Effects of Experimental Cryptorchidism on Sperm Motility and Testicular Endocrinology in Adult Male Rats

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1 Journal of Reproduction and Development, Vol. 52, No. 2, 2006 Full Paper Effects of Experimental Cryptorchidism on Sperm Motility and Testicular Endocrinology in Adult Male Rats Longquan REN 1,2,3), Mohamed S. MEDAN 2,4), Mariko OZU 2), Chunmei LI 1,2), Gen WATANABE 1,2) and Kazuyoshi TAYA 1,2) 1) Department of Basic Veterinary Science, The United Graduate School of Veterinary Sciences, Gifu University, Gifu , 2) Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo , Japan, 3) Department of Veterinary Medicine, Yanbian University, Longjing, Jilin province , China, and 4) Department of Theriogenology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt Abstract. The effect of induced cryptorchidism on testicular function and sperm motility was investigated. Bilateral cryptorchidism was created surgically in adult male rats (treated group), and sham-operated rats were used as a control group. Five rats from each group were sacrificed on days 1, 3, 5, and 7 after surgery. The percentage of motile spermatozoa began to decrease 1 day after the operation, followed by an abrupt decline 3 and 5 days later in cryptorchid rats. Furthermore, there were significant decreases in the other sperm motility parameters 5 days after inducement of cryptorchidism. In cryptorchid rats, plasma concentrations of LH, FSH, testosterone, and inhibin B were significantly lower than in the control group 1 day after the operation. Thereafter, plasma concentrations of LH, FSH, and testosterone gradually increased in the cryptorchid rats. On the other hand, plasma concentrations of inhibin B showed a further decline from day 3 after the operation onward. Concentrations of immunoreactive (ir)-inhibin, but not testosterone, in testicular interstitial fluid were remarkably increased until 3 days after surgery in the cryptorchid rats, and declined thereafter. Testicular response to human chorionic gonadotropin (hcg) for testosterone release was decreased in the cryptorchid rats compared with the control rats, indicating that heat stress to testes resulted in a reduction of the activity of Leydig cells and Sertoli cells. These results clearly indicate that heat stress to the testes resulted in a significant reduction of sperm activity within 3 days, and this was followed by changes in testicular endocrine function. Key words: Cryptorchidism, Heat stress, Inhibin B, Sperm motility, Testosterone (J. Reprod. Dev. 52: , 2006) ormal testicular descent is dependent on a series of complex endocrine and mechanical interactions [1]. In most mammalian species, the testis is kept approximately 4 5 C below body temperature. This lower temperature has been shown to be important for normal germ cell Accepted for publication: December 1, 2005 Published online: January 16, 2006 Correspondence: K. Taya ( taya@cc.tuat.ac.jp) development and testicular function [2, 3]. A slight increase in testicular temperature for a short period results in a rapid loss of mature germ cells [4, 5], and the number of primary spermatocytes and spermatids drops rapidly [6]. In the field of animal production, summer sterility is a serious problem especially in male domestic animals, such as pigs, cattle, and stallions. In the rat, testicular damage caused by cryptorchidism is reversible, if the

2 220 REN et al. abdominal testis is surgically descended during early sexual maturation [7]. High temperatures induce an increased synthesis of several proteins [8, 9] and a decreased synthesis of many others [10, 11]. Several enzymatic activities are affected [12 15], and the lipid composition of plasma membranes is modified [16]. However, the effect of temperature on testicular function, especially sperm motility and sperm motion, are unclear. Therefore, in the present study, the sperm quality of male rats was investigated by computer-assisted sperm motility analysis, using an experimentallyinduced cryptorchidism model. In addition, endocrinological changes in testicular function were investigated in the cryptorchid rats. Materials and Methods Animals and procedures Adult male Wistar Imamichi strain rats weighing between 250 g and 350 g and aged 3 4 months were used in this study. Cryptorchidism was produced by pulling the testes into the abdomen and closing the internal inguinal ring with a purse-string suture [17]. Briefly, the rats were anesthetized using ether, and the peritoneal cavity was opened using a lower incision. Cryptorchidism was then induced by dividing the distal gubernaculum on each side, displacing the testis inside the abdominal cavity, and then suturing the inguinal canal. The control group of rats underwent a sham operation. The abdomen was closed, and the location of the testis was verified at postoperative examination. The rats were returned to their cages, with controlled temperature, humidity, and lighting (14 h light, 10 h dark; light on at 0500 h) conditions, and were watered and fed ad libitum with a commercial rat food. They were decapitated at intervals of 1, 3, 5, and 7 days postoperation. Blood samples were collected from each animal in individual heparinized centrifuge tubes, and plasma samples were immediately obtained by centrifugation at 1,700 g for 15 min at 4 C. The plasma samples were stored at 20 C until assayed for FSH, LH, immunoreactive (ir)-inhibin, inhibin B, and testosterone. All procedures were carried out in accordance with the guidelines established by the Tokyo University of Agriculture and Technology. Testicular interstitial fluid The rats were decapitated, and the testicular interstitial fluid was collected before and 0.5, 1, 3, 5, 7, 10, and 14 days after operation. The interstitial fluid was collected using the method described by Maddocks and Setchell [18]. Following collection, the interstitial fluid was stored at 20 C until assayed for ir-inhibin and testosterone. Testicular response to human chorionic gonadotropin (hcg) The ability of the Leydig cells of testes to secrete testosterone after induced cryptorchidism was tested by measuring plasma concentrations of testosterone in response to hcg. One week after operation, 10 IU hcg (2200 IU/mg; Sankyo Zoki Co., Tokyo, Japan) dissolved in 0.85% (W/V) NaCl was injected into the tail vein of the rats at 0900 h. Individual 0.5 ml blood samples were collected from the jugular veins of the rats from each group under ether anesthesia at the following times: 0, 1, 3, 6, 12, 24, 48, and 72 h after administration of hcg. Individual plasma samples were obtained after centrifugation and stored at 20 C until assayed for testosterone. Computer-assisted sperm motility analysis Semen from the cauda epididymidis was collected into 1.5 ml tubes containing 1 ml of modified Tyrode s medium (3 µl semen sample from the cauda epididymis diluted with 1 ml modified Tyrode s medium). Sperm motility was measured by computer-assisted sperm analysis (CASA) using a C-IMAGING C-MEN System (C- IMAGING System, Compix, Inc. Tualatin, OR, USA). Briefly, diluted sperm suspensions were placed in prewarmed slide chambers at a depth of 20 µm. The slides were viewed using an Olympus microscope (Olympus BX50F, Olympus Optical Co., Ltd. Tokyo, Japan) equipped with a 4 dark field optic-stet and video camera (CCD XC77, Sony Corporation, Tokyo, Japan) connected to personal computer. The temperature of the microscope stage was maintained at 37 C throughout the observation using a stage warmer (MP-10DM, Kitazato Supply Co., Tokyo, Japan). CASA was performed using the C-IMAGING C-MEN System operating with the C-IMAGING software. Our CASA system was based upon the analysis of 15 consecutive, digitalized photographic images obtained from a single field. These 15 consecutive

3 CRYPTORCHIDISM IN ADULTS RATS 221 photographs were taken in a time lapse of 0.5 s. Two to three separate fields were taken for each sample. The percentage of motile spermatozoa (%), straight line velocity (VSL, µm/s), curvilinear velocity (VCL, µm/s), linearity index (ratio of the straight line distance to the actual tracked distance), amplitude of lateral head displacement (ALH, µm), and beat frequency of the centroids crossing the average trajectory (BCF, Hz) were determined. Radioimmunoassay of FSH, LH, immunoreactive (ir) - inhibin, and testosterone Concentrations of FSH, LH, ir-inhibin, and testosterone in the plasma were determined by specific RIA for each. The iodinated preparations were rat FSH-I-7 and LH-I-7. The antisera used were anti-rat FSH-S-11 and LH-S-10. The results were expressed in terms of NIDDK rat FSH-RP-2 and LH-RP-2. The intra- and inter-assay coefficients of variation were 3.4 and 5.3% for FSH, and 7.2 and 11.2% for LH, respectively. Plasma concentrations of ir-inhibin were measured as described previously [19]. The iodinated preparation used was 32 kda bovine inhibin, and the antiserum used was rabbit antiserum against bovine inhibin (TNDH-1). The results were expressed in terms of 32 kda bovine inhibin. The intra- and inter-assay coefficients of variation were 8.8 and 14.4%, respectively. Concentrations of testosterone were determined by a double-antibody RIA system with 125 I-labeled radioligands as described previously [20]. The antiserum against testosterone (GDN 250) was kindly provided by Dr. G.D. Niswender (Colorado State University, Fort Collins, CO, USA). The intraand inter-assay coefficients of variation were 6.3 and 7.2%, respectively. Enzyme-linked immunosorbent assay of inhibin B The plasma concentration of inhibin-b was determined using commercially available SEROTEC assay kits (Oxford, United Kingdom) [21]. The intra- and inter-assay coefficients of variation were 4.2 and 8.7%. Statistical analysis All data are expressed as means ± SEM. Oneway ANOVA was performed, and the significance between means was determined by Student s t-test. A value of P<0.05 was considered statistically significant. Fig. 1. Weight of the testis (A), prostate (B), and seminal vesicles + coagulating glands (C) in rats after induced cryptorchidism (solid bar) or sham-operation (open bar). Each point represents the mean ± SEM of five animals. *P<0.05; **P<0.001 compared with the shamoperation value. Results Weights of the testis, prostate and seminal vesicle + coagulating glands The weight of testes in the cryptorchidism group showed a significant decrease from 3 days postoperation compared with the control group (Fig. 1A). By seven days postoperation, the mean weight of the testes in the cryptorchidized rats had diminished to about 50% of the weight in the

4 222 REN et al. Fig. 2. Changes in epididymal sperm motility parameters: percentages of motile spermatozoa (A), straight velocity (B), curvilinear velocity (C), amplitude of lateral displacement mean (ALH) (D), linearity index (E), and beat frequency of centroids crossing the average trajectory (BCF, Hz) (F) after induced cryptorchidism (solid bar) or sham-operation (open bar). Each point represents the mean ± SEM of five animals. *P<0.05 compared with the sham-operation value. control group. On the other hand, there were no significant changes in the weight of prostate and seminal vesicles + coagulating glands between the cryptorchidism and control groups (Figs. 1B and C). Sperm motility The data from the computer-assisted sperm motility analysis is shown in Fig. 2. No sperm was available in the cauda epididymis for motility parameter analysis after 7 days in the cryptorchidism group, and therefore no results are shown for after 7 days. In the cryptorchidism group, the percentage of motile spermatozoa began to decrease 1 day after induced cryptorchidism was induced, although the difference was not significant (Fig. 2A). Three days later, it decreased significantly and reached about 50% of the control group level on day 5 postoperation. Five sperm motility parameters, the mean straight line velocity

5 CRYPTORCHIDISM IN ADULTS RATS 223 Fig. 3. Changes in plasma concentrations of luteinizing hormone (LH) (A), folliclestimulating hormone (FSH) (B), testosterone (C), and inhibin B (D) in rats after induced cryptorchidism (solid bar) or sham-operation (open bar). Each value is expressed as the mean ± SEM of five animals. *P<0.05 compared with the sham-operation value. (VSL), curvilinear velocity (VCL), head oscillation (ALH), linearity index, and beat frequency of centroids crossing the average trajectory (BCF) were unchanged 3 days after the operation (Fig. 2B- F). Thereafter, those five sperm motility parameters were significantly decreased in the cryptorchidism group as compared with the controls on day 5 after operation. Plasma concentrations of LH and FSH Plasma concentrations of LH were significantly lower in the cryptorchid rats on day 1 postoperation than in the control group (Fig. 3A). Plasma concentrations of LH increased gradually in the cryptorchid rats on day 3, and became higher than the control group on days 5 and 7 postoperation, although the differences were not statistically significant. Plasma concentrations of FSH were significantly lower in the cryptorchid rats on day 1 postoperation than in the control group, as was seen for LH (Fig. 3B). Plasma concentrations of FSH increased gradually on days 3 and 5 postoperation in the cryporchid rats, reaching a significantly higher level on day 7 postoperation in comparison with the control group. Plasma concentrations of testosterone and inhibin B There was a significant decrease in plasma concentration of testosterone and inhibin B on days 1 and 3 postoperation in the cryptorchid rats as compared with the control rats (Figs. 3C and D). Plasma concentrations of testosterone recovered on day 5 postoperation in the cryptorchid rats to a level comparable to the controls (Fig. 3C). However, plasma concentrations of testosterone in the cryptorchid rats significantly decreased again 7 days after the operation as compared with the control rats. On the other hand, plasma concentrations of inhibin B showed a further decrease on days 5 and 7 postoperation in the cryptorchid rats (Fig. 3D). Ir-inhibin and testosterone concentrations in testicular interstitial fluid The concentrations of ir-inhibin in the testicular interstitial fluid clearly increased in the cryptorchid rats as compared with the control rats on day 0.5 after the operation, reaching a peak level on day 1 after the operation, followed by an abrupt decline thereafter (Fig. 4A). On days 10 and 14 after the operation, the concentrations of ir-inhibin in the testicular interstitial fluid was significantly low as compared with the control group. On the other hand, there were no significant changes in the concentration of testosterone, except on day 3 after the operation, in the testicular interstitial fluid (Fig. 4B).

6 224 REN et al. Fig. 4. Changes in concentrations of ir-inhibin (A) and testosterone (B) in the testicular interstitial fluid of rats after induced cryptorchidism ( ) or sham-operation ( ). Each value is expressed as the mean ± SEM of five animals. *P<0.05 compared with the sham-operation value. Effect of cryptorchidism on testicular response to hcg In response to hcg, plasma concentrations of testosterone were markedly increased in the control group. However, testicular response to hcg for testosterone release was significantly suppressed in the cryptorchid rats as compared with the control rats (Fig. 5). Plasma concentrations of testosterone increased significantly in control rats compared with saline-treated control rats 1 h after the hcg treatment (Fig. 5A). Plasma concentrations of testosterone in the rats sharply increased 3 h after hcg treatment, and the levels remained high until 12 h later, followed by an abrupt decline by 24 h later. Significantly high levels of testosterone were maintained until 48 h later (Fig. 5A). In the cryptorchid animals, plasma concentrations of testosterone also significantly increased 1 h after treatment and reached a peak level 3 h after hcg treatment. However, the levels of testosterone began to decline after 12 h, with the level declining to the saline-treated control level by 24 h later (Fig. 5B). Fig. 5. Effect of the i.v. injection of the saline or hcg on plasma testosterone levels in sham-operated or cryptorchid rats at 0, 1, 3, 6, 12, 24, 48, and 72 h. Panel A shows the plasma testosterone levels of shamoperated rats injected with hcg ( ) or saline ( ). Panel B shows the plasma testosterone levels of cryptorchid rats injected with hcg ( ) or saline ( ). Each point represents the mean ± SEM of five animals. *P<0.05; **P<0.001; ***P< compared with the control value. Discussion The present study demonstrated that experimentally induced cryptorchidism in adult male rats decreased the epididymal percentage of motile spermatozoa within 3 days, followed by an abrupt decline in five sperm motility parameters by 5 days after the operation. Testicular weight also decreased on day 3 after the operation, whereas the weights of the prostate and seminal vesicles were unchanged. In addition, the present study demonstrated that induced cryptorchidism caused a temporal increase in concentrations of ir-inhibin in the testicular interstitial fluid during the three days after the operation, followed by an abrupt decline thereafter, although circulating inhibin B showed a constant decline after the operation. The results of the present study showed degenerative

7 CRYPTORCHIDISM IN ADULTS RATS 225 changes (vacuoles) in the cytoplasm of Sertoli cells (data not shown). In the hcg challenge test to examine testicular (Leydig cells) functions in detail, cryptorchidism significantly inhibited Leydig cell response to hcg for testosterone release, indicating that the function of Leydig cells is also decreased by heat stress, as are Sertoli cells. However, there were no remarkable changes in the concentrations of testosterone in the testicular interstitial fluid and circulation. These results clearly suggest that heat stress to the testes resulted in acute impairment in Sertoli cells. Although the function of the Sertoli cells was stimulated during the first step of heat stress, it was reduced abruptly thereafter. The increase in Sertoli cell inhibin production at this time is not an isolated function of the Sertoli cell [22], since a similar increase in androgen binding protein (ABP) secretion has been reported 2 days after cryptorchidism [23] and incubation of testicular minces at 37 C also increases ABP production [24]. Likewise, cryptorchid rats showed a significant increase in plasma immunoreactive α- subunit levels 1 week after surgery, and then remained at a lower level than the controls [25]. However, in the present study, the plasma inhibin B level was significantly lower in the cryptorchid rats than in the sham-operation control group starting on day 3 after inducement of cryptorchidism. The weight of cryptorchid testes showed a rapid decrease starting on day 3 after the surgery, and this may be due to the loss of germ cells (data not shown). This is in agreement with a previous study demonstrating that loss of germ cells started on days 3 4 in cryptorchid rats [26]. The morphological changes in cryptorchid testes suggests that the decrease in DNA polymerase beta and gamma activities might be related to the deleterious effects of elevated temperature on spermatogenesis [11]. Also, cryptorchidism exposes the testis to an elevated temperature, which causes an increased incidence of germ cell apoptosis [27]. Cryptorchidism has been associated with degenerative changes in both Sertoli cells and germ cells [28, 29]. Testis energy is provided mostly by glucose, which is the preferred substrate in mammals [30, 31], and most germ cell protein synthesis is under the control of glucose metabolism [32]. Degenerative changes in the cryptorchid testis may be associated with decreased Glut 3 (glucose transporter)-mediated glucose transport in the seminiferous tubules and spermatogonia [12]. In these cells, no ATP is generated through lipid degradation [33], and the low glucose concentration in the adluminal compartment of the seminiferous tubules [34] is not sufficient to feed the glycolytic pathway. Either the germ cells located in the adluminal compartment of the seminiferous tubules are unable to use the lactate produced by Sertoli cells, or production is lowered at high temperatures, as demonstrated in a previous paper [26]. On the other hand, it is known from scrotal animal models that an elevated temperature adversely affects not only testicular functions, but also epididymal functions [35]. The sperm storage functions of the cauda epididymidis, as well as its ability to support sperm survival, are severely impaired at abdominal temperatures, even when this situation is associated with a normal scrotal testis [36]. Elevation of the caudal temperature in vivo is accompanied by a change in protein profiles of the cauda epididymal fluid and water and ion flux across the cauda epithelium [37]. In addition, impaired detoxication of reactive oxygen species and concomitant oxidative stress may be implicated in the biochemical mechanisms responsible for testicular dysfunction in cryptorchidism [14]. Furthermore, decreased intratesticular testosterone, elevated temperature, and high oxidative stress following cryptorchidism probably affects cell viability and spermatid maturation [11, 38, 39]. Therefore, it is possible that the motility of a sperm and the number of sperm decreased in the rats with cryptorchidism. The present study clearly showed early changes in the hormones in the rats with cryptorchidism. There were significant decreases in plasma levels of LH, FSH, inhibin B, and testosterone one day after inducement of cryptorchidism. However, concentrations of ir-inhibin in the testicular interstitial fluid were significantly higher than in the control group until 3 days after the operation. Surgical [40, 41], physical [42 47], and social [48 50] stress can result in an acute decline in plasma levels of gonadotropins, inhibin, and/or testosterone in adult male rats [51, 52]. However, it must be pointed out that the expected reciprocal relationship between plasma FSH and inhibin B levels following early cryptorchidism in mature rats is clear. Plasma concentrations of inhibin B significantly decreased in the cryptorchid rats. The plasma inhibin B level is an accurate marker of the

8 226 REN et al. function of Sertoli cells and also of spermatogenesis [53, 54], indicating that this hormone is useful in the clinical evaluation of male subfertility. Because Sertoli cell function is reflected by plasma inhibin B, the effect of cryptorchidism on Sertoli cell function can thus be evaluated. Low inhibin B levels indicate impairment of Sertoli cell function, which is mainly responsible for the increased FSH secretion in cryptorchid rats. As a direct secretory product of the Sertoli cell, inhibin B is more sensitive than testosterone as a parameter for evaluation of spermatogenesis [53, 54]. In conclusion, the present results demonstrate that induced cryptorchidism in adult male rats remarkably decreased sperm motility and inhibin B secretion. The low inhibin B levels observed in the cryptorchid rats indicate that Sertoli cell function can be affected by heat stress. Moreover, cryptorchidism led to impairment of the function of Leydig cells and decreased sperm production. These results may partially explain changes such as summer sterility in male domestic animals due to heat stress. Acknowledgments We are grateful to the National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, CA, USA, and Dr A. F. Parlow for providing rat LH and FSH RIA materials, and to Dr G.D. Niswender (Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO, USA) for the generous gift of antiserum to testosterone (GDN 250). This work was supported in part by a Grant-in-Aid for Scientific Research (No.03338) from the Japan Society for the Promotion of Science and a Grant-in- Aid for Scientific Research (The 21 st Century Center of Excellence Program, E-1) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References 1. Husmann DA, Levy JB. Current concepts in the pathophysiology of testicular undescent. Urology 1995; 46: Chowdhury AK, Steinberger E. A quantitative study of the effect of heat on germinal epithelium of rat testes. Am J Anat 1964; 115: Davis JR, Firlit CF. The germinal epithelium of cryptorchid testes experimentally induced in prepubertal and adult rats. Fertil Steril 1966; 17: Henriksen K, Hakovirta H, Parvinen M. In-situ quantification of stage-specific apoptosis in the rat seminiferous epithelium: effects of short-term experimental cryptorchidism. Int J Androl 1995; 18: Sailer BL, Sarkar LJ, Bjordahl JA, Jost LK, Evenson DP. Effects of heat stress on mouse testicular cells and sperm chromatin structure. J Androl 1997; 18: Hochereau-de Reviers MT, Locatelli A, Perreau C, Pisselet C, Setchell BP. Effects of a single brief period of moderate heating of the testes on seminiferous tubules in hypophysectomized rams treated with pituitary extract. J Reprod Fertil 1993; 97: Karpe B, Ploen L, Hagenas L, Ritzen EM. Recovery of testicular functions after surgical treatment of experimental cryptorchidism in the rat. Int J Androl 1981; 4: Dix DJ. Hsp70 expression and function during gametogenesis. Cell Stress Chaperones 1997; 2: Biggiogera M, Tanguay RM, Marin R, Wu Y, Martin TE, Fakan S. Localization of heat shock proteins in mouse male germ cells: an immunoelectron microscopical study. Exp Cell Res 1996; 229: Cataldo L, Mastrangelo MA, Kleene KC. Differential effects of heat shock on translation of normal mrnas in primary spermatocytes, elongated spermatids, and Sertoli cells in seminiferous tubule culture. Exp Cell Res 1997; 231: Fujisawa M, Matsumoto O, Kamidono S, Hirose F, Kojima K, Yoshida S. Changes of enzymes involved in DNA synthesis in the testes of cryptorchid rats. J Reprod Fertil 1988; 84: Farooqui SM, Al-Bagdadi F, O Donnell JM, Stout R. Degenerative changes in spermatogonia are associated with loss of glucose transporter (Glut 3) in abdominal testis of surgically induced unilateral cryptorchidism in rats. Biochem Biophys Res Commun 1997; 236: Hoffmann AM, Bergh A, Olivecrona T. Changes of testicular cholesteryl ester hydrolase activity in experimentally cryptorchid rats. J Reprod Fertil 1989; 86: Ahotupa M, Huhtaniemi I. Impaired detoxification of reactive oxygen and consequent oxidative stress

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