Quantitative (stereological) study of incomplete spermatogenic suppression induced by testosterone undecanoate injection in rats
Zheng-Wei Yang1, Yang Guo1, Li Lin2, Xing-Hai Wang3, Jian-Sun Tong3, Gui-Yuan Zhang4
Research Laboratory, North Sichuan Medical College, Nanchong 637007, China
Asian J Androl 2004 Dec; 6: 291-297
Keywords: Leydig cells; rat; spermatogenesis; spermatogenic cells; stereology; testis; testosterone
Aim: To evaluate the key lesions in spermatogenesis suppressed partially by testosterone undecanoate (TU) treatment. Methods: Adult male SD rats were treated with vehicle or TU (19 mg/kg) injection (i.m.) every 15 days for 130 days. The numbers of all types of cells (nuclei) in the seminiferous tubules and the interstitial tissue were estimated using a contemporary stereological tool, the optical disector. Results: In response to TU treatment, the numbers of non-type B spermatogonia, type B spermatogonia and late elongated spermatids per testis were reduced to 51 %, 66 % and 14 % of the controls, respectively. The conversion ratios from type B spermatogonia to early spermatocytes and pachytene spermatocytes were not significantly affected and the ratios to the later germ cell types fell to 51 % - 65 % of the controls. Less than 1.0 % of immature round spermatids were seen sloughing into the tubule lumen, 4.0 % of elongated spermatids retained in the seminiferous epithelium, and about half of the elongated spermatid nuclei appreciably malformed. Leydig cells were atrophied but their number and the peritubular myoid cell number per testis were unchanged. Conclusion: Double inhibition of spermatogenesis (i.e. inhibition at spermiation and spermatogonial conversion to type B spermatogonia), a scenario seen in the monkey and human following gonadotrophin withdrawal, was not sufficiently effective for a complete spermatogenic suppression in the rat after TU treatment, probably due to ineffective inhibition of the Leydig cell population and therefore the intra-testicular testosterone levels.
It has been well established that testosterone and estradiol-filled implants (TE) treatment in the rats for 12 weeks, which causes slightly supraphysiological circulating testosterone (T) levels that suppress LH (luteinizing hormone) and intra-testicular T (to ~3 % of normal levels) but not follicle stimulating hormone (FSH), induces a complete spermatogenic arrest at the round spermatids: the number of round spermatids per testis is reduced to less than 20 % of control and elongated spermatids are absent . Besides, such treatment for 16 weeks led to a 47 % reduction in the Leydig cell number . Only a partial suppression of the rat spermatogenesis, however, was obtained by testosterone undecanoate (TU) injection for 130 days (i) at a dose of 19 mg/kg every 15 days, where a considerable number of sperm (5.6 % of control) remained in the epididymis (data not reported), or (ii) at a dose of 30 mg/kg every month, where the T levels in the testicular interstitial fluid fell to 7.2 % of control and sperm (8.6 % of control) remained in the epididymis . In the humans, TU treatment resulted in azoospermia or severe oligozoospermia in 299 out of 308 men . However, the combined use of TU and levonor-gestrel at lower doses induced azoospermia or severe oligozoospermia in only 7 of 16 men . While the incomplete suppression of spermatogenesis following T treatment may be primarily ascribed to the partial or inadequate inhibition of testicular T levels, it would be of significance to study the partially suppressed spermatogenesis in terms of the numbers and conversion of different stages of spermatogenic cells. These data would help clarify the mechanism, in terms of spermatogenesis, of failure of induction of azoospermia, a sufficient, although not absolutely necessary, requirement for effective contraception with T or T plus progestin combi-nations. Yet such data, as obtained with contemporary unbiased stereological methods, were lacking.
We recently carried out studies using different doses (8 - 30 mg/kg every 15 days or one month) of TU injection for 60 - 130 days in the rats [3, 6]. Dosages of 30 or 19 mg/kg every 15 days or 30 mg/kg every month for 130 days, which raised circulating T levels by 12 % - 65 % on the average, were the most effective ones in terms of sperm counts measured in the sperm wash obtained by cutting the cauda epididymis into small pieces. The sperm counts for the three dosages were very similar, averaging 5.6 % - 8.3 % of control at the end of treatment, and in no case was azoospermia induced (data not reported). In addition, elongated spermatids (~10 % of the controls), the only testicular cell type whose number was estimated previously, were still developing at 130 days after the treatment (19 mg/kg every 15 days) . Reutilizing the same sample, this study was futher undertaken to estimate the numbers of all testicular cell types including spermatogenic cells, Sertoli cells, Leydig cells and others, thus determining comprehensively the morphological basis for the incomplete suppression of spermatogenesis.
2 Materials and methods
The same testicular sections as we used previously  were re-used in the current study. Briefly, they were obtained from animals (adult male SD rats, 5 in each group) after treatment with the vehicle (control) or TU injection (i.m., 19 mg/kg every 15 days) for 130 days.
2.2 Cell identification and grouping
Spermatogenic cells in the seminiferous epithelium were classified as late elongated spermatids (corres-ponding to steps 15 - 19 spermatids as described by Clermont ), middle stage elongating spermatids (steps 9-14 spermatids ) and early round spermatids (steps 1-8 spermatids ), secondary spermatocytes, middle and late stage primary spermatocytes (mostly pachytene spermatocytes), early primary spermatocytes (prelep-totene, leptotene plus zygotene spermatocytes), type B spermatogonia and other non-type B spermatogonia (Figure 1). Such grouping of cells (nuclei) and their histological identification, except that of the non-type B spermatogonia, were previously described in detail for monkeys  or rabbits . The non-type B spermatogonia in the rats consist of different stages of type A spermatogonia and intermediate type spermatogonia ; they were grouped as one type in the current study due to difficulty in their accurate differentiation in cell counting.
Figure 1. Schematic illustration of spermatogenic cells (nuclei) classified and counted in the current study. Different columns represent different stages of rat seminiferous epithelium, but do not correspond to stages defined previously . The left 7 columns are defined in the current study as pre-stages (before elongated spermatids are released) and the right 3 columns as post-stages (after elongated spermatids are released). Sg: type A spermatogonia; ISg: intermediate-type spermatogonia; BSg: type B spermatogonia; ESc: early primary spermatocytes; Sc: middle and late stage primary spermatocytes; SSc: secondary spermatocytes; ESt: early round spermatids; MSt: middle stage elongating spermatids; LSt: late elongated spermatids.
Cells (nuclei) in the interstitial tissue were readily classified as peritubular myoid cells, Leydig cells and other cells (not including anuclear erythrocytes). The nuclei of the myoid cells are squamous and attenuated in shape, lining the basement membrane of the seminiferous tubules. The nuclei of Leydig cells are relatively large in size compared to other nuclei and often circular or elliptic in shape, but may be rod shaped or even dented. More typically, the Leydig cell nucleus is characterized by coarse granules of chromatin that are uniform in size, distributed evenly along the nuclear membrane and also scattered in the nucleoplasm (Figure 2). Apparently identifiable leukocyte nuclei inside the blood vessels were separately recorded when counted.igure 2. Typical micrographs taken on 25-µm-thick methacrylate-embedded sections of the control (A) and TU-treated (B) rat testes with a ×100 oil lens, showing typical nuclei of various testicular cells. Le, Leydig cell; My, myoid cell; Ser: Sertoli cell; Sc, middle stage primary spermatocyte; ESt, early round spermatid; LSt, late elongated spermatid; LSt', malformed elongated spermatid. Scale bar represents 10 µm.
Nuclei were counted using the contemporary unbiased stereological tool, the optical disector, as we previously described [9, 10]. On the average, 3 046, 381 and 762 disectors (each with size 17 µm × 22 µm × 10 µm) per testis were used to count non-type B spermatogonia, spermatids and other spermatogenic cells, respectively, with a total of 960 nuclei being counted per testis. Detached round spermatids or spermatocytes that were scattered inside the tubule lumen (Figure 3a & b) were separately recorded when counted. For the nuclear counting of Sertoli cells and cells in the interstitium, 1944 disectors (each with size 13 µm × 15 µm × 10 µm) per testis were used and a total of 488 nuclei were counted per testis.
Figure 3. Typical micrographs taken on 25-µm-thick methacrylate-embedded sections of TU-treated rat testis with a ×40 objective lens, showing spermatogenic cell sloughing. Micrograph (A): 2 detached round spermatids (R); (B): a group of detached round spermatids (R) and a group of detached late elongated spermatids (L); (C): a large mass of seminiferous epithelium that appears to be sloughing or bulging into tubule lumen. Note, in the mass, besides seemingly normal pachytene spermatocyte (P) and round spermatid nuclei, there are some darker forms of these nuclei indicating degeneration. Scale bar represents 25 µm.
In TU-treated rats, many other forms, besides detachment or sloughing, of spermatogenic cells (primarily the elongated spermatids) were also observed: retained ones that were located unusually close to the basement membrane compared to the normal  and malformed ones such as pyknotic or disorientated (Figure 4). To have a rough (semiquantitative) idea about the frequency of these cells seen in the seminiferous tubules, all the testicular sections were re-observed and measured in a similar way as we used previously . Briefly, 30 (per testis) round or approximately round seminiferous tubule profiles with a clear lumen were sampled using a rectangular sampling frame and the presence or absence of the cells was recorded and their number counted in the tubules.
Figure 4. Typical micrographs taken on 25-µm-thick methacrylate-embedded sections of TU-treated rat testis with a×100 oil lens, showing malformed elongated spermatid nuclei. 1, retained; 2, pyknotic; 3, curviform; 4, disorientated (inside out). Note that most of elongated spermatid nuclei are malformed (cf. Fig. 2A for normal forms). Scale bar represents 10 µm.
2.4 Statistical analysis
Data in the text and Table 1 are shown as mean±SEM (n = 5). Statistical difference between groups was detected with the t-test and significance set at P < 0.05.
In response to TU treatment, the testicular volume and absolute volume of the interstitial tissue were significantly reduced to 43.2 % and 38.0 % of the controls, respectively (Table 1). Leydig cell nuclei became smaller in size and their numerical density in the testis increased significantly to 208 % (there appeared to be more of them on sections, see Figure 2), but their total number per testis was unchanged (Table 1). The peritubular myoid cell number per testis was unchanged either, while other cells (nuclei) in the interstitium dropped significantly to 63.7 %. Of these cells other than Leydig cells and myoid cells, leukocytes accounted for about 0.37 % (control) and 0.12 % (treated group) only.
Table 1. Volumes of testicular structures and number of testicular cells (mean ± SEM).
Non-BSg: non-type B spermatogonia; BSg: type B spermatogonia; ESc: early primary spermatocytes; Sc: middle and late stage primary spermatocytes; SSc: secondary spermatocytes; ESt: early round spermatids; MSt: middle stage elongating spermatids; LSt: late elongated spermatids. bP < 0.05 compared to control (unpaired t-test).
Spermatogenic cell numbers, as percentages
of control, decreased to 51.3 % in non-type B spermatogonia, 65.7 % in
type B spermatogonia, 84.6 % in early primary spermatocytes (P >
0.05), 71.2 % in middle and late stage primary spermatocytes, 45.7 % in
In terms of conversion from progenitor cells to daughter cells, the conversion ratio from non-type B spermatogonia to type B spermatogonia, from type B spermatogonia to early spermatocytes or from early spermatocytes to middle and late stage spermatocytes was not significantly changed, but the ratios to the later germ cell types were significantly decreased: to 65.4 % of control from the middle and late stage spermatocytes to round spermatids, 50.7 % from round spermatids to elongating spermatids and 56.9 % from elongating spermatids to elongated spermatids (Table 1).
In control animals, only one detached round spermatid was counted inside the tubule lumen, accounting for 0.08 % of the total number of round spermatids. In TU-treated animals, about 0.58 % of round spermatids and 0.15 % of pachytene primary spermatocytes were detached.
With respect to the epithelial appearance evaluated with randomly sampled seminiferous tubules, 73.7 % ± 2.1 % of the tubule profiles were in the pre-stages (Figure 1) in TU-treated animals, not significantly different from the control (75.3 % ± 2.5 %). In control animals, few detached, retained or pyknotic spermatids were observed. In the TU-treated group, (i) the total number of elongated spermatids per pre-stage tubule was 50.8 ± 13.5 (30 - 102). (ii) An average of 2 (1 - 9) detached round spermatids per tubule were observed in 12.7 % ± 2.9 % of all tubules, accounting for 0.86 % ± 0.27 % of the total elongated spermatid number. Large masses of the seminiferous epithelium that appeared to be sloughing or bulging into the tubule lumen (Figure 3C) were observed in 8.3 % ± 4.5 % (0 - 23.3) of all tubules. (iii) Retained elongated spermatids were seen in 58.2 % ± 7.8 % of the pre-stage tubules and 26.7 % ± 9.0 % of the post-stage tubules. An average of 2.7 (1 - 8) retained elongated spermatids were observed in these tubules with the retained spermatids accounting for 4.0 % ± 0.8 % of the total elongated spermatids in all the tubules. (iv) Numerous appreciably malformed elongated spermatid nuclei, including retained, pyknotic or disorientated ones (Figure 2B and Figure 4), were observed, accounting for 50.0 % ± 11.3 % of total elongated spermatids.
The present study demonstrated that, in response to the TU injection in the rat, non-type B (type A and intermediate type) spermatogonial division and conversion to type B spermatogonia was suppressed in view of the significant reduction in the numbers of both cell types. The larger (although non-significant) numerical ratios between type B and non-type B spermatogonia and between early spermatocytes and type B spermatogonia in the TU-treated group might be the result of an impairment in the division and conversion of both type B spermatogonia (mitosis) and primary spermatocytes (meiosis). Probably, not all continually developed type B spermatogonia and primary spermatocytes proceeded to the mitotic and meiotic processes, respectively, and those left were removed (e.g. by apoptosis) at a relatively slower speed. Impairment in meiosis was also supported by the finding that the number of secondary spermatocytes, an indicator of meiotic dynamics, was decreased by 82 % while the number of middle and late stage primary spermatocytes decreased by 29 % only.
Following the TU treatment, numbers of round, elongating and elongated spermatids decreased significantly and progressively. This pattern of progressive reduction of advanced (adluminal) spermatids, as was also observed following TE treatment , might be partly the result of immature spermatids sloughing off the seminiferous epithelium. Detachment of round spermatids from the seminiferous epithelium was found to be a prominent lesion 6 weeks after T withdrawal . It was also observed in the present study that single or small groups of detached round spermatids were seen in 13 % of tubules and large mass of seminiferous epithelium appeared to be detaching in some tubules as well. With the sloughing of round spermatids, sloughing of immature elongated and elongating spermatids must have occurred concomitantly. The fact that detached round spermatids accounted for less than 1 % of total round spermatids would not necessarily suggest that few round spermatids sloughed off. Very few elongated spermatids are seen free in the tubule lumen according to our experience in the normal monkeys, humans, rabbits or rats, although elongated spermatids are constantly maturing in large numbers. So mature spermatids, and perhaps round spermatids, once released or sloughing into the lumen, would soon flow into the epididymis .
Spermatid degeneration observed in the present study or apoptosis observed previously  was likely another factor for the reduction of spermatid numbers. Half of elongated spermatid nuclei after TU treatment appeared morphologically abnormal. The analysis of epididymal sperm wash showed that the percentage of malformed sperms was 26 times more in the treated than in the control groups and the percentage of motile sperm was only 8 % of control (data not reported). So a dramatic reduction in the number of step 8 round spermatids compared to that of step 7 spermatids [1, 13] might indicate cessation of conversion of step 7 to step 8 spermatids , and/or loss of relatively more step 8 spermatids resulting from spermatid degeneration or apoptosis  and spermatid sloughing.
Despite multi-stage spermatogenic lesions described above, spermatogenesis was not completely arrested at any stage after the TU treatment. This is in contrast to the TE treatment that resulted in complete depletion of elongated spermatids . The incomplete inhibition of spermatogenesis may well be due to the insufficient withdrawal of testicular T (refer to the Introduction section). In response to the TE treatment in the rat, the cytoplasm of Leydig cells was markedly atrophied and the cell number decreased by 47 % . In the present study, however, the Leydig cell number was unchanged although the cells were atrophied to some degree, a considerable indirect evidence of an inadequate withdrawal of intra-testicular T levels. So why could the TE implants obtain a more profound inhibition of spermatogenesis than the TU injection? The reasons might be that (i) implants achieves a more stable circulating T levels and therefore a more persistent feedback inhibition of LH than TU injection, and/or (ii) combination with estradiol enhances the inhibition of the Leydig cell population. The later speculation might be of importance and needs further study, for it has been speculated that exogenous T in combination with an estrogen or progestin may be a potentially better regimen for a hormonal contraceptive  while the theoretical basis was unclear.
Following gonadotrophin withdrawal in the monkeys and humans, we previously found that the key lesion was the double inhibition of spermatogenesis, i.e. the spermatogenic process was primarily suppressed at both its starting point (conversion from type A to type B spermatogonia) and end point (releasing of elongated spermatids, i.e. spermiation) [11, 12]. Later studies in the monkeys and humans confirmed the findings [15, 16]. It was further shown in the rats that spermiation failure is a major contributor to early spermatogenic suppression caused by T and/or FSH withdrawal . Double inhibition of a continual developmental process at its two ends should be an effective inhibition of the process and the effectiveness of the double inhibition may well determine the efficacy of a hormonal contraceptive [11, 12]. In the current rat study, the double inhibition of spermatogenesis was not effective enough at both ends of the spermatogenic process. Retaining of elongated spermatids, although present, was insufficient, and a considerable number of type B spermatogonia remained. This was probably due to the inadequate inhibition of testicular T and failure of sufficient or significant inhibition of FSH. As a result, some spermatozoa kept developing and releasing into the epididymis.
The study was supported by grants from the Sichuan Youth Foundation of Science and Technology (Chuan Ke Ji  2) and the Sichuan Committee of Family Planning (#99-4-2), and by a ?th Five-Year?National Key Grant of Science and Technology (969040401).
RI, O扗onnell L,
Meachem SJ, Stanton PG, de Kretser DM, Pratis K, et al.
Identification of specific sites of hormonal regulation in spematogenesis
in rats, monkeys, and man. Recent Prog Horm Res 2002; 57: 149-79.
to: Dr. Zheng-Wei Yang,
Professor & Director, Morphometric Research Laboratory, North Sichuan
Medical College, 234 Fujiang Road, Nanchong, Sichuan 637007, China.