BIOLOGY 468/568

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PRINCIPLES AND APPLICATIONS OF ELECTRON MICROSCOPY

STUDENT NAME:

Mike Giblin

PROJECT TITLE:

A Comparative Study of the Ultrastructural Characteristics of Spermatogenesis in three Species of Gastropod Mollusc: Littorina littorea, Littorina planaxis, and Littorina scutulata.

Species Identification

• Kingdom: Animalia
  
  
• Phylum: Mollusca
  
  
• Class: Gastropoda
  
  
• Order: Prosobranchia
  
  
• Family: Littorinacae
  
  
• Genus: Littorina
  
  
• Species: littorea

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ABSTRACT:

A comparative study of spermatogenesis in three species of the Gastropod Mollusc family Littorinacae has shown that the ultrastructural aspects of the processes are essentially similar for each species. For the most part, only superficial differences in the size of certain structures have been discovered. However, some subtle differences in ultrastructure have been found. The overriding similarities between these species confirm initial expectations, which were based on the fact that these species occupy similar ecological niches and are in fact members of the same genus. This supports the view that significant differences in spermatozoon ultrastructure among members of the same genus are extremely rare. .

Key Words:

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INTRODUCTION

Certain gastropod molluscs of the genus Littorina have been found to be almost identical with respect to the process of spermatogenesis. However, the process of Spermatogenesis in other members of this genus is known to differ in certain respects.

In this study we compare the process of spermatogenesis in Littorina littorea, an Atlantic intertidal species, to that occurring in two Pacific coast splash zone snails, Littorina planaxis and the well characterized Littorina scutulata. Certain species of Littorina, including Littorina scutulata, have been exhaustively characterized with regards to sperm production by investigators John Buckland-Nicks and Fu-Shiang Chia. Their studies will be referred to often in this comparative study.

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MATERIALS AND METHODS

Specimens of Littorina planaxis and Littorina scutulata (Allen, 1976) were collected along the coast of Southern California at Alamitos Bay during the spring of 1990. Specimens of Littorina littorea were collected in Boston harbor in Boston, Mass., at the same time of year.

The seminiferous tubules and gonads of the males of these three species were removed by dissection and fixed in 6% gluteraldehyde in filtered seawater/0.05 M sodium cacodylate buffer, pH = 7.2, for one hour at room temperature. The tissues were then washed in the same cacodylate buffer and post-fixed for one hour at 4 oC with 2% osmium tetroxide in 0.05 M cacodylate buffer. The tissue was then dehydrated using a graded ethanol series and embedded in low viscosity resin (Spurr, 1969). Gold/silver sections were obtained on a LKB Nova ultramicrotome and mounted on 180 mesh copper grids.

Sections were double stained using saturated solutions of uranyl acetate and lead citrate for 3.5 and 3 minutes, respectively. Micrographs were taken on a JEOL 1200 EXII electron microscope operated at 80 kV. For light microscopy,sections of a purple interference color were cut and stained with filtered toluidine blue in 1% borax and photographed using either phase contrast or phase interference optics on an Olympus BH2 light microscope.

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RESULTS

Fig. 1:



Gonad tissue of L. littorea. Adjacent to the basal membrane lie nurse cells with lipid droplets within. Nascent sperm cells are shunted toward the lumen of the tubule as they develop. Spermatogonia, spermatocytes, and spermatids are visible. X 1150, scale bar = 10 microns.

Fig. 2:


Grazing cross section of a testicular tubule in L. scutulata. X 490, scale bar = 20 microns.

Fig. 3:



Section through gonad tissue of L. planaxis. Lipid droplets are visible within the nurse cells at the border of the tubule. Progressing inward toward the lumen of the tubule one notices the mitotic and meiotic figures in the nuclei of cells in various stages of development. Spermatids nearing maturity can be seen near the center of the tubule. X 1200, scale bar = 10 microns.

Fig. 4:



Oblique section through gonad tissue of L. littorea. X 1250, scale bar = 10 microns.


Fig. 5:



Phase interference photo of a group of spermatozeugmata of L. scutulata. X 500, scale bar = 10 microns.

Fig. 6:



Phase contrast photo of a spermatozeugma dissected from the seminal vesicle of L. scutulata. Bright circle is refraction of the liquid media. X 370, scale bar = 20 microns.

Fig. 7:



A group of early spermatogonia from L. littorea. X 7K, scale bar = 2 microns.

Fig. 8:



A group of spermatogonia from L. planaxis. X 6900, scale bar = 2 microns.

Fig. 9:



Primary spermatocytes from L. littorea in late leptotene or early zygotene. X 9600, scale bar = 1 micron.

Fig. 10:



Primary spermatocyte from L. littorea, in diplotene. X 9000, scale bar = 1 micrometer.

Fig. 11:



Late spermatogonia from L. planaxis. X 6900, scale bar = 2 microns.

Fig. 12:



Late spermatogonia from L. scutulata. X 9000, scale bar = 1 micron.

Fig. 13:



Primary spermatocytes from L. planaxis, in early zygotene. X 6900, scale bar = 1 micron.

Fig. 14:



Spermatogonia from L. planaxis. X 9500, scale bar = 1 micron.

Fig. 15:



Late spermatogonium from L. littorina. X 4800, scale bar = 2 microns.

Fig. 16:



Stage B spermatid from L. littorea. Note condensation of chromatin into characteristic pattern and aggregation of mitochondria at base of nucleus. X 14K, scale bar = 500nm.

Fig. 17:



Interdigitating nurse cells from L. littorea. Note lipid droplet and clear vesicle in one pseudopodium and lysosome in an adjacent one. X 14K, scale bar = 1 micron.

Fig. 18:



The mature nurse cell is here shown to have lost its pseudopodia. The size of secretion droplets has increased, and spermatids have become unilaterally attached by their acrosomal caps. X 4800, scale bar = 2 microns.

Fig. 19:



Stage C spermatid showing acrosomal granule in place at apex of nucleus. The intranuclear canal is developing and the mitochondria are beginning to elongate. X 18K, scale bar = 500nm.

Fig. 20:



Stage C spermatid of L. planaxis showing golgi body migrating to apex of nucleus. Mitochondria are gradually elongating. Spiralling chromatin of other spermatids is visible at right. X 12K, scale bar = 1 micron.

Fig. 21:



Stage C L. planaxis spermatid with golgi depositing pro- acrosomal granule and interstitial granule. X 48K, scale bar = 200nm.

Fig. 22:



Small electron dense granules both anterior and posterior to the L. planaxis Golgi. X 60K, scale bar = 100nm.

Fig. 23:



Nearly mature spermatozoons of L. littorea. Note acrosomal cone, acrosomal rod supporting it, central fibers issuing from the basal body along the flagellar shaft. The lower of the two spermatozoons is still shedding residual cytoplasm.X 96K, scale bar = 100nm.

Fig. 24:



Two nearly mature spermatozoons of L. littorea, note the flexibility of the acrosome where it meets the nurse cell. X 70K, scale bar = 100nm.

Fig. 25:



A developed acrosome meets a nurse cell. Notice the acrosomal vesicle and the remnants of the interstitial granule appearing disc-like below the acrosomal cone. The basal body is also visible. X153K, scale bar = 50 nm.

Fig. 26:



A close-up of figure 25 illustrating the small projections or "blebs" at the tip of the acrosome which form the connection between spermatozoon and nurse cell in all three species. From L. littorea. X 262K, scale bar = 50 nm.

Fig. 27:



The nuclear-mitochondrial junction in L. littorea. Note the pitch of the mitochondrial spiral, which strongly indicates the presence of five rather than four Nebenkerne. X 60K, scale bar = 200nm.

Fig. 28:



Nuclear-mitochondrial junction in L. planaxis. X 60K, scale bar = 200 nm.

Fig. 29:



Developing mitochondrial sheath of L. planaxis. Note the six mitochondria involved. Stage C spermatid of the same type is seen in longitudinal section at right. X22,500; scale bar = 400nm.

Fig. 30:



Two developing sheaths from L. littorea, Notice that one has seven, the other six mitochondria. In the sheath at right some mitochondria are in the process of fusing; notice the shared membrane (arrows). X48K, scale bar = 200nm.

Fig. 31:



Developing sheath with seven mitochondria, alongside transverse sections of lamellar nuclei and end pieces. X 31K, scale bar = 400nm.

Fig. 32:



Further development of the mitochondrial sheath in L. littorea. Seven mitochondria are shown here being reduced to the five present in the developed spermatozoon. No enveloping common membrane is yet visible. Two of the mitochondria appear to be getting forced out of the sheath in a manner more suggestive of sloughing off than of fusion. The condition of the cytoplasm suggests this could be a fixation artifact. X 35K, scale bar = 400nm.

Fig. 33:



Mitochondrial-tail junction in L. littorea, The ring centriole is visible as two dark spots on either side (arrows). Note the glycogen particles in the tail region, and the invagination of the plasma membrane at the site of the annulus. X 48 K, scale bar = 200nm.

Fig. 34:



Longitudinal view of the ring centriole, appearing as two dense bands. Nebenkerne spiral toward the centriole from lower left. X 60K, scale bar = 200nm.

Fig. 35:



Cross section through the nuclei of two L. littorea spermatids. Radial spokes are clearly visible radiating out from the central two microtubules. X 73K, scale bar = 200nm.

Fig. 36:



TS illustrating the stages of lamellar plate formation in developing Littorina sperm. Chromatin condenses into maze-like bands which later assemble into concentric rings in the stage D spermatid. X 31K, scale bar = 400nm.

Fig. 37:



Close up view of lamellar plate formation. The inner and outer dynein arms and the radial spokes can be clearly seen. X 91K, scale bar = 100nm.

Fig. 38:



Cross-section through various levels of developing sperm. The nuclear section shows a dense circle of chromatin (the nuclear tube) surrounding the 9+2 arrangement of microtubules encompassing the flagellum. The small microtubule outside the nuclear tube has been described in L. scutulata and now in L. littorea. However, the apparent vesicle along the inside of the nuclear tube (arrows) is in a different position and of a different extent than has been described for L. scutulata. Other sections represent typical cross sections through the mitochondrial and glycogen-rich tail regions. X 75K, scale bar = 200nm.

Fig. 39:



Cross section through the end piece of L. planaxis, below the glycogen containing tail piece. X 171K, scale bar = 100nm.

The general layout of the gonad and seminiferous tubule tissue is identical in all three species. Gonad tubules are found interlaced with digestive tissue in the visceral complex of the animals. Mature sperm bundles pass down the tubules into the seminiferous tubules, which appear as thick, white convoluted bands on the ventral surface of the animals at the base of the visceral complex.

Within the gonadal tubule, the pattern of cell division is also the same. In all three species the basal membrane of the tubule is lined with nurse cells which contain numerous lipid droplets. Nurse cells provide support and sustenance for the germ cells which will ultimately develop into mature spermatozoans. The developing sperm show a characteristic concentric organization within the tubule. As these cells divide and develop into spermatozoans, later developmental stages are forced inward toward the lumen of the tubule. Thus spermatogonia, the initial developmental stage, lie nearest the basement membrane at the periphery of the tubule. These divide mitotically to form primary spermatocytes, which are thereby crowded closer to the lumen of the tubule. Primary spermatocytes grow in preparation for the meiotic division which takes place in the transformation into secondary spermatocytes, which are again pushed further into the lumen of the tubule. Secondary spermatocytes divide mitotically to become spermatids, which themselves progress through four stages of maturation prior to being ultimately released into the seminiferous tubule as the nurse cell/sperm bundle known as the spermatozeugma. This general overview of spermatogenesis is shown pictorially in figures 1-6.

Although the process of spermatogenesis is essentially the same in the three species studied, the relative sizes of the tubules involved differs. The gonad tubules of Littorina scutulata and Littorina planaxis are approximately the same size, averaging from 80-140 micrometers in diameter. But the gonad tubule of Littorina littorea is significantly larger, averaging 160-200 micrometers in diameter. The differences in tubule size do not seem to be carried over into significant size differences among developing cell types. The cells in figures 7, 8, and 15 are all spermatogonia from Littorina Planaxis and Littorina littorea, and all show the same sized cell body and nucleus as described in Littorina scutulata and Littorina sitkang (Buckland-Nicks, 1974). Primary spermatocytes from Littorina littorea are again approximately ,the same size as those described for Littorina scutulata.They also show the same pattern of cell division, including the leptotene (where chromatin begins to condense, as in fig. 9) and diplotene (where chromosomes are paired, shown in fig. 10) stages of the first meiotic division. In certain cases some slight size differences were found in comparison to previously published reports (Buckland-Nicks,1974; Buckland-Nicks and Chia,1976). For example, the spermatogonia shown in figures 11 and 12 from Littorina planaxis and Littorina scutulata, respectively, have nuclei that are about 1 micrometer smaller than was expected. This could be anomalous or it could be an artificial result caused by an oblique sectioning plane. The primary spermatocytes in early zygotene shown in figure 13 are from Littorina planaxis, and are just as large as those from Littorina scutulata.. Figure 14 shows L. planaxis spermatogonia which appear rather gargantuan, but again this is an artifact due to the plane of sectioning.

The three species of Littorina discussed here show profound similarity throughout their development. The stage B spermatid of L. littorea for example is of an identical size and structure as that of both other species (fig. 16). The chromatin assumes its accustomed "doughnut-shape", and the mitochondria have migrated to the area outside the nucleus where they will later fuse into the mitochondrial sheath.

Another similarity lies in the maturation and ultimate fate of the nurse cell. In L. littorea, as in the other species, nurse cells line the gonad tubules and send out pseudopodia to contact ,developing spermatozoons. Having the same cytoplasmic inclusions as the pseudopodia of the other species, including lipid droplets, lysosomes, mitochondria, and glycogen (fig. 17), it is assumed that they provide the same functions of support, both physical and nutritional, that they do for the other species. As the nurse cell matures, it detaches from the wall of the tubule, loses its pseudopodia and becomes roughly spherical. Its nucleus begins to degrade, secretion droplets increase in size, and mature sperm remain attached to it only by their acrosomal caps (figs. 18,5,6).

The development of the acrosome begins in all three species in the stage C spermatid. At this stage the Golgi body, which has migrated to the apex of the nucleus, has released both the acrosomal and interstitial granules, which form the basis for the completed acrosome. The nucleus has a homogeneous appearance due to the evenly dispersed chromatin within it, and the mitochondria have assembled and started to elongate about the flagellar shaft. The mitochondria have not however begun to spiral or fuse together (figs. 19,20). Several small electron-dense granules are produced by the golgi of L. littorea both anteriorly and posteriorly, just as with the other species (Figs. 21,22). The mature acrosome in Littorina littorea appears to be smaller than that in Littorina scutulata, The developed acrosome of L. scutulata has been previously identified as being 0.7 micrometers in length (Buckland-Nicks, 1973), while that of L. littorea is only 0.5 micrometers long. The structure, however, remains identical, with the acrosomal cone supported by acrosomal ,rods and enclosed by an acrosomal membrane (Fig. 23). The acrosome in L. littorea is shown to have great flexibility as in the other species (Fig. 24), and retains the same connection to the nurse cell in the form of small "blebs" on the acrosomal tip (Fig. 25,26). As to the number of Nebenkerne involved in the formation of the flagellar shaft in L.littorea, only circumstantial evidence has been obtained here. It has previously been stated (BucklandNicks, 1973) that only four Nebenkerne are involved ultimately in the structure of the mitochondrial tube surrounding the flagellum of L. scutulata. Figure 27 shows the nuclear-mitochondrial junction in L. littorea. The pitch of the mitochondrial spiral shown here is more suggestive of a structure involving five, not four, Nebenkerne. However, measurements of the width of individual Nebenkerne as well as of the distance of the mitochondrial sheath from the nucleus (approximately 100 and 23 nanometers, respectively) agree well with the observations for L. scutulata (figs. 27,28).

The mitochondrial sheath is shown cross-sectionally during its development in figures 29-32. A cross section through the developing sheath of L. planaxis shows six mitochondria at that stage (Fig. 29). The developing sheath of L. littorea was observed in cross section to have as many as seven and as few as five mitochondria, but never fewer than five (Figs. 30-32). The final positioning and structure of the ring centriole or "annulus" is, as should be expected, identical among these species. Longitudinal sections reveal it as two dark spots in the invagination at the mitochondrial-tail junction (Fig. 33); in grazing sections it appears as two electron-dense bands (Fig. 34). The similarity between these species is also evident in cross sections down the length of the developing sperm.

In cross-sections through nearly developed sperm, the characteristic 9+2 arrangement of microtubules can be seen within the intranuclear canal, surrounded by condensed chromatin and residual cytoplasm that remains to be sloughed off (Fig. 35). In all three species the chromatin condenses into lamellar plates which are at first disorganized in form but later condense further into concentric rings (Figs. 36, 37).

Cross sections through the basal portion of the nucleus also reveal in L. littorea a single microtubule about 12 nanometers in diameter lying outside of the nuclear tube, exactly as was found in L. scutulata (Buckland-Nicks, 1974). Sections here also reveal however a vesicle overlying two of the outer nine doublets; this is a situation different from that described for L. scutulata (Fig. 38). Further down the flagellar shaft the cross-sectional appearance is the same for all three species. Sections are shown through the mitochondrial region and through the tail, where glycogen granules are present, as well as through the end piece, below the level at which glycogen is present (Figs. 38, 39).

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DISCUSSION

It is evident from the preceding that the process of spermatogenesis in Littorina scutulata, Littorina planaxis, and Littorina littorea is identical in most respects and similar in all, despite the geographical and phylogenetic separation of the species.

The general layout of the gonad is the same, as can be seen by comparing testicular cross sections in Figs. 1-3. Some differences do exist in the size of these tubules however, which is not surprising given that L. littorea is approximately three times the size of either of the other species.

All stages of early sperm development can be observed interchangeably in any of the three species. Minor differences in cell size seen in some micrographs are probably artifacts due to tangential orientation of the sectioning plane and cell shrinkages during fixation rather than actual differences. This conclusion is also supported by measurements taken from the light micrographs in Figs. 1 and 3. Although differences in nurse cell structure have been noted among some members of the Littorinidae, specifically L. angulifera (ref. Buckland-Nicks and Chia, 1977), none were found among these three species. Contents of both juvenile and mature nurse cells were identical among species. The unilateral arrangement of spermatids on the nurse cell forming the spermatozeugma was also retained throughout. The formation and appearance of the acrosome was also similar, although the acrosome of L. littorea appeared shorter than that of L. scutulata. It is not impossible that this is a spurious result based on the orientation of the acrosomes viewed, but it seems more likely that it reflects an actual difference. As to the number of mitochondrial constituents in the ,developed sheath, the present evidence strongly suggests that L. littorea possesses five Nebenkerne. Whether this uncovers a major difference between L. littorea and L. scutulata remains to be determined. One interesting note: Buckland-Nicks' assertion that L. scutulata possesses four Nebenkerne (Buckland-Nicks,1973) actually predates his doctoral thesis statement that it possesses "four, or five" Nebenkerne.

Given the degree of similarity in the process of spermatogenesis between the three species studied, it seems likely that all three species in fact posses five Nebenkerne. Other aspects of the developed sheath are unquestionably identical, including the presence in all species of an invagination at the mitochondria-tail junction that is important to flagellar motion and sperm motility. The fact that these closely related species are so similar in regards to spermatogenesis is not surprising. Their geographical and phylogenetic isolation is obviously not as important to this process as a number of other factors. Firstly, both the Atlantic and Pacific species occupy a similar environmental niche. L. littorea is an intertidal organism, while the others reside in the splash zone. Secondly, all practice internal fertilization, which minimizes environmental pressures on their mode of reproduction. Finally, it has long been known that differences in spermatogenesis between species in the same genus are generally much less important than their similarities.

More work is still to be done though, especially in regards to the number of Nebenkerne, the microtubulary structure, and the origin of the centrioles among all the Littorinidae.

Abbreviations Used:

ac = acrosomal cone; ag = acrosomal granule; am = acrosomal membrane; ar = acrosomal rod; bb = basal body; bm = basement membrane; cf = central fibers of flagellum; ch = chromatin; ctv = connective tissue vesicular cells; f = flagellum; g = golgi body; gy = glycogen; ig = interstitial granule; inc = intranuclear canal; li = lipid; ls =lysosome; m = mitochondrion; n = nucleus; nc = nurse cell; nu =nucleolus; pf = peripheral fibers of flagellum; pm = plasma membrane; rc = ring centriole; SC = spermatocyte; sd = secretion droplet; sg = spermatogonia; st = spermatids. Symbols are in both upper and lower case



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CITATIONS



1. Allen, Richard K., Common Intertidal Invertebrates of Southern California, Revised Edition, 1976, Peek Publications. Pg. 133, 160.

2. Berril, N.J., and Gerald Karp; Development, McGraw-Hill inc., 1976. PP. 80-92.

3. Buckland-Nicks, J.A.: The fine structure of the spermatozoan of Littorina (Gastropoda: Prosobranchia), with special reference to sperm motility. Z. Zellforsch. 144, 111-129 (l973).

4. Buckland-Nicks, J. A.; Thesis submitted to the Department of Zoology, University of Alberta, Canada; 1974.

5. Buckland-Nicks, J.A. and Chia, Fu-Shiang: spermatogenesis of a marine snail, Littorina sitkana. Cell and Tissue Res.; 170, 455- 475 (1976).

6. Buckland-Nicks, J. A. and Chia, Fuo-Shiang; ibid, 179, 347-356 (1977).

7. Jaramillo, Roberto, et al. : Ultrastructural analysis of spermiogenesis and sperm morphology in Chorus giganteus (Lesson, 1829)(Prosobranchia: Muricidae). The Veliger, 29(2):217-225 (1986).

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zedmason@csulb.edu