CYTOLOGY AND KINETICS OF SPERMATOGENESIS IN THE RABBIT E. E. SWIERSTRA and R. H. FOOTE Department of Animal Husbandry, Cornell University, Ithaca, New York, U.S.A. {Received 21st August 1962) Summary. The cycle of the seminiferous epithelium of the rabbit was divided into eight stages, using as criteria the shape of the spermatid nucleus, the location of the spermatids and spermatozoa in regard to the basement membrane, the presence of meiotic figures and the release of spermatozoa from the lumen. The relative duration (frequency) of Stages 1 to 8 were 27-7, 13-4, 7-3, 11-0, 4-1, 15-7, 12-2 and 8-6%, respectively. Each stem cell (Type A spermatogonium) divided to produce two Type A spermatogonia. One of these was the starting cell for the next genera¬ tion, while the other gave rise to two intermediate-type spermatogonia. Three more spermatogonial divisions followed, producing sixteen primary spermatocytes from one Type A spermatogonium, as is characteristic for the bull and the ram, but unlike the rat, mouse and hamster. It was estimated that only 3-1 spermatids were generated from one primary spermatocyte, suggesting that in the rabbit there is considerable degeneration of spermatogenic cells during the two maturation divisions. INTRODUCTION Since the end of the last century, it has been known that well-defined cellular associations succeed one another in time in any one area of the semini¬ ferous tubules, and that along the tubules a more or less regular pattern of cell populations exists (Brown, 1885; Benda, 1887; von Ebner, 1888). This succession of cellular associations at any one location in the seminiferous tubules led to the concept of the cycle of the seminiferous epithelium defined by Leblond & Clermont (1952b) as that "series of changes occurring in a given area of the seminiferous epithelium between two successive appearances of the same cellular association". The cycle of the seminiferous epithelium has been divided into a number of distinct stages. Leblond & Clermont (1952a, b), Clermont & Leblond (1955) and Oakberg (1956) used the development of the acrosome and head cap as revealed by the periodic acid-Schiff technique to divide this cycle into twelve or fourteen stages. Curtis (1918), Roosen-Runge & Giesel (1950) and Ortavant (1954) divided this cycle into eight stages using as criteria the shape of the spermatid nucleus, the location of the spermatids and spermatozoa in regard to the basement membrane, the presence of meiotic figures and the release of spermatozoa in the lumen of the seminiferous tubule. The number of cycles of the seminiferous epithelium which occur between 309 Downloaded from Bioscientifica.com at 10/07/2021 03:59:08AM via free access 310 E. E. Swierstra and R. H. Foote the time that stem cells (Type A spermatogonia) divide and the time that the spermatozoa derived from these stem cells are free in the lumen of the tubule varies between species. Four cycles of the seminiferous epithelium occur in the rat (Leblond & Clermont, 1952a), the mouse (Oakberg, 1956), the ram and bull (Ortavant, 1959), and six cycles occur in the monkey (Clermont & Leblond, 1959). The number of primary spermatocytes generated from one stem cell (coefficient of efficiency) also depends upon the species. Twenty-four primary spermatocytes are generated by one stem cell in the rat (Clermont & Leblond, 1953) and the hamster (Clermont, 1954), sixteen in the ram (Ortavant, 1954) and bull (Orgebin, Courot & Ortavant, 1958), eight in the guinea-pig (Cleland, 1951) and monkey (Clermont & Leblond, 1959), and apparently four in the duck (Clermont, 1958). Roosen-Runge (1955) observed that degenerating cells occurred regularly during spermatogenesis in the rat. He estimated that the actual number of spermatozoa formed from one preleptotene primary spermatocyte was 22% lower than the expected number, assuming that one preleptotene primary spermatocyte gave rise to four spermatozoa. Similarly, Oakberg (1956) estimated that in the mouse the number of young spermatids was only 87% of what would be expected. Since detailed information on the cytology and kinetics of spermatogenesis was not available for the rabbit, a study was undertaken to investigate these phenomena in this important laboratory animal. MATERIALS AND METHODS Six normal male Dutch-Belted rabbits averaging 2022 g (range 1776 to 2174 g) in body weight and averaging 36 weeks of age (range 32 to 40 weeks) were used in this study. One day prior to euthanasia a semen sample was collected from each rabbit by means of an artificial vagina to get a gross indication of active spermatogenesis. Epididymal spermatozoa were checked for motility and abnormalities at the time the testes were removed. Pieces of tissue from six randomly chosen areas of the testis were fixed in Allen's fixative (Gray, 1958). Histological sections 4 and 8 µ thick were prepared from each area. All slides were stained with the periodic acid-Schiff-haematoxylin technique (pas) (Swierstra, 1962). The cycle of the seminiferous epithelium was divided into eight stages on the basis of criteria which were similar to those used by other workers (Curtis, 1918; Roosen-Runge & Giesel, 1950; Ortavant, 1959). Eighth-thick sections were used. Stage 1. Extends from the absence of spermatozoa in the lumen to the begin¬ ning of the elongation of the spermatids. Stage 2. Extends from the beginning of elongation to the end of elongation of the spermatids. Stage 3. Extends from the end of elongation of the spermatids to the begin¬ ning of the first maturation division of the primary spermatocytes. Downloaded from Bioscientifica.com at 10/07/2021 03:59:08AM via free access Spermatogenesis in the rabbit 311 Stage 4. Extends from the beginning of the first maturation division to the end of the second maturation division. Stage 5. Extends from the end of the second maturation division to the time the spermatid nuclei show a dusty appearance. Stage 6. Extends from the time the spermatid nuclei show a dusty appearance to the time all the spermatozoa have left the Sertoli cells and move towards the lumen. Stage 7. Extends from the beginning to the end of the movement of the spermatozoa towards the lumen. Stage 8. Extends from the time the spermatozoa line the lumen to their complete disappearance from the lumen. The relative duration (frequency) of these eight stages was determined by classifying the first twenty cross-sections of seminiferous tubules observed microscopically at each of the six locations sampled. This resulted in a total of 1440 classified tubules (six animals X two testes six locations X twenty tubules). These data were analysed according to the procedure outlined by Henderson (1959). In this analysis it is assumed that the eight stages (S) and the testes ( ) are fixed classifications, and that the six animals (A) and the six locations within each testis (L) are random classifications. A colon is used to denote a nested or 'within' classification. Thus, A:S denotes animals within stages, T:AS denotes testes within animals and stages, and L:ATS denotes locations within animals, testes and stages. The renewal of spermatogonia and subsequent divisions of spermatogenesis were studied in 4^-thick sections. All whole nuclei and fragments of nuclei were classified and counted in twenty-four tubular cross-sections at each of the eight stages (two tubular cross-sections per stage per testis in each of six animals). Thus, whole nuclei and fragments of nuclei were classified and counted in a total of 192 cross-sections of seminiferous tubules. All 'raw' counts were transformed to nuclear points by an adaptation of Abercrombie's formula (Abercrombie, 1946). Abercrombie defined a nuclear point as "any geometrical point of the same relative position in all nuclei". t"L X L+T D In this formula, = the average number of nuclear points per cross-section, C the crude count of the number of whole and cut nuclei in the cross-section, — ... = section thickness in microns, L = the diameter of the nuclei in microns, and D = the diameter of the seminiferous tubule in microns. All tubules were corrected to a standard diameter of 190 µ. Since nuclear points have no dimensions they can be used to compare populations of nuclei with different diameters, characteristic of the nuclear populations in the seminiferous tubules. The diameter, L, of the different types of nuclei at each of the eight stages of the cycle of the seminiferous epithelium was obtained by measuring twelve whole nuclei with a filar micro¬ meter in 10-µ-thick sections. Because of the greater variability in size of Type A spermatogonia, twenty-four nuclei of this cell type were measured. Each Downloaded from Bioscientifica.com at 10/07/2021 03:59:08AM via free access 312 E. E. Swierstra and R. H. Foote nucleus was measured in two directions, once parallel to the major axis of the nucleus and once parallel to the minor axis. The average of the two measure¬ ments (Text-fig. 1) was used for the factor L in the formula. Because two generations of primary spermatocytes are present during approximately half of the cycle of the seminiferous epithelium, the following terminology is used to distinguish between them. The term preleptotene primary spermatocyte is used for the newly-formed primary spermatocytes ' ' 1 ' 2 3 4 5' 6 '7 8' 1 '2'3' Stages of the cycle of the seminiferous epithelium Text-fig. 1. Nuclear diameters of the different spermatogenic cells during the eight stages of the cycle of the seminiferous epithelium. of Stage 8, the term 'young primary spermatocytes' denotes the youngest generation of primary spermatocytes in Stages 1, 2, 3 and 4, and the term 'old primary spermatocytes' denotes the older generation of primary sperm¬ atocytes from the time they enter Stage 5 until they give rise to secondary spermatocytes.