Burkhart 1
1 Ultrastructure of Spermiognesis in the Yellow-Bellied Sea Snake, Pelamis platurus 2 (Squamata: Elapidae: Hydrophiinae) 3
4 Brenna M. Burkhart
5
6 Department of Biology, Wittenberg University, Springfield, Ohio 45501
7 Number of pages: 24
8 Number of plates: 5
9 Short Title: Spermiogenesis in Pelamis platurus
10 *Correspondence: Kevin M. Gribbins, Department of Biology, Wittenberg University,
11 PO Box 720, Springfield, OH 45501-0720
12 Email: [email protected]
13 Burkhart 2
14 Abstract
15 Within the order Squamata, only a few studies have been completed on the
16 morphological characteristics of developing spermatids as they undergo spermiogenesis,
17 including a recent study in 2010 on Cottonmouths (Gribbins, et al., 2010). To date there have
18 been no studies on the spermiogenesis within the sea snakes of the subfamily Hydrophiinae that
19 consists of 17 genera and 62 species of venomous snakes. Testicular tissue samples of three
20 male Pelamis platurus were captured in Costa Rica in July of 2009. Cellular analysis, through
21 the use of light and transmission electron microscopy, was performed on developing spermatids
22 in the three phases of spermiogenesis: acrosome formation, nuclear elongation, and chromatin
23 condensation. Transmission electron microscopy was used to determine the ultrastructure of
24 these sperm cells for comparison with the other snakes studied to date. Spermatids of P. platurus
25 possesses some notable differences such as a more prominent central lacuna in the nucleus,
26 radiating arrays of the outer longitudinal manchette microtubules, and a shorter epinuclear lucent
27 zone when compared to the Cottonmouth and other snakes studied to date. The majority of the
28 spermatid morphology is conserved during the phases of spermiogenesis. The minute differences
29 that do exist in the Yellow-bellied Sea Snake spermatids may help us understand the
30 phylogenetics and evolution of aquatic snakes from their terrestrial ancestors. However, data on
31 snake spermatids is lacking at this time and many more species of snakes have to be studied
32 before we have a robust understanding of spermiogenesis in the taxa of squamates.
33 Key Words: spermiogenesis; ultrastructure; Pelamis platurus; germ cell development
34 Introduction
35 Taxonomists still widely disagree upon the classification of sea snakes in relation to the
36 Elapidae family. Sea snakes can be found in the literature in their own family, Hydrophiidae, but Burkhart 3
37 more commonly and recently are seen as a subfamily of the Elapidae family, Hydrophiinae (Ray-
38 Chaudhuri et al., 1971; Gutierrez and Bolanos, 1980). Because spermiogenesis is a very specific
39 biological tool that can aid in the determination of phylogenetic relationships, the analysis of the
40 ultrastructure of developing spermatids during spermiogenesis in sea snakes would be useful for
41 determining the classification of these snakes (Gribbins and Rheubert, 2011).
42 Pelamis platurus, the Yellow-bellied Sea Snake is the most widely distributed species of
43 sea snake, and for this reason was chosen for completing the first complete study in the
44 Hydrophiinae subfamily on the ultrastructure of developing spermatids (Sever, et al., 2012). The
45 intraabdominal testis of these snakes are closely associated with the kidneys, as with most
46 reptiles (Sever and Freeborn, 2012). The germ cell development takes place within the male
47 reproductive tract and spermatogenesis occurs in the seminiferous tubules of the testis.
48 Spermiogenesis is the last phase of spermatogenesis and the longest part of sperm
49 development. Spermiogesis consists of three phases and includes acrosome vesicle
50 development, nuclear elongation and flagellar formation, and condensation of the DNA within
51 the nucleus. The only complete ultrastructural study that currently exists of this developmental
52 process in a snake was completed on the Cottonmouths, Agkistrodon piscivorus (Gribbins, et al.,
53 2010).
54 Spermiogenic events and morphologies should lead to characteristics displayed in the
55 mature spermatozoa, such as the acrosome, perforatorium, and flagellum. Analysis of these
56 characteristics can lead to the identification of species-specific structures that could be used to
57 enhance phylogenetic matrices (Gribbins and Rheubert, 2011). An understanding of spermatid
58 development ultrastructurally could provide a robust morphological matrix that could be Burkhart 4
59 combined with our current understanding of sperm ultrastructure to perform preliminary
60 phylogenetic and toxicological analysis on spermatogenesis in snakes and squamates.
61 Spermiogenic ultrastructure as a histopathological tool for the study of heavy metal
62 poisoning in the marine and freshwater environments is another possibility for such data
63 (Gribbins and Rheubert, 2011). Due to bioaccumulation and the abundant amounts of lipids in
64 the gonads of snakes, abnormalities in the mature or developing sperm cells may provide
65 indication to an unhealthy marine environment (Haubruge, et al., 2000). Additionally, many
66 organisms experience sertoli cell apoptosis when subjected to pollutants in the gonads, leading to
67 a decreased sperm count (Haubruge, et al., 2000).
68 Thus, the aim of this study is to provide the first complete ultrastructural analysis of
69 developing spermatids within a snake from the subfamily Hydrophiinae. A thorough description
70 of the developing spermatids is provided with a focus on the three phases of spermiogenesis.
71 From the present spermiogenic characteristics a comparison can be made with the morphology of
72 spermatids in the Cottonmouths for a better understanding of their relationships to one another
73 within Viperidae. With additional research in the Hydrophiinae subfamily, an increased
74 phylogenetic tree could theoritically be created for true sea snakes and possibly other closely
75 related squamates. The results of this study will not only be the first published literature and data
76 on spermiogenesis within the sea snakes, but these data will help to increase the understanding
77 of germ cell development in squamates as a whole.
78 Lastly, toxicological examination could be performed of the coral reef environment based
79 upon the presence of pollutants and their effect on the testis and spermiogenesis in Yellow-
80 bellied Sea Snakes. These sea snakes are widely dispersed among tropical pacific ocean
81 ecosystems and would be easily accessible for study. Since coral reefs are highly endangered Burkhart 5
82 ecosystems, with more than fifty percent of the surviving reefs today at risk of collapse (Bonnet,
83 2012), spermiogenesis is an excellent way to monitor pollutant concentrations in the ocean
84 environment over time.
85 Materials and Methods
86 Animal Collection and Dissection
87 Three male Pelamis platurus were collected on July 10, 2009 approximately 12
88 kilometers south of Playa del Coco, off of the coast of Costa Rica through the use of dip nets,
89 were placed in large bins full of seawater for no more than 12 hours, and euthanized by a lethal
90 injection of 10 % sodium pentobarbital in 70 % ethanol (Sever and Freeborn, 2012).
91 Reproductive tracts were removed and the left reproductive tracts were placed in 10 % neutral
92 buffered formalin (NBF) for light microscopy. The right reproductive tracts were placed in
93 Trump’s fixative in 0.1M sodium cacodylate buffer for transmission electron microscopy (Sever
94 and Freeborn, 2012).
95 Tissue Preparation
96 Tissues that were fixed in the Trump’s fixative by Dr. David Sever were rinsed with DI
97 water, postfixed in 2 % osmium tetroxide, and dehydrated through the use of ethanol series. The
98 samples were cleaned in propylene oxide and then embedded in epoxy resin (Sever and
99 Freeborn, 2012). The embedded blocks were then sent to Dr. Kevin Gribbins and Brenna
100 Burkhart for sectioning, staining, and analysis of the spermiogenesis.
101 Ultrastructural Analysis
102 Samples were first viewed with light microscopy after sectioning with a glass knife and a
103 Leica UC7 Ultramicrotome. This allowed for the determination of reproductive activity for the
104 snake tissue samples and allowed for the determination of whether spermiogenesis was occurring Burkhart 6
105 at this time. Confirmation was also provided through this analysis that there were seminiferous
106 tubules in the tissue sample and not a duct of the epididymis.
107 To perform ultrastructural analysis of the tissues with the use of transmission electron
108 microscopy, tissues were sectioned with a Leica UC7 ultramicrotome and a diamond knife to
109 create 90 nm sections for TEM (Gribbins, et al., 2010). Sections were then placed on copper
110 grids and stained with uranyl acetate and lead citrate. These tissue samples were then viewed
111 using a Jeol JEM-1200EX II transmission electron microscope (Jeol, USA). Micrographs were
112 also taken of spermatids and their ultrastructural components through the use of a Gatan 785
113 Erlangshen digital camera. Lastly, analysis took place using Adobe Photoshop CS, which also
114 was utilized to create composite plates and to perform analysis of the spermatid characteristics.
115 Results
116 Inside the testis are coiled seminiferous tubules that are surrounded by a tunica albuginea
117 connective tissue layer (Gribbins and Rheubert, 2011). The space between the tubules is filled
118 with interstitial cells, blood vessels, leukocytes, collagen fibers, and lymph (Gribbins et al.,
119 2010). The seminiferous tubules are continuous with the anterior ducts, where sperm exit the
120 testis. The anterior testicular ducts are responsible for transporting sperm from the seminiferous
121 tubules to the ductus deferens (Sever and Freeborn 2012). Inside the seminiferous tubules is a
122 hollow, centrally located lumen, which is where mature sperm cells are spermiated. The tubules
123 are lined with seminiferous epithelia that is made of Sertoli cells and is where spermatogenic
124 development occurs.
125 Spermiogenesis takes place within the seminiferous tubules of the testis and specifically
126 in the seminiferous epithelium. This epithelium is highly layered with developing germ cells and
127 at any point in time there can be up to five layers of germ cells containing eight or nine Burkhart 7
128 generations of spermatids (Gribbins and Rheubert, 2011). Developing spermatids are closely
129 associated with the Sertoli cells that are located in the seminiferous epithelium and they provide
130 nutrients to the developing spermatids (Gribbins and Rheupert, 2011). Sertoli cells can increase
131 their contact with developing sperm cells through desmosome junctions and their long
132 cytoplasmic processes that they wrap around developing cells. This increases their contact with
133 the developing cells and helps them to provide nutrient and energy molecules to the spermatids.
134 As the sperm cells mature they are pushed towards the centrally located lumen, or hollow cavity
135 within the tubules, where they are released during spermiation. The earliest spermatids are found
136 along the outermost parts of the seminiferous tubules, while the mature spermatids can be found
137 centrally.
138 The acrosome is the structure on the mature sperm cell that is responsible for the release
139 of enzymes to aid the sperm in breaking down the egg layers. These enzymes help the sperm to
140 penetrate and fertilize the female egg. During acrosome formation secretory vesicles are released
141 from the Golgi apparatus (Fig 1A,D) and they begin to fuse with one another to form the
142 acrosome vesicle near the nucleus, as seen in Figure 1A. As more transport vesicles fuse with
143 one another, the acrosome will grow in size (Gribbins and Rheubert, 2011). An acrosome
144 granule will also form and it results from the concentration of the proteins inside the transport
145 vesicles of the Golgi. The granule is centrally and basally located near the nucleus of the cell
146 (Gribbins, et al., 2010). The granule can be seen in Pelamis platurus after the acrosome vesicle
147 fuses with the nucleus, as in Fig. 1B. The nuclear indentation that results from the fusion of the
148 acrosome vesicle and the nucleus is a characteristic of spermiogenesis in all vertebrates
149 (Gribbins and Rheubert, 2011). It also leads to the formation of a subacrosomal space, which Burkhart 8
150 can be seen in the mature spermatozoa and in Fig. 3E. The subacrosomal space is the gap
151 between the nuclear membrane and the acrosomal vesicle membrane.
152 Also, the principle piece of the flagellum can be seen in Fig. 1C, and it displays how
153 short the flagellum is at this stage of maturation. The proximal and distal centrioles can be seen
154 at the base of the principle piece. Early dense collar proteins also exist at this stage of
155 maturation, which are structures that help to connect the flagellum to the nucleus of the cell.
156 Following acrosome formation and the fusion of the nucleus and the acrosome vesicle,
157 nuclear elongation takes place in the spermatids of Pelamis platurus (Fig. 2). The acrosome
158 vesicle now becomes the acrosome complex and starts to flatten and envelop the nuclear head.
159 The nucleus begins to move apically and becomes cylinder shaped. The nucleus can reach
160 lengths of up to 30 micrometers or more in length within reptiles. This longitudinal growth is
161 aided by a structure called the manchette, seen in Fig. 3F,G. The manchette is a series of parallel
162 and circumcylindrical fibers that run along and around the nucleus. These fibers provide added
163 structure for the cell and aid in the longitudinal growth of the nucleus. For the spermatids of
164 Pelamis platurus, the manchette possesses radiating arrays of the outer longitudinal manchette
165 that vary from those of the Cottonmouth. The manchette fibers can be seen in Fig. 3 surrounding
166 the nucleus but they are not found around the acrosome complex, which reaffirms that it is a
167 structure solely for the nucleus and nuclear growth.
168 Additionally the chromatin begins to become very condensed and it can be seen in both
169 the sagittal section of the cell (Fig. 2A) and the cross-sectional cut of the cell (Fig. 2B). Nuclear
170 lacuna can also be seen in Figure 2B as well. One difference between the maturing cells of P.
171 platurus and the Cottonmouth is the presence of a prominent central lacuna. Burkhart 9
172 Step 5 spermatids are just beginning nuclear elongation. They are not yet
173 compartmentalized or at maximum length, which is better seen in Fig 3. The perforatorium is
174 hypothesized to be a structure that contains supportive fibers that allow the nucleus to penetrate
175 the egg cell once the acrosome breaks down the egg layers with enzymes. Through actin
176 filaments, the perforatorium can push through the cell membrane in order to release the genetic
177 information from the sperm into the egg, thus allowing for fertilization of the egg.
178 During late nuclear elongation flagellar development takes place. The flagella display
179 the 9+2 microtubule arrangement, that is seen in most squamates, and it evident in the cross
180 sectional photos of Fig. 4. Additionally, most reptiles possess enlarged peripheral fibers located
181 at doublets 3 and 8 that are laterally located along the flagellum. They are most easily seen in
182 Fig. 4C and D. They aid in the stability of the flagella. Also during late nuclear elongation, the
183 mitochondria will begin to concentrate along the sides of the flagella. These mitochondria
184 provide ATP that is essential for the movement of the flagella and ultimately the motility of the
185 sperm in the female reproductive tract. Mitochondrial ratios can differ between species of
186 snakes, which is one way that spermiogenesis can be used for comparison between different
187 species of snake.
188 Located at the top of the flagella are proximal and distal centrioles, which can be seen in
189 Figure 4 at 90 degree angles to one another. They help to connect the flagellum to the nucleus
190 and are surrounded by dense collar protiens that also serve as structural elements that help the
191 flagella to stay attached to the nucleus. The fibrous sheath can be seen in these sagittal sections,
192 which are a series of circum-cylindrical fibers that surround the flagellar microtubules. They act
193 as protective and supportive structures and can be found in the midpiece and principle piece. Burkhart 10
194 The end piece can easily be identified from the rest of the flagellum because it does not
195 possess the fibrous sheath around the axoneme, which is visible in Figure 4. The midpiece is
196 characterized by the presence of mitochondria and dense bodies that surround the flagella
197 (Gribbins and Rheubert, 2011). Mitochondria provide ATP and the dense bodies can provide
198 energy molecules and nutrients to the flagella as it moves. Cross-sections of the flagella are
199 shown in Figure 5. The most anterior cross section is D and they then progress down the
200 flagella’s midpiece (C), principle piece (B), and endpiece (A).
201 The last phase of spermiogenesis is chromatin condensation (Gribbins, et al., 2007).
202 Although chromatin continually condenses throughout spermiogenesis, a large amount of the
203 chromatin becomes highly concentrated after nuclear elongation. Figure 2 shows chromatin
204 condensation taking place during a step five spermatid in the nuclear elongation phase. The
205 condensation is very prominent also during the late nuclear elongation phase. Chromatin will
206 condense in a spiral fashion, allowing excess nucleoplasm to be reduced (Gribbins and Rheubert,
207 2011). The sperm cell will then reduce the cytosol and excess material to gain a more
208 hydrodynamic shape and to allow the sperm cell to more easily navigate through the female
209 reproductive tract.
210 At this point in maturation, sperm cells gain their characteristic filliform shape for
211 reptiles (Gribbins, et al., 2010). The spermatids will simply become curved in shape and are
212 released as mature spermatozoa to the lumina of the seminiferous tubules. Mature sperm will exit
213 the testis through the anterior ducts, which are continuous with the lumen of the seminiferous
214 tubules. They then continue to the epididymis, where they will remain until ejaculation. The
215 ductus deferens allows the sperm cells to exit the rostral and caudal epididymis and to continue
216 to the urethra, exemplifying one reason for the snake testis close association with the kidney Burkhart 11
217 (Gribbins and Rheubert, 2011). Then the cells will combine with fluid from the kidney and
218 seminal vesicles to create semen. The semen travels to the ampulla and then out the hemi-penis
219 upon ejaculation (Gribbins and Rheupert, 2011).
220 Discussion
221 Through ultrastructural analysis a complete study of the spermiogenesis in Pelamis
222 platurus was completed. This study significantly contributes to the little published research that
223 exists on snakes and squamates for spermiogenesis. For the subfamily Hydrophiinae, this is the
224 first study completed for spermiogenesis.
225 Each of the phases of spermiogenesis were observed with transmission electron
226 microscopy. Acrosome formation, nuclear elongation, and chromatin condensation appeared to
227 be highly conserved between the P. platurus, Cottonmouth snakes, and other squamates. There
228 were only a few differences in the morphology of the spermatids in this sea snake and this was
229 expected of snakes that are not closely related to one another.
230 During the ultrastructural analysis several defining characteristics for the species were
231 identified when comparing the observations to spermiogenesis of Cottonmouths. Although a lot
232 of the mature spermatozoa structure was conserved, three prominent differences were found
233 between the two species. The sea snakes possessed a shorter epinuclear lucent zone, radiating
234 arrays of the manchette, and a prominent central nuclear lacuna that was not observed in the
235 Cottonmouth.
236 Some of these character differences could be useful in future phylogenetic studies
237 between snakes if more spermiogenic data is completed for snakes and other squamates in the
238 near future. With additional ultrastructural analysis of spermiogenesis in the family
239 Hydrophiinae, a better understanding of the relationship between sea snakes and sea kraits could Burkhart 12
240 also occur. Studies of snakes in the Elapinae subfamily of terrestrial snakes may also help to
241 determine if true sea snakes should be placed in a subfamily of the family Elapidae or if they
242 should be placed in their own family.
243 There are very few articles to compare the results of this study. As a result, most of the
244 differences between the Cottonmouths and sea snakes are not definitive defining characteristics
245 between these two species. With an increase in research the importance of these characteristics
246 will become more evident once other species are studied for spermiogenic characters. With the
247 completion of spermiogenetic analysis of additional snakes in the Elapidae family a better
248 understanding of the relationship between sea snakes, sea kraits, and terrestrial snakes including
249 cobras could be determined.
250 Spermiogenesis can also be used for toxicology. Due to the fact that sea snakes inhabit
251 the marine environment, particularly coral reefs, an increase in the research of spermiogenesis
252 could provide toxicological information on how pollutants affect spermiogenesis.
253 Bioaccumulation is a phenomena that occurs through the accumulation of toxins like heavy
254 metals and pesticides in the lipids of organisms. As sea snakes ingest more contaminated prey
255 items, these harmful toxins can accumulate and magnify in their lipids and specifically their
256 gonads (where lipid content is high). Since the testis have a lot of lipids for providing energy
257 and the production of hormones, they are frequently subjected to pollution and the accumulation
258 of toxins (Haubruge, et al., 2000).
259 Until more research is completed, we lack knowledge of the implications that pollutants
260 and bioaccumulation have on spermiogenesis. With additional research on more individual males
261 of P. platurus and other sea snakes, intraspecies differences in spermiogenesis and genera
262 differences could potentially be examined. Sperm cells of individuals can be compared with one Burkhart 13
263 another to determine which characters may give an individual male a reproductive advantage
264 over others when it comes to mating and sperm. The hope is that this study will provide the
265 basic framework by which other scientist can study spermiogenesis in other snake species to help
266 provide data for the questions posed within this study.
267 Acknowledgements
268 Dr. Kevin Gribbins and Brenna Burkhart would like to thank Dr. David Sever for providing the
269 tissue samples for analysis. Additionally, we would like to thank Wittenberg University and
270 Saint Louis University for funding this research project and for providing the necessary
271 equipment to complete this research.
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378 Figure Legends
379 Figure 1: The acrosome formation of spermatids in early spermiogenesis takes place in the
380 seminiferous epithelium. (A) The acrosome vesicle (AV) begins to form near the nucleus (NU).
381 (D) The Golgi apparatus (Black Arrow) can be seen with the highly folded cisternae releasing
382 secretory vesicles (White Star) that fuse to become the acrosome vesicle. (B) When more
383 transport vesicles from the Golgi begin to fuse with the acrosome vesicle it grows in size and due
384 to the proximity with the nucleus it makes contact with the nuclear surface. As the proteins
385 concentrate within the acrosome vesicle from the Golgi, the acrosome granule forms, and is
386 marked by the white triangle. The mitochondria (Black Triangle) can also be seen near the
387 nucleus. (C) The principle piece, consisting of a proximal (PC) and distal centriole (DC), can be
388 seen and is relatively small compared to the nucleus. The dense collar proteins (White Ring) can
389 also be seen in the principle piece and can be observed in the mature spermatozoa. Flagellar
390 development has not yet seen in these spermatids.
391
392 Figure 2: Step 5 spermatids can be seen in these micrographs and display the early steps of
393 nuclear elongation. (A) A sagittal section of a developing elongating spermatid can be viewed.
394 The nucleus appears to be rod-shaped. The acrosome vesicle (AV), condensing chromatin (CC),
395 and nuclear shoulders (NS) are also visible in this elongating cell. The acrosome vesicle is also
396 enveloping the nuclear apex at this step. (B) A cross-sectional cut of the spermatid reveals
397 nuclear lacuna (NL) along with the condensing chromatin. There is a prominent central lacuna
398 that is also evident (B).
399 Burkhart 19
400 Figure 3: Nuclear elongation is characterized by high amounts of compartmentalization. Cross-
401 sections of the nucleus are represented by photos A-C and D-F. (A) represents the top-most
402 cross-section of the tip of the acrosome complex, running towards the nucleus through (C) and
403 continuing down the acrosome complex (D) until reaching the nucleus and machette only in (F).
404 The acrosome vesicle (AV) and sertoli cell (SC) can be seen in (A). In (B) the perforatorium
405 (PE) can also be seen centrally located in the acrosome vesicle. The sertoli cell membrane
406 (SCM), subacrosome space (SAS), epinuclear lucent zone (ELZ), acrosomal lucent ridge (ALR)
407 can all be seen in (C). (D) represents a sagittal cut of the acrosome complex (AC) in which the
408 sertoli cell can be seen, the perforatorium, the basal plate (BP), the epinuclear lucent zone, the
409 acrosomal lucent ridge (ALR), and the acrosomal vesicle shoulder (AVS) can all also be seen
410 within the acrosome complex. (G) shows a sagittal section of the elongating nucleus with high
411 amounts of compartmentalization in the acrosome complex The peak of nuclear elongation
412 occurs when the acrosome fully envelops the nuclear apex, as is most evident in Fig. 3, photo G.
413 The high amounts of compartmentalization are evident in Fig. 3, photo D and G.
414
415 Figure 4: This micrograph shows the flagellum of developing spermatids in sagittal cuts. The
416 midpiece (MP) is the part of the flagellum that contains mitochondria and a fibrous sheath
417 around the microtubules. Dense collar proteins (White Ring) can be seen in (A) and (B). The
418 proximal centriole (PC) is more anteriorly located and closer to the nucleus than the distal
419 centriole (DC). The annulus (AN) is located at the end of the midpiece and marks the beginning
420 of the principle piece (PP). The principle piece is after the midpiece and is only surrounded by a
421 fibrous sheath. Last is the endpiece (EP) that does not possess a fibrous sheath or mitochondria
422 and dense bodies. It is located furthest from the nucleus. Burkhart 20
423 Figure 5: Cross-sectional cuts of the flagellum can be seen in this figure. (A) is a cross-
424 sectional cut that is in the end piece of the flagella. This is evident in the photo because there is
425 not a fibrous sheath (FS) surrounding the microtubule doublets and singlets. In (A) the enlarged
426 peripheral fibers are marked at doublets 3 and 8. (B) this is a cross-sectional cut of the principle
427 piece of the flagella. This section is marked by the presence of a fibrous sheath surrounding the
428 microtubule doublets and singlets. (C) shows a cross-section of the midpiece, which possesses a
429 fibrous sheath, but also mitochondria (Black Triangle) and dense bodies (DB) around the fibrous
430 sheath. (D) shows the most anteriorly located cross-section, that contains a dense collar protien
431 that is represented by the white ring. Additionally the peripheral fibers (PF) can be seen
432 surrounding the microtubules.
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445 Burkhart 21
446 Plate 1:
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455 Burkhart 22
456 Plate 2:
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467 Burkhart 23
468 Plate 3:
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470 Plate 4:
471 Burkhart 24
472 Plate 5:
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