Serum Hormone and Cellular Proliferation Changes of Testis in Rats with Experimental Orchitis Induced by Lipopolysaccharide

Journal of Reproduction & Contraception

http://www.RandC.cn

2007 Sep; 18(3):181-186

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Serum Hormone and Cellular Proliferation Changes of Testis in Rats with Experimental Orchitis Induced by Lipopolysaccharide Li-ying XUE1, Xiang-hong ZHANG1, Ling-xiao XING1, Jie LI2, Geng-xin WANG1 1. Hebei Medical University, Shijiazhuang 050011, China 2. Hebei Provincial People’s Hospital, Shijiazhuang 050051, China

Objective To explore the changes of serum male hormone, androgen binding protein (ABP) expression as well as the proliferation of testicular cells in rats with experimental orchitis induced by bacterial lipoplysaccharide (LPS) in vivo and to elucidate the putative mechanism of LPS on spermatogenesis of testis. Methods The serum testosterone (T), luteinizing hormone (LH) levels were detected with magnetic enzyme immunoassay. The expression of proliferating cell nuclear antigen (PCNA) and ABP expression at mRNA level of testis were studied with immunohistochemical staining and in situ hybrydization respectively. Results The serum T level in rats with experimental orchitis was significantly higher than that in the rats of control (P<0.05) and ABP mRNA expression in Sertoli cells of testis was significantly increased (P<0.05) while PCNA expression in seminiferous epithelium in experimental rats significantly was decreased as compared with that of the control (P<0.05). No significant change in serum LH level was seen between experimental orchitis and control groups (P>0.05). Conclusion The serum level of T and ABP expression significantly increased in rats with experimental aspecific orchitis induced by LPS, and at the same time inhibition of cellular proliferation of seminiferous epithelium can be detected, which may be the possible mechanism of male infertility in inflammatory process. Key words: orchitis; LPS; PCNA; ABP; testosterone Orchitis may be caused by many factors, and it was aspecific infection in male genital system. Severe orchitis led possibly to male infertility[1]. In testis there are many kinds of cells for Corresponding author: Jie LI; Tel: +86-311-86265561; E-mail: [email protected] 181

example Leydig cells, Sertoli cells, spermatogenic cells, and macrophages, etc. These cells are activated each other directly or indirectly by their secretion, by which testes are kept in a normal state. When testes are infected by antigen, damaged or exposed to immune reaction, testes lesion and testicular itself unbalance would be seen. Furthermore, some immune complex deposits in testes likely result in spermatogenic handicap[2]. Lipoplysaccharide(LPS) is one kind of bacterial endotoxin and can induce the whole body immune reaction by injected ip, then influnce multiorgan including testes. Therefore, the present study has studied the changes of serum LH and T level with magnetic enzyme immunoassay, androgen binding protein (ABP) biosynthesis at mRNA level and proliferating cell nuclear antigen(PCNA)express in testes with experimental aspecific orchitis induced by LPS, in order to comprehend possibly damaged mechanism of testes with inflammatory factors.

Materials & Methods Experimental animals Twenty healthy adult male Wistar rats weighted about 180-250 g were obtained from Experimental Animals Center in Beijing Military Scientical Academy. Reagents LPS was obtained from Sigma, and immunohistochemical reagents and PCNA antibody were purchased from Beijing Zhongshan Biotechnological Company. In situ hybrydization materials were bought from Wuhan Boster Biological Technology Company. Experimental procedue The rats were divided randomly into control group and experimental group. The experimental group rats were injected ip with saline containing 1mg LPS/kg, once every 2 d (5 times), for 10 d according to the improved way as O’Bryan report[3]. And the rats in control group were treated with pyrogen-free saline instead of LPS. The animals were killed for collection of tissues as described below. At appropriated time intervals after injection, rats were anesthetized with ether and the abdomen was exposed via a midline incision. A sample of blood was collected via inferior vena cava puncture in order to examine serum LH and T level by magnetic enzyme immunoassay[4]. Subsequently testes were removed and put in Bourin’s fixative. After the testes tissue were fixationed, embeded and sectioned as usual methods of light microscope, they were involved to examine the changes of PCNA. Testicular samples used in in situ hybrydization technique were obtained after testicular artery douche with Bourin’s fixative containing 1/1 000 diethypyrocarbonate (DEPC), and were put in Bourin’s for 2 h fixation, then embeded and sectioned for study of ABP mRNA. PCNA immunohistochemical staining Procedue was performed as described by illustrative paper of SP way. 182

In situ hybrydization of ABP mRNA The sections were deparaffinized in xylene and washed in ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. Ag retrieval was performed in pepsine K at 37℃ for 3 min. Slides were then incubated with prehybridization fluid in a humidified chamber at 40℃ for 4 h, and with hybridization fluid to overnight at 42℃, then washed with 2 × SSC for 5 min twice, 0.5 × SSC for 15 min once, 0.2 × SSC for 15 min once. After tissue was blocked at 37℃ for 30 min, sections were incubated with a biotinylated anti-rat Ab in a humidified chamber for 60 min, followed by incubation with SABC for 20 min and with avidin-peroxidase for 20 min. Finally, stained cells were visualized using diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. Positive standard and statistical analysis Brown granules in slides were regarded as positive express, 20 seminiferous tubules stored full were selected randomly in every slide, and followed by countting stained cells or nucleolus. Comparison between two means was performed by t-test (α<0.05).

Results The changes of serum T and LH level Serum T level in experimental animals(3.765±1.120 ng/ml) was significantly increased compared with that of the control rats (5.766 ± 1.141 ng/ml) (P<0.05). While serum LH level in rats with administration of LPS did not change obviously between experimental group and control group (1.136 ± 0.251 mIU/ml vs 0.988 ± 0.123 mIU/ml). In situ hybrydization of ABP mRNA The positive productions were the brown granules and mainly located at the nuclear of Sertoli cells (Figure 1). The expression of ABP mRNA in experimental group (21.59±2.68) was increased distinctly than that of the control (16.80±1.98), and differences between two groups was significant (P<0.05). Immunohistochemistry staining of PCNA The positive productions were brown granules and located at the nuclear of spermatogonium and a part of spermatocytes. On occasion, myroid cells were also stained, however spermatid and spermatozoon were not stained (Figure 2).The positive expression of PCNA in seminiferous epithelium in exprimental rats significantly decreased: after LPS injection, the positive cells in every seminiferous in which only spermatogenous cells were stained were 59 ± 5, it was significant compared with that of the control (80 ± 6) (P<0.05). While the percentage of seminiferous tubules in which spermatogonia were only stained in animals with LPS administration increased in experimental group compared with that in the control (67.3 ± 5.4% vs 58.7 ± 4.4%) (P<0.05).

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Figure 1 In situ hybrydization of ABP mRNA in the control (400 × )

Figure 2 Immunohistochemistry staining of PCNA in experimental group (SP × 100)

Discussion Testis is regulated by hormone that is secreted by hypothalamus-pituitary-gonad, meanwhile is influenced by its internal environment. Many cells in testes and their secretion participate possibly in testicular action, and maintain balance of testicular internal microenvironment. Studies have reported that inflammatory factors or other stimulators could regulate T production by Leydig cells. IL-1 could disorder male hormone secretion by incretion or paracrine at the level of hypothalamus and pituitary in rats exposed to infection or inflammation[5]. After macrophages were depleted by the way of liposome-encapsulated clodronate, T level within that testis was lowered[6]. But Brigham, et al. did not find the same outcome[7]. Rats treated bilaterally with liposomes containing dichloromethylene diphophonate (C12MDP) showed an absence of testicular macrophages, whereas serum LH and T levels were increased[8]. Normally intratesticular T level was higher about 100 times than serum T level because of ABP, therefore, intratesticular T level undulating obviously could be detected when serum T changed lightly. In the present study increased serum T levels in 184

exprimental rats indicated that Leydig cells might be induced directly or indirectly with presence of LPS. Otherwise, difference of serum LH levels between two groups was not significant, and reasons may be that: 1) cytokines secreted by activated macrophages stimulated directly Leydig cells to synthesis and secret male hormone, however had not effect on sexual gland axis; 2) hypothalamus-pituitary-sex gland axis was possibly activated by some inflammatory factors earlier, but when serum T level goes to a certain level, negative feedback would be observed and serum LH level cameback to a normal state; 3) of course, there were other reasons to study more and more. Sertoli cell was one of body cells in testis and could provide essential nutrition and high androgen environment for seminiferous cells. Furthermore, they attend to blood-testis barrier and phagocytosis degeneration cells. If Sertoli cells were damaged, spermatogenesis was certainly impaired too. Riccioli detected that Sertoli cells activated by inflammatory cytokines were able to produce surface adhesion molecules such as ICAM-1, ICAM-2, VCAM-1 and IL-6, and the results suggested the presence of both direct and paracrine mechanisms of interaction between Sertoli and immune-competent cells, possibly involved in the control of immune reactions in the testis[9]. The macrophage activation was accompanied by downregulation of IL-1alpha mRNA expression in Sertoli cells[10]. Increased numbers of CD68-positive macrophages directly (via phagocytosis) or indirectly (via paracrine actions exerted through their secretory products) are involved in the regulation of steroidogenesis, Sertoli cell activity, germ cell survival, and, in consequence, in the pathogenesis or maintenance of infertility states in the human testes[11]. Sertoli cell and ABP deficiency resulted possibly in progressive infertility[12]. Therefore, more or less ABP production might influence hormone level in seminiferouse tubules, and then spermatogenesis was damaged. Increased ABP expression at mRNA level in Sertoli cells in rats treated with LPS suggested that Sertoli cells secreted more ABP at the level of transcription, furthermore, raised androgen level in microenvironment could be detected. PCNA as one kind of cyclin proteins was acidic nucleoprotein with relative molecular weight of 36 000, and was an essential molecule at the initiation of DNA duplication[13]. Salama believed that PCNA is a useful molecular marker for assessing germ cell kinetics[14], and the staining intensity could evaluate the testes spermotogenic function of male infertility[15]. PCNA expression is more at the end of G1 and around the S phase of the cell cycle, especially is maximal around the S phase. And PCNA had been used to marked S phase cells in basic and clinic study, besides that, PCNA staining intensity could evaluate proliferation of the cells. In this study positive productions were located at the nuclear of spermatogonium and primary spermatocyte, and less PCNA expression in epithelium showed that DNA synthesis in spermatogenic cells was decreased and testes were damaged in rats treated with LPS. All in all, in the present study serum T level and ABP produce at mRNA level were raised with experimental orchitis induced by LPS, however the proliferation of testicular 185

cells was disturbed in rats. The outcome possibly showed that spermatogonium proliferation might be hampered by higher T level in seminiferous tubules. Therefore, it was considered that changed microenvironmental androgen level could damage spermiogenesis in aspecific inflammatory testis induced by LPS, which was one of the important reasons why orchitis could result in immunity infertility. This investigation likely availed comprehending putative mechanism in spermatogenesis handicap under condition of inflammatory state, which might provide some theoretic evidence for male immunity infertility.

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