Amino acid content of L5178Y and L5178Y/L-ASE cells after L-asparaginase treatment

The amino acid contents of tumor cells that are either sensitive or resistant to treatment with L-asparaginase were measured. These amino acid concentrations were measured as a function of incubation time with L-asparaginase or as a function of the L-asparaginase dose. The cell types compared were the mouse leukemia lines L5178Y (sensitive to L-asparaginase treatment) and L5178Y/L-ASE (resistant to L-asparaginase treatment). Upon L-asparaginase treatment both cell lines lost most of their cellular asparagine but, whereas the resistant cells exhibited the ability to rebound to about 50% of initial values, the sensitive cells did not. While previous work had suggested that asparagine-dependent glycine synthesis was essential for sensitive cells (but not in resistant cells), we found no difference in the glycine content of either of the two cell lines as a function of either time or dose that would support this hypothesis. Major differences between the two cell lines were seen in the content of the essential amino acids before treatment with L-asparaginase. After incubation without L-asparaginase the contents of the two cell lines became similar. These results are discussed in terms of possible mechanisms of L-asparaginase sensitivity and resistance.     

In vitro fertilization in the rabbit after delayed ovum recovery

Rabbit ovum donors were superovulated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Ova were recovered 16-17 h post-hCG from oviducts immediately after killing and from excised oviducts held in saline 30 min at 33 degrees or 38 degrees C prior to ovum recovery. In vivo-capacitated spermatozoa were used to inseminate both groups of ova. Data revealed a decrease in fertilization rates following a 30-min delay at 38 degrees C in ovum recovery. Thus, 64% (44/69 ova) were fertilized with rapid recovery, whereas 43% (39/90 ova) were fertilized following a 30-min delay. The decrease in fertilization imposed by delay in ovum recovery was apparently overcome when oviduct storage was at 33 degrees C. Under these conditions, 69% of inseminated ova were fertilized. Ova inseminated with in vitro-capacitated sperm showed a similar response to delayed ovum recovery. Embryonic development in culture of ova obtained from mated does was not affected by delay in recovery at 33 degrees or 38 degrees C provided mated does had been injected only with hCG. Ova from mated does receiving both PMSG and hCG were adversely affected by a 38 degrees C delay. The data emphasize the importance of rapid ovum recovery from oviducts and suggest the possibility of altering conditions to overcome damaging effects of delayed recovery.     

PHENOTYPIC CONSEQUENCES OF FORCING GERMINATION: A GENERAL PROBLEM OF INTERVENTION IN EXPERIMENTAL DESIGN

Many evolutionary and ecological studies of plants with seed dormancy are plagued by design problems related to seed germination. On the one hand, traits of interest, especially life history traits like early growth rate or time to flowering, may not be independent of dormancy phenotype. On the other hand, germination-inducing treatments are likely to affect these traits of interest. Consequently, researchers often have had to use confounded designs without information about the consequences. To examine this problem, we studied how early growth in California poppy (Eschscholzia californica) is affected by treating seeds with giberellic acid (GA) to stimulate germination. The dose used was sufficient to slightly but significantly advance emergence time in treated seeds, but it appeared to cause variable growth responses among treated seeds. Experiments using GA to stimulate germination may thus be misleading, at least for the populations and dose studied. Perhaps more importantly, the experimental and statistical approach we used can be employed to study the effects of this confounded design for other doses and species.     

Molecular phylogenetics at the population/species interface in cave spiders of the southern Appalachians (Araneae:Nesticidae:Nesticus)

This paper focuses on the relationship between population genetic structure and speciation mechanisms in a monophyletic species group of Appalachian cave spiders (Nesticus). Using mtDNA sequence data gathered from 256 individuals, I analyzed patterns of genetic variation within and between populations for three pairs of closely related sister species. Each sister-pair comparison involves taxa with differing distributional and ecological attributes; if these ecological attributes are reflected in basic demographic differences, then speciation might proceed differently across these sister taxa comparisons. Both frequency-based and gene tree analyses reveal that the genetic structure of the Nesticus species studied is characterized by similar and essentially complete population subdivision, regardless of differences in general ecology. These findings contrast with results of prior genetic studies of cave-dwelling arthropods that have typically revealed variation in population structure corresponding to differences in general ecology. Species fragmentation through both extrinsic and intrinsic evolutionary forces has resulted in discrete, perhaps independent, populations within morphologically defined species. Large sequence divergence values observed between populations suggest that this independence may extend well into the past. These patterns of mtDNA genealogical structure and divergence imply that species as morphological lineages are currently more inclusive than basal evolutionary or phylogenetic units, a suggestion that has important implications for the study of speciation mechanisms.      

Laparoscopic oviductal transfer of in vitro matured and in vitro fertilized bovine oocytes

The objective of this study was to obtain normal pregnancy following laparoscopic oviductal transfer of in vitro matured and fertilized bovine oocytes. Methods for in vitro maturation and in vitro fertilization were similar to those previously reported (1). Primary oocytes judged to be potentially viable were cultured for 26 h in modified TCM 199 supplemented with heat-treated fetal calf serum (20% v/v), 5mug/ml FSH (USDA-bFSH-B-1), and 1mug/ml estradiol 17-beta. Oocyte cumulus complexes were microscopically evaluated for maturation (first polar body formation) following a brief treatment with hyaluronidase. Mature oocytes were inseminated with heparin-treated spermatozoa and incubated at 39 degrees C under paraffin oil and moist 5% CO(2), 5% O(2), 90% N(2). In this work, 450 oocytes were recovered at slaughter from ovaries of 42 random cows of unknown reproductive status and 336 oocytes (74.7%) with compact cumulus were selected for culture. Of these, 322 (95.4%) matured in vitro. Of 218 inseminated oocytes, 198 (90.8%) were penetrated by sperm and 83 (38.1%) cleaved, with 102 (46.6%) of the embryos reaching four- to eight-cell stages. None of 40 oocytes not exposed to sperm and none of 30 oocytes inseminated with untreated sperm showed signs of activation. In a control experiment with hormones added, 105 of 115 (91.3%) oocytes matured in vitro and 20 of 105 (19.5%) cleaved following in vitro insemination. Laparoscopy was performed on four synchronized recipients under local anesthesia. A catheter containing three embryos in the two to four cell stages was passed through the operating channel of a direct viewing bronchoscope for deposition in the oviduct ipsilateral to the recipients developing corpus luteum while the fimbria and the mesovarium were manipulated with Semm's forceps. A normal term pregnancy confirmed in vitro fertilization and provides feasibility data for use of laparoscopic methodology developed in this work for testing viability of bovine oocytes and embryos. These results are encouraging for the application of in vitro maturation and in vitro fertilization for overcoming infertility in domestic and endangered species.     

Chronic stress and its effects on adrenal cortex apoptosis in pregnant rats

The model of chronic intermittent stress by immobilization during pregnancy may produce alterations in the mechanisms that maintain adrenal gland homeostasis. In earlier investigations using this model, significant variations in plasma prolactin and corticosterone levels, and adrenal gland weights were observed. We hypothesized that chronic stress causes changes in apoptosis in the adrenal glands of pregnant rats. We identified and quantified apoptotic cells in the adrenal cortex and examined their ultrastructural characteristics using transmission electron microscopy. Adrenal glands of pregnant rats at gestation days 12, 17 and 21 were studied for control and experimental (stressed) rats. Immunolabelling techniques, stereological analysis and image quantification of adrenal gland sections were combined to determine differences in apoptosis in the different cell populations of the adrenal cortex. The apoptotic index of the experimental rats showed a significant reduction at gestation day 17, while at days 12 and 21 there were no differences from controls. Moreover, the apoptotic index of the reticular zones in control and experimental animals showed a significant increase compared to the glomerular and fascicular zones at the three gestation times studied. Chronic stress by immobilization reduced the caspase-dependent apoptotic index at gestation day 17, which may be related to variations in plasma concentrations of estrogens and prolactin.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Dihydrotestosterone induces a sexual dimorphism in estrogen uptake by specific anterior pituitary cell types in vivo

Summary Castrated male and female rats pretreated with dihydrotestosterone (DHT) were injected i.v. with ³H-estradiol (E₂). Nuclear uptake and retention of ³H-E₂ was measured in each of five cell types of the anterior pituitary gland using a combined quantitative autoradiographic and immunocytochemical procedure. In non-pretreated groups, each cell type bound a characteristic amount of ligand but no sex differences were apparent. DHT pretreatment, however, caused a significant decrease in ³H-E₂ retention by gonadotrophs in both males and females. The treatment also caused a decrease in binding by lactotrophs and somatotrophs, but only in the females. No other cell types were altered. Thus, androgen appears to modulate E₂ binding and retention by pituitary cells in both a cell-type and sexdependent manner. Our results also indicate that the inhibitory effects of androgens on E₂ binding by the pituitary gland is more complex than can be explained by simple competition for the estrogen receptor.