ADRENAL INDEPENDENCE OF FLUID AND ELECTROLYTE REABSORPTION IN THE DUCTULI EFFERENTES TESTIS OF THE RAT

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1 Experimental Physiology (1997), 82, Printed in Gr-eat Britain ADRENAL INDEPENDENCE OF FLUID AND ELECTROLYTE REABSORPTION IN THE DUCTULI EFFERENTES TESTIS OF THE RAT S. Y. MAN, J. CLULOW, L. A. HANSEN AND R. C. JONES Department of Biological Sciences, University of Newcastle, NSW 2308, Australia (MANUSCRIPT RECEIVED 16 JULY 1996, ACCEPTED 27 NOVEMBER 1996) SUMMARY The ductuli efferentes testis (efferent ducts) of the rat were studied to determine whether fluid and electrolyte reabsorption by the ducts is under the control of adrenal mineralocorticoids. Testicular fluid output and the rate of fluid reabsorption by the ducts were determined in sham-operated controls and in rats which had been adrenalectomized 10 days previously, adrenalectomized 10 days previously and given aldosterone therapy (75 jug kg-' day-' s.c.) from day 3 to day 10 post adrenalectomy, and in rats given injections of the aldosterone antagonist, spironolactone, for 10 days (10 mg kg-' day-' s.c.). The values for testicular fluid output and fluid flow from the efferent ducts (means + S.E.M.) in the sham-operated rats were and Jul h-', respectively, resulting in an estimate of fluid reabsorption by the efferent ducts of % of the testicular fluid output. None of the treatments resulted in a significant change in testicular fluid output or in fluid reabsorption from the efferent ducts. Similarly, the treatments did not significantly alter the osmolality or electrolyte concentrations in fluid samples from the rete testis or the distal end of the efferent ducts (sham-operated values for rete testis and efferent duct fluid, respectively, were: osmolality, and mosmol kg-'; [Na+], and mmol 1'; [ClF], and mmol 1-'; [K+], and mmol 1-'). It is concluded that, like the homologous proximal tubule of the metanephric kidney, fluid reabsorption from the efferent ducts is independent of adrenal mineralocorticoid control. INTRODUCTION The ductuli efferentes testis (efferent ducts) play a key role in reproductive function of mammals by reabsorbing most of the fluid leaving the testes. In the rat they reabsorb more than 95 % (Jones & Jurd, 1987; Clulow, Jones & Hansen, 1994) of the testicular fluid output of around jdl h-' (Tuck, Setchell, Waites & Young, 1970; Free & Jaffe, 1979; Clulow et al. 1994). The remaining luminal fluid is delivered to the initial segment of the epididymis with a constant solute composition, and a flow rate which varies only within narrow limits (mean + 2 S.D., sl h-'; Clulow et al. 1994). As a result, a stable milieu is established for sperm maturation and storage in the epididymis. The control of fluid reabsorption by the efferent ducts is poorly understood, having been the subject of a limited number of studies. Recent reports indicate there are both acute and chronic mechanisms regulating reabsorption in the ducts. Luminal flow rate (Clulow, Hansen & Jones, 1996) and solute composition are two important contributors to the acute control of reabsorption, with alterations in flow rate or solute composition inducing substantial changes in reabsorption of a perfused duct within minutes. On the other hand, systemic sex steroid status of an animal affects the chronic flow rate and sperm concentration of fluid leaving the efferent ducts, an effect which is mediated by changes in the reabsorptive capacity of the efferent duct epithelium (L. A. Hansen, J. Clulow 1532

2 284 S. Y. MAN, J. CLIJLOW. L. A. HANSEN AND R. C. JONES & R. C. Jones, unpublished observations). Other control mechanisms for regulating fluid reabsorption in the efferent ducts remain to be identified. Since the main solutes transported by the efferent duct mucosa are Na' and Cl-, the studies reported in this paper were undertaken to determine whether the adrenal cortex is important in maintaining fluid reabsorption in the ducts. Aldosterone, the principal mineralocorticoid in the rat and the rabbit, acts primarily by stimulating Na+ reabsorption and K+ secretion in the distal nephron (cortical collecting ducts especially; O'Neil & Helman, 1977; Schwarz & Burg, 1978; Stokes, Ingram, Williams & Ingram, 1981; Horisberger & Diezi, 1983; Morel & Doucet, 1986; Bonvalet, 1987; Marver, 1992). The ductus epididymidis is homologous to the distal nephron and maintenance of fluid reabsorption in the ductus epididymidis is reported to be under adrenal control (Au, Ngai, Yeung & Wong, 1978; Wong & Lee, 1982; Turner & Cesarini, 1983). However, there have been no reports of studies undertaken to determine whether the efferent ducts are dependent on adrenal control. In this study, luminal composition and fluid reabsorption in the efferent ducts were assessed following adrenalectomy and aldosterone replacement therapy. In addition, the effects on the efferent ducts of the aldosterone antagonist, spironolactone, were investigated. METHODS Animals anid tr-eatmenits Six- to nine-month-old outbred Wistar rats were assigned to one of four treatment groups: (i) shamoperated (controls); (ii) bilateral adrenalectomized; (iii) adrenalectomized with aldosterone therapy; and (iv) spironolactone therapy. Bilateral adrenalectomy and sham operations were performed under halothane anaesthesia (3 % v/v). Sham operations were performed by exposing the adrenal glands before closing and suturing the abdominal incision. Adrenalectomized rats were maintained on drinking water containing 0.91 % saline. All animals were maintained for 10 days after surgery or the commencement of spironolactone treatment before, in the same animal, assessing efferent duct function, and determining testis and accessory gland weights. Aldosterone (Sigma) and spironolactone (Sigma) were prepared for injection in a vehicle of' 10% v/v benzyl benzoate (Ajax Chemicals, Sydney, NSW, Australia) and 90 % v/v sunflower oil (Meadow Lea Foods, Mascot, NSW, Australia) at a concentration of 150,ug ml-1 and 20 mg ml-1, respectively. Aldosterone was injected subcutaneously from 3 days post adrenalectomy at 75 jug (kg body wt)-' day-' as a volume of ml. Spironolactone was administered subcutaneously at a dose of 10 mg (kg body wt)- 'day-' in a similar volume. The sham-operated animals were injected with vehicle only. Before weighing accessory glands, luminal fluid was squeezed out to avoid confounding organ weight with luminal contents. Determination of effects of treatments on efferenit duct reabsorption in situ A procedure described by Clulow et al. (1994) for the estimation in intact animals of testicular fluid output and the rate of fluid reabsorption by the efferent ducts was used to compare the effects of the treatments. In rats anaesthetized with sodium pentobarbitone (60 mg kg-' I.P., supplemented via a jugular cannula to maintain anaesthesia; Nembutal, Abbott Laboratories, Sydney, Australia), the flow rate of fluid from the terminal end of the efferent ducts (Fla, where la refers to zone la of the ductus epididymidis; Reid & Cleland, 1957) was determined by continuous collection of fluid in vivo into a microcannula in the initial segment of the epididymis close to its junction with the efferent ducts. Subsequently, micropuncture samples of rete testis fluid were collected. Samples of the fluids entering and leaving the efferent ducts were used to determine the concentration of electrolytes and sperm, and osmotic pressure. At the conclusion of experiments, animals were killed with an overdose of anaesthetic. The rate of testicular fluid output was calculated by comparing the change in sperm concentration between the rete testis ([sperm]rt) and epididymal cannula ([sperm]1). Equations for calculating daily sperm production (DSP), testicular fluid output (TFO) and reabsorption in the eflerent ducts (RED) are

3 ADRENAL INDEPENDENCE OF THE EFFERENT DUCTS given by Clulow et al. (1994). In the study described in this report, values for TFO and RED were calculated for each animal and the means then calculated from the sum of the individual animal values, whereas Clulow et al. ( 1994) calculated single estimates for TFO and RED from overall means. Assays and statistical analyses Procedures for the collection of blood and the collection and separation of luminal fluids from sperm were as described by Clulow et al. (1994). Osmotic pressure determinations were carried out using a Clifton nanolitre osmometer (Clifton Technical Physics, New York, USA) and sodium, chloride and potassium concentrations were determined by X-ray microanalysis of microdroplets as described by Clulow et al. (1994, 1996). Analysis of variance of data for parameters of fluid reabsorption and luminal composition were performed using the General Linear Models (GLM) procedure of the SAS statistics program (SAS Institute, Cary, NC, USA) using the estimate of variance between animals as the denominator in the F tests. The data for estimates of TFO, RED and DSP, which were the most variable parameters, were log transformed before analysis. Means are presented + S.E.M., where the S.E.M. was calculated from the variance between animals. 285 RESULTS Testis and accessory gland masses The mean masses (± S.E.M., n = 7) of the testis, seminal vesicles and prostate glands for the sham-operated control group were , and g, respectively, and there was no statistically significant effect of any of the treatments on the means. Testicular fluid output and fluid reabsorption in the efferent ducts There was no statistically significant effect of treatment on estimates of testicular fluid and sperm output and fluid reabsorption by the efferent ducts (Table 1), which indicates that fluid reabsorption in the efferent ducts is independent of adrenal control. Concentration of electrolytes and osmotic pressure of blood and luminal fluids There was no statistically significant effect of treatment on Na+, K+ or Cl- concentrations, osmotic pressure in blood plasma (Table 2) or fluids from the rete testis (RTF) or epididymal cannula (Epid,C) (Table 3) in control and treated animals. There was also no statistically Table 1. Effect of adrenalectomy (Adx), adrenalectomy with aldosterone therapy (Adx + Aldo) and spironolactone administration on testicular fluid production and efferent duct reabsorption Treatment TFO [sperm]rt DSP Fia [sperm],, RED RED (I'lh-') (x104 /tl 1) (xl16day ') (,1lh-') (xlji5l1 1) (#I h-') (M) Control ± ± Adx ± Adx + Aldo ± 0.3 Spironolactone TFO, testicular fluid output; [sperm]rt, sperm concentration in rete testis fluid; DSP, daily sperm production; F,a and [spermr],,, flow rate and sperm concentration, respectively, of fluid leaving the efferent ducts; RLI), estimate of fluid reabsorption in the efferent ducts in situ expressed as microlitres of TFO per hour and as a percentage of TFO. Values are means + S.E.M. from 7 animals.

4 286 S. Y. MAN, J. CLULOW, L. A. HANSEN AND R. C. JONES Table 2. Effects of adrenalectomy (Adx), adrenalectomy with aldosterone therapy (Adx + Aldo) and spironolactone administration on blood plasma electrolyte concentrations and osmotic pressure Treatment Osmotic pressure [Na+] [K+] [Cl-] (mosmol kg-1) (mmol 1-) (mmol 11) (mmol 1-) Control Adx ± Adx+Aldo Spironolactone ± 4.0 Values are means + S.E.M. from 5 animals. Table 3. Effects of adrenalectomy (Adx), adrenalectomy with aldosterone therapy (Adx + Aldo) and spironolactone administration on the composition of rete testis fluid (RTF) and fluid flowing from the end of the efferent ducts into the epididymal (zone la) cannula (Epidia) Treatment Site Osmotic pressure [Na+] [K+] [Cl-] (mosmol kg-') (mmol 1') (mmol 1') (mmol 1-) Control RTF ± b a Epidia ± b * Adx RTF _9b a Epid,a b * Adx + Aldo RTF ± ± b ±53a Epidia b * Spironolactone RTF ±05b a Epidia b * Values are means ± S.E.M. from 5 animals. Superscripted letters indicate a significant difference between means for blood plasma and RTF or Epid,a within the same treatment: a p < 0.05, b p < * Significant difference between RTF and Epidia means within the same treatment: P < significant effect of treatment on any of the blood-lumen differences in concentrations of electrolytes, where these existed in controls. That is, [K+] remained higher in RTF and Epid,1 fluids, and [Cl-] remained higher in rete testis fluid, than in blood. The significant decrease in [Cl-] between RTF and the EpidSa cannula was unaffected by the treatments. The absence of any significant change in these parameters between RTF and Epidla fluids in control and experimental groups indicates that the maintenance of overall isotonic fluid and Na+ transport in the efferent ducts was unaffected by treatments. DISCUSSION This study demonstrated that, in the rat, fluid and sperm output from the testis, and fluid and solute reabsorption by the efferent ducts, are independent of adrenal control. Flow rate and composition of the luminal fluids entering and leaving the efferent ducts were unaffected by removal of adrenal support or the action of the aldosterone antagonist, spironolactone. The absence of any effect of treatments on epithelial transport or ionic gradients across the efferent

5 ADRENAL INDEPENDENCE OF THE EFFERENT DUCTS 287 duct epithelium indicates that the principal mechanisms of solute and water transport are independent of adrenal control. The absence of an effect of aldosterone contrasts with the perturbation offea which occurs when the systemic sex steroid status of male rats is altered. For example, L. A. Hansen, J. Clulow & R. C. Jones (unpublished observations) demonstrated that treatment with 17/?-oestradiol elevated meanfea from1-8 to 4.6 jul h-. Thus, flow rate and solute dependence (local, acute control) and systemic sex steroid status (chronic control) remain the only identified regulatory systems which have been shown to affect fluid transport in the efferent ducts and, consequently, the flow rate of fluid and the concentration of sperm entering the epdidymis. The absence of an effect of the experimental treatments on efferent duct function requires consideration of the appropriateness of the treatments and doses employed in the study. period post adrenalectomy (10 days) employed before the assessment of efferent duct function in this study was similar to or longer than the period post adrenalectomy in other studies on the rat in which adrenal and aldosterone dependency were demonstrated in target tissues such as the renal collecting ducts (El Mernissi & Doucet, 1983a; Horisberger & Diezi, 1983) and the ductus epididymidis (Wong & Yeung, 1977; Au et al. 1978; Turner & Cesarini, 1983). Similarly, the dose (1O mg kg-1 day-1) and duration of administration (10 days) of spironolactone were similar to or greater than values reported to inhibit fluid reabsorption and Na+ and K+ transport in the ductus epididymidis of the rat (Wong & Lee, 1982; Jenkins, Lechene & Howards, 1983; Turner & Cesarini, 1983). The absence of an effect of adrenalectomy or spironolactone treatment on the masses of the testis and accessory glands in this study is in agreement with published reports in the rat which show that adrenalectomy (Feek, Tuzi & Edwards, 1989) and spironolactone treatment (Menard, Stripp & Gilette, 1974) do not affect chronic circulating testosterone levels, or mass of the testes or seminal vesicles (Wong & Lee, 1982). Turner & Cesarini (1983), however, did report a small but significant effect on seminal vesicle (but not testis) weight. Although Corvol, Michaud, Menard & Freifeld (1975) reported a weak anti-androgenic effect of spironolactone in the rat at doses which affect mineralocorticoid activity, major anti-androgenic effects require doses around 10 times the dose employed in this study (Basinger & Gittes, 1974). The rate of flow of fluid from the efferent ducts (FEa) is the most sensitive indicator of efferent duct function since it directly measures the rate of fluid release from the efferent ducts estimated into the epididymis. Estimates of TFO are more variable since they are indirectly from several parameters (Fa, [sperm]rt, [sperm]ed). Even though the mean TFO estimates were 10-20,ul h-1 higher in adrenalectomized and spironolactone-treated rats than in the controls, there is no reason to believe that the elevation is due to anything other than the inherent variability of TFO estimates. For example, the elevated adrenalectomy mean ( #l h-1) was due to one out of seven replicates. Without the elevated value, the mean of the remaining six replicates was ,ul h-', which is The close to the control mean of al h-. RED is similarly variable since it is estimated from the difference between TFO and Fla. The dependence of RED on TFO is the reason for the parallel increase in the RED mean and S.E.M. within the adrenalectomized animals. consistent with the The independence of the efferent ducts from mineralocorticoid control is common embryonic origin of the efferent ducts and the proximal kidney tubules (the efferent ducts are derived from embryonic mesonephric kidney tubules: Torrey, 1943; Du Bois, 1969). The proximal tubules do not have specific receptors for aldosterone (Scholer, Mishina & Edelman, 1979; Doucet & Katz, 1981), and Na+-K+-ATPase activity in the proximal tubule

6 288 S. Y. MAN, J. CLULOW, L. A. HANSEN AND R. C. JONES of the rabbit is unaffected by adrenalectomy or aldosterone supplementation (Garg, Knepper & Burg, 1981; El Mernissi & Doucet, 1983 b, 1984), and only slightly depressed by adrenalectomy in the rat (presumably by indirect effects from factors such as a reduced glomerular filtration rate: El Mernissi & Doucet, 1983a). After some initial controversy, it is also now generally accepted that mineralocorticoids do not directly affect water and electrolyte reabsorption in the proximal tubule. Several early reports (Hierholzer, Wiederholt & Stolte, 1966; Wiederholt, Stolte, Brecht & Hierholzer, 1966; Stumpe & Ochwaldt, 1968; Hierholzer & Stolte, 1969; Stolte, Wiederholt, Fuchs & Hierholzer, 1969) on the rat concluded that mineralocorticoids enhanced proximal tubule reabsorption after observation of reduced reabsorption in adrenalectomized or salt-loaded rats which was restored with aldosterone, but not dexamethasone, therapy. However, as a result of other studies on the rat (Cortney, 1969; Martin & Berliner, 1969), dog (Wright, Knox, Howards & Berliner, 1969; Lynch, Schneider, Willis & Knox, 1972) and rabbit (Knepper & Burg, 1981), which found no evidence of direct mineralocorticoid stimulation of reabsorption, it is accepted by most reviewers (Sharp & Leaf, 1973; Burg, 1976; Bonvalet, 1987) that proximal tubule reabsorption is independent of mineralocorticoid control. It has been suggested (Lynch et al. 1972; Sharp & Leaf, 1973; Burg, 1976; Knepper & Burg, 1981) that the apparent stimulation of proximal tubule reabsorption by aldosterone indicated by some studies is due to indirect systemic effects, including changes in extracellular volume and glomerular filtration rate, and potassium depletion, which result from perturbation of mineralocorticoid status. The adrenal independence of fluid reabsorption by the efferent ducts (whose function is not as directly linked to systemic haemodynamic factors as the homologous proximal tubules) supports this interpretation. The absence of a response to adrenalectomy also indicates that glucocorticoids do not affect volume reabsorption in the rat efferent ducts. However, further studies are required to determine whether they affect luminal acidification as in the proximal tubule (Morel & Doucet, 1986). REFERENCES Au, C. L., NGAI, H. K., YEUNG, C. H. & WONG, P. Y. D. (1978). Effect of adrenalectomy and hormone replacement on sodium and water transport in the perfused rat cauda epididymidis. Journal of Endocrinology 77, BASINGER, G. T. & GITTES, R. F. (1974). Antiandrogenic effect of spironolactone in rats. Journal of Urology 111, BONVALET, J.-P. (1987). Binding and action of aldosterone, dexamethasone, 1-25(OH)2D3, and estradiol along the nephron. Journal of Steroid Biochemistry 27, BURG, M. B. (1976). The renal handling of sodium chloride. In The Kidney, 1st edn, vol. 1, chap. 7, ed. BRENNER, B. M. & RECTOR, F. C., pp W. B. Saunders, Philadelphia. CLULOW, J., HANSEN, L. A. & JONES, R. C. (1996). In vivo microperfusion of the ductuli efferentes testis of the rat: flow dependence of fluid reabsorption. Experimental Physiology 81, CLULOW, J., JONES, R. C. & HANSEN, L. A. (1994). Micropuncture and cannulation studies of fluid composition and transport in the ductuli efferentes testis of the rat: comparisons with the homologous metanephric proximal tubule. Experimental Physiology 79, CORTNEY, M. A. (1969). Renal tubular transfer of water and electrolytes in adrenalectomized rats. American Journal of Physiology 216, CORVOL, P., MICHAUD, A., MENARD, J., FREIFELD, M. & MAHODEAU, J. (1975). Antiandrogenic effect of spirolactones: mechanism of action. Endocrinology 97, DOUCET, A. & KATZ, A. I. (1981). Mineralocorticoid receptors along the nephron: [H']aldosterone binding in rabbit tubules. American Journal of Physiology 241, F Du Bois, A. M. (1969). The embryonic kidney. In The Kidney, Morphology, Biochemistry, Physiology, vol. 1, ed. ROUILLER, C. & MULLER, A. F., pp Academic Press, New York.

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