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C J Flickinger

Publications and source records attributed to C J Flickinger.

At least 73 records · Page 4Linked to original sources
PubMed1988

Testicular alterations are linked to the presence of elevated antisperm antibodies in Sprague-Dawley rats after vasectomy and vasovasostomy.

The relationship between alterations in testicular histology and antisperm antibodies was studied after vasectomy and vasovasostomy in Sprague-Dawley rats, which are immunologically relatively non-responsive to vasectomy. Testes were prepared for histologic study at intervals up to seven months after vasectomy, vasectomy followed three months later by vasovasostomy, or sham operations. Antisperm antibodies were assessed with an ELISA. Testicular alterations, which were observed in a minority of animals after vasovasostomy, consisted mainly of depletion of germ cells. Mean serum antisperm antibody levels were greater for animals with altered testes than for rats with normal testicular histology. In addition, the proportion of rats that showed a positive antisperm antibody response was greater among animals with testicular changes than among those with unaltered testes. When the present results on Sprague-Dawley rats were compared with previous findings on the highly responsive Lewis strain, it was evident that the incidence of testicular changes and the proportion of positive antibody responders were greater in the Lewis strain. However, elevated antisperm antibodies and testicular alterations appeared to be more tightly linked in the less responsive Sprague-Dawley rats.

Animals↗
PubMed1988

Effects of granuloma formation at site of vasovasostomy.

Spermatic granulomas forming after vasectomy reversal have been thought to mechanically compromise the anastomosis. We have studied the physiologic effects of vasectomy and vasovasostomy in rats. Following delayed microsurgical vasovasostomy, fluid flow through the anastomosis and cauda epididymidal hydrostatic pressure are not significantly different in tracts that form, or do not form, sperm granulomas at the anastomosis. Given a properly performed microsurgical vasectomy reversal, a sperm granuloma arising from a small leak does not appear to mechanically compromise the anastomosis in the rat. Fertility after vasovasostomy is not significantly reduced in rats with granulomas.

Animals↗
PubMed1988

Post-vasectomy sperm autoimmunogens in the Lewis rat.

Vasectomy was performed on inbred Lewis rats to induce anti-sperm autoantibodies and to identify their cognate autoantigens. Different detergent extracts of cauda epididymal spermatozoa were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted to nitrocellulose, and probed with sera from pre-vasectomy, post-vasectomy, and hyperimmunized animals to detect isologous sperm antigens. Nonreduced SDS-soluble autoantigens at greater than 200, 86, 43, and 26 kDa were bound by post-vasectomy antisera. Reduction of SDS-soluble antigens resulted in increased staining of the 86, 72-63, and 43 kDa autoantigens. Laemmli extraction of SDS insoluble pellets with beta-mercaptoethanol generated the largest repertoire of autoantigens including several autoantigens found in SDS-soluble extracts. Therefore, to analyze the entire repertoire of post-vasectomy autoantigens, whole sperm were extracted with Laemmli buffer under reducing conditions. Autoantiserum from most vasectomized animals bound Laemmli-extracted reduced autoantigens of approximately 86 (89-78), 63, 43, and 20 (21-16) kDa. Testicular extracts, reduced and separated by SDS-PAGE, contained autoantigens of 76, 60, and 42 kDa that were also recognized by hyperimmune and post-vasectomy antisera. The repertoire of sperm antigens recognized by pooled serum from hyperimmunized animals was similar to the cumulative repertoire recognized by post-vasectomy sera. These studies define several major sperm autoimmunogens recognized by post-vasectomy antisera and indicate that many of these peptide autoimmunogens are disulfide-bonded complexes.

Animals↗
PubMed1987

The influence of vasovasostomy on testicular alterations after vasectomy in Lewis rats.

The occurrence of alterations in testicular weight and morphology after vasectomy and vasectomy reversal by vasovasostomy was studied in Lewis rats. Animals were studied 3, 4, and 7 months after bilateral vasectomy or a vasectomy followed 3 months later by vasovasostomy. Other rats served as sham-operated controls. The weights of the testes in vasectomy and vasovasostomy animals fell into two groups-small testes weighing less than 0.88 g and normal-sized testes of 1.2 g or more. When the extent of testicular alterations was estimated in sections for light microscopy by use of a semiquantitative testicular biopsy score count (TBSC), the morphology of the testes corresponded closely to the testis weight (r = .94), small testes having correspondingly low TBSC scores. In severely altered small testes, the seminiferous tubules were narrower than in sham-operated rats, and numbers of germ cells were greatly depleted. Many tubules contained only Sertoli cells and spermatogonia, although spermatocytes were present in a minority of tubules. A few seminiferous tubules contained multinucleate spermatids. Electron microscopy of severely altered tubules revealed closely apposed processes of Sertoli cells, which contained filaments, microtubules, and endoplasmic reticulum. In contrast, testes with normal weight in vasectomy and vasovasostomy groups resembled those of the sham-operated animals. Comparison of distributions of testicular biopsy score counts demonstrated differences between vasectomy and vasovasostomy groups as time after operation increased. At the 3-4-month intervals, approximately one-third of the testes were severely altered in both vasectomy and vasovasostomy groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

The relationship between the morphology of cell organelles and kinetics of the secretory process in male sex accessory glands of mice.

Two male sex accessory glands of the mouse, seminal vesicle and coagulating gland, were compared with the aim of relating differences in the morphology of organelles to the kinetics of the secretory process. The epithelial cells of the two glands were assessed by morphometric analysis, cytochemical staining, and electron-microscopic autoradiography after administration of a labeled amino acid. The rough endoplasmic reticulum of the seminal vesicle comprised narrow parallel cisternae, while that of the coagulating gland was greatly distended and occupied a much larger percentage of the cytoplasmic volume. Radioactively labeled products were secreted much more rapidly in the seminal vesicle than in the coagulating gland. The primary point of difference in kinetics of intracellular transport between the two glands was in exit of material from the rough endoplasmic reticulum. The more rapid drainage of the rough endoplasmic reticulum may be related to its relatively greater membrane surface density and lesser internal volume. In contrast, similarities in size and cytochemical staining in the Golgi apparatus of the two glands were accompanied by similar kinetics of intracellular transport of secretory protein through this organelle.

Animals↗
PubMed1987

The relation between antisperm antibodies and testicular alterations after vasectomy and vasovasostomy in Lewis rats.

The relationship between antisperm antibodies as determined by enzyme-linked immunosorbent assay (ELISA) and the occurrence of alterations in testicular weight and histology was studied following vasectomy in Lewis rats. The effects of vasovasostomy on antisperm antibody levels were also examined. At 1, 3, and 4 months after vasectomy, the mean absorbance values in an ELISA for sera from animals with altered testes was significantly greater than that from animals lacking testicular alterations. However, animals showing positive antisperm antibody responses were represented both in the group with testicular alterations and among those that lacked testicular damage. Levels of antisperm antibody in both vasectomy and vasovasostomy groups significantly exceeded that for sham-operated animals, but the level of antisperm antibodies in vasovasostomized animals with positive responses was similar to vasectomized animals one and four months after reanastomosis. It is suggested that persistence of antisperm antibodies or testicular alterations, or both, may play roles in limiting the restoration of fertility after vasovasostomy.

Animals↗
PubMed1986

Incorporation of 3H-fucose and the secretion of glycoproteins in the coagulating gland of the mouse.

The coagulating gland of rodents, which is part of the prostatic complex, secretes components of semen. Although possessing some ultrastructural features of other exocrine glands, the mechanism of secretion by these cells has been problematic. In the present study the pathway, kinetics, and mode of secretion in the coagulating gland of the mouse were studied by light and electron microscope autoradiography at intervals between 10 minutes and 3 hours after injection of 3H-fucose. The majority of silver grains overlay the Golgi apparatus at the initial interval, but in addition, more than a third of the grains were associated with extremely distended cisternae of the rough endoplasmic reticulum. At later intervals, radioactivity of the Golgi apparatus and the endoplasmic reticulum declined, while labeling of secretory granules increased greatly. Luminal contents became labeled 1 hour after administration of precursor. The results indicate that the pathway for secretion of glycoproteins proceeds through the Golgi apparatus to secretory granules and the glandular lumen, as in many other cells. In particular, heavy labeling of secretory granules at later intervals indicates that merocrine secretion is the most likely mechanism in the coagulating gland. However, the unusual observation that a significant proportion of grains overlay the rough endoplasmic reticulum at the initial interval raises the possibility that some fucose is incorporated into glycoproteins in the endoplasmic reticulum, as has been reported for other cell types with similarly configured endoplasmic reticulum.

Animals↗
PubMed1986

Intracellular pathway and kinetics of protein secretion in the coagulating gland of the mouse.

The coagulating gland of male rodents is part of the prostatic complex. Various mechanisms of secretion have been postulated, in part because organelles commonly involved in the secretory process possess unusual features, such as extreme distension of the rough endoplasmic reticulum. In the present study, the pathway, kinetics, and mode of secretion in the coagulating gland of the mouse were studied by electron microscope autoradiography at intervals between 5 min and 8 h after administration of 3H-threonine. The percentage of grains associated with the rough endoplasmic reticulum was initially high and generally decreased throughout the experiment, while a pronounced rise in the proportion of grains associated with the Golgi apparatus and secretory granules was observed 6 h after injection of precursor. In addition, there was a smaller elevation in the percentage of grains over the Golgi apparatus and secretory granules between 1 and 4 h, and radioactive material first reached the lumen of the gland 4 h after injection of the precursor. Although the general pathway of intracellular transport of secretory protein resembles that in other cells, the results indicate that there are several unusual aspects to the secretory process in the coagulating gland. First, the rate of transport was markedly slower than in most other exocrine gland cells, since the bulk of the labeled protein did not reach the Golgi apparatus and secretory granules until 6 h after administration of precursor. This reflected prolonged retention of secretory products in the endoplasmic reticulum. Second, in addition to the major bolus of labeled material that traversed the cells at about 6 h, a smaller wave of radioactivity appeared to pass through the Golgi apparatus and secretory granules and reach the lumen earlier, within the first few hours after the injection. Finally, the primary mode of secretion in the coagulating gland appears to be merocrine because the secretory granules contained much labeled protein.

Animals↗
PubMed1986

An enzyme-linked immunosorbent assay for measuring antisperm autoantibodies following vasectomy in Lewis rats.

An indirect enzyme-linked immunosorbent assay (ELISA) was devised to measure antisperm auto-antibodies in the Lewis rat following vasectomy. The assay system was validated by employing prevasectomy sera and postvasectomy antisera, previously demonstrated to contain antisperm antibodies by indirect immunofluorescence. A standardized ELISA protocol was developed employing 10(5) sperm per microtiter plate well and sucrose-polyvinylpyrrolidone as a postcoat stabilizer solution. The ELISA was shown to yield significant detectable antibody at dilutions of 1/512 or greater in the most reactive sera. A standard for scoring positive titers was adopted: 1.96 standard deviations above the mean of the preimmune value. Using the criterion, 88% of 7-week postvasectomy samples could be discriminated from preimmune samples at a 1:16 dilution, which was adopted for subsequent assays. The ELISA demonstrated 73% and 91% reproducibility for an intraassay analysis of single prevasectomy and postvasectomy serum samples (7 weeks postvasectomy) tested in 160 determinations on a standard sperm pool. When this single antigen pool was employed in 35 determinations at 0, 1, and 4 weeks in an interassay study, 56% and 70% reproducibility was found for pre- and postvasectomy sera respectively. A correlation (r = 0.75) was made between a single absorbance value and the endpoint titer of the same sera, which indicated that single absorbance values could be used to predict serum titer and single dilutions could be used for general screening of a large number of samples. The ELISA described provides a rapid, sensitive, and reliable method that discriminated between samples taken before and after vasectomy.

Animals↗
PubMed1985

Radioautographic analysis of the secretory pathway for glycoproteins in principal cells of the mouse epididymis exposed to [3H] fucose.

The secretory process for glycoproteins in principal cells of the mouse caput epididymis was studied by electron microscope radioautography at intervals after exposure to [3H] fucose in vitro. The large Golgi apparatus showed very heavy labeling at the initial interval, followed by a steady decline in percent of grains and relative grain concentrations. Conversely, the epididymal lumen and the apical cell surface began low and increased in radioactivity at the 30-min interval. The extensive sparsely granulated endoplasmic reticulum showed modest increases in percent of grains and relative grain concentrations 30 min after administration of the percursor. Subdivision of the sparsely granulated reticulum into "intermediate" profiles (some ribosomes attached to the membranes) and "smooth" profiles (lacking ribosomes) showed that this increase was due to silver grains assigned to the smooth portions. After the initial interval, high relative grain concentrations were calculated for vesicles. The results indicate that glycosylation of epididymal secretory glycoproteins occurs in the Golgi apparatus, which is, therefore, not bypassed as its morphological features had suggested. The kinetics of the secretory process in the principal cells includes 15 to 30 min for synthesis of the polypeptide parts of secretory products and addition of sugars in the Golgi apparatus, and a similar time for subsequent release from the Golgi apparatus, transport to the apical end of the cell and discharge to the lumen. Ribosome-studded (intermediate) portions of the sparsely granulated endoplasmic reticulum are probably involved in synthesis of polypeptide parts of secretory products, while vesicles or smooth portions of the sparsely granulated reticulum may play a role in intracellular transport of glycoproteins.

Animals↗
PubMed1984

The secretory pathway in the mouse epididymis as shown by electron microscope radioautography of principal cells exposed to monensin.

The secretory pathway in principal cells of the mouse epididymis was studied using in vitro labeling and electron microscope radioautography of tissue exposed to the ionophore monensin. After a 5-minute pulse of 3H-leucine, control samples of caput epididymidis were incubated in a modified Krebs-Ringer solution (MKRH medium), while experimental specimens were placed in the same medium, to which 1 microM monensin had been added. At intervals between 5 minutes and 4 hours, samples were fixed and prepared for electron microscope radioautography. Analysis of control specimens revealed heaviest labeling of the rough and the sparsely granulated endoplasmic reticulum early in the experiment followed by a fall in radioactivity, maximal labeling of the Golgi apparatus at 30 minutes, and a pronounced rise in the percentage of grains associated with the apical cell surface and the epididymal lumen beginning 1 hour after administration of precursor. In monensin-treated epididymides, radioactive material accumulated in the Golgi region while the normal increase in labeling of the apical surface and the lumen was completely inhibited for at least 2 hours. The percentage of grains attributed to coated vesicles was also reduced in samples exposed to monensin. In contrast, labeling patterns of the abundant, sparsely granulated, endoplasmic reticulum and the rough endoplasmic reticulum were very similar in monensin-treated and control specimens. The concomitant alterations in labeling of the Golgi apparatus and the lumen demonstrate that the Golgi apparatus participates in intracellular transport of secretory proteins in epididymal principal cells, and is not bypassed as previously suggested. The percentage of grains associated with the sparsely granulated endoplasmic reticulum suggests that much of the synthesis of secretory protein in the principal cells occurs in this organelle, and the lack of alteration of its labeling in the presence of a monensin-induced block at the level of the Golgi apparatus indicates that the sparsely granulated endoplasmic reticulum lies before the Golgi apparatus in the secretory pathway. It is speculated that vesicles play a role in transport of secretory protein from the Golgi apparatus to the lumen.

Animals↗
PubMed1984

Altered labelling of the cell surface and intracellular organelles with [3H]mannose in enucleated amoebae.

The production, transport, and disposition of material labelled with [3H]mannose were studied in microsurgically enucleated and control amoebae. Cells were injected with the precursor and samples were prepared for electron-microscope radioautography at intervals, up to 24 h later. Control cells showed heavy labelling of the rough endoplasmic reticulum and the Golgi apparatus at early intervals after injection. Later, labelling of groups of small vesicles increased, and the percentage of grains over the cell surface peaked 12 h after administration of the precursor. Two major changes were detected in enucleate amoebae. First, the kinetics of labelling of cell organelles with [3H]mannose were altered in the absence of the nucleus. The Golgi apparatus and cell surface both displayed maximal labelling at later intervals in enucleates, and the percentage of grains over the rough endoplasmic reticulum varied less with time in enucleated than in control cells. Second, the distribution of radioactivity was altered. A greater percentage of grains was associated with lysosomes in enucleates than in control cells. The change in the kinetics of labelling of the endoplasmic reticulum, Golgi apparatus and cell surface indicates that intracellular transport of surface material was slower in the absence of the nucleus. It is suggested that this is related to the decreased motility of enucleate cells.

Amoeba↗
PubMed1983

Secretory process in Brunner's glands during recovery from stimulation with a single dose of pilocarpine.

The secretory pathway and kinetics of the secretory process were studied in Brunner's glands of mice after stimulation of secretion with a parasympatho-mimetic drug. Adult male mice were injected with pilocarpine, while unstimulated controls received saline. The animals were subsequently administered an intravenous injection of 3H-threonine, and tissue was prepared for electron microscope autoradiography at intervals ranging from 5 minutes to 2 hours after injection of the radioactive precursor. Stimulation with pilocarpine resulted in discharge of secretory granules, which was reflected in a significantly lower percentage of the cell volume occupied by granules. In both control and stimulated mice, at 5 minutes after injection of 3H-threonine, the highest percentage of silver grains was found over the rough endoplasmic reticulum. The proportion of silver grains over the rough endoplasmic reticulum declined at later intervals, and a peak of labeling was observed over the Golgi apparatus at 1 hour. Labeling of the secretory granules increased in the 1- and 2-hour samples from both control and stimulated mice, although the relative concentration of radioactivity in both Golgi-associated and apical secretory granules was greater in stimulated than control glands at 1 hour. The results suggest that the secretory protein produced by Brunner's glands was synthesized by the rough endoplasmic reticulum and transported to the Golgi apparatus where secretory granules were formed in both stimulated and control glands. Depletion of secretory granules by prior stimulation resulted in no change in the kinetics of arrival of radioactivity in the cell organelles involved in the secretory process. However, the drainage of the radioactive label from the rough endoplasmic reticulum was significantly slower in the stimulated glands than in the controls. The existence of two subcompartments within the rough endoplasmic reticulum is among the possible explanations for the latter observation.

Animals↗
PubMed1982

The fate of sperm after vasectomy in the hamster.

Young adult male hamsters were subjected to bilateral vasectomy. The reproductive tracts were studied by light and electron microscopy at intervals up to 1 year after the operation. Sperm continued to be produced, since testicular alterations were focal. Spermatic granulomas were associated with the excurrent ducts of all animals 5 months or 1 year after vasectomy and with those of one of four hamsters 2 weeks after the operation. Phagocytosis of sperm in the lumina of the efferent ducts and proximal parts of the epididymis, and disintegration of membranous components of intraluminal sperm occurred in approximately three-fourths of the animals studied 5 months or more after vasectomy. The results indicate that after vasectomy in the hamster sperm are disposed of by phagocytosis in spermatic granulomas, intraluminal phagocytosis, and dissolution in the lumen of the male ducts, although the latter process may be incomplete.

Animals↗
PubMed1982

Proliferative activity in the rat epididymis during postnatal development.

The proliferative activity of the rat epididymis during postnatal development was investigated with the use of autoradiography. Animals at 14, 21, 28, 35 and 56 days of age were sacrificed 2 hours following the administration of 3H-thymidine. Cell types that showed a significant labeling index were columnar cells in 14- and 21-day-old animals; principal and basal cells in 28, 35, and 56 day old rats. During this period of development, the pattern of cellular activity in the initial segment differed from the middle and terminal segments in having a peak of activity on day 28. The middle and terminal segments had similar proliferative patterns. In two additional experiments, 10- and 21-day-old rats were given 3H-thymidine and killed 1 week later on day 16 and 28. Labeled narrow cells were present in day 16 animals, whereas labeled narrow, principal, and basal cells were found in day 28 rats. It was concluded that columnar cells are the precursor to narrow, principal and basal cells.

Animals↗
PubMed1982

Distribution of phospholipids labeled with 3H-choline and relationship between membranous organelles in amoebae, as studies by electron-microscopic radioautography.

Amoebae were injected with a solution of 3H-choline, and samples were prepared for electron-microscopic radioautography at intervals between 15 min and 24 h thereafter. At the earliest interval, the rough endoplasmic reticulum was the most heavily labeled organelle. At subsequent intervals, the proportion of silver grains over the rough endoplasmic reticulum decreased rapidly, while that associated with other membranes increased. Most notably this involved a rapid rise in labeling of vacuoles, up to 1 h, and a more gradual increase in plasma membrane labeling up to 2 h. The results suggest that phospholipids are synthesized in the rough endoplasmic reticulum and are then transferred to other cellular membranes. A sequence of transfer steps suggested by the order of increases in labeling of different types of membranes is rough endoplasmic reticulum, smooth membranes and nuclear membranes, vacuoles, and the plasma membrane.

Amoeba↗
PubMed1982

Intracellular localization of Prostatic Binding Protein (PBP) in rat prostate by light and electron microscopic immunocytochemistry.

Extra- and intracellular distribution of Prostatic Binding Protein (PBP) was studied in the different genital organs of the male rat by immunocytochemistry at the light and electron microscopic levels. PBP was extracted from cytosols of rat ventral prostate and used for immunization of rabbits. The specificity of the antiserum raised was tested by "western blotting" and immunoelectrophoresis. From the different fixatives tested for optimal structural and antigenic preservation of the ventral prostate a mixture containing 2.5% paraformaldehyde, 0.5% glutaraldehyde and 0.5% CaCl2 in cacodylate buffer, 0.05 M, pH 7.3 was selected. Using the immunofluorescence technique and the unlabeled antibody enzyme method PBP-immunoreactivity was detected at the light microscopic level in the luminal secretions of the ventral prostate. No reaction was observed with the seminal vesicle, the coagulating gland, the dorsal and lateral prostates, the epididymis and the testis. Intracellular secretory granules reacting with PBP antiserum were exclusively found in the secretory cells of the ventral prostate. Insufficiently fixed cells showed a diffuse generalized reaction of the cytoplasm indicating a leakage of the antigen from the secretory granules. Such artifacts were common in tissue sections processed with the preembedding-staining procedure. At the ultrastructural level therefore mostly the postembedding staining method was performed using both the unlabeled antibody enzyme method and the ferritin-labeled immunoglobulin technique in osmicated, Epon-embedded tissue. Labeling with either method was intense in the secretory granules and the condensing vacuoles, while the labeling density of the rough endoplasmic reticulum and the Golgi cisternae was in the background range. Castration experiments showed that secretory material displaying PBP immunoreactivity was retained within the acinar lumen of the gland for several days after castration, but was absent from most secretory cells already by four days after castration. Immunocytochemistry of PBP therefore is a very sensitive method for analysing the secretory activity and its androgen dependence of the prostate of the rat.

Androgen-Binding Protein↗