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Biomedical subjects

J K Koehler

Publications and source records attributed to J K Koehler.

At least 19 recordsLinked to original sources
PubMed1998

Semen parameters and electron microscope observations of spermatozoa of the red wolf, Canis rufus.

Semen parameters were evaluated on ejaculates of a captive population of red wolves (Canis rufus) sampled over two consecutive mating seasons. A total of 31 samples from 15 animals yielded mean sperm motility of 69.6 +/- 19.4%, mean sperm density of 131 +/- 124 x 10(6) ml-1, mean total number of spermatozoa of 470 +/- 465 x 10(6) and mean percentage morphologically abnormal spermatozoa of 35 +/- 11.8%. Restricting the data to animals sampled three times or more or limiting the samples to proven breeders resulted in statistically non-significant differences in these numbers (P < 0.05). When compared with data from other canines the seminal parameters of red wolves are at the lower extremes of the range. In particular the proportion of morphologically abnormal spermatozoa (35%) is approximately twice that seen in other canine species. Light microscopic analysis of abnormal forms revealed that almost half (45%) were bent defects, another 40% were secondary defects (coiled, detached and immature) and 15% were primary defects. Electron microscopy confirmed the presence of substantial numbers of morphologically abnormal forms including double-headed and double-flagellar cells, bent or kinked forms especially in the neck region, acrosomal abnormalities and bizarre spermatids. Approximately one-third of the samples also showed the presence of white blood cells, in some cases demonstrating sperm phagocytosis (spermophagy). These results are consistent with the concept of declining sperm parameters associated with restricted gene pools in numerically limited populations. However, alternative explanations are also explored.

Animals↗
PubMed1997

Failure of oocyte activation after intracytoplasmic sperm injection using round-headed sperm.

OBJECTIVE: To examine the outcome of intracytoplasmic sperm injection (ICSI) with round-headed sperm (globozoospermia). DESIGN: Retrospective analysis. SETTING: In vitro fertilization laboratory with extensive ICSI experience. PATIENT(S): A patient couple with infertility because of globozoospermia seeking ICSI treatment. MAIN OUTCOME MEASURE(S): Fertilization, cleavage, and pregnancy rates. INTERVENTION(S): Intracytoplasmic sperm injection and calcium ionophore. RESULT(S): This couple experienced only 7% fertilization after ICSI in their first cycle. Treatment of the unfertilized oocytes with calcium ionophore 20 hours after ICSI-induced fertilization and cleavage of 70% of the oocytes. Embryo quality was fair to good. On the second cycle, 8 of the injected oocytes were treated with ionophore immediately after ICSI and the remaining 20 oocytes were untreated. Normal fertilization was achieved in 75% of the treated and 10% of the untreated oocytes. Treatment of these unfertilized oocytes with ionophore 20 hours after ICSI resulted in fertilization in 73%. Pregnancy was not achieved after either ICSI cycle. Ultrastructural analysis indicated multiple structural abnormalities in the sperm. CONCLUSION(S): These results indicate that the round-headed sperm from this patient were incapable of oocyte activation after ICSI. This may be the reason for the frequent ICSI fertilization failure seen with this condition. Current ICSI procedures may not always overcome the infertility associated with globozoospermia, and further study of the etiology of this condition is needed.

Adult↗
PubMed1996

Activation of the HIV long terminal repeat and viral production by H2O2-vanadate.

The long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) contains sequences required for the initiation of gene transcription. Among the substances known to activate the HIV-1 LTR is hydrogen peroxide (H2O2). We report here that H2O2-induced activation of the LTR in the macrophage cell line THP-1 and the lymphocyte cell line, Jurkat, is greatly increased by vanadate. Activation of the LTR by phorbol myristate acetate, tumor necrosis factor alpha, lipopolysaccharide, or Staphylococcus epidermidis extract was not increased by vanadate, indicating some selectivity for H2O2. H2O2 and vanadate also acted synergistically to increase the production of HIV-1 virions by the latently infected macrophage cell line U-1 as determined by p24 antigen release and the detection of intact virions by electron microscopy. Effects were observed at H2O2 and vanadate concentrations down to 3 x 10(-6) M, with high concentrations leading to cell toxicity. Catalase was strongly inhibitory when added prior to the interaction of H2O2 and vanadate, but was considerably less inhibitory when the H2O2 and vanadate were allowed to preincubate prior to the catalase addition. H2O2 reacts with vanadate to form peroxides of vanadate that have potent biological effects. Our findings suggest that among these is the activation of the HIV-1 LTR.

Catalase↗
PubMed1994

Spermophagy in semen in the red wolf, Canis rufus.

The red wolf (Canis rufus) is an endangered species with 194 individuals remaining in the wild and in various captive facilities. Breeding efforts at the Graham, WA site (Point Defiance Zoo and Aquarium) have involved artificial insemination with fresh or frozen semen in an effort to increase population and maximize the genetic potential of the stock. Electron microscopic observations were made in semen specimens obtained by electro-ejaculation from mature males prior to their use in an effort to determine semen parameters that might be useful in guiding breeding procedures. Sperm samples were either fixed immediately or treated with capacitating media and fixed after 4 to 7 hr of incubation. Many of the specimens examined were pyospermic (white cell in semen) and showed evidence of spermophagy, primarily by neutrophils. Of the six animals surveyed, only one showed little evidence of spermophagy, and three had extensive pyospermia and spermophagy but this finding was not correlated with fertility. Samples fixed immediately as well as those incubated for several hours showed evidence of spermophagy, indicating that the phagocytosis was not the result of culture. Gene pool restriction and/or captive stress may be contributing factors of reduced semen quality.

Animals↗
PubMed1992

Cytokine activation of human macrophages infected with HIV-1 to inhibit intracellular protozoa.

Peripheral blood mononuclear cells (PBMCs) from HIV-seronegative donors were infected in vitro with HIV-1. Infection was monitored by cytopathology, supernatant p24 antigen, and by immunocytochemical staining. After 14 days in culture, approximately 70-90% of the cells became infected with HIV, as indicated by cell fusion and immunostaining for virus. At this time, recombinant HuIFN-gamma was added to the cultures, followed by infection 24 h later with the intracellular protozoan parasites Toxoplasma gondii, Trypanosoma cruzi, or Leishmania chagasi. Percentages of intracellular parasites were determined at various points thereafter. Using a system capable of detecting both virus and parasite infection, we determined that (a) cells infected with HIV were capable of ingesting and/or being infected by each of these parasitic protozoa, (b) HIV-infected macrophages could be activated to inhibit the replication of all three parasites following treatment with IFN-gamma, and (c) cultures of HIV-infected macrophages could respond to IFN-gamma with increased oxidative burst activity. The degree of parasite infection or inhibition observed in infected cells was not significantly different from that observed in non-HIV-infected cells. From these observations, we concluded that HIV-1 infection does not render macrophages unresponsive to IFN-gamma activation for microbicidal activity.

Animals↗
PubMed1991

Further observations on the phagocytosis of Candida albicans by hamster and human oocytes.

Pathogenic yeast, Candida albicans, were incubated with hamster and human oocytes for up to 21 hours in order to determine the nature and time course of phagocytosis of these organisms. Aliquotes of the interacting cells were taken at various time intervals for electron microscopic examination. Some specimens had their zona pellucidae enzymatically removed prior to incubation with yeast, and these specimens showed the most extensive interaction and phagocytosis of Candida. The zona pullucida appears to be an effective barrier to yeast, at least over the time span studied. The observations are consistent with the hypothesis of an initial attachment of yeast via a surface component to oocyte microvilli followed by phagocytic uptake into an endosome. There is no compelling evidence of lysosomal degradation of the yeast over the time course of this study; however, the oocytes appear to undergo some degenerative changes at long incubation times.

Animals↗
PubMed1991

Fertility parameters in men infected with human immunodeficiency virus.

The effect of human immunodeficiency virus type 1 (HIV) infection on semen parameters that assess fertility was investigated in 50 semen specimens from 21 asymptomatic or minimally symptomatic HIV-seropositive men and 3 specimens from 3 men with AIDS. HIV was isolated from 15 (30%) of 50 specimens from asymptomatic or minimally symptomatic persons and from 1 of 3 specimens from patients with AIDS. The men with AIDS all had pyosemia and grossly abnormal sperm. In contrast, semen specimens from other seropositive men did not differ significantly from semen specimens from healthy seronegative semen donors. No abnormality in sperm count, morphology, numbers or types of leukocytes in semen, or other seminal parameters was associated with HIV shedding in semen. Zidovudine therapy did not affect sperm morphology or seminal characteristics. Thus, although patients with AIDS had abnormal semen, the laboratory parameters that assess fertility were not affected by shedding of HIV in semen or concomitant therapy with zidovudine.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Observations of hamster sperm-egg fusion in freeze-fracture replicas including the use of filipin as a sterol marker.

We have extended the observations of previous transmission electron microscopy studies of sperm-egg fusion to include those of freeze-fracture replicas showing sperm-egg interactions before, during, and following sperm head fusion with the egg membrane. Hamster eggs were incubated with hamster sperm under polyspermic conditions and were observed after a period of 5-30 minutes. After fixation, the eggs and sperm were exposed to filipin, which binds beta-OH-sterols to form visible complexes in freeze-fracture replicas. Filipin can act as a marker for egg plasma membrane wherein it is abundant, while filipin is relatively scarce in the acrosome-reacted hamster sperm membrane, found only in the plasma membrane of the equatorial segment. The earliest sperm-egg interactions are observed between the egg microvilli and the perforatorium and the equatorial segment of the sperm, and the initial fusion between egg and sperm occurs in the vicinity of the equatorial segment. At later stages of fusion involving the postacrosomal segment, a clear line of demarcation is observed between the filipin-rich egg membrane and the filipin-poor sperm postacrosomal segment, suggesting that filipin binding lipids from the egg intercalate into the sperm membrane following membrane fusion. The anterior segment of the sperm does not fuse with the egg but is instead incorporated into a cytoplasmic vesicle derived from both sperm and egg membranes. In this latter step, filipin-sterol complexes are not found in sperm-derived membranes suggesting that there may be barriers to the movement of filipin binding lipids from the egg into these sperm membranes.

Animals↗
PubMed1988

Does phospholipase C inhibit fusion between hamster sperm and zona-free eggs?

Previous studies (Hirao and Yanagimachi: Gamete Res. 1:3-12, 1978) have found that phospholipase C (PLC) preparations inhibit sperm-egg fusion. We have attempted to duplicate these results with PLC, as well as with a more specific enzyme, phosphatidylinositol-specific PLC. PLC preparations were applied externally to zona-free hamster eggs prior to incubation with sperm. Phosphatidylinositol-specific PLC did not inhibit sperm penetration. The degree of sperm-egg fusion observed after egg exposure to PLC, however, was dependent upon the purity of the commercial preparation. An impure sample of PLC inhibited sperm penetration, while a more purified preparation did not. The morphology of eggs was unaffected by exposure to phosphatidylinositol-specific PLC and the more purified PLC preparation. The impure preparation, however, was disruptive primarily to the egg plasma membrane as well as to internal organelle organization. The degree of damage by the impure PLC preparation was concentration dependent. The results suggest that as purity of the PLC preparation is increased, the adverse effects of PLC on sperm-egg fusion become negligible.

Animals↗
PubMed1987

Phagocytosis of yeast by human oocytes: fine structural observations.

Fine-structure observations have been made on the interaction between invasive yeast cells and human oocytes. The yeast appear to make their way through the zona pellucida and once in the perivitelline space are incorporated into phagocytic vacuoles by surface activity of the oocyte. The yeast attach to the vitelline membrane via fuzzy surface material on the cell wall, and incorporation appears to be aided by oocyte microvillar activity. Coated pits in the oocyte plasma membrane are incorporated into the phagosomes, but no lysosomal activity is seen, and neither oocytes nor yeast cells appear to undergo degeneration in the time frame investigated.

Cells, Cultured↗
PubMed1985

Freeze-fracture observations on mammalian oocytes.

Freeze-fracture studies on mammalian oocytes have been hampered by the relatively small numbers of cells available at a given time as well as by difficulties encountered in effectively freezing these large, watery cells. We have nevertheless pursued this area because of the benefits of visualizing membrane faces involved in various fusion reactions by the freeze-fracture method. Our observations indicate no overall change in intramembranous particle (IMP) distribution before and after sperm penetration, although the question of possible alterations of these structures at the precise locus of sperm attachment remains open. Preliminary statistical analysis indicates that there is a much higher IMP density on the P face than on the E face of the plasma membrane and that the microvillar membranes bear more IMPs than those of the intermicrovillus regions. Probes of lipid subclasses were used to determine the distribution of cholesterol and anionic lipid in the egg plasma membrane. Filipin and tomatin showed extensive complex formation in microvillus as well as nonmicrovillus regions, whereas anionic lipids (using polymyxin B) have been difficult to detect on the oocyte surface. These results are discussed relative to current views of membrane fusion mechanisms.

Animals↗
PubMed1985

Light and electron microscopic studies on the localization of steroid-binding protein (SBP) in rabbit spermatozoa.

Light (fluorescence) and electron microscopic studies were carried out to localize steroid-binding protein (SBP) in rabbit spermatozoa. Both nonpermeabilized and permeabilized (with Tween 20, saponin, or cold acetone) spermatozoa showed fluorescence following treatment with antirabbit SBP (anti-rSBP) and subsequently with rabbit antisheep immunoglobulin G-fluorescein isothiocyanate. While the ejaculated spermatozoa were positive, epididymal sperm were observed to be negative. Although the pattern of localization of rSBP was variable, the occurrence of a negative equatorial region as well as the presence of an intense positive spherical profile ("spot") at the junction of the head and midpiece were notably consistent. The intensity of labeling with the probe, both at light and electron microscopic level, was maximal following permeabilization with cold acetone. A possible role of SBP as a steroid carrier protein across the plasma membrane of the sperm has been suggested.

Animals↗
PubMed1984

Ultrastructural sperm tail defects associated with sperm immotility.

Four individuals with a complaint of infertility were evaluated after a semen analysis had demonstrated no motility. The semen analyses were otherwise normal, with the exception of a low count in one subject. All had normal percentages of living sperm. One of the individuals had chronic respiratory disease, and two others had a previous history of genitourinary infection and/or testicular injury. Three subjects possessed antisperm antibodies. Electron microscopy revealed a multiplicity of sperm tail structural defects seen in all specimens. Some of these data support the concept of acquired immotile sperm syndrome(s) with ultrastructural defects, as contrasted with the usual congenital forms of the immotile-cilia syndrome.

Adult↗
PubMed1982

Restricted domains of the sperm surface.

The plasma membrane of the sperm is a mosaic, with different moieties restricted to specific areas of the head or tail. Some of these heterogeneously distributed surface components are inserted into the membrane during spermatogenesis while others are acquired during post-testicular maturation, as the sperm passes through the epididymis. A variety of surface probes have been used to examine sperm, with lectins and antibodies proving to be effective at relating quantitative and qualitative changes to specific areas of the sperm surface. More recently, monoclonal antibodies have been used in conjunction with surface labeling procedures to study the distribution, time and site of origin and possible roles of sperm components. The use of such highly specific probes in conjunction with high resolution SEM instruments and new molecular labeling techniques offer considerable promise in addressing these problems.

Acrosome↗
PubMed1981

Lectins as probes of the spermatozoon surface.

Studies involving the use of lectins to probe the spermatozoon surface are reviewed, with particular emphasis on mammalian spermatozoa and their various transformations during maturation, capacitation, and the acrosome reaction. Although the numbers of lectin bindings sites on spermatozoa are generally comparable to those of many somatic cells, the distribution of most lectins thus far studied is highly restricted to certain domains of the cell surface. These domains are often related to specific underlying morphological entities such as the acrosome, postacrosomal region, mitochondria associated with the middle piece, and so on. Changes in the patterns of lectin binding have been noted during sperm maturation in the epididymis, during capacitation, and after the acrosome reaction, and, in some instances, these studies contribute to the understanding of mechanism that may mediate these transformations. This is particularly true of situations where lectin labeling or binding studies have been used in conjunction with biochemical or other correlative methods to synthesize a more detailed picture of the sperm surface.

ABO Blood-Group System↗
PubMed1980

A collagen-binding protein on the surface of ejaculated rabbit spermatozoa.

A rabbit antibody to mouse 3T3 cell fibronectin was used in conjunction with a fluorescein-tagged second antibody to detect fibronectin-like activity on the surface of rabbit spermatozoa. Only ejaculated sperm displayed an intense and highly localized fluorescence over the acrosomal region. Cauda epididymal sperm of the rabbit as well as several other species did not exhibit any reaction. The fluorescent activity could be eliminated by trypsin treatment but was re-established by incubation in cell-free seminal fluid. Sperm recovered from females 10-12 h after mating showed a reduction or absence of antifibronectin fluorescence, suggesting that this component's loss could be a factor in sperm capacitation. Because fibronectins show strong binding to collagen, mixtures of ejaculated sperm and collagen were examined in the light and electron microscope. Living sperm appear to have a strong affinity for collagen and quickly adhere to the filaments by their heads, while continuing vigorous flagellations. Surface labeling of sperm with the galactose-oxidase-NaB[3H]4 technique, extraction with urea-detergent mixtures and affinity chromatography of extracts on gelatin-Sepharose revealed a single radioactive band of mot wt approximately 40,000 after SDS polyacrylamide gel electrophoresis and fluorography.

Animals↗
PubMed1979

Tight junctions in the choroid plexus epithelium. A freeze-fracture study including complementary replicas.

The tight junctions of the choroid plexus epithelium of rats were studied by freeze-fracture. In glutaraldehyde-fixed material, the junctions exhibited rows of aligned particles and short bars on P-faces, the E-faces showing grooves bearing relatively many particles. A particulate nature of the junctional strands could be established by using unfixed material. The mean values of junctional strands from the lateral, third, and fourth ventricles of Lewis rats were 7.5 +/- 2.6, 7.4 +/- 2.2, and 7.5 +/- 2.4; and of Sprague-Dawley rats 7.7 +/- 3.4, 7.4 +/- 2.3, and 7.3 +/- 1.6. Examination of complementary replicas (of fixed tissue) showed that discomtinuities are present in the junctional strands: 42.2 +/- 4.6% of the length of measured P-face ridges were discontinuities, and the total amount of complementary particles in E-face grooves constituted 17.8 +/- 4.4% of the total length of the grooves, thus approximately 25% of the junctional strands can be considered to be discontinuous. The average width of the discontinuities, when corrected for complementary particles in E-face grooves, was 7.7 +/- 4.5 nm. In control experiments with a "tighter" tight junction (small intestine), complementary replicas revealed that the junctional fibrils are rather continuous and that the very few particles in E-face grooves mostly filled out discontinuities in the P-face ridges. Approximately 5% of the strands were found to be discontinuous. These data support the notion that the presence of pores in the junctional strands of the choroid plexus epithelium may explain the high transepithelial conductance in a "leaky" epithelium having a high number of junctional strands. However, loss of junctional material during fracturing is also considered as an alternative explanation of the present results.

Animals↗