Fluid Movement in the Lumen of the Rat Epididymis: Effect of Vasectomy and Subsequent Vasovasostomy

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1 Journal of Andrology, Vol. 11, No. 5, September/October 1990 Copyright 0 American Society of Andrology Fluid Movement in the Lumen of the Rat Epididymis: Effect of Vasectomy and Subsequent Vasovasostomy T. T. TURNER,*t J. L. GLEAVY,* AND J. M. HARRS* ntralumlnal fluid movement rate was measured in four regions of the rat epididymis. The fastest flow occurred in the proximal caput epididymis (18.5 ± 3.7 mm/hour) and the slowest in the distal cauda (2.5 ± 0.5 mm/hour). Vasectomy significantly reduced caput fluid flow rates unless a sperm granuloma was present at the vasectomy site. Thirty days after vasovasostomy, caput fluid movement remained reduced in animals unless a granuloma was present. Failures of this or other aspects of epididymal biology to return to normal after vasovasostomy could play a role in the frequent infertility which persists after the operation. Key words: vasovasostomy, vasectomy, epididymis, fluid movement J Androl 1990;11: Spermatozoa are exposed to the intraluminal epididymal microenvironment for a number of days which have been estimated in several species, including the human (see Robaire and Hermo, 1988). The epididymal transit times (5 to 15 days, depending on species) are governed by the rate of fluid movement within the epididymal tubule. This, in turn, is influenced by hydrostatic pressure gradients (Johnson and Howards, 1976), peristaltic-like contractions of the epididymal tubule (Jaak- This work was supported by NH grants HD09490, HD18252, and HD Correspondence to:. T. Turner, Department of Urology, University of Virginia, Health Sciences Center, Charlottesville, VA Received for publication January 12, 1990; accepted for publication March 29, From the *Depa,.tment of Urology and the tdepartment of Anatomy and Cell Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia kola and Tab, 1982; Phoipramool et a!, 1984), and regional variations in fluid reabsorption (Levine and Marsh, 1971). Only one previous paper has been found which reports specific observations on fluid movement within the various zones of the epididymis. Jaakkola (1983) reported that proximal regions of the rat epididymis exhibited greater intraluminal fluid movements than did the distal epididymis, and estimated that fluid transit times through the proximal caput was 6 to 7 hours. Transit through the remaining caput and corpus was estimated to require approximately 3 days and through the cauda epididymidis to require approximately 2 days. These estimates gave a considerably shorter total epididymal transit time (approximately 5.3 days) than had previous estimates in the rat (8.5 to 15.0 days) using different techniques (Macmillan and Harrison, 1955; Grant, 1958; Robb et al, 1978; Sujarit and Pholpramool, 1985). Fluid movement rate through an actively secreting or reabsorbing tubule has a profound effect on the constitution of the intraluminal fluid, thus conditions which significantly alter fluid flow rates could have a significant effect on the epididymal 422

2 No. 5 FLUD MOVEMENT N THE EPDDYMS. Turner, Gleavy, and Harris 423 microenvironment to which sperm are exposed during maturation. Certain clinical conditions can be expected to alter intraluminal fluid flow rate. Among these conditions are vasectomy, congenital absence of the vas deferens, and vasal obstruction due to injury or disease. n the case of vasectomy, tubal patency is often re-established by vasovasostomy, but in 40 to 70% of cases the return to patency is not accompanied by a return to fertility (Yarbro and Howards, 1987). t has not been established whether alterations within the epididymis play a role in this persistent infertility, and in particular, whether or not epididymal lumen flow rates return to normal after re-establishment of tubal patency. The present investigation was performed to: i) determine intraluminal flow rates in four regions of the rat epididymis, ii) to make qualitative observations on the pattern of intraluminal flow, and iii) to determine the effect of vasectomy and vasovasostomy on flow rates in the rat caput epididymidis. nitial Segment Diet. Corpus Prox. Cauda nitial Segment 61,0cm, (19%) Caput Epidid. 95.1cm. (29%) Corpus Epidid cm. (10%) Cauda Epidid cm. (4 1%) Materials and Methods Diet. Cauda TOTAL TUBULE 3.23m Adult, male, Sprague-Dawley rats ( g) were anesthetized with mactin (100 mgfkg body weight, ; Byk Guilden Konstanz, Hamburg, Germany); their epididymides were prepared for in vivo micropuncture in a 35#{176}C agar jacket. A mineral oil covered window to the epididymis allowed for micropuncture and observation (Turner, 1988). Additionally, one epididymis was extirpated, placed in 0.9% saline in a Petri dish, and subjected to microdissection of the entire epididymal tubule for estimation of length of tubule within each epididymal zone. A sharpened glass micropipette (30-44) p.m tip diameter) was attached to a syringe via a PE-50 polyethylene cannula, and was pre-loaded with Sudan black stained mineral oil. The pipette was directed with a micromanipulator into an epididymal tubule in four different regions of different epididymides: proximal caput (zone 2, see Fig 1), proximal corpus (zone 4), proximal cauda (zone 6), and distal cauda (zone 8). A sufficient volume of stained mineral oil was injected into the epididymal lumen to form a single droplet just capable of completely spanning the epididymal lumen and forming a single, moveable, visible fluid unit in the tubule lumen. Beginning with the time of oil droplet injection, observations were made every 5 minutes for 75 minutes in order to determine and record the location of the oil droplet relative to its point of origin in the tubule. Movement was estimated to the nearest millimeter with the visual aid of a transparent gauge viewed through the dissecting microscope. At the end of the 75 minute period, the epididymides were quiddy removed. The length of tubule between the oil injection point and its terminal point were determined after dissection of the tubule in saline Fig 1.-Micropuncture zones (1-8) of the rat epididymis; es. timated length of the initial segment, caput, corpus, and cauda epididymides, and estimated total epididymal tubule length of the rat (and percent of total length). and measurement with the transparent metric gauge. Care was taken to avoid stretching the tubule lengths during the measurement procedure. Tubules were not placed in fixative during dissection because preliminary experiments showed that this caused tubule contractions and further movement of the intraluminal fluid drop. Observation of the position of the oil drop in the tubules in the present study confirmed that careful dissection in vitro did not cause appreciable movement of the oil drop from its terminal location in vivo. The net distance the oil drop moved along the epididymal tubule was expressed in mm/hour and was determined in a minimum of five epididymides for each of the four zones studied. A separate group of rats were vasectomized using standard techniques (D Addario et a!, 1980) that incorporated an approach through a laparatomy incision. ntraluminal fluid movement rates were studied (as described above) in the proximal caput epididymdis 30 days after vasectomy. A third group of rats was vasectomized for 30 days then subjected to vasovasostomy (VV) using the modified two-layer microsurgical procedure of Howards (1980). ntraluminal fluid movement rates were studied in these animals 30 days after VV. At the time of the study of the vasectomized and VV animals, vasa and epididymides were examined for the presence of sperm granulomata. Average net fluid

3 424 Journal of Andrology September/October 1990 Vol. 11 movement rates were calculated for each group and data were compared by analysis of variance followed by Duncan s Multiple Range test (P < 0.05). Patency of vasa deferentia 30 days after VV was determined by the presence of fluid flow through a portion of excised tubule which included the VV site. The rate of fluid flow through the reanastomosed vas deferens was determined under conditions of high and low hydrostatic pressure (Carey et al, 1988). n brief, the reanastomosed vas deferens was excised, including the VV site and any sperm granuloma present. Under a dissecting microscope, the proximal (epididymal) end of the vas was cannulated with a 24 gauge irrigation cannula (Edward Wick & Co. Long sland, NY), and this cannula was advanced into the lumen of the vas until the tip rested approximately 0.5 cm proximal to the VV suture line. The vas distal to the suture line was transected at a point also 0.5 cm from the VV suture line. This left a 1.0 cm portion of the excised, cannulated vas deferens extending from the tip of the irrigation cannula, with the anastomosis site in the middle of the 1 cm section. The cannula was connected by polyethylene tubing to a reservoir of 0.9% saline which was positioned to provide high pressure in the cannula (90 cm H20) or low pressure (40 cm H2O) in the cannula. The 1 cm strip of vas deferens was flushed at high pressure to remove the viscous, sperm debris-filled luminal contents. The escape of saline from the distal extremity of the vas segment was considered proof of patency across the VV site. Rate of flow of the 0.9% saline solution through the anastomosed vasa deferentia were carried out first at 90 cm H20, and then at 40 cm H20. The volume of fluid traversing the anastomosis was determined during a 2- minute period. This was done three times at each pressure, and an average flow rate (mi/minute) was determined for each vas deferens examined. Results ntraluminal Fluid Movement in the Normal Rat Epididymis Net intraluminal fluid movement rates in the proximal zones of the rat epididymis were significantly greater than those in the distal zones (Table 1), but movement of individual oil drops within the same zone of different epididymides could be widely variant (Table 1). This was especially evident in the proximal caput and distal cauda. Qua!- itative observation of fluid movement within the tubules showed that it was not unidirectional toward the vas. n all zones the oil droplet was observed to move progressively forward, then some distance backwards, then forward again with a gradual step-wise advance toward the distal regions. The coiling of the small, proximal tubule was such that estimates of the movement of a single drop within the tubule lumen was impossible Region Table 1. Oil droplet movement in the lumen of the rat epididymis Zone Net movement (mm/h) n Max:Min (mm/h) Proximal caput ± 3.7t 6 31:5 Proximal corpus ± 1.4t 5 20:11 Proximal cauda ± 1.4f 5 12:5 Distal cauda ± O :0 * Maximum and minimum net distances traveled from point of injection by oil drops in different animals. t5 Means ± SE sharing the same superscript are not significantly different (P <.05). to follow over the entire experimental time period. However, it was possible to follow the movements of individual drops in the tubules of distal caudal epididymides. Four patterns of oil drop movement was seen in the distal cauda epididymidis (Fig 2). Some oil drops experienced long periods of short, bidirectional movements leading to no or little net forward movement (Fig 2A, 2B). Other drops were injected into tubules with activity causing large bidirectional movements but resulting in relatively large net forward movements (Fig 2C), whereas others exhibited relatively straight-forward unidirectional movement (Fig 2D). The patterns of movement illustrated in Fig 2A did not occur in the caput and corpus epididymidis, but the type of movement illustrated in Fig 2B, and especially 2C, did contribute to the variations in the net intraluminal fluid movement observed in those regions. f the average movement rates found for zones 2 and 4 are used as approximations of fluid movement rates in the entire caput and corpus epididymidis, then transit times through those regions can be estimated to be 2.1 days and 0.8 days, respectively. f the average intraluminal fluid movement rate in the cauda epididymis is estimated to be the average of those flow rates determined for the proximal and distal cauda ( = 5.7 mm/hour), then transit time through the cauda epididymidis can be estimated to be 9.8 days, without ejaculation. Effects of Vasectomy and Vasovasostomy on Ca put ntraluminal Fluid Movement Net intraluminal fluid movement in the proximal caput epididymides of rats 30 days after vasectomy were significantly reduced from the control value (Table 2). t was noted, however, that the presence of a granuloma at the vasectomy site

4 No. 5 FLUD MOVEMENT N THE EPDDYMS. Turner, Gleavy, and Harris 425 A. TOTAL NET Table 2. Effect of vasectomy and vasovasostomy on oil droplet movement (mm/hour) in the lumen of the rat caput epididymidis Group Total Granuloma No granuloma Control 18.5 ± 3.74: (6) - - Vasectomy 12.3 ± 1.8 (7) 15.4 ± 1.8 (4) 8.2 ± 0.7 (3) Vasec/ vasovast 13.3 ± 2.8 (8) 18.6 ± 2.3 (5) 6.2 ± 0.6 (3) * Measurement done 30 days after vasectomy. O 3.0 t Measurement done 30 days after vasovasostomy 30 days after vasectomy. f Means ± SE sharing the same superscript are not significantly different (P <.05). D = ::i C-) Distance Traveled (mm) (#{247}) Fig 2.-Four patterns of bulk, intraluminal fluid movement in the rat cauda epididymidis. The point of oil drop injection is indicated by the closed circle, and the terminal position of the oil drop is noted by the arrowhead. An estimated total and net distance traveled is indicated in each panel. Each line shift indicates the position of the oil drop at 5 minute intervals. n some cases, drops did not move during a 5 minute period; no movement is indicated by a larger line shift in the y axis. seemed to preserve normal fluid flow rates, whereas absence of a granuloma was associated with reduced intraluminal flow rate (Table 2). Thirty days after vasovasostomy in animals who had had a 30-day vasectomy, the overall intraluminal flow rates had not returned to normal (Table 2). VV animals also divided into those with and those without granulomata at the VV site (Table 2), and, whereas the number of animals was not distributed in a manner allowing statistical analysis, it again appeared that the presence of a granuloma at the VV site was associated with normal intraluminal fluid flow rates in the caput epididymidis and its absence with extremely retarded flow rates (Table 2). One hundred percent of the VV animals had patent vasa deferentia at the time of examination, whether or not granulomata were present. Fluid flow rates ( ± SE) through unoperated vasa were 2.1 ± 0.08 m/minute and 4.15 ± 0.14 m/minute at 40 and 90 cm water, respectively (Table 3). Fluid flow through VV vasa was not significantly different from control values at either high or low pressures, and fluid flow rate was not affected by the presence or absence of a sperm granuloma (Table 3). Estimated transit times of fluid through the caput epididymidis in control animals was 2.2 days. This estimate was increased to 3.2 days in vasectomized animals and was not reduced by VV if all data are considered together (Table 4). nterestingly, transit times in vasectomized and VV animals with granulomata were apparently normal while transit times in the animals without granulomata were two to three times normal (Table 4). Discussion n the only previous paper found on luminal fluid transport rates in the epididymis, Jaakkola (1983) measured the forward travel of micro-oil droplets injected into ten different regions of the rat epididymis. Regions V, V, V, and X of that study appear roughly analgous to zones 2 (proxi- Table 3. Effect of vasectomy and subsequent vasovasostomy (VV) on fluid flow rate (milmin) through the W site on the rat vas deferens Group Control W (total) W (granuloma) W (no granuloma) Flow when pressure at 40 mm H20 90 cm H ± ± ± ± ± ± ± ± 0.19

5 426 Journal of Andrology. September/October 1990 Vol. 11 Table 4. Effect of vasectomy or vasectomy/vasovasostomy (VV) on calculated transit times (days) of lumen content through the rat caput epididymidis Group Total Granuloma No granuloma Control Vasectomy VV mal caput), 4 (proximal corpus), 6 (proximal cauda), and 8 (distal cauda) of the present study. The two studies are consistent in that Jaakkola (1983) also reported wide variations in oil droplet movement, but the median intraluminal movement reported by Jaakkola (1983) for the proximal caput (zone 2 of the present study) was 64.2 mml2 hours, or approximately 32 mm/hour, and this was not significantly altered in what would be zone 4 (proximal corpus) of the present study. These times are approximately twice those found for the intraluminal fluid flow rates in the present study (18.5 mm/hour and 16.5 mm/hour for caput and corpus, respectively), but are consistent in finding that flow rates in the proximal caput and proximal corpus are not significantly different from each other. n the proximal cauda epididymidis, the median movement rate found by Jaakkola (1983) was 45.7 mm/2 hours or approximately 23 mm/hour, a movement rate more than twice that found in the present study (approximately 9 mm/hour) n the distal cauda epididymdis, the median rate of transport reported by Jaakkola (1983) was 25.5 mm/2 hours or approximately 13 mm/hour, a rate approximately five times the fluid movement rate in the distal cauda epididymidis found in the present study (approximately 2.5 mm/hour). The more rapid transport rates reported by Jaakkola (1983) might be due to the differing size of oil droplets injected in that study. n the present study, a drop sufficiently large to fill the epididymidal lumen was used in order to measure bulk flow, ie, native content could not move around the oil drop and it is not likely the oil drop could move independently of bulk intraluminal fluid movement. The much smaller microdroplets injected by Jaakkola (1983) were said to have dispersed along the epididymal tubule wall. t is possible that these microdroplets responded more easily to both major and minor contractions of the tubule wall and were dispersed at a rate faster than the bulk flow of intraluminal content. n addition, Jaakkola (1983) did not detail how the epididymides were prepared and maintained during the experimental period in that study; and these factors might also cause differences. The total epididymal tubule length (including the initial segment) measured in the present study was 3.23 m, which appears to be similar to the lengths (approximately 3.60 m) dissected by Tao et al (1979) and used by Jaakkola (1983), but zonal comparisons of length are not possible. Jaakkola (1983) estimated a total transit time for the rat caput and corpus epididymidis to be approximately 2.9 days. This is identical to the present estimate of 2.9 days (2.1 days for caput and 0.83 days for corpus), even though the actual rate of intraluminal fluid movement reported in the present study is much less than those reported by Jaakkola (1983). This similarity of net results might be due to discrepancies in the measurements of intrazonal lengths. There is a great difference in the estimation of transit time through the cauda epididymidis. The present estimate is approximately 10 days, absent ejaculation, while Jaakkola (1983) estimated a cauda transit time of only 2.4 days. The present estimate of total epididymal transit time based on bulk flow in four regions of the rat epididymis is 13.5 days, similar to the total transit times found by MacMillan and Harrison (1955) and Grant (1958) when studying intraluminal movement of radioopaque media or trypan blue, respectively, through the rat epididymis. The total transit time estimate from Jaakkola s work (1983) is 5.3 days, much shorter than even the 8.5 day estimate proposed by Robb et al (1978) for mature rats after using a calculation based on epididymal sperm reserves and rat testicular daily sperm production, and the estimate of 8 days by Sujarit and Pholpramool (1985) after observing the passage of 3Hthymidine labelled sperm through the epididymis. No other independent estimates of epididymal transit time in the rat have been found. Both the present report and that of Jaakkola (1983) are consistent with regard to the decreasing speed of fluid movement as the epididymal content moves from the proximal to distal tubule and in reporting the pendular movements of intraluminal content. This oscillatory movement presumably provides considerable opportunity for mixing of intraluminal sperm and fluid. Effect of Vasectomy and the Vasovasostomy on ntraluminal Fluid Movement in the Proximal Ca put Epididymidis Vasectomy obstructs the vas deferens and blocks

6 No. 5 FLUD MOVEMENT N THE EPDDYMS. Turner, Gleavy, and Harris 427 the male excurrent duct system without halting spermatogenesis (Plante, 1973; Bedford, 1976), thus forcing the epididymis to become the site of sperm disposal rather than the site of sperm maturation and storage. n the past, damage to the epididymal epithelium by vasectomy was seen by many as an irrelevant issue since vasectomized men expected to be permanently infertile. n the last decade, however, increasing numbers of Vasectomized males have been requesting reanastomosis of their vasa in order to continue their fertile life. This has led to the development of microsurgical vasovasostomy techniques which allow reestablishment of a patent vas deferens (Silber, 1978; Howards, 1980). Literature surveys show most centers report post-vv patency rates as being from 75 to 95%, but pregnancy rates are reported to be between 43 to 70% (Yarbro and Howards, 1987). Since only experienced centers generally report results in the literature, it is supposed that in North America a realistic estimate of the average pregnancy rate from all VV patients with patent vasa is likely to be 40% or less. The reason for the discrepancy between patency rates and pregnancy rates is unclear, but considerable research has focused on the role of antisperm antibodies in post-vv infertility (Fuchs and Alexander, 1983; Parslow et al, 1983; Sutherland et al, 1984; Herr et al, 1987; Herr et al, 1989). Unfortunately, the association of antisperm antibodies and infertility is problematical (Haas, 1987), and close association between post-vv antisperm antibodies and continued infertility has been difficult to demonstrate experimentally. This has led us to investigate other factors that might influence post-vv sperm function. n the normal adult male, the epididymis is responsible for sperm maturation and storage. While obstructed by a vasectomy, the epididymis becomes distended with sperm debris and infiltered with leukocytes (Bedford, 1976). The intraluminal microenvironment is potentially altered by the more permeant blood-epididymal barrier (Turner et al, 1979) and the modified epithelial transport mechanisms such as those for glucose, or its unmetabolizable analog, 3-0-methyl-D-glucose (Turner et al, 1980). These alterations, or other changes in epididymal physiology, persisting over a long period, might contribute to male infertility after vasovasostomy if they do not return to normal. The function of the epididymis after vasovasostomy has been relatively unexplored. n this initial study, we have examined intraluminal caput epididymidal fluid flow rates before and during vasectomy and after vasectomy repair by VV. As mentioned previously, fluid flow rates are fundamental to the construction of an intratubular microenvironment. When all animals in the experimental groups were considered, vasectomy caused a significant decrease in caput epididymidal flow rates which did not return to normal after vasovasostomy (Table 2), despite the reestablishment of patency and normal fluid flow rates through the VV site (Table 3). nterestingly, however, the response to vasectomy and \ V appeared to be binomial, depending on the presence or absence of a granuloma at the site of vasectomyfvv (Table 2). Normal flow appeared to occur in animals with granulomas; severely reduced flow was found in those animals without granulomas, even after VV (Table 2). This finding of a normal epididymal process with the occurrence of granuloma is in concert with previous reports that testicular weights of rats were better maintained in the presence of a vas granuloma after vasectomylvv (Flickinger et al, 1986). t has also been reported that the results of VV in human males are better when granulomas are observed intraoperatively (Silber, 1977; Belker, 1983). Our experience leads us to believe that the VV epididymides without granulomata were probably obstructed despite the presence of a patent vas. n the epididymis obstructed by vasectomy, the intraluminal content can become a very thick semisolid which may require some weeks, or even months, after VV to clear. Edema at the VV site might prolong the functional obstruction of the vas deferens past the time of the VV as well. Thus, month after VV in these rats, perhaps even longer in humans, the epididymis can remain obstructed with thick lumen content awaiting dissolution. A granuloma occurring soon after vasectomy may circumvent the problem by not allowing the thick debris to build-up in the lumen in the first place. The reduction of intraluminal fluid movement rates in the vasectomized and VV groups (Table 2) would theoretically cause an increase in transit times through the epididymis, and based on the movement rates observed, caput transit times were increased by vasectomy and not returned to normal by VV unless a granuloma was present (Table 4). Thus, patency and normal fluid flow through the surgical site is not necessarily indicative of normal flow through the epididymis; other factors persistent after VV reduced sperm transit

7 428 Journal of Andrology. September/October 1990 Vol. i through the epididymis when no granuloma is present. Fortunately for the preservation of fluid flow, most humans and experimental animals develop sperm granulomas after vasectomy, but there is no information available about the reoccurrence of granulomata after VV in humans. t is possible that, in a significant number of patients, post-vv fluid flow remains reduced through the epididymis even though the vas is patent. Such altered fluid flow likely results in an alteration of the microenvironment required for sperm maturation. Acknowledgment The careful reading of this manuscript and helpful comments by Dr. Stuart S. Howards is gratefully acknowledged. References Bedford JM. Adoptations of the male reproductive tract and the fate of spermatozoa following vasectomy in the rabbit, Rhesus monkey, hamster, and rat. 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Daily sperm production and epididymal sperm reserves of pubertal and adult rats. Reprod Fertil. 1978; 54: Silber Si. Microscopic vasectomy reversal. Fertil Steril. 1977; 28: Silber Si. Vasectomy and its microsurgical reversal. Urol Clin North Am. 1978; 5: Sujarit S and Pholpramool C. Enhancement of sperm transport through the rat epididymis after castration. Reprod Fertil. 1985; 24: Sutherland PD, Matson PL, Mastus JRW, and Piyor JP. Association between infertility following reversal of vasectomy and the presence of sperm agglutinating activity in semen. mt Androl. 1984; Tab A, Jaakkola UM, Viitanen MV. Spontaneous electrical activity of the rat epididymis in vitro. Reprod Fertil. 1979; 54: Turner U. Transepitheliab movement of 3H-androgen in seminiferous and epididymal tubules: a study using in vivo micropuncture and in vivo microperifusion. Biol Reprod. 1988; 7: Turner U, D Addario DA, Howards SS. Effects of vasectomy on the blood-testis barrier of the hamster. Reprod Fertil. 1979; 55: Turner U, D Addano DA, Howards SS. [3H]-3-0-methyl- 0-glucose transport from blood into the lumina of the seminiferous and epididymal tubules in intact and vasectomized hamsters. J Reprod Fertil. 1980; 60: Yarbro ES, Howards SS. Vasovasostomy. Urol Clin North Am. 1987; 14: