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