MICROPUNCTURE AND MICROANALYTIC STUDIES OF THE RAT TESTIS AND EPIDIDYMIS*

Transcription

1 F'ERTII.JTY AND STERILITY Copyright " 1975 The American Fertility Society Vol. 26, No. 1, January 1975 Printed in U.S.A. MICROPUNCTURE AND MICROANALYTIC STUDIES OF THE RAT TESTIS AND EPIDIDYMIS* STUARTS. HOWARDS, M.D., ANNE JOHNSON, M.S., AND SANDRA JESSEE, B.S. Male Reproductive Micropuncture Laboratory, Department of Urology, Charlottesville, Virginia Since the pioneering studies of Richards, 1 micropuncture and microanalytic techniques have been utilized to study normal renal function and renal pathophysiology. These methods have allowed the investigator to obtain ultramicro samples in vivo from specific locations in individual nephrons and to analyze these specimens for the concentration of ions, protein, sugars, and labeled compounds. It has also been possible to monitor in vivo the potential difference across, and the hydrostatic pressure in the individual renal tubule. Such studies have contributed greatly to our current understanding of renal physiology. Considering the anatomic, embryologic, and physiologic similarities between the kidney and the testis and the epididymis, we felt that micropuncture and microanalysis could be adapted for the study of the male reproductive tract. We are a ware of only two studies of this nature in the literature. 2 3 The purpose of this paper is to describe our techniques and findings using micropuncture and microanalysis in the normal rat. MATERIALS AND METHODS Male Sprague-Dawley rats, weighing Received November 15, *Supported in part by NIH-NICHD contract and the American Urological Association Research Fund. from 300 to 350 gm, were obtained from Flow Research Animals (Dublin, Va). They were allowed to acclimate to their new surroundings for at least one week 13 prior to the day of the study. The rats were anesthetized with an intraperitoneal injection of sodium 5-ethyl-5- (1-methyl propyl)-2- thiobarbiturate (Inactin) in a dose of 100 mg/kg body weight. A tracheostomy was performed and a PE 240 tube was inserted into the trachea. The body temperature of the animal was maintained at 37.5 ± 0.5 C with a specially fabricated, constant heat, small animal table (Alfred Zwies, Long Island City, NY). Micropipettes for collection of samples were drawn on a vertical pipette puller (David Kopf Instruments, Tujunga, Calif) from constant-bore flint glass tubing with an outside diameter of 0.72 mm and an inside diameter of 0.22 mm. This tubing had previously been boiled for 15 minutes, three times, in distilled water to remove contaminants. Since the tubing had a smaller bore and a thicker wall than that used in renal micropuncture, centrifugation of a sample could be performed directly in the constant-bore section of the pipette. Sample transfers and resultant possibilities of sample loss were eliminated. In order to facilitate penetration of the tubule wall, the pipette tips were sharpened on a rotating wet

2 14 HOWARDS ET AL January 1975 stone grinder (Alfred Zwies) to a diameter of 120 to 150 JL. This tip size, larger than that used in renal micropuncture, was required to aspirate the viscous fluid from the tubules of the testes and epididymides. After they had been ground, the pipettes were cleaned with chloroform and distilled water, rendered hydrophobic by rinsing in a 1% aqueous solution of Siliclad (Clay Adams, Parsippany, NJ), and dried for 15 minutes in an oven at l10 C. The testis, epididymis, and vas deferens were approached through a scrotal incision and were bathed with waterequilibrated mineral oil to prevent surface dehydration. Brightly illuminated with a fiber optic light system, the testicle and epididymis were viewed under a Nikon stereo zoom microscope at a magnification of 10 to 20 times. The tunica albuginea and epididymal capsule were slit to expose the tubules for micropuncture. Care was taken to avoid the capsular blood vessels since blood would obscure the tubules and would prevent accurate puncture. The puncture pipette, attached to a 10 ml glass syringe with PE 50 tubing, was filled with water-equilibrated mineral oil colored with Sudan black, and placed in a Leitz micromanipulator. After the pipette had been guided into the tubule, a small drop of the Sudanblack mineral oil was injected to confirm the intraluminal location of the tip. Samples up to 300 nl in volume (1 nl=10-6 ml) could be aspirated from the tubules of the caput and cauda epididymidis. It was difficult to obtain more than 120 nl of fluid from a seminiferous tubule because the tubule would collapse around the pipette after 70 to 120 nl had been withdrawn. In rats prepared as outlined above, four distinct regions as defined by tubule size could be distinguished in the epididymis (Fig. 1). It is well known that changes in sperm morphology and metabolism, as well as changes in tubular physiology, occur along the tubules of the epididymis. Therefore, great care was taken to obtain samples from similar areas in each animal. The samples from the caput and cauda epididymides were taken from surface tubules in the shaded portions of areas B and C, respectively (Fig. 1). In comparison, the seminiferous tubule samples were obtained from random surface segments of the tubules. The ductuli efferentes and the rete testis were avoided. Immediately after the removal of the pipette from the tubule, water-equilibrated mineral oil was aspirated into the pipette tip to prevent evaporation of the sample. To determine the spermatocrit, the tip of the collection pipette was broken off, leaving the sample sandwiched between columns of Sudan-black mineral oil in the constant diameter body of the pipette. After one end of the pipette had been sealed with Eastman 910 adhesive, it was inserted into a standard capillary hematocrit tube. The sample was spun at 11,500 rpm (13,460 g) for 15 minutes in a microcapillary centrifuge (International Model MB). The centrifuged sample was then viewed with a Nikon stereo zoom microscope at x 20, and the lengths of the total sample column and the cellular column were determined with a stage micrometer. The spermatocrit was expressed as the percentage of the total volume of the sample occupied by cells. No correction was made for plasma trapping. Cellular morphology was examined in samples not used for spermatocrit determinations. The sample was expelled from the collection pipette into a drop of physiologic saline on a glass slide, which was rotated to disperse the cells. The sample was air dried and stained with

3 Vol. 26, No.1 MICROPUNCTURE STUDIES. I 15 Caput Epididymidis Corpus Epididymidis Cauda Epididymidis FIG. 1: Schematic diagram of the rat epididymis showing the four distinct regions, A, B, C, and D, based on tubule size and translucence and the shaded areas ofb in the caput and C in the cauda, from which samples of tubule fluid were taken. 1% rose bengal. The slides were viewed at x 450 with a Leitz compound microscope. One hundred cells were examined from each specimen on two separate occasions. The types and frequencies of abnormalities were recorded and the total percentage of abnormal spermatozoa was calculated as the mean of the two observations.. Spermatozoa without heads were counted as abnormal cells. RESULTS The in vivo spermatocrit in the tubules of the normal rat testis, caput epididymidis, and cauda epididymidis was ± (N=34), ±0.019 (N=30), and ± (N=31), respectively (Table 1). The spermatocrit in the cauda was significantly higher than the spermatocrit in either the seminiferous tubules (P<O.OOOOl) or the caput <P < ). There was no significant difference between the spermatocrit in the seminiferous tubules and in the caput epididymidis (P<0.1). As indicated by the smaller standard deviation, the variation in the spermatocrit in the caput (SD ± 10.5) was less than the variation in the seminiferous tubules (SD ± 16.6) and the cauda (SD ± 15.6). The spermatozoa aspirated in vivo from the caput and cauda of the rat epididymis had similar morphology. In contrast, the appearance of cells aspirated from the seminiferous tubules varied greatly from tubule to tubule within a given rat testis. The tubules were found to contain in varying numbers one or more of the following: mature spermatozoa, immature spermatozoa, spermatids, spermatid tails, cellular debris, and spermatozoa with damaged heads and/or tails. Compared with the spermatozoa from the epididymis, the spermatozoa from the seminiferous tubules had a more distinct and wavy midpiece. Their heads were often surrounded by cellular debris which was assumed to be Sertoli cell cytoplasm. The percentages of abnormal spermatozoa in the caput and cauda epididymides, 5.5% ±.7% (N=14) and 4.3% ±.6% (N=13), respectively, were not statistically different (Table 2). Those spermatozoa counted as abnormal included those which were with abnormal or without heads and tails or those which were broken. It is not meaningful to present the percentage of abnormal cells in the seminiferous tubules

4 16 HOWARDS ET AL January 1975 TABLE 1. Spermatocrit of Seminiferous Tubules, Caput Epididymidis, and Cauda Epididymidis of the Normal Rat Sample N021 N022 N023 NOll N012 N013 N014 N031 N032 N033 N034 N041 N042 N043 N044 N051 N052 N053 N054 N061 N062 N063 N071 N072 N073 N081 N082 N083 N091 N092 N093 NlOl N102 N103 Mean SD SE Seminiferous Caput Cauda tubules epididymidis epididymidis because of the variation in the morphologic findings from tubule to tubule. DISCUSSION This work, together with two other preliminary studies, 2 3 demonstrates that micropuncture and microanalysis can be adapted to study the physiology of the seminiferous tubules and the ductus epididymidis. Since these tubules and the fluid which they contain are not identical to renal tubules and renal tubule fluid, the modifications previously outlined of the standard techniques of renal micropuncture were developed. TABLE 2. Percentage of Abnormal Spermatozoa in the Caput and Cauda Epididymidis of the Normal Rat Sample NOll N021 N022 N031 N041 N051 N061 N071 N072 N081 N082 N091 N092 NlOl Mean SD SE Caput %abnormal Sample Cauda % abnormal N021 4 N022 3 N031 6 N041 5 N051 2 N061 3 N071 3 N072 3 N081 7 N091 3 N092 7 NlOl 2 N102 8 Mean 4.3 SD 2.1 SE 0.6 These techniques should facilitate and accelerate research in the field of male reproductive physiology. Using these methods, we were able to determine the in vivo concentration of cells in the seminiferous tubules and the tubules of the caput and cauda epididymidis. The spermatocrit is defined as the percentage of the total volume of the aspirated sample composed of solid material. It is an arbitrary index which depends on the conditions under which the sample is collected and centrifuged. The absolute value of this index decreases if centrifugation is longer or faster and if corrections are made for plasma trapping. Nevertheless, if all samples are obtained and handled m a similar fashion, statistically significant differences in spermatocrit are meaningful. Previous histologic studies indicate that usually very few free cells are in the seminiferous tubule fluid. Thus, cells m seminiferous samples are, in part, aspirated from the seminiferous epithelium. In contrast to the spermatocrit of samples obtained from various areas in the epididymis, the spermato-

5 Vol. 26, No.1 MICROPUNCTURE STUDIES. I 17 crit of the seminiferous tubule sample is not an accurate index of the in vivo concentration of free cells in the tubule fluid. The aspiration of cells from the epithelium also results in a more heterogeneous population of cellular material in the seminiferous tubule samples than the epididymal samples. We postulate that this heterogeneity results from obtaining samples from different portions of the wave of the seminiferous epithelium in the rat.4 The length of the wave has been estimated to be to 3.25 em. Thus, to cover an entire wave we would have to obtain cells from approximately 3 em of the tubule. Our average sample size is 120 nl. Since the mean spermatocrit is 35%, we obtain approximately 80 nl of fluid. The internal diameter of the rat seminiferous tubule in vivo is roughly 80 f.l. At this internal diameter, there are 5 nl of fluidlmm of tubule length. Thus, the fluid in a given sample comes from 1.6 em of tubule length, or approximately one half of a wave. There are few free spermatozoa in this fluid; the suctioned cells probably come from an even more limited area, representing only a fraction of a wave. The cytologic findings in a given seminiferous tubule sample thus depend on the site of puncture. The spermatocrit in the cauda epididymidis was statistically higher than in either the caput epididymidis (P<O.OOOOl) or the testis (P< ). This higher concentration of spermatozoa in a cauda reflects its storage function. No difference was found between the concentration of cells in the seminiferous tubules and in the area of the caput epididymidis from which we obtained samples, although the concentration of mature spermatozoa was clearly higher in the caput. There was greater variability in the spermatocrit from the seminiferous tubule than from the caput, which may reflect the seminiferous sample collection problems outlined above. The greater variability of the spermatocrit in the cauda than in the caput may reflect differences in elapsed time since the last ejaculation. In addition, despite their gross morphologic similarity, the punctured areas in the cauda may have been physiologically heterogeneous. Crabo and Gustafsson 6 studied the concentration of spermatozoa in the epididymis of the slaughtered bull. Their sections C and F correspond roughly with the shaded areas of sections B and C, respectively, of the rat epididymis from which we obtained samples. They found that the spermatocrit in section C was 44%, a value similar to our 35.5% for the shaded area of section B. However, their value of 49% for section F, lower than our value of 73% for section C, indicates that the bull has a lower concentration of spermatozoa in the cauda than has the rat. This difference may be related to natural species variation or to differing technical procedures. Tuck et al 2 reported that the spermatocrit of free-flow fluid collected by micropuncture from the rat seminiferous tubules was 13% with a range of 6% to 20%. Levine and Marsh, 3 using techniques similar but not identical to ours, found the spermatocrit in the rat seminiferous tubules, caput, and cauda to be ± (N=30), ± (N=23), and ± (N=19), respectively. The value for the caput is similar to the one noted in this study, while the values for the seminiferous tubules and cauda are different. As discussed above, the spermatocrit is an arbitrary number, and variations in experimental technique and site of collection may explain these discrepancies. Levine and Marsh 3 calculated from these data that approximately 46% of the fluid formed in the seminiferous

6 18 HOWARDS ET AL January 1975 tubules is resorbed before reaching the caput epididymidis, and an additional 23% is resorbed between the caput and the cauda. Our data indicate that 78% of the fluid is resorbed between the sites we punctured in the caput and cauda epididymidis. However, we feel that calculations of fluid resorption from the testis to the caput are not meaningful because, as previously mentioned the spermatocrit of a seminiferous tubule sample is not an accurate index of the in vivo concentration of free cells in that tubule. In fact, Tuck et al 2 found the spermatocrit to be 1% in rat rete testis fluid. Using this number a much higher fractional resorption between the testis and the caput would have been calculated than was computed by Levine and Marsh. The samples prepared for light microscopy were dried and thereby subjected to an osmotic insult which may have created artifacts. If, indeed, significant artifacts are induced by this method, they could be avoided by fixing the specimens in glutaraldehyde or osmium tetroxide, and by utilizing phase microscopy coupled with photography to obtain a permanent record of the appearance of the cells. Nevertheless, all samples were treated identically; consistent differences from area to area thus reflect either actual variation in morphology, or underlying physiologic differences which produce characteristic morphologic changes secondary to the fixation process. As outlined by Orgebin-CrisF and others, 8 9 physiologic changes which have been observed in spermatozoa of various species as they pass along the tubules of the testes and epididymides include: increasing capacity for fertility, changes in motility, progressive dehydration of the cytoplasm, decreased resistance to cold shock, changing metabolism, and variations in membrane permeability. Morphologic changes noted as the spermatozoa travel from the testis to the cauda include caudal migration of the cytoplasmic droplet and changes in the size and shape of the acrosome. 9 ' 13 Since we are not consistently able to see the cytoplasmic droplet or the acrosome, we cannot comment on changes in those structures. We did observe consistent differences between the midpieces of the mature spermatozoa from the seminiferous tubules and those from the epididymides. The mean percentages of abnormal spermatozoa observed in the caput and cauda epididymidis were similar. The approximately 5% abnormal spermatozoa noted after the in vivo aspiration of fluid from individual rat epididymal tubules is significantly lower than the 11% to 19% abnormal spermatozoa reported in studies of slaughtered bulls.t4-t6 SUMMARY Micropuncture techniques developed for the study of renal physiology have been adapted for investigation of the male reproductive tract. Ultramicro specimens were obtained in vivo from the tubules of the rat testis and epididymis. These samples were analyzed for sperm morphology and concentration. The new methods developed to conduct these studies are discussed in detail. The mean in vivo spermatocrits were ± 0.029, ± 0.019, and ± in the seminiferous tubule, caput epididymidis, and cauda epididymidis, respectively. The caudal spermatocrit was significantly higher than the spermatocrit in the caput or seminiferous tubule. The percentages of abnormal spermatozoa in the caput and cauda were 5.5% ±.7% and 4.3% ±.6%, respectively.

7 Vol. 26, No.1 MICROPUNCTURE STUDIES. I 19 REFERENCES 1. Richards AN: Kidney function. Harvey Lect 16:163, Tuck RR, Setchell BP, Waites GMH, et al: The composition of fluid collected by micropuncture and catheterization from the seminiferous tubules and rete testis of rats. Pflugers Arch 318:225, Levine N, Marsh DJ: Micropuncture studies of the electrochemical aspects of fluid and electrolyte transport of individual seminiferous tubules, the epididymis and the vas deferens in rats. J Physiol 213:557, Perey B, Clermont Y, Leblond CP: The wave of the seminiferous epithelium in the rat. Am J Anat 108:47, von Ebner V: Zur spermatogenese bei den Saugetieren. Arch Mikrobiol Anat Entwickl 31:236, Crabo B, Gustafsson B: Distribution of sodium and potassium and its relation to sperm concentration in the epididymal plasma of the bull. J Reprod Fertil 7:337, Orbebin-Crist MC: Maturation of spermatozoa in the rabbit epididymis: fertilizing ability and embryonic mortality in does inseminated with epididymal spermatozoa. Ann Bioi Anim Biochem Biophys 7:373, Gaddum P: Sperm maturation in the male reproductive tract: development of motility. Anat Rec 161:471, Hamilton DW: The mammalian epididymis. Reprinted from the Excerpta Medica Monograph, Reproductive Biology. Edited by H Balin, S Glasser, Amsterdam, Fawcett DW, Hollenberg RD: Changes in the acrosome of guinea pig spermatozoa during passage through the epididymis. Zeitschrift fy Zellforsch 60:276, Bedford JM: Morphological changes in rabbit spermatozoa during passage through the epididymis. J Reprod Fertil 5:169, Bedford JM, Nicander L: Ultrastructural changes in the acrosome and sperm me~branes during maturation of spermatozoa m the testis and epididymis of the rabbit and monkey. J Anat 108:527, Leblond CP, Clermont Y: Spermiogenesis of rat, mouse, hamster and guinea pig as revealed by the "periodic acid-fuchsin sulphurous acid" technique. Am J Anat 90:1,167, Branton C, Salisbury GW: Morphology of spermatozoa from different levels of the reproductive tract of the bull. J Anim Sci 6:154, Amann RP, Almquist JO: Reproductive capacity of dairy bulls. VII. Morphology of epididymal sperm. J Dairy Sci 45:1516, Igboeli G, Foote RH: Maturation changes in bull epididymal spermatozoa. J Dairy Sci 51:1703, 1968