Fetal fluid steroids and their relationship to gonadal steroid secretion in single and twin bovine fetuses

Steroid concentrations in the fetal fluids of 153 single and 69 twin bovine pregnancies, ranging in age from 35 to 125 d of gestation, were studied to compare gonadal steroid secretions in vitro with the concentrations found in amniotic and allantoic fluids during the early stages of sex differentiation. Among the steroids measured in fetal fluids, only the testosterone level showed a correlation with the amount secreted by the gonads. Significantly higher concentrations of testosterone were associated with male fetuses than with female fetuses. The concentrations of androstenedione, estradiol and estrone in both fetal fluid compartments were generally correlated with age, reflecting the extra-gonadal source of steroids in these fluids. Androstenedione levels in fetal fluids were significantly higher in twins than in singletons, suggesting that this parameter may be useful for the diagnosis of fetal sex and/or type of pregnancy.     

Tracing paternal ancestry in mice, using the Y-linked, sex-determining locus, Sry

The molecular evolution of mammalian Y-linked DNA sequences is of special interest because of their unique mode of inheritance: most Y- linked sequences are clonally inherited from father to son. Here we investigate the use of Y-linked sequences for phylogenetic inference. We describe a comparative analysis of a 515-bp region from the male sex- determining locus, Sry, in 22 murine rodents (subfamily Murinae, family Muridae), including representatives from nine species of Mus, and from two additional murine genera--Mastomys and Hylomyscus. Percent sequence divergence was < 0.01% for comparisons between populations within a species and was 0.19%-8.16% for comparisons between species. Our phylogenetic analysis of 12 murine taxa resulted in a single most- parsimonius tree that is highly concordant with phylogenies based on mitochondrial DNA and allozymes. A total evidence tree based on the combined data from Sry, mitochondrial DNA, and allozymes supports (1) the monophyly of the subgenus Mus, (2) its division into a Palearctic group (M. musculus, M. domesticus, M. spicilegus, M. Macedonicus, and M. spretus) and an Oriental group (M. cookii++, M. cervicolor, and M. caroli), and (3) sister-group relationships between M. spicilegus and M. macedonicus and between M. cookii and M. cervicolor. We argue that Y- chromosome DNA sequences represent a valuable new source of characters for phylogenetic inference.      

Modulation of cisPlatin cytotoxicity by interleukin-1α and resident tumor macrophages

The modulation of cisPlatin cytotoxicity by interleukin-1 (IL-1α) was studied in cultures of SCC-7 tumor cells with and without tumor macrophages to examine potential mechanisms for the synergistic antitumor activity of cisPlatin and IL-1α in SCC-7 solid tumors. Neither IL-1α nor tumor macrophages affected the survival of clonogenic tumor cells and IL-1α had no direct effect on tumor cell growthin vitro. Macrophages had no direct effect on cisPlatin sensitivity (IC₉₀=6.0 μM), but, the addition of IL-1α (500–2000U/ml) to co-cultures of cisPlatin pretreated tumor cells and resident tumor macrophages increased cell killing (IC₉₀=3.1 μM). Similar responses were seen in primary cultures treated with cisPlatin before IL-1α. The modulation of cisPlatin cytotoxicity by IL-1α exhibited a biphasic dose response that paralleled the IL-1α dose dependent release of H₂O₂by resident tumor macrophages. Further, IL-1α modification of cisPlatin cytotoxicity was prompt and inhibited by catalase. CisPlatin and exogenous H₂O₂ (50 μM) produced more than additive SCC-7 clonogenic cell kill and hydroxyl radicals played an important role in the response. Interleukin-1 modulation of cisPlatin cytotoxicity was schedule dependent. IL-1α treatment for 24 hrs, before cisPlatin, produced drug resistance (IC₉₀=11.1 μM). Our study shows that IL-1α can stimulate tumor macrophages to release pro-oxidants that modify cellular chemosensitivity in a schedule and dose dependent fashion. Our findings may also provide a mechanistic explanation for the synergistic antitumor activity of cisPlatin and IL-1αin vivo.     

Active and passive cation transport and L antigen hertogeneity in low potassium sheep red cells

Several lines of experimental evidence are presented suggesting that the L antigens in low potassium (LK) sheep red cells are associated with separate Na(+)K(+) pump flux is distinct from the action of anti-L(l) on K(+) leak flux, implying that K(+) leak transport sites may not be converted into active pumps by the L antiserum. Treatment of LK red cells with trypsin completely abolished both the stimulation of K(+) pump flux and the enhancement of the rate of ouabain binding brought about by anti- L. That this effect is due to a total destruction of the L(p) determinant associated with the LK pump was evident from the complete failure of anti-L(p) to bind to trypsinized LK red cells. The L(p) antigen can be effectively protected against the trypsin attack by prior incubation with anti-L, indicating that the sites for antibody binding and trypsin action may be closely adjacent at the structural level. Trypsin treatment, however, did not interfere with anti-L(l) reducing ouabain insensitive K(+) leak influx, nor did it prevent binding of anti-L(ly), the hemolytically active L antibody which is probably identical with anti-L(l). The functional independence of the L(p) and L(l) sites was documented by the observation that anti-L(l) still reduced K(+) leak influx in LK cells with experimentally induced high potassium concentrations, at which K(+) pump flux is fully suppressed, whether or not anti-L(p) was binding to the L(p) antigen associated with the LK pump.     

Nature of leukemia-associated antigenicity of dinitrophenylated human lymphocytes and lymphoblasts

Using dinitrophenylated human lymphocytes and phytohaemagglutinin-stimulated human lymphoblasts as antigens, antibodies were produced in rabbits. The immunological reactivities of the antisera so produced were tested against various types of leukemic cells after absorbing the sera with pooled normal leukocytes. Both the sera showed reactivity with all types of leukemic cells and no specific affinity for lymphoid leukemic cells was seen. This may suggest the presence of some common antigens on all types of leukemic cells or that dinitrophenylation brings about similar changes on all types of normal leukocytes.     

Membranes in the miotic apparatus of barley cells

Membranes in the mitotic apparatus have been investigated ultrastructually in dividing cells of barley (Hordeum vulgare). After osmium tetroxide- potassium ferricyanide or ferrocyanide postfixation (OsFeCN) of material that had been fixed in glutaraldehyde in the presence of Ca(++), the nuclear envolope (NE)-endoplasmic reticulum (ER) complex is selectively stained, permitting observations on the cellular pattern and structural ramifications of this membrane system that have not been previously recognized. Specifically, it is observed that membrane system that have not been previously recognized. Specifically, it is observed that during mitosis the NE-ER forms a continuous membrane system that ensheathes and isolates the mitotic apparatus (MA). Elements of ER progressively accumulate in the region of the spindle pole, becoming most concentrated by early anaphase. Within the MA itself, there are striking spindle- membrane associations; in particular, tubular elements of predominantly smooth NE-ER invade the spindle interior selectively along kinetochore microtubules. The membrane elements at the pole and surrounding the MA consist of tubular reticulum and fenestrated lamellae. Membranes of the MA thus resemble in considerable detail the tubular network and fenestrated elements of the sarcoplasmic reticulum of muscle. It is suggested that the NE-ER of the dividing barley cell may function in one or both of the following ways: (a) to control the concentration of free Ca(++) in the MA and (b) to serve as an anchor to chromosome motion.     

Saccharomyces cerevisiae DNA Ligase IV Supports Imprecise End Joining Independently of Its Catalytic Activity

DNA ligase IV (Dnl4 in budding yeast) is a specialized ligase used in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation in vitro and NHEJ activity in vivo, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that dnl4-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast) at DSBs was observed in wild-type as well as dnl4 point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5′ resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.     

Impacts of cage culture on physico-chemical and bacteriological water quality in Lake Volta,Ghana

The effects of cage fish farming on physico-chemical and bacteriological water quality in Lake Volta, Ghana, were investigated in 2013–2014. Farmed and unfarmed (control) areas of the lake were selected for monitoring. Nutrients, temperature, dissolved oxygen, conductivity, turbidity, pH, total coliforms, Pseudomonas and Vibrio spp. in the water were monitored monthly. Analyses of the water samples were carried out according to standard procedures. Physico-chemical quality of the water in both farm and control sites were within ranges typical of minimally impacted water and did not vary significantly between the two contrasting sites. The bacteriological analysis, however, revealed contamination of the lake water by fish farming. The bacterial counts at the farmed sites were significantly higher (p < 0.05) than those of the control sites, with figures at the farmed sites ranging from 132 to 1 708 cfu 100 ml?1 for total coliforms, 514 to 5 170 cfu 100 ml?1 Pseudomonas spp. and 14 to 516 cfu 100 ml?1 for Vibrio spp. The results suggested that cage fish farming has increased bacterial loads in the lake water, but has had minimal impact on its physico-chemical quality.     

Use of somatic cell nuclear transfer to study meiosis in female cattle carrying a sex-dependent fertility-impairing X-chromosome abnormality

Animal models have played an important part in establishing our knowledge base on reproduction, development, and the occurrence and impact of chromosome abnormalities. Translocations involving the X chromosome and an autosome are unique in that they elicit sex-dependent infertility, with male carriers rendered sterile by synaptic anomalies during meiosis, whereas female carriers conceive but repeatedly abort. Until now the limited access to relevant fetal oocytes has precluded direct study of meiotic events in female carriers. Because somatic cell nuclear transfer (SCNT) circumvents meiotic problems associated with fertility disturbances in translocation carriers, we used SCNT to generate embryos, fetuses, and calves from a cell line derived from a deceased subfertile X-autosome translocation carrier cow to study the meiotic configurations in carrier oocytes. Data from 33 replicates involving 2470 oocyte-donor-cell complexes were assessed for blastocyst development and of these, 42 blastocysts were transferred to 21 recipients. Fourteen pregnancies were detected on day 35 of gestation. One of these was sacrificed for ovary retrieval on day 94 and three went to term. Features of oocytes from the fetal ovary and from the newborn ovaries were examined. Of the pachytene spreads analyzed, 16%, 82%, and 1.5% exhibited quadrivalent, trivalent/univalent, and bivalent/univalent/univalent structures, respectively, whereas among the diakinesis/metaphase I spreads, 16% ring, 75% chain, and 8.3% bivalent/bivalent configurations were noted, suggesting that the low fertility among female carriers may be related to synaptic errors in a predominant proportion of oocytes. Our results indicate that fibroblasts carrying the X-autosome translocation can be used for SCNT to produce embryos, fetuses, and newborn clones to study such basic aspects of development as meiosis and to generate carriers that cannot easily be reproduced by conventional breeding.     

Synaptic pattern of sex complements and sperm head malformation in X-autosome translocation carrier bulls

Testicular activity and semen characteristics of bulls carrying an X-autosome translocation t(Xp +;23q-) revealed all stages of spermatogenesis although their semen consisted of few and, exclusively, of malformed spermatozoa. Chromosome painting on metaphase spreads of their mother and synaptonemal complex analysis on these and normal bulls were carried out to test whether the location and meiotic pairing behaviour of the rearranged segments could have contributed to the sperm head malformation and oligospermia in our X-autosome translocation (X-AT) carrier bulls. Spermatocytes of X-AT carriers displayed the rearranged chromosomes in a univalent-trivalent association, with 23q- always remaining as a univalent and Xp + in synapsis with normal chromosome 23 and the Y chromosome. Chromosome painting studies to test whether the total absence of meiocytes showing a quadrivalent is due to the non-reciprocal nature of this translocation, identified Xp sequence homology with the distal end of 23q- confirming its relocation to the terminal segment of 23q-. Our synaptonemal complex analyses also confirmed that the bovine pseudo-autosomal region (PAR) is at the distal ends of Xq and Yp and further revealed that over 85% of spermatocytes of X-AT carriers (and up to 13% of spermatocytes of normal bulls) sustain a Y-axis break adjacent to the PAR. Although the exact cause of a Y-axis break in bovine spermatocytes is not known at present, we believe that the break and possible loss of Yq in such high proportions of spermatocytes of X-AT carriers could have contributed to the sperm head malformation and oligospermia in our X-AT carrier bulls.