SECRETIONS OF THE TESTIS AND EPIDIDYMIS B. P. SETCHELL Biochemistry Department, A.R.C. Institute of Animal Physiology, Babraham, Cambridge In recent years, it has become apparent that the testis produces, in addition to the androgens, considerable amounts of another type of secretion which flows from the lumina of the seminiferous tubules through the rete testis into the epididymis. Here a large part of the fluid part of the secretion is reabsorbed, but additional compounds are added as the spermatozoa pass down the epididymal duct. In this paper, attention will be largely confined to the most recent findings concerning this latter secretion, as the earlier observations have been reviewed by Setchell (1970a) and Setchell & Waites (1971). Rate offlow and ionic composition The fluid which flows from the rete testis of all species so far examined (ram, bull, boar, rat, hamster, wallaby) is a low-protein fluid, isotonic with plasma but containing about three times as much potassium, less sodium and bicarbonate and more chloride than plasma or testicular lymph. The fluid leaves the testis at a rate of between 10 and 20 \g=m\l/g testis/hr, i.e. about 40 ml/day from a 200-g testis of a ram (Voglmayr, Scott, Setchell & Waites, 1967), but to keep these figures in proportion it should be remembered that this rate of flow is exceeded five- to tenfold by the flow of lymph from the testis (Lindner, 1963; Cowie, Lascelles & Wallace, 1964) and 1000-fold by the flow of blood through the testis (Setchell & Waites, 1964). Fluid leaves the rete testis continually and at a steady rate and normally contains between 0-3 and 1 IO6 spermatozoa/ml. The number of spermatozoa can be dramatically reduced about 20 days after heating the testis to about body temperature for 1 to 3 hr without affecting or only slightly reducing the flow of fluid, and without apparently affecting its composition (Setchell, Voglmayr & Hinks, 1971; Setchell & Waites, 1972). However, the fluid leaving the rete testis is not exactly representative of the fluid inside the seminiferous tubules. Fluid removed from the tubules by micro- puncture contains more spermatozoa and potassium and less sodium and chloride than rete testis fluid (Tuck, Setchell, Waites & Young, 1970; Levine & Marsh, 1971). Furthermore, the fluid which is secreted into a droplet of oil in a seminiferous tubule (so-called 'primary fluid') contains even more potassium and less sodium and chloride, i.e. its composition approaches that ofintracellular fluid (Tuck et al., 1970). It would therefore appear that two fluid secretions are involved. Firstly, in the seminiferous tubules, there is a potassium- and bicarbon¬ ate-rich secretion which is then mixed with that from the rete testis epithelium. This second secretion probably contains sodium and chloride in plasma-like concentrations ; if this assumption is made, then it can be calculated from the 165 Downloaded from Bioscientifica.com at 09/29/2021 11:33:00AM via free access 166 . P. Setchell potassium or sodium concentrations of primary and rete testis fluid that the ratio of the volume of tubular to rete secretion is about 1:10. A value of this order for this ratio is also suggested by the concentration of spermatozoa in tubular and rete testis fluid in the rat (Tuck et al., 1970; Levine & Marsh, 1971 ; Setchell & Waites, 1972) and also from the calculated concentration of sper¬ matozoa in the total fluid trapped inside the testis by efferent duct ligation (Setchell, Duggan & Evans, 1973). Mechanism of secretion and its control The earlier observations by Barack (1968) and Setchell & Linzell (1968) suggested that the flow ofrete testis fluid was not affected by gonadotrophins and secretion is not immediately affected by hypophysectomy (Setchell, 1970b). However, Burgos & Vitale-Calpe (1969) reported that ovine luteinizing hor¬ mone (LH), injected into anaesthetized hamsters in enormous doses, caused increases in flow from a catheter in the rete testis. We were not able to confirm their observations, reproducing the dose of LH and other conditions as closely as possible, but using the weight gain of the testis after efferent duct ligation to measure fluid secretion (Setchell et al., 1973), and it would appear that Burgos & Vitale-Calpe's results were probably due to the expression of pre-formed fluid. In any case, they found that flow from their rete testis catheters declined during their periods of observation, in contrast to our observations of continuing and steady flow offluid in animals of many other species, both anaesthetized and conscious. Vitale-Calpe & Burgos (1970a, b) also suggested, from electron microscopical observations, that LH caused shedding of spermatozoa from the germinal epithelium. Again using a different technique, our results do not support this suggestion (Setchell et al., 1973). We counted the numbers of spermatozoa in rat and hamster testes, the efferent ducts of one testis of each animal being ligated (EDL) 10 hr previously, at which time half the animals were injected with LH. If LH were causing the release of spermatozoa from the germinal epithelium, then one would expect that there would be fewer spermatozoa in the unligated testes of the LH-injected animals than in the unligated testes of the albumin-injected controls, but no difference between the EDL testes, and there¬ fore a larger difference between the EDL and ligated testes after LH injection. This was not the case, and therefore we conclude that Vitale-Calpe & Burgos' results must have been due to unrepresentative sampling, always a problem when quantitative conclusions are drawn from the tiny samples examined under the electron microscope. Text-fig. 1. A diagram of the optical density along a disc gel after separating and staining the proteins of blood plasma, testicular lymph and rete testis fluid from a ram. Approxi¬ mately 50 /ig protein in 50 µ\ fluid were applied to the top of a 70 4 mm cylindrical 5-6% acrylamide gel pH 7-4 containing 0-5% sodium dodecyl sulphate which was run for 90 min at 60 V and 6 mA/gel. The gel was stained overnight with 0-25% naphthalene black in 25% isopropyl alcohol and 10% acetic acid, destained electrophoretically in 5% iso propylalcohol-7-5% acetic acid for 70 min, and then the optical density recorded with a Vitratron Densitometer (R. W. Evans, B. T. Hinton and B. P. Setchell, unpublished observations). Downloaded from Bioscientifica.com at 09/29/2021 11:33:00AM via free access 167 Relè testis fluid 2 3 Dislance along gel (cm) Downloaded from Bioscientifica.com at 09/29/2021 11:33:00AM via free access 168 . P. Setchell Proteins of rete testisfluid and seminiferous tubularfluid One of the most obvious features of rete testis fluid (RTF) is that, in contrast to testicular lymph and blood plasma, it contains very little protein. Moreover, it was apparent that the proteins which were present seemed to be partly similar to proteins in blood plasma and partly specific to RTF (Johnson & Setchell, 1968). These observations made with comparatively crude techniques have now been extended by Kormano, Koskimies & Hunter (1971) who examined both rete testis fluid and seminiferous tubular fluid, using step gel electrophoresis. They showed that many of the individual plasma proteins do not appear in rete testis fluid of the rat and that tubular fluid and to a lesser extent rete testis fluid contain a variety of specific proteins so that albumin constitutes a comparatively small fraction of the proteins in tubular fluid. This difference in the protein composition supports the idea of a dual origin of the fluids of the testis originally deduced from the different ionic composition (see above and Tuck et al., 1970), and it also means that immunological damage to the testis may be caused by antibodies entering the tubules, not across the blood-testis barrier which normally excludes proteins (Setchell, Voglmayr & Waites, 1969) but through the rete testis and the luminal surface of the germinal epithelium (Johnson, 1970, 1972). This idea is supported by the observation that the concentration of immuno¬ globulin is low in rete testis fluid (Johnson & Setchell, 1968) but is even lower in tubular fluid (Koskimies, Kormano & Lahti, 1971). Patterns for the proteins of Text-fig. 2. The enzyme activities per unit of protein in blood plasma and rete testis fluid compared with those from an aqueous extract of the testis in the ram. Note that the specific activity of many enzymes is higher in rete testis fluid than in blood plasma and the ratio ofthe activities in the fluid to that in the extract varies widely. Data from Suominen & Setchell (1972). AcG: N-acetyI-/3-glucosaminidase. AcP: acid phosphatase. ArS: arylsulphatase. AlkP: alkaline phosphatase. /3G1: /J-glucuronidase. G6PDH: glucose-6- phosphate dehydrogenase. G1DH : glutamate dehydrogenase. GOT : glutamate oxalacetate transaminase. GPT: glutamate pyruvate transaminase. ICDH: isocitrate dehydrogenase. LDH: lactate dehydrogenase. MDH: malate dehydrogenase. PK: pyruvate kinase. SDH: sorbitol dehydrogenase. Am pep: aminopeptidase. Amyl: -amyiase. Est: non¬ specific esterases. Hyal : hyaluronidase. Downloaded from Bioscientifica.com at 09/29/2021 11:33:00AM via free access Secretions of the testis and epididymis 169 human tubular fluid are similar to those for rat tubular fluid and show a number of specific proteins (Koskimies, Kormano & Alfthan, 1973). Rete testis fluid from rams and boars has also been analysed using 'disc', step gel and 'gradipore' electrophoresis and the presence of a number of specific proteins confirmed (R. W. Evans, B. T. Hinton and B. P. Setchell, unpublished observations, Text-fig. 1). Enzymes in rete testisfluid Some observations have also been made on the enzymic activity ofthe proteins of ram rete testis fluid. Most of the enzymes normally found in blood plasma are also found in rete testis fluid, but in lower concentrations than in plasma because of the low protein concentration.