Evaluation of epididymal semen quality using the Hamilton-Thorne analyser indicates variation between the two caudae epididymides of the same bull

Epididymal semen is being more often considered as a potential source of valuable genes for genome resource banks. To utilize this resource as efficiently as possible, storage and freezing fertility and preservation characteristics of epididymal semen have to be examined. Because semen quality should be assessed as objectively as possible, we introduced computer assisted sperm analysis (CASA) of epididymal bull semen. The aims of this study were: to determine the quality of fresh cauda epididymal bull sperm, conventionally and by CASA (Hamilton-Thorne Ceros 12.1); to compare epididymal sperm movement with the motion characteristics of ejaculated semen; and to investigate whether equality of semen characteristics exists between both caudae epididymides of the same bull. In experiment 1, it is shown that epididymal sperm has a lower motility (total: 48.7% versus 79.9%, p < 0.0001 and progressive: 34.4% versus 58.4%, p < 0.0001) and moves less straight (80.5% versus 84.5%, p < 0.0009) with a higher amplitude (6.1 microm versus 5.0 microm, p < 0.0001) than ejaculated semen. The epididymal straight line velocity (85.2 microm/s versus 98.3 microm/s, p < 0.0001) is lower, but the curvilinear velocity (173.5 microm/s versus 156.4 microm/s, p < 0.0001) is higher than those of ejaculated semen. The data in experiment 2 are analysed to determine equality, rather than to find a difference. They illustrate that mean differences, for most semen parameters, between the semen from paired caudae epididymides, deviated more than 20% from the average values of these parameters from all bulls; the exceptions (those parameters within 20% of the average for all bulls) were the percentage of live spermatozoa, the linearity of sperm movement, the weights of testis and epididymis, the weights of the cauda epididymis alone, the volumes, and the amplitudes of movement of the semen (p < 0.05). The mean differences between the percentage of live spermatozoa and the amplitude of movement of the epididymal semen of both epididymides of one bull, were the only values smaller than 10% of the average value of this parameter (p < 0.05). This implies that sperm from one cauda epididymis should not be used as a control for the other because, for most of the semen parameters (concentration, morphology, motility, and beat cross frequency), equality between caudae epididymides of the same bull could not be established.     

Xenotransplantation in immunodeficient mice to study ovarian follicular development in domestic animals

Nowadays, in vitro study of follicular dynamics of primordial and primary follicular stages is limited because in vitro culture systems for these follicles are lacking, both in domestic animal species and in human. Therefore, additional insights might be generated by grafting ovarian tissue into immunodeficient mice to study activation and maturation of early follicular stages. A considerable amount of data has already been gathered in laboratory animals and through clinical application of human assisted reproduction technologies where live births were reported recently after the use of (cryopreserved) ovarian grafts. However, given that human preantral follicles are difficult to obtain and that there are many similarities between the bovine and human species with regard to ovarian physiology, the bovine model offers exciting additional prospects and is therefore discussed in more detail. This review will focus on recent developments related to preantral follicle and (repeated) ovarian tissue retrieval and xenotransplantation of (bovine) ovarian tissue strips to immunodeficient mice as a model to study preantral follicular dynamics. Different grafting strategies will be discussed as well as the consequences of this procedure on the viability and dynamic behavior of the grafted tissue and follicles.     

Characterization of rainbow trout cell lines using microsatellite DNA profiling

Ten microsatellite loci (Omy27DU,Omy325(A3)UoG, OmyFGT5TUF,OmyFGT14TUF, OmyFGT15TUF,OmyFGT23TUF, Omy77DU,Ssa20.19NUIG, Ots1BML, andOne18ASC) were amplified using the polymerase chain reaction to create genetic profiles for nine cell lines (RTG-2, RTH-149,RTL-W1,RTgill-W1, RTS-11, RTS-34st, RTP-2, RTP-91E and RTP-91F) from rainbow trout(Oncorhynchus mykiss) and one cell line (CHSE-214) from Chinook salmon (O. tschawytscha). A cell line (PHL) from anon-salmonid, the Pacific herring (Clupea harengus pallasi), was included as a control. The ten loci clearly revealed the uniqueness of each cell line, except for two cell lines (RTP-91E andRTP-91F) from the same fish. RTP-91E and RTP-91F were identical at all loci except Ssa20.19NUIG. The most useful locus for demonstrating uniqueness was Ots1BML. The information was used to demonstrate that an uncharacterized rainbow trout cell line (Clone 1A)was in fact CHSE-214, illustrating the usefulness of multiplexed microsatellites for the creation of genetic profiles for salmonid cell lines and for the testing of cell line cross-contamination. This revised version was published online in August 2006 with corrections to the Cover Date.     

Follicle survival and growth to antral stages in short-term murine ovarian cortical transplants after Cryologic solid surface vitrification or slow-rate freezing

This study was designed to asses murine preantral follicle survival and growth, after cryopreservation of ovarian tissue by two different methodologies, solid-surface vitrification by the Cryologic vitrification method (CVM) and slow-rate freezing (SRF). Cryotreated tissue was stored in liquid nitrogen for 24h, and upon warming follicle viability was assessed by live/dead fluorescent probes, and by 7-day autotransplantation of both cryotreated tissue types to the left and right kidney capsule of the donor animals (n=16). The live/dead assay immediately upon tissue warming did not allow a distinction to be made in terms of follicle viability between the CVM and SRF cryoprocedure. In grafted tissue, follicular survival and growth was assessed by conventional histological examination and proliferating cell nuclear antigen immunohistochemistry. In each experimental group (control, CVM and SRF), follicles were classified according to developmental stage, and a comparison of the proportions of follicle stages between the three groups was executed by statistical analysis of variance. The fraction of primordial follicles in CVM and SRF grafts significantly decreased as compared to control tissue, whereas intermediary and primary follicles significantly increased. The proportion of secondary and antral follicles after SRF was significantly larger than after CVM, but did not differ significantly between CVM and control tissue. The observed massive follicle activation is a typical transplantation effect, but testifies to the survival of cryopreserved follicles. In both types of cryotreated tissue, growing follicles, including antral stage, were present in grafts from all recipient animals. The significantly more abundant further developed stages in SRF treated tissue, however, suggest that CVM treated tissue may have suffered a growth disadvantage. To our knowledge, this is the first time that the CVM technique has been utilized to vitrify preantral follicles.     

Specific glycosidase activity isolated from a random phage display antibody library

Carbohydrates serve as key receptor sites in various cellular events such as viral attachment, tumor formation, and tissue inflammation. A potential route to control these events is to manipulate targeted carbohydrate structures in vivo using specifically designed glycohydrolases. Here we show that a stereospecific catalytic activity designed toward a particular sugar and linkage can be readily isolated from a phage display antibody library derived from a nonimmunized host. The activity was isolated using a transition-state analogue mimicking an alpha-glucosidasic linkage as antigen and showed a 20-fold specificity for that sugar and linkage. The DNA sequence, however, contains a large deletion in the antibody gene, which also changes the downstream reading frame, resulting in a translated sequence containing only 57 amino acids that has a predominantly hydrophobic amino terminal and a strongly hydrophilic carboxy terminal. The isolated catalytic activity has a strong pH dependence, attributable to one or more of the numerous potentially charged groups in the carboxyl terminal. While the protein readily forms more stable multimers, the 7.3-kD monomer represents by far the smallest glycosidase enzyme reported to date and can provide substantial new information toward understanding and modifying glycosidase activity.     

Accurate determination of rate constants of very slow, tight-binding competitive inhibitors by numerical solution of differential equations, independently of precise knowledge of the enzyme concentration

This paper is concerned with the determination of rate constants characterizing the binding and release of a slow binding inhibitor to and from an enzyme, here almond beta-glucosidase. We demonstrate the inability of the conventional method to yield reliable rate constants when one or more of these is less than 1 x 10(-4) per second. Instead one must use the much more accurate fitting of rate constants of the set of simultaneous differential equations characterizing the kinetic model. This procedure has the added advantage, when properly used, that the rate constants found pertaining to the inhibitor are largely insensitive to the particular value used for the enzyme concentration; i.e., the same data set may be fitted using a range of enzyme concentrations with no change in the resulting parameters. Hence the method can be used when little is known about the enzyme, except for the value of K(m), which is readily determined. Also, we report the somewhat unexpected finding that the association rate constant for the substrate (4-nitrophenyl-beta-d-glucopyranoside) is about one-third of the value of the corresponding rate constant for the inhibitor. The method is used to determine rate constants at several temperatures for the strong, slow binding inhibitor 2-phenethylglucoimidazole 1, enabling us to compute standard thermodynamic functions. The identity of these functions with those of isofagomine (2) reported earlier leads us to argue that the two compounds share a common binding mechanism, involving the same groups, whereas the different stabilities of the enzyme-inhibitor complexes must reside in those parts of the molecules that are not identical.     

Preferential induction of apoptosis in the rainbow trout macrophage cell line, RTS11, by actinomycin D, cycloheximide and double stranded RNA

The rainbow trout macrophage cell line RTS11 was found to be considerably more sensitive than rainbow trout fibroblast (RTG-2) and Chinook salmon epithelial (CHSE-214) cell lines to killing by macromolecular synthesis inhibitors, actinomycin D (AMD) and cycloheximide (CHX), a synthetic double stranded RNA (dsRNA), polyinosinic:polycytidylic acid (poly IC), and combinations of poly IC with AMD or CHX. Exposures of 24-30 h to AMD or CHX alone killed RTS11, but not CHSE-214 and RTG-2, in basal medium, L-15, with or without fetal bovine serum (FBS) supplementation. A two-week exposure to poly IC killed RTS11 in L-15, whereas RTG-2 and CHSE-214 remained viable. At concentrations that caused very little or no cell death, CHX or AMD pretreatments or co-treatments sensitized RTS11 to poly IC, causing death within 30 h. In all cases death was by apoptosis as judged by two criteria. H33258 staining revealed a fragmented nuclear morphology, and genomic degradation into oligonucleosomal fragments was seen with agarose gel electrophoresis. With AMD- or CHX-induced death, killing seemed caspase-independent as the pan caspase inhibitor, z-VAD-fmk, failed to block killing. By contrast, z-VAD-fmk almost completely abrogated killing by co-treatments of poly IC and low concentrations of AMD or CHX, suggesting caspase dependence. Killing by both types of treatments was blocked by 2 aminopurine (2-AP), which suggests the involvement of dsRNA-dependent protein kinase (PKR). The sensitizing of RTS11 to poly IC killing by AMD or CHX could be explained by a decrease in the level of a short-lived anti-apoptotic protein(s) and/or by the triggering of a ribotoxic stress.     

Processing glucosidase inhibition by 1-azafagomine

Natural azasugars have the ring oxygen substituted by nitrogen. They show potent inhibitory activity against glycosidases. The effect of substituting the ring carbon with nitrogen was examined with 1-azafagomine. 1-Azafagomine exhibited similar activity against processing glucosidase to that of fagomine.     

Identification and bioactivities of IFN-gamma in rainbow trout Oncorhynchus mykiss: the first Th1-type cytokine characterized functionally in fish

IFN-gamma is one of the key cytokines in defining Th1 immune responses. In this study, an IFN-gamma homologue has been identified in rainbow trout Oncorhynchus mykiss, and its biological activities have been characterized. The trout IFN-gamma cDNA is 1034 bp in length and translates into a 180-aa protein. The first intron of the trout IFN-gamma gene contains highly polymorphic GACA minisatellites and 44-bp DNA repeats, giving rise to at least six alleles. IFN-gamma is structurally conserved among vertebrates, and a signature motif has been identified. A nuclear localization sequence known to be crucial for IFN-gamma biological activities is also present in the C-terminal region of the trout IFN-gamma. The IFN-gamma expression was induced in head kidney leukocytes by stimulation with PHA or poly(I:C) and in kidney and spleen of fish injected with poly(I:C). rIFN-gamma produced in Escherichia coli significantly stimulated gene expression of IFN-gamma-inducible protein 10 (gammaIP-10), MHC class II beta-chain, and STAT1, and enhanced respiratory burst activity in macrophages. Deletion of 29-aa residues from the C terminus containing the nuclear localization sequence motif resulted in loss of activity with respect to induction of gammaIP-10 in RTS-11 cells. Moreover, IFN-gamma-induced gammaIP-10 expression was completely abolished by the protein kinase C inhibitor staurosporine, and partially reduced by U0126, a specific inhibitor for ERKs. Taken together, the present study has demonstrated for the first time a functional IFN-gamma homologue in a fish species, strongly suggesting a conserved Th1 immune response is most likely present in lower vertebrates.