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Prof. Yi-Fei WANG,

Cycloheximide prevents production of arresting, a fraction of 30-50 kDa obtained from seminiferous tubule conditioned medium

Asian J Androl  2004 Dec; 6: 359-364         

Keywords: cycloheximide; arresting; conditioned medium; seminiferous tubule; sperm production; transillumination

Abstract

Aim: To evaluate the effect of a protein synthesis inhibitor cycloheximide on arresting activity in spermatogenesis and sperm count in male rats. Methods: The study used seminiferous tubule (ST) segments from adult rats cultured in vitro with or without cycloheximide to condition culture media, which have been concentrated, size fractioned (30-50 kDa) and administered 7 days to adult rats by intraperitoneal injections. The effects on testicular and epididymal weights, spermatogenesis and epididymal sperm count were determined. Results: The fraction (30-50 kDa), named arresting, obtained from the culture without cycloheximide decreased testicular and epididymal weights (P<0.01) and reduced the epididymal sperm count significantly. Study of the spermatogenic cycle by transillumination showed spermatogenic arrest at stage VII in rats treated with arresting compared to that observed in controls. The length of stage VII in the group receiving the seminiferous tubules culture media with cycloheximide (30-50 KDa CHX-STCM fraction) was similar to control. Conclusion: The difference in the effect may be the result of the presence or absence of arresting, a protein secreted by the tubules.

1 Introduction

Although gonadotrophins and testosterone are required for normal spermatogenesis [1], it is believed that local control factors also participate in its regulation [2].

We have previously demonstrated that a >30 kDa fraction of medium conditioned for 2 days by adult rat seminiferous tubules inhibited inhibin secretion by cultured tubules, arrested spermatogenesis and reduced fertility of male rats [3]. Thereafter, a fraction 30-100 kDa obtained from seminiferous tubules of adult male rats in culture resulted in low epididymal sperm count when administered to adult male rats. This effect was observed without changes in serum LH or testosterone levels [4]. The substance responsible for spermatogenic arrest has been named arresting [4].

Arresting is possibly a secreted protein with 30-50 kDa molecular weight. Protein synthesis should be required for seminiferous tubules for the production of arresting. Since cycloheximide treatment inhibits protein synthesis, production of arresting by seminiferous tubules could be reduced. The present study was designed to explore the effects of 30-50 kDa factor(s) or component(s) of the conditioned medium by adult rat seminiferous tubules in the presence or absence of cycloheximide, a protein synthesis inhibitor, on stages of the spermatogenetic cycle and sperm production in vivo in adult male rats.

2 Materials and methods

Adult intact male Holtzman rats aged 90 days obtained from our own colony were used for all studies described below. Animals were housed under conventional and controlled conditions. The study was approved by the Institutional Review Board (IRB) at the Universidad Peruana Cayetano Heredia.

Details of the preparation of the seminiferous tubule (ST) segments and STCM were published previously [3]. In brief, the rats were killed by decapitation, the testes removed and washed in a culture plate containing Dulbecco's phosphate buffer (DPB). The seminiferous tubules were dissected free of interstitial tissue using fine forceps. The ST segments of each testis were placed in individual wells of a tissue culture plate (Corning, NY, USA) containing 7 mL of sterile Dulbecco's modified Eagle's medium (DMEM). The medium was supplemented with 1 g/L bovine serum albumin (BSA, Fraction V; Sigma, MO, USA), 0.06 g/L penicillin-G and 0.1 g/L streptomycin sulphate. The ST segments were incubated at 32 ℃ in a 95 % air-5 % CO2 humidified environment. After two days of incubation, the media were removed by aspiration and centrifuged at 1000 g for 15 min. The seminiferous tubule culture medium (STCM) were kept frozen at -20 ℃ until collecting a pool to carry out the experiment. The STCM (10 mL) was submitted to ultra filtration through Biomax 50 membranes (MW cut off 50 kDa) (Millipore Corporation, Ma, USA) at 4 ℃ for 4 hours. The <50 kDa supernatant fraction was submitted to a second filtration with Biomax 30 membranes (MW cut off 30 kDa) and centrifuged for 20 min. The retentate 30-50 kDa fraction of STCM (30-50 kDa STCM) was collected in vials containing 1 mL each and stored at -20 ℃.

To establish de novo synthesis of the factor(s) secreted by ST, the STCM was prepared as above. The ST segments were cultured in DMEM supplemented with 1 mg/mL cycloheximide (Sigma, St. MO, USA). This STCM was denoted as CHX-STCM and was also size fractionated. The protein concentration of the STCM fractions was determined by the Bradford's method [5].

The fractions of STCM or CHX-STCM were injected daily to adult male rats by intraperitoneal route in a dose of 0.025 mg of protein per kg body weight (mg protein/kg B.W). Treatment lasted 7 days and rats were killed by decapitation at day 8.

The study included three groups of 6 rats each. The first group received vehicle DMEM (control), the second group received seminiferous tubules culture media without cycloheximide (30-50 kDa STCM fraction) and the third group received the seminiferous tubules culture media with cycloheximide (30-50 kDa CHX-STCM fraction).

One day after the last treatment, the testes and epididymis were excised and weighted. The right epididymis was sectioned and homogenized for spermatozoa head count as described previously [6] with some modifications. Homogenization was performed in 5 mL saline (NaCl 0.9 %). Modifications included refrigeration at 4 ℃ for 24 hours of homogenized epididymal preparation to allow sperm be released from the walls. Number of spermatozoa was determined by counting the homogenisation-resistant spermatozoa heads in an hemocytometer. Sperm count was referred as total count per epididymis.

Assessment of lengths and frequencies of seminiferous tubule stages were made by transillumination in freshly isolated unstained seminiferous tubules of 1) control, 2) group treated with seminiferous tubule culture media without cycloheximide (STCM) and 3) group treated with culture media with cycloheximide (CHX-STCM). Tubules were observed under an inverted stereomicroscope at ×40 magnification as previously described [3]. Seminiferous tubules were assessed in total lengths of about 100 cm. The mean length of each seminiferous stage(s) from control group was considered as 1 and the length of stages in the group treated with culture media without cycloheximide and the CHX-STCM-treated group were assessed relatively to the control group. This is named relative length as described previously [7].

Testes from the control and the arresting-treated groups were also fixed in Bouin's solution for histological study. Tissues were mounted in paraffin and 4 mm sections were cut and stained by the periodic acid-Schiff (PAS)-haematoxylin method. Seminiferous tubules within a cross-section of the testis were examined at ?00 magnification.

Data were analysed using the statistical package STATA (version 8.0) for personal computer (Stata Corporation, USA). Data were expressed as means ±
S.E.M. Statistical analysis was performed by ANOVA test followed by a comparison with the values of the control-treated animals using unpaired student's t-test. The differences with P<0.05 were considered significant.

Intraperitoneal treatment of the 30-50 kDa fraction obtained from the culture media without cycloheximide to intact adult male rats significantly decreased the testicular and epididymal weights (P<0.01). Treatment with CHX-STCM resulted in a significantly higher testicular (P<0.01) and right epididymal weight (P<0.05) than in the group treated with culture media without cyclo-heximide. Left epididymal weight in the CHX-STCM treated group was not different to that obtained in the group treated with culture media without cycloheximide (P:NS) (Table 1). Testicular weight and epididymal weight in the CHX-STCM treated group were closer to weights of the controls, but a significant difference is still observed (P<0.05).

Table 1. Testicular and epididymal weights, and epididymal sperm count in rats treated for 7 days with 30-50 kDa fraction STCM, vehicle (control) or with CHX-STCM. ^bP<0.05 vs control; ^cP < 0.01 vs control. ^eP<0.05, ^fP<0.01with respect to the group treated with culture media without cycloheximide. STCM: Seminiferous tubule culture media without cycloheximide. CHX-STCM: seminiferous tubule culture media with cycloheximide.

Epididymal sperm counts were significantly decreased after treatment with the 30-50 kDa fraction STCM from 279.50±6.09 (mean±S.E.M) to 145.87±14.05 sperm ×10⁶/epididymis (P<0.01). Epididymal sperm counts in rats treated with CHX-STCM were significantly higher (236.00±14.33 sperm ×10⁶/epididymis) than in the rats treated with culture media without cycloheximide (P<0.05), but they were lower as compared to the controls (P<0.05).

Transillumination study demonstrated that treatment with culture media without cycloheximide resulted in arrest of spermatogenesis at stages VII, VIII, IX-XI and XIII-XIV as lengths of these stages were significantly reduced (Table 2). Concomitantly an increase in stages I-V was observed.

Table 2. Lengths of stages (cm) of epithelium seminiferous tubules in adult rats treated with STCM, vehicle (control) or CHX-STCM. (n = 6), ^bP < 0.05 vs control. STCM: Seminiferous tubule culture media without cycloheximide. CHX-STCM: Seminiferous tubule culture media with cycloheximide.

This effect of culture media without cycloheximide was not observed in rats treated with CHX-STCM. Lengths of stages of seminiferous tubules in rats treated with CHX-STCM were similar to the controls (Table 2). Relative lengths showed arrest in spermatogenesis in rats treated with culture media without cycloheximide, whereas rats treated with CHX-STCM showed values similar to the control group (Figure 1).

Figure 1. Relative lengths of stages of seminiferous tubule epithelium in male rats treated during 7 days with vehicle (control), STCM (arresting) or CHX-STCM. STCM: Seminiferous tubule culture media without cycloheximide. CHX-STCM: Seminiferous tubule culture media with cycloheximide. Diamond: Control. Square: STCM. Triangle: CHX-STCM.

Histological picture indicated the presence of mature spermatids at the luminal edge of stage VIII in the controls (Figure 2a), whereas in the group treated with culture media without cycloheximide, the number of cells, mainly late spermatids, was reduced (Figure 2b).

Figure 2a. Photomicrograph control adult rat at stage VIII (×400).

Figure 2b. Photomicrograph of stage VIII of a rat treated with seminiferous culture media (arresting) (×400).

4 Discussion

The purpose of this study was to determine the in vivo effect of intraperitoneal administration of 30-50 kDa fractions obtained from the seminiferous tubule conditioned media of adult male rats on stages of the seminiferous tubules cycle and sperm production in intact adult male rats and whether the effect was inhibited when cycloheximide, a protein synthesis inhibitor, was added to the culture medium.

A reduction in testicular weight, and epididymal sperm count was observed after short-term course of treatment with culture media without cycloheximide. The rapid effect on reduction in epididymal sperm count coincided with reduction of stages VII and VIII. In stage VIII is occurring spermiation [8]. Our data suggested an initial effect of the fraction 30-50 kDa reducing the release of sperm to epididymis (spermiation) and also the lengths of the stages VII and VIII. This confirms previous finding with a fraction greater 30 kDa [3] or a fraction 30-100 kDa [4].

Germ cells are known to regulate Sertoli cells and testicular function possibly through released factor(s) or via cell-cell contact [9, 10]. Our results demonstrate that a soluble factor(s) with 30-50 kDa is able to arrest spermatogenesis when administered to normal adult male rats.

At this time it is difficult to ascertain if 30-50 kDa fraction activity proceeds from germ, Sertoli or myoid cells. It is known that proteases were involved in the regulation of spermatogenesis. Proteases are produced in Sertoli cells but these are mainly located on Sertoli cells membranes and are marginally present in Sertoli cell conditioned supernatants [11]. Then, it is negated that 30-50 kDa fraction activity may be proteases. Myoid cells are also a component of seminiferous tubules. Dysfunction of myoid cells may result in abnormal spermatogenesis [12]. Further studies are required to determine probably cellular source of the 30-50 kDa fraction.

Arresting activity almost disappeared in the STCM obtained in the presence of cycloheximide The results suggest that at least partially the production of arresting is dependent of de novo protein synthesis. In other studies, cycloheximide was unable to abolish the stimulatory effect of FSH on stem cell factor (SCF) mRNA levels in vitro [13]. Cycloheximide treatment partially abolished the effect of arresting on testis and epididymis weight, while it completely blocked its effect on seminiferous tubules. It is possible that critical levels of arresting is necessary for the activity in seminiferous tubules.

The concentration of cycloheximide used in the present study is high, but it is without significant deleterious effect on cell viability.

Seminiferous tubules in culture will release numerous germ cell-derived proteins due either to their secretion or leakage or resulting from some germ cell degeneration [14, 15]. It is well established that some of these proteins will induce autoimmune responses, which, in turn, will cause some degree of orchitis that will then lead to all of the changes reported.

It is possible that our results may be due to an autoimmune effect, but there are some arguments against this. First, in the presence of CHX-STCM the injection is without effect despite that the amount of protein injected was the same. Previously we have also demonstrated that removal of the >30 kDa fraction at day 2 of culture and replenishment with fresh medium and subsequent incubation of the tubules for a further 4 days resulted in a pattern of inhibin secretion similar to that observed in the control group [3]. Therefore, In this experiment the obtained results could be discarded as an immunological effect. Treatment of adult male rats with the 30-50 kDa STCM fraction for periods of 7 days resulted in arrest of spermatogenesis due to significant reduction of frequencies of stages VII and VIII but an increase in the frequency of stages I, II-III and IV-V. This is not observed in the presence of CHX-STCM.

Data on transillumination and histological studies reveal that effect of the 30-50 KDa fraction is at the spermatid level. For such reason lengths of stages VII and VIII (stages of spermiation) were reduced. Once late spermatids are released from the seminiferous epithelium they would be rapidly transported into the epididymis [16]. As epididymal sperm count was significantly reduced with treatment with culture media without cycloheximide, it is suggested that sperm entry to epididymis would be reduced.

It will be useful to demonstrate how the 30-50 KDa fraction is acting to affect progression of spermato-genesis. Effect on apoptosis or in oxidative stress [17] should not be ruled out. Bcl-2 family members are key regulator of apoptosis [18]. In the adult rat testis, both Bcl-w mRNA and protein in Sertoli cells expressed stage specific pattern. Stage-specific patterns of signals for Bcl-w mRNA were observed with more intense signals at stage II-VI and less intensity at stages VII-VIII [18]. Data on inhibin resembles those from Bcl-w mRNA. In fact, inhibin secretion was lower is stages VII-VIII and highest in stages II-VI [19]. The 30-100 KDa fraction from the seminiferous tubules culture media reduced secretion of inhibin in vitro [3]. Therefore, it is suggested that this fraction obtained from the seminiferous tubules in culture may also be acting on apoptosis.

In summary, our data suggest that the activity present in a 30-50 kDa fraction obtained from the seminiferous tubules in culture almost disappears after their incubation with cycloheximide, a protein synthesis inhibitor. The difference in the effect observed in the present study may be the result of the presence or absence of arresting, a protein secreted by the seminiferous tubules.

The project was founded by a grant from the Vicer-rectorate of Investigation at the Universidad Peruana Cayetano Heredia. Authors acknowledge to Eduardo Gazzo and Daniela Parodi by their help at the beginning of the study. Authors thank to Julio Rubio for his technical assistance during the development of the project.

References

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Correspondence to: Dr. Gustavo F. Gonzales. Universidad Peruana Cayetano Heredia. P.O Box 1843. Lima, Peru.
Tel/Fax: +51-1-482 1195
E-mail: iiad@upch.edu.pe
Received 2003-09-26   Accepted 2004-10-08