J. Physiol. (1977), 269, pp. 1-15 With 10 text-figure8 Printed in Great Britain

1

IN VITRO MEASUREMENT OF RATE OF FLUID SECRETION IN RAT ISOLATED SEMINIFEROUS TUBULES: EFFECTS OF METABOLIC INHIBITORS AND IONS

BY Y. M. CHEUNG, J. C. HWANG AND P. Y. D. WONG From the Department of Physiology, Medical Faculty, University of Hong Kong, Hong Kong (Received 29 April 1976) SUMMARY

1. An in vitro technique for measuring secretary rate in rat isolated seminiferous tubules is described. 2. The basal rate of fluid secretion was 0-44 + 0-06 nl. cm-' min-' (s.E.) (n = 21). The rate was found to be inhibited by cooling, addition of metabolic inhibitor 2,4-dinitrophenol (2.5 x 10-4 M) and removal of glucose from the incubating solution. This indicates that fluid secretion in isolated rat seminiferous tubules is an energy dependent process. 3. Removal of K+ from the incubating medium inhibited the secretary rate in the isolated seminiferous tubules, whereas a fivefold increase in [K+]o to 23-5 mm had no effect. The secretary rate was also unaffected by the absence of Cl- in the peritubular fluid. 4. Removal of Ca2+ from the peritubular medium caused a rise in the secretary rate. 5. Ouabain (10-3 M) and acetazolamide (4 x 10-5 M) caused a fall in the rate of fluid secretion in isolated seminiferous tubules. 6. These results are discussed in relation to the nature of the ionic secretion produced in the tubules. INTRODUCTION

It has been shown by Tuck, Setchell, Waites & Young (1970) that a considerable amount of fluid can be collected from the rete testis of rats. The fluid is secreted from the Sertoli cells of the seminiferous tubules (Setchell, 1969) and is reabsorbed as it enters the epididymis. The primary secretion is principally an isotonic solution of potassium bicarbonate (Setchell & Waites, 1971). However, the mechanism underlying the process of fluid secretion is still obscure. In order to study the ionic basis of fluid secretion we have developed an in vitro technique to measure the rate of fluid secretion in isolated seminiferous tubules of rats. 1-2

Y. M. CHEUNG, J. C. HWANG AND P. Y. D. WONG Part of this work has been presented in preliminary form, at a meeting of the Physiological Society (Cheung, Hwang & Wong, 1976). 2

METHODS

Adult Sprague-Dawley rats weighing between 200 and 350 g were decapitated. The testes were rapidly extirpated and immersed in cold oxygenated Krebs bicarbonate solution. The encapsulating tunica albuginea was removed under a dissecting microscope and individual seminiferous tubules were freed from the interstitial tissues with the aid of fine stainless-steel forceps. A segment of the seminiferous tubules about 2 cm long was then teased out and transferred to a specially designed chamber containing a grooved platform (Fig. 1). The ends of the isolated segment of seminiferous tubule were then clamped into the fine grooves on the platform by

in

Fig. 1. Diagram of chamber and platform used for clamping rat isolated seminiferous tubules.

S.T.

Clamp

Clamp

Krebs soln.

Sperms

Oil droplet

Oil

Slit

l

Slit

I

2nd clamp at slit

I I I I

Krebs soln.

Sperms/

I'W

I

I~l

W

D

I

I

'

-

1W

Fig. 2. Operational procedure for preparing a segment of rat isolated seminiferous tubules (S.T.) for measurement of the rate of fluid secretion. See text for application.

SECRETION IN RAT TESTES

3

tightly fitting pieces of stainless-steel wire (Fig. 2). With the aid of ultra-fine iridectomy scissors, incisions were made in the segment at points close to the clamped ends. A fine polyethylene cannula with tip diameter between 60 and 80 ,tm was introduced into the lumen through one incision and the tubule was flushed free of spermatozoa with Ringer solution using a tuberculin syringe. A fine droplet of paraffin oil was then introduced by the same technique into the lumen of the seminiferous tubule at about the midpoint of its length. Subsequently one end of the tubule was entirely occluded and the other end was left open to the bathing medium. To estimate the rate of fluid secretion, the bath was placed on a microscope stage (Olympus). The temperature of the bathing solution was maintained at 350 C using a glass heat-exchanger as shown in Fig. 1. The temperature was monitored by using a thermister probe. The tubule was examined in transmitted light with a magnification of 40 x . The system was allowed to equilibrate for about 20 min before measurement was made. When fluid was secreted into the enclosed segment, the oil droplet was pushed in a direction towards the open end. The initial secreting lengths of the seminiferous tubules (between the oil droplet and the clamped end) were kept between 1-00 and 2 -00 mm. Measurements of the movement of the oil droplet were made at intervals of 5 min either with a calibrated eye-piece micrometer or from photomicrographs or from a TV monitor screen through a CCTV system (ITC-5000 series). The volume of fluid secreted during the 5 min period was calculated from the distance displaced by the oil droplet and the luminal diameter of the tubule as measured from the size of the oil droplet. The secreting length was taken to be the distance between the closed end and the oil droplet at the beginning of each 5 min measurement period. The secretary rate was expressed in nl. cm-1 min'. The composition of the different bathing solutions used to incubate the tissue are given in Table 1. Solution B was considered the normal solution and was the solution in which tubules were incubated at the beginning of the experiments. TABLE 1. Composition of solutions (m-mole/l.)

NaCl Na isethionate KCl K2SO4 Choline Cl

CaCl2 MgSO4 NaH2PO4 NaHCO3 KHCO3 Sucrose Glucose pH Gas* Osmolarity (m-osmole)

High K+ Cl--free Ca2+-free High Ca2+solution solution solution solution

K+-free solution

KrebsHCO3-

(A)

(B)

(C)

122-7

118-0

99-2

(D)

(E)

(F)

120-6

110-0

120-4

4-7 -

2-56 1-13 1-17 25-0

2-56 1-13 1-17 25-0

4.7

23-5

-

2-56 1-13 1-17 25-0

2-56 1-13 1-17 25-0

4.7 -

2-35 1-13

1*17 25-0

10-24 1-13 1-17 25-0

-

11.1

7-4

11.1 7.4

7-4

7.4

O2/CO2

O2/CO2

O2/CO2

O2/CO2

021CO2

319

319

318

316

326

11.1

02/CO2 319 *

11.1

11.1 7-4

11.1 7-4

02/CO2 refers to 95% 02 and 5% C02.

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Y. M. CHEUNG, J. C. HWANG AND P. Y. D. WONG RESULTS

Basal rate offluid secretion In twenty-one experiments the starting length of the secreting tubule, that is the length from the closed end to the oil droplet, was 1*19 + 0412 mm (s.E.) (n = 21). In these experiments the oil droplet moved a distance of 0*29 + 0*03 mm (mean + S.E., n = 21) per hour. The mean luminal diameter determined from the size of the oil droplet together with the value of its velocity indicated a secretary rate of approximately 0 44 + 0-06 nl.cmmin-'. mm-1.~~06

E E

,- 04 0

, 0-2

10

20

30 40 Time (min)

50

60

70

Fig. 3. Effect of removal of glucose on the rate of fluid secretion in rat isolated seminiferous tubules. The open circles represent secretary rate in normal tubules and the closed circles represent secretary rate in tubules which were deprived of glucose during the whole experiment (glucose was also absent during the 20 min preincubation period). Each open circle shows the mean + s.E. from eight experiments and each filled circle shows the mean + s.E. from three experiments.

Effects of 2,4-dinitrophenol, glucose deprivation and cooling on secretary rate in seminiferous tubules To discover how far the secretary rate was dependent on cellular metabolism the effects of a number of inhibitors and procedures were determined, since it is known for some species that fluid secretion is dependent on glucose and oxygen (Linzell & Setchell, 1969). When glucose was completely removed from the incubating mediums,

SECRETION IN RAT TESTES

5

"I-.

I)

4.

0-6

E

)E 04

0

0 v

v V)

0

U

35

o

25

(4

E

15

._

0-2C

co

20

40

80 60 Time (min)

I

120

100

140

I

I

E

E 0*15 _ E I) S-

'4.

, 0*05 V

0 Time (min)

Fig. 4. A and B, effect of cooling on the rate of fluid secretion in rat isolated seminiferous tubules. The upper tracing shows the temperature of the bath and the lower tracing shows the secretary rate.

6 Y. M. CHEUNG, J. C. HWANG AND P. Y. D. WONG the secretary rate fell to zero within 65 min of the measurement period (Fig. 3) (glucose was also absent during the 20 min pre-incubation period). In contrast, the control rate of secretion declined by 37 % over a 1 hr period when glucose was present. This suggests that the rate of fluid secretion is dependent on the supply of substrate. DNP (2.5 x 10-4 M)

E

1*

E

DNP (2-5x10-4 M)

C

I

A-

0

V,.I- 0.

In vitro measurement of rate of fluid secretion in rat isolated seminiferous tubules: effects of metabolic inhibitors and ions.

J. Physiol. (1977), 269, pp. 1-15 With 10 text-figure8 Printed in Great Britain 1 IN VITRO MEASUREMENT OF RATE OF FLUID SECRETION IN RAT ISOLATED SE...
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