Quantitative (stereological) study on the spermatozoal storage capacity of epididymis in rats and monkeys¹

Xiao-Hong WEN, Zheng-Wei YANG

Morphometric Research Laboratory, North Sichuan Medical College, Nanchong 637007, China.

Asian J Androl  2000 Mar; 2: 73-77

Keywords: epididymis; spermatids; spermatozoa; stereology; testis

Abstract

1 Introduction

2 Materials and methods

2.1 Sections

The testes and epididymides were collected from six normal adult rats (SD, body weight 190-275 g) provided by the Animal Center of West China University of Medical Sciences (Chengdu, China). The same testicular sections from 6 normal adult monkeys (Macaca fascicularis; body weight 3.8-7.4 kg) previously used by Yang et al^[5] were employed in this study to estimate the late spermatid number in the testis by means of a different stereology set (see below). Epididymides from these monkeys were also used in this study.

Left or right testis and epididymis were removed and immersion-fixed in Bouins fluid overnight and then stored in 70% ethanol. Organs were weighed and their volumes calculated^[5]. The testis and epididymis were cut into pieces orthogonal to their long axis and then 3 testicular blocks and 5 (monkey) or 3 (rat) alternate epididymal blocks from each organ were sampled systematically. After being dehydrated in ethanol and butanol the blocks were embedded in methacrylate resin (Technovit 7100, Heraeus Kulzer GmbH, Wehrheim/Ts, Germany). One thick (25 m) section was cut from each block using a semi-automatic microtome (RM2145, Leica Instruments GmbH, D-69226 Nussloch, Germany) and stained with hemotoxylin as previously described^[5].

2.2 Stereological Estimation

Length of epididymal tubules Sections were observed using 4 objective lens (NA 0.13, UPlanFI, Olympus) on a video screen at a final magnification of 130 (Figure 1). Fields were systematically sampled with a computer-assisted motorized stage (Sichuan University, Chengdu, and Smart Image Technology, Beijing, in cooperation with Zheng-Wei YANG) with a distance of 1.13-1.56 mm between fields along the X or Y axis. A test system with 20 test points (each with an area of 48 m²) and a frame with an area of 0.36 (monkey) or 0.72 (rat) mm² at the lower left corner of the field was generated and superimposed on the field by a software package (Smart Image Technology, Beijing, in cooperation with Zheng-Wei YANG). Test points hitting the different structures were counted to estimate their volume fractions and then their absolute volumes estimated by multiplying the volume fractions by the epididymal volume. Round or elliptical profiles of the efferent ductules (ductuli efferentes) and the ductus epididymidis (epididymal duct) with a clear lumen were sampled using the test frame and their diameters (short axes) measured. The tubule length was calculated by dividing the total volume of the tubules per epididymis by the cross-sectional area of the tubules as previously described^[5]. An average number of 134 (monkey) or 24 (rat) fields were sampled, 401 (monkey) or 102 (rat) points hitting the lumen counted, and 56 (monkey) or 34 (rat) tubules measured per epididymis.

Figure 1.  Micrographs showing the monkey (A) and rat (B) efferent ductules (1) and ductus epididymidis (2): epididymal fluid full of densely packed spermatozoa. The width of each micrograph is 355 m.

The cells counted in this study included (i) late spermatids in the testis i.e. steps 13-14 (monkey) or 15-19 (rat) spermatids in stages I-VI (monkey) or I-VIII (rat) seminiferous tubules^[7]; the apparently free spermatids or spermatozoa in the tubular lumen were also included in this group, and (ii) spermatozoa in the epididymis.

As previously described, the cell numerical density is estimated by directly counting the nuclei in certain volume of the section according to the optical disector principle (Figure 2) and then the absolute number per organ is estimated in combination with the organ volume^[4-6]. The section was observed on the video screen using a 100 oil immersion lens (NA1.30, UplanFI, Olympus) at a final magnification of 3286 for the epididymis or 2676 for the testis. Fields were systematically sampled with the automatic stage with a space of about 0.75-1.00mm (epididymis) or 0.44 mm (testis) between fields along the X or Y axis. On each field were superimposed a set of counting frames: 2 for monkey epididymis (each with an area of 36 m²), 12 for rat epididymis (area: 48 m²), 6 for monkey testis (area: 168 m²) and 12 for rat testis (area: 81 m²) for convenience of counting. The thick section was optically sectioned along the Z axis with a distance of 0.25 m (epididymis) or 0.5 m (testis) between the focusing planes (optical sections) using the computer-assisted stage. Nuclei within the section of 10 m in depth were counted. The average number of frames i.e. their upper left corners (points) hitting the testicular sections or the epididymal tubular lumen and the average number of the late spermatids (in testis) or spermatozoa (in epididymis) counted per animal were: 75 and 183 for the monkey epididymis, 169 and 148 for the rat epididymis, 862 and 244 for the monkey testis and 2072 and 231 for the rat testis, respectively.

Figure 2.  Three (1, 2 and 3) serial focussing planes (optical sections) focussing down the same field on a monkey (A) and two rats (B and C) epididymal sections with a distance of 2 m between adjacent section planes. : the new nuclei which come into focus in the counting frames when focusing down from plane A to C and therefore should be counted according to the optical disector principle. The area of each counting frame is 36 m² (A) or 48 m² (B and C).

2.3 Cell count

Data were presented as means.

3 Results

The volume of each epididymis was 2.95 cm³ (monkey) or 0.39 cm³ (rat) on the average, which was 16.9% (monkey) or 29.8% (rat) of the testicular volume (Table 1). The epididymal fluid in the ductus epididymidis occupied 78% (monkey) or 81% (rat) of the luminal space in the tubules and 14.7%3.5%(monkey) or 33.5%9.4% (rat) of the epididymal volume. The length of the ductus epididymidis per epididymis was 15.3 m (monkey) or 7.0 m (rat). Efferent ductules were observed in the sections from only 3 (monkey) or 1 (rat) of the 6 epididymides, and their total length was 0.6% (monkey) or 2.9% (rat) of the length of the ductus epididymidis.

The total number of late spermatids per testis was 2902 (monkey) or 179 (rat) million, with 7.16%5.81% (monkey) or 3.48%2.38% (rat) of them being noticed apparently free in the tubular lumen.

The total number of spermatozoa per epididymis was 3235 (monkey) or 241 (rat) million (Table 1). Almost all profiles of the ductus epididymidis were full of densely packed spermatozoa, which, however, were only sparsely seen in the efferent ductules (Figure 1). A total of only 1 (monkey) or 17 (rat) non-sperm round nuclei were observed in cell counting from 1 (monkey) or 5 (rat) of the 6 animals, and only 1 (monkey) or 6 (rat) non-sperm round nuclei were counted with the optical disectors, i.e., the overwhelming majority (more than 99.9% in the monkey or more than 99.3% in the rat) of the cells in the epididymal tubule lumen were spermatozoa.

Table 1. Various results (means).

4 Conclusions

This is the first study to report the absolute number of spermatozoa in the epididymis in comparison with that of late spermatids in the testis. The ratio between the two numbers was estimated to be 1.190.68 (monkey) or 1.340.36 (rat). Considering the duration of stages I-VI (monkey) or I-VIII (rat) of the seminiferous cycle (4.84 days in monkeys^[8] or 8.43 days in rats^[7]), the above results suggested that the total transit time of spermatozoa through the lumen of the seminiferous tubules, efferent ductules and ductus epididymidis, assuming that spermatozoal production (in testis) and transport (in epididymis) was a steady and continual process regulated by certain mechanism, was about 5.4 days (monkey) or 11.4 days (rat) on the average. Frequency of ejaculation may influence the transit time. Interestingly, with a tritiated-thymidine labeling technique and by observation of the labeled sperms first appearing in the ejaculate, such transit time (for the fastest sperms) was estimated to be about 2 days in the chimpanzee^[9].

Only a small percentage of late spermatids were noticed apparently free in the lumen of the seminiferous tubules in the current study, and the situation was the same in previous monkey and human studies^[5,6] (data not presented). This suggested that once late spermatids were released from the seminiferous epithelium they would be rapidly transported into the epididymis. Spermatozoa were sparsely observed in the efferent ductules while the ductus epididymidis was full of densely packed spermatozoa with the latter tubule having a much greater length than the former. This indicated that spermatozoa also passed through the efferent ductules rapidly and the ductus epididymidis was the storehouse for spermatozoa. So, the transit time for spermatozoa to pass through the lumen of the seminiferous tubules and efferent ductules constituted a small portion of the above total transit time and the epididymal transit time should be about 5 (monkey) or 11 (rat) days.

The number of spermatozoa per ejaculate in the monkey (Macaca fascicularis) was around 330 million^[10], or 70-80 million^[11-13], which was less than 5% of the total spermatozoa stored in the two epididymides as estimated in this study, indicating a large spermatozoal storage capacity of the epididymis. A similar occurrence may also be seen in men. In accordance with the duration (6.60 days) of stages I-II of the seminiferous cycle^[7] and the number of late (steps 7-8) spermatids (1153 million per testis) in stages I-II seminiferous epithelium, a pair of testes would yield a total of 2446 million spermatozoa per week, which is much more than the number of spermatozoa (397 million) per ejaculate^[6].The large spermatozoal storage capacity of the epididymis is partly owing to its high concentrating ability, i.e., spermatozoa are very densely arranged in the ductus epididymidis. In the cynomolgus monkey the sperm count was about 155-267 (million) per mL of ejaculate^[14], being less than 5% of the numerical density (9136 million/mL) of spermatozoa in the epididymal fluid as estimated in this study. Similarly in the rat, the sperm count (94 million) per mL of semen yielded by para-chloroamphetamine-induced ejaculation^[15] was also less than 5% of the numerical density (2313 million/mL) of spermatozoa in the epididymal fluid indicated in this study. It may thus be assumed that the epididymal secretion would account for less than 5% of the ejaculated seminal fluid.

The spermatozoal number per epididymis in the adult SD rat estimated with the hemocytometer method^[1] was about 170 million, which was about 70% of the estimate obtained in this study. The difference might be partly attributed to broken-down of sperm heads during the tissue homogenizing process in that study. The sperm count of the caudal epididymal fluid in 12-13 week-old SD rats determined microscopically or by FCM was about 1300 million/mL^[2], which was about a half of the value estimated in this study. Explanation for a part of the difference could be: (i) the epididymal fluid taken out of the epididymis might be relatively diluted compared to the highly concentrated fluid inside the ductus epididymidis, and/or (ii) shrinkage of the epididymal fluid might have happened during fixation or dehydration in this study. It should be noted that the absolute cell numbers obtained in situ in this study by stereological methods were unbiased in methodology.

Using the same monkey sections, the number of the same late spermatids per testis estimated in this study (2902749 million; total number of late spermatids counted per testis: 244) was comparable with a previous estimate (28761767 million; total number of late spermatids counted per testis: 121)^[5]. This served as an accuracy check for the results obtained in this study.

References

[1] McLachlan RI, Wreford NG, Tsonis C, de Kretser DM, Robertson DM. Testosterone effects on spermatogenesis in the gonadotropin-releasing hormone-immunized rat. Biol Reprod 1994; 50: 271-80.
[2] Yamamoto T, Mori S, Yoneyama M, Imanishi M, Takeuchi M. Evaluation of rat sperm by flow cytometry: simultaneous analysis of sperm count and sperm viability. J Toxicol Sci 1998; 23: 373-8.
[3] Gundersen HJG, Bagger P, Bendsten TF, Evans SM, Korbo L, Marcussen N, et al. The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS 1988; 96: 857-81.
[4] Wreford NG. Theory and practice of stereological techniques applied to the estimation of  cell number and nuclear volume in the testis. Microscopy Research and Technique 1995; 32: 423-36.
[5] Yang ZW, McLachlan RI, Bremner WJ, Wreford NG. Quantitative (stereological) study of the normal spermatogenesis in the adult monkey. J Androl 1997; 18: 681-7.
[6] Yang ZW, Wreford NG, Royce P, de Kretser DM, McLachlan RI. Stereological evaluation of human spermatogenesis after suppression by testosterone treatment: heterogeneous pattern of spermatogenic impairment. J Clin Endocrinol Metab 1998; 83: 1284-91.
[7] Sharpe RM. Regulation of spermatogenesis. In: Knobil E, Neill JD, editors. The Physiology of Reproduction. v 1. New York: Raven Press; 1994. p 1363-434.
[8] Fouquet JP, Dadoune JP. Renewal of spermatogonia in the monkey (Macaca fascicularis). Biol Reprod 1986; 35: 199-207.
[9] Smithwick EB, Gould KG, Young LG. Estimate of epididymal transit time in the chimpanzee. Tissue Cell 1996; 28: 485-93.
[10] Akhtar FB, Weinbauer GF, Nieschlag E. Acute and chronic effects of a gonadotropin-releasing hormone antagonist on pituitary and testicular function in monkeys. J Endocrinol 1985; 104: 345-54.
[11] Weinbauer GF, Surmann FJ, Akhtar FB, Shah GV, Vickery BH, Nieschlag E. Reversible inhibition of testicular function by a gonadotropin-releasing hormone antagonist in monkeys (Macaca fascicularis). Fertil Steril 1984; 42: 906-14.
[12] Weinbauer GF, Surmann FJ, Nieschlag E. Suppression of spermatogenesis in a nonhuman primate (Macaca fascicularis) by concomitant gonadotropin-releasing hormone antagonist and testosterone treatment. Acta Endocrinol (Copenh) 1987; 114: 138-46.
[13] Weinbauer GF, Limberger A, Behre HM, Nieschlag E. Can testosterone alone maintain the gonadotropin-releasing hormone antagonist-induced suppression of spermatogenesis in the non-human primate. J Endocrinol 1994; 142: 485-95.
[14] Okamoto M. Annual sperm concentration variation in semen collected by electroejaculation in the cynomolgus monkey. Jikken Dobutsu 1994; 43: 25-31 (Article in Japanese).
[15] Saito TR, Hokao R, Wakafuji Y, Igarashi N, Agematsu Y, Takahashi KW. Para-chloroamphetamine (PCA)-induced ejaculation in aged rats, semen analysis and artificial insemination. Lab Anim 1996; 30: 332-6.

¹Financially supported by the Sichuan Committee of Education.
Correspondence to: Zheng-Wei YANG, Professor/Director, Morphometric Research Laboratory, North Sichuan Medical College, 234 Fujiang Road, Nanchong 637007, Sichuan, China.
Received 1999-11-29     Accepted 2000-02-30