The presence of metabolites of 3-H-testosterone in the lumen of the cauda epididymidis of the rat.

Following the administration of 3-H-testosterone to adult male rats radioactivity appeared in epididymal fluid and at 60 minutes was in excess of the level in tissue of the cauda epididymidis and blood plasma. Ligation of the arterial blood supply to this region caused a significant decline in the radioactive content of epididymal fluid and cauda tissue. It is concluded that direct transfer occurs from the systemic circulation into the cells of the cauda epididymidis and thence into the lumen of the duct. The major radiometabolite of 3-H-testosterone identified in chloroform extracts of epididymal tissue (60.6%) and epididymal fluid (72.8%) was 17-beta-hydroxy-5-alpha-androstan-3-one.     

Factors affecting the entry of antibiotics into Escherichia coli

Factors affecting the entry into Escherichia coli of diverse antibacterial agents, especially beta-lactams were investigated. Agents of greater than a critical molecular weight (approximately 600 Daltons) penetrated extremely poorly. However, there was little correlation between penetrative ability and molecular weight for substances below the critical size. Within classes of related antibiotics (e.g. cephalosporins) penetrative ability was highly dependent on hydrophobicity. The relationship was parabolic rather than linear in nature. The proposal that the envelope of E. coli preferentially excludes hydrophobic molecules is to some extent an artefact arising from pre-selection of the agents used. For unrelated antibiotics hydrophobic nature was a poor guide to penetrative ability. A rather empirical property, diffusion ability through agar, was found to show good inverse correlation with penetrative ability for many unrelated antibiotics.     

Electrical activity and intraluminal pressure of the cauda epididymidis of the rat

Smooth muscle electrical activity was recorded simultaneously with intraluminal pressure in the cauda epididymidis and epididymal vas of the rat in vitro. Spontaneous electrical activity spread over distances of several centimetres and was associated with a pressure wave recorded through an open-ended catheter in the distal cauda and epididymal vas. Transmission of pressure wave was gradually damped by filling of the catheter with spermatozoa.     

GPX5 is present in the mouse caput and cauda epididymidis lumen at three different locations

In mice, GPX5 is a secreted protein abundantly synthesized by the caput epididymidis. The protein is secreted as early as the initial segment of the caput and is found subsequently associated with the sperm plasma membrane in a sub-acrosomic localization. We show here that GPX5 is present in the caput and cauda epididymides lumens in three different locations: either free as a soluble protein in the caput epididymal fluid, weakly bound to caput sperm membranes, or, finally, associated to lipid-containing structures conferring to the protein a protective effect against proteolytic digestions. Within the cauda epididymidis, the amount of free GPX5 is low compared to the caput and the association with sperm membranes proved to be more solid. In both caput and cauda sperm samples, the association of GPX5 with the sperm membrane protects GPX5 from proteolytic cleavages. Protection against proteolytic digestions can be overcome by physical treatments of epididymal fluid and sperm samples such as ultrasounds or very acidic pH. These data suggest that complex phenomena and structures participate in the transfer and binding of the caput-secreted GPX5 protein to the sperm plasma membrane.     

Adenylyl cyclase isoforms in rat testis and spermatozoa from the cauda epididymidis

Expression of adenylyl cyclase genes in rat testis and spermatozoa from the cauda epididymidis was investigated using RT-PCR analysis. Genes encoding the transmembrane adenylyl cyclases (tmAC) II, III, IV, V, VI, VII, and VIII were expressed in the testis, whereas only the gene for tmAC III was expressed in caudal spermatozoa. Immunocytochemistry was used to investigate which tmAC were translated into putative, functional proteins in spermatozoa. Indirect immunofluorescence localized the tmAC II enzyme to a region on the head occupied by the acrosome. The tmAC III enzyme was localized to the posterior margin of the head and to the flagellum, whereas tmAC V and/or VI was localized to the region where the ventral surface of the acrosomal equatorial segment is located. The tmAC VII and VIII enzymes were localized to the convex margin of the head, covering the dorsal region of the acrosomal crescent. To our knowledge, this is the first demonstration that five apparently different tmAC enzymes are localized to discrete subcellular regions of mammalian spermatozoa. These findings provide a fundamental basis for future studies, to determine the physiological roles of tmAC in testis and mature spermatozoa.This work was supported by a grant from the Australian Research Council/Department of Education, Training and Youth Affairs (ARC A40001141)     

A method for estimating the concentration of spermatozoa in the rat cauda epididymidis

A method for estimating the concentration of spermatozoa in the rat cauda epididymidis is described. Treatment of a sperm suspension with 0.05% collagenase for 20-60 min or 0.025% trypsin for 1-2 min at 34-37 degrees C was found to result in consistently homogeneous sperm. Sperm concentration ranged from 152.5 to 230.0 X 10(7) spermatozoa/ml, with a mean of 187.7 (+/- 5.6 SEM) X 10(7) spermatozoa/ml.     

Influence of abdominal temperature and luminal contents on the cauda epididymidis of the rat

In an attempt to determine if changes previously described in the epididymides of cryptorchid testes were related to the elevated environmental temperature or to the absence of normal luminal constituents, rats were divided into four test groups. Group I animals were made unilaterally cryptorchid. Animals in Group II had only the cauda epididymidis of one side maintained within the abdominal cavity (cryptepididymal) while the caput epididymides and testes remained in the scrotum. The testes of animals in Group III remained in the scrotum but had their efferent tubules ligated on one side. Testes of unoperated rats and contralateral testes of the test animals served as controls. The histochemical demonstration of sorbitol dehydrogenase (SDH) was used to determine differences in functional activity and light and electron microscopy were used to determine structural changes. SDH activity could not be demonstrated in the cauda epididymidis of cryptorchid and efferent tubule-ligated animals; animals in which the luminal contents were obviously changed. These same groups of animals showed abnormal folding of the basal surface of the epididymal epithelium at the ultrastructural level. Activity of SDH could be demonstrated in control epididymides and in those that contained normal luminal contents but were maintained at the temperature of the abdominal cavity. The basal surface of the epididymal epithelium was not unusual in these animals. The results indicate that the epididymis is influenced to a greater extent by changes in luminal contents than by temperature elevation.     

Changes in surface ATPase of rat spermatozoa in transit from the caput to the cauda epididymidis.

Rat spermatozoa from the cauda epididymidis were found to have a lower activity of the surface ATPase than the spermatozoa from the caput region. The enzyme from spermatozoa of both regions had the same Michaelis constant (Km) for ATP of 5 X 10(-4) M. It was partly inhibited by ouabain and fluoride, but strongly inhibited by Cu2+, Zn2+,p-chloromercuribenzoate, 8-anilino-1-naphthalenesulphonate Triton X-100, Lubrol-PX, urea, guanidine hydrochloride, sodium dodecyl sulphate and glycerylphosphorylcholine. The enzyme of the spermatozoa from the cauda epididymidis was more sensitive to inhibition by ouabain and fluoride but less sensitive to inhibition by Cu2+ than that of the cells form the caput region. The Arrhenius plot of the temperature dependence of enzymatic activity varied for the cells from the caput and cauda epididymidis. The differences in the enzyme properties of spermatozoa from the two regions of the epididymis suggested that the decline in the activity during epididymal maturation may reflect changes in the lipids and sulphydryl groups of the sperm membrane.     

Genetically defined lysosomal acetylesterase EC 3.1.1.6 in the cauda epididymidis of mouse, rat, and man.

After inhibition by bis-p-nitrophenyl phosphate and subsequent staining for esterase using naphthol AS-D acetate as the substrate, a strong lysosomal esterase was demonstrated in the cauda epididymidis of mouse, rat, and man. Owing to its behaviour towards the classifying inhibitors eserine, diisopropyl fluorophosphate, bis-p-nitrophenyl phosphate, and p-chloromercuriphenylsulphonate, this lysosomal esterase was shown to be an acetylesterase (EC 3.1.1.6). Control experiments involving isoelectric focusing revealed that this acetylesterase was identical with the genetically defined homologues ES-17, ES-6, and ES-A4 in mouse, rat, and man, respectively.     

Changes in lipase and phosphatase activities of rat spermatozoa in transit from the caput to the cauda epididymidis.

Rat spermatozoa from the cauda epididymidis, freed from their cytoplasmic droplets and acrosomes, were found to have a lower lipid content and to incorporate [14C]glucose into their glycerides and glycerophosphatides at a lower rate than spermatozoa from the caput epididymidis. Against the background of the activities of some glycolytic enzymes which remained constant the activity of alkaline phosphatase decreased in spermatozoa migrating through the epididymis, whereas the activity of monoglyceride lipase increased. The corresponding enzyme activities of non-flagellate germ cells of the testis were measured for comparison. The triglyceride lipase of non-flagellate germ cells and of spermatozoa from both caput and cauda epididymidis was activated by cyclic 3':5'-AMP.     

Influence of age on the production of rat spermatozoa, on their concentration in the cauda epididymidis, and on FSH, LH and testosterone plasma levels

Testis samples were taken from young (3 months), middle-aged (12 months) and aged (24 months) male rats, processed, stained and examined via a light microscope. There were no prominent abnormal germinal epithelium and interstitial tissue. However, the aging process promoted a significant decrease in the mean amount of spermatids 19 per cross tubular section, and in the amount of Sertoli cells per cross tubular section in 24-month-old rats. The concentration of spermatozoa in the cauda epididymidis showed a gradual decrease from 3 to 12 and 24 months. After hCG injection all groups of animals exhibited an increase in plasma testosterone level, although the response was smaller in 12- and 24-month animals compared to the young mature (3 months) ones.     

Genetically defined lysosomal acetylesterase EC 3.1.1.6 in the cauda epididymidis of mouse,rat, and man

After inhibition by bis-p-nitrophenyl phosphate and subsequent staining for esterase using naphthol AS-D acetate as the substrate, a strong lysosomal esterase was demonstrated in the cauda epididymidis of mouse, rat, and man. Owing to its behaviour towards the classifying inhibitors eserine, diisopropyl fluorophosphate, bis-p-nitrophenyl phosphate, and p-chloromercuriphenylsulphonate, this lysosomal esterase was shown to be an acetylesterase (EC 3.1.1.6). Control experiments involving isoelectric focusing revealed that this acetylesterase was identical with the genetically defined homologues ES-17, ES-6, and ES-A4 in mouse, rat, and man, respectively.     

The influence of body temperature and castration on the protein composition of fluid in the rat cauda epididymidis

Polyacrylamide gel electrophoresis under reducing and non-reducing conditions, and immunoelectrophoretic analysis, revealed that several characteristic proteins disappear from the luminal fluid of the rat cauda epididymidis when it is maintained at body temperature. On SDS-PAGE gels prepared under reducing conditions, one Coomassie-blue staining band of Mr 18,000 disappeared and another of 52,000 was significantly reduced after only 6 days; bands of Mr 23,000, several in the Mr 34-38,000 range, one of Mr 48,000, and others of Mr 100-200,000 were eliminated or markedly reduced after 15 days at body temperature. Some were glycoprotein, as judged by their affinity for FITC-ConA. At 15 days after castration there was a broadly similar but rather more extensive disappearance of macromolecules, and of glycoproteins in particular, from caudal fluid. The fact that several similar proteins are diminished in or disappear from fluid or the cauda epididymidis maintained at body temperature, or after androgen withdrawal, raises the possibility that one or more such proteins play a role in sperm storage there.