Ultrastructure of Spermiognesis in the Yellow-Bellied Sea Snake, Pelamis platurus (Squamata: Elapidae: Hydrophiinae) Brenna M.

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1 Burkhart Ultrastructure of Spermiognesis in the Yellow-Bellied Sea Snake, Pelamis platurus (Squamata: Elapidae: Hydrophiinae) Brenna M. Burkhart Department of Biology, Wittenberg University, Springfield, Ohio Number of pages: 24 Number of plates: 5 Short Title: Spermiogenesis in Pelamis platurus *Correspondence: Kevin M. Gribbins, Department of Biology, Wittenberg University, PO Box 720, Springfield, OH kgribbins@wittenberg.edu 13

2 Burkhart Abstract Within the order Squamata, only a few studies have been completed on the morphological characteristics of developing spermatids as they undergo spermiogenesis, including a recent study in 2010 on Cottonmouths (Gribbins, et al., 2010). To date there have been no studies on the spermiogenesis within the sea snakes of the subfamily Hydrophiinae that consists of 17 genera and 62 species of venomous snakes. Testicular tissue samples of three male Pelamis platurus were captured in Costa Rica in July of Cellular analysis, through the use of light and transmission electron microscopy, was performed on developing spermatids in the three phases of spermiogenesis: acrosome formation, nuclear elongation, and chromatin condensation. Transmission electron microscopy was used to determine the ultrastructure of these sperm cells for comparison with the other snakes studied to date. Spermatids of P. platurus possesses some notable differences such as a more prominent central lacuna in the nucleus, radiating arrays of the outer longitudinal manchette microtubules, and a shorter epinuclear lucent zone when compared to the Cottonmouth and other snakes studied to date. The majority of the spermatid morphology is conserved during the phases of spermiogenesis. The minute differences that do exist in the Yellow-bellied Sea Snake spermatids may help us understand the phylogenetics and evolution of aquatic snakes from their terrestrial ancestors. However, data on snake spermatids is lacking at this time and many more species of snakes have to be studied before we have a robust understanding of spermiogenesis in the taxa of squamates. Key Words: spermiogenesis; ultrastructure; Pelamis platurus; germ cell development Introduction Taxonomists still widely disagree upon the classification of sea snakes in relation to the Elapidae family. Sea snakes can be found in the literature in their own family, Hydrophiidae, but

3 Burkhart more commonly and recently are seen as a subfamily of the Elapidae family, Hydrophiinae (Ray- Chaudhuri et al., 1971; Gutierrez and Bolanos, 1980). Because spermiogenesis is a very specific biological tool that can aid in the determination of phylogenetic relationships, the analysis of the ultrastructure of developing spermatids during spermiogenesis in sea snakes would be useful for determining the classification of these snakes (Gribbins and Rheubert, 2011). Pelamis platurus, the Yellow-bellied Sea Snake is the most widely distributed species of sea snake, and for this reason was chosen for completing the first complete study in the Hydrophiinae subfamily on the ultrastructure of developing spermatids (Sever, et al., 2012). The intraabdominal testis of these snakes are closely associated with the kidneys, as with most reptiles (Sever and Freeborn, 2012). The germ cell development takes place within the male reproductive tract and spermatogenesis occurs in the seminiferous tubules of the testis. Spermiogenesis is the last phase of spermatogenesis and the longest part of sperm development. Spermiogesis consists of three phases and includes acrosome vesicle development, nuclear elongation and flagellar formation, and condensation of the DNA within the nucleus. The only complete ultrastructural study that currently exists of this developmental process in a snake was completed on the Cottonmouths, Agkistrodon piscivorus (Gribbins, et al., 2010). Spermiogenic events and morphologies should lead to characteristics displayed in the mature spermatozoa, such as the acrosome, perforatorium, and flagellum. Analysis of these characteristics can lead to the identification of species-specific structures that could be used to enhance phylogenetic matrices (Gribbins and Rheubert, 2011). An understanding of spermatid development ultrastructurally could provide a robust morphological matrix that could be

4 Burkhart combined with our current understanding of sperm ultrastructure to perform preliminary phylogenetic and toxicological analysis on spermatogenesis in snakes and squamates. Spermiogenic ultrastructure as a histopathological tool for the study of heavy metal poisoning in the marine and freshwater environments is another possibility for such data (Gribbins and Rheubert, 2011). Due to bioaccumulation and the abundant amounts of lipids in the gonads of snakes, abnormalities in the mature or developing sperm cells may provide indication to an unhealthy marine environment (Haubruge, et al., 2000). Additionally, many organisms experience sertoli cell apoptosis when subjected to pollutants in the gonads, leading to a decreased sperm count (Haubruge, et al., 2000). Thus, the aim of this study is to provide the first complete ultrastructural analysis of developing spermatids within a snake from the subfamily Hydrophiinae. A thorough description of the developing spermatids is provided with a focus on the three phases of spermiogenesis. From the present spermiogenic characteristics a comparison can be made with the morphology of spermatids in the Cottonmouths for a better understanding of their relationships to one another within Viperidae. With additional research in the Hydrophiinae subfamily, an increased phylogenetic tree could theoritically be created for true sea snakes and possibly other closely related squamates. The results of this study will not only be the first published literature and data on spermiogenesis within the sea snakes, but these data will help to increase the understanding of germ cell development in squamates as a whole. Lastly, toxicological examination could be performed of the coral reef environment based upon the presence of pollutants and their effect on the testis and spermiogenesis in Yellowbellied Sea Snakes. These sea snakes are widely dispersed among tropical pacific ocean ecosystems and would be easily accessible for study. Since coral reefs are highly endangered

5 Burkhart ecosystems, with more than fifty percent of the surviving reefs today at risk of collapse (Bonnet, 2012), spermiogenesis is an excellent way to monitor pollutant concentrations in the ocean environment over time. Materials and Methods Animal Collection and Dissection Three male Pelamis platurus were collected on July 10, 2009 approximately 12 kilometers south of Playa del Coco, off of the coast of Costa Rica through the use of dip nets, were placed in large bins full of seawater for no more than 12 hours, and euthanized by a lethal injection of 10 % sodium pentobarbital in 70 % ethanol (Sever and Freeborn, 2012). Reproductive tracts were removed and the left reproductive tracts were placed in 10 % neutral buffered formalin (NBF) for light microscopy. The right reproductive tracts were placed in Trump s fixative in 0.1M sodium cacodylate buffer for transmission electron microscopy (Sever and Freeborn, 2012). Tissue Preparation Tissues that were fixed in the Trump s fixative by Dr. David Sever were rinsed with DI water, postfixed in 2 % osmium tetroxide, and dehydrated through the use of ethanol series. The samples were cleaned in propylene oxide and then embedded in epoxy resin (Sever and Freeborn, 2012). The embedded blocks were then sent to Dr. Kevin Gribbins and Brenna Burkhart for sectioning, staining, and analysis of the spermiogenesis. Ultrastructural Analysis Samples were first viewed with light microscopy after sectioning with a glass knife and a Leica UC7 Ultramicrotome. This allowed for the determination of reproductive activity for the snake tissue samples and allowed for the determination of whether spermiogenesis was occurring

6 Burkhart at this time. Confirmation was also provided through this analysis that there were seminiferous tubules in the tissue sample and not a duct of the epididymis. To perform ultrastructural analysis of the tissues with the use of transmission electron microscopy, tissues were sectioned with a Leica UC7 ultramicrotome and a diamond knife to create 90 nm sections for TEM (Gribbins, et al., 2010). Sections were then placed on copper grids and stained with uranyl acetate and lead citrate. These tissue samples were then viewed using a Jeol JEM-1200EX II transmission electron microscope (Jeol, USA). Micrographs were also taken of spermatids and their ultrastructural components through the use of a Gatan 785 Erlangshen digital camera. Lastly, analysis took place using Adobe Photoshop CS, which also was utilized to create composite plates and to perform analysis of the spermatid characteristics. Results Inside the testis are coiled seminiferous tubules that are surrounded by a tunica albuginea connective tissue layer (Gribbins and Rheubert, 2011). The space between the tubules is filled with interstitial cells, blood vessels, leukocytes, collagen fibers, and lymph (Gribbins et al., 2010). The seminiferous tubules are continuous with the anterior ducts, where sperm exit the testis. The anterior testicular ducts are responsible for transporting sperm from the seminiferous tubules to the ductus deferens (Sever and Freeborn 2012). Inside the seminiferous tubules is a hollow, centrally located lumen, which is where mature sperm cells are spermiated. The tubules are lined with seminiferous epithelia that is made of Sertoli cells and is where spermatogenic development occurs. Spermiogenesis takes place within the seminiferous tubules of the testis and specifically in the seminiferous epithelium. This epithelium is highly layered with developing germ cells and at any point in time there can be up to five layers of germ cells containing eight or nine

7 Burkhart generations of spermatids (Gribbins and Rheubert, 2011). Developing spermatids are closely associated with the Sertoli cells that are located in the seminiferous epithelium and they provide nutrients to the developing spermatids (Gribbins and Rheupert, 2011). Sertoli cells can increase their contact with developing sperm cells through desmosome junctions and their long cytoplasmic processes that they wrap around developing cells. This increases their contact with the developing cells and helps them to provide nutrient and energy molecules to the spermatids. As the sperm cells mature they are pushed towards the centrally located lumen, or hollow cavity within the tubules, where they are released during spermiation. The earliest spermatids are found along the outermost parts of the seminiferous tubules, while the mature spermatids can be found centrally. The acrosome is the structure on the mature sperm cell that is responsible for the release of enzymes to aid the sperm in breaking down the egg layers. These enzymes help the sperm to penetrate and fertilize the female egg. During acrosome formation secretory vesicles are released from the Golgi apparatus (Fig 1A,D) and they begin to fuse with one another to form the acrosome vesicle near the nucleus, as seen in Figure 1A. As more transport vesicles fuse with one another, the acrosome will grow in size (Gribbins and Rheubert, 2011). An acrosome granule will also form and it results from the concentration of the proteins inside the transport vesicles of the Golgi. The granule is centrally and basally located near the nucleus of the cell (Gribbins, et al., 2010). The granule can be seen in Pelamis platurus after the acrosome vesicle fuses with the nucleus, as in Fig. 1B. The nuclear indentation that results from the fusion of the acrosome vesicle and the nucleus is a characteristic of spermiogenesis in all vertebrates (Gribbins and Rheubert, 2011). It also leads to the formation of a subacrosomal space, which

8 Burkhart can be seen in the mature spermatozoa and in Fig. 3E. The subacrosomal space is the gap between the nuclear membrane and the acrosomal vesicle membrane. Also, the principle piece of the flagellum can be seen in Fig. 1C, and it displays how short the flagellum is at this stage of maturation. The proximal and distal centrioles can be seen at the base of the principle piece. Early dense collar proteins also exist at this stage of maturation, which are structures that help to connect the flagellum to the nucleus of the cell. Following acrosome formation and the fusion of the nucleus and the acrosome vesicle, nuclear elongation takes place in the spermatids of Pelamis platurus (Fig. 2). The acrosome vesicle now becomes the acrosome complex and starts to flatten and envelop the nuclear head. The nucleus begins to move apically and becomes cylinder shaped. The nucleus can reach lengths of up to 30 micrometers or more in length within reptiles. This longitudinal growth is aided by a structure called the manchette, seen in Fig. 3F,G. The manchette is a series of parallel and circumcylindrical fibers that run along and around the nucleus. These fibers provide added structure for the cell and aid in the longitudinal growth of the nucleus. For the spermatids of Pelamis platurus, the manchette possesses radiating arrays of the outer longitudinal manchette that vary from those of the Cottonmouth. The manchette fibers can be seen in Fig. 3 surrounding the nucleus but they are not found around the acrosome complex, which reaffirms that it is a structure solely for the nucleus and nuclear growth. Additionally the chromatin begins to become very condensed and it can be seen in both the sagittal section of the cell (Fig. 2A) and the cross-sectional cut of the cell (Fig. 2B). Nuclear lacuna can also be seen in Figure 2B as well. One difference between the maturing cells of P. platurus and the Cottonmouth is the presence of a prominent central lacuna.

9 Burkhart Step 5 spermatids are just beginning nuclear elongation. They are not yet compartmentalized or at maximum length, which is better seen in Fig 3. The perforatorium is hypothesized to be a structure that contains supportive fibers that allow the nucleus to penetrate the egg cell once the acrosome breaks down the egg layers with enzymes. Through actin filaments, the perforatorium can push through the cell membrane in order to release the genetic information from the sperm into the egg, thus allowing for fertilization of the egg. During late nuclear elongation flagellar development takes place. The flagella display the 9+2 microtubule arrangement, that is seen in most squamates, and it evident in the cross sectional photos of Fig. 4. Additionally, most reptiles possess enlarged peripheral fibers located at doublets 3 and 8 that are laterally located along the flagellum. They are most easily seen in Fig. 4C and D. They aid in the stability of the flagella. Also during late nuclear elongation, the mitochondria will begin to concentrate along the sides of the flagella. These mitochondria provide ATP that is essential for the movement of the flagella and ultimately the motility of the sperm in the female reproductive tract. Mitochondrial ratios can differ between species of snakes, which is one way that spermiogenesis can be used for comparison between different species of snake. Located at the top of the flagella are proximal and distal centrioles, which can be seen in Figure 4 at 90 degree angles to one another. They help to connect the flagellum to the nucleus and are surrounded by dense collar protiens that also serve as structural elements that help the flagella to stay attached to the nucleus. The fibrous sheath can be seen in these sagittal sections, which are a series of circum-cylindrical fibers that surround the flagellar microtubules. They act as protective and supportive structures and can be found in the midpiece and principle piece.

10 Burkhart The end piece can easily be identified from the rest of the flagellum because it does not possess the fibrous sheath around the axoneme, which is visible in Figure 4. The midpiece is characterized by the presence of mitochondria and dense bodies that surround the flagella (Gribbins and Rheubert, 2011). Mitochondria provide ATP and the dense bodies can provide energy molecules and nutrients to the flagella as it moves. Cross-sections of the flagella are shown in Figure 5. The most anterior cross section is D and they then progress down the flagella s midpiece (C), principle piece (B), and endpiece (A). The last phase of spermiogenesis is chromatin condensation (Gribbins, et al., 2007). Although chromatin continually condenses throughout spermiogenesis, a large amount of the chromatin becomes highly concentrated after nuclear elongation. Figure 2 shows chromatin condensation taking place during a step five spermatid in the nuclear elongation phase. The condensation is very prominent also during the late nuclear elongation phase. Chromatin will condense in a spiral fashion, allowing excess nucleoplasm to be reduced (Gribbins and Rheubert, 2011). The sperm cell will then reduce the cytosol and excess material to gain a more hydrodynamic shape and to allow the sperm cell to more easily navigate through the female reproductive tract. At this point in maturation, sperm cells gain their characteristic filliform shape for reptiles (Gribbins, et al., 2010). The spermatids will simply become curved in shape and are released as mature spermatozoa to the lumina of the seminiferous tubules. Mature sperm will exit the testis through the anterior ducts, which are continuous with the lumen of the seminiferous tubules. They then continue to the epididymis, where they will remain until ejaculation. The ductus deferens allows the sperm cells to exit the rostral and caudal epididymis and to continue to the urethra, exemplifying one reason for the snake testis close association with the kidney

11 Burkhart (Gribbins and Rheubert, 2011). Then the cells will combine with fluid from the kidney and seminal vesicles to create semen. The semen travels to the ampulla and then out the hemi-penis upon ejaculation (Gribbins and Rheupert, 2011). Discussion Through ultrastructural analysis a complete study of the spermiogenesis in Pelamis platurus was completed. This study significantly contributes to the little published research that exists on snakes and squamates for spermiogenesis. For the subfamily Hydrophiinae, this is the first study completed for spermiogenesis. Each of the phases of spermiogenesis were observed with transmission electron microscopy. Acrosome formation, nuclear elongation, and chromatin condensation appeared to be highly conserved between the P. platurus, Cottonmouth snakes, and other squamates. There were only a few differences in the morphology of the spermatids in this sea snake and this was expected of snakes that are not closely related to one another. During the ultrastructural analysis several defining characteristics for the species were identified when comparing the observations to spermiogenesis of Cottonmouths. Although a lot of the mature spermatozoa structure was conserved, three prominent differences were found between the two species. The sea snakes possessed a shorter epinuclear lucent zone, radiating arrays of the manchette, and a prominent central nuclear lacuna that was not observed in the Cottonmouth. Some of these character differences could be useful in future phylogenetic studies between snakes if more spermiogenic data is completed for snakes and other squamates in the near future. With additional ultrastructural analysis of spermiogenesis in the family Hydrophiinae, a better understanding of the relationship between sea snakes and sea kraits could

12 Burkhart also occur. Studies of snakes in the Elapinae subfamily of terrestrial snakes may also help to determine if true sea snakes should be placed in a subfamily of the family Elapidae or if they should be placed in their own family. There are very few articles to compare the results of this study. As a result, most of the differences between the Cottonmouths and sea snakes are not definitive defining characteristics between these two species. With an increase in research the importance of these characteristics will become more evident once other species are studied for spermiogenic characters. With the completion of spermiogenetic analysis of additional snakes in the Elapidae family a better understanding of the relationship between sea snakes, sea kraits, and terrestrial snakes including cobras could be determined. Spermiogenesis can also be used for toxicology. Due to the fact that sea snakes inhabit the marine environment, particularly coral reefs, an increase in the research of spermiogenesis could provide toxicological information on how pollutants affect spermiogenesis. Bioaccumulation is a phenomena that occurs through the accumulation of toxins like heavy metals and pesticides in the lipids of organisms. As sea snakes ingest more contaminated prey items, these harmful toxins can accumulate and magnify in their lipids and specifically their gonads (where lipid content is high). Since the testis have a lot of lipids for providing energy and the production of hormones, they are frequently subjected to pollution and the accumulation of toxins (Haubruge, et al., 2000). Until more research is completed, we lack knowledge of the implications that pollutants and bioaccumulation have on spermiogenesis. With additional research on more individual males of P. platurus and other sea snakes, intraspecies differences in spermiogenesis and genera differences could potentially be examined. Sperm cells of individuals can be compared with one

13 Burkhart another to determine which characters may give an individual male a reproductive advantage over others when it comes to mating and sperm. The hope is that this study will provide the basic framework by which other scientist can study spermiogenesis in other snake species to help provide data for the questions posed within this study. Acknowledgements Dr. Kevin Gribbins and Brenna Burkhart would like to thank Dr. David Sever for providing the tissue samples for analysis. Additionally, we would like to thank Wittenberg University and Saint Louis University for funding this research project and for providing the necessary equipment to complete this research. Literature Cited Al-Dokhi OA Electron microscopic study of sperm head differentiation in the Arabian Horned Viper Cerastes cerastes (Squamata, Reptilia). J Biol Sci 2: Al-Dokhi OA Ultrastructure of sperm head differentiation in the lizard Acanthodactylus boskinus (Squamata, Reptilia). Journal of Zoological Research 1: Al-Dokhi OA, Al-Onazee YZ, Mubarak M Light and electron microscopy of the testicular tissue of the snake Eryx jayakari (Squamata, Reptilia) with a reference to the dividing germ cells. J Biol Sci 3: Bonnet, X Long-term Field Study of Sea Kraits in New Caledonia: Fundamental Issues and Conservation. Integrative and Comparative Biology 52, Butler RD, Gabri MS Structure and development of the sperm head in the lizard Podarcis (Lacerta) taurica. J Ultrastructure Res 88: Clark AQ Some aspects of spermiogenesis in a lizard. American Journal of Anatomy 121:

14 Burkhart Conant R, Collins JT Reptiles and Amphibians, Eastern/Central North America. Houghton Mifflin Company, MA. Pp Cunha LD, Tavares-Bastos L, Báo SN. Ultrastructural description and cytochemical study of the spermatozoon of Crotalus durissus (Squamata, Serpentes). Micron 39: Da Cruz-Landim C, Da Cruz-Hofling MA Electron microscope study of lizard spermiogenesis in Tropidurus torquatus (Lacertilia). Caryologia 30: Dehlawi GY, Ismail MF, Hamdi SA, Jamjoom MB Ultrastructure of spermiogenesis of a Saudian reptile. The sperm head differentiation in Agama adramitana. Arch Androl 28: Del Conte E The subacrosomal granule and its evolution during spermiogenesis in a lizard. Cell and Tissue Research 171: Ferreira A, Dolder H Ultrastructural analysis of spermiogenesis in Iguana iguana (Reptilia: Sauria: Iguanidae). Eur J Morphol 40: Ferreira A, Dolder H Sperm ultrastructure and spermatogenesis in the lizard, Tropidurus itambre. Biocell 27: Gribbins KM, Mills EM, Sever DM Ultrastructural examination of spermiogenesis within the testis of the ground skink, Scincella laterale (Squamata, Sauria, Scincidae). J Morphol 268: Gribbins, KM, Rheupert JL, Anzalone M, Siegel DS, Sever DM Ultrastructure of Spermiogenesis in the Cottonmouth, Agkistrodon piscivorus (Squamata: Viperidae: Crotalinae). Journal of Morphology 271: Gribbins, KM and Rheupert, JL The Ophidian Testis, Spermatogenesis, and Mature Spermatozoa. Reproductive Biology and Phylogeny of Snakes 9:

15 Burkhart Gribbins KM, Rheubert JL, Siegel DS, Sever DM Histological analysis of spermatogenesis and the germ cell development strategy within the testis of the male western cottonmouth snake, Agkistrodon piscivorus leucostoma. Ann Anat. 190: Gutierrez, J. and Bolanos, R Karyotype of the Yellow-Bellied Sea Snake, Pelamis platurus. Journal of Herpetology 14, Haubruge, E., Petit, F. and Gage, M Reduced Sperm Counts in Guppies (Poedilia reticulate) Following Exposure to Low Levels of Tributyltin and Bisphenol A. Proceedings: Biological Sciences 267, Harding HR, Aplin KP, Mazur M Ultrastructure of spermatozoa of Australian blindsnakes, Ramphotyphlops spp. (Typhlopidae, Squamata): first observations on the mature spermatozoon of scolecophidian snakes. In: Jamieson, BGM, Ausio J, Justine JL (Eds.). Advances in spermatozoal phylogeny and taxonomy. vol Mémoires du Musséum National d;histoire Naturelle. pp Healy JM, Jamieson BGM The ultrastructure of spermatogenesis and epididymal spermatozoa of the Tuatara Sphenodon punctatus (Sphenodontidae, Amniota). Philosophical Transactions: Biological Sciences 344: Jamieson BGM Fish evolution and systematics: Evidence from spermatozoa. Cambridge, UK: Cambridge University Press. Hondo E, Kurohmaru M, Toriba M, Hayashi Y Seasonal changes in spermatogenesis and ultrastructure of developing spermatids in the Japanese rat snake, Elaphe climacophora. J Vet Med Sci 56: Jamieson BGM. Ausio J, Justine JL Advances in spermatozoal phylogeny and taxonomy. Mémoires du Musséum National Histoire Naturelle. 166:1-565.

16 Burkhart Jamieson BGM, Oliver SC, Scheltinga DM The ultrastructure of the spermatozoa I. Scincidae, Gekkonidae, and Pygopididae (Reptilia). Acta Zool. 77: Jamieson BGM, Scheltinga DM The ultrastructure of spermatozoa of the Australian skinks, Ctenotus taeniolatus, Carlia pectoralis, and Tiliqua scincoides scincoides (Scincidae, Reptilia). Memoirs of the Queensland Museum 37: Lin M, Jones RC Spermiogenesis and spermiation in the Japanese quail (Coturnix coturnix japonica). J Anat 183: Lin M, Jones RC Spermiogenesis and spermiation in a monotreme mammal, the platypus, Ornithorhynchus anatinius. J Anat 196: McIntosh JR, Porter KR Microtubules in the spermatids of the domestic fowl. J Cell Bio 35: Newton WD, Trauth SE Ultrastructure of the spermatozoon of the lizard Cnemidophorus sexlineatus (Sauria: Teiidae). Herpetologica 48: Ray-Chaudhuri, S., Singh, L. and Sharma, T Evolution of Sex-chromosomes and Formation of W-chromatin in Snakes. Chromosoma 33, Russell LD, Ettlin RA, Hikim AMP, Cleff ED Histological and Histopathological Evaluation of the Testis. Clearwater, FL: Cache River Press. Pp Sever, D. and Freeborn, L Observations on the Anterior Testicular Ducts in Snakes with Emphasis on Sea Snakes and Ultrastructure in the Yellow-Bellied Sea Snake, Pelamis platurus. Journal of Morphology 273, Sever, D., Rheupert, J., Gautreaux, J., Hill, T. and Freeborn, L Observations on the Sexual Segment of the Kidney of Snakes with Emphasis on Ultrastructure in the Yellow-Bellied Sea Snake, Pelamis platurus. The Anatomical Record 295,

17 Burkhart Soley JT Nuclear morphogenesis and the role of the manchette during spermiogenesis in the ostrich (Struthio camelus). J Anat 190: Sprando RL, Russell LD Spermiogenesis in the red-ear turtle (Pseudemys scripta) and the domestic fowl (Gallus domesticus): A study on cytoplasmic events including cell volume changes and cytoplasmic elimination. J Morphol 198: Talbot P Compartmentalization in the acrosome. In: Bacetti B, editor. Comparative Spermatology-20 Years After. New York: Raven Press. Pp Tavares-Bastos L, Cunha LD, Colli GR, Báo SN Ultrastructure of spermatozoa of scolecophidian snakes (Lepidosauria, Squamata). Acta Zoologica 88: Tavares-Bastos L, Teixeira RD, Colli GR, Báo SN Polymorphism in the sperm ultrastructure among four species of lizards in the genus Tupinambis (Squamata: Teiidae). Acta Zooloogica 80: Teixeira, RD, Vieira GHC, Colli GR, Bao SN Ultrastructural study of spermatozoa of the neotropical lizards, Tropidurus semitaeniatus and Tropidurus torquatus (Squamata, Tropiduridae). Tissue and Cell 31: Ventela S, Toppari J, Parvinen M Intercellular organelle traffic through cytoplasmic bridges in early spermatids of the rat: Mechanism of haploid gene product sharing. Mol Biol Cell 14: Vieira GHC, Colli GR, Báo SN The ultrastructure of the spermatozoon of the lizard Iguana iguana (Reptilia, Squamata, Iguanidae) and the variability of sperm morphology among iguanian lizards. J Anat 204: Wiens JJ The role of morphological data in phylogeny reconstruction. Syst Biol 53:

18 Burkhart Figure Legends Figure 1: The acrosome formation of spermatids in early spermiogenesis takes place in the seminiferous epithelium. (A) The acrosome vesicle (AV) begins to form near the nucleus (NU). (D) The Golgi apparatus (Black Arrow) can be seen with the highly folded cisternae releasing secretory vesicles (White Star) that fuse to become the acrosome vesicle. (B) When more transport vesicles from the Golgi begin to fuse with the acrosome vesicle it grows in size and due to the proximity with the nucleus it makes contact with the nuclear surface. As the proteins concentrate within the acrosome vesicle from the Golgi, the acrosome granule forms, and is marked by the white triangle. The mitochondria (Black Triangle) can also be seen near the nucleus. (C) The principle piece, consisting of a proximal (PC) and distal centriole (DC), can be seen and is relatively small compared to the nucleus. The dense collar proteins (White Ring) can also be seen in the principle piece and can be observed in the mature spermatozoa. Flagellar development has not yet seen in these spermatids Figure 2: Step 5 spermatids can be seen in these micrographs and display the early steps of nuclear elongation. (A) A sagittal section of a developing elongating spermatid can be viewed. The nucleus appears to be rod-shaped. The acrosome vesicle (AV), condensing chromatin (CC), and nuclear shoulders (NS) are also visible in this elongating cell. The acrosome vesicle is also enveloping the nuclear apex at this step. (B) A cross-sectional cut of the spermatid reveals nuclear lacuna (NL) along with the condensing chromatin. There is a prominent central lacuna that is also evident (B). 399

19 Burkhart Figure 3: Nuclear elongation is characterized by high amounts of compartmentalization. Crosssections of the nucleus are represented by photos A-C and D-F. (A) represents the top-most cross-section of the tip of the acrosome complex, running towards the nucleus through (C) and continuing down the acrosome complex (D) until reaching the nucleus and machette only in (F). The acrosome vesicle (AV) and sertoli cell (SC) can be seen in (A). In (B) the perforatorium (PE) can also be seen centrally located in the acrosome vesicle. The sertoli cell membrane (SCM), subacrosome space (SAS), epinuclear lucent zone (ELZ), acrosomal lucent ridge (ALR) can all be seen in (C). (D) represents a sagittal cut of the acrosome complex (AC) in which the sertoli cell can be seen, the perforatorium, the basal plate (BP), the epinuclear lucent zone, the acrosomal lucent ridge (ALR), and the acrosomal vesicle shoulder (AVS) can all also be seen within the acrosome complex. (G) shows a sagittal section of the elongating nucleus with high amounts of compartmentalization in the acrosome complex The peak of nuclear elongation occurs when the acrosome fully envelops the nuclear apex, as is most evident in Fig. 3, photo G. The high amounts of compartmentalization are evident in Fig. 3, photo D and G Figure 4: This micrograph shows the flagellum of developing spermatids in sagittal cuts. The midpiece (MP) is the part of the flagellum that contains mitochondria and a fibrous sheath around the microtubules. Dense collar proteins (White Ring) can be seen in (A) and (B). The proximal centriole (PC) is more anteriorly located and closer to the nucleus than the distal centriole (DC). The annulus (AN) is located at the end of the midpiece and marks the beginning of the principle piece (PP). The principle piece is after the midpiece and is only surrounded by a fibrous sheath. Last is the endpiece (EP) that does not possess a fibrous sheath or mitochondria and dense bodies. It is located furthest from the nucleus.

20 Burkhart Figure 5: Cross-sectional cuts of the flagellum can be seen in this figure. (A) is a crosssectional cut that is in the end piece of the flagella. This is evident in the photo because there is not a fibrous sheath (FS) surrounding the microtubule doublets and singlets. In (A) the enlarged peripheral fibers are marked at doublets 3 and 8. (B) this is a cross-sectional cut of the principle piece of the flagella. This section is marked by the presence of a fibrous sheath surrounding the microtubule doublets and singlets. (C) shows a cross-section of the midpiece, which possesses a fibrous sheath, but also mitochondria (Black Triangle) and dense bodies (DB) around the fibrous sheath. (D) shows the most anteriorly located cross-section, that contains a dense collar protien that is represented by the white ring. Additionally the peripheral fibers (PF) can be seen surrounding the microtubules

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