Reproduction (2001) 121, 239–247 Research A comparative study of sperm production in two species of Australian arid zone rodents (Pseudomys australis, Notomys alexis) with marked differences in testis size E. J. Peirce and W. G. Breed Department of Anatomical Sciences, University of Adelaide, Adelaide, South Australia, 5005, Australia The plains rat, Pseudomys australis, and the spinifex hop- 2.6 ؋ 107 spermatozoa per gram of testis and epididymal ping mouse, Notomys alexis, show marked differences in transit of spermatozoa took approximately 9 days (caput the size of their testes and in the number of spermatozoa 0.8 days; corpus 1.5 days; cauda 6.5 days). In contrast, in within the epididymides. In the present study, the dynam- the hopping mouse, the mean duration of the cycle of the ics of sperm production and the duration of sperm transit seminiferous epithelium was 14 days, the duration of along the male excurrent ducts were compared between spermatogenesis was 56 days and daily sperm production these two species. The durations of the cycle of the sem- per gram of testis was < 1.0 ؋ 107. Epididymal transit of iniferous epithelium, spermatogenesis and sperm transit spermatozoa was completed in about 4 days (caput + were determined by tracking cells using autoradiography corpus < 1 day; cauda 3 days); however, spermatozoa after [3H]thymidine incorporation. Daily sperm produc- may be stored for an additional 1.5–2.0 days in the vas tion was determined from counts of testicular spermatids deferens. These results indicate that, in addition to small after homogenization and further estimates of sperm testes, the hopping mouse shows a low efficiency of transit were obtained by dividing sperm reserves within sperm production, a relatively long duration of spermato- the various regions of the extratesticular ducts by the genesis and rapid passage of spermatozoa through the daily sperm production of the attached testis. In the epididymis, all of which contribute to low epididymal plains rat, the mean duration of the cycle of the semin- sperm counts. These data are considered in relation to iferous epithelium was 11.2 days, the duration of sper- interspecific differences in sperm competition. matogenesis was 45 days, daily sperm production was reflects not only the rate of sperm production, but also the Introduction duration of sperm transit, which in turn is influenced by In males, the number of viable spermatozoa available the length and width of the duct and its storage capacity. for fertilizing oocytes relates to the amount of sperm- Among the conilurine rodents of Australia, there are producing tissue and the rate of sperm production by the marked differences in the size of the testes and the numbers testes, as well as the duration of sperm transit and storage of epididymal spermatozoa. The plains rat, Pseudomys capacity of the extratesticular ducts. Although the size of australis, and the spinifex hopping mouse, Notomys alexis, the testis and especially the volume of the testis occupied are two closely related species of Australian rodents (Watts by the seminiferous tubules play a major role in determin- et al., 1992) which inhabit the arid zone, but nevertheless ing the absolute number of spermatozoa produced, the ef- represent two extremes with respect to testes mass relative ficiency of sperm production is influenced by the duration to body weight (Breed, 1982, 1986, 1997a; Peirce and of the cycle of the seminiferous epithelium and the capac- Breed, 1989). Such differences in the size of the testes may ity of developing germ cells to survive and complete sper- relate, at least in part, to differences in intermale sperm matogenesis. Therefore, high sperm production per unit competition and breeding system (Short, 1979; Harcourt time is achieved by having relatively large testes com- et al., 1981; Kenagy and Trombulak, 1986). The relatively posed of predominantly spermatogenic tissue, a short sper- small testes of the hopping mouse are possibly due to low matogenic cycle, a high efficiency of spermatogenesis, or intermale sperm competition as a result of a single male any combination of these factors. Similarly, the number breeding system. However, the social organization of of spermatozoa within the epididymis and vas deferens hopping mice in the laboratory indicates group living, in which multiple matings at oestrus could sometimes occur (Breed and Washington, 1991). Tests set up to inves- Email: [email protected] tigate this hypothesis did not reveal multiple paternity, © 2001 Journals of Reproduction and Fertility 1470-1626/2001 Downloaded from Bioscientifica.com at 09/23/2021 08:22:21PM via free access 240 E. J. Peirce and W. G. Breed although it was evident in the plains rat (Breed and at each of 4 days 1 h, 5 days 1 h, 15 days, 15 days 1 h and Adams, 1992). In hopping mice, females are more aggres- 16 days 1 h after [3H]thymidine administration. Three hop- sive than males, thus it has been suggested that, in the ping mice were killed at 1 h, two animals were killed at natural environment, the behaviour of the female, rather each of 3 days 22 h, 7 days 1 h, 8 days, 12 days 4 h, than that of the male, minimizes the chances of multiple 13 days 1 h, 14 days 1 h, 15 days and 16 days 1 h, and matings at oestrus (Breed, 1990; Breed and Taylor, 2000). one animal at 26 days after [3H]thymidine administration. In cases where the intensity of sperm competition has in- After the animals were killed, testes were removed, fluenced the numbers of spermatozoa produced in males weighed and fixed by immersion in either TEM fixative of a particular species, it is possible that differences have (3% (w/v) formaldehyde and 3% (w/v) glutaraldehyde) or also evolved in the dynamics and efficiency of sperm pro- Bouin’s solution for 24 h. The tissue was processed using duction and the duration of sperm transit. It has been standard methods, embedded in Paraplast and sectioned at shown that the organization of the testicular seminiferous 5 µm. Sections were hydrated, coated by dipping in Kodak epithelium varies between the plains rat and the hopping NTB-2 nuclear track emulsion (Eastman Kodak, Rochester, mouse (Peirce and Breed, 1987), but how this difference NY) or Illford K2 nuclear research emulsion (Illford relates to the dynamics of sperm production and differ- Australia, Mt Waverley, Victoria) diluted with an equal ences in the number of epididymal spermatozoa between volume of double-distilled water, and dried flat for 24 h at the species is not known. Therefore, in the present study, 20ЊC. The sections were transferred to light-tight black the duration and efficiency of sperm production by the plastic boxes containing desiccant and were stored at 4ЊC testes and the duration of sperm transit along the epi- in a refrigerator. After 4–12 weeks of exposure to the didymides in these two species of Australian rodents were emulsion, batches of slides were developed with Kodak determined. In particular, comparisons were made be- Dektol developer (Kodak Australasia, Coburg, Victoria), tween (i) the duration of the cycle of the seminiferous fixed in 30% (w/v) sodium thiosulphate and stained with epithelium and that of spermatogenesis; (ii) daily sperm either Harris’ haematoxylin or haematoxylin and eosin. production and the efficiency of sperm production; and (iii) the duration of sperm transit along the epididymides. Frequency of cycle stages. The relative frequency of each stage of the cycle of the seminiferous epithelium was deter- mined from plastic-embedded toluidine blue-stained sec- Materials and Methods tions of testes from 12 plains rats and 14 hopping mice. The Animals cycle of the seminiferous epithelium was divided into 11 stages in the plains rat and eight stages in the hopping Sexually mature male plains rats, Pseudomys australis, mouse, according to differences in the acrosomal and nu- and spinifex hopping mice, Notomys alexis, aged 3– clear morphology of the younger generation of spermatids. 15 months, were obtained from colonies bred in the animal The criteria used to determine the stages of the cycle were house of the Medical School, Adelaide University, or from modified from those described by Leblond and Clermont local suppliers. For each species, groups of four or five (1952) and Russell et al. (1990) for the laboratory rat, to take males were housed together in standard rat or mouse into account species differences in the degree of spread of cages under conditions of regulated temperature (18–25ЊC) the acrosome over the nucleus and the elongation and and lighting (12 h light:12 h dark). Animals were fed a diet shape of the spermatid head. Sections of testes were exam- of mouse pellets (Milling Industries, Mile End) supple- ined at either ϫ 400 or ϫ 1000 magnification. Each cross- mented twice a week with mixed seed, fresh apple and sectional profile of a seminiferous tubule was classified carrot, and water was available at all times. All experimen- according to the stage of the cycle observed. For each ani- tal procedures were approved by The Animal Ethics mal, approximately 200 tubular cross-sections from at least Committee, Adelaide University. four testicular locations were scored, a total of 2059 sem- iniferous tubules for the plains rat and 3157 seminiferous Duration of the cycle of the seminiferous epithelium tubules for the hopping mouse. In cases in which cross- Administration of [3H]thymidine and preparation of sections of seminiferous tubules of the hopping mouse con- sections. Plains rats and hopping mice (n = 20 per tained more than one stage of the cycle (see Peirce and species) were given a single i.p. injection of [methyl- Breed, 1987), each stage was assigned a score correspond- 3H]thymidine (specific activity 43 Ci mmol–1; Amersham, ing to the fraction of the cross-sectional profile occupied by Buckinghamshire) at a dose rate of 1.0–1.5 µCi g–1 body that stage.