TIMING OF SPERMATOGENESIS IN FOUR NONHUMAN PRIMATE SPECIES*

Transcription

1 FERTILITY AND STERILITY Copyright 1973 by The Williams & Wilkins Co. Vol. 24, No.5, May 1973 Printed in U.S.A. TIMING OF SPERMATOGENESIS IN FOUR NONHUMAN PRIMATE SPECIES* ARNOLD B. BARR, M.D. Department of Medicine, United States Public Health Service Hospital, Staten Island, New York Spermatogenesis is a continuous dynamic process by which primitive germinal cells proliferate and mature into spermatozoa. The duration of spermatogenesis has been determined by the tritiated thymidine technic in many mammalian species, including the rat, 1 mouse, 2 man, 3 and one monkey species, Macacus speciosa. 4 Arsenieva et al. 5 investigated the duration of spermatogenesis in Macacus mulatta by a radiation technic. Several unique characteristics of spermatogenesis make this determination possible. In all mammalian testes, a specific type of spermatogonium is always associated with a given type of spermatocyte and a given type of spermatid, thus forming cellular groupings of constant composition characteristic for a species called "stages of the cycle of the seminiferous epithelium." With the aid of the periodic acid-schiff (PAS) staining technic, which simplifies the precise identification of early spermatids, Clermont 6 identified 12 "stages of the cycle" in the rhesus monkey (see Fig. 1). In vivo, "stages" appear in sequence with time over a particular area in a given seminiferous tubule, forming a "cycle." The "cycle of the seminiferous epithelium" has been defined as "a complete series of the successive cellular associations (or stages) appearing in anyone area of a seminiferous tubule." 1 Thymidine-methyl-H3 is an ideal substance with which to time spermatogene- Received December 11, * Supported by Federal Health Programs Service, Department of Health, Education and Welfare, Grants P and P sis, since it is specifically incorporated into the nuclei of cells undergoing deoxyribonucleic acid (DNA) reduplication in preparation for mitosis or meiosis. 2 In mammalian spermatogenesis, the most advanced cell type undergoing DNA reduplication is the preleptotene ("resting") primary spermatocyte. 2 By doing testicular biopsies at appropriate time intervals after the intravenous injection of this tracer, examining the specimens after radioautographic processing, and identifying the "oldest" labeled cell type, it can be determined how long it takes ea:ch germinal cell type to develop and to determine the length of the spermatogenic cycle in any mammalian species. This technic was used to study the duration of spermatogenesis in four subhuman primates, the squirrel monkey, baboon, rhesus monkey, and African Green monkey. MATERIALS AND METHODS Initial Experiment. One baboon (Papio cynocephalus) (identified as B-1), one rhesus monkey (Macacus mulatta) (Rh-17), one African Green monkey (Cercopithecus sabaeus) (Ag-1), and two squirrel monkeys (Saimiri sciureus) (S-5 and S-7) were selected for study. Their exact ages were unknown. In order to get an estimate of spermatogenic activity, semen was obtained in all animals by electroejaculation by a rectal probe technic. 7 Sperm motility was then determined and sperm counts were done by hemocytometer. If the ejaculate formed a thick plug, the specimen was first incubated for 1 hr. at 37 C., allowing

2 382 BARR Vol. 24 enough of the clot to lyse so that a count could be made. In order to establish the "stages of the cycle of the seminiferous epithelium" for each of these species, a unilateral orchiectomy was performed in the African Green monkey, the baboon, the rhesus monkey, and squirrel monkey S-7. Thin slices were cut from each specimen with a sharp razor blade. Half of the slices were fixed in Zenker's formol, processed, and then stained by the PAS technic; the rest were fixed in Bouin's solution, processed, and stained with hematoxylin. Squirrel monkey S-7 was exsanguinated shortly after this operative procedure. Thymidine-methyl-H3, 0.5 mc./kg., was then injected intravenously into the four remaining animals. Testicular biopsies were done by the method of Heller and Rowley 8 3 hr., 3 days, 23 days, and 30 days later in each animal; orchiectomies were done on the 47th day after injection. The specimens were fixed in Bouin's solution. After histologic processing, radioautography was done using NTB2 emulsion by the dipping technic of Kopriwa and Leblond. 9 The exposure time was 2-3 months. Histologic processing and staining were then done. Final Experiment. Four adult male squirrel monkeys (identified as S-9, S-l1, S-13, and S-15) were chosen for study. Semen was obtained in all animals by electroejaculation. No control testicular biopsies were done. Each animal was injected intravenously with thymidinemethyl-h3, 0.8 mc./kg. Testicular biopsies were then done according to the following schedule: S-9, left biopsy at 4 days; S-15, right biopsy at 4 days; S-9, right biopsy at 10 days; S-l1, left biopsy at 10 days; S-l1, right biopsy at 11 days; S-9, S-l1, S-13, left orchiectomies at 31 days; S-9, S-l1, S-13, right orchiectomies at 32 days. The specimens were fixed, processed, and examined by the technics described elsewhere in this report. Examination of the Specimens. The nomenclature of the germinal cell types and the identification of each "stage ofthe cycle" used here are those described previously by Clermont 6 (see Fig. 1). When the processed slides were examined, a cell was called "labeled" if four or more silver grains overlay its nucleus. Examples of labeled cells can be seen in Figs. 2 and 3. The only notation made in the tables and charts in this report is that of the most advanced or "oldest" labeled cell type for each specimen. This determination required judgment, because the label was usually "weakest" at this point in time and often only a few tubules of the total number in the section in that particular stage contained labeled cells. As an example, in the specimen from squirrel monkey S-5 obtained 3 days after tritiated thymidine injection, the oldest cells labeled were clearly the zygotene primary spermatocytes in Stage 11. However, only about a third of the tubular cross sections in Stage 11 contained weakly labeled zygotene primary spermatocytes; most of the zygotene spermatocytes in Stage 10 were strongly labeled (many more silver grains per nucleus than over the zygotene spermatocytes in Stage 11). In most cases, it was assumed that the labeled "wave front" had progressed to the midpoint of the alloted time span for a particular stage. No attempt was made to more precisely estimate this point in time because of the relatively small number of tubules in most of the testicular biopsies. RESULTS Determination of the "Stages of the Cycle of the Seminiferous Epithelium." PAS-stained sections from control testes for each of the four monkey species were examined. The cellular associations of specific types of late spermatogonia, spermatocytes, and spermatids ("stages of the cycle of the seminiferous epithelium") were for all practical purposes identical in

3 May 1973 TIMING OF SPERMATOGENESIS 383 ~ o~~, 9 10 II FIG. 1. Drawing illustrating the cellular associations of the 12 stages of the "cycle of the seminiferous epithelium" in the rhesus monkey. Each column numbered with a Roman numeral shows the cell types present in one of the cellular associations found in cross-sections of seminiferous tubules. The cellular associations or "stages of the cycle" succeed one another in time in any given area of seminiferous tubule,.. according to the sequence indicated from left to right in the figure. Following cellular association XII, cellular association I reappears, so that the sequence starts over again. The "stages of the cycle" were identified by means of the first 12 of the 14 steps of spermiogenesis (Numbers 1-14). These steps were defined by the changes observed in the nucleus and in the acrosomic structure (acrosome and head cap are seen closely applied to the surface of the nucleus) in sections stained with the PAS technic. Steps 10,11, and 12 spermatids are depicted as seen from their flat surface and from the side; Steps 13 and 14 are shown as seen from their flat surface only. Associated with Step 14, the residual body (RB) is illustrated. AI, A., represent type Al and A. spermatogonia; B I, B., generations of type B spermatogonia; PI, preleptotene primary spermatocyte; L, leptotene spermatocyte; Z, zygotene spermatocyte; P, pachytene spermatocyte; II, secondary spermatocyte. (The above figure and explanation reproduced from Clermont, Y., AmerJ Anat 126:70,1969, with permission from the Wistar Institute Press and Dr. Clermont.) each of the four species and corresponded exactly to those previously reported by Clermont for the rhesus monkey alone (see Fig. 1). Sperm Counts in Each Animal. The results of the sperm counts are given in Table 1. Semen analyses in squirrel monkeys presented several difficulties that could easily lead to inaccuracies in counting. The volume of the ejaculate was small, averaging about 0.04 cm. 3 The semen immediately coagulated after ejaculation, forming a thick plug that lysed only partially after 1-2 hr. of incubation at 37 C. Monkeys are known to masturbate fre ~ quently. If an animal did this often enough, his sperm counts would be greatly lowered. This could explain in part the great variability in sperm counts. Duration of the "Cycle of the Seminiferous Epithelium" in the Squirrel Monkey. These data are best demonstrated by referral to Table 2 and Fig. 4. It can be seen that it takes between 30 and 31 days (30.5 ± 1/2 days) for the labeled "wave front" to progress from the pre leptotene primary spermatocyte in Stage 7 to the Stage 6 late spermatid; this time interval constitutes exactly 3 "cycles of the seminiferous epithelium." Therefore, the duration of 1 "cycle" is 10.2 ± 0.2 days. The data show that it takes 4 days for the "wave front" to progress from the Stage 7 pre leptotene spermatocyte to the zygotene spermatocyte in Stage 12. The life span of the

4 384 BARR Vol. 24 FIG. 2, A and B. Radioautographed section of a testicular biopsy from squirrel monkey (S-9) obtained 10 days after the intravenous injection of tritiated thymidine. The two photomicrographs are of the same field in two different planes of focus. The tubule shown is in Stage 6; the pachytene primary spermatocytes are clearly labeled. The abbreviations are the same as in Fig. 4. Original magnification x 1000.

5 May 1973 TIMING OF SPERMATOGENESIS 385 FIG. 3, A and B. Radioautographed section of a testicular biopsy from squirrel monkey (S-l1) obtained 31 days after the intravenous injection of tritiated thymidine. The two photomicrographs are of the same field in two different planes of focus. The tubule shown is in Stage 6; the late spermatids are clearly labeled. Labeled spermatozoa in the rete testis were also seen in this specimen. The abbreviations are the same as in Fig. 4. Original magnification x 1000.

6 386 BARR Vol. 24 TABLE 1. Sperm Counts in Semen Specimens of Experimental Animals in Millions per Milliliter* SQUIRREL Animal Specimen Specimen Specimen Specimen B Ag Rh * Abbreviations: 8 refers to squirrel monkey, B to baboon, Ag to African Green monkey, and Rh to rhesus monkey. TABLE 2. Results of Determination of the "Oldest" Labeled Cell Type in the Squirrel Monkey* Time after Animal "Oldest" cell thymidine number labeled 2 hours 8-5 PI (7) 3 days 8-7 Z (11) 4 days 8-9 Z (12) 4 days 8-15 Z (12) 10 days 8-9 P (7) 10 days 8-11 P (7) 11 days 8-11 P (8) 23 days 8-7 E8 (8) 30 days 8-7 L8 (5) 31 days 8-9 RT 31 days 8-11 RT 31 days 8-13 RT 32 days 8-9 RT 32 days 8-11 RT 32 days 8-13 RT * Abbreviations: PI, preleptotene primary spermatocyte (p.s.); Z, zygotene p.s.; P, pachytene p.s.; E8, early spermatid; L8, late spermatid; RT, labeled sperm in the rete testis or epididymis. The numbers in parentheses refer to the stage of the cycle containing the labeled cell. No labeled cells were seen in the 47-day specimens. pachytene spermatocyte is 1 whole cycle or 10 days. Thus, on the reasonable assumption that the life span of the secondary spermatocyte is only a few hours, the estimated life span of the primary spermatocyte is 14 days. The probable duration of spermiogenesis is 30 minus 14 or 16 days. SUGES OF THE CYCLE FIG. 4. Diagram illustrating the position of the most advanced labeled cells within the "cycle of the seminiferous epithelium" at the indicated time intervals after the intravenous injection of tritiated thymidine into squirrel monkeys. The construction of the diagram is similar to that of Fig. 1, except that all spermatogonia other than type B4 have been grouped together as "Stem Cells." The arrows indicate that, following cellular association 12, the "wave front" progresses to Stage 1 again. The stars correspond to the most advanced labeled cells at the indicated time intervals after injection. The wavy lines indicate where mitoses occur. Legend: B4, type B4 spermatogonia; R, L, Z, P, resting (or preleptotene), leptotene, zygotene, and pachytene primary spermatocytes, respectively; II, secondary spermatocytes; S 1 to S 14, spermatids in the indicated steps of spermiogenesis; lumen corresponds more accurately to the rete testis, since few free sperm are seen in the lumen of a tubule. It is possible to extrapolate these data to estimate the total duration of spermatogenesis. The onset of spermatogenesis will be arbitrarily assigned to the spermatogonial cell type that is no longer involved in the "renewal cycle"; 10 in the monkey this is the Bl spermatogonium, which first appears in Stage 10. Clermont's data on the rhesus monkey 6 (see Fig. 1) show that the life span of the three types of B spermatogonia is from Stage 10 in 1 "cycle" to Stage 6 of the next "cycle." Referral to Fig. 2 shows that this time interval is 10.5 minus an estimated 2 days, or about 8.5 days. The total duration of spermatogenesis in the squirrel monkey would therefore be approximately 30.5 plus 8.5, or 39 days. No labeled cells were seen in the 47-day specimens. Duration of the "Cycle of the Seminiferous Epithelium" in the African Green Monkey. The data for the one animal

7 May 1973 TIMING OF SPERMATOGENESIS studied are best demonstrated by referral to Fig. 5. It can be seen that it takes between 30 and 31 days (30.5 ± l/z days) for the "wave front" to progress from the preleptotene primary spermatocyte to the Stage 6 late spermatid. Therefore, the duration of 1 "cycle" is 10.2 ± 0.2 days. Comparison of Fig. 3 with Fig. 4 shows that the results obtained in the African Green monkey are identical to those in the squirrel monkeys, with one exception. After 3 days in the African Green monkey, the label progressed to the Stage 10 zygotene spermatocyte, while after 3 days in the squirrel monkeys the label had reached the Stage 11 zygotene spermatocyte. However, the size of the 3-day biopsy specimen obtained from the one African Green monkey studied was small; by chance very few cross-sections of seminiferous tubules in Stage 11 were found. Thus, the apparent difference between these two species at this particular time interval could be due to a sampling error. No labeled cells were seen in the 47 -day specimen. Duration of the "Cycle of the Seminiferous Epithelium" in the Baboon. The data for the one animal studied are demonstrated by referral to Fig. 6. The results obtained after 3 days, 23 days, and 30 days are identical to those found in the squirrel monkeys after the same time interval. The duration of 3 "cycles" in the baboon is 30.5 ± l/z days, and the duration of 1 "cycle" is 10.2 ± 0.2 days. Duration of the "Cycle of the Seminiferous Epithelium" in the Rhesus Monkey. The data for the one animal studied are presented in Fig. 7. It can be seen that the duration of spermatogenesis is shorter in this animal than in the other 3 species studied. A reasonable assumption would be that the duration of 3 "cycles" in the rhesus monkey is 28.5 ± 1 day, and that the duration of 1 "cycle" is 9.5 ± 0.3 days. Using the same reasoning given under the section on Duration of the "Cycle of the AFRICAN GREEN LUlU I SIll 591 su I 51l130=;:~1 =5 I4~Y-r'...,...-r~'---' I 51 I 5! Izf~ 1 5, i pip I pip I pip I pip I pip I p ~ IIJ' i c\\el~ ~B41" t R Izrl L I L 1 3 : D 1 I I I l' L- ~S~TA~GE~S~DI~T~HE~CY~CL~E ~ FIG. 5. Diagram illustrating the position of the most advanced labeled cells at the indicated time intervals after the intravenous injection of tritiated thymidine into an African Green monkey. BABOON LUMEN ~ III 5 III SIll 5 Il 13::[ 51 15! I I J Izi~1 591 sw I 5 f 1l::;~I::s 14~J_/',..~-r-r-r1 l1l 5 ~I [p j pip I pip I pip I pip I pip ~ IIj' [ C\?L~ i "I B4 t R I R I L I L II 13;~1 zj' ~ ~S~TA~CE~S~DI~T~HE~CY~CL~E ~ FIG. 6. Diagram illustrating the position of the most advanced labeled cells at the indicated time intervals after the intravenous injection of tritiated thymidine into a baboon. RHESUS l 5 III 5 III su I 5 LUlU Il ::5 1l~1=5 ~141~/'..,30r~-r-r-r::rr-' r 5 L 15! Is J ,15 II 11~ [, I pi pi p pip I pip I pip I p Lll' [NL\~ ib4 "t R Izrl L I L I I 13;~1 11' L- ~S~TA~GE~S~DI~T~IE~C'~CL~E ~ FIG. 7. Diagram illustrating the position of the most advanced labeled cells at the indicated time intervals after the intravenous injection of tritiated thymidine into a rhesus monkey. Seminiferous Epithelium" in the Squirrel Monkey and by referral to Fig. 7, the total duration of spermatogenesis, timed from the first appearance of the Bl spermatogonium, is estimated to be 9.5 days minus 2 plus 28.5 or 36 days.

8 388 BARR Vol. 24 No labeled cells were seen in the 47 -day specimen. Some data are available from the work of Clermont and Leblond 6 on the relative duration of each "stage of the cycle" in the rhesus monkey; however, it is felt that this information is not precise enough to further refine the figure given of 9.5 ± 0.3 days for the duration of 1 "cycle." In retrospect, testicular biopsies 28 and 29 days after thymidine injection were needed in this species. DISCUSSION The results obtained demonstrate that the kinetics of spermatogenesis are similar in the four primate species studied. The "stages of the cycle of the seminiferous epithelium" are identical in all. The duration of 1 "cycle" is 10.2 ± 0.2 days in the squirrel monkey (Saimiri sciureus), the baboon (Papio cynocephalus), and the African Green monkey (Cercopithecus sabaeus) and is 9.5 ± 0.5 days in the rhesus monkey (Macacus mulatta). The total duration of spermatogenesis is approximately 39 days in the squirrel monkey, baboon, and African Green monkey, and 36 days in the rhesus monkey. Arsenieva et al. 5 determined the duration of spermatogenesis in the rhesus monkey (Macacus mulatta) by the technic of interrupting mitotic activity with X-ray therapy. They found the length of 1 "cycle" to be 10.5 days. However, it is generally agreed by workers in the field that the tritiated thymidine method used in this report is a more accurate way to time spermatogenesis. 11 Antar,4 working in Clermont's laboratory, investigated the duration of spermatogenesis by the tritiated thymidine technic in Macacus speciosa. By analyzing specimens obtained 3 hr. and 12 days after thymidine injection, the duration of 1 "cycle" was estimated to be 11.6 days and the duration of spermatogenesis 44 days. To date, only an abstract of this work has been published; the reasons for the differences between Antar's findings and our results in a different rhesus species, Macacus mulatta, may become evident when his complete report is published. Although few animals were studied in this experiment, the data for the six squirrel monkeys, the one baboon, and the one African Green monkey are consistent with each other. Data for the rat 12 indicate that the duration of 1 "cycle" is a constant for each member of a species and is not dependent on the age of the animal or on any hormonal influence. All present evidence indicates that data from a few animals are valid for an entire species. However, more rhesus monkeys need to be studied. It is known that squirrel monkeys in the wild are seasonal breeders. The best information on this subject has been compiled by Dumond. 13 Testicular biopsies obtained by him in animals in a wild state during the September breeding season showed active spermatogenesis; during the birth season in February there was no active spermatogenesis. There is one report by Lancaster14 that the testes of rhesus monkeys in the wild are larger during the breeding seasons and become smaller during the birth seasons; there is no confirmation of these findings. However, all available evidence from experienced monkey handlers indicates that spermatogenic activity in monkeys in captivity is constant and is not cyclical. Full spermatogenic activity was proven by biopsy in all monkeys in the present study. The duration of 1 "cycle" in the human was found by Heller and Clermont 3 to be 16 ± 1 days. This is considerably longer than that found in the four subhuman primates studied here. The kinetics of spermatogenesis for the true simians (the orangutan, guerilla, and chimpanzee) have not been studied; it is possible that...

9 May 1973 TIMING OF SPERMATOGENESIS 389 the duration of spermatogenesis in one of these species more closely approximates that in man. SUMMARY The kinetics of spermatogenesis were determined in four subhuman primates, the squirrel monkey, the African Green monkey, the baboon, and the rhesus monkey. The cellular compositions of the "stages of the cycle of the seminiferous epithelium" were found to be identical in all. Using the tritiated thymidine technic, the duration of 1 "cycle of the seminiferous epithelium" in the squirrel monkey, the African Green monkey, and the baboon was found to be 10.2 ± 0.2 days. The total duration of spermatogenesis in the squirrel and African Green monkeys and the baboon, timed from the first appearance of B spermatogonia, was estimated to be 39 days. The duration of 1 "cycle" in the rhesus monkey was found to be 9.5 ± 0.3 days; the duration of spermatogenesis was estimated to be 36 days. Acknowledgments. I wish to thank all of the personnel of the New York University primate colony (LEMSIP), Sterling Forest, New York, for their excellent assistance and cooperation in handling the monkeys; Drs. John MacLeod, Edward Michals, and Martin Dym for editing the manuscript; and Virginia McConnon for typing it. REFERENCES 1. PEREY, B., CLERMONT, Y., AND LEBLOND, C. P. The wave of the seminiferous epithelium in the rat. Amer J Anat 108:47, MESSIER, B., AND LEBLOND, C. P. Cell proliferation and migration as revealed by radioautography after injection of thymidine-h' into male rats and mice. Amer J Anat 106:247, HELLER, C. G., AND CLERMONT, Y. Kinetics of the germinal epithelium in man. Recent Progr Hormone Res 20:545, ANTAR, M. Duration of the cycle of the seminiferous epithelium and of spermatogenesis in the monkey (Macaca speciosa). Anat Rec 169:268, ARSENIEVA, N. A., DUBININ, N. P., ORLOVA, N. N., AND BAKULINA, E. D. A radiation analysis of the duration of the meiosis phases in the spermatogenesis of Macaca mulatta (Russian). Dokl Akad Nauk SSSR 141:1486, CLERMONT, Y., AND LEBLOND, C. P. Differentiation and renewal of spermatogonia in the monkey, Macacus rhesus. AmerJ Anat 104:237, FREUND, M. Liquefaction of guinea pig semen. Proc Soc Exp Bioi Med 98:538, ROWLEY, M. J., AND HELLER, C. G. The testicular biopsy: Surgical procedure, fixation, and staining technics. Fertil Steril17:177, KOPRIWA, B. M., AND LEBLOND, C. P. Improvements in the coating technique of radioautography. J Histochem Cytochem 10:269, CLERMONT, Y. Two classes of spermatogonial stem cells in the monkey (Cercopithecus aethiops). Amer J Anat 126:57, COUROT, M., HOCHEREAU-DE-REVERS, M., AND ORTAVANT, R. "Spermatogenesis." In The Testis (Vol. I) Johnson, A. P., Ed. Academic Press, 1970, pp CLERMONT, Y., AND HARVEY, S. C. Duration of the cycle of the seminiferous epithelium of normal, hypophysectomized and hypophysectomizedhormone treated albino rats. Endocrinology 76:80, DUMOND, F. V. "The Squirrel Monkey in a Seminatural Environment." In The Squirrel Monkey, Rosenblum, L. A., Ed. Academic Press, London, 1968, pp LANCASTER, S. B., AND LEE, R. B. "The Annual Reproductive Cycle in Monkeys and Apes." In Primate Behavior. Field Studies of Monkeys and Apes, DeVore, 1., Ed. Holt, New York, 1965, pp