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CYTOLOGY AND KINETICS OF 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 nucleus, the location of the 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 (Type A ) 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 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 , 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 , 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 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 . 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 . 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 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 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 ¬ atocytes from the time they enter Stage 5 until they give rise to secondary spermatocytes. In terms of pre-meiotic phases, the time interval delineated by the term 'young' includes leptotene and zygotene, and the time interval delineated by the term 'old' includes pachytene, diplotene and diakinesis.

RESULTS Each of the eight stages of the cycle of the seminiferous epithelium had characteristic cell populations. Stage 1 (PI. 1, Fig. 1). Type A spermatogonia and Sertoli cells are present along the basement membrane. The Type A spermatogonia have oval to round nuclei with 'dustlike' and a nuclear membrane which can easily be observed in stained nuclei. Two generations of primary spermatocytes are present, a 'young' generation and an 'old' generation. The young primary spermatocytes are in the preleptotene phase and are located near the basement membrane ; the old primary spermatocytes are in pachytene and are scattered between the basement membrane and the lumen of the tubule. In the very beginning of Stage 1, the nuclei of the young primary spermatocytes are

Downloaded from Bioscientifica.com at 10/07/2021 03:59:08AM via free access Spermatogenesis in the rabbit 313 round with rather faintly staining chromatin, and a nuclear membrane which is visible in pas stained preparations. Soon the membrane of these leptotene nuclei becomes less visible and the chromatin starts to stain very intensely. The old primary spermatocytes have larger pachytene nuclei and the nuclear membrane of these cells is indistinct. The spermatids have round nuclei with a distinct nuclear membrane. Sections fixed in Allen's fixative and stained with the pas technique reveal a darkly staining acrosome which covers about half of the nucleus. Inside the nucleus one or more darkly staining karyosomes are present. Stage 2 (PI. 1, Fig. 2). The characteristic feature of this stage is the elongation of the spermatid nuclei. In the beginning of Stage 2, the nuclei are slightly oval, and in the end they have completely elongated. Two generations of primary spermatocytes are present. Both generations have an indistinct nuclear membrane. The appearance of the nuclear chromatin for both genera¬ tions is similar to that described under Stage 1. Sertoli cells and Type A spermatogonia line the basement membrane. The number of Type A spermatogonia has increased over the number present in Stage 1. Stage 3 (PI. 1, Fig. 3). Sertoli cells and Type A spermatogonia continue to line the basement membrane. Again two generations of primary spermatocytes are present. The nuclei of the young primary spermatocytes are still small and in the zygotene phase, and those of the old primary spermatocytes are in the pachytene phase. The spermatids are elongated. Stage 4 (PI. 1, Fig. 4). The composition of the cell population lining the basement membrane depends upon the extent to which this stage has progressed. During Stage 4, Type A spermatogonia start to divide and as a tesult intermediate-type spermatogonia as well as Type A spermatogonia and Sertoli cells can be observed along the basement membrane. The intermediate- type spermatogonia have ovoid nuclei with the long axis parallel to the basement membrane. The chromatin of these nuclei is in the form of coarse granules. In the early part of Stage 4, two generations of primary spermatocytes can be observed. The nuclei of the young generation, located close to the basement membrane, are in the pachytene phase and the chromatin stains intensely. The nuclei of the older generation of primary spermatocytes are very large and are in the diplotene or diakinesis phase. At the beginning of Stage 4, these old primary spermatocytes start to undergo the first maturation division and give rise to secondary spermatocytes. The secondary spermatocytes have spherical nuclei with a distinct nuclear membrane and the chromatin is in the form of granules connected by filaments. The diameter of these nuclei is 7-1 µ as compared to 9-6 µ for the diameter of the old primary spermatocytes and about 5-5 µ for the diameter of the newly formed spermatids. The life span of the secondary spermatocytes is relatively short. They are only present in Stage 4. Soon after their formation they undergo the second maturation division and form spermatids. The spermatids have spherical nuclei with a distinct nuclear membrane. The elongated spermatids from Stage 3 will be called spermatozoa in Stage 4 since a new generation of spermatids is formed. In addition to the nuclei described for this stage, one often can observe metaphase configurations of the dividing primary and secondary spermatocytes.

Downloaded from Bioscientifica.com at 10/07/2021 03:59:08AM via free access 314 E. E. Swierstra and R. H. Foote Stage 5 (PI. 2, Fig. 5). During this stage the spermatozoa move towards the Sertoli cells. Many Type A spermatogonia divide to give rise to intermediate- type spermatogonia, and in the latter part of this stage intermediate types divide once to give rise to more intermediates. In contrast to Stages 1, 2, 3 and early Stage 4, where there are two generations of primary spermatocytes present, in Stages 5, 6, 7 and half of Stage 8 only one generation of primary spermatocytes is present. The chromatin of the nuclei of these cells start to spread out and the nuclei move away from the basement membrane. The spermatids are spherical and have a distinct nuclear membrane. Stage 6 (PI. 2, Fig. 6). Sertoli cells, Type A spermatogonia and intermediate- type spermatogonia line the basement membrane. Towards the end of this stage the intermediate-type spermatogonia start to divide and form Type spermatogonia. Type spermatogonia have spherical nuclei and a distinct nuclear membrane. The chromatin of these cells is in the form of darkly staining granules. The nuclei of the primary spermatocytes have increased in Table 1 relative duration (frequency) of the eight stages of the cycle of the seminiferous epi¬ thelium in the rabbit

Mean percentage of tubules Stage in each stage ±s.e. 27-7 ±1-2 13-4 ±0.4 7-3 ±0-7 11-0 ±0-9 41 ±0-6 15-7 ±1-2 12-2 ±0-9 8-6 ±0-9

size during the long pachytene phase. The spermatid nuclei are spherical and they establish contact with the Sertoli cells during this stage. Stage 7 (PI. 2, Fig. 7). Sertoli cells and spermatogonia line the basement membrane. The type of spermatogonia present depends upon how far this stage has progressed. Intermediate-type spermatogonia can be observed in the early part of Stage 7. Soon these cells divide and give rise to Type sperm¬ atogonia. As a result one can observe many Type spermatogonia in Stage 7. In addition a few Type A spermatogonia are also present throughout Stage 7. The pachytene nuclei of the primary spermatocytes have further increased in size and the chromatin is in the form of a dense network. The spermatid nuclei are spherical and reveal a nuclear membrane. In pas preparations a distinct acrosome covering about one-third of the anterior portion of the spermatid nucleus is visible. Spermatozoa move towards the lumen. Stage 8 (PL 2, Fig. 8). The composition of the cell population lining the basement membrane depends upon the degree to which this stage has progressed. Sertoli cells and Type A spermatogonia are present at all times.

Downloaded from Bioscientifica.com at 10/07/2021 03:59:08AM via free access Spermatogenesis in the rabbit 315 Type spermatogonia are present during the first half of Stage 8. During the second half they undergo mitoses and produce primary spermatocytes. These newly formed primary spermatocytes in the preleptotene stage have spherical Table 2

analysis of variance of the different stages of the seminiferous epithelium in the normal rabbit

Variance component Source of Mean variations* d.f. square Per cent F Level of Expectation of mean square Actual of total ratio probability S 7 147-05 «¿ + °i:.,.+ 12°*.+72o? 2-01 47-3 72-08 /><0005

„2 , „2 A:S 40 204 Jw + CTl:ats- -002 (0) <1 T:AS 48 2-26 Jw + CTl:ats^ 0-25 L:ATS 480 2-24 «,+ ·. 2-24 52-7

* Stages (S) = 8 (Fixed) ; Animals (A) = 6 (Random) ; Testes ( ) = 2 (Fixed) ; Locations (L) = 6 (Random). nuclei which resemble Type spermatogonia. However, the nuclei of the preleptotene primary spermatocytes are smaller than those of the Type spermatogonia (average nuclear diameters of 6-0 µ versus 7-1 µ), they stain

2

4 INT ~INT

5] INT INT INT INT b

BB88BBBB

pppppp pppppppppp

Text-fig. 2. Proposed pattern of spermatogonial divisions in the rabbit. A = Type A spermatogonia; INT = intermediate-type spermatogonia; = Type spermatogonia; = preleptotene primary spermatocytes. The space given to each stage on the ordinate is proportional to its relative duration. less intensely and have a less distinct nuclear membrane. The pachytene nuclei of the old generation of primary spermatocytes are relatively large and the chromatin is in the form of a network. The spermatid nuclei are spherical and reveal an acrosome in pas preparations. The spermatozoa line the lumen.

Downloaded from Bioscientifica.com at 10/07/2021 03:59:08AM via free access 316 E. E. Swierstra and R. H. Foote

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Downloaded from Bioscientifica.com at 10/07/2021 03:59:08AM via free access 318 E. E. Swierstra and R. H. Foote The relative duration (frequency) of the eight stages is presented in Table 1 and the statistical analysis of these data is shown in Table 2. A total of 50,155 whole and cut nuclei were classified in twenty-four tubules of each of the eight stages in order to determine the kinetics of spermatogenesis in the rabbit. The mean number of whole and cut nuclei for each stage is presented in Table 3. The nuclear diameters of the different cell types during Table 5

mean diameters of the seminiferous tubules in which nuclei were COUNTED

Stage Mean diameter ± s.E. (µ) 192* ±3-7 190 ±30 193 ±4-4 191 ±40 190 ±3-4 199 ±2-7 195 ±3-6 194 ±4-2

* Mean diameter of twenty-four semi¬ niferous tubules. the eight stages are shown in Table 4 and the average diameter of the tubules in which nuclei were measured is given in Table 5. The data given in Tables 3, 4 and 5 were substituted in Abercrombie's formula to calculate the data presented in Table 6. The data of Table 6 were used to construct the proposed scheme of spermatogonial divisions in the rabbit (Text-fig. 2). DISCUSSION In this study the cycle of the seminiferous epithelium of the rabbit was divided into eight stages using as criteria the shape of the spermatid nuclei, the location of the spermatids and spermatozoa in regard to the basement membrane, the presence of meiotic figures and the release of spermatozoa into the lumen (Curtis, 1918; Roosen-Runge & Giesel, 1950; Ortavant, 1959). The pas staining technique was employed as an additional aid in identifying certain stages more rapidly. By this technique, the eight stages can be recognized at low magnifications by the experienced technician. It should be noted that pas staining is not a necessity for recognizing the different stages. Furthermore, not all fixatives give good results with the pas stain, e.g. Carnoy, Flemming and 95% alcohol dissolve PAS-positive material (Clermont & Leblond, 1955). The relative duration of the eight stages presented in Table 1 is similar to that reported from the ram and bull (Ortavant, 1959) and differs from that reported for the boar (Ortavant, 1959) and for the rat (Roosen-Runge & Giesel, 1950). The observations on the rabbit support the view that the duration of the different stages of the cycle of the seminiferous epithelium is a function of the species under consideration. The analysis of variance (Table 2) reveals that

Downloaded from Bioscientifica.com at 10/07/2021 03:59:08AM via free access Spermatogenesis in the rabbit 319 the total experimental variation was about equally divided between 'stages' (S=47-3%) and 'locations within animals, testes and stages' (L:ATS=52-7%). The large amount of variation between L:ATS is partly due to the small number of tubules classified per location. The seminiferous tubules are strongly coiled, and as a result many tubules are in the same stage in a certain area of a histological section. Increasing the number of tubules classified for an area (increasing sample size) reduces the variation between areas considerably. Variation between testes can be reduced by either increasing the number of classified tubules per area or by increasing the number of areas. Table 2 reveals Table 6

MEAN NUMBER OF NUCLEAR POINTS FOR SPERMATOGONIA, SPERMATOCYTES AND SPERMATIDS PER CROSS-SECTION OF THE SEMINIFEROUS TUBULES AT THE EIGHT STAGES OF THE CYCLE OF THE SEMINIFEROUS EPITHELIUM

Stage Type of cell

Spermatogonia Type A 1-1-* 2-1 2-1 1-8* 1-7 1-5 1-3 1-1 i Intermediates 0-2 0-8 2-2 20 1-0 01 - 1 Type 0-9 5-4 2-2 Spermatocytes Preleptotene 9-6t Young primaries 14-8 131 13-6 12-9 Old primaries 16-8 16-5 15-7 16-6 Old primaries 20-2 15-6 15-7 Varies due to 1 Secondary spermatocytes 48-7 46-1 48-3 50-8 Spermatids 46-3 44-7 Spermatozoa

symbolizes cell divisions and symbolizes differentiation. * Part of the Type A spermatogonia have divided and some of the intermediate-type spermatogonia may have divided to produce more intermediate-type spermatogonia. t These become young primary spermatocytes in Stage 1 of the next cycle of the seminiferous epithelium (see text for details). that the mean square of : AS is about equal to the mean square of L:ATS versus 2-24, indicating that a total of 120 classified tubules per testis (six locations twenty tubules per location) was sufficient to reduce the variance component due to testes to 0. Other studies by the authors suggest that approximately eighty classified tubules per testis will reduce the testes com¬ ponent to approximately 0. The analysis also reveals that there was no difference between animals with respect to the frequency of the stages. This supports the view that the duration of the stages of the cycle of the seminiferous epithelium is a biological constant within a species. During normal spermatogenesis, the spermatogonia must maintain their

Downloaded from Bioscientifica.com at 10/07/2021 03:59:08AM via free access 320 E. E. Swierstra and R. H. Foote numbers as well as give rise to primary spermatocytes. The number of primary spermatocytes generated by one Type A spermatogonium varies from species to species. For the rabbit it appears that about sixteen primary spermatocytes are generated from one Type A spermatogonium. The proposed scheme by which this takes place is presented in Text-fig. 2. The data on which this scheme is based is presented in Table 6. It should be noted that the composition of the cell population for each stage as given in Table 6 is an average ofcell populations (2-26 of twenty-four tubules. Any one tubule belonging to a certain stage may deviate considerably from this average, e.g. tubules in early Stage 8 have Type spermatogonia and no preleptotene primary spermatocytes, but those in late Stage 8 have preleptotene primary spermatocytes and no Type spermatogonia. However, the table is useful in determining the location and the number of cell divisions which occur in the spermatogenic cycle of the rabbit. It can be seen that Type A spermatogonia are present in all eight stages. The number varies from stage to stage. At the end of Stage 1, the Type A spermatogonia divide (IT in Stage 1 versus 2T in Stage 2). About half of the Type A spermatogonia present in Stage 2 stay behind as stem cells, and are the starting cells for the next generation of Type A spermatogonia. The rest, namely 2-1—1-1 = 1 spermatogonium, divide mainly in Stages 3, 4 and 5, and give rise to intermediate-type spermatogonia. These in turn divide to produce more intermediate-type spermatogonia. This conclusion is based on the figures listed under Stage 5 in Table 6. It can be seen that 2-2 intermediate- type spermatogonia were counted. In addition, it is assumed that 0-6 Type A spermatogonia (1-7 had not yet divided. These 0-6 Type A spermatogonia — 1-1) have the potential of producing 2 xO-6 = 1-2 intermediate-type spermatogonia. Thus, the total intermediate-type spermatogonia (those already formed and those yet to be produced by the Type A spermatogonia) in Stage 5 is 2-2 -4- 1-2 = 3-4. These 3-4 intermediate-type spermatogonia are thought to be produced from one type A spermatogonium (2-1 in Stage 2—1-1 in Stage 1) by two divisions. The first division is the one which produces intermediate- type spermatogonia and the second division is the one in which these newly formed intermediates divide to produce more intermediate-type spermatogonia. These in turn undergo in late Stage 6 and early Stage 7 and give rise to the Type spermatogonia. The Type spermatogonia divide in Stage 8 and produce preleptotene primary spermatocytes. Preleptotene primary spermatocytes undergo a morphological change and become leptotene primary spermatocytes in early Stage 1. During this change the amount of deoxyribo- nucleic acid in the nucleus doubles (Swierstra, 1962). The average number of primary spermatocytes per stage as given in Table 6 is 15-6, and these have originated from one Type A spermatogonium (2T in Stage 2—1-1 in Stage 8 = 1 spermatogonium). The variation observed in the number of primary spermatocytes of the different stages is attributed to sampling variation. The 15-6 primary spermatocytes undergo two maturation divisions in Stage 4, and are expected to give rise to 62-4 spermatids. Table 4 reveals that on the average 47-5 were counted per generation per stage. Thus, about 24% fewer spermatids were counted than were expected from theoretical considerations. This seems to indicate that during normal spermatogenesis in the rabbit there

Downloaded from Bioscientifica.com at 10/07/2021 03:59:08AM via free access Spermatogenesis in the rabbit 321 is a considerable degeneration of spermatogenic cells during the two matura¬ tion divisions. However, it is possible that even in 4^-thick sections the smaller, more lightly stained spermatids were somewhat underestimated in the counting procedure. This would result in an over estimate of the number of degenerating cells. An indication that this may have been the case is the observation that the number of young primary spermatocytes (small nuclei) in Stages 1, 2 and 3, is lower than the number of old primary spermatocytes (large nuclei) in the same stages. Theoretically one would expect the same number or fewer old primary spermatocytes since these cells are older and some may have degenerated. The proposed scheme of spermatogonial divisions for the rabbit is similar to the one presented by Ortavant (1959) for the ram and bull, and differs from the pattern reported for other species (Clermont & Leblond, 1953, 1959). The coefficient of efficiency (number of primary spermatocytes generated from one stem cell) for the rabbit is the same as that for the ram and bull, namely sixteen. It is different from the coefficient of efficiency for the rat, hamster, guinea-pig, monkey and duck. During spermatogenesis there is a considerable loss of spermatogenic cells in the rabbit. Twenty-four per cent fewer spermatids were counted than expected from theoretical considerations. Most of this loss seems to occur during and immediately after the two maturation divisions.

ACKNOWLEDGMENTS The authors wish to thank the Lalor Foundation whose grant in part supported this study. Gratitude is extended to Dr C. R. Henderson for his assistance in analysing the data. REFERENCES Abercrombie, M. ( 1946) Estimation of nuclear population from microtome sections. Anat. Ree. 94, 239. Benda, C. (1887) Untersuchungen über den Bau des funktionirenden Samekanälchens einiger Säugethiere und Folgerungen für die Spermatogenèse dieser Wirbelthierklasse. Ark. mikr. Anat. 30, 49. Brown, H. H. (1885) On spermatogenesis in the rat. Quart. J. micr. Sei. 25, 343. Cleland, K. W. (1951) The spermatogenic cycle of the guinea pig. Aust. J. sci. Res. B, 4, 344. Clermont, Y. (1954) Cycle de l'épithélium séminal et mode de renouvellement des spermatogonies chez le hamster. Rev. Cañad. Biol. 13, 208. Clermont, Y. (1958) Structure de l'épithélium séminal et mode de renouvellement des spermatogonies chez le canard. Arch. Anat. micr. Morph. exp. 47, 47. Clermont, Y. & Leblond, C. P. (1953) Renewal of spermatogonia in the rat. Amer.J. Anat. 93, 475. Clermont, Y. & Leblond, C. P. (1955) of , monkey, ram, and other as shown by the periodic acid—Schiff technique. Amer. J. Anat. 96, 229. Clermont, Y. & Leblond, C. P. (1959) Differentiation and renewal of spermatogonia in the monkey, Macacus rhesus. Amer. J. Anat. 104, 237. Curtis, G. M. (1918) The morphology of the mammalian seminiferous tubule. Amer. J. Anat. 24, 339. Ebner, V. von (1888) Zur Spermatogenèse bei den Säugethieren. Ark. mikr. Anat. 31, 236. Gray, P. (1958) Handbook of basic microtechnique, 2nd edn., p. 74. McGraw-Hill, New York. Henderson, C R. (1959) Design and analysis of animal husbandry experiments. Techniques and Procedures in Animal Production Research. American Society of Animal Production, Beltsville. Leblond, C. P. & Clermont, Y. (1952a) Spermiogenesis of rat, mouse, hamster and guinea pig as revealed by the 'periodic acid-fuchsin sulfurous acid' technique. Amer. J. Anat. 90, 167. Leblond, C. P. & Clermont, Y. (1952b) Definitions of the stages of the cycle of the seminiferous epithelium in the rat. The Biology of the Testes. Ed. R. W. Minor. Ann. N. T. Acad. Sci. 55, 548. Oakberg, E. F. (1956) A description of spermiogenesis in the mouse and its use in the analysis of the cycle of the seminiferous epithelium and renewal. Amer. J. Anat. 99, 391.

Downloaded from Bioscientifica.com at 10/07/2021 03:59:08AM via free access 322 E. E. Swierstra and R. H. Foote Orgebin, M. C, Courot, M. & Ortavant, R. (1958) Unpublished data cited by R. Ortavant. Reproduction in domestic animals, vol. 2, p. 1. Ed. H. H. Cole and P. T. Cupps. Academic Press, New York. Ortavant, R. (1954) Etude des générations spermatogoniales chez le Bélier. C.R. Soc. Biol, Paris, 148, 1958. Ortavant, R. (1959) Spermatogenesis and morphology of the . Reproduction in Domestic Animals, vol. 2, p. 1. Ed. H. H. Cole and P. T. Cupps. Academic Press, New York. Roosen-Runge, E. C. (1955) Untersuchungen über die Degeneration samenbildender Zellen in der normalen Spermatogenèse der Ratte. - Ze^\f0,scn- 41, 221. Roosen-Runge, E. C. & Giesel, L. O. (1950) Quantitative studies on spermatogenesis in the albino rat. Amer. J. Anat. 87, 1. Swierstra, E. E. (1962) The cytology and kinetics of spermatogenesis in the rabbit, and the desoxyribonucleic acid content of spermatogenic cells. Ph.D. Thesis, Cornell University.

EXPLANATION OF PLATES Cross-sections of the seminiferous tubules of the rabbit representative of the different stages described in the text. All photographs 720. A= Type A spermatogonium, I = intermediate-type spermatogonium, = Type spermatogonium, M = basement membrane, = preleptotene primary spermatocyte, Ó = 'old' primary spermatocyte, Y = 'young' primary spermatocyte, R = secondary spermatocyte, U = metaphase of secondary spermatocyte, = spermatid, = sperm¬ atozoon, S = .

Downloaded from Bioscientifica.com at 10/07/2021 03:59:08AM via free access PLATE 1

Fig. 1. Stage 1. Fig. 2. Stage 2. Fig. 3. Stage 3. Fig. 4. Stage 4.

(Facing p. 322)

Downloaded from Bioscientifica.com at 10/07/2021 03:59:08AM via free access PLATE 2

Fig. Stage 5. Fig. 6. Stage 6. Fig. Stage 7. Fig. 8. Stage 8.

Downloaded from Bioscientifica.com at 10/07/2021 03:59:08AM via free access