RESUMO
Livestock that result from biotechnology have been a part of agricultural science for over 30 years but have not entered the market place as food or fiber. Two biotechnologies are at the forefront as challenges to the world's systems for regulating the market place: animal clones and transgenic animals. Both technologies have come before the Food and Drug Administration in the United States and it appears that action is imminent for clones. The FDA has asserted principles for evaluation of clones and asserts that "... remaining hazard(s) from cloning are likely to be subtle in nature." The science-based principles recognize that in some areas related to developmental biology and gene expression in clones, additional scientific information would be useful. The role of science then is to use the genomic tools that we have available to answer questions about epigenetic regulation of development and reprogramming of genes to the state found in germ cells. Transgenics pose additional challenges to regulators. If the transgenics are produced using cloning from modified cells then the additional scientific information needed will be related to the effects of insertion and expression of the transgenes. Other approaches such as retrovirally vectored transgenesis will elicit additional questions. These questions will be challenging because the science will have to be related to the expression and function of each gene or class of genes. For the promises of animal biotechnology to be fulfilled, scientists will have to resolve many questions for regulators and the public but tools to answer those questions are rapidly becoming available.
Assuntos
Animais Geneticamente Modificados , Pesquisa Biomédica/tendências , Biotecnologia/legislação & jurisprudência , Animais , Animais Domésticos/genética , Animais Domésticos/imunologia , Clonagem de Organismos/legislação & jurisprudência , Alimentos , Têxteis , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Standard protocols aimed at identifying subclones of interest from bacterial artificial chromosomes (BACs) include the use of hybridization methods that are time consuming and often require the use of radioactive isotopes. Through our efforts to identify microsatellites in BACs from rainbow trout (Oncorhynchus mykiss) we have developed a nonradioactive polymerase chain reaction (PCR)-based screening technique to select microsatellites containing subclones for marker development. Two BACs were subcloned and screened by PCR using a vector-specific primer and a mix of microsatellite repeat primers. The subclones were then sequenced to evaluate the efficiency of the PCR screening method. Correlation between positive PCR amplification and presence of microsatellites varied between the two BACs (21.9% and 71.4%), but still a sufficient number of subclones were identified to enable design and optimization of microsatellite markers.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Repetições de Microssatélites/genética , Oncorhynchus mykiss/genética , Reação em Cadeia da Polimerase/métodos , Animais , Primers do DNA/química , Biblioteca GênicaRESUMO
Calpains are calcium-dependent neutral proteases responsible for many cellular functions. The two forms of calpain ubiquitously expressed in mammalian tissues are known as mu-calpain and m-calpain. We report here the identification of a novel calpain that is similar to but distinct from the mu- and m-calpains in rainbow trout. The cDNA of the novel gene is 2623 bp in length with a single open reading frame. The predicted protein (676 amino acids) contains the conserved calpain characteristic domains that include: domain I (pro peptide), II (cysteine catalytic site), III (electrostatic switch), and IV (calmodulin-like) with five Ca(2+)-binding EF hands. Northern blot and RT-PCR analyses demonstrated that the novel calpain gene is predominantly expressed in rainbow trout gills. Comparison of the novel protein with the ubiquitously expressed calpains and several mammalian tissue-specific calpains revealed that the novel calpain is an orthologue of the mammalian digestive tract specific calpain (calpain 9).
RESUMO
In this study, we describe the development and characterization of the first high-density single nucleotide polymorphism (SNP) genotyping array for rainbow trout. The SNP array is publically available from a commercial vendor (Affymetrix). The SNP genotyping quality was high, and validation rate was close to 90%. This is comparable to other farm animals and is much higher than previous smaller scale SNP validation studies in rainbow trout. High quality and integrity of the genotypes are evident from sample reproducibility and from nearly 100% agreement in genotyping results from other methods. The array is very useful for rainbow trout aquaculture populations with more than 40 900 polymorphic markers per population. For wild populations that were confounded by a smaller sample size, the number of polymorphic markers was between 10 577 and 24 330. Comparison between genotypes from individual populations suggests good potential for identifying candidate markers for populations' traceability. Linkage analysis and mapping of the SNPs to the reference genome assembly provide strong evidence for a wide distribution throughout the genome with good representation in all 29 chromosomes. A total of 68% of the genome scaffolds and contigs were anchored through linkage analysis using the SNP array genotypes, including ~20% of the genome assembly that has not been previously anchored to chromosomes.
Assuntos
Variação Genética , Técnicas de Genotipagem/métodos , Oncorhynchus mykiss/classificação , Oncorhynchus mykiss/genética , Polimorfismo de Nucleotídeo Único , Animais , Genética Populacional/métodos , Reprodutibilidade dos TestesRESUMO
Expressed sequence tag (EST) projects have produced extremely valuable resources for identifying genes affecting phenotypes of interest. A large-scale EST sequencing project for rainbow trout was initiated to identify and functionally annotate as many unique transcripts as possible. Over 45,000 5' ESTs were obtained by sequencing clones from a single normalized library constructed using mRNA from six tissues. The production of this sequence data and creation of a rainbow trout Gene Index eliminating redundancy and providing annotation for these sequences will facilitate research in this species.
Assuntos
DNA Complementar/genética , Bases de Dados Genéticas/tendências , Biblioteca Gênica , Genes/genética , Oncorhynchus mykiss/genética , Análise de Sequência de DNA/veterinária , Animais , Arabidopsis/genética , Peixes-Gato/genética , Bovinos , Galinhas/genética , Análise por Conglomerados , DNA de Plantas/genética , Bases de Dados Genéticas/estatística & dados numéricos , Etiquetas de Sequências Expressas , Genes/fisiologia , Genes de Plantas/genética , Genes de Plantas/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , Ratos , Análise de Sequência de DNA/estatística & dados numéricos , Suínos/genética , Peixe-Zebra/genéticaRESUMO
The capacity of structurally modified analogs of prostaglandin F2 alpha (PGF2 alpha) to inhibit binding of [3H]PGF2 alpha to receptors on ovine luteal cells was evaluated by radioreceptor assay using dispersed, viable, ovine luteal cells. Binding assays were conducted at pH 5.75, since binding to both high (Kd 17.4 +/- 2.3 nM) and low (Kd 409 +/- 166 nM) affinity sites was enhanced markedly at reduced pH. The capability to compete with [3H]PGF2 alpha for binding was evaluated for different prostaglandin analogs having modifications in the C-8 "upper" side-chain, in the cyclopentane ring, or in the C-12 "lower" side-chain. Prostaglandin J2 was a surprisingly potent competitor for binding to the PGF2 alpha receptor. Several phenyl-substituted analogs exhibited receptor-binding potency greater than or equal to native PGF2 alpha, while most other analogs had reduced capacity to compete with native PGF2 alpha for binding. Several 17-azidophenol PGF2 alpha analogs were synthesized and tested, but analogs having hydroxyl groups on the aryl ring had low affinity for receptors. However, 17-(4-azidophenyl)-18,19,20-trinor-PGF2 alpha as well as 17-(3-iodo-4-azidophenyl)-18,19,20-trinor-PGF2 alpha exhibited binding affinities that were approximately 10% of native PGF2 alpha, and the radioiodinated analogs of PGF2 alpha may be useful as probes of the PGF2 alpha receptor.
Assuntos
Dinoprosta/metabolismo , Receptores de Prostaglandina/metabolismo , Marcadores de Afinidade , Animais , Ligação Competitiva , Corpo Lúteo/metabolismo , Dinoprosta/análogos & derivados , Feminino , Técnicas In Vitro , Estrutura Molecular , Fotoquímica , Preservação Biológica , Ensaio Radioligante , OvinosRESUMO
Serotonin (5-HT)-induced stimulation or progesterone (P4) production by bovine luteal cells was characterized with respect to the receptor subtype mediating this response, the steroidogenic response to 5-HT metabolites, the role of adenylate cyclase, and the 5-HT concentration of bovine luteal tissue. Addition of 5-HT (10(-5) M) stimulated the production of P4 (P less than 0.05) and this stimulation was inhibited by the 5-HT antagonist mianserin at a concentration of 10(-5) M (P less than 0.05), but not at a mianserin concentration of 10(-7) M. Additionally, the response to 5-HT could not be inhibited by ketanserin (10(-5) M), a 5-HT2 receptor antagonist. Incubation of luteal cells with a specific 5-HT1 agonist, (+/-)-8-hydroxydipropylaminotetralin HBr (DPAT) (10(-4) M), stimulated the production of P4 (P less than 0.05) and this response could not be blocked by mianserin at 10(-7) M or by ketanserin, but was inhibited by mianserin at 10(-5) (P less than 0.05). The addition of the 5-HT metabolite 5-methoxytryptamine (5-MTA) stimulated P4 production (P less than 0.05) and this response could be inhibited by mianserin (10(-5) M, P less than 0.05). Neither, N-acetyl-5-HT nor 5-methoxytryptophan significantly affected P4 production. The addition of the phosphodiesterase inhibitor 3-isobutyl-methylxanthine (IBMX, 0.1 mM) potentiated the effects of 5-HT and DPAT (P less than 0.05), but this effect was additive rather than synergistic. In contrast, the addition of luteinizing hormone (10 ng/ml) plus IBMX resulted in a significant synergistic response (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Corpo Lúteo/fisiologia , Progesterona/biossíntese , Receptores de Serotonina/fisiologia , Serotonina/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , 8-Hidroxi-2-(di-n-propilamino)tetralina , Animais , Bovinos , Estro , Feminino , Técnicas In Vitro , Ketanserina/farmacologia , Mianserina/farmacologia , Receptores de Serotonina/classificação , Relação Estrutura-Atividade , Tetra-Hidronaftalenos/farmacologiaRESUMO
Transgenic pigs and sheep have been produced by the microinjection of single-cell zygotes and two-cell ova with linear molecules of mouse metallothionein I (MT) promoter/regulator fused to either the human growth hormone (hGH) or bovine growth hormone (bGH) structural genes. The foreign genes integrated into the chromosomes of 3 of 111 lambs or fetuses and 31 of 341 pigs or fetuses examined. Immunoreactive hGH or bGH was present in the plasma of two transgenic lambs and 19 transgenic pigs. The hGH concentration in plasma varied greatly among pigs and was unrelated to the number of gene copies that had integrated. Rate of growth was not enhanced in any of the transgenic pigs in comparison to their littermate controls. However, bGH and hGH exerted definite biological effects in transgenic pigs as evidenced by significantly depressed backfat measurements, elevated levels of insulin-like growth factor (IGF-I), stimulation of mammary development (by hGH) and reduction in porcine growth hormone (pGH) to nondetectable levels in plasma. Five of six founder transgenic pigs transmitted the MT-hGH gene construct to one or more progeny. Three progeny of a boar that expressed hGH also expressed the foreign gene.
Assuntos
Criação de Animais Domésticos/métodos , Engenharia Genética , Hormônio do Crescimento/genética , Ovinos/genética , Suínos/genética , Animais , Animais Geneticamente Modificados , Bovinos , Feminino , Hormônio do Crescimento/biossíntese , Hormônio do Crescimento/sangue , Humanos , Masculino , Camundongos , Microinjeções , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/genética , Ovinos/crescimento & desenvolvimento , Suínos/crescimento & desenvolvimentoRESUMO
The effect of catecholamine depletion within three brain regions on subsequent food intake and body weight gain were investigated in two week-old chicks. Two levels of pargyline (P; 50 mg/kg and 100 mg/kg intraperitoneally) and three levels of six-hydroxydopamine (6-OHDA; 0, 200 micrograms (136 free base) and 300 micrograms (204 free base) intracerebroventricularly) were administered to 36 chicks (n = 6 per treatment). The greatest reduction in food intake and body weight gain occurred in the group given 100 mg/kg P, 300 micrograms 6-OHDA. These chicks displayed an average aphagic response of 4.5 days. Chicks given the higher drug doses were hyperactive when handled and displayed difficulty in orienting their bills when attempting to consume pellets. Biogenic amines were determined in three brain regions (striatum, hypothalamus and brainstem) using high pressure liquid chromatography with electrochemical detection. Dopamine (DA) was reduced (p less than 0.05) in the striatum of all groups treated with 6-OHDA when compared to their respective controls. Significant depletions of norepinephrine (NE) occurred in all three brain regions in all groups treated with 6-OHDA when compared with their respective control groups (p less than 0.05). In contrast, striatal epinephrine levels were elevated in chicks given 100 mg P plus 6-OHDA. It is proposed that depletions of both DA and NE contribute to the aphagic or anorexic response of chicks following administration of the neurotoxin 6-OHDA.
Assuntos
Química Encefálica/efeitos dos fármacos , Dopamina/análise , Ingestão de Alimentos/efeitos dos fármacos , Epinefrina/análise , Hidroxidopaminas/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Tronco Encefálico/efeitos dos fármacos , Galinhas , Corpo Caloso/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Masculino , Oxidopamina , Pargilina/farmacologiaRESUMO
The efficiency of developing polymorphic microsatellite markers from 2 repeat enriched libraries was evaluated. Thirty-six polymorphic microsatellite markers were developed for rainbow trout, 27 of which were informative in a mapping family. The ability of each marker to amplify genomic DNA from other salmonids was also observed.
RESUMO
The aims of the present study were: (1) to characterize alkaline phosphatase (AP) activity in bovine oocytes and embryos with intact or removed zona pellucida (ZP); and (2) to evaluate the effect of culture medium constituents on AP activity. Alkaline phosphatase activity in non-matured and matured oocytes was most evident nearest the plasma membrane and perivitelline space. In more than 90% of two- to 16-cell embryos, AP activity was observed in the perivitelline space and at blastomere contacts. In blastocysts, AP activity was localized to the trophectoderm. Only after immunodissection was AP activity detected in the inner cell mass. Removal of the ZP by pronase or mechanical means reduced AP activity. Alkaline phosphatase activity was detected in evacuated ZP after mechanical removal. Specific constituents comprising the embryo culture medium altered AP activity. Alkaline phosphatase activity was reduced in eight- to 16-cell embryos and evacuated ZP cultured in CR1aa + 0.4% bovine serum albumin compared with embryos cultured in CR1aa alone or embryos co-cultured on a monolayer of Buffalo rat liver cells in the presence of 10% fetal bovine serum. The presence of AP activity at blastomere contacts and in evacuated ZP limits its usefulness as a marker for the differentiation of embryonic cells comprising the early cleavage-stage embryo.
Assuntos
Fosfatase Alcalina/análise , Blastocisto/efeitos dos fármacos , Meios de Cultura/farmacologia , Oócitos/efeitos dos fármacos , Zona Pelúcida , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores/análise , Blastocisto/enzimologia , Blastocisto/ultraestrutura , Bovinos , Fase de Clivagem do Zigoto/efeitos dos fármacos , Fase de Clivagem do Zigoto/enzimologia , Fase de Clivagem do Zigoto/ultraestrutura , Meios de Cultura/química , Feminino , Oócitos/enzimologia , Oócitos/ultraestruturaRESUMO
Pig epiblast cells that had been separated from other early embryonic cells were cultured in vitro. A three-step dissection protocol was used to isolate the epiblast from trophectoderm and primitive endoderm before culturing. Blastocysts collected at 7 to 8 days postestrus were immunodissected to obtain the inner cell mass (ICM) and destroy trophectodermal cells. The ICM was cultured for 2 to 3 days on STO feeder cells. The epiblast was then physically dissected free of associated primitive endoderm. Epiblast-derived cells, grown on STO feeders, produced colonies of small cells resembling mouse embryonic stem cells. This primary cell morphology changed as the colonies grew and evolved into three distinct colony types (endodermlike, neural rosette, or complex). Cell cultures derived from these three colony types spontaneously differentiated into numerous specialized cell types in STO co-culture. These included fibroblasts, endodermlike cells, neuronlike cells, pigmented cells, adipogenic cells, contracting muscle cells, dome-forming epithelium, ciliated epithelium, tubule-forming epithelium, and a round amoeboid cell type resembling a plasmacyte after Wright staining. The neuronlike cells, contracting muscle cells, and tubule-forming epithelium had normal karyotypes and displayed finite or undefined life spans upon long-term STO co-culture. The dome-forming epithelium had an indefinite life span in STO co-culture and also retained a normal karyotype. These results demonstrate the in vitro pluripotency of pig epiblast cells and indicate the epiblast can be a source for deriving various specialized cell cultures or cell lines.
Assuntos
Blastocisto/citologia , Suínos/embriologia , Animais , Blastocisto/ultraestrutura , Células Cultivadas , Cromossomos/ultraestrutura , Ectoderma/citologia , Ectoderma/ultraestrutura , Endoderma/citologia , Endoderma/ultraestrutura , Células Epiteliais , Epitélio/ultraestrutura , Fibroblastos/citologia , Fibroblastos/ultraestrutura , Cariotipagem , Macrófagos/citologia , Macrófagos/ultraestruturaRESUMO
Secondary macrophage cell cultures were generated from the primary culture of epiblasts of 8-d-old pig blastocysts. The epiblast-derived macrophagelike (EDM) cells have a morphology and ameboid behavior that is typical of tissue histocytes. The cells reacted positively with monoclonal antibodies specific for pig granulocyte-macrophage lineage cells, and were not reactive with monoclonal antibodies specific for pig B and T lymphocytes. Marked phagocytic behavior and the formation of phagosomes were demonstrated following incubation with FITC-labeled bacteria. The EDM cells stained positively for nonspecific acid esterase that was not inhibited by sodium fluoride. DiI-acetylated-LDL was rapidly taken up by the cells. Transmission electron microscopy of the EDM cells showed phagolysosomes, numerous cytoplasmic vacuoles, large, lobed nuclei, and numerous pseudopods or filopodia at the cell surface. Strong reactivity of the cells with anti-CD14 monoclonal antibody was observed. Further, cytotoxic activity was produced from the EDM cells after exposure to lipopolysaccharide in a concentration and time-dependent manner. The cultures could be maintained and expanded for several months on STO co-culture. Their derivation from the epiblast of the pig demonstrates the possibility of obtaining hemopoietic cell cultures from the preimplantation blastocysts of all mammals.
Assuntos
Macrófagos/imunologia , Acetilação , Animais , Linfócitos B/imunologia , Carbocianinas/química , Células Cultivadas , Testes Imunológicos de Citotoxicidade , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Receptores de Lipopolissacarídeos/imunologia , Lipoproteínas LDL/farmacocinética , Macrófagos/citologia , Microscopia Eletrônica , Naftol AS D Esterase/imunologia , Fagocitose/imunologia , Receptores Imunológicos/imunologia , Suínos , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Continuous cultures of pluripotent parenchymal hepatocytes were derived from the epiblasts of 8-day-old pig blastocysts. The cells were polygonal and had phase-contrast dark, granular cytoplasm with prominent nuclei and nucleoli. These feeder-dependent cell cultures differentiated into large, multicellular, secretory, duct-like structures or formed small canaliculi between individual cells. Alternatively, the cells accumulated droplets that stained intensely with Oil Red O, a lipid-specific stain. Alpha-fetoprotein (AFP), albumin, and beta-fibrinogen mRNAs were expressed as the cells differentiated in culture. Serum-free medium that was conditioned by the cells contained transferrin, AFP, and albumin. The growth and viability of the cells were inhibited by transforming growth factor beta 1 (TGF beta 1) at concentrations > or = 1 ng/ml. The cell cultures grew slowly with doubling times of 2 to 3 d. One of the cultures, pig inner cell mass-19 (PICM-19), was passaged continuously for over 2 yr [> 100 population doublings (PD)] and appears to be an infinitely self-renewing cell population. The stem cell characteristics of the epiblast-derived fetal hepatocytes indicate that the cells may be unique for investigations of liver differentiation and organogenesis.
Assuntos
Fígado/embriologia , Animais , Northern Blotting , Diferenciação Celular , Divisão Celular , Núcleo Celular/ultraestrutura , Células Cultivadas , Meios de Cultura , Meios de Cultivo Condicionados , Citoplasma/ultraestrutura , Fibrinogênio/genética , Cobaias , Immunoblotting , Fígado/ultraestrutura , RNA Mensageiro/metabolismo , Albumina Sérica/genética , Suínos , Fator de Crescimento Transformador beta/farmacologia , alfa-Fetoproteínas/genéticaRESUMO
The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and beta-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.
Assuntos
Fígado/embriologia , Animais , Canalículos Biliares/citologia , Diferenciação Celular , Separação Celular , Células Cultivadas , Meios de Cultivo Condicionados , Fibrinogênio/genética , Expressão Gênica , Metabolismo dos Lipídeos , Fígado/citologia , RNA Mensageiro/metabolismo , Albumina Sérica/genética , Albumina Sérica/metabolismo , Suínos/embriologia , Transferrina/metabolismo , alfa-Fetoproteínas/genéticaRESUMO
This study examined whether development of bovine in vitro produced (IVP) blastocysts in the sheep uterus resulted in morphologically and karyotypically normal elongation stage bovine blastocysts. Seven day IVP bovine blastocysts, resulting from either in vitro maturation and fertilization, nuclear transfer (NT), or parthenogenic activation, were surgically transferred at the blastocyst stage into sheep uteri. Sheep were sacrificed after 7-9 days, and blastocysts were flushed from their uteri. One of each kind of IVP bovine blastocyst was recovered from sheep uteri for analysis by transmission electron microscopy, and nine NT blastocysts were used to establish cell cultures that were analysed for chromosome complement. TEM analysis of in vivo-derived elongation stage bovine and ovine blastocysts was done for comparative purposes. Most ultrastructural features of the 13-19 day blastocysts were similar to earlier stage blastocysts except that distinct alternative mitochondrial morphologies were found between epiblast and trophectoderm cells. Monociliated cells, presumably nodal cells, were observed in the bovine epiblast and hypoblast, and retrovirus-like particles were elaborated by cells in these same areas. Development in the sheep uterus of IVP bovine blastocysts resulted in the presence of crystalloid bodies in the trophectoderm cells, and apoptotic and necrotic cells were observed in the epiblast tissue. Thus, in vivo incubation in the sheep uterus allowed nearly normal development to the elongated blastocyst stage and may be useful for assessment of NT bovine blastocyst developmental competence. Cell cultures derived from the NT blastocysts had normal chromosome complements suggesting that activation by ionomycin and 6-dimethyl-aminopurine did not cause detrimental changes in ploidy in those blastocysts that developed.
Assuntos
Transferência Embrionária , Embrião de Mamíferos/fisiologia , Fertilização in vitro , Ovinos/fisiologia , Animais , Blastocisto/fisiologia , Blastocisto/ultraestrutura , Bovinos , Células Clonais , Embrião de Mamíferos/ultraestrutura , Desenvolvimento Embrionário e Fetal , Feminino , Cariotipagem , Masculino , Microscopia Eletrônica , Técnicas de Transferência Nuclear , PartenogêneseRESUMO
RNA was extracted from single or small groups of ovine ovarian follicles after treatment of ewes with FSH and/or LH. The content of mRNA for the alpha-inhibin and beta A-inhibin subunits was analyzed by hybridization with specific cDNA probes. All ewes were treated with progestin vaginal pessaries to suppress spontaneous preovulatory follicle maturation and ewes were given three intramuscular injections of gonadotropins at 8-hr intervals starting 24 hr prior to collection of ovaries. In experiment I, both Schering-FSH and NIDDK-oFSH-17 (oFSH) significantly increased alpha- and beta A-inhibin mRNA per ewe in 2-5 mm follicles and tended to increase alpha- and beta A-inhibin mRNA in large (greater than 5 mm) follicles. In experiment II, oFSH and NIDDK-oLH-25 (oLH) were administered in a 2X2 factorial arrangement. Separate administration of oFSH or oLH increased (P less than .05) the alpha-inhibin mRNA concentration in large follicles. alpha-inhibin mRNA concentration in 4-5 mm follicles was also increased by oFSH but was decreased by oLH. Concomitant treatment with oFSH and oLH did not change alpha-inhibin mRNA concentrations from those measured in oFSH treated ewes. In experiment II, beta A mRNA concentrations followed a pattern similar to that of alpha A mRNA, but the differences were not statistically significant. We conclude that, in the ewe, exogenous FSH increases the concentration of inhibin mRNA in the whole follicle. The ability of exogenous oLH to alter expression of the inhibin subunit genes may depend upon the stage of follicle maturation.
Assuntos
Gonadotropinas/farmacologia , Inibinas/genética , Folículo Ovariano/fisiologia , RNA Mensageiro/análise , Ovinos/genética , Animais , Sequência de Bases , Northern Blotting , Feminino , Hormônio Foliculoestimulante/farmacologia , Hormônio Luteinizante/farmacologia , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Progestinas/farmacologia , Sondas RNA/química , RNA Mensageiro/química , Ovinos/fisiologiaRESUMO
Three experiments were conducted to determine whether coculture of early sheep eggs with oviductal cells would improve the ability of eggs to survive in culture. Eggs recovered from superovulated ewes were cultured in Ham's F-10 medium supplemented with 10% fetal calf serum (F10FCS) at 37.5 degrees C in 95% air:5% CO(2). In Experiment 1, eggs with one to eight cells were either transferred into recipient ewes immediately after collection or were cultured for 24 h in 5 ml Ham's F10 medium supplemented with 10% fetal calf serum (F10FCS), 5 ml F10FCS on a confluent monolayer of oviductal cells; in 25 ml of fresh F10FCS; or in 25 ml of F10FCS removed cultures of oviductal cells, 25 mul of fresh F10FCS or 25 mul of F10FCS removed from cultures of oviductal cells. After 24 h, the cultured eggs were transferred to recipient ewes synchronous with donors and subsequently recovered at necropsy on Day 8 post estrus. Coculture of sheep eggs with oviductal cells improved (P < 0.05) the development of transferred eggs compared to culture in F10FCS alone. In Experiment 2, eggs recovered from superovulated ewes on Days 3 to 6 after estrus had undergone 1.8 cleavages by Day 3 and 4.1 cleavages by Day 6. In Experiment 3, single-cell eggs were cultured for 3 d in 5 ml F10FCS, cocultured with ovine trophoblastic vesicles or cultured on a confluent monolayer of oviductal cells. Coculture of eggs in F10FCS on a monolayer of oviductal cells supported in vitro egg cleavage to a greater degree than did F10FCS alone or F10FCS with trophoblastic vesicles (P < 0.05). In Experiment 4, single-cell eggs were cultured for 3 d then transferred to recipients. Eggs were cultured in 5 ml F10FCS on confluent monolayers of oviductal cells from luteal or estrous ewes or on cells that had been frozen after recovery from a culture of oviductal cells. After culture, the eggs were transferred to oviducts of recipients and recovered 3 d later at necropsy. Coculture of eggs for 72 h with oviductal cell monolayers did not increase the in vitro, or subsequent in vivo, cleavage rate regardless of the type of oviductal cells.
RESUMO
Three experiments were conducted to identify, sources of loss of fertilized single-cell sheep eggs microinjected with DNA. In the first experiment, immediate transfer of eggs into synchronous recipients resulted in 86% of embryos developing (>32 cells) at Day 7. Incubating eggs in microdrops of Ham's F-10 medium+10% fetal calf serum for 5 h at 37 degrees C in an atmosphere of 95% air: 5% CO2 before transfer reduced development (65%>32 cells). Removing eggs from drops for 30 min of microscopic inspection, simulating manipulation during microinjection, caused no additional reduction in development (63%>32 cells). However, injection of eggs with buffer was detrimental to subsequent development (42%>32 cells). In Experiment 2, injection of buffer or injection of DNA in buffer into the pronuclei before transfer of eggs into recipient ewes resulted in 29 and 19%, respectively, of embryos developing to >32 cells at Day 7. In Experiment 3, more eggs developed when held in 5 ml of medium than in microdrops (P=0.07). No difference in development was found between eggs held in bicarbonate-buffered BMOC or in phosphate-buffered saline with added fetal bovine serum. The development of sheep eggs appears to be greatly reduced after microinjection, but until alternate procedures are found, a high rate of loss of injected eggs may be an unavoidable cost of inserting foreign genes into sheep.