Scanning Electron and Light Microscopy of the Equine Seminiferous Tubule*

FERTILITY AND STERILITY Copyright © 1978 The American Fertility Society

Vol. 29, No.2, February 1978 Printed in U.s.A.

SCANNING ELECTRON AND LIGHT MICROSCOPY OF THE EQUINE SEMINIFEROUS TUBULE*

LARRY JOHNSON, M.S. RUPERT P. AMANN, PH.D.t B. W. PICKETT, PH.D.:!: Animal Reproduction Laboratory, Colorado State University, Fort Collins, Colorado 80523

Changes within the equine seminiferous tubules during the cycle of the seminiferous epithelium were studied with light and scanning electron microscopy (SEM). Once observed with BEM, tubules were sectioned and staged using light microscopy. As viewed by SEM,the web like, spongy cytoplasm of germ cells or Sertoli cells in stages I and I[extended over the entire height of the germinal epithelium. The cytoplasm of the basal portion of the germinal epithelium in stages III to VIII was similar to that in stages I and II. However, the cytoplasm which occupied the luminal third of the epithelium in stages III to VII was smooth in appearance. The smooth-surfaced, periluminal cytoplasm diminished in stage VIII. Principal pieces of flagella from spermatids extended into the tubular lumina in all stages, whereas the middle pieces extended into the lumen only in stage VIII. Later in stage VIII, the middle pieces, which were thickened with cytoplasm, were connected to the germinal epithelium by stalks. After spermiation, the diameter of the middle pieces was similar to that of ejaculated spermatozoa. Thus, the cytoplasm within the thickened middle pieces contributed to the formation ofthe cytoplasmic droplets.

Scanning electron microscopy (SEM) permits observation of structures in three dimensions. With other types of microscopy, these structures must be visualized by the reconstruction of serial sections. In capitalizing on this advantage, SEM has been employed to observe structures within the testis of the dog,I-3 rabbit,3 monkey,4 rat,5.6 and hamster.7 Changes within the seminiferous tubules during the cycle of the seminiferous .epithelium have not. been documented for any species with the use of SEM. The objective of this qualitative study was to describe differences within the germinal epithelium during different stages of the cycle of the

seminiferous epithelium as observed by SEM. Light microscopy was incorporated to permit staging of the same seminiferous tubules which had been observed by SEM.

MATERIALS AND METHODS

Immediately after castration, testes from five adult stallions were perfused via the spermatic artery with a solution containing 0.15 M NaCI, O.4%(w/v) procaine HCI, and 10 IU/ml of sodium heparin. Once the blood was cleared from the testis, the perfusion continued with 4% (v/v) glutaraldehyde in 0.2 M s-collidine buffer (pH 7.4). .After 1 hour of fixation, thin slices (approximately 3 x 5 x 10 mm) were removed from four regions of the testis (two near and two away from the central vein) and. immersed in the same fixative for an additional hour. Tissues were washed in 0.2 M s-collidine buffer, further fixed in 2% (w/v) OS04 in 0.2 M s-collidinebuffer for 1 hour, dehydrated in acetone, infiltrated

Received June 27, 1977; revised August 8, 1977; accepted August 10, 1977. *Portions of these data are from a thesis to be submitted by.Larry Johnson to the Graduate Faculty of Colorado State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. tPresent address: Dairy Breeding Research Center, Pennsylvania State University, University Park, Pa. 16802. :!:To whom reprint requests should be addressed.

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with Freon 113, critical-point dried8 in Freon 13, coated with carbon and gold, and observed with an Hitachi HHS-2R scanning electron microscope. Photographed tubules were charted on low-magnification, scanning electron micrographs. Dried tissues, which had been observed

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with SEM, were infiltrated with a 1:1 mixture of propylene oxide-Epon 812 and embedded in Epon 812. Serial sections (1 I1-m) were cut, mounted on glass slides, and stained with toluidine blue. Since this was a qualitative and not a quanti-

FIG. 1. Scanning electron micrograph of a cross-section of an equine testis containing seminiferous tubules (ST), blood vessels (V), Leydig cells CL), and connective tissue (CT) (x440). FIG. 2. Photomicrograph (2A) and scanning electron micrograph (28) of the same tubule of §tages I a) and II al). Both stages have two generations of primary spermatocytes (P) and one generation of spermatids. The spermatid nuclei are round (JlS) in stage I and elongated (fES) in stage II (x440).

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tative study, only 100 tubular cross-sections were photographed in SEM. However, several thousand were observed. Only tubules which had excellent preservation, were free from surface debris, and were cut in cross-section were photographed in SEM. The identical tubules which had been photographed in SEM were located in the Epon sections and photographed under bright-field microscopy. The location and stage(s) of each cross-section were established jointly by two observers by using the Epon sections. The germinal epithelium was classified into eight stages on the basis of cellular association of germ cells plus the shape and orientation of spermatids. 9 Paired light and scanning electron micrographs of the same seminiferous tubules are presented. Slight differences in the shapes of the tubules can be observed in the light and SEM micrographs because it was difficult to section in precisely the same plane. Most cross-sections of the equine seminiferous tubules contained a single stage of the cycle of the seminiferous epithelium. 9 Occasionally, cross-sections containing two or three stages were viewed. There was no difference in the appearance of the germinal epithelium for a given stage whether or not it occupied a portion of the entire cross-section. Therefore, tubules containing multiple stages are presented to conserve space. RESULTS

When viewed by SEM, a cross-section of the equine testis contained seminiferous tubules with associated peritubular tissue, clusters of interstitial cells, blood vessels, and connective tissue (Fig. 1). Flagella of spermatids were

observed in the lumina of all seminiferous tubules. The germinal epithelium in stages I and II was characterized by two generations of primary spermatocytes and one generation of spermatids (Fig. 2A). The spermatid nuclei were round in stage I and elongated in stage II. The method of preparation for SEM· exposed the interior of most cells constituting the germinal epithelium. As viewed by SEM, this internal cytoplasm of cells presumed to be spermatocytes, spermatids, and Sertoli cells was similar in appearance and homogeneous over the entire height of the germinal epithelium (Fig. 2B). Flagella extending from round spermatids were seen in the tubular lumina in stages I (Fig. 3) and II (Fig. 4). Presented in Figure 5A is a tubular cross-section representing stages III, IV, and V. Stage III was identified by bundle formation of spermatid nuclei along with two generations of primary spermatocytes. Stage IV contained meiotic metaphase figures and/or secondary spermatocytes, while stage V was characterized by two generations of spermatids and the absence of type B spermatogonia. Dark-staining cytoplasm occupied the luminal third (periluminal region) of the germinal epithelium in these three stages. Neither bundle formation in stage III nor specific cell types in stages IV or V was distinguishable by SEM (Fig. 58). The germinal epithelium in the periluminal region was smoothsurfaced (Fig. 58). This region corresponded to the dark-staining cytoplasm in the light micrograph of the same tubule (Fig. 5A). The smooth outer surface of Sertoli cells was exposed where germ cells were removed (Fig. 58). Stages VI and VII (Fig. 6A) were characterized by the presence of type B spermatogonia, one

FIG. 3. Luminal portion of the germinal epithelium in stage I. The developing flagellum of a round spermatid (JlS) is attached to the nucleus and extends into the lumen (arrow). The gold coat (aG) stains the luminal surface

of this tubule (x 1500). FIG.

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4. Luminal portion of the germinal epithelium in stage II containing slightly elongated spermatid nuclei

fES) with attached flagella. One flagellum (arrow), sectioned almost sagittally, extends into the lumen (xI500).

FIG. 5. Photomicrograph (SA) and scanning electron micrograph (58) of the same tubule of stages III (JII), IV (IV), and V (V). Stage III is characterized by two generations of primary spermatocytes <1') and deeply embedded bundles of elongated spermatid nuclei fES). In stage IV, the epithelium contains metaphase figures (M) and secondary spermatocytes (SS). Stage V contains newly formed round spermatids (JlS) and older, elongated, spermatid nuclei. Cytoplasm (C) which occupies the luminal third of the germinal epithelium stains darkly with toluidine blue. In 58, the region corresponding to this dark-staining cytoplasm (G) is smooth in appearance. The smooth, outer surfaces of Sertoli cells (arrows), where germ cells had been removed during tissue preparation, are seen (x440). FIG. 6. Photomicrograph (6A) and scanning electron micrograph (68) of the same tubule of stages VI (VI) and VII (VII). Both stages are characterized by elongated spermatid nuclei fES), round spermatid nuclei (JlS), and primary spermatocytes <1'). In stage VI, bundles of elongated spermatid nuclei are deeply embedded, while in stage VII the elongated spermatids have begun to migrate toward the lumen. Dark-staining cytoplas~ (G) occupies the luminal third of the epithelium. This dark-staining cytoplasm corresponds to the smooth-surfaced cytoplasm (G) of elongated spermatids in 68. The outside surface of several round spermatids (arrow) is exposed (x440).

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generation of primary spermatocytes, and two generations of spermatids. However, in stage VI the older generation of spermatids was deeply embedded in the germinal epithelium, while in stage VII these elongated spermatids had begun to migrate toward the lumen. As viewed

February 1978 by light microscopy and SEM, the appearance of the cytoplasm of cells in the periluminal region in these two stages was similar to that found in stages III to V. The smooth outer surfaces of several cells, presumed to be round spermatids on the basis of their location in the tubule,

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were exposed during the cleavage of the tissue (Fig. 68). Only the principal pieces of flagella of older spermatids extended into the lumina of seminiferous tubules in stage VII (Fig. 7). In early stage VIII, the middle pieces of older spermatids extended into the tubular lumen (Fig. 8). Later in stage VIII, the smooth-surfaced, periluminal cytoplasm had diminished and the head portion of the spermatids was exposed to the lumen (Fig. 9). When the entire spermatid was exposed to the lumen, it remained attached only by cytoplasmic stalks (Figs. 10 and 11). One or two of these stalks connected the thickened middle piece of each spermatid with the germinal epithelium. Prior to spermiation, the cytoplasm had concentrated at the proximal end of the middle pieces, forming cytoplasmic droplets (Fig. 11).

DISCUSSION

When the germinal epithelium was viewed by SEM, the cytoplasm was either rough and web like or smooth. We interpreted the weblike cytoplasm (Figs. 2B, 7 and 9) as cytoplasm of the interior of cells cleaved during tissue preparation. The smooth cytoplasm (Figs. 5B, 7, and 9) represents outer surfaces of the plasma membrane. The cytoplasm of Sertoli and germ cells was elevated around germ cell nuclei, but nuclei per se were rarely seen. Occasionally, the smooth outer surface of round spermatids was exposed. Leydig cells were in clusters and did not appear to contain microvilli as do Leydig cells of the rat.s In stages I and II, spongy, weblike cytoplasm extended the entire height of the germinal epithelium (Fig. 2B). In stages III to VII, the periluminal region of the germinal epithelium

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was stained darkly under light microscopy (Figs. 5A and 6A) and appeared smooth when viewed by SEM (Figs. 5B, 68, and 7), whereas the basal two-thirds was web like as in stages I and II. Cleavage of the periluminal region apparently revealed either the cell membrane of Sertoli cells, which envelope the elongated spermatids,IO-13 or the cell membrane of spermatids. The region occupied by smooth-surfaced cytoplasm had virtually disappeared by stage VIII (Fig. 9), and the smooth-surfaced cytoplasm was not conspicuous until the following stage III (Fig. 5). Flagella were seen in the lumina of all seminiferous tubules regardless of stage (Figs. 1,2, and 5 to 9). Developing flagella from round spermatids in stage VIII were observed in Epon sections and by SEM (Fig. 10). Similar projections of spermatid flagella were observed by SEM in the canine seminiferous tubule. 1 Although development of flagella was observed as early as stage VIII (Fig. 10), with more extensive development in stages I and II (Figs. 3 and 4), the middle pieces of these sperm at ids were not exposed to the tubular lumen until the following stage VIII (Fig. 8). The usefulness of SEM in studying spermatogenesis is limited by the fact that individual germ-cell types and thus specific cellular associations cannot be distinguished. However, the 3-dimensional appearance produced by SEM was especially useful in interpreting the events occurring during spermiation. Spermiation is the process by which mature spermatids are released as testicular spermatozoa into the lumina of the seminiferous tubules. Aspects of spermiation have been reported for the bandicoot,lO, 11 ram,l1 guinea pig, 13 chinchilla,13 hamster, 14-17 and mouse 18 with transmission electron microscopy and for the dog,! monkey,4 and hamster7 with SEM.

FIG. 7. An SEM view of the germinal epithelium in stage VII revealing spongy cytoplasm (SC) and smooth cytoplasm (C) found in the luminal third of the epithelium. Only the principal pieces (PP) of the spermatids extend into the lumen (x880). FIG. 8. An SEM view of the tubular lumen in early stage VIII. The exposed middle pieces (MP) of spermatids are thickened with cytoplasm (x 1760) FIG. 9. Scanning electron micrograph of the germinal epithelium in late stage VIII, with the entire length of elongated spermatids exposed to the lumen. A Sertoli cell (S) and primary spermatocytes (P) are revealed. The smooth-surfaced periluminal cytoplasm (C) is scant, and the spongy cytoplasm occupies a larger portion of the germinal epithelium as compared with that in stage VII (x880). FIG. 10. The luminal region of the tubule shown in Figure 9. Cytoplasmic stalks (arrows) connect the middle piece (MP) of each spermatid with the germinal epithelium. The middle pieces of attached spermatids are enlarged with cytoplasm. In the released spermatozoa, the portion of the middle piece (open arrow) which is."not covered by the cytoplasmic droplet (CD) is smaller in diameter than the middle piece of attached spermatids. Flagella (ji') of the younger generation of spermatids extend into the lumen (x5280).

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FIG. 11. An SEM view of the luminal region of germinal epithelium in late stage VIII which contains spermatids attached by cytoplasmic stalks (arrows) to the epithelium. Formation of the cytoplasmic droplets (CD) is partially complete (x5280) .

Like the nuclei of spermatids of other species,10-18 the nuclei of equine spermatids became elongated before the cells migrated to the lumen. Cytoplasmic stalks (Figs. 10 and 11) connected the middle piece of elongated spermatids to their residual bodies. Similar cytoplasmic stalks were present in other species during spermiation.1, 4. 7.12-15.17.18 The middle pieces of elongated spermatids in the stallion (Figs. 10 and 11) and in other species. l l • 15. 17. 19-21 were thickened with cytoplasm prior to migration into the lumen and formation of the cytoplasmic droplet. After formation of the cytoplasmic droplet, the residual cytoplasm was scant in the remaining portion of the middle pieces,u-13. 17. 19. 21 Immediately after spermiation, the difference in diameter between the distal portion of the middle piece and the principal piece of released spermatozoa (Figs. 9 and 10) was less distinct and more typical of ejaculated spermatozoa than of attached spermatids. Therefore, we concluded that cytoplasm migrated from more distal portions of the middle piece and became concentrated at the proximal end. It is difficult to determine from

static micrographs whether the major contributor to the cytoplasmic droplet is cytoplasm from these swollen middle pieces or cytoplasm from the cytoplasmic stalk. 13 REFERENCES 1. Connell CJ: A SEM study of spermiogenesis in the canine

testis (abstr). J Cell BioI 67:78a, 1975 2. Connell CJ: A scanning electron microscope study of the interstitial tissue of the canine testis. Anat Rec 185:389, 1976 3. Gondos B, Connell CJ: Transmission and scanning electron microscopy study of the developing testis. In Proceedings of the VIIIth International Congress on Animal Reproduction and Artificial Insemination, Vol III, Krakow, July 12-16, 1976, p 50 4. Flechon JE, Hafez ESE: Spermiation and epididymal maturation of spermatozoa in the bonnet macaque (Macaca radiata) as viewed by scanning electron microscopy. Fertil Steril 26:1219, 1975 5. Clark RV: Three-dimensional organization of testicular interstitial tissue and lymphatic space in the rat. Anat Rec 184:203, 1976 6. Eddy EM, Kahri AI: Cell associations and surface features in cultures of juvenile rat seminiferous tubules. Anat Rec 185:333, 1976

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7. Gravis CJ: A scanning electron microscopic study of hamster Sertoli cells during spermiation. Anat Rec 187:593, 1977 8. Gould KG: Preparation of mammalian gametes and reproductive tract tissues for scanning electron microscopy. Fertil Steril 24:448, 1973 9. Swierstra EE, Gebauer MR, Pickett BW: Reproductive physiology of the stallion. I. Spermatogenesis and testis composition. J Reprod Fertil 40:113, 1974 10. Sapsford CS, Rae CA, Cleland KW: Ultrastructural studies in maturing spermatids and on Sertoli cells in the bandicootPerameles nasuta geoffroy (Marsupialia). Aust J Zool 17:195, 1969 11. Sapsford CS, Rae CA: Ultrastructural studies on Sertoli cells and spermatids in bandicoot and ram during the movement of mature spermatids into the lumen of the seminiferous tubule. Aust J ZooI1'7:415, 1969 12. Fawcett DW: Ultrastructure and function of the Sertoli cell. In Handbook of Physiology, Edited by DW Hamilton, RO Greep, Sect 7: Endocrinology, Vol 5: Male Reproductive System. Washington DC, American Physiological Society, 1975, p 21 13. Fawcett DW, Phillips DM: Observations on the release of spermatozoa and on changes in the head during passage through the epididymis. J Reprod Fertil [Suppl] 6:405, 1969

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14. Vitale-Calpe R, Burgos MH: The mechanism of spermiation in the hamster. I. Ultrastructure of spontaneous spermiation. J Ultrastruct Res 31:381,1970 15. Vitale-Calpe R, Burgos MH: The mechanism of spermiation in the hamster. ll. The ultrastructural effects of coitus and LH administration. J Ultrastruct Res 31:394, 1970 16. Fouquet JP: Le mecanisme de la spermiation chez Ie hamster: signification des relations entre cellules de Sertoli et spermatids. CR Acad Sci [D] (Paris) 275:2025, 1972 17. Fouquet JP: La spermiation et la formation des corps residuels chez Ie hamster: role des cellules de Sertoli. J Microsc (Paris) 19:161, 1974 18. Ross MH: The Sertoli cells' junctional specialization during spermiogenesis and at spermiation. Anat Rec 186:79, 1976 19. Burgos MH, Vitale-Calpe R, Aoki A: Fine structure of the testis and its functional significance. In The Testis, Vol 1, Edited by AD Johnson, WR Gomes, NL Vandemark. New York, Academic Press, 1970, p 551 20. Bloom G, Nicander L: On the ultrastructure and development of the protoplasmic droplet .of spermatozoa. Z Zellforsch Mikrosk Anat 55:833, 1961 21. Phillips DM: Spermiogenesis. New York, Academic Press, 1974, p 55