Optimization and Standardization of Male Gonad Weight Determinations in Rats

Transcription

1 TOXICOLOGIC PATtIOLOCY lssn: Copyright by the Society of Toxicologic Pathologists VOl. 10, No. 1,1982 Printed in U.S.A. Optimization and Standardization of Male Gonad Weight Determinations in Rats A. G. ADKINS, B.A., M.B.A., C. L. ALDEN, D.V.M., M.S., AND R. L. KANERVA, B.A., M.S. Miami VaIIey Laboratories, The Procter and Gamble Company, Cincinnati, OHIO ABSTRACT In order to develop an optimal method for measuring male gonad weight, one that is both statistically dependable (i.e. not subject to wide fluctuations within similarly treated groups) and appropriate with respect to end uses of data, testis, epididymis, and epididymal fat pad weights were obtained from normal adult male rats and subjected to statistical analyses. The results underscore the necessity for omitting the epididymal fat pad from gonad weight measurements since there appeared to be no relationship between its weight and the weight of either the testis or epididymis. Furthermore, the ~nclusion of epididymal fat resulted in large variation in total unit weight. An interdependent relationship was shown for testis and epididymis weights, indicating it may be appropriate to report a combined value for these entities, though this decision should be based on the intent of the study. A significant saving in time was effected by obtaining the weight of the testis alone. In studies which require male gonad weight assessment, therefore, the testis, excluding epididymis and epididymal fat, should be weighed to maximize the combined factors of time/cost effectiveness and scientific validity. I NT RO D UCTIO N Studies involving male gonad toxicity are frequently reported in the literature using gonad weight as one parameter in the evaluation of function. Agents such as busulfan (1,2), WIN (2), hydroxyurea (3), cadmium (4), and caffeine (5), have been shown to have degenerative effects on the rat testis with a corresponding and significant loss of organ weight. A description, however, of what is includ~d in such weights is often lacking in the literature. Furthermore, an investigation into male gonad weight de~erminations asperformed by several independent laboratbk$fs revealed wide variation in the interpretation of the word gonad, ranging from the testis alone to a group of tissues including the testis, epididymis, and epididymal fat pad. This technique variation among laboratories together with the lack of specificity in published reports precludes inter-lab comparability of toxicity study results. More importantly, values may be generated which have the poten- 33 tial of maskifig or mimicking real changes in the testis. Clearly, a method for obtaining male gonad weights which maximizes data utility and minimizes statistical variation is needed. It was the intent of this study to determine the inter-relationships inherent among weights of the testis, epididymis, and epididymal fat pad in normal, mature male rats in order to define the most pertinent method for determining male gonad weight. Throughout this report the term gonad unit is used collectively to refer to the testis, epididymis and epididymal fat pad. MATERIALS AND METHODS Fifteen male retired breeder Sprague-Dawley rats were used. The animals ranged in weight from 440 to 590 g (mean = k g) and were approximately 47 wk. old. The acclimatization period was 4 days during which each rat was housed individually in a stainless steel, wire-bottomed cage with unlimited access to tap water and a dry com-

2 34 ADKINS, ALDEN, AND KANERVA TOXICOLOGIC PATHOLOGY mercial diet. The light cycle was 12 hrs of light and 12 hrs of darkness; temperature was 72 F -C 10 F and humidity 50% 2 10%. On the fourth day after arrival all rats were weighed, anesthetized by ether inhalation, and exsanguinated via the posterior vena cava. Removal of Gonad Units. Immediately after exsanguination the testes were gently manipulated from the scrotum through the inguinal canal and into the abdomen. Figure 1 shows the exposed testes after displacement into the abdominal area and anatomical orientation of relevant organs/tissues. Each unit was removed by first excising the mesenteric attachment to the tunica vaginalis, or the inner lining of the scrotum. The vas deferens was then severed at the tail of the epididymis and the entire unit freed by excising the internal spermatic artery and vein at the point of their junction with the epididymal fat pad (Figure 2). The unit thus obtained consisted of the testis, epididymis, and epididymal fat. Next note the separation of epididymal fat from the epididymis, the line of demarcation between the two becqming easily distinguishable upon examination (Figures 3 and 4). The epididymis was then separated from the testis as shown in Figures 5 and 6. Each of the three components of the gonad unit was rinsed in 0.9% saline, gently blotted on a paper towel, and weighed; RESULTS Total body weights and weights of each component of the right and left gonad unit are summarized in Table I. The coefficient of variation (standard deviation/mean) makes it possible to compare variability among data groups having different means. Based on an average of right and left corresponding tissues, these coefficients of variation are 12.5%, 7.5%, and 30.3% for the testis, epididymis, and epididymal fat pad, respectively. The coefficient of correlation (Table 11) is used to describe how two measures vary together; however, it does not imply causation. As the testis weight varied from one animal to the next, the epididymal weight changed in a positive, linear fashion (r = 0.742; p co.001); however, neither of these two organ weights was related to the weight of the fat pad (r = and for the testis and epididymis, respectively; p <.001). Furthermore, fat pad weight was positively correlated with total body weight (r = 0.660; p <0.01) while neither the testis nor the epididymis demonstrated a significant positive correlation (r = and for the testis and epididymis, respectively; p = <0.001). This agrees with data obtained by.others who found that for 125-day-old Wistar rats (sexually mature in terms of testis weight and sperm production) the correlation between body weight and paired testes weight was insignificant (r = 0.24; p > 0.05; N = 31) (6). DlscussloN The basic organ of interest when performing male gonad weight determinations for use in toxicity testing is the testis. The present FIG. l-exposed testes (T) after displacement into the abdomen. Note anatomical interrelationships of vas deferens (VD), epididyrnis (E), epididymal fat pad (EFP).

3 Vol. 10, No. 1, 1982 MALE GONAD WEIGHT 35 FIG. 2-The gonad unit [testis (TO, epididymis (E), and epididymal fat pad (EFP)] is removed in toto. The vas deferens (VD) has been transected and the prosector is preparing to transect the spermatic vessels (SV). FIG. 3-The gonad unit resected from the body. The prosector is separating epididymal fat pad (EFP) from the epididymis. study was designed to explore some of the implications involved in the practice of including the epididymis and the epididymal fat pad in these measurements, as is commonly FIG. 4-Epididymis and testicle separated from the epididymal fat pad. done by some independent laboratories at sacrifice. The present results justify the omission of epididymal fat pad weight from such data for two reasons, First, epididymal fat pad weight (approximately 3 times greater than

4 36 ADKINS, ALDEN, AND KANERVA TOXICOLOGIC PATHOLOGY FIG. 5-The testis (T). epididymis (E) is separated from the FIG. 6-The testis (T). epididymis (E) is separated from the the weight of. the testis) is not related to testis weight, but rather, is related to total body weight. The latter relationship has also been found to be true by other workers (7,8). Therefore, including fat pad weight would result in a gonad unit weight which is more reflective of total body weight than of testis weight. This effect could lead to the occasional masking (or mimicking) of test-related changes in the testis. Secondly, the coefficient of variation for epididymal fat pad weights is greater than twice that for testis weights. This indicates a meaningful increase in reproducibility of weight data would be associated with the omission of epididymal fat. The present results do not necessaril3f.iustify the omission of the epididymis from mhle. gonad weight determinations based on statistical evidence alone. Epididymal weights were directly related to testis weights and had a relatively small coefficient of variation. Furthermore, the epididymis is dependent upon androgen production for functional maturation(9) and its weight, in contrast to that of the fat pad, has value as an indicator of testic- TABLE I-Mean Weights and Variabilities of Body and Gonad Unit Segments* Mean Standard Coefficient Tissue Weight Error of of Variation (g) themean (%) Total body Right testis Right epididyrnis Right epididyrnal fat pad Left testis Left epididyrnis Left_ epididymal fat Pad * N = 15 animals. ular function (10). In view of these relationships, the negative implications of including the epididymis in routine male gonad weight determinations would be minimal; however, the appropriateness of including epididymal weight would necessarily depend on the individual study objective and associated time/ cost factors discussed below.

5 Vol. 10, No. 1,1982 MALE GONAD WEIGHT 37 TABLE 2-Correlations among Individual Gonad Unit Segment Weights and Body Weight Organ Relationship Coefficient of Correlation Testis weight-epididyrnis weight * Testis weight-epididyrnal fat pad weight Epididymis weight-epididyrnal fat pad weight Testis weight-body weight Epididyrnis weight-body weight Epididyrnal fat pad weight-body weight * Asterisks denote significance at P < a n = 15 animals. Since it was determined that data from the left side of the body was not significantly different from data from the right side of the body, data representing corresponding right and left weights are averaged. The process of separating the gonad unit into three separate pieces (as described above) prior to weighing took an average of 2 min. per unit (N = 8). This means that if the weights of both testis and epididymis were desired, an additional 4 min. of trimming time per animal would be needed at necropsy. In contrast, it is estimated that the separation of the epididymis (with the fat pad attached) from the testis, in order to isolate the latter for weighing purposes, would take approximately 30 seconds, or approximately 1 min. per animal. In view of the statistical evidence generated by this study and the time associated with the methods presented, it is judged most appropriate to weigh the testis alone as a standard practice whenever weight is to be used as an indicator of male gonadal function in toxicity studies. REFERENCES 1. Debeljuk L, Arimura A, and Schally AV: Pituitary and serum FSH and LH levels after massive and selective depletion of the germinal epithelium in the rat testis. Endocrinology 92:48-54, Gomcs WR, Hall RW, Jain SK, Boots RL Serum gonadotropin and testosterone levels during loss and recovery of spermatogenesis in rats. Endocrinology 93: , Mecklenburg RS, Hetzel WD, Gulyas BJ, Lipstet; MB: Regulation of FSH secretion: Use of hydroxyurea to deplete germinal epithelium. Endocrinology 96~ , Davis JT and Coniglio JG: The effect of cryptorchidism, cadmium and antispermatogenic drugs on fatty acid composition of rat testis. J Reprod Fertil 14~ , Weinberger MA, Friedman L, Farber TM, Moreland FM, Peters EL, Gilmore CE, Khan MA: Testicular atrophy and impaired spermatogenesis in rats fed high levels of the mcthylxanthines caffeine, theobromine, or theophylline. J Environ Pothol Toxicol 1~ , Robb GW, Amann RP, and Killian GJ: Daily sperm production and epididymal sperm reserves of pubertal and adult rats. J Reprod Fertil 54: , Kirtland J and Gurr MI: The effect of different dietary fats on fat cell size and number in rat epididyma1 fat pad. Br J Nutr , a. DiGirolamo M and Mendlinger S: Role of fat cell size and number in enlargement of epididymal fat pads in three species. Am J Physiol 221: , Setty BS and Jehan Q: Functional maturation of the cpididymis in the rat. J Reprod Fertil 49: , Ribelin WE: Atrophy of rat testis as index of chemical toxicity. Arch Pathol 75: , 1963.