Ovarian and placental morphology and endocrine functions in the pregnant giraffe (Giraffa camelopardalis).

Gross, histological and immunocytochemical examinations carried out on maternal and fetal reproductive tissues from two pregnant giraffes at an estimated 8 and 13.5 months of gestation (term=15 months) revealed a typically ruminant macrocotyledonary placenta with binucleate trophoblast cells scattered sparsely in the placentome where they stained intensely with a prolactin antiserum. Binucleate cells were present in greater numbers in the intercotyledonary allantochorion where they did not stain for prolactin whereas the uninucleate trophoblast still did. A single large corpus luteum of pregnancy and several small luteinised follicles were present in the maternal ovaries while the fetal ovaries at 13.5 months gestation showed an assortment of enlarging antral follicles and partially and completely lutenised follicles, the granulosa and luteal cells of which stained positively for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17,20 lyase, prolactin, progesterone receptor and androgen receptor, but negatively for aromatase. The uninucleate trophoblast of the placentome and intercotyledonary allantochorion, the epithelium of the maternal endometrial glands, the seminiferous epithelium in the fetal testis at 8 months of gestation and the zonae fasciculata and reticularis of the fetal adrenal at 13.5 months also stained positively for 3ß-HSD and negatively for aromatase. Endocrinologically, it appears that the giraffe placenta is more similar to that of the sheep than the cow with a placental lactogen as the likely driver of the considerable degree of luteinisation seen in both the maternal and the fetal ovaries.

Species differences in the sensitivity of avian embryos to methylmercury.

We injected doses of methylmercury into the air cells of eggs of 26 species of birds and examined the dose-response curves of embryo survival. For 23 species we had adequate data to calculate the median lethal concentration (LC(50)). Based on the dose-response curves and LC(50)s, we ranked species according to their sensitivity to injected methylmercury. Although the previously published embryotoxic threshold of mercury in game farm mallards (Anas platyrhynchos) has been used as a default value to protect wild species of birds, we found that, relative to other species, mallard embryos are not very sensitive to injected methylmercury; their LC(50 )was 1.79 microg/g mercury on a wet-weight basis. Other species we categorized as also exhibiting relatively low sensitivity to injected methylmercury (their LC(50)s were 1 microg/g mercury or higher) were the hooded merganser (Lophodytes cucullatus), lesser scaup (Aythya affinis), Canada goose (Branta canadensis), double-crested cormorant (Phalacrocorax auritus), and laughing gull (Larus atricilla). Species we categorized as having medium sensitivity (their LC(50)s were greater than 0.25 microg/g mercury but less than 1 microg/g mercury) were the clapper rail (Rallus longirostris), sandhill crane (Grus canadensis), ring-necked pheasant (Phasianus colchicus), chicken (Gallus gallus), common grackle (Quiscalus quiscula), tree swallow (Tachycineta bicolor), herring gull (Larus argentatus), common tern (Sterna hirundo), royal tern (Sterna maxima), Caspian tern (Sterna caspia), great egret (Ardea alba), brown pelican (Pelecanus occidentalis), and anhinga (Anhinga anhinga). Species we categorized as exhibiting high sensitivity (their LC(50)s were less than 0.25 microg/g mercury) were the American kestrel (Falco sparverius), osprey (Pandion haliaetus), white ibis (Eudocimus albus), snowy egret (Egretta thula), and tri-colored heron (Egretta tricolor). For mallards, chickens, and ring-necked pheasants (all species for which we could compare the toxicity of our injected methylmercury with that of published reports where methylmercury was fed to breeding adults and was deposited into the egg by the mother), we found the injected mercury to be more toxic than the same amount of mercury deposited naturally by the mother. The rank order of sensitivity of these same three species to methylmercury was, however, the same whether the methylmercury was injected or maternally deposited in the egg (i.e., the ring-necked pheasant was more sensitive than the chicken, which was more sensitive than the mallard). It is important to note that the dose-response curves and LC(50)s derived from our egg injections are useful for ranking the sensitivities of various species but are not identical to the LC(50)s that would be observed if the mother bird had put the same concentrations of mercury into her eggs; the LC(50)s of maternally deposited methylmercury would be higher.

Plasticity in the adrenocortical response of a free-living vertebrate: the role of pre- and post-natal developmental stress.

Optimal functioning of the hypothalamo-pituitary-adrenal (HPA) axis is paramount to maximizing fitness in vertebrates. Research in laboratory mammals has suggested that maternally-induced stress can cause significant variation in the responsiveness of an offspring's HPA axis involving both pre- and post-natal developmental mechanisms. However, very little is known regarding effects of maternal stress on the variability of offspring adrenocortical functioning in free-living vertebrates. Here we use an experimental approach that independently lowers the quality of both the pre- and post-natal developmental environment to examine programming and plasticity in the responsiveness of the HPA axis in fledglings of a free-living passerine, the European starling (Sturnus vulgaris). We found that mimicking a hormonal signal of poor maternal condition via an experimental pre-natal increase in yolk corticosterone decreased the subsequent responsiveness of the HPA axis in fledglings. Conversely, decreasing the quality of the post-natal developmental environment (by decreasing maternal provisioning capability via a maternal feather-clipping manipulation) increased subsequent responsiveness of the HPA axis in fledglings, apparently through direct effects on nestling body condition. The plasticity of these responses was sex-specific with smaller female offspring showing the largest increase in HPA reactivity. We suggest that pre-natal, corticosterone-induced, plasticity in the HPA axis may be a 'predictive adaptive response' (PAR): a form of adaptive developmental plasticity where the advantage of the induced phenotype is manifested in a future life-history stage. Further, we introduce a new term to define the condition-driven post-natal plasticity of the HPA axis to an unpredictable post-natal environment, namely a 'reactive adaptive response' (RAR). This study confirms that the quality of both the pre- and post-natal developmental environment can be a significant source of variation in the responsiveness of the HPA axis, and provides a frame-work for examining ecologically-relevant sources of stress-induced programming and plasticity in this endocrine system in a free-living vertebrate, respectively.

On the relationship between rabbit age and lens dry weight: improved determination of the age of rabbits in the wild.

PURPOSE: Eye lens dry weights are commonly used for estimating the age of animals in the wild, but reported relationships are variable. The purpose of this study was to determine why different relationships have been reported using data available for the same species of rabbits. METHODS: Published results that relate lens weight to age for wild European rabbits from four locations in Australia and cottontail rabbits from two locations in the United States have been reexamined. In addition, the effects of variations in lens preparation have been tested with fresh eyes. RESULTS: It was found that, in previous studies, the logistic type relationship between lens weight and age was interpreted inappropriately through the use of age constants, which imply that lens growth commences before conception. Using gestational time as the constant yields a single formula for each species, and this is consistent with most of the published data. Variations in fixation and drying conditions may be responsible for small differences between different populations. CONCLUSIONS: When using lens dry weights as a measure of age, it is recommended that eyes be fixed for at least four weeks and the fixed lenses be dried for two weeks at 85 degrees C or, preferably, three days at 100 degrees C. Any formula, relating age and lens dry weight for any species, must take into account the fact that lens growth commences during gestation not before or after.

Morphometric abnormalities in brains of great blue heron hatchlings exposed in the wild to PCDDs.

Great blue heron hatchlings from colonies in the Strait of Georgia, British Columbia, Canada are being monitored for environmental contaminant exposure and effects by the Canadian Wildlife Service. The contaminants of concern are polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), primarily derived from kraft pulp mill effluent. The levels of PCDDs and PCDFs in eggs from the most contaminated colonies peaked in 1988 and 1989 and dropped dramatically through 1990 to 1992. Brains of heron hatchlings (taken as eggs from the wild and hatched in the laboratory) were analyzed for gross morphological abnormalities. Brains from highly contaminated colonies (Crofton, British Columbia and University of British Columbia Endowment Lands) in 1988 exhibited a high frequency of intercerebral asymmetry. The frequency of this abnormality decreased in subsequent years as the levels of TCDD and TCDD-TEQs (toxic equivalence factors) decreased. The asymmetry was significantly correlated with the level of TCDD and TCDD-TEQs in eggs taken from the same nest. Yolk-free body weight negatively correlated and the brain somatic index positively correlated with the TCDD level in such pair-matched eggs. These results indicate that gross brain morphology, and specifically intercerebral asymmetry, may be useful as a biomarker for the developmental neurotoxic effects of PCDDs and related chemicals.

Cryopreservation of gametes and embryos of non-domestic species.

Many species of mammals are threatened or endangered. Methods of assisted reproduction that are being used with increasing frequency to produce offspring of domestic animals and humans are often viewed as offering innovative ways to reproduce non-domestic species as well. Uncounted millions of live young of domestic or laboratory species have been produced from gametes and embryos stored at -70 degrees C or below, sometimes for as long as 25 to 35 yrs. Such methods of cryopreservation are now being applied with increasing frequency and urgency to preserve gametes and embryos of non-domestic and threatened species to establish "genome resource banks" or "frozen zoos." But levels of success to produce live young from such cryopreserved gametes or embryos vary considerably from species to species, as well as from individual to individual. It is sometimes thought that differences among species in fundamental characteristics of their gametes may determine the efficacy of cryopreservation and the production of live young. However, it may not be that ineffective cryopreservation is responsible for low success rates. Rather, the limiting factor may be insufficient information and knowledge of the most basic reproductive biology of such non-domestic species. Even standard methods of cryopreservation may be completely adequate to act as a "temporary" expedient to preserve germplasm of non-domestic species to permit time to acquire a fuller understanding of the biology and behavior of non-domestic species.

[The creation of genetic cryobanks and the use of the methods of developmental biology as a means for preserving rare animal species. II. The obtaining and cryopreservation of the embryos of wild mammals]. / Sozdanie geneticheskikh kriobankov i ispol'zovanie metodov biologii razvitiia kak sposob sokhraneniia redkikh vidov zhivotnykh. II. Poluchenie i kriokonservatsiia zarodyshei dikikh mlekopitaiushchikh.

A review of published data on obtaining, cryoconservation, and subsequent development of the embryos of wild animals. Development of the embryos until birth after freezing-thawing and transplantation of the embryos to recipient females was followed in eight species. Studies are mostly carried out in the zoos and some universities of USA, Canada, and UK. The Red Data Book species and artificially bred species are the main objects. In these studies, diverse methods of developmental biology were used: induced oocyte maturation, artificial fertilization and cultivation of the embryos in vitro, transplantation of the embryos to foster mothers of the same or closely related species, and obtaining of chimeras.

[The creation of genetic cryobanks and the use of the methods of developmental biology as a means for preserving rare animal species. III. The outlook, problems and limits: selection, modifications and mutations]. / Sozdanie geneticheskikh kriobankov i ispol'zovanie metodov biologii razvitiia kak sposob sokhraneniia redkikh vidov zhivotnykh. III. Perspektivy, problemy i ogranicheniia: selektsiia, modifikatsii i mutatsii.

A review of our own and literature data about perspectives, problems and limitations in the use of cryoconservation techniques and methods of developmental biology for the conservation of genetic resources is presented. It is demonstrated that the use of these methods may result in the selection and possibly leads to modifications and mutations in germ cells and embryos and in this way may change the genetic structure of restored populations as compared with the original ones. The need to improve current techniques of cryoconservation and accompanying biotechnological procedures in order to abolish these undesirable genetic effects is emphasized.

Establishment of germline-competent embryonic stem cell lines from the MSM/Ms strain.

MSM/Ms is an inbred mouse strain established from the Japanese wild mouse, Mus musculus molossinus, which has been phylogenetically distinct from common laboratory mouse strains for about 1 million years. The nucleotide substitution rate between MSM/Ms and C57BL/6 is estimated to be 0.96%. MSM/Ms mice display unique characteristics not observed in the commonly used laboratory strains, including an extremely low incidence of tumor development, high locomotor activity, and resistance to high-fat-diet-induced diabetes. Thus, functional genomic analyses using MSM/Ms should provide a powerful tool for the identification of novel phenotypes and gene functions. We report here the derivation of germline-competent embryonic stem (ES) cell lines from MSM/Ms blastocysts, allowing genetic manipulation of the M. m. molossinus genome. Fifteen blastocysts were cultured in ES cell medium and three ES lines, Mol/MSM-1, -2, and -3, were established. They were tested for germline competency by aggregation with ICR morulae and germline chimeras were obtained from all three lines. We also injected Mol/MSM-1 ES cells into blastocysts of ICR or C57BL/6 x BDF1 mice and found that blastocyst injection resulted in a higher production rate of chimeric mice than did aggregation. Furthermore, Mol/MSM-1 subclones electroporated with a gene trap vector were also highly efficient at producing germline chimeras using C57BL/6 x BDF1 blastocyst injection. This Mol/MSM-1 ES line should provide an excellent new tool allowing the genetic manipulation of the MSM/Ms genome.

[Late embryonic development of the vomeronasal complex of the cat (Felis silvestris)]. / Zur spätembryonalen Entwicklung des Vomeronasalkomplexes der Katze (Felis silvestris).

An examination of 2 feline embryos in different stages of development (overall length 60 and 115 mm respectively) reveals a well developed vomeronasal complex in each case. Jacobson's Organs embedded within the paraseptal cartilage form long blind tubes at the base of the septum nasi. The cartilage is caudally tub-shaped and embraces rostrally completely the organ over a considerable length. In this manner a long, nearly tunnel-like tube is formed which represents a modified form of the original outer bar and which has not been described so far in cats. It stretches rostro-ventrally across the branching region of the paraseptal cartilage as far as the mouth of Jacobson's Organ. The dorsal branch of the cartilago paraseptalis on the other hand forms a vertically oriented strip which connects to the lamina transversalis anterior. The ductus nasopalatinus passing through the palate is laterally supported by a cartilago ductus nasopalatini which rostrally to the mouth of Jacobson's Organ forms a unified element with the ventral branch of the cartilago paraseptalis. In the case of the younger cat embryo, this cartilago ductus nasopalatini is yet weakly developed. The ductus nasopalatini of the embryos studied are in an amazingly retarded state of development. The ductus, which are blocked in the early stages of the embryonic development during secondary palate formation, form predominantly solid strands of epithelium. By dissolving the cemented epithelium, the ductus are open. But even in the case of the older embryo of the cat, this process is not completed yet. The short duct connecting Jacobson's Organ with the ductus nasopalatinus is also still closed in both embryos. Such cemented sections of epithelium of the younger embryo reveals an interesting relation between the ductus nasopalatinus and the ductus nasolacrimalis which so far has not been pointed out for mammals. From the point of view of phylogenetics, the locally specialized vomeronasal complex of cats exhibits all the criteria of a progressive development of characteristics.

Interspecific cell markers and lineage in mammals.

Study of cell lineage in the mammalian embryo has relied heavily on the use of chimeras to follow the fate of genetically marked cells in later development. Such studies have often been limited by the types of genetic markers available; there are very few markers that allow analysis of the spatial distribution of individual cells at all stages of development. We have developed a marker system that is based on the identification of cells of Mus musculus origin in M. musculus-M. caroli chimeras by in situ DNA-DNA hybridization using a cloned probe to M. musculus satellite DNA. This provides the first ubiquitous in situ cell marker system for mammalian chimeras. We have recently refined the system by the use of biotin-labelled probes and detection of hybridization by streptavidin-peroxidase binding. This increases both the speed and the resolution of the assay. We have used the marker for cell lineage analysis in both embryonic and adult chimeras and results from analysis of the derivatives of early cell lineages in later development and study of coherent growth versus cell mixing in the postimplantation embryo are presented. The importance of understanding embryonic cell lineages as a prelude to molecular studies is emphasized.

Alterations in development of reproductive and endocrine systems of wildlife populations exposed to endocrine-disrupting contaminants.

Wildlife and human populations are affected by contaminants in natural settings. This problem has been a growing concern over the last decade with the realization that various environmental chemicals can alter the development and functioning of endocrine organs, cells and target tissues. Documented disruptions or alterations in reproductive activity, morphology or physiology in wildlife populations have been correlated with contaminant-induced modifications in endocrine system functioning. Alterations of the endocrine system are complex, and not limited to a particular organ or molecular mechanism. For instance, contaminants have been shown to (1) act as hormone receptor agonists or antagonists, (2) alter hormone production at its endocrine source, (3) alter the release of stimulatory or inhibitory hormones from the pituitary or hypothalamus, (4) alter hepatic enzymatic biotransformation of hormones, and (5) alter the concentration or functioning of serum-binding proteins, altering free hormone concentrations in the serum. This review focuses on two of these alterations, altered hormone synthesis and hepatic biotransformation, as a number of recent studies indicate that these actions are important components of endocrine disruption in developing organisms. The possible role of contaminants in altering sex determination mechanisms is also examined.