Cycle of the Seminiferous Epithelium of the Guinea Pig

Cycle of the Seminiferous Epithelium of the Guinea Pig A Method for Identification of the Stages Yves Clermont, Ph.D. IN THE GUINEA PIG, the cells of the seminiferous epithelium are arranged in definite cellular associations of which there are only a limited number. These, over a period of time and in a given area of the seminiferous epithelium, follow each other in an orderly sequence to give the so-called "cycle of the seminiferous epithelium." In this respect, the seminiferous epithelium of the guinea pig is not different from that of most other mammalian species. 8,16 Although spermiogenesis in the guinea pig has been repeatedly investigated,5-7, 9-11, 15 no attempt has been made to classify the various cellular associations into a series of well-defined, easily identifiable "stages of the cycle." A detailed description of the stages of the cycle facilitates understanding of the continuously changing seminiferous epithelium. It also permits the identification of every step of spermatogenesis from the Type A stem spermatogonium to the spermatozoon. A classification of the cell associations into clear-cut stages may also be useful for those studying the specific effect of agents such as radiation, heat, nutritional deficiencies, etc., on the various germ cells of the guinea pig testis. Indeed, if a given cell type is missing from a cell association where it should be found we can safely conclude that these cells have been selectively destroyed by the agent. From the Department of Anatomy, McGill University, Montreal, Canada, 563

564 CLERMONT Fertility & Sterility In the present article we shall propose a classification of 12 such stages for the various cell associations appearing during one cycle of the seminiferous epithelium of the guinea pig. THE STEPS OF SPERMIOGENESIS The stages of the cycle have been identified in several mammalian species by making use of the steps of development of the spermatids. 1, 3, 7, 12 Of the germ cells that compose the seminiferous epithelium, the spermatids are certainly the elements that undergo the most striking series of cytological changes. These yield morphological criteria that may be used to define clearcut steps of spermiogenesis, which in tum constitute useful means for the identification of the stages of the cycle. The same approach is taken in the present description of the stages of the cycle of the guinea pig; thus steps of spermiogenesis are defined by considering the characteristic changes of the acrosomic system and nucleus of the spermatids. Sections of Orth's or Zenker-formol-fixed testes, stained with the periodic acid-schiff-hematoxylin technic were used for this purpose. The periodic acid-schiff deeply stained the acrosomic structures (proacrosomic and acrosomic granules, acrosome, and head cap) while hematoxylin outlined the nucleus. Spermiogenesis of the guinea pig as revealed by the PAS technic has already been described in detail,7 In this description, spermiogenesis is divided into four main phases. In the first or Golgi phase, the Golgi zone elaborates the proacrosomic granules from which a single acrosomic granule arises. In the second or cap phase, the acrosomic granule becomes the acrosome and the head cap appears and covers part of the nucleus. In a third or acrosome phase the acrosome becomes conical and the nucleus flattens and takes a discoid shape. In the last or maturation phase the spermatid completes its morphological evolution to become a spermatozoon. Each of these phases are subdivided into steps totaling 17. In an attempt to determine workable criteria for the identification of the stages of the cycle, we have had to modify the early description of spermiogenesis; we now propose to subdivide spermiogenesis into 15 steps (Fig. 1). Of these, the first 12 steps will be used to identify the 12 corresponding cellular associations or stages of the cycle. (The division of spermiogenesis into four phases may be main-

eej,!!'. Fig. 1. Cellular composition of the 12 stages of the cycle of the seminiferous epithelium in the guinea pig. 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. These cellular associations or stages of the cycle were identified by means of the first 12 of the 15 steps of spermiogenesis (Numbers 1-15). The steps were defined by the changes observed in the nucleus (delineated by a gray line) and in the acrosomic structure (shown in black or gray) in sections stained with the periodic acid-schiff-hematoxylin technic. From Step 11 onward the spermatids are depicted as seen from their flat surface (left) and from the side (rl,ght). A, represents Type A spermatogonia; AP, Type A spermatogonia in prophase; In, intermediate type spermatogonia; B, Type B spermatogonia; R, resting primary spermatocytes; L, leptotene primary spermatocytes; Z, zygotene primary spermatocytes; P, pachytene primary spermatocytes; Vi, diakinesis of primary spermatocytes; II, secondary spermatocytes. The changes taking place during spermatogenesis are in sequence from left to right, starting with the bottom row.

566 CLERMONT Fertility & Sterility tained; the Golgi phase includes Steps 1-4; the cap phase, Steps r!r-7; the acrosome phase, Steps 8-12; and the maturation phase, Steps 13-15.) THE STAGES OF THE CYCLE OF THE SEMINIFEROUS EPITHELIUM The definition of each stage of the cycle will be preceded by a description of the criterion used to identify the stage, i.e., one of the first 12 steps of spermiogenesis. Then the remaining germ cells, which together with the spermatids of Steps 1-12 form typical cell associations, will be described in the following order: spermatids in Maturation phase (Steps 13-15) whenever present, spermatogonia, and spermatocytes. The reader should refer continually to Fig. 1, which illustrates with drawings of the various cell types the cellular composition of each stage of the cycle. It should be noted that the stages of the cycle are numbered with Roman numerals to avoid confusion with the steps of spermiogenesis which are numbered with Arabic numerals. Stage I is identified by the presence of newly formed spermatids (Fig. 1, 1 ). In the Golgi zone of these cells, which is close to the spherical nucleus, many (4 or more) small PAS-positive proacrosomic granules may be seen. Associated with Step 1 spermatids we find maturing spermatids at Step 13 Fig. 1, 13). Their flattened nucleus has a discoid shape. The acrosome forms a large crescent expanding on the lateral edges of the nucleus, but not spreading over its dorsal and ventral surfaces, covered only by the head cap. In a side view of the cell the acrosome is lenticular (Fig. 1, 13 right). These cells are more or less grouped in bundles between the spermatids of the younger generation. Along the basement membrane two types of spermatogonia are found at this stage: the Type A and the intermediate type spermatogonia (Fig. 1, A, In). The Type A spermatogonia have an ovoid nucleus containing a palestained granular chromatin. Two or three nucleoli are seen attached to the nuclear membrane. (These Type A spermatogonia are found at all stages of the cycle.) The intermediate type spermatogonia have an ovoid nucleus containing both a pale-staining granular chromatin distributed throughout the nucleus, and thin flakes of intensely stained chromatin applied to the nuclear membrane. Only one generation of spermatocytes is present at this stage. These are at the early pachytene step of the meiotic prophase (Fig. 1: P). The chromosomes are thick, but still long, tortuous, and heavily stained.

Vol. 11. No.6. 1960 SEMINIFEROUS EPITHEliUM CYCLE 567 Stage II begins when the spermatids show only two or three large spherical proacrosomic granules in their Golgi zone (Fig. 1, 2). The generation of maturing spermatids is still at Step 13, but these cells are now more deeply inserted in the seminiferous epithelium than at Stage I. The Type A and intermediate type spermatogonia as well as the early pachytene spermatocytes are also present at this stage. Stage III is characterized by spermatids that have only one large spherical acrosomic granule in their Golgi zone (Fig. 1, 3). This acrosomic granule probably forms by coalescence of the proacrosomic granules. The maturing spermatids have proceeded to Step 14 of spermiogenesis (Fig. 1, 14). The large crescentic acrosome subdivides into two zones, a pajestained zone proximal to the nucleus and a more intensely stained apical zone. In a side view of the cell the acrosome is more Hattened than at the previous step. On the basement membrane, Type A and intermediate type spermatogonia are found. The latter cell type shows a more deeply stained nucleus with larger chromatin Hakes attached to the nuclear membrane. At the end of this stage and at the beginning of the next, spermatogonial mitoses are frequently observed along the basement membrane. These are interpreted as divisions of intermediate type spermatogonia. At this stage the generation of spermatocytes is still at the early pachytene step. Stage IV begins with the Hattening of the acrosomic granule at the surface of the spermatid nucleus (Fig. 1, 4). The acrosomic granule becomes hemispherical and may now be referred to as the acrosome. The Step 14 spermatids, seen at this stage, are still very deeply inserted in the seminiferous epithelium. Type A spermatogonia are present along the basement membrane with a new type of spermatogonia referred to as Type B (Fig. 1, B). These are numerous and apparently arise from divisions of intermediate type spermatogonia. The nucleus of Type B cells tends to become spherical and shows large Hakes of heavily stained chromatin on the nuclear membrane. The early pachytene spermatocytes are also present. Stage V is characterized by the early formation of the head cap (Fig. 1, 5), which is a delicate membranous structure extending from the edges of the hemispherical acrosomic granule onto the nuclear membrane. In a side view of the cell the head cap is seen as two small lateral projections on both

568 CLERMONT Fertility & Sterility sides of the acrosome. Stage V is considered to last until the acrosome and head cap cover approximately one third of the nuclear surface. Step 14 spermatids have a tendency to retract from the seminiferous epithelium in the direction of the tubular lumen. Types A and B spermatogonia are seen on the basement membrane. The latter often show some change in the chromatin suggesting early prophase. The nuclei of pachytene spermatocytes contain shorter and thicker chromosomes with more hazy outlines than at Stage IV. They are regarded as entering the late pachytene step of the meiotic prophase (Fig. 1, D). Stage VI begins when the acrosomic system of the spermatids (acrosome and head cap) covers more than one third of the nuclear surface and ends when this system extends over half of the nucleus (Fig. 1,6). The growing acrosome remains hemispherical. The older generations of spermatids has reached Step 15 (Fig. 1, 15); these cells are seen lining the luminal surface of the seminiferous epithelium and are in the process of shedding their residual cytoplasm. Their large crescentic acrosome is clearly subdivided into two zones and, in a side view of the cell, takes a globular boxing-glove shape. Along the basement membrane the Type A spermatogonia are present as well as the Type B cells, which are seen at various phases of mitotic division. The cells arising from these divisions constitute a new generation of spermatocytes. The older generation is at the late pachytene step. Stage VII begins when the acrosomic system of the spermatid covers more than one half of the nuclear surface. The growing acrosome also changes from a hemispherical to a crescentic shape when the spermatid is viewed from the side (Fig. 1, 7). During this stage the spermatids of the older generation (Step 15) are being released from the seminiferous epithelium into the lumen of the tubule. Type A spermatogonia are present on the basement membrane along with the new generation of primary spermatocytes produced by the divisions of Type B spermatogonia. These so-called resting spermatocytes (Fig. 1, R) have a small spherical nucleus containing a darkly stained granular chromatin as well as larger chromatin flakes on the nuclear membrane. The older generation of spermatocytes is at the late pachytene step of meiotic prophase. Stage VIII begins when the acrosomic system of the spermatids comes in contact with the cytoplasmic membrane. This movement is accompanied by a rotation of the nucleus so that the acrosomic system orients itself towards the basement membrane of the seminiferous tubule (Fig. 1, 8).

Vol. II, No.6, 1960 SEMINIFEROUS EPITHELIUM CYCLE 569 Along the basement membrane Type A spermatogonia are present and some of them show dark-staining granules along the nuclear membrane. These may be considered as early prophases of Type A mitoses, which are seen at the end of Stage VIII and during the following Stage IX. The young generation of primary spermatocytes enters the leptotene step of meiotic prophase (Fig. 1, L). In their nuclei, which enlarge progressively, the darkly stained chromatin is forming thin coiled and beaded Hlaments. No change is noticed in the generation of late pachytene primary spermatocytes except for a progressive increase of nuclear volume. Stage IX starts when the acrosome of the spermatid becomes a pointed cone. In a side view of the cell, the height of the cone is less than the width at the base (Fig. 1, 9). Some Type A spermatogonia are seen dividing at the beginning of this stage. The younger generation of primary spermatocytes is at the leptotene step and the older generation at the late pachytene step of the meiotic prophase. Stage X begins when the acrosome of the spermatid has elongated to the extent that the height of the acrosome cone is greater than its width at the base (Fig. 1, 10). The tip of the acrosome is deeply inserted in the Sertoli cytoplasm. Type A spermatogonia divide toward the end of the stage. The younger generation of primary spermatocytes usually enter the zygotene step of meiotic prophase (Fig. 1, Z). In their nuclei, the thin filamentous, homologous chromosomes pair and form long loops. The older generation of spermatocytes is still at the late pachytene step. Stage XI begins as the nucleus and the acrosome of the spermatids start to flatten (Fig. 1, 11 ). The nucleus condenses and progressively takes a discoid appearance. Seen from the flat surface the acrosome has the shape of a wide spearhead. Seen from the side, the pointed acrosome is narrowel than at the preceding Step 10. Along the basement membrane the Type A spermatogonia are found as well as the young generation of pachytene spermatocytes, still at the zygotene step. The older generation of primary spermatocytes enters diakinesis (Fig. 1, Vi). The thick chromosomes partially split in nuclei which have reached their maximal volume. Stage XII begins when the acrosome seen from the side (Fig. 1, 12) takes a lenticular shape in spermatids. Seen from the flat surface the acrosome assumes the shape of a wide, pointed crest.

570 CLERMONT Fertility & Sterility This stage however is usually identified by the presence of maturation divisions of primary and secondary spermatocytes or of secondary spermatocytes in interphase. The latter are characterized by nuclei containing numerous clumps of darkly stained chromatin (Fig. 1, II). The younger generation of primary spermatocytes enter the early pachyte~e step of the meiotic prophase (Fig. 1, P). The heavily stained chromosomes become thick but remain long and do not fill the whole nuclear space. These cells are found along the basement membrane with Type A spermatogonia, which are seen to divide during the present stage. Following Stage XII of the cycle Stage I reappears and a new cycle begins as a new generation of spermatids is formed from the second maturation divisions of spermatocytes. SPERMATOGENESIS OF THE GUINEA PIG The cellular composition of the stages of the cycle being known, as well as the order in which these stages appear at a given locus of the seminiferous tubule, it becomes possible to review briefly the whole spermatogenesis; that is, the complete series of changes observed in the germ cells from the Type A stem spermatogonia to the spermatozoon. In Fig. 1, this is accomplished by reading from left to right, starting on the bottom row and continuing on the successive rows above. The Type A spermatogonia, seen at Stages I-VII of the cycle, may be considered as "dormant" stem cells from which all other germ cells derive. Pertinent discussion of the mode of renewal of spermatogonia in rat and monkey may be found in Clermont and Leblond. 2,3 These Type A cells divide at Stages VIII-IX and at Stage X to give more Type A spermatogonia. The latter divide again at Stage XII and produce two categories of cells: new Type A cells, which will provide later for new generations of germ cells, and intermediate type spermatogonia. The latter type of cell divides at Stages III and IV of the cycle to produce Type B spermatogonia, which finally divide at Stage VI to produce a generation of primary spermatocytes (R, Fig. 1). Counts of resting and dividing cells at the various stages of the cycle confirmed the existence of five distinct peaks of spermatogonial divisions in the guinea pig, as in the rat. The primary spermatocytes remain resting during Stages VII and VIII of the cycle; they then enter the long meiotic prophase. They go successively through the leptotene (Stages IX and X), the zygotene (Stages X and XI) and the long pachytene (Stages XII, I-XI) steps of meiosis. Finally, after

Vol. 11. No.6. 1960 SEMINIFEROUS EPITHELIUM CYCLE 571 the diplotene step and diakinesis, they complete the first maturation division to give rise to secondary spermatocytes (Stage XII). The latter, after a short interphase, go through the second maturation division to produce spermatids. The spermatids begin the series of changes referred to as spermiogenesis, which terminates with the departure of the spermatids from the seminiferous epithelium as free cells-the spermatozoa. It should be noted (Fig. 1) that if one considers the spermatogonial division of Type A cells at Stages VIII and IX as the starting point of spermatogenesis, then the whole phenomenon extends over four consecutive cycles. COMMENTS To understand the various histological images seen in sections of seminiferous tubules it is necessary to perceive clearly the phenomenon referred to here as the cycle of the seminiferous epithelium. Indeed, the wide variety of cell types and multiplicity of their associations is somewhat confusing to the uninitiated since it is difficult at first to establish an exact correlation between these images. It is only after an analysis of the various cell associations that such images can be related in a sequence of events that repeats itself in cyclic manner. Thus, in the seminiferous epithelium there are one or two generations of spermatids associated with one or two generations of spermatocytes and of spermatogonia. Each generation is formed by a group of germ cells that appear at approximately the same time and develop synchronously. The development of anyone generation of spermatogonia, spermatocytes, and spermatids is so closely integrated with the developmental changes of the other generations that a limited number of typical cell associations appears in the tubules. Generally only one, sometimes two, such cell associations are seen per tubular cross section. These typical cell associations-12 were depicted here for the guinea pig-follow each other in a rigid sequence, at any given point or area of seminiferous tubules. For example, if an observer. could watch the changes taking place in a transverse slice of a living tubule, he would see the typical cellular associations (called here "stages") succeeding each other smoothly in the order given above. (Following Stage XII, Stage I would reappear, and so on.) This cycle of the seminiferous epithelium is a time concept and should not be confused with the distribution of the various cellular associations along tubules. It is obvious then, that the cycle cannot be directly observed in a fixed preparation of the testes, but can be ascertained by the nature of the cellular associations themselves as observed in the various tubular cross sections.

572 CLERMONT Fertility & Sterility The duration of the cycle in the guinea pig has not yet been determined, but if one examines the duration of the cycle obtained for several mammalian species (mouse, 8.6 days;13 ram, 10.5 days;14 rat, 12.0 days4 it is likely that for the guinea pig the duration of the cycle may also have to be calculated in days. A subdivision of the cycle into as many as 12 stages may be considered by some as an unnecessary refinement too elaborate to be useful in a study of the seminiferous epithelium. We believe, however, that a qualitative or quantitative analysis of the cellular components of the seminiferous epithelium, especially in experimental conditions resulting in a destruction of given cell types, requires a classification refined enough to include every important step of development of the germ cells. The classification proposed here, on the basis of experience in quantitative studies on spermatogonia, appeared to fulfil this purpose, and was found to be simple enough to be used, after a few days of initiation, by any conscientious technical assistant. SUMMARY A classification into 12 stages is proposed for the various cellular associations appearing during the cycle of the seminiferous epithelium of the guinea pig. The first 12 of a series of 15 steps, described for the development of the spermatids, have been used as a means of identification of the stages. These steps of spermiogenesis are characterized by the changes observed in the nucleus and acrosomic structure of the spermatids seen in sections stained with the periodic acid-schiff-hematoxylin technic. The proposed classification was devised to be useful in qualitative and quantitative studies of the seminiferous epithelium in normal and experimental animals. REFERENCES 1. CLERMONT, Y. Cycle de l'epitmlium seminal et mode de renouvellement des spermatogonies chez Ie hamster. Rev. Can. de Biol. 13:208, 1954. 2. CLERMONT, Y., and LEBLOND, C. P. Renewal of spermatogonia in the rat. Am.]. Anat. 93:475, 1953. 3. CLERMONT, Y., and LEBLOND, C. P. Differentiation and renewal of spermatogonia in the monkey. Macacus rhesus. Am. J. Anat. 104:237, 1959. 4. CLERMONT, Y., LEBLOND, C. P., and MESSIER, B. Duree du cycle de l'epithelium seminal du rat. Arch. Anat. micro 48:37, 1959. 5. FAWCETT, D. W., and ITO, S. Observations on the cytoplasmic membranes of testicular cells examined by phase contrast and electron microscopy. ]. Biophys. & Biochem. Cytol. 4: 135, 1958. 6. GATENBY, J. G., and WOODGER, J. H. The cytoplasmic inclusions of the germ cells. Part IX. On the origin of the Golgi apparatus on the middle-piece of the ripe sperm of Cavia and on the development of the acrosome. Quart. J. micro Sci. 65:265, 1920.

Vol. 11. No.6. 1960 SEMINIFEROUS EPITHELIUM CYCLE 573 7. LEBLOND, C. P., and CLERMONT, Y. Spermiogenesis of rat, mouse, hamster and guinea pig as revealed by the periodic acid-fuchsin sulfurous acid technique. Am. I. Anat. 90: 167, 1952. 8. LEBLOND, C. P., and CLERMONT, Y. Definition of the stages of the cycle of the 9. 10. 11. 12. 13. 14. 15. 16. seminiferous epithelium in the rat. Ann. New York Acad. Sc. 55:549, 1952. VON LENHOSSEK, M. Untersuchungen i.iber Spermatogenese. Arch. f. mikr. Anat. 51 :215, 1898. VON MEVES, F. Dber Struktur und Histogenese des Samenfaden des Mierschweinchen. Arch. f. mikr. Anat. 54:329, 1899. NIESSING, G. Die Betheiligung von Centralkorper und Sphare am Aufbau des Samenfadens bei Saugethieren. Arch. f. mikr. Anat. 48: Ill, 1897. OAKBERG, E. F. A description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am. I. Anat. 99:391, 1956. OAKBERG, E. F. Duration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium. Am.]. Anat. 99:507, 1956. ORTAVANT, R. Autoradiographie des cellules germinales du testicule de belier. Duree des phenomenes spermatogenetiques. Arch. Anat. micro 45: 1, 1956. PAPANICOLAOU, G. N., and STOCKARD, C. R. The development of the idiosome in the germ cells of the male guinea pig. Am. J. Anat. 24:37, 1918. ROOSEN-RuNGE, E. C. Kinetics of spermatogenesis in mammals. Ann. New York Acad.Sc.55:574,1952.