1 Introduction

2 Materials and methods

2.1 Animals and treatment

Adult SD (Sprague-Dawley) rats (body weight 220-250 g) were provided by the Experimental Animal Center of Jiangsu Province and housed under a 12-hour light/dark cycle with free access to food and water. TU, dissolved in tea seed oil (125 mg/mL), was the product of the Xianju Pharmaceutical Co., Ltd., Zhejiang, China.

A pilot study demonstrated that serum T levels (measured by RIA) of rats prior to and 14 and 27 days after a single TU injection (19 mg/kg) were 5.85, 6.55 and 4.60 nmol/L, respectively. Thus the dose of 19 mg/kg every 15 days was chosen as the regimen to maintain supraphysiological serum testosterone levels. The low dosage and the very large dosage were arbitrarily determined.

Sixty animals were randomly divided into 3 groups of 20 animals each, and were designated as 60-Day Group, 130-Day Group and 250-Day Group. The numerals are the duration of treatment; 250 signifies 130 days of treatment followed by 120 days of rest (without the treatment). Each group was further divided into 4 subgroups (5 animals each) of different treatments, designated as control, T8, T19 and T625. The numerals are the TU doses (mg/kg) used. The controls were injected physiological saline. Injection was given with a micro-syringe (minimum scale 10 L). At the end of treatment (at the end of the rest period in case of 250-Day Group) one testis was removed from each animal. Testis was immediately weighed and immersion-fixed in Bouin's solution for 24 h before beingstored in 70% ethanol^[11].

2.2 Stereological estimation

2.2.1 Section preparation  

Three systematic testicular blocks and 25 m-thick methacrylate-embedded sections stained with hematoxylin were prepared from each testis as previously described^[11].

2.2.2 Spermatid number

Sections were observed using oil lens on a computer monitor and the number of elongated late spermatids (steps 15-19) per unit volume of testis or per testis was estimated with the optical dissector stereological tool^[11]. Eight counting frames (each with an area of 81 m²) were superimposed on each field, and a total of 749 frames were measured and a total of 92 elongated spermatid nuclei counted per animal on the average.

2.2.3 Volume of testicular structures

Test points (the upper left corners of counting frames) hitting the seminiferous tubular lumen, seminiferous tubules (including the basement membrane) or interstitial tissue were counted on the first focusing plane, while counting the spermatids mentioned above. Then the volume fractions (percentage volumes) of these components in testis and their absolute volumes were estimated as previously described^[11].

2.2.4 Tubule diameter and length    

Sections were observed using a 4 objective lens on monitor, and the diameter of the seminiferous tubules and their length per testis were estimated in the same way as previously used for the diameter and length estimation of the epididymal tubules^[11]. An average of 51 tubules were sampled and measured per animal.

2.3 Statistics

3 Results

Nothing special was noted with the animals during the observation period. Upon histological examination, it was found that in one animal from the T19 and one from the T625 subgroups of the 250 Day Group, the testicular volume (0.73-0.84 cm³), the percentage volume of the seminiferous tubules in testis (17.3%-44.3%) and the tubular diameter (181-186 m) were much lower than those of the other animals (1.13-1.66 cm³, 65.2%-84.8% and 247-293 m, respectively). Furthermore in these two animals marked interstitial edema was observed, mainly around the seminiferous tubules, and only spermatogonia (mostly type A) and Sertoli cells were present in the seminiferous epithelium. The spermatogenesis of other animals from the two subgroups appeared normal. Consequently the data of these two animals were discarded.

3.1 Testicular volume

The mean testicular volumes of the three control groups were 1.56-1.62 cm³. In the 60-Day Group, the testicular volumes in T19 and T625 subgroups (83% of control) were significantly reduced compared to the control and T8 subgroups. In the 130 Day Group, the volume in T19 (43% of control) was significantly smaller than each of the other three subgroups. In the 250 Day Group, the testicular size in T19 was recovered after a rest period of 120 days and the size in T625 was significantly smaller than that in the control and T8 subgroups. However the size in T625 of the 250 Day Group was not significantly different from that in T625 of the 130 Day Group (t-test, P>0.05) (Figure 1).

Figure 1. Testicular volume, diameter and length of the seminiferous tubules, and number of late (steps 15-19) elongated spermatids in animals receiving vehicle (empty) and testosterone undecanoate injection  (i.m. every 15 days) at doses of 8 mg/kg (stippled), 19 mg/kg (striped) and 625 mg/kg (shaded). The symbol indicates the pair of data being significantly different (P<0.05, one-way ANOVA in conjunction with the Student-Newman-Keuls method) at the corresponding time point. day 60 and day 130: 60 and 130 days after treatment; day 250: 130 days of treatment followed by a rest period of 120 days without treatment.

3.2 Volumes of testicular structures

The percentage volume of the seminiferous tubule in the testis was 77.4%-80.5% in the three control groups, with no significant differences being tested between control and other treated groups at each time point. The tubular volumes per testis were 1.22-1.28 cm³ in the three controls. In the 60 Day Group, the tubular volume (82% of control) in T19 was significantly smaller than that in the control or T8 subgroup. In the 130 Day Group, the tubular volume (45% of control) in T19 was significantly reduced compared to each of the other three groups.

In the three controls, the percentage volume of the tubular lumen in the testis was 5.6%-7.1% and the luminal volume per testis was 0.09-0.11 cm³. In the 130 Day Group, the percentage volume (38% of control) and the luminal volume (17% of control) in T19 were significantly reduced compared to each of the other three subgroups.

3.3 Tubular diameter and length

In the three controls, the tubular diameter and length per testis were 278-299 m and 17.9-21.0 m, respectively. In the 130 Day Group, the tubular diameter (73% of control) in T19 was significantly smaller compared to each of the other three subgroups; the tubular length (83% of control) in T19 was significantly shorter than that in T625 but not the control (Figures 1 and 2).

3.4 Number of late spermatids

The number of late spermatids per mm³ of testis was 0.14-0.19 million in the three controls. A considerable number of late spermatids could still be seen even in the most seriously suppressed animals (T19 subgroup of the 130 Day Group), but the number was greatly reduced with a numerical density (23% of control) significantly smaller than that in the other three subgroups (Figure 2).

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