Different mechanisms are responsible for 3H-androgen movement across the rat seminiferous and epididymal epithelia in vivo.

Mechanisms involved in the maintenance of the microenvironment of the seminiferous and epididymal tubules were examined in a series of experiments utilizing in vivo microperifusion, microperfusion, and micropuncture. Intraluminal 3H-androgen concentrations in seminiferous tubules increased linearly as interstitial 3H-androgen concentrations increased from 10 nM to 2,000 nM, but in caput epididymidal tubules, intraluminal 3H-androgen concentrations increased hyperbolically across the same range of peritubular 3H-androgen concentrations. Intraluminal 3H-androgen concentrations in the caput epididymidis did not rise above approximately 340 nM even if peritibular 3H-androgen concentrations exceeded 2,000 nM. Perifusion of caput tubules with 0.1 mM dinitrophenol or potassium cyanide or 100 micrograms/ml cyclohexamide significantly reduced proluminal 3H-androgen movement, but tubules perifused with control medium would not support antigrade 3H-androgen movement in the absence of native lumen fluids which contain androgen-binding protein. Antigrade proluminal 3H-androgen movement was not inhibited by competition with estradiol at ten-times 3H-androgen concentrations. Thus, energy-requiring protein synthesis is necessary for antigrade 3H-androgen movement in the caput epididymidis, but the mechanism for the interaction of intracellular protein(s) and 3H-androgen movement remains undetermined.     

Transepithelial movement of 3H-androgen in seminiferous and epididymal tubules: a study using in vivo micropuncture and in vivo microperifusion

In vivo micropuncture and a new system of in vivo microperifusion were used to examine the movement of 3H-androgen from blood to lumen and from interstitum to lumen in the rat testis and epididymis. Movement of 3H-androgen into the seminiferous tubule lumen was restricted, with intraluminal isotope concentrations plateauing at approximately 15% of extratubular isotope concentrations whether the 3H-androgen originated in the vascular or interstitial compartments. In the caput epididymidis, intraluminal 3H-androgen plateaued at approximately 35% of serum concentrations, but when 3H-androgens were presented directed to the basal aspect of the caput epididymidal epithelium, 3H-androgen was transported into the lumen against a concentration gradient. Intraluminal isotope concentrations were greater than 200% of those in the epididymal interstitial compartment. Similar results were found for the cauda epididymids. Factors controlling the proluminal movement of 3H-androgens in the rat testis and epididymis were therefore fundamentally different.     

Proluminal movement of 3H-androgen across the epididymal epithelium in the rat after hypophysectomy and gonadotropin supplementation

The effects of hypophysectomy and gonadotropin replacement on transepithelial movement of 3H-androgen in the rat epididymis were examined by in vivo microperifusion of 3H-testosterone followed by in vivo micropuncture to obtain peritubular and intraluminal fluid. In the caput epididymidis of normal rats, intraluminal 3H-androgen concentrations were approximately 300% of those in the interstitial space. In contrast, proluminal movement of 3H-androgen into rat caput epididymal tubules was significantly decreased 10 days after hypophysectomy. 3H-Testosterone movement across the caput epididymal epithelium was completely returned to normal by supplementation with 24 micrograms/day follicle-stimulating hormone (FSH) or 24 micrograms/day luteinizing hormone (LH). However, neither 0.12 micrograms/day FSH nor 250 micrograms/day prolactin returned proluminal androgen movement to normal. It is speculated that epididymal uptake of peritubular testosterone is mediated by androgen-binding protein, which is known to be secreted by Sertoli cells after stimulation by FSH or testosterone.     

On the proluminal movement of 3H-androgens across the rat epididymal epithelium

3H-Androgens in rat epididymal interstitium have previously been shown to move into the epididymal lumen against a concentration gradient. This is true especially in the caput epididymidis. The present investigation used the technique of in vivo epididymal perifusion and tubule micropuncture to demonstrate that the proluminal movement of 3H-androgens is subject to competitive inhibition (unlabeled testosterone in the perifusion fluid at 10 times and 100 times the concentration of 3H-testosterone significantly reduced proluminal movement of isotope) and is not energy-dependent (1 mM 2,4-dinitrophenol in perifusion fluid did not reduce the proluminal movement of isotope). Additionally, dry-mount autoradiography demonstrated high intraluminal concentrations of isotope relative to interstitial concentrations after caput tubule incubation in 3H-dihydrotestosterone (3H-DHT), and showed that the high intraluminal concentrations of isotope were not dependent on the presence of spermatozoa, i.e. proluminal movement of 3H-androgens was not due to binding to intraluminal spermatozoa. Isolation of caput epididymidal sperm on filters followed by 3H-DHT binding experiments also failed to demonstrate the presence of specific binding of this androgen to spermatozoa. Finally, it was confirmed that electrophoresed epididymal lumen fluid contains a single 3H-DHT binding peak that is at its highest concentration in the caput epididymal fluid. These data are consistent with the conclusion that intraluminal androgen-binding protein is an important factor in transepithelial androgen movement.     

Transport of carnitine by the epididymis of the cynomolgus macaque (Macaca fascicularis)

Unravelled tubules from the monkey caput and cauda epididymidis were perfused through the lumen in vitro during immersion in an organ bath kept at scrotal temperature and containing [3H]carnitine and [14C]inulin. The specific transport of carnitine from the bath to the lumen was constant for 4 h and reached a steady-state value of about 90 pmol/30 min per cm perfused length in the cauda and about 30 pmol/30 min/cm in the caput. These regional variations in carnitine transport differ from those found in the rat epididymis but may be relevant to human epididymal physiology.     

Resorption versus secretion in the rat epididymis

Micropuncture samples were taken from the rete testis, caput epididymidis and cauda epididymidis of anaesthetized adult rats and assayed for total protein, sodium and potassium concentrations. Intraluminal sperm concentrations were determined and used to calculate the amount of fluid resorbed from the efferent duct and epididymal lumen. It was demonstrated that large amounts of protein (30.2 mg/ml cauda volume) and sodium (241.8 mequiv./l) and smaller amounts of potassium (19.4 mequiv./l) are resorbed from the rat epididymal lumen between the caput and corpus epididymidis. This occurs despite increases in intraluminal concentrations of protein (from 22 to 28 mg/ml) and potassium (from 16 to 50 mequiv./l). Resorption is an important aspect of epididymal control of the intraluminal environment.     

Protein transport to the epididymal lumen

Summary Experiments were performed to clarify the debate over the entry of circulating proteins into the epididymal lumen by use of the marker horseradish peroxidase (HRP). Epididymal tubules from the caput epididymidis of the rat were immersed in medium TC 199 containing HRP (3.5 mg/ ml) for 5 min to 3 h at 33° C. Sections were examined for the presence of tracer within the epithelial cells by electron microscopy. From 5 min to 3 h, vesicles containing peroxidase reaction products were found throughout the cytoplasm of the principal cells. Vesicles occurred close to both the basal and apical membranes, and many were found opening into the interstitial space and lumen, depending on the length of incubation. By 5 min labelled vesicles were infrequently found in the apical part of the cells. Reaction product was observed in the epididymal lumen adhering to the microvilli from 30 min of incubation onwards. At all periods of incubation peroxidase was present at the base of the epithelium and between the cells, but it was never found within the tight junctional complexes, and no reaction deposits were found within epithelial cells of tubules incubated in the absence of peroxidase. It is concluded that large molecules leaving the capillaries may enter the epididymal lumen in the caput by means of fluid-phase endocytosis.     

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.     

Organization of tubules in the human caput epididymidis and the ultrastructure of their epithelia

The structure of the human caput epididymidis was examined by gross morphological and light and electron microscopic techniques. There were at least seven types of tubules, each characterized by a different epithelium. These tubules were connected with one another by at least eight types of junctions to form a network. Most of the caput epididymidis was composed of efferent ducts. Within these, five types of tubules, each with a different ciliated epithelium, were found in different regions; and four types of junctions between the efferent ducts and the epididymal tubule were observed. The efferent ducts left the testis, initially as parallel straight tubules containing both ciliated and non-ciliated cells in an epithelium of irregular height. Each efferent duct then coiled tortuously into lobules that folded over one another. These efferent ducts then branched out as thin tubules to join a network of dark tubules which were lined by a regular epithelium containing prominently vacuolated, non-ciliated cells. These tubules anastomosed via common cavities characterized by a ciliated cuboidal epithelium and sometimes joined tubules exhibiting a non-vacuolated ciliated epithelium. The latter, as well as typical efferent ducts, made connection with the epididymis proper in both end-to-end and end-to-side junctions. In the more distal junctions with the epididymis, the efferent ducts joined to a transitional epididymal ductule before joining to the side of the epididymis proper. Post-junctional epithelia in the beginning of the epididymis occasionally contained patches of cells characteristic of efferent ducts. Tall cells with long stereocilia constituted a discontinuous "initial segment"-like region of the epididymis. This is the most detailed study so far of the epithelia and the tubule organization in the caput epididymidis of any species, and most of the results are reported for the first time for the human. Although the pattern of the tubule network resembles that of some domestic species, the rich variety of epithelia has not been appreciated before.     

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.     

Rapid development of vasopressin-induced hydroosmosis in kidney collecting tubules measured by a new fluorescence technique.

The pre-steady-state kinetics of the vasopressin-induced increase in collecting tubule osmotic water permeability (Pf) has been measured by a new fluorescence technique. Isolated cortical collecting tubules (CCT) from rabbit kidney were perfused with physiological buffers containing the impermeant fluorophores fluorescein sulfonate (FS) and pyrenetetrasulfonic acid (PTSA). Tubules were subject to a 120 mOsm bath-to-lumen osmotic gradient in the presence and absence of 250 microU/ml vasopressin. The magnitude of transepithelial volume flow was determined from the self-quenching of FS, or from the ratio of PTSA/FS fluorescence, measured at 380 nm excitation and 420 +/- 10 nm (PTSA) and greater than 530 nm (FS) emission wavelengths. Pf was calculated from the magnitude of transepithelial volume flow, lumen and bath osmolarities, lumen perfusion rate, and tubule geometry. The instrument response time for a change in bath osmolality was less than 3 s. At 37 degrees C, CCT Pf was (in units of cm/s x 10(4] 13 +/- 2 (mean +/- SE, 16 tubules) before, and 227 +/- 10 after addition of vasopressin to the bath. CCT Pf began to increase in 23 +/- 3 s after vasopressin addition and was half-maximal after 186 +/- 20 s. At 23 degrees C, Pf was 9 +/- 1 (seven tubules) before, and 189 +/- 12 after vasopressin addition. Pf began to increase in 40 +/- 4 s and was half-maximal after 195 +/- 35 s. After vasopressin removal from the bath, Pf decreased to its baseline value with a half-time of 14 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Androgens and androgen-binding protein in the rat epididymis

The levels of testosterone, dihydrotestosterone (DHT), 5alpha-androstan-3alpha,17beta-diol and androgen-binding protein (ABP) were measured in various segments of the epididymis from adult rats which had been unilaterally orchidectomized for 4 weeks. On the 'intact' side, ABP concentrations were highest in the caput region. The segmental distribution of DHT closely followed that of ABP with the highest concentration in the caput (40 ng/g tissue) and lowest in the cauda (10 ng/g tissue) epididymidis. There was a high degree of correlation (r = 0.98) between the concentration of DHT in the epididymis and ABP levels. 'Castration' completely abolished the DHT gradient. The levels of testosterone and androstanediol were lower than those of DHT; most was present in the corpus epididymidis. The relative differences were reduced after 'castration'. It is concluded that ABP in the rat epididymis is the primary factor for determining the concentration of DHT in the epididymal fluid.     

Tissue and cell specificity of immobilin biosynthesis

The mechanisms for the initiation of sperm motility have been poorly understood until recently. Immobilin is a novel mucin glycoprotein of high molecular weight found in the cauda epididymis of the rat that, at concentrations equivalent to those found in native cauda epididymal fluid, reversibly inhibits sperm motility. In this study, immobilin was purified from rat cauda epididymal fluid to apparent homogeneity and used to generate polyclonal antibody in rabbits. The antibody was characterized by immunoblotting, and immunofluorescence was used to localize immobilin in paraffin sections of components of the reproductive system of adult male rats. Immobilin was not detectable in the efferent duct and was first detectable in the apical portion of some epithelial cells of the initial segment of the caput epididymis. Immobilin was detectable intracellularly only in cells of the caput epididymis. In the corpus and cauda epididymis immobilin was detectable only in the lumen of the tubules. Immunoprecipitation of immobilin radiolabeled in vitro confirmed that immobilin biosynthesis in the adult rat is restricted to the caput epididymis. Principal cells in the caput epididymis synthesize immobilin and secrete it into the lumen of the tubules to travel with the sperm into the cauda.     

Immunohistochemical demonstration of androgen-binding protein in the rat prostatic gland.

Androgen-binding protein (ABP) is one of the best-characterized products of synthesis by the Sertoli cells in the rat. Although the exact physiological role of ABP remains to be determined, it has been widely used to study Sertoli cells and testicular function in this species. Since this protein is the principal carrier for testosterone in rat testis and epididymis, we decided to investigate ABP immunoreactivity (ABP-I) in androgen-dependent organs, including testicle, epididymides, prostate, and seminal vesicles. The location of ABP was investigated by immunohistochemistry using specific antisera against rat ABP. As previously described in the testis, rat ABP-I was identified in the seminiferous tubules within the cytoplasm of the Sertoli cells and the tubular luminae. The epididymis showed ABP-I only in epithelial cells of the proximal caput. We demonstrated ABP-I in the apical portions of epithelial cells of the rat prostate. Short-term castration and/or ligation of the efferent ducts did not suppress prostatic ABP-I. ABP-I was not present in seminal vesicles of control rats nor under any of the experimental conditions used throughout this study. The results also indicate the presence of ABP-I in prostatic epithelium, probably because of a mechanism similar to that described in epididymis. Our data support and enhance the concept that ABP may serve as a transmembrane carrier protein for androgens in androgen target organs in the male reproductive tract.     

The influence of unilateral castration on testicular morphology and function in adult rams

An experiment was designed to investigate the mechanisms controlling testicular compensatory hypertrophy in rams. Endocrine and histological events were examined, with special attention to Sertoli cell hyperplasia and hypertrophy as contributing factors to the compensatory process. Fifteen sexually mature yearling Targhee rams were allotted to intact control (C, n = 5) and unilateral castrate (UC, n = 10) treatment groups in June. Approximately 150 days after UC, testicular tissue was collected in November after efferent duct cannulation and rete testis fluid (RTF) collection or perfusion fixation. Unilateral castration increased mean testis weight by 56% (p = 0.01) and mean epididymal weight by 15% (p = 0.05). Although the mean volume of RTF collected more than doubled after UC (1.55 +/- 0.86 vs. 0.63 +/- 0.10 ml for UC and C rams, respectively), the difference was not statistically significant. By 150 days after UC, the concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) in jugular venous blood did not differ between the two treatment groups. The concentrations of T. dihydrotestosterone (DHT), and androgen-binding protein (ABP) in RTF were also similar for UC and C rams. However, since the observed mean RTF volume was increased, the amounts of T, DHT, and ABP exiting the testes of these UC rams via the RTF were approximately doubled, although this difference was not statistically significant. UC increased the mean diameter of seminiferous tubules by 21% (p less than 0.01) and of their lumina by 51% (p less than 0.01), but did not significantly increase mean height of seminiferous epithelium or estimated length of seminiferous tubules per testis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epididymal functions and their hormonal regulation

The epididymis is a complex organ which maintains a specific intraluminal environment thought to be important for effecting sperm maturation in proximal regions and sperm storage in distal regions of the duct. The composition of the internal milieu is achieved both by transport between blood and lumen (and vice versa) and by synthesis and secretion into the lumen. Several low-molecular weight organic molecules achieve high concentration in the epididymal lumen, but their functions in the events of sperm maturation and storage still remain unclear. Metabolic processes occurring within epididymal tissue and the absorptive and secretory activity of the epididymal epithelium are regulated by androgens. The synthesis of some, but not all, secretory proteins is also androgen-dependent. In addition to androgens, other hormones and local testicular factors may influence epididymal function. There is now increasing evidence that epididymal-specific and androgen-dependent secretory proteins play a fundamental role in modifying the surface characteristics of sperm in preparation for the events of fertilization.     

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)     

Physiological role for androgen binding protein-steroid complex in testis?

The concentration of $\text{unbound}$ androgens in the lumen of rat testis seminiferous tubules is not dependent on the presence of the extra-cellular androgen binding protein (ABP); other parameters such as permeability properties, fluid dynamics and metabolic activities in different testicular compartments appear to have a much greater influence. It has been shown that in other target tissues the metabolic effects of steroids on cells depend on the concentration of the steroids which are not bound to extra-cellular proteins. It is therefore unlikely that the physiological function of ABP is related to the accumulation of androgens around germinal cells.