Lactate dehydrogenase C4 in male sex accessory glands of normal mice and in testes of sex-reversed mice.

Lactate dehydrogenase (LDH) C4 activity was observed in testis extracts of sex-reversed mice (Sxr) and in male sex accessory gland (seminal vesicle and prostate) extracts from C3H/He and C57BL/Go mice. These results reflect either (1) the presence of low concentrations of germinal cells in these tissues; or (2) the synthesis of LDH-C4 by somatic cells in Sxr testes and normal male sex accessory glands.     

XY pair associates with the synaptonemal complex of autosomal male-sterile translocations in pachytene spermatocytes of the mouse (Mus musculus)

Analysis of the chromosome behaviour at pachytene has been performed by means of the silver staining technique visualizing the synaptonemal complexes (SCs) in male mice heterozygous for the male-sterile translocations T(5;12)31H, T(16;17)43H and T(7;19)145H, respectively. The T(9;17)138Ca male heterozygotes and T43H/T43H homozygous males were used as fertile controls. The sterile mice displayed a high frequency (about 60%) of pachytene spermatocytes with autosomal translocation configuration located in close vicinity of the XY pair. The dense round body (XAB), normally located near the X-chromosome axis in fertile males, exhibited abnormal affinity to translocation configuration in the sterile translocation heterozygotes. The incomplete synapsis of autosomes involved in translocation configuration was observed in more than 70% of the pachytene spermatocytes with the male-sterile translocations but in less than 20% of the cells from T138Ca fertile male.s. A hypothesis relating the spermatogenic arrest of carriers of male-sterile rearrangements to the presumed interference with X chromosome inactivation in male meiosis is discussed.     

Modulation of T cell functions by sperm-specific lactate dehydrogenase: fluorescence analysis of immune competent cells and local graft versus host reaction.

Immune responses to a well-defined sperm-specific isogenic lactate dehydrogenase-C4 (LDH-C4) have been studied in C57Bl/Ks (H-2d) mice after immunization through intra-rectal route. Presence of anti-LDH-C4-antibodies in the sera of females immunized in presence or absence of adjuvant suggested that the immune system of mice becomes exposed to sperm antigens following intrarectal insemination. LDH-C4 primed lymphocytes from both males and females, when transferred in F1 hybrids, suppressed stimulation index of local graft versus host reaction. However, contrary to females, male counterparts which did not elicit measurable anti-LDH-C4-antibody titer, showed the presence of a higher proportion of Ly2+ and Ia+ fluorescence labelled cells in the spleen of LDH-C4 administered mice. Results suggest that males are more susceptible for immune suppression of T cell functions through generation of T suppressor cells. Sex differences in relation to immune deviation by intra-rectal administration of sperm-specific LDH-C4 in mice and their consequences in AIDS and AIDS-related complex diseases are described.     

Differential activity and synthesis of lactate dehydrogenase isozymes A (muscle), B (heart), and C (testis) in mouse spermatogenic cells

Spermatogenic cells isolated from adult and prepubertal mice by unit gravity sedimentation were used to examine enzyme activities and synthesis of the lactate dehydrogenase (LDH) isozymes during spermatogenesis. The synthesis and activity of LDH-C4, the germ cell-specific isozyme, was detected earliest in isolated preleptotene and leptotene/zygotene spermatocytes prior to the mid-pachytene stage of meiosis reported previously. The LDH-C4 isozyme was prominent in pachytene spermatocytes, round spermatids, and condensing spermatids, whereas spermatozoa contained only the LDH-C4 isozyme. In addition, somatic-type LDH isozymes consisting primarily of LDH-B subunits were present in germ cells throughout spermatogenesis. This is in contrast to a previous report that the LDH-B subunit was not synthesized in germ cells. Sertoli cells were further shown to exhibit comparable amounts of five tetrameric LDH isozymes formed by combination of muscle-type LDH-A and heart-type LDH-B subunits.     

Chinese hamster ovary cells with reduced hexokinase activity maintain normal GDP-mannose levels

Parental Chinese hamster ovary (CHO) cells were mutagenized and subjected first to a mannose suicide selection technique and second to a screen of individual colonies grown on polyester discs for reduced mannose incorporation into protein. The incorporation of radioactivity for the selection and the screen was conducted at 41.5 degrees C instead of the normal growth temperature of 34 degrees C in order to allow for the isolation of temperature-sensitive lesions. This selection/screening procedure resulted in the isolation of M15-4 cells, which had three- to five-fold lower incorporation of [2-3H]mannose into mannose 6-phosphate, mannose 1-phosphate, GDP-mannose, oligosaccharide-lipid, and glycoprotein at 41.5 degrees C. We detected no difference in the qualitative pattern of mannose-labeled lipid-linked oligosaccharides compared to parental cells. M15-4 cells synthesized dolichol. The defect of M15-4 cells was determined to be in hexokinase activity; crude cytosolic extracts were eight- to nine-fold lower in hexokinase activity in M15-4 cells compared to parental cells. As a result of this defect, incorporation of labeled mannose from the medium was significantly decreased. However, the level of GDP-mannose in M15-4 cells was 70% of normal. The phenotype of M15-4 was a lower specific activity of labeled GDP-mannose, not a substantial reduction in the level of GDP-mannose. Consistent with these results, no alterations in the glycosylation of a model glycoprotein, G protein of vesicular stomatitis virus, were observed. These cells grew slower than parental cells, especially in low-glucose medium.     

Correction of chlorophyll-defective male-sterile winter oilseed rape (Brassica napus) through organelle exchange: Characterization of the chlorophyll deficiency

Jarl, C. I., Ljungberg, U. K. and Bornman, C. H. 1988. Correction of chlorophyll-defective male-sterile winter oilseed rape ( Brassica napus ) through organelle exchange: Characterization of the chlorophyll deficiency. - Physiol. Plant. 72: 505–510.
As is known, the introduction of male-sterile Raphanus sativus L. cytoplasm into Brassica napus L. results in male-sterile oilseed rape plants, which display a temperature-related chlorophyll defect. The influences of temperature and irradiance on this defect were investigated. Compared to a line of normal (green phenotype) male-fertile oilseed rape, the male-sterile line had reduced chlorophyll content, fewer chloroplasts per cell, an altered ultrastructure of the chloroplasts and reduced activities of both photosystems, although the relative amounts of the photosystems and the chlorophyll a/b ratio were similar. The lower activity of the photosystems is explained by a decreased functional antennae size and a reduced efficiency in the interactions between the nuclear-encoded light-harvesting proteins and the reaction centres coded for by the plastome. Some thylakoid polypeptides differed in proportion between the male-fertile line with green phenotype and the male-sterile line with chlorotic phenotype. Characters, in which the two lines exhibited differences, are ascribed to difficulties in molecular communication between the oilseed rape nucleus and the radish cytoplasm, which are combined in the deficient male-sterile line.     

Seasonal changes in antioxidant defence systems in seminal plasma and fluids of the boar reproductive tract

This study aimed to analyze seasonal variations in the antioxidant defence systems of the seminal plasma and fluids of the cauda epididymis and vesicular glands of the boar. The analyzed antioxidants included superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total L-glutathione (GSH+GSSG). Seasonal changes in total protein content and total antioxidant status (TAS) of the seminal plasma and reproductive fluids were also analyzed. Compared with the spring-summer period, total protein content in the seminal plasma was significantly higher during the autumn-winter period. Among the antioxidants analyzed, only SOD activity showed marked seasonal variations, being significantly higher during the spring-summer period. Likewise, the fluid of the cauda epididymis exhibited greater SOD and CAT activity during the spring-summer period, whereas TAS levels were markedly higher during the autumn-winter period. Neither GPx activity nor total GSH+GSSG content in the cauda epididymal fluid was significantly affected by the seasonal periods. The vesicular gland fluid exhibited an approximately 4-fold greater level of SOD activity during the autumn-winter period, as compared with the spring-summer period. By contrast, greater CAT and GPx activity, and a higher level of total GSH+GSSG were observed in the vesicular gland fluid during the spring-summer period. In conclusion, the findings of this study indicate that seasonal variations could have varying effects on the antioxidant defence systems in the seminal plasma and fluids of the boar reproductive tract.     

Age and strain dependence of O6-methylguanine DNA methyltransferase activity in mice

The activity of the DNA repair protein O6-methylguanine DNA methyltransferase (MT) was compared in liver extracts from female ICR and male C57BL/6 mice at various ages (3-130 weeks old). Similar patterns of overall enzyme activity were observed in both strains with O6-MT activity being relatively low in young mice (3 or 8 weeks old). However, the activity significantly increased after adolescence (middle age), thereafter decreasing with old age (over 100 weeks old) to a level equivalent to that found in young mice. In an additional strain difference study, O6-MT activities in liver extracts from 4 strains of mice were compared at 5 and 30 weeks of age. Although a similar age-associated increase of enzyme activity in adolescence was confirmed in all 4 strains investigated, the closed-colony ICR mice differed from the inbred strains in demonstrating significantly higher levels of O6-MT activity in females than in males. However, the same tendency was also observed in a comparison of the sexes in 30-week-old C3H/HeN, C57BL/6 and BALB/c mice.     

The sterols of normal and male-sterile maize tassels during development

The steroids of normal and male-sterile (Texas type) genotypes of maize were investigated during tassel development. A bioassay for estrogen activity of the normal meiotic and postmeiotic tassels was negative, indicating estrogen activity (estrone equivalent) much less than one ng/g of plant tissue. The sterols found were cholesterol, campesterol, stigmasterol, sitosterol, and probably isofucosterol, stigmast-7-enol, and 24-methylenecholesterol. In the premeiotic, meiotic, and postmeiotic stages of both genotypes between 300 and 400 μg of C₂₈ and C₂₉ free sterols per g tassels (wet wt) were found, the proportions of the sterols being ca 45% sitosterol, 30% stigmasterol, and 13% campesterol, with less than 5% each of the remaining sterols. In all three stages before saponification more free sterols were found in the normal than in the male-sterile tassels. The differences were significant at the 95% level in the meiotic and post-meiotic stages. The amounts of these sterols derived from esters decreased from approximately 140 μg/g in the premeiotic stage to 50 μg/g in the meiotic stage, and to an undetectable amount in the postmeiotic stage. After application of cholesterol-[4-¹⁴C] to the normal and male-sterile maize leaves for 3 days at meiosis, the label was found in the free sterols and steryl esters of the leaves but only in the free sterols of the tassels.     

Differential effects of (+)- and (-)-gossypol enantiomers on LDH-C4 activity of hamster spermatogenic epithelium in vitro

Tubular fragments (spermatogenic epithelium) from immature hamsters were isolated and cultured with low doses of (+)- and (-)-gossypol enantiomers. The activity of lactate dehydrogenase isoenzyme LDH-C4 was estimated as a marker for spermatogenic cell development and alpha-ketoisovalerate was used as the substrate. In the absence of gossypol, the specific activity of LDH-C4 in the tubular fragments was increased 40% during incubation for 48 h. This developmental increase was suppressed by gossypol. The specific activity of LDH-C4 in the tubular fragments was lowered by gossypol, after 48 h of culture in the presence of low doses of racemic gossypol (1-4 microM) and 1% fetal calf serum. In this in-vitro system the (-)-enantiomer but not the (+)-enantiomer of gossypol affected the LDH-C4 activity. This is in agreement with other reports showing that only the (-)-enantiomer induces infertility. The observed action of gossypol on LDH-C4 activity in the tubular fragments may reflect gossypol-induced degeneration of spermatogenic cells. The present in-vitro system can be used to estimate the actions of gossypol derivatives, other investigational antifertility agents, and toxic agents on the spermatogenic epithelium.     

A reciprocal autosomal translocation which causes male sterility in the mouse also impairs oogenesis

A quantitative histological analysis of ovaries from 3- and 5-day-old female mice heterozygous for the male-sterile reciprocal autosomal translocation, T(11;19)42H, revealed a marked reduction (by 65%) in the number of oocytes as compared to controls. These findings call into question the widely held view that chromosomal anomalies causing spermatogenic failure have no effect on oogenesis. It is suggested that during meiosis in males and females there is a mechanism operating which tends to eliminate cells which had incomplete chromosome pairing at the pachytene stage.     

Interleukin-21 Is a Critical Cytokine for the Generation of Virus-Specific Long-Lived Plasma Cells

Long-lived plasma cells that reside in the bone marrow constitutively produce antibody in the absence of antigen and are the cellular basis of durable humoral immunity. The generation of these long-lived plasma cells depends upon a series of highly orchestrated interactions between antigen-specific CD4 T cells and B cells and the formation of germinal centers (GCs). In this study, we have examined the role of the cytokine interleukin-21 (IL-21) in regulating humoral immunity during acute viral infections. Using IL-21 receptor-deficient (IL-21R^−/−) mice, we found that virus-specific CD4 T cells were generated after infection with lymphocytic choriomeningitis virus (LCMV) and that these CD4 T cells differentiated into T follicular helper (T_FH)-like cells in the absence of IL-21 signaling. There was also no defect in the formation of GCs, although after day 15 these GCs disappeared faster in IL-21R^−/− mice than in wild-type mice. Isotype switching and the initial LCMV-specific IgG response were normal in IL-21R^−/− mice. However, these mice exhibited a profound defect in generating long-lived plasma cells and in sustaining antibody levels over time. Similar results were seen after infection of IL-21R^−/− mice with vesicular stomatitis virus and influenza virus. Using chimeric mice containing wild-type or IL-21R^−/− CD4 T cells and B cells, we showed that both B and CD4 T cells need IL-21 signaling for generating long-term humoral immunity. Taken together, our results highlight the importance of IL-21 in humoral immunity to viruses.     

Reversible male sterility in transgenic tobacco carrying a dominant-negative mutated glutamine synthetase gene under the control of microspore-specific promoter

Metabolic engineering was used to disrupt glutamine metabolism in microspores in order to block pollen development. We used a dominant-negative mutant (DNM) approach of cytosolic glutamine synthetase (GS1) gene under the microspore-specific promoter NTM19 to block glutamine synthesis in developing pollen grains. We observed partial male sterility in primary transgenic plants by using light microscopy, FDA, DAPI and in vitro pollen germination test. Microspores started to die in the early unicellular microspore stage, pollen viability in all primary transgenic lines ranged from 40-50%. All primary transgenics produced seeds like control plants, hence the inserted gene did not affect the sporophyte and was inherited through the female germline. We regenerated plants by in vitro microspore embryogenesis from 4 individual lines, pollen viability of progeny ranged from 12 to 20%, but some of them also showed 100% male sterility. After foliage spray with glutamine, 100% male-sterile plants were produced viable pollen and seed set was also observed. These results suggested that mutated GS1 activity on microspores had a significant effect on normal pollen development. Back-cross progenies (T2) of DH 100% male-sterile plants showed normal seed set like primary transgenics and control plants.     

Comparison of damage to Eucalyptus caused by Amorbus obscuricornis and Gelonus tasmanicus

Amorbus obscuricornis (Westwood) and Gelonus tasmanicus (Le Guillou) (Heteroptera: Coreidae) are specific to Eucalyptus (Myrtaceae). A. obscuricornis feeds almost exclusively upon apical shoots and causes a characteristic wilting and necrosis. By comparison, the feeding activities of G. tasmanicus result in no obvious phytotoxicosis. Salivary gland extracts from both species exhibited sucrase activity but no pectinmethylesterase (PME) activity. Saliva from A. obscuricornis also exhibited considerable oxidase activity. Sucrase activity was significantly higher in extracts derived from G. tasmanicus than from A. obscuricornis, but this could not explain the observed differences in phytotoxic symptoms. It is suggested that differences in plant damage are attributable to the site of feeding activity (i.e. young versus mature tissue), which predetermines the reactivity of host tissues, and/or the quantity of salivary enzymes injected.     

Induction of cell-mediated cytotoxic immunity to sperm-specific lactate dehydrogenase-C4 in SJL/J female mice

SJL/J female mice were tested for a cell-mediated cytotoxic response to sperm-specific lactate dehydrogenase C4 (LDH-C4). We demonstrated this response with a 51chromium release assay. Mouse LDH-C4 was coupled to EL-4 tumor cells. These cells were then labeled with 51Cr and mixed with splenocytes from LDH-C4-immune and -nonimmune mice. Specific lysis of the tumor cells by splenocytes from LDH-C4-immune mice was detected at 7 days and 14 days following a single footpad immunization. Since LDH-C4 is present on the surface of sperm, these results support the suggestion that cytotoxic removal of sperm from the female reproductive tract is one of the mechanisms whereby fertility is reduced following immunization with this enzyme.     

Cutting edge: functional characterization of the effect of the C3H/HeJ defect in mice that lack an Lpsn gene: in vivo evidence for a dominant negative mutation

A point mutation in the Tlr4 gene, which encodes Toll-like receptor 4, has recently been proposed to underlie LPS hyporesponsiveness in C3H/HeJ mice (Lpsd). The data presented herein demonstrate that F1 progeny from crosses between mice that carry a approximately 9-cM deletion of chromosome 4 (including deletion of LpsTlr4) and C3H/HeJ mice (i.e., Lps0 x Lpsd F1 mice) exhibit a pattern of LPS sensitivity, measured by TNF activity, that is indistinguishable from that exhibited by Lpsn x Lpsd F1 progeny and whose average response is "intermediate" to parental responses. Thus, these data provide clear functional support for the hypothesis that the C3H/HeJ defect exerts a dominant negative effect on LPS sensitivity; however, expression of a normal Toll-like receptor 4 molecule is apparently not required.     

Muscular dystrophy by merosin deficiency decreases acetylcholinesterase activity in thymus of Lama2dy mice

Half of congenital muscular dystrophy cases arise from laminin alpha2 (merosin) deficiency, and merosin-deficient mice (Lama2dy) exhibit a dystrophic phenotype. The abnormal development of thymus in Lama2dy mice, the occurrence of acetylcholinesterase (AChE) in the gland and the impaired distribution of AChE molecules in skeletal muscle of the mouse mutant prompted us to compare the levels of AChE mRNAs and enzyme species in thymus of control and Lama2dy mice. AChE activity in normal thymus (mean +/- SD 1.42 +/- 0.28 micromol acetylthiocholine/h/mg protein, U/mg) was decreased by approximately 50% in dystrophic thymus (0.77 +/- 0.23 U/mg) (p = 0.007), whereas butyrylcholinesterase activity was little affected. RT-PCR assays revealed variable levels of R, H and T AChE mRNAs in thymus, bone marrow and spinal cord. Control thymus contained amphiphilic AChE dimers (G2A, 64%) and monomers (G1A, 19%), as well as hydrophilic tetramers (G4H, 9%) and monomers (G1H, 8%). The dimers consisted of glycosylphosphatidylinositol-anchored H subunits. Western blot assays with anti-AChE antibodies suggested the occurrence of inactive AChE in mouse thymus. Despite the decrease in AChE activity in Lama2dy thymus, no differences between thymuses from control and dystrophic mice were observed in the distribution of AChE forms, phosphatidylinositol-specific phospholipase C sensitivity, binding to lectins and size of AChE subunits.     

Niemann-Pick disease, Type C: evidence for the deficiency of an activating factor stimulating sphingomyelin and glucocerebroside degradation

1) Qualitative lipid analyses by thin-layer chromatography of 4 Niemann-Pick type C spleens confirmed sphingomyelin accumulation together with increase in the amount of glucocerebroside. 2) In the presence of crude sodium taurocholate as detergent, sphingomyelin degradation rates of normal and Niemann-Pick type C-cultured fibroblasts were fairly close under standard conditions at pH 5.0. In the absence of sodium taurocholate, sphingomyelinase activity was optimal at pH 4.0. Sphingomyelinase activities of fibroblasts from two patients with Niemann-Pick disease type C measured without detergent, were about 30% of that of controls. 3) Extracts from Gaucher spleen heated to 90 degrees C and devoid of sphingomyelinase activity stimulated at the optimal pH of 4.0 sphingomyelin degradation by cultured normal fibroblasts (2--4-fold, Niemann-Pick type C fibroblasts (5--9-fold), whereas similarly treated extracts from Niemann-Pick type C spleen showed no stimulation of sphingomyelin catabolism. Heated extracts from normal human spleen exhibited a smaller stimulation than that shown by Gaucher spleen. This stimulating effect could not be observed in fibroblasts from patients suffering from Niemann-Pick type B (sphingomyelinase defect). 4) Heat-treated extracts of Gaucher spleen were fractionated by ion exchange chromatography, isoelectric focusing and gel filtration. The active fractions obtained by these procedures stimulated sphingomyelin as well as glucocerebroside degradation and were absent from the corresponding Niemann-Pick type C preparations. Enriched activator preparations of Gaucher spleen stimulated sphingomyelinase activity of Niemann-Pick type C fibroblasts 25--38-fold and that of normal cells 3-fold. 5) The activating factor had an isoelectric point of 4.0 and an apparent molecular weight, as estimated by gel filtration, of 25000. Treatment with pronase E abolished its activity.     

Daily variation in antioxidant enzymes lipid peroxidation in thyroid and plasma level thyroxine and triiodothyronine levels of a tropical bird Perdicula asiatica during reproductively active and inactive phases

The aim of this work was to study the variations in the interference of neuroendocrine pineal gland and metabolically active thyroid gland in a tropical bird, Perdicula asiatica. Maximum pineal gland activity (pineal weight and melatonin level), minimum thyroid gland activity (weight, T3/T4 and thymidine kinase activity) along with less oxidative load (MDA level, SOD, CAT and ABTS activity) were observed during reproductively inactive phase (RIP) was observed. Further, a robust and significant rhythmicity was noted in melatonin levels during RIP and RAP, but no significant rhythmicity was noted in T4/T3 level by cosinor analysis. Overall, melatonin and thyroid circadian profile suggested that melatonin might be acting as an antioxidant molecule with time of the day effect in rescuing thyroid gland from free radical load in birds.