-muscle cultures develop three times as much CAT as do 7-day spinal cord-muscle plates, prepared in the same (gentle) manner. More than 50% of the relatively large 4-day neurons innervate nearby myotubes. Thus, both methods are useful first steps toward the complete isolation of motoneurons. Both methods should facilitate study of the development of cholinergic neurons and of nerve-muscle synapse formation.
Assuntos
Separação Celular/métodos , Células Cultivadas , Neurônios Motores/citologia , Medula Espinal/citologia , Animais , Contagem de Células , Sobrevivência Celular , Centrifugação com Gradiente de Concentração , Embrião de Galinha , Colina O-Acetiltransferase/metabolismo , Potenciais da Membrana , Junção Neuromuscular , SinapsesRESUMO
Chick ciliary ganglion neurons have a membrane component that shares an antigenic determinant with the main immunogenic region (MIR) of nicotinic acetylcholine receptors from skeletal muscle and electric organ. Previous studies have shown that the component has many of the properties expected for a ganglionic nicotinic acetylcholine receptor, and that its distribution on the neuron surface in vivo is restricted predominantly to synaptic membrane. Here we report the presence of a large intracellular pool of the putative receptor in embryonic neurons and demonstrate that it is associated with organelles known to comprise the biosynthetic and regulatory pathways of integral plasma membrane proteins. Embryonic chick ciliary ganglia were lightly fixed, saponin-permeabilized, incubated with an anti-MIR monoclonal antibody (mAb) followed by horseradish peroxidase-conjugated secondary antibody, reacted for peroxidase activity, and examined by electron microscopy. Deposits of reaction product were associated with synaptic membrane, small portions of the pseudodendrite surface membrane, most of the rough endoplasmic reticulum, small portions of the nuclear envelope, some Golgi complexes, and a few coated pits, coated vesicles, multivesicular bodies, and smooth-membraned vacuoles. No other labeling was present in the neurons. The labeling was specific in that it was not present when the anti-MIR mAb was replaced with either nonimmune serum or mAbs of different specificity. Chick dorsal root ganglion neurons thought to lack nicotinic acetylcholine receptors were not labeled by the anti-MIR mAb. Substantial intracellular populations have also been reported for the muscle acetylcholine receptor and brain voltage-dependent sodium channel alpha-subunit. This may represent a general pattern for multisubunit membrane proteins during development.
Assuntos
Gânglios Parassimpáticos/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Anticorpos Monoclonais , Compartimento Celular , Membrana Celular/metabolismo , Embrião de Galinha , Gânglios Parassimpáticos/citologia , Complexo de Golgi/metabolismo , Técnicas Imunológicas , Membranas Intracelulares/metabolismo , Microscopia Eletrônica , Receptores Nicotínicos/imunologia , Membranas Sinápticas/metabolismoRESUMO
We have investigated the uptake and release of [3H]gamma-aminobutyric acid (GABA) by embryonic chick spinal cord cells maintained in culture. Cells dissociated from 4- or 7-d-old embryos were studied between 1 and 3 wk after plating. At 3 degrees C, [3H]GABA was accumulated by a high affinity (Km approximately equal to 4 microM) and a low affinity (Km approximately equal to 100 microM) mechanism. The high affinity transport was markedly inhibited in low Na+ media, by ouabain, at 0 degrees C, and by 2,4-diaminobutyric acid. Autoradiography, after incubation in 0.1 microM [3H]GABA, showed that approximately 50% (range = 30-70%) of the multipolar cells were labeled. These cells were neurons rather than glia; action potentials and/or synaptic potentials were recorded in cells subsequently found to be labeled. Non-neuronal, fibroblast-like cells and co-cultured myotubes were not labeled under the same conditions. The fact that not all of the neurons were labeled is consistent with the suggestion, based on studies of intact adult tissue, that high affinity transport of [3H]GABA may be unique to neurons that use GABA as a neurotransmitter. Our finding that none of fifteen physiologically identified cholinergic neurons, i.e., cells that innervated nearby myotubes, were heavily labeled after incubation in 0.1 microM [3H]GABA is significant in this regard. The newly taken up [3H]GABA was not metabolized in the short run. It was stored in a form that could be released when the neurons were depolarized in a high K+ (100 mM) medium. As expected for a neurotransmitter, the K+-evoked release was reversibly inhibited by reducing the extracellular Ca++/Mg++ ratio.
Assuntos
Neurônios/metabolismo , Ácido gama-Aminobutírico/metabolismo , Aminobutiratos/farmacologia , Animais , Embrião de Galinha , Técnicas de Cultura , Neurônios Motores/metabolismo , Músculos , Ouabaína/farmacologia , Sódio/farmacologia , Medula EspinalRESUMO
In organ culture, alpha-[(125)I]bungarotoxin bound to extrajunctional receptors of denervated muscle is lost from the tissue at a more rapid rate than the toxin bound to the junctional receptors of normal muscle. The rapid loss of toxin from denervated muscle can be blocked by inhibitors of energy production and protein synthesis, and may reflect turnover of the toxin-receptor complex in the membrane.
Assuntos
Bungarotoxinas/metabolismo , Denervação Muscular , Músculos/metabolismo , Receptores Colinérgicos/efeitos dos fármacos , Animais , Sítios de Ligação , Cicloeximida/farmacologia , Diafragma , Radioisótopos do Iodo , Cinética , Proteínas Musculares/biossíntese , Músculos/inervação , Junção Neuromuscular/metabolismo , Técnicas de Cultura de Órgãos , Puromicina/farmacologia , RatosRESUMO
Synaptic activation of the transcription factor CREB and downstream gene expression usually depend on calcium influx aided by voltage-gated calcium channels. We find that nicotinic signaling, in contrast, activates CREB and gene expression in ciliary ganglion neurons both in culture and in situ only if voltage-gated channels are silent. The nicotinic response requires calcium influx and release from internal stores and acts through CaMK and MAPK pathways to sustain activated CREB. Voltage-gated channels mobilize CaMK to activate CREB initially, but they also enable calcineurin and PP1 to terminate the activation before transcription is affected. L-type voltage-gated channels dominate the outcome and block the effects of nicotinic signaling on transcription. This demonstrates a novel aspect of activity-dependent gene regulation.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica/fisiologia , Ativação do Canal Iônico/fisiologia , Neurônios/metabolismo , Receptores Nicotínicos/fisiologia , Animais , Células Cultivadas , Embrião de Galinha , Relação Dose-Resposta a Droga , Estimulação Elétrica , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Fosforilação/efeitos dos fármacos , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Neuronal membrane components that bind alpha-bungarotoxin with high affinity can increase intracellular levels of free calcium, demonstrating the components function as nicotinic receptors. Though such receptors often contain the alpha 7 gene product, which by itself can produce ionotropic receptors in Xenopus oocytes, numerous attempts have failed to demonstrate an ion channel function for the native receptors on neurons. Using rapid application of agonist, we show here that the native receptors are ligand-gated ion channels which are cation selective, prefer nicotine over acetylcholine, and rapidly desensitize. Much of the calcium increase caused in neurons by the receptors under physiological conditions appears to result from their depolarizing the membrane sufficiently to trigger calcium influx through voltage-gated channels.
Assuntos
Bungarotoxinas/farmacologia , Canais Iônicos/fisiologia , Neurônios/fisiologia , Nicotina/farmacologia , Receptores Colinérgicos/fisiologia , Animais , Bungarotoxinas/metabolismo , Cádmio/farmacologia , Células Cultivadas , Embrião de Galinha , Potenciais Evocados/efeitos dos fármacos , Feminino , Gânglios Simpáticos/fisiologia , Ativação do Canal Iônico , Canais Iônicos/efeitos dos fármacos , Cinética , Potenciais da Membrana/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Receptores Colinérgicos/biossíntese , Receptores Colinérgicos/metabolismo , Tetrodotoxina/farmacologia , Xenopus laevisRESUMO
The acetylcholine receptor (AChR) alpha 5 gene has been classified as a member of the AChR gene family based on sequence homology. Expression studies, however, have yet to identify a function for the alpha 5 gene product or even to demonstrate an interaction with known AChR subunits. We report here that the alpha 5 gene product is identical to the 49 kd protein previously found on immunoblots of AChRs purified from brain and ciliary ganglia. In brain the alpha 5 gene product is present both in alpha 3- and in alpha 4-based receptor subtypes, while in the ganglion it is found in an alpha 3-based receptor subtype concentrated in postsynaptic membrane. Immunoprecipitation experiments with subunit-specific monoclonal antibodies indicate that some native AChRs are likely to have at least three kinds of subunits, with two being of the alpha type. These findings support new views about the construction of AChRs in neurons.
Assuntos
Encéfalo/fisiologia , Família Multigênica , Receptores Colinérgicos/biossíntese , Receptores Colinérgicos/genética , Animais , Anticorpos Monoclonais , Embrião de Galinha , Órgão Elétrico/fisiologia , Eletroforese em Gel de Poliacrilamida , Electrophorus , Gânglios Parassimpáticos/fisiologia , Immunoblotting , Substâncias Macromoleculares , Músculos/fisiologia , Biossíntese de Proteínas , Receptores Colinérgicos/análise , Proteínas Recombinantes de Fusão/análiseRESUMO
A family of genes encoding neuronal acetylcholine receptor (AChR) subunits has been identified and cloned from vertebrates. Expression studies have implied that as few as one or two kinds of subunits may be sufficient to construct neuronal AChRs and that multiple pair-wise combinations of the gene products are capable of generating functional receptors. We show here that a class of AChRs with a predominantly synaptic location on neurons contains receptors having at least three types of subunits and that the subunits are encoded by the alpha 3, beta 4, and alpha 5 AChR genes. In addition, we show that a class of extrasynaptic AChRs on the same neurons contains the alpha 7 subunits but lacks the alpha 3, beta 4, and alpha 5 subunits. The results demonstrate that native AChRs on neurons are more complex in composition than previously appreciated and suggest that constraints on subunit interactions limit the kinds of receptor species produced.
Assuntos
Neurônios/metabolismo , Receptores Colinérgicos/metabolismo , Animais , Anticorpos Monoclonais , Bungarotoxinas , Embrião de Galinha , Técnicas Imunológicas , Testes de Precipitina , Proteínas/metabolismo , Receptores Colinérgicos/genéticaRESUMO
Nicotinic acetylcholine receptors are widely distributed throughout the nervous system, but their functions remain largely unknown. One of the most abundant is a class of receptors that contains the alpha 7 gene product, has a high relative permeability to calcium, and binds alpha-bungarotoxin. Here, we report that receptors sensitive to alpha-bungarotoxin, though concentrated in perisynaptic clusters on neurons, can generate a large amount of the synaptic current. Residual currents through other nicotinic receptors are sufficient to elicit action potentials, but with slower rise times. This demonstrates a postsynaptic response for alpha-bungarotoxin-sensitive receptors on neurons and suggests that the functional domain of the postsynaptic membrane is broader than previously recognized.
Assuntos
Bungarotoxinas/farmacologia , Neurônios/metabolismo , Receptores Colinérgicos/efeitos dos fármacos , Receptores Colinérgicos/fisiologia , Sinapses/fisiologia , Acetilcolina/farmacologia , Potenciais de Ação , Animais , Bungarotoxinas/antagonistas & inibidores , Embrião de Galinha , Inibidores da Colinesterase/farmacologia , Condutividade Elétrica , Neurônios/efeitos dos fármacosRESUMO
Many populations of vertebrate neurons have a membrane component that binds alpha-bungarotoxin and cholinergic ligands. Despite the abundance of this component and its similarities to nicotinic receptors, its function has remained controversial. Using a fluorescence assay, we show here that activation of the component elevates the intracellular concentration of free Ca2+, demonstrating a receptor function for the toxin-binding component. Whole-cell voltage-clamp and intracellular recordings did not detect a significant current resulting from receptor activation, possibly because the currents were small or the receptors rapidly desensitized. The rise in intracellular free Ca2+ caused by the receptor was prevented by Ca2+ channel blockers. This suggests a signaling cascade likely to have important regulatory consequences for the neuron.
Assuntos
Bungarotoxinas/metabolismo , Cálcio/metabolismo , Neurônios/metabolismo , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/fisiologia , Animais , Bungarotoxinas/farmacologia , Cádmio/farmacologia , Cálcio/análise , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Células Cultivadas , Embrião de Galinha , Relação Dose-Resposta a Droga , Fluorescência , Neurônios/química , Neurônios/ultraestrutura , Níquel/farmacologia , Nicotina/metabolismo , Nicotina/farmacologia , Nifedipino/farmacologia , Tubocurarina/farmacologiaRESUMO
A chicken genomic clone encoding a portion of the neuronal acetylcholine receptor (AChR) alpha 3 subunit was used to identify homologous mRNA in embryonic chick ciliary ganglia. In situ hybridization indicated that the mRNA was neuronal. Northern blot analysis revealed a major hybridizing species of 3.5 kb. Protection experiments confirmed that ganglionic RNA contained material indistinguishable by RNAase digestion from the 300 nucleotide probe used. No transcripts were detected by in situ hybridization or Northern blot analysis for chick neuronal AChR alpha 2 or alpha 4 genes. alpha 3 transcripts were present at all times examined (E6 to 1 year posthatch). Both postganglionic axotomy and preganglionic denervation of ciliary ganglia in newly hatched chicks produced declines in alpha 3 mRNA levels, implying regulation of neuronal AChR mRNA by cell-cell interactions.
Assuntos
Gânglios Parassimpáticos/metabolismo , Expressão Gênica , Neurônios/metabolismo , RNA Mensageiro/genética , Receptores Colinérgicos/genética , Animais , Northern Blotting , Embrião de Galinha , Galinhas , Clonagem Molecular , Sondas de DNA , Regulação da Expressão Gênica , Substâncias Macromoleculares , Hibridização de Ácido NucleicoRESUMO
The temporal relationships among oocyte maturation, gamete transport and fertilisation following the pre-ovulatory luteinsing hormone surge in red deer were established; and secondly, early preimplantation development to the blastocyst stage in relation to the onset of oestrus was determined for red deer. In the first series of observations, oestrus was synchronised in April (N=22), for the fixed time recovery of gametes from 0 to 36 h after the estimated pre-ovulatory LH peak. Matings were observed and the time of the LH peak was determined from the retrospective analysis of blood plasma collected at 3h intervals. Gametes were recovered surgically and the meiotic status of follicular and ovulated oocytes assessed. Spermatozoa were recovered from the oviduct and their motility analysed by videomicroscopy. Nineteen of 22 hinds exhibited a pre-ovulatory LH surge and were observed to mate. Oocyte metaphase I occurred between 11 and 18 h, and metaphase II was completed within the follicle between 20 and 25 h following the pre-ovulatory LH peak. Fertilised ova were recovered from 30 to 36 h in both the ampulla and isthmic portions of the oviduct. Motile spermatozoa were first recovered from the isthmus and the ampulla at 13 and 21 h, respectively, after the LH peak. Hyperactive spermatozoa were observed in both the isthmus and the ampulla flushings but only from the eight hinds that had ovulated. In the second series of observations, 16 mature hinds were synchronised and allocated to groups for embryo collection on days 3, 5 and 7 after oestrus. Eight embryos were recovered; an 8-cell at 90 h, 3 morulae at 137, 138 and 186 h, and 4 blastocysts at 180, 182 and 190 h post-mating. Blastocysts were only recovered from the uterine horns and the mean+/-S.E.M. number of nuclei per blastocyst was 93.5+/-10.0 with a range of 66-114 cells. The results of this study will improve the application of assisted reproductive technologies to red deer as they indicate that oocyte maturation, fertilisation and early embryonic development of the red deer is similar to other domestic ruminants with the exception that the red deer embryo enters the uterus at the blastocyst stage.
Assuntos
Cervos/fisiologia , Desenvolvimento Embrionário/fisiologia , Hormônio Luteinizante/fisiologia , Oócitos/fisiologia , Animais , Blastocisto/fisiologia , Blastocisto/ultraestrutura , Cervos/embriologia , Estro/fisiologia , Sincronização do Estro/fisiologia , Feminino , Hormônio Luteinizante/sangue , Masculino , Microscopia de Vídeo/veterinária , Oócitos/crescimento & desenvolvimento , Oócitos/ultraestrutura , Gravidez , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/fisiologia , Espermatozoides/ultraestruturaRESUMO
Nicotinic acetylcholine receptors (AChRs) on vertebrate neurons represent a family of receptors distinct from the well-characterized AChR of skeletal muscle. New probes for neuronal AChRs are now being used to examine the regulation of receptor number and function. The results suggest that neuronal AChRs differ from muscle AChRs in regulation by presynaptic input and by at least one second messenger system, and that neuronal AChRs are additionally regulated by retrograde signals from the synaptic target tissue. The forms of regulation provide potential mechanisms by which cell-cell interactions could stabilize synaptic contacts on neurons and modulate synaptic function.
Assuntos
Músculos/metabolismo , Neurônios/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Músculos/fisiologia , Neurônios/fisiologia , Receptores Nicotínicos/fisiologiaRESUMO
Nicotinic synapses employ acetylcholine to activate ligand-gated ion channels that are cation-selective in vertebrates. Although the resulting nicotinic cholinergic transmission is famously excitatory at the neuromuscular junction, it plays many additional roles in the CNS. Most prevalent is that of modulation, usually involving calcium and signal transduction. Because of this, it is becoming increasingly important not only to understand the mechanisms that guide nicotinic receptors to appropriate locations but also to identify the postsynaptic machinery making possible the requisite signal transduction. Clearly, the kinds of components tethered in the vicinity of the receptor will assume a major role in determining the consequences of receptor activation. One of the most abundant and interesting nicotinic receptors in this respect is the species comprised of the alpha7 gene product (Broide and Leslie, 1999). These alpha7 homopentameric nicotinic acetylcholine receptors (alpha7 nAChRs) have a high relative permeability to calcium, rivaling that of NMDA receptors. But unlike NMDA receptors, alpha7 nAChRs promote calcium influx without requiring a coincident event such as membrane depolarization. As a result, the receptors are well equipped to regulate calcium-dependent events in neurons, particularly when depolarization might be occluded.
Assuntos
Corpo Ciliar/fisiologia , Nicotina/farmacologia , Receptores Nicotínicos/fisiologia , Transdução de Sinais/fisiologia , Animais , Embrião de Galinha , Corpo Ciliar/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Interneurônios/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Receptores Nicotínicos/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/fisiologiaRESUMO
Nicotinic acetylcholine receptors containing alpha7 subunits are widely expressed in the nervous system. The receptors are cation-selective, relatively permeable to calcium, and avid binders of alpha-bungarotoxin. Although the receptors can act both pre- and postsynaptically, their physiological significance is unclear. Using whole-cell patch-clamp analysis of chick ciliary ganglion neurons in situ, we show that the receptors are required for reliable synaptic transmission early in development. Stimulation of the presynaptic nerve root elicited a biphasic synaptic current, including a large rapidly decaying component generated by alpha7-containing receptors. Selective blockade of alpha7-containing receptors by perfusing the ganglion with alpha-bungarotoxin induced failures in synaptic transmission. One-half of the ciliary neurons that were tested failed when stimulated synaptically at 1 Hz, and two-thirds failed at 25 Hz. Failing cells missed, on average, 80% of the trials during a test train of stimuli. The ability to fire synaptically evoked action potentials after toxin treatment was correlated positively with the amplitude of the remaining synaptic current, suggesting that alpha7-containing receptors were needed to augment synaptic responses. Consistent with patch-clamp analysis, toxin blockade reduced the amplitude of the synaptically evoked compound action potential in the postganglionic nerve; it also desynchronized the firing of the remaining units. Methyllycaconitine, another antagonist of alpha7-containing receptors, mimicked alpha-bungarotoxin blockade. Toxin blockade had less impact on transmission in ganglia at the end of embryogenesis. The ability of the receptors to synchronize and sustain population firing, together with their ability to deliver calcium, may influence early developmental events such as target innervation and neuronal survival.