Failure of spermatozoa from T/t mice to fertilize in vitro is overcome by zona drilling

Failure of epididymal spermatozoa from T/t mutant mice, but not from t/t individuals, to fertilize oocytes in vitro was partially overcome by opening a small aperture in the zona pellucida with acidified Tyrode's solution to permit direct access of the spermatozoon to the vitellus. This study provides a model system to evaluate requirements for successful zona drilling in the treatment of human infertility and further insights into the effects of the t complex on sperm fertility.     

Pregnant mares' serum gonadotropin source influences fertilization and fresh or thawed embryo development,but the effect is genotype specific

The influence of the source of pregnant mares' serum gonadotropin (PMSG) on the num ber, quality, and in vitro development of mouse embryos before and after freezing was evaluated among three genotypes: N:NIH(S), C57BL/6N, and C3H/HeN-MTV^?. Immature females were given PMSG from one of five commercial sources. Following col lection ( 116 hr later), embryos were evaluated for stage of development, and four-to eight-cell embryos were pooled within genotype and assigned to standardized fresh or freeze-thaw culture trials. Different PMSG sources stimulated the production of different num bers of total embryos (P < 0.05) but not necessarily more embryos suitable for freezing. Differences in embryo production among genotypes indicated that absolute embryo num bers using a single mouse genotype may not accurately reflect the potency of a specific gonadotropin source. The PMSG source also affected the ability of an embryo to survive in culture either immediately after collection or after frozen storage. The effect, however, was genotype specific, with some mouse strains being relatively insensitive to PMSG source, whereas gonadotropin source played a major role in determining in vitro viability in others. Development rates for freshly collected embryos differed, often inconsistently, from those of thawed embryos regardless of the PMSG source used, demonstrating that fresh embryo development cannot be used to estimate expected post-thaw survival. In vitro development of thawed embryos is influenced not only by genotype, but also the source of the gonadotropin used to promote follicular development and oocyte maturation. These findings may explain, in part, the wide variation in embryo viability and culture rates reported among laboratories and intraspecies animal populations.     

In vitro capacitation and fertilizing ability of ejaculated rabbit sperm treated with lysophosphatidylcholine

Four experiments were replicated 1) to establish dose-response relationships between lysophosphatidylcholine (LPC), sperm motility, and the acrosome reaction (AR), 2) to evaluate the influence of rabbit serum (RS) on these endpoints, 3) to compare buck differences in induction of the AR, and 4) to examine fertilizing ability in vitro of sperm tested under the first three objectives. Semen was collected from Dutch-belted rabbits, washed once by centrifugation, resuspended, and preincubated for 2 or 4 hr in a chemically defined medium (DM), DM plus 20% RS, or BSA-free DM plus 20% RS at 37°C. At the end of preincubation LPC was added to the preincubated sperm at concentrations of from 0 to 100 μg/ml. Sperm were examined .5–4 hr later for AR and sperm motility. For in vitro fertilization, sperm and ova were coincubated in DM up to 24 hr after insemination and in a more complex medium for another 24 hr. Addition of LPC to 4-hr-preincubated sperm was more effective for inducing the AR than addition to 2-hr-preincubated sperm. A significant increase (P < .05) in the AR occurred in 15 and 30 min following exposure to 100 and 80 μg of LPC per ml, respectively, but the higher concentration of LPC decreased sperm motility. Addition of 20% RS to DM with or without BSA surprisingly inhibited the AR but maintained sperm motility, as expected. Bucks differed (P < .05) in the initial percentage and the induced percentage of AR sperm. For the AR the optimal concentration of LPC per ml was 80 μg, but for in vitro fertilization 60 μg tended to be superior.     

Production of monoclonal antibodies against avian LHRH-I

Summary Production of antibodies against peptides or poorly antigenic proteins by conventional methods often requires either large quantities of the native immunogen or some chemical modification to increase their antigenicity. In this study an in vivo and in vitro immunization protocol has been used to generate monoclonal antibodies against the decapeptide luteinizing hormone-releasing hormone (LHRH). Two injections of 100 μg of avian LHRH-I into BALB/c mice were given 7 d apart. Dissociated splenocytes were collected under sterile conditions. They were incubated with 100 μg of the immunogen in 75-cm² tissue culture flasks in thymocyte-conditioned media. After 5 to 8 d exposure to the antigen, splenocytes were fused with SP2/O myeloma cells by polyethylene glycol. The cells were plated into 24 wells and then incubated in hypoxanthine aminopterin and thymidine selective media. After 14 d an initial screening was done by enzyme immunoassay. The positive wells (6/24) were expanded into 96-well plates and rescreened. Selected lines were cloned out 3 times by limiting dilution and the most positive expanded for ascites production. The antibody was affinity purified in a protein A column. The antibody cross-reacted with LHRH-I and II but preferentially to LHRH-I, as shown by competitive assay. A hypothalamic extract from a mature chick showed a higher response than preparations from whole brain explants of 1- to 3-d posthatched chicks, mature quail, and mature mouse. This work was funded by the Maryland Agricultural Experiment Station artical no. A4975, contribution no. 8019.     

Conservatism of sites of tRNA loci among the linkage groups of severalDrosophila species

Summary The sites of seven tRNA genes (Arg-2, Lys-2, Ser-2b, Ser-7, Thr-3, Thr-4, Val-3b) were studied by in situ hybridization.¹²⁵I-labeled tRNA probes fromDrosophila melanogaster were hybridized to spreads of polytene chromosomes prepared from fourDrosophila species representing different evolutionary lineages (D. melanogaster, Drosophila hydei, Drosophila pseudoobscura, andDrosophila virilis). Most tRNA loci occurred on homologous chromosomal elements of all four species. In some cases the number of hybridization sites within an element varied and sites on nonhomologous elements were found. It was observed that both tRNA ₂ ^Arg and tRNA ₂ ^Lys hybridized to the same site on homologous elements in several species. These data suggest a limited amount of exchange among different linkage groups during the evolution ofDrosophila species.     

Poly- and Monoclonal Antibodies Against Recombinant Rat Brain Calbindin D-28K Were Produced to Map Its Selective Distribution in the Central Nervous System

Many processes in the CNS depend on calcium. The calcium signal is transduced into an intracellular response via Ca2(+)-binding proteins, including calbindin D-28K. In many laboratories, polyclonal antibodies against chicken intestinal calbindin D-28K have been used to study its localization in the brain (normal and degenerated) of various species, including humans, but some of these antisera cross-reacted with other proteins, including calretinin. We purified recombinant rat brain calbindin D-28K to raise antisera in rabbits and purified a recombinant rat-chicken calbindin D-28K hybrid protein to immunize mice for the generation of monoclonal antibodies. These antisera were highly specific for calbindin D-28K, as demonstrated by two-dimensional Western blotting analysis. Immunohistochemical analyses combined with in situ hybridization studies demonstrated that calbindin D-28K in the Purkinje cells of the cerebellum is independent of vitamin D. The antibodies described here will be important tools for studying the regulation of expression of calbindin D-28K and its biological function in the brain and in the PNS.     

In Vivo Labeling of Serotonin Uptake Sites with [3H]Paroxetine

Previous work has shown that [3H]paroxetine is a potent and selective in vitro label for serotonin uptake sites in the mammalian brain. In the present study, [3H]paroxetine was tested in mice as an in vivo label for serotonin uptake sites. Maximum tritium concentration in the whole brain (1.4% of the intravenous dose) was reached 1 h after injection into a tail vein. Distribution of the tracer at 3 h after injection followed the distribution of serotonin uptake sites known from previous in vitro binding studies (r = 0.85). The areas of highest [3H]paroxetine concentration, in decreasing order, were: hypothalamus greater than frontal cortex greater than olfactory tubercles greater than thalamus greater than upper colliculi greater than brainstem greater than hippocampus greater than striatum greater than cerebellum. Preinjection of carrier paroxetine (1 mg/kg) significantly decreased [3H]paroxetine concentration in all areas except in the cerebellum, which is known to contain a relatively low number of specific binding sites. Kinetic studies showed highest specific [3H]paroxetine binding (tissue minus cerebellum) at 2 h after injection and slow clearance of activity thereafter (half-time of dissociation from the hypothalamus, 215 min). The specificity of in vivo [3H]paroxetine binding was studied by preinjecting monoamine uptake blockers or receptor antagonists 5 min before administration of [3H]paroxetine. Serotonergic or muscarinic cholinergic receptor antagonists and dopamine or norepinephrine uptake blockers did not reduce the in vivo binding of [3H]paroxetine. In contrast, there was an excellent correlation (r = 0.99) between the in vivo inhibitory potencies of serotonin uptake blockers in this study and previously published in vitro data on inhibition of [3H] serotonin uptake in brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

大熊猫与金黄地鼠体外异种受精的研究

在大熊猫精子与地鼠卵的体外异种受精中,发现大熊猫精子穿入地鼠卵后可以激活受精卵产生极区,释放第二极体,受精卵内雌性原核形成。与此同时,地鼠卵的胞质也能促使大熊猫精子头发育成雄性原核,异种精卵间的相互作用与同种受精的相似。 细胞松弛素B能阻抑大熊猫雄性原核从地鼠卵皮层迁移到卵的中央,实验表明大熊猫雄性原核的迁移也受异种卵的微丝的控制。