Burkhart 1

1 Ultrastructure of Spermiognesis in the Yellow-Bellied Sea , Pelamis platurus 2 (: 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 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 (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 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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