ABSTRACT
Purpose. Transplantation of stem, progenitor, or precursor cells has resulted in photoreceptor replacement and evidence of functional efficacy in rodent models of retinal degeneration. Ongoing work has been directed toward the replication of these results in a large animal model, namely, the pig. Methods. Retinal progenitor cells were derived from the neural retina of GFP-transgenic pigs and transplanted to the subretinal space of rhodopsin Pro347Leu-transgenic allorecipients, in the early stage of the degeneration and the absence of immune suppression. Results. Results confirm the survival of allogeneic porcine RPCs without immune suppression in the setting of photoreceptor dystrophy. The expression of multiple photoreceptor markers by grafted cells included the rod outer segment-specific marker ROM-1. Further evidence of photoreceptor differentiation included the presence of numerous photoreceptor rosettes within GFP-positive grafts, indicative of the development of cellular polarity and self-assembly into rudiments of outer retinal tissue. Conclusion. Together, these data support the tolerance of RPCs as allografts and demonstrate the high level of rod photoreceptor development that can be obtained from cultured RPCs following transplantation. Strategies for further progress in this area, together with possible functional implications, are discussed.
ABSTRACT
The aims of this study were to examine the role of transcription and the coincident involvement of type I and type II protein kinase A (PKA) in the resumption of meiosis in murine cumulus-oocyte complexes (COCs) using the transcriptional inhibitors 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and alpha-amanitin. The first series of experiments was designed to: (i) characterize the role of transcription in gonadotrophin-mediated and spontaneous maturation of murine oocytes; (ii) examine the roles of specific gonadotrophins (FSH versus hCG) and cumulus cells in transcriptionally mediated oocyte maturation; and (iii) determine the reversibility of the transcriptional arrest of meiosis. In the presence of FSH, transcriptional inhibitors arrested germinal vesicle breakdown (GVBD) (DRB: 2 +/- 2% and control: 76 +/- 2%; alpha-amanitin: 4 +/- 4% and control: 70 +/- 4%). Furthermore, cumulus cells were required for transcriptional inhibitors to arrest GVBD (DRB with cumulus cells: 0 +/- 15%; DRB without cumulus cells: 94 +/- 13%; alpha-amanitin with cumulus cells: 15 +/- 2%; alpha-amanitin without cumulus cells: 99 +/- 2%). Thus, in mice, FSH-mediated GVBD uses a transcriptional mechanism, which probably occurs within the cumulus cell compartment. In a second series of experiments, the role of transcription in mediating the resumption of meiosis after activation of either type I or type II PKA was examined. Activation of type I PKA in murine COCs resulted in an arrest of GVBD that was independent of a transcriptional event (with DRB: 7 +/- 9% GVBD; without DRB: 11 +/- 9% GVBD). In contrast, activation of type II PKA resulted in a resumption of meiosis, which required the occurrence of gene transcription (with DRB: 12 +/- 9% GVBD; without DRB: 80 +/- 9% GVBD). As FSH binding to cumulus cells activates the PKA second messenger system, our results indicate that, in cultured murine COCs, FSH binding to cumulus cells results in the activation of type II PKA, which, in turn, mediates a downstream transcriptional event required for the initiation of GVBD.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme Activation/physiology , Oogenesis/physiology , Transcription, Genetic/physiology , Amanitins/pharmacology , Animals , Cells, Cultured , Chorionic Gonadotropin/pharmacology , Cyclic AMP-Dependent Protein Kinase Type II , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Dichlororibofuranosylbenzimidazole/pharmacology , Enzyme Inhibitors/pharmacology , Female , Follicle Stimulating Hormone/pharmacology , Isoenzymes/metabolism , Mice , Mice, Inbred Strains , Transcription, Genetic/drug effectsABSTRACT
Retinitis pigmentosa (RP), a type of retinal degeneration involving first rod and then slow cone photoreceptor degeneration, can be caused by any of a number of mutations in different genes. In the cases of mutations affecting rod-specific genes such as rhodopsin, it is unclear how the mutations may cause degeneration of cones. We have used the porcine retina, which is rod-dominated and has an abundance of cones, to study the mutation-induced changes in both rod and cone photoreceptors. Like patients with the same mutation, rhodopsin P347L transgenic swine manifest rod-cone degeneration. In addition, the rod bipolar cells fail to form synaptic connections with rods; instead, they form ectopic synapses with cones. The mechanisms that prevent the formation of the rod-rod bipolar cell synaptic connection are not known. We used specific antibodies and immunocytochemistry to show that the synaptic protein, PSD-95, is present in both normal and transgenic porcine retinas. During neonatal development, however, PSD-95 is lost from rod terminals in the transgenic swine. This loss is virtually complete (90%) by postnatal day 5, at a time when greater than 80% of rod cell bodies still remain. Furthermore, the remaining rods retain their outer segments and their gross morphology appears relatively normal. In contrast, PSD-95 expression continues in cone terminals, even in 10-month-old transgenic swine, where the rods have all disappeared and the cones show signs of severe degeneration. These results suggest that loss of PSD-95 may not be a general consequence of the deteriorating cell. Rather, the very early and selective loss of PSD-95 from the rod terminals may be causally related to the absence of rod-rod bipolar cell synapses in the rhodopsin P347L transgenic retina.
Subject(s)
Nerve Tissue Proteins/metabolism , Retinal Rod Photoreceptor Cells/metabolism , Rhodopsin/genetics , Synapses/metabolism , Animals , Animals, Genetically Modified , Animals, Newborn , Antibodies , Disease Models, Animal , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/immunology , Retinal Rod Photoreceptor Cells/chemistry , Retinal Rod Photoreceptor Cells/pathology , Retinitis Pigmentosa/genetics , Retinitis Pigmentosa/metabolism , Retinitis Pigmentosa/pathology , Swine , Synapses/chemistryABSTRACT
Transgenic animals, especially mice, have been used quite extensively as models for various human diseases. At first, the level of scientific inquiry was driven by the need to establish the model. In many cases, these models may be considered quite crude because of their limitations. More recently, transgenic models of disease have become more refined and are currently being used to study the pathological mechanisms behind the disease rather than to just provide a model of the disease. Using some examples from the recent literature, we will document the current level and complexity of inquiry using transgenic animals. New techniques and techniques that may prove promising will be discussed.
Subject(s)
Animals, Genetically Modified/genetics , Disease Models, Animal , Animals , Genetic Techniques , Genetic Vectors , HumansABSTRACT
In addition to rod photoreceptor loss, many mutations in rod photoreceptor-specific genes cause degeneration of other neuronal types. Identifying mechanisms of cell-cell interactions initiated by rod-specific mutations and affecting other retinal cells is important for understanding the pathogenesis and progression of retinal degeneration. Here we show in animals with rod and cone degeneration due to mutations in the genes encoding rhodopsin and cGMP phosphodiesterase beta-subunit (PDE-beta) respectively, that rod bipolar cells received ectopic synapses from cones in the absence of rods. Thus, synaptic plasticity links certain rod-specific mutations to retina-wide structural alterations that involve different types of neurons.
Subject(s)
Mutation/physiology , Neuronal Plasticity/physiology , Phosphoric Diester Hydrolases , Retinal Cone Photoreceptor Cells/physiology , Retinal Rod Photoreceptor Cells/physiology , Synapses/physiology , 3',5'-Cyclic-GMP Phosphodiesterases/genetics , 3',5'-Cyclic-GMP Phosphodiesterases/metabolism , Animals , Animals, Genetically Modified , Cyclic Nucleotide Phosphodiesterases, Type 6 , GTP-Binding Proteins/metabolism , Mice , Nerve Degeneration/genetics , Nerve Degeneration/metabolism , Retina/physiology , Retinal Cone Photoreceptor Cells/cytology , Retinal Rod Photoreceptor Cells/cytology , Rhodopsin/genetics , Rhodopsin/metabolism , SwineSubject(s)
Mutation/genetics , Retinal Degeneration/genetics , Rhodopsin/genetics , Animals , Animals, Genetically Modified , Swine , TomographyABSTRACT
Rod-specific photoreceptor dystrophies are complicated by the delayed death of genetically normal neighboring cones. In transgenic (Tg) swine with a rod-specific (rhodopsin) gene mutation, cone photoreceptor physiology was normal for months but later declined, consistent with delayed cone cell death. Surprisingly, cone postreceptoral function was markedly abnormal when cone photoreceptor physiology was still normal. The defect was localized to hyperpolarizing cells postsynaptic to the middle wavelength-sensitive cones. Recordings throughout postnatal development indicated a failure of cone circuitry maturation, a novel mechanism of secondary cone abnormality in rod dystrophy. The results have implications for therapy for human retinal dystrophies and raise the possibility that rod afferent activity plays a role in the postnatal maturation of cone retinal circuitry.
Subject(s)
Retinal Cone Photoreceptor Cells/growth & development , Retinal Cone Photoreceptor Cells/physiology , Retinal Rod Photoreceptor Cells/growth & development , Retinal Rod Photoreceptor Cells/physiology , Rhodopsin/genetics , Amino Acid Substitution , Animals , Animals, Genetically Modified , Dark Adaptation/physiology , Electroretinography , Gene Expression Regulation, Developmental , Humans , Membrane Potentials/physiology , Neural Pathways , Retinitis Pigmentosa/genetics , Retinitis Pigmentosa/physiopathology , Swine , Synapses/physiologyABSTRACT
PURPOSE: To further characterize the retinas of Pro3471Leu rhodopsin transgenic pigs, a model for human retinitis pigmentosa. METHODS: Retinas from normal and transgenic pigs, newborn to 20 months old, were processed for light and electron microscopic immunocytochemical examination. RESULTS: At birth, rod numbers were normal in the transgenic retinas, but their outer segments were short and disorganized and their inner segments contained stacks of rhodopsin-positive membranes. The newborn rod synapses lacked synaptic vesicles and ribbons and had numerous rhodopsin-positive, filopodia-like processes that extended past the cone synapses into the outer plexiform layer. Rod cell death was apparent by 2 weeks and was pronounced in the mid periphery and central regions by 6 weeks. Far peripheral rods were initially better preserved, but by 9 months virtually all rods had degenerated. Cones degenerated more slowly than rods, but by 4 weeks the cone synapses were shrunken and some mid peripheral cones had lost their immunoreactivity for phosphodiesterase-gamma, arrestin, and recoverin. From 9 months to 20 months, the cone outer segments shortened progressively, and more cones lost immunoreactivity for these proteins. CONCLUSIONS: The rhodopsin transgenic pig retina shares many cytologic features with human retinas with retinitis pigmentosa and provides an opportunity to examine the earliest stages in photoreceptor degeneration, about which little is known in humans. The finding of abnormal rhodopsin localization in newborn rods is consistent with misrouting of mutant rhodopsin as an early process leading to rod cell death. Novel changes in the photoreceptor synapses may correlate with early electrophysiological abnormalities in these retinas.
Subject(s)
Animals, Genetically Modified/genetics , Disease Models, Animal , Photoreceptor Cells/pathology , Retinitis Pigmentosa/pathology , Rhodopsin/genetics , Swine , Animals , Animals, Newborn , Cell Count , Cell Death , Eye Proteins/metabolism , Fluorescent Antibody Technique, Indirect , Neuroglia/metabolism , Neuroglia/pathology , Neuroglia/ultrastructure , Photoreceptor Cells/metabolism , Photoreceptor Cells/ultrastructure , Retinal Pigments/metabolism , Retinitis Pigmentosa/genetics , Retinitis Pigmentosa/metabolism , Rhodopsin/metabolismABSTRACT
Epidermal growth factor (EGF) and insulin like growth factor-I (IGF-I) were evaluated for their effects on in vitro maturation and fertilization in presence or absence of gonadotropin and porcine follicular fluid. Four groups were made with the addition of growth factors: none (control), EGF, IGF-I or EGF+IGF-I. Each group underwent four predefined treatments with gonadotropin (FSH and LH), follicular fluid, a combination of both, or none (as control). Porcine cumulus-oocyte complexes (COCs) were matured in media containing the above-mentioned treatments for 42-44 h prior to fertilization with fresh sperm capacitated for 2.5 h. At the end of the fertilization period, the presumable embryos were fixed, stained and examined as whole-mounts to ascertain their nuclear status. The addition of EGF alone or in combination with IGF-I, significantly increased the proportion of monospermic oocytes forming 2 normal pronuclei. Also, supplementation with both growth factors together enhanced the percentages of pronucleus formation and total penetration. In addition, treatments with EGF+IGF-I significantly decreased (P<0.01) the incidence of degeneration in fertilized oocytes. However, no significant differences in the proportions of COCs undergoing polyspermy were observed among all treatments. These results suggest a stimulatory effect of tested growth factors in maturation and fertilization of pig oocytes. Furthermore, gonadotropins and follicular fluid can be replaced by the addition of EGF and IGF-I to the maturation media with positive effects on fertilization rate.
Subject(s)
Epidermal Growth Factor/pharmacology , Follicular Fluid/physiology , Gonadotropins, Pituitary/pharmacology , Insulin-Like Growth Factor I/pharmacology , Oocytes/growth & development , Swine , Animals , Cells, Cultured , Culture Media , Female , Fertilization in Vitro/veterinary , Follicle Stimulating Hormone/pharmacology , Luteinizing Hormone/pharmacologyABSTRACT
Patients with retinitis pigmentosa (RP) typically develop night blindness early in life due to loss of rod photoreceptors. The remaining cone photoreceptors are the mainstay of their vision; however, over years or decades, these cones slowly degenerate, leading to blindness. We created transgenic pigs that express a mutated rhodopsin gene (Pro347Leu). Like RP patients with the same mutation, these pigs have early and severe rod loss; initially their cones are relatively spared, but these surviving cones slowly degenerate. By age 20 months, there is only a single layer of morphologically abnormal cones and the cone electroretinogram is markedly reduced. Given the strong similarities in phenotype to that of RP patients, these transgenic pigs will provide a large animal model for study of the protracted phase of cone degeneration found in RP and for preclinical treatment trials.
Subject(s)
Retina/physiopathology , Retinal Cone Photoreceptor Cells/physiopathology , Retinitis Pigmentosa/genetics , Swine/genetics , Animals , Animals, Genetically Modified , Blotting, Southern , Disease Models, Animal , Electroretinography , Embryo Transfer , Gene Expression Regulation/genetics , Genetic Engineering , Microscopy, Electron , Molecular Sequence Data , Phenotype , Polymerase Chain Reaction , Retina/pathology , Retina/ultrastructure , Retinal Cone Photoreceptor Cells/ultrastructure , Retinal Degeneration/physiopathology , Retinitis Pigmentosa/physiopathology , Rhodopsin/chemistry , Rhodopsin/genetics , Swine/embryology , TransgenesABSTRACT
PURPOSE: Transgenic pigs with rhodopsin (Pro347Leu) mutation exhibited rod-cone degeneration. We compared the pathologic characteristics of the rod degeneration versus those of the cone cells. METHODS: The posterior and peripheral retinas of these transgenic pigs of age 4, 6, 8, 12, 24 and 33 weeks and normal pigs of age 4 and 8 weeks were studied by light and EM and morphometry. RESULTS: The pathologic changes observed in the posterior and peripheral retinas of the transgenic pigs could be conveniently described in 3 phases: I) an initial phase of rapid and extensive degeneration of the rod cells in the first 6 weeks of age; II) an acute phase of cone cell degeneration involving approximately half of the population and lingering rod degeneration in the 6 to 12 weeks of age; and III) a partial cone recovery to be followed by a chronic degenerative phase of the remaining cones cells from 12 to 33 weeks of age. CONCLUSION: Our study showed that the degenerative changes of rod cells could be differentiated from those of the cone cells. Cone and rod populations degenerated along different time schedule with different pathologic features. Hence, treatment for retinitis pigmentosa might vary with the different stages of the disease.
Subject(s)
Animals, Genetically Modified/genetics , Photoreceptor Cells/pathology , Point Mutation , Retinal Degeneration/pathology , Rhodopsin/genetics , Swine/genetics , Animals , Apoptosis , Photoreceptor Cells/ultrastructureABSTRACT
Genetic models of human disease can lead to new insights concerning disease aetiology or suggest novel therapeutic interventions. Livestock species, especially pigs, cows, sheep and horses, are often good animal models of human disease. However, genetic models in livestock species have included only the study of spontaneous mutations. Production of transgenic livestock is now possible but owing to a low efficiency of production, it is very expensive and its application limited. Anticipated application of improved technologies such as embryonic stem cells and homologous recombination will allow for increased sophistication of experimental design and wider use of genetically modified livestock. In all cases, the appropriate species for a genetic model of human disease should be the species which best models the physiology of the organ or system under consideration. In the future, livestock will play an increasingly more important role in biomedical research through the application of genetic engineering methodologies.
Subject(s)
Animals, Genetically Modified/genetics , Genetic Diseases, Inborn/genetics , Models, Genetic , Acromegaly/genetics , Animals , Eye Diseases/genetics , Humans , SwineABSTRACT
Photoreceptors of transgenic mice expressing a mutant rhodopsin gene (Pro347-->Ser) slowly degenerate. The mechanism of degeneration was studied by aggregation of embryos of normal and transgenic mice to form chimeras. In these chimeras, mosaicism was observed in the coat color, retinal pigment epithelium, and retina. In the retina, the genotype of adjacent patches of normal and transgenic photoreceptors was determined by in situ hybridization with a transgene-specific RNA probe. Photoreceptors in the chimeric retina degenerated uniformly, independent of the genotype and similar to the photoreceptors in transgenic mice. However, the chimeric retinas showed varying proportions of normal and transgenic cells. The chimeric retina with a nearly even proportion of normal and transgenic photoreceptors displayed uniform but slower degeneration than that observed in a transgenic mouse of the same age. Our results demonstrate non-autonomy of gene action for the mutated rhodopsin gene and imply that cellular interactions between photoreceptors in the retina probably play a role in degeneration.
Subject(s)
Photoreceptor Cells/pathology , Retinal Degeneration/pathology , Rhodopsin/physiology , Amino Acid Sequence , Animals , Chimera , Crosses, Genetic , Female , Genotype , In Situ Hybridization , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mosaicism , Phenotype , Point Mutation , RNA, Messenger/biosynthesis , RNA, Messenger/metabolism , Retinal Degeneration/genetics , Retinal Degeneration/physiopathology , Rhodopsin/geneticsABSTRACT
Undefined follicular factors that may influence nuclear maturation and/or cytoplasmic maturation are required during in vitro maturation of pig oocytes. Epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and dialysed porcine follicular fluid (dpFF) were evaluated for their effects on porcine oocyte nuclear maturation in vitro. In Experiment I, eight different maturation media were made in a split-plot factorial design with dpFF (0% vs. 10% v/v dialyzed pFF) as the whole plot component, and EGF (0.0 vs. 50 ng/ml) and/or IGF-I (0.0 vs. 100 ng/ml) as the factorial subplot component. Experiment II was a complete factorial design with dpFF and EGF. Pig follicular granulosa-cumulus-oocyte complexes (GCOC) were obtained from slaughterhouse ovaries, washed, and cultured at 38.5 degrees C in a humidified incubator with 5% CO2 in air for 42 h. Following culture, GCOC were mechanically stripped of granulosa-cumulus cells and evaluated for nuclear maturation by light microscopy. In Experiment I, the percentage of Metaphase II oocytes for control, IGF-I, EGF, and IGF-I+EGF treatments without pFF were 50.7%, 52.6%, 80.9%, and 84.3% (control and IGF-I groups significantly less, P < .001). The same treatments in the presence of pFF were similar and high (84.2, 84.9, 82.1, and 86.8%, respectively). Experiment II gave similar results. These results demonstrate that EGF, in the absence of pFF, promotes a similar level of oocyte nuclear maturation as does pFF alone or pFF with EGF and/or IGF-I. IGF-I does not appear to influence nuclear maturation of GCOC.
Subject(s)
Epidermal Growth Factor/pharmacology , Follicular Fluid , Insulin-Like Growth Factor I/pharmacology , Oogenesis/drug effects , Animals , Cell Nucleus/drug effects , Culture Techniques , Female , SwineABSTRACT
Pig embryos can be cultured using a number of different strategies including complex approaches like culture in vivo in a surrogate oviduct (rabbit, sheep, mouse), culture in mouse oviducts in organ culture, and co-culture of embryos with cells in addition to simple approaches like culture in defined media or salt solutions. Addition of serum to medium has been of particular importance where blastocyst development and hatching are required. Pig conceptuses (day 10-15), embryonic discs or cell lines derived from conceptuses can be cultured in complex media like Eagle's minimal essential medium or Dulbecco's modified Eagle's medium with serum, although embryonic discs can be cultured in the absence of serum. In contrast, early stage pig embryos (one-cell to blastocyst) are best cultured in simpler media such as those used for mouse embryos. The media that have been used are all relatively similar in composition. They contain salts and one or more energy sources such as glucose, lactate, or pyruvate with BSA as a macromolecular component. Early attempts to culture pig embryos were not very successful, but some embryos did develop to the blastocyst stage. More recent reports indicate a much higher rate of development with relatively little or no change in media composition. Some workers have reported improved development in medium lacking glucose, which is consistent with findings with laboratory animals such as hamsters. Glutamine can serve as the sole exogenous energy source in medium lacking glucose, lactate and pyruvate. Addition of taurine and hypotaurine to culture medium enhances development of pig embryos in vitro. We suggest, where possible, adoption of a standard medium that could be used by different laboratories and, perhaps, with different species. Use of one medium for different species would simplify experimental protocols, enhance studies of comparative embryonic physiology and metabolism, and expedite transfer of information obtained in different species.
Subject(s)
Embryonic and Fetal Development , Swine/embryology , Animals , Cells, Cultured , Culture Media , Fallopian Tubes , Female , Mice , Organ Culture TechniquesABSTRACT
Porcine embryos at Day 13 (Day 0 = first day of oestrus) were collected surgically and embryonic discs were isolated microsurgically. The discs were washed and cultured in Dulbecco's modified Eagle's medium without serum, with either 14C-leucine alone or 14C-leucine plus insulin-like growth factor-I (IGF-I) (100 ng/ml) at 37 degrees C for 48 h in 5% CO2 in air. After incubation, discs were morphologically evaluated, frozen in liquid nitrogen and stored at -70 degrees C. No statistical differences in morphology were observed between embryonic discs cultured in medium with IGF-I and those cultured in medium alone (control). Although more radioactivity was incorporated by embryonic discs in the presence of IGF-I than by those cultured in medium without the growth factor, the difference between the two groups was not significant. From two-dimensional gel electrophoresis, it was observed that IGF-I selectively stimulated the synthesis of four new proteins with Mr of 24,000, 70,000, 77,000 and 95,000, respectively and pI between 5.5 and 6.5. At least 90% of the other proteins in the gels was synthesized in greater amount by embryonic discs cultured in the presence of IGF-I than in the controls. These results show that IGF-I can stimulate protein synthesis in pig embryonic discs cultured in vitro and suggest that this growth factor may play an important role in regulating early development.
Subject(s)
Embryo, Mammalian/metabolism , Embryonic and Fetal Development/drug effects , Insulin-Like Growth Factor I/pharmacology , Protein Biosynthesis , Swine/embryology , Animals , Cells, Cultured , Electrophoresis, Gel, Two-Dimensional , Embryo, Mammalian/chemistry , Embryo, Mammalian/drug effects , Molecular Weight , Proteins/analysis , Swine/metabolismABSTRACT
To determine the relative survival of porcine embryos after co-culture with cells producing an avian retrovirus, four-cell stage embryos were obtained from sows following synchronization with altrenogest and superovulation with gonadotropins. These embryos were randomly assigned to the following treatments: no manipulation (zona-intact); zona removed with acidified Tyrode's solution (zona-free); and zona removed followed by co-culture with D-17 canine cells producing an avian retrovirus vector derived from spleen necrosis virus (zona-free + co-culture). The survival rates of four-cell stage embryos to morulae or early blastocysts during a 48-h culture period were 93.3, 80.0 and 57.7% in zona-intact, zona-free and zona-free + co-culture groups, respectively. Following embryo transfer, the development of embryos to fetuses at six weeks of gestation was 37.5, 30.0 and 11.7% in zona-intact, zona-free and zona-free + co-culture groups. These results indicate that early preimplantation porcine embryos can develop to apparently normal fetuses following co-culture with cells producing a retrovirus, and the feasibility of this method for retrovirus-mediated gene transfer in pigs was demonstrated.
ABSTRACT
The in vitro motility of caudal epididymal spermatozoa from four hamsters, four rats, and four mice was compared in modified Tyrode's medium (TLP-PVA) prepared with water of three qualities: (1) Sigma tissue culture water, 18 m omega, high quality (HQ); (2) deionized distilled water, 4.5 m omega prior to distillation, intermediate quality (IQ); and (3) tap water, low quality (LQ). The objective was to evaluate the in vitro bioassay potential of spermatozoa from these species, in terms of relative sensitivities to toxins in different qualities of water. An average sperm motility index (SMI) was calculated per treatment at 2, 4, and 6 hr, where SMI = fpm2 x % motility. Hamster SMI could be used to discriminate between HQ and IQ media at 4 and 6 hr (P less than 0.001), while rat SMI could be used to discriminate between HQ and IQ media at 6 hr (P less than 0.05). Mouse SMI did not differ between HQ and IQ media. The ability to discriminate between extremes in quality. HQ or IQ vs LQ, was equal between species (P less than 0.001). These results suggest that hamster spermatozoa provide the more sensitive in vitro bioassay model, while rat and mouse spermatozoa may be used for assay of extremes in water quality.
Subject(s)
Sperm Motility , Water , Animals , Cricetinae , Culture Media , Male , Mice , Rats , Time FactorsABSTRACT
Pig embryos at the 1- or 2-cell stage (before the 'block' to development in vitro) were cultured in 8 different media derived from Krebs'-Ringer-bicarbonate medium. A 2 x 2 x 2 factorial arrangement was used for the treatments, with glucose, glutamine and phosphate being the major effects tested. Embryos were obtained from sows approximately 44-48 h after the observation of oestrus, with the majority being at the 1-cell stage. Embryos from each female were randomly assigned to each treatment. After in-vitro culture, all embryos were scored for the stage of development attained and stained to determine final cell number. Significant effects were evident due to female, glucose, glutamine, a phosphate x glucose interaction and a glutamine x glucose interaction. None of the media components tested was inhibitory to embryo development. The greatest development (45-60% morula or blastocyst) was achieved with glucose and glutamine (both alone and in combination) in the media, demonstrating that an amino acid can serve as the sole energy source for complete preimplantation embryonic development in vitro.
Subject(s)
Embryonic and Fetal Development/physiology , Glucose/metabolism , Glutamine/metabolism , Phosphates/metabolism , Swine/physiology , Animals , Blastocyst/metabolism , Culture Media , Culture Techniques/methodsABSTRACT
Oviductal fluid (OVF) was harvested chronically from 5 sows beginning on Day 1 of the estrous cycle (Day 0 of estrous cycle = day of detected estrus) and used for embryo culture (Day 3 OVF only). Two experiments were conducted to investigate in vitro development of 1-cell and 2-cell porcine embryos in a modified Kreb's Ringer bicarbonate medium (culture medium, CM), early luteal phase OVF or CM supplemented with OVF (CM-OVF, 25% OVF v/v in CM) with or without transfer to fresh CM. In Experiment 1, 1-cell and 2-cell embryos were harvested from sows (n = 7) approximately 44 h after detected estrus. In Experiment 2, 1-cell embryos were collected from 5 sows treated with altrenogest and gonadotropins, approximately 50 h after injection of human chorionic gonadotropin. The volume of OVF (ml) declined progressively throughout the 4 days of collection (24 h, 8.44 +/- 0.28; 48 h, 6.88 +/- 1.78; 72 h, 4.96 +/- 0.35; 96 h, 4.64 +/- 0.25 after onset of estrus; p less than .01). In both experiments, development to blastocyst stage was lowest among embryos cultured in OVF and highest among those cultured in CM-OVF (Experiment 1: CM, 27.3; OVF, 10; CM-OVF, 63.6; Experiment 2: CM, 26.7; OVF, 0; CM-OVF, 82.4; % blastocyst formation).(ABSTRACT TRUNCATED AT 250 WORDS)