Department of Urology University of Virginia School of Medicine Charlo ttesville, Virginia 22908

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1 BIOLOGY OF REPRODUCTION 36, (1987) Experimental Varicocele does not Affect the Blood-Testis Barrier, Epididymal Electrolyte Concentrations, or Testicular Blood Gas Concentrations' T. T. TURNER,' C. E. JONES, and M. S. RODDY Department of Urology University of Virginia School of Medicine Charlo ttesville, Virginia ABSTRACT It has been previously shown that 30-day experimental left varicocele (EL V) in adult rats produces a bilateral increase in testicular blood flow and temperature, as well as a concomitant decrease in epididymal sperm count and motility. In the present study, adult male rats with induced EL V were subjected to a variety of studies to determine the mechanism by which unilateral EL V causes a bilateral testicular response. The results demonstrate that ELV does not alter the blood-testis barrier (BTB) to 3H-inulin (MW SOOO), it being largely excluded from entry into the tubule lumen in both control and EL V animals. Neither left nor right cauda epididymidal temperature was altered by ELV. Intraepididymal Na+ and K+ concentrations in the left caput epididymidis were 81.3 * 3.8 meq/l and 26.3 * 1.5 meq/l, respectively. From the cauda epididymidis, these values were 25.0 f 2.2 meq/l and 46.8 * 1.0 meq/l, respectively. These values were similar on the right side and in the left and right epididymis of ELV animals. Left testis arterial ph was 7.3 f 0.1, and Po, and Pco, were f 6.4 mm of mercury and mm of mercury, respectively. Left testicular venous values were 7.3 f 0.1 (PH), and 52.6 f 2.2 mm of mercury and 49.9 f 2.0 mm of mercury. These values were similar for right control testicles and left and right testicles of ELV animals. These results indicate that the mechanism by which unilateral ELV produces a bilateral change in testicular or epididymal function is not by altering the BTB, epididymal temperature or electrolyte concentrations, or testicular blood gas concentrations. I NTR ODUCTI ON Varicocele, abnormal dilatation of the spermatic vein, is a common pathological finding in human males with the clinical complaint of infertility (Dubin and Amelar, 1978; Sherins and Howards, 1978). The abnormal testicular histology and seminal aberrations seen in subfertile men with varicocele have been well described (MacLeod, 1965; McFadden and Mehan, 1978; Rodriguez-Rigau et al., 198l), but the causeeffect relationship between varicocele and male infertility is uncertain. In fact, between 10 and 50% of untreated infertility patients with varicocele prove to be fertile (Stewart, 1974; Rodriguez-Rigau et al., 1978; Cockett et al., 1979), a fact that adds to the Accepted November 18, Received June 3, 'This work supported by NIH Grant HD 'Reprint requests: T. T. Turner, Ph.D., Department of Urology, Box 422, University of Virginia Medical School, Charlottesville, VA confusion about the pathophysiology of the lesion. Nevertheless, approximately 40,000 men per year (Mali, 1984) undergo a surgical repair procedure in hopes of removing the putative ill-effects of the usually unilateral, left-sided varicocele. Despite many claims about the anti-testicular effect(s) of varicocele and hypotheses about the underlying mechnism(s) behind the effect(s) (see Turner, 1982), it was not until the development of an appropriate model in experimental animals (Kay et al., 1979; Al-Juburi et al., 1979) that it was possible to investigate the lesion specifically. It has been demonstrated that in the adult male rat, experimental left-sided varicocele causes a bilateral increase in testicular blood flow and temperature (Saypol et al., 1981), and a subsequent decrease in cauda epididymidal sperm concentrations and motility (Turner et al., 1982). The present report and a companion paper (Rajfer et al., 1986) detail experiments designed to determine whether some other facets of testicular and epididymal physiology are altered by unilateral experimental varicocele in the rat.

2 EXPERIMENTAL VARICOCELE 927 The Varicocele Model MATERIALS AND METHODS The varicocele model was constructed as described previously (Saypol et al., 1981; Green et al., 1984). Briefly, adult male Sprague-Dawley rats ( grams) were anesthetized with inspired halothane and subjected to mid-ventral laparotomy. The viscera were retracted to reveal the left kidney and attendant venous architecture. The left renal vein was exposed and circumscribed by blunt dissection medial to the insertion of the left adrenal vein, and a 4-0 silk ligature was used to restrict the diameter of the left renal vein to a distance of approximately 1.0 mm. This procedure restricted renal vein diameter by approximately 50% at the position of the ligature and resulted in an increase of left intrarenal vein pressure. This pressure increase was also transmitted to the left spermatic vein and caused the development of a varicosity of that vein. The sham operations were performed exactly as described above, except that the ligature was passed around the left renal vein and then removed. The laparotomy incision was closed, the animal was allowed to recover, and a period of 30 days was allowed to elapse before experimental use. Diameter of the left spermatic vein was measured under direct observation through a dissecting microscope before surgery and 30 days afterwards in all animals. Occasional vessels less than 0.2 mm could not be measured on our scale, but for purposes of data analysis were assigned the value of 0.2 mm, the smallest measurable quantity. In five separate groups of animals (n=5 ea) left spermatic vein diameter, left and right testis weights, and paired seminal vesicle weight were obtained at 0, 2, 4, 8, and 12 wk after establishment of the experimental left varicocele. All between-week measurements were compared by analysis of variance (ANOVA) followed by Duncan s Multiple Range test (DMRT) (p<0.05). Bl o o d- Testis and Blo o d- Ep ididy ma1 Barriers The blood-testis barrier and blood-epididymal barrier experiments were carried out using micropuncture techniques, as described previously (Howards et al., 1976; Turner et al., 1981). Briefly, adult male rats were allotted to 3 groups: unoperated controls (n=3), sham operation (n=5), and 30-day experimental varicocele (n=5). At the time of the bloodtestis and blood-epididymal barrier experiments, animals were anesthetized with i.p. injections of urethane (1 mg/kg body weight), and a single priming dose of 0.33 mci 3H-inulin (specific activity 250 mci/g; New England Nuclear Corporation, Boston, MA) in 0.33 ml of 0.9% saline was infused over a 10-min period via ajugular cannula. A single sustaining dose of 0.33 mci in 1.0 ml of 0.9% saline was continuously infused during the remaining 3-h time period of the experiment. Samples of seminiferous tubule fluid (SNF) and cauda epididymidal fluid (CEF) and carotid artery blood were collected at the beginning of the sustaining infusion and every 30 min thereafter for 3 h. All samples were centrifuged at 10,000 X g to obtain cell-free fluids, and 100-nl aliquots were assayed for isotope concentrations in a scintillation spectrophotometer. Counts per minute (cpm) in the intraluminal fluids were divided by the cpms per same-unit volume of blood serum and multiplied by 100. This product reflected the percentages of blood isotope concentrations appearing in intraluminal fluids at each 30-min time period. Data from the 3 groups of animals were compared by the Kruscal-Wallis test for nonparametric data, and by the Wilcoxin rank-sum test (p<0.05). Epididymal Temperature and Intraluminal Sodium and Potassium Concentrations Animals were divided into 2 groups (n=5 ea): unoperated controls and 30-day experimental varicoceles. Animals were anesthetized with urethane, as previously described, and a carotid cannula was installed. Bilateral caput and cauda epididymidal temperatures were obtained directly by use of a needle thermister probe attached to a Model BAT-4 thermometer (Bailey Instruments, Saddlebrook, NJ). Left and right epididymal temperatures were compared to rectal temperatures by ANOVA, followed by DMRT (p<0.05). The testis and epididymis were exposed, and intraluminal fluids were collected from the caput and cauda epididymidis by in vivo micropuncture, as described previously. A carotid artery blood sample was collected before each caput and cauda epididymal collection. Samples were centrifuged at 10,000 X g to obtain cell-free fluids, and each sample was analysed in triplicate for sodium and potassium concentrations on a flame photometer (IL model 143, Instrumentation Laboratories, Boston, MA). Blood serum and epididymal electrolyte concentrations were compared by ANOVA and DMRT (p<0.05), as described above. Within each fluid type,

3 928 TURNER ET AL. electrolyte concentrations in control and varicocele animals were compared by Student's t-test (p<0.05). Testicular Vascular Po2, Pco2, p~ Animals were divided into 3 groups: unoperated controls, sham-operated animals, and animals with 30-day varicocele. Animals were anesthetized with urethane, as previously described, and a cannula was installed into the carotid artery. Testicular arterial and venous blood (50 p1 per sample) was collected with sharpened, heparinized, glass micropipettes. Similar volumes of carotid blood were collected immediately prior to each testicular blood collection to serve as a reference sample. Blood samples were immediately transferred to a standard hematocrit capillary tube and the sample was aspirated into the analysis chamber of an IL System 1302 ph and blood gas.analyzer (Instrumentation Laboratories). Po,, Pco,, and ph electrodes were calibrated every day. Barometric pressure changes were monitored every day by the instrument, and internal calculations allowed direct readout of Pco2, Po,, and ph. Mean values for each parameter within each fluid were compared by ANOVA followed by DMRT (p<0.05). The Varicocele Model RESULTS The left internal spermatic vein in control rats averaged approximately 0.2 mm in diameter (Fig. 1A). Dilatation essentially plateaued by 4 wk after the partial renal vein obstruction (Fig. 1A) at a spermatic vein diameter of approximately 0.8 mm. Neither ipsilateral nor contralateral testis nor empty seminal vesicle weights were ever significantly decreased from control values (Fig. 1 B). Blood-Testis and Blood-Epididymal Barrier Seminiferous tubules and cauda epididymidal tubules of control and sham-operated animals essentially excluded 'H-inulin (intratubular 'H-inulin was always <5% of serum concentration; Figs. 2A, B). Experimental left varicocele did not significantly alter the proluminal movement of 3H-inulin into seminiferous or epididymal tubules (Fig. 2C). Ipsilateral and contralateral data were not significantly different, so data were pooled for illustration in Figure 2C. Ep id idy ma 1 Te mp e ra tu res and Sodium and Potassium Concentrations Cauda epididymidal temperatures in control rats were 4-5 C below rectal temperatures (Table 1). Intraepididymal temperatures in control rats and in those with 3 0-day varicocele were not significantly different (Table l), but within the control animal group, right intraepididymal temperatures were significantly higher than left epididymal temperatures. Left and right intraluminal caput epididymidal sodium concentrations in control animals were significantly lower than in blood serum and significantly higher than left and right intraluminal cauda sodium concentrations. Similar values were obtained from animals with 30-day varicocele (Table 2). Left and right caput epididymidal potassium concentrations were higher than concentrations in blood serum and significantly lower than in left and right cauda epididymides. Intraluminal potassium h I.o E E Y i 0.8 C V.- '. 0.4 E L Q) g 0.2 A 0 Y $ 2.0 c 0 P i -. " I I 0.0 L. Test is [3 Seminal Vesicles OR. Testis Duration of Varicocele (wks) FIG. 1. A) Left internal spermatic vein diameter in adult, male Sprague-Dawley rats weeks after surgery to impose experimental, left varicocele. B) Left and right testis weights and paried, empty seminal vesicle weights of adult, male Sprague-Dawley rats 0-12 weeks after surgery to impose unilateral, left-sided varicocele.

4 30i A. Control (3) 2ol O / I 0 -- * t I I I I I I I B. Sham (5) - *-/ *-----a i C. day -Varicocele(5) 2ol 10 I I I I I I I Time (min) FIG. 2. Blood-testis and blood-epididymal barrier to 3H-inulin in Sprague-Dawley rats with and without varicocele. 0-0; % serum isotope appearing in seminiferous tubule fluid. o-----o; % serum isotope appearing in cauda epididymidal fluid. A) Unoperated controls. B) Sham-operated controls 30 days after surgery. C) Varicocele animals 30 days after surgery to impose the varicocele. concentrations in animals with 30-day varicocele were not significantly different from controls (Table 2). Testicular Vascular ph, Po2, Pco, Carotid arterial and testicular arterial and venous EXPERIMENTAL VARICOCELE 929 blood phs in control animals ranged between 7.25 and The phs of blood collected from all three vessels were statistically identical, and no differences were induced by 2-wk and 4-wk varicocele (data not shown). Mean testicular arterial and venous Po2 and Pco2 values were within normal values (Table 3). There were no significant differences between left - and right testis, nor were there any significant dif- #- 0 ]I --- ferences associated with the 2-wk and 4-wk varicocele. The ph, Po2, and Pco2 from each artery and vein of each animal were also calculated as percentages of ph, Po2, or Pco2 of carotid arterial samples taken immediately before each testicular vascular sample. This calculation eliminated varying ventilation status within and between animals as a potential cause of data variance. Left testis arterial ph, Po2, and Pco2 in control animals were f 0.2%, f 2.6%, and 93.1 k 4.4% of carotid arterial values obtained immediately prior to the testicular samples, for example. Right testicular artery values were similar, and no changes were induced by 2 and 4-wk varicocele (data not shown). Mean absolute arterial-venous Poz differences in right and left testes range between 61.7 and 70.1 mm Hg in all groups (Table 4). The data for mean percentage of oxygen extraction by left and right testis tissue demonstrate a significant difference in the right testis of 2-wk varicocele animals (Table 4). DISCUSSION Experimental left varicocele in the rat and dog have been shown to cause a bilateral increase in testicular temperature and blood flow (Saypol et al., 1981). and to cause a significant alteration in testicular histology (Saypol et al., 198 1) or cauda epididymidal sperm concentration and motility (Turner et al., 1982). Experimental varicocele in these studies was defined as a visually apparent dilatation of the left spermatic vein, extending from the left renal vein through the pampiniform plexus. The time course of development and eventual diametric extent of the TABLE 1. Rectal and intracaudal epididymidal temperatures ( C) of control rats and those with 30-day varicocele. Group Rectal Left cauda epididymidis Right cauda epididymidis Control Varicocele 37.2 t 0.3a 36.7? 0.3a 31.0 f 0.7b 31.7 t O.Sb 32.8 t 0.4c 31.9 f 0.3b a b cwithin rows, means sharing the same superscript are not significantly different (p<o.os).

5 ~~ 930 TURNER ET AL. TABLE 2. Intraluminal sodium and potassium concentrations in the epididymis of control rats and those with 30-day left varicoele. Carotid Caput epididymidis Concentration blood serum Left Right Left Cauda eoididvmidis Right Sodium Control f 3.5a (5)* 81.3 t 3.8b (4) 67.5 t 3.2c (5) 25.0 f 2.2d (5) 25.0 f 2.2d (5) Varicocele f 8.7a (8) 80.0 t 4.3b (6) 77.5 f 7.2b (4) 25.6 t 4.lC (8) 21.7 f 2.SC (6) Potassium Control 7.0f 0.8a (5) 26.3 f 1.5b (4) 27.5 f 2.2b (5) 46.2 f 1.3c (5) 48.0 t 1.8c (5) Varicocele 8.8 f 1.3a (8) 27.0 f 0.6b (6) 26.3 f 1.7b (4) 46.8 f l.oc (8) 49.1 f 3.6c (7) * Numbers in parentheses refer to the number of samples analysed. a9b c %ithin rows, means sharing the same superscript are not significantly different. varicocele in the rat model has not been determined, however. Neither have testis nor seminal vesicle weights been followed over an extended course of time. In the present study, the development of experimental varicocele has been determined to be underway by the second week after surgery to establish the varicocele. The extent of the dilatation essentially plateaus by 30 days after this surgery (Fig. 1A). Consistent with previous studies at 30 days (Saypol et al., 1981; Green et al., 1984), rat testicular weights did not change significantly with varicocele, even in the long term (Fig. 1B). Green et al. (1984) have shown that varicocelectomy by high ligation of the rat spermatic vein returns blood flow and temperature to normal 30 days after the varicocelectomy. By 100 days after varicocelectomy, rat testicular blood flow, temperature, and cauda epididymidal sperm concentrations and motility are all returned to normal (Hurt et al., 1986). Thus, in the rat, experimental left varicocele is associated with a bilateral anti-testicular effect that is amenable to correction by varicocelectomy. These findings have led us to attempt to determine the more specific mechanism by which altered blood flow or temperature are deleterious to normal testis or epididymal function. The effects of varicocele on the testis could be due primarily to alterations in either the tubular or extratubular compartment. In the tubular compartment, Sertoli cells have as a major function the maintenance of the blood-testis barrier. Sertoli cells maintain cell-cell tight junctions that prevent the movement of many blood-borne macromolecules from entering the luminal environment of the seminiferous tubule. Cameron et al. (1980) used a static morphological method to examine the blood-testis barrier of human males with varicocele and found no evidence for disruption. Using in vivo micropuncture technique to follow a more dynamic process of molecular movement over time, the present study finds a similar absence of effect of varicocele on the blood-testis and blood-epididymal barrier in the rat model (Fig. 2). The reports of Wong et al. (1982) and Rasweiler and Bedford (1982) that increased temperatures could alter epididymal epithelial transport processes TABLE 3. Arterial and venous blood gas partial pressures (mmhg) from right and left testis of control rats and those two or four weeks after surgery to establish experimental left varicocele. Group T. artery T. vein Testis po2 pc02 po2 pc02 Control left f 6.4 (7)* 44.3 f 3.2 (7) 52.6f 2.2 (7) 49.9 f 2.0 (7) 2-wk. varicocele left t 4.3 (4) 43.1 f 2.1 (4) 56.3 f 3.6 (5) 51.4 f 1.1 (5) 4-wk. varicocele left f 4.7 (7) 44.5 f 1.7 (7) 50.3 f 1.4 (8) 54.3 f 1.6 (8) Control right f 5.1 (7) 42.2f 3.6 (7) 49.7 k 3.3 (6) 54.9 f 2.9 (6) 2-wk. varicocele right f 5.2 (5) 43.6 f 2.2 (5) 48.2 t 2.6 (5) 50.9f 2.0 (5) 4-wk. varicocele right f 4.5 (7) 44.2 f 2.0 (7) 52.6f 2.2 (8) 53.6f 4.0 (8) *Numbers in parentheses refer to number of samples analyzed.

6 TABLE 4. Mean arterial-venous Poz differences (mmhg) in control rat testes and those with two- and four-week varicoce1e.a Group Left testis Right testis Control 63.4 (52.8) 70.1 (56.3) 2-wk. varicocele 73.3 (55.4) 77.8 (61.7)* 4-wk. varicocele 61.7 (55.2) 68.4 (53.1) * Significantly different from all other values (p<o.os). anumbers within parentheses = % oxygen extraction by testicular tissue. raised the possibility that varicocele might induce changes in epididymal temperature and thereby affect the epididymal microenvironment on which spermatozoa depend for maturation. The present results demonstrate that cauda epididymidal temperatures ( C) are lower than testicular temperatures measured similarly in our system (34-35 C; Green et al., 1984). Our results are consistent with the results of others (Brooks, 1973). Cauda epididymidal temperatures were not affected 30 days after surgery to impose ELV. Sodium and potassium concentrations in control rat serum and epididymal lumen fluids are consistent with previous reports (Levine and Marsh, 1971; Jenkins et al., 1980), except for the unusual value for sodium in the right control caput epididymidis. The right control caput sodium concentrations (67.5 k 3.2 meq/l) were significantly lower (p<0.05) than the left control caput sodium concentrations (81.3 f 3.8 meq/l), but the differences between sides were not repeated in the cauda epididymidis of control animals or for any other value in the caput or cauda varicocele animals. There were no varicocele-induced differences in electrolyte concentrations in any fluid studied. This is consistent with the lack of temperature effect already mentioned. An alteration in blood flow to an organ suggests the possibility of altered blood ph or partial pressures of 02 or C02. This has been recognized previously with regard to varicocele (Donahue and Brown, 1969; Netto et al., 1977), but these investigators have not found significant changes in blood ph, Po2, or Pco2 associated with varicocele in human patients. These previous studies of ph, Po2, and Pco2 have been from patients where blood sampling was possible only from the spermatic vein in sites remote from the testis itself; therefore, it remained possible that vessels within the testis of the rat model might EXPERIMENTAL VARICOCELE 93 1 exhibit ph or blood gas alterations not detectable in the less direct studies of the human testis. The present analyses of rat testicular blood demonstrated that blood ph and blood gases at the level of the testis are not altered by 30-day varicocele. It was possible that by analyzing only partial pressure data a real difference in oxygen extraction by testicular tissue might be overlooked. Thus, within each animal, arterial-venous differences in Po2 were calculated and percentage of oxygen extraction was determined. There was no significant alteration of oxygen extraction by testicular tissues between control animals and those with two- or four-wk varico cele. The present study has documented the gradual development of a varicosity of the left spermatic vein of adult male rats subjected to partial occlusion of the left renal vein. Previous studies have documented significant increases in testicular blood flow and temperature due to this experimental varicocele. The present results have shown that experimental varicocele does not cause its anti-testicular effect through disruption of the blood-testis barrier, alteration of epididymal temperatures or electrolyte transport, or alteration in testicular vascular ph, Po2, or pc02. 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