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B T Hinton

Publications and source records attributed to B T Hinton.

At least 37 records · Page 2Linked to original sources
PubMed1992

Clusterin (SGP-2) in epididymal luminal fluid and its association with epididymal spermatozoa in androgen-deprived rats.

Clusterin is a heterodimeric glycoprotein synthesized and secreted by rat Sertoli cells and epididymal epithelium. The goal of this study was to determine the presence of clusterin in the luminal fluid of the cauda epididymides and its association with the membranes of developing spermatozoa in the presence and absence of androgen. We have previously demonstrated by two-dimensional (2-D) Western blot probing for clusterin that in epididymal fluid the amounts of clusterin were: caput greater than corpus greater than cauda. Luminal fluid from cauda epididymides was collected from control and orchiectomized rats (6 and 12 days) and orchiectomized animals that received testosterone implants. Equal volumes of fluid were analyzed by 2-D Western blot probing for clusterin. Following orchiectomy, there was an increase in clusterin in the luminal fluid after 6 days and maximal amount after 12 days compared with control cauda fluid. Orchiectomized animals which received testosterone treatment showed levels of clusterin comparable to that of controls. Serum clusterin was detected in fluid of orchiectomized animals with and without testosterone. Western blots of cauda sperm membrane extracts of control animals and orchiectomized animals treated with testosterone had a very low level of epididymal clusterin, whereas extracts collected from orchiectomized animals revealed high levels of clusterin. We suggest that, in the normal animal, clusterin is secreted into the lumen of the proximal epididymis where it binds to the sperm membrane. In the distal epididymis, clusterin dissociates from sperm and is processed (proteolysis/endocytosis). We hypothesize that, in the absence of androgen, the processing and regulation of clusterin is disrupted.

Animals↗
PubMed1991

Expression and activity of gamma-glutamyl transpeptidase in the rat epididymis.

Following Northern analysis, GGT mRNA was found predominantly within the caput epididymides and kidney. The size of mRNAs for kidney, caput, corpus, and ductus deferens were 2.2, 2.3, 2.2, and 2.3 kb, respectively, whereas cauda showed a doublet of 2.2 and 2.3 kb. GGT transpeptidation and hydrolytic activity within epididymal luminal fluids collected by micropuncture showed caput = corpus greater than cauda and corpus greater than caput greater than cauda, respectively. Caput luminal GGT transpeptidation activity was significantly inhibited by serine-borate and was optimal at pH 8.0. The calculated Km and Vmax values for hydrolysis of GSH by caput luminal GGT were 0.06 microM and 2.19 nmoles/min/microliters luminal fluid at pH 8.5 compared to 0.49 microM and 0.49 nmoles/min/microliters luminal fluid, respectively, at the physiological pH 6.5 of caput fluid. These studies would suggest that the epididymis can control the activity of luminal GGT by pH. Lower Km (0.12 microM) and higher Vmax (1.13 nmoles/min/microliters luminal fluid) values were also calculated when GSSG was used compared to GSH. Results from Triton X-114 partitioning experiments suggest that luminal GGT probably exists in both membrane bound and nonmembrane bound forms. Western blot analysis of proteins within epididymal luminal fluids revealed both subunits of GGT in all epididymal regions studied. However, two lower molecular bands, approximately 22 kDa and 21 kDa, were also observed in cauda fluid. It is suggested that as GGT is transported along the epididymal duct it undergoes degradation, which accounts for its loss of activity in the distal epididymal regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

In vivo secretion and association of clusterin (SGP-2) in luminal fluid with spermatozoa in the rat testis and epididymis.

Clusterin (sulfated glycoprotein-2) is a heterodimeric glycoprotein synthesized and secreted by rat Sertoli cells. An antigenically similar form is synthesized and secreted by the epididymis. The goal of this study was to define the epididymal regions in which clusterin is present and the regions in which clusterin is secreted and interacts with developing spermatozoa. Seminiferous tubule (STF), caput, corpus, and cauda fluids were collected by micropuncture and/or microperfusion and two-dimensional Western blot analysis was performed with a polyclonal antibody directed against Sertoli cell clusterin. Clusterin was found in both STF and epididymal fluid. STF contained predominantly the clusterin heavy chain (45 kd); however, a 70 Kd heterodimer was present under nonreducing conditions. Two subunits of clusterin with lower molecular weights (41 kd, heavy chain; 32 kd, light chain) and higher isoelectric points were present in the luminal fluid of all epididymal regions. The intraluminal levels of the heavy and light chains decreased from caput to cauda. Analysis by two-dimensional gel electrophoresis of proteins secreted directly into the epididymal luminal fluid revealed that clusterin was secreted by caput epithelium and not by the corpus and cauda epithelium. Western blots of membrane extracts from testicular, caput, and cauda spermatozoa revealed that testicular clusterin was associated with testicular sperm and epididymal clusterin with predominantly caput sperm. Our findings suggest that clusterin is secreted into the caput epididymal lumen, where it binds to sperm and then dissociates from sperm to be endocytosed by cells of the distal epididymal epithelium.

Animals↗
PubMed1987

Changes in binding of a 27-kilodalton chimpanzee cauda epididymal protein glycoprotein component to chimpanzee sperm.

Motility patterns of caput epididymal chimpanzee sperm, caput epididymal chimpanzee sperm incubated in vitro with chimpanzee cauda epididymal fluid, and cauda epididymal chimpanzee sperm were assessed quantitatively. Sperm recovered from the caput epididymis showed no motility, whereas sperm recovered from cauda epididymis showed progressive forward motility. After incubation in cauda fluid, approximately 25% of caput epididymal sperm showed some motile activity. Electrophoretic analysis of 125I-labeled sperm plasma membrane preparations revealed that the surface of caput epididymal sperm, incubated in cauda fluid, was modified by the appearance of a major protein-glycoprotein surface component with an apparent molecular weight of 27 kilodaltons (kD). THis 27-kD component was not detected on caput epididymal sperm incubated in buffer or in caput fluid. However, it was present in cauda fluid and on cauda epididymal sperm. Binding to caput epididymal sperm was cell specific in that chimpanzee erythrocytes incubated in cauda fluid did not bind this 27-kD cauda fluid component. Motility patterns of ejaculated chimpanzee sperm and of ejaculated chimpanzee sperm incubated in the uterus of adult female chimpanzees also were assessed quantitatively. Ejaculated sperm showed progressive forward motility, whereas in utero incubated ejaculated sperm showed hyperactivated motility typical of capacitated sperm. Electrophoretic analysis of 125I-labeled sperm plasma membrane preparations revealed the loss of a 27-kD component from the surface of ejaculated sperm after in utero incubation. No significant change in the 125I-distribution pattern was detectable when ejaculated sperm were incubated in buffer. These results suggest that the lumenal fluid component, which becomes adsorbed to the surface of chimpanzee sperm during maturation in the epididymis and which is removed from the surface of mature chimpanzee sperm in the female reproductive tract, affects sperm motility.

Animals↗
PubMed1987

Neutral amino acid absorption by the rat epididymis.

The technique of stopped-flow/split-drop microperfusion was used to study the absorption of the neutral amino acid alpha-aminoisobutyric acid (AIB) from different epididymal regions of the rat. Absorption of AIB from the lumen of the caput, corpus, and cauda was saturable and time-dependent. The apparent Km values for each of the regions studied were similar (approximately 6 mM), whereas the Vmax values were progressively higher from caput, corpus, and cauda, respectively. Absorption of AIB from the lumina of the caput, corpus, and cauda epididymidis was linear over 60 min. The absorption of AIB from the lumen of the caput was sodium-dependent and inhibitable by 2-methyl-alpha-aminoisobutyric acid (MeAIB), a specific inhibitor of neutral amino acid transport. Similarly, absorption of AIB from the lumen of the corpus epididymidis was sodium-dependent; however, uptake was not significantly reduced in the presence of MeAIB. Absorption of AIB from the lumen of the cauda epididymidis was neither sodium-dependent nor inhibitable by MeAIB. It is suggested that neutral amino acid absorption involves different transport carriers in different epididymal regions. These findings also support our previous observations that there exists a selective permeability barrier from lumen to blood along the epididymal duct.

Absorption↗
PubMed1985

Binding of [3H]aldosterone to a single population of cells within the rat epididymis.

Using the dry-mount autoradiographic technique, a single population of cells within the rat epididymis, the clear cells, have been shown to bind [3H]aldosterone at a nuclear site. Competitive binding experiments demonstrated that aldosterone was more potent than desoxycorticosterone than testosterone in reducing the nuclear uptake of radioactive aldosterone. Furthermore, the other epididymal cells (principal and basal cells) in all regions of the epididymis were not significantly labelled; occasional labelling was noted in some endothelial and stromal cells. It is suggested that aldosterone may play a role in controlling the intracellular and transcellular movement of ions and water necessary for concentrating absorbed macromolecules in the clear cell.

Aldosterone↗
PubMed1985

Surface changes in chimpanzee sperm during epididymal transit.

Intact chimpanzee caput and cauda epididymal sperm, sperm cell lysates, and caput and cauda epididymal fluid were radiolabeled by enzymatic iodination with lactoperoxidase and Na125 I and were compared by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Caput epididymal sperm showed nine labeled macromolecular components of 90, 64, 56, 48, 38, 31, 20, 18 and 16 Kd and cauda epididymal sperm showed eleven macromolecular components of 90, 64, 55, 47, 42, 33, 27, 18, 17, 15 and 11 Kd. Six of the components labeled on caput sperm (90, 64, 56, 48, 18 and 16 Kd) were detected in equal amounts of cauda sperm and two (38 and 20 Kd) were detected at greatly reduced labeling intensities. In the cauda epididymidis, four new components (33, 27, 17 and 11 Kd) became prominent features of the sperm surface. Analysis of labeled caput and cauda sperm cell lysates resolved components distinct from those detected on sperm surfaces. Electrophoresis of caput epididymal fluid showed five labeled components of 66, 56, 47, 41 and 37 Kd, while electrophoresis of cauda epididymal fluid showed eight labeled components of 92, 66, 56, 48, 31, 27, 24 and 11 Kd. Three components (66, 56 and 47 Kd) were present in both caput and cauda fluid, two (41 and 37 Kd) in caput fluid only, and five (92, 31, 27, 24 and 11 Kd) in cauda fluid only. Components of 37 Kd were labeled in caput fluid and on caput sperm but not on cauda sperm, whereas components of 27 Kd and 11 Kd were labeled in cauda fluid and on cauda sperm but not on caput sperm. These data show that chimpanzee sperm undergo extensive surface modifications during epididymal maturation and that some of these modifications may be related to exogenous proteins/glycoproteins in epididymal fluids.

Animals↗
PubMed1984

Morphological evidence for a blood-epididymis barrier and the effects of gossypol on its integrity.

Ultrastructural and micropuncture techniques were used to obtain morphological evidence for a blood-epididymis barrier ( BEB ) in the rat and to determine whether gossypol, an oral male contraceptive, alters the permeability of the BEB or blood-testis barrier ( BTB ) in rats made infertile with gossypol. Rats were treated by gavage with 20 mg/kg per day of gossypol for 6 weeks; control animals received the vehicle alone. For electron microscopy the components of the BEB were analyzed in each region of the epididymis with intravascularly perfused lanthanum nitrate. Throughout the epididymis in both control and gossypol-treated animals it was found that the zonula occludens at the apicolateral surface of the epididymal epithelial cells was the sole and ultimate structural component of the rat BEB ; the flow of intravascularly perfused lanthanum was not significantly impeded by the vascular endothelium, the peritubular myoid layer or other lateral cell surface specializations. For micropuncture, control and treated rats were administered 0.3 mCi [3H]inulin via the jugular vein. Radioactivity was determined in samples collected from the seminiferous tubules, caput and cauda epididymidis, and carotid artery. Results showed that [3H]inulin entry in seminiferous tubules, caput and caudal luminal fluid from blood was similar for control and treated groups. It was concluded that gossypol treatment does not alter the permeability properties of the BTB and BEB to macromolecules such as inulin or to small electron-dense tracers such as lanthanum.

Animals↗
PubMed1984

Alteration in surface charge of chimpanzee sperm during epididymal transit and at ejaculation.

To further investigate changes in the sperm surface occurring during epididymal transit and after ejaculation, the surface negative charge on the head of chimpanzee sperm recovered from the caput (n = 4) and cauda epididymis (n = 3) and from the ejaculate (n = 4) was measured. Washed sperm were exposed to colloidal iron at pH 1.6, washed, mounted on carbon plates, and examined in a scanning electron microscope equipped with an energy dispersive X-ray analyzer. This technique permits evaluation of individual sperm in a population and provides information not available when the entire population is measured as a whole. The results demonstrate a net increase in negative charge during epididymal transit and after ejaculation and a change in charge distribution within the population from a unimodal to a tetra modal distribution. Caput sperm showed a unimodal distribution with a mode of 20 +/- 2%. Cauda sperm showed three modes at 23 +/- 1, 34 +/- 3, and 41 +/- 3%. Ejaculated sperm showed four modes at 21 +/- 3, 31 +/- 4, and 40 +/- 2, and 46 +/- 2%. The sperm population in the chimpanzee epididymis and ejaculate is not homogeneous, and this technique will aid in future measurement of fertility of subpopulations of sperm in the ejaculate.

Animals↗
PubMed1983

Evidence for protein absorption from the lumen of the seminiferous tubule and rete of the rat testis.

As luminal fluid moves from the seminiferous tubule and enters the rete testis, its protein concentration declines from approximately 6 mg/ml to 1 mg/ml. It was therefore suggested that protein is either 1) utilized by the spermatozoa, 2) transported across the epithelium of the terminal segment of the seminiferous tubule, the tubuli recti or rete testis, or 3) absorbed and degraded by the epithelium. Horseradish peroxidase (HRP), a protein marker, was microperfused into single seminiferous tubules or perfused directly into the rete. After fixation, the HRP was localized histochemically and the tissue observed under the light- and electron microscope. HRP was taken up via pinocytotic vesicles into the cytoplasm of the Sertoli cells and germ cells but did not permeate extracellularly beyond the tight junctions. Similar results were obtained in the cells lining the terminal segment and the tubuli recti. The rete epithelium showed uptake of HRP into coated and noncoated vesicles, while some cells additionally revealed diffuse cytoplasmic distribution of HRP. The terminal segment, tubuli recti, and rete testis may be important routes by which proteins may leave the testicular fluid either to be degraded or to enter the blood.

Absorption↗
PubMed1982

Electrophysiological analysis of rat renal sugar and amino acid transport. II. Dependence on various transport parameters and inhibitors.

Transepithelial and cellular electrical potential changes were measured in response to luminal perfusion of D-glucose and related substrates in micropuncture experiments on rat kidney in vivo. By studying the dependence of the potential response on various experimental parameters, some insight was obtained into the mechanism of Na+ coupled glucose absorption. The experiments confirm the driving forces for glucose absorption in the living cell to be: a) the Na concentration gradient, b) the electrical potential gradient and c) the glucose concentration gradient across the brush-border membrane. Furthermore they describe the substrate specificity of the cotransport mechanism and the mechanism of inhibition of D-glucose transport by various inhibitors, such as phlorizin, harmaline and ouabain. The latter experiments suggest that the active Na+ pump in the peritubular cell membrane, which establishes the Na+ ion gradient and the electrical potential gradient across the brushborder, contributes a measurable partial conductance to the overall electrical conductance of the peritubular cell membrane.

Acetazolamide↗
PubMed1982

Rat testis and epididymis can transport [3H] 3-O-methyl-D-glucose, [3H] inositol and [3H] alpha-aminoisobutyric acid across its epithelia in vivo.

The in vivo movement of [3H] 3-0-methyl-D-glucose ([3H] 30MG), [3H] L-glucose, [3H] inositol and [3H] alpha-aminoisobutyric acid ([3H] alpha AIB) into and across the rat testicular and epididymal epithelium was investigated. After systemic infusion of [3H] 30MG, both epididymal and testicular tissue concentrations of the isotope were approximately 50% lower than blood plasma levels, even 24 h after infusion. Similar results were observed for caput and seminiferous tubule fluid. There was a significant reduction of [3H] 30MG caput lumen to blood ratio when the blood glucose concentration was increased to 2-5 times that of normal. A similar reduction of [3H] 30MG caput lumen to blood ratio was observed when 1000 mumol of 2-methylaminoisobutyric acid (meAIB) was infused with the isotope. Caput luminal [3H] L-glucose concentration never exceeded 10% of blood levels. The data suggest that glucose is transported across the testicular and epididymal epithelium via a facilitated diffusion mechanism (carrier mediated) and that the carrier is situated on the basolateral membrane. This study did not demonstrate either a glucose countertransport system or a glucose-inositol exchange system across the epithelium of the caput epididymidis. Epididymal tissue (except cauda) and caput luminal fluid concentrations of [3H] inositol exceeded that of blood plasma within 1 h after infusion of isotope. The proximal regions of the epididymis demonstrated significantly higher transporting activity than either cauda or testis. Systemic infusion of 100 mumol myo-inositol significantly reduced the transport of [3H] inositol into caput luminal fluid and epididymal and testicular tissue. It is suggested that there is a carrier situated on the basolateral membrane which is able to transport inositol against a concentration gradient (probably active transport). Epididymal tissue (except cauda), testicular tissue and caput luminal fluid concentrations of [3H] alpha AIB also exceeded that of blood plasma but only 24 h after infusion of isotope. The initial segment and corpus regions of the epididymis had a significantly higher transporting activity compared to the remainder of the epididymis and the testis. A dose of 500 mumol meAIB reduced epididymal uptake of [3H] alpha AIB. These results suggest that there is a carrier situated in the basolateral membrane which can transport the amino acid against a concentration gradient (probably active transport). The differences in transport activities of the compounds investigated along the reproductive tract probably indicate differences in the affinity or quantity of the carriers. These epididymal transporting systems regulate the movement of material across its epithelium and may help to protect the epididymis from fluctuations in the blood concentration of each compound.

3-O-Methylglucose↗
PubMed1981

The concentration of some inorganic ions and organic compounds in the luminal fluid of the human ductus deferens.

The concentrations of some inorganic ions (sodium, potassium, chloride and phosphate) and organic compounds (carnitine, myo-inositol, glycerophosphocholine, phosphocholine and total phosphate) were estimated in the luminal fluid collected from sections of human ductus deferens removed at vasectomy. From the data presented it would appear that in contrast to non-primate species, in the human the inorganic ions contribute more to the total osmolarity of the luminal fluid than do the organic compounds.

Adult↗
PubMed1981

Effects of carnitine and some related compounds on the motility of rat spermatozoa from the caput epididymidis.

Spermatozoa were collected from the rat caput epididymidis by micropuncture and their motility assessed after dilution in physiological saline containing carnitine or related compounds. L(+)-Carnitine caused, 2 min after dilution, a transient stimulation of the motility of spermatozoa with low initial motility. No stimulatory effects were seen on spermatozoa which had high initial motility. The D-isomer inhibited the motility of spermatozoa with high initial motility after 2 min and all compounds tested appeared to inhibit, at 20 min after dilution, the motility of spermatozoa with high initial motility. Acetyl-L-carnitine and acetyl-D-carnitine stimulated the motility of spermatozoa with low initial motility. This study suggests that carnitine may be important in the development by spermatozoa of the potential for motility and also to maintain mature spermatozoa in a quiescent state.

Acetylcarnitine↗
PubMed1981

Permeability characteristics of the epithelium in the rat caput epididymidis.

Micropuncture techniques were used to study the in-vivo transfer of radioactive compounds of different molecular weights across the epithelium of the rat caput epididymidis. Small molecular weight compounds such as 3H2O and [14C]urea equilibrated in less than 20 min and 2 h respectively after intravenous injection. Compounds of larger molecular weight ([3H]polyethylene glycol, [3H]inulin and 125I-labelled bovine serum albumin) entered the lumen more slowly and reached less than 5% of blood plasma concentrations 3 h after injection. The luminal concentration of [3H]inulin remained below 7% of the blood level for 18 h. These results demonstrate that a permeability barrier exists in the epithelium of the rat caput epididymidis.

Animals↗