Embryonic Developmental Study on Vomeronasal Organ of Montpellier Snake (Malpolon monspessulana)

World Journal of Zoology 10 (2): 70-77, 2015 ISSN 1817-3098 IDOSI Publications, 2015 DOI: 10.5829/idosi.wjz.2015.10.2.9371 Embryonic Developmental Study on Vomeronasal Organ of Montpellier Snake (Malpolon monspessulana) Eman Y. Salah EL-Din and Ahmed Imam Dakrory Zoology Department, Faculty of Science, Cairo University, Egypt Abstract: The development of vomeronasal organ was studied in Montpellier snake (Malpolon monspessulana) at 1.2, 1.5, 1.9, 4, 5, 5.5, 6.5, 7 and 8 cm Snout vents lengths. The vomeronasal organ started its development as a vomeronasal placode. The latter continued in its growth (vomeronasal pit-vomeronasal sac) until reached the dome shape. The vomeronasal epithelium became differentiated into thick sensory epithelium and thin non sensory epithelium. The developing dome shape vomeronasal organ opened into the oral cavity through vomeronasal duct. Accessory nasolacrimal duct opened medially into vomeronasal duct. The sensory epithelium of the organ gave off nerve fibers which form the vomeronasal nerve. Finally, the vomeronasal organ was supported by a cartilaginous capsule and membrane bones. Key words: Malpolon monspessulana Vomeronasal organ Vomeronasal duct Accessory nasolacrimal duct Vomeronasal nerve INTRODUCTION were little studies on its ontogenetic development. There were many ontogenetic studies on the vomeronasal organ The vomeronasal or Jacobson's organ (VNO) is a pair of both amphibians [25, 26] and mammals [10, 27-31]. The of blind-ended cavities or tubules present in many all sense organs, the vomeronasal organ originated from vertebrates [1-4]. It is absent in fishes and some mammals the cranial placode. It arose in common with the main as chiropterans, cetaceans and sirenians [5-10]. It is olfactory organ from the olfactory placode. The cranial believed that the vomeronasal first evolved in amphibians placodes vitally contributed to the formation of the paired [11-13]. Among reptiles, the vomeronasal shows a wide sense organs and to the cranial sensory ganglia. These variation in occurrence, structure and development placodes are specialized areas of the head ectoderm of [14-16]. Moreover, the highest variability and the vertebrate embryos that typically first became diversification were found in recent reptiles among the apparent as patches of thickened columnar epithelial cells members of order Squamata [17-18-19-20]. Chamaeleonids [32]. From the available reptilian literatures, it appeared showed the most extreme degree of reduction of the organ that, there are many anatomical and physiological studies [21, 22]. Snakes are mostly dependent on their smell and on this organ, but its ontogenetic studies or studies on its touch senses for detection [23], which appeared to be morphogenesis were rare or scarce. Hence, the present essential component to the snakes for a perfect work aimed to study the embryonic development of this accommodation of its way of life. The development of the organ in the successive embryonic stages. Another aim nasal chemical senses of snakes is of particular interest was to compare the results as well as those of other since evidence suggested that the neonatal reptiles use snakes and lizards and with the other vertebrate taxa. both their olfactory and vomeronasal system [24]. Embryologically, the vomeronasal organ derived from the MATERIALS AND METHODS olfactory placode and is both morphologically and physiologically similar to the main olfactory system [15]. The snake used in this investigation is the Although, there are many anatomical and physiological Montpellier snake; Khodari Malpolon monspessulana studies on the vomeronasal organ of snakes, yet there (Family: Colubridae, Suborder: Ophidia, Order: Squamata). Corresponding Author: Eman Y. Salah EL-Din, Zoology Department, Faculty of Science, Cairo University, Egypt. E-mail: e_youssri@yahoo.com. 70

Table 1: The Snout vents lengths (SVL cm) of the studied stages of Malpolon monspessulana. stages 1 2 3 4 5 6 7 8 9 SVL (cm) 1.2 1.5 1.9 4 5 5.5 6.5 7 8 According to [33], it distributed from North Africa to stage 1 (SVL; snout vent length: 1.2 cm(. Shortly latter on, south western Asia. In Egypt, the Montpellier inhabits at stage 2 (SVL: 1.5 cm (, this placode invaginated forming western Mediterranean coastal desert, Nile Delta, lower a shallow olfactory pit which soon became deeper at stage valley and Siwa oasis. It found in the semi-desert, sandy 3 (SVL: 1.9 cm (forming an olfactory pouch and the areas of northern Delta, around vegetated salt marshes olfactory sac. and in cultivated land. It feeds on small mammals, lizards, Stage 4 (SVL: 4 cm (: The medial part of the olfactory frogs and birds. Variations in its color include reddish placode (at the olfactory sac stage) became more individuals. thickened forming the vomeronasal placode (Fig.1, VO.P), The different developmental stages were available in which gradually sunked down forming a shallow the fertilized collecting eggs. The latter had been getting vomeronasal pit (Fig. 2, VO.PI). In this stage (Fig. 2), the with the help of specialist. Embryos were carefully taken vomeronasal sensory epitheliums gave off a nerve fiber out of the shells and their snout vents lengths (SVL) were which forms the vomeronasal nerve primordium estimated. The Snout vents lengths (SVL cm) of the (VO.N.PR). This primordium adhered to the main olfactory studied stages of Malpolon monspessulana are listed in nerve primordium (OL.N.PR). Table 1. Stage 5 (SVL: 5 cm(:the vomeronasal pit (Fig. 3, Live healthy four embryos from each stage were VO.PI) became more deeper forming a tubular; immediately fixed in aqueous Bouin s fixative for 24-48 vomeronasal sac (VO.SC) which lied medial to the main hours according to the size of the embryos. olfactory canal (OL.CN) near its posterior end (Fig. 4). Large four embryos were treated with EDTA solution While at its anterior end, the vomeronasal pit opened into for decalcification. Taken time for decalcification process the main olfactory canal. ranged from 30 to 40 days depending on the size of the Stage 6 (SVL: 5.5 cm (: The vomeronasal sac opened embryo, during which the EDTA solution was changed into the olfactory canal from its medial side. The every 4 days. This was followed by washing the embryos vomeronasal sac increased in size. Its lumen acquired a several times with 70% ethyl alcohol. crescentic shape (Fig.5). Here, the sensory epithelium of Embryos were treated with ascending series of ethyl the organ was undifferentiated. The mushroom body alcohol and then cleared with xylene. Thereafter, the primordium (M.B.PR) appeared along the ventral side of specimens were embedded in a paraffin wax. This was the organ consisting of undifferentiated mesenchymal followed by sectioning embryos transversely at 10 tissue. Here (Fig. 6), the vomeronasal sac opened by a microns thickness using Reichert microtome. short vomeronasal duct primordium (VO.D.PR) together The sections of each specimen were mounted serially with the main olfactory canal by a common duct (CO.D). on microscopic slides and prepared for staining. The latter The latter opened into the palate by the internal naris was carried out by haematoxylin and eosin. (IN.NR), which appeared as an anteroposterior cleft in the The vomeronasal organ is examined in these palate lateral to the organ. The vomeronasal nerve (VO.N) sections. Several sections were chosen for appeared as few nerve bundles, which formed from the photomicrography using Zeiss photomicroscope supplied nerve arising from the dorsal, lateral and medial sensory by Canon digital camera to demonstrate twice different epithelium. This nerve appeared entering the accessory developmental changes of the organ and its relation to the olfactory bulb from its medial side (Fig. 7). The posterior different neighboring structures. part of the common duct elongated forming the nasopharyngeal duct (Fig. 5, NP.D) of the main olfactory RESULTS organ. Stage 7 (SVL: 6.5 cm (: The vomeronasal organ In Malpolon monspessulana, the vomeronasal organ (Fig. 8) became completely separated from the main originated from medial part of the olfactory placode after olfactory organ (Chamber). It grew large and became dome its invagination to form olfactory sac. The olfactory shape structure. The epithelium lining its lumen became placode appeared as a thickened area of the ectoderm in differentiated into a dorsal thick sensory epithelium (V.SE) a position lateral to the anterior part of the and a ventral thin non sensory epithelium (V.NSE) prosencephalon and anterior to the developing eyes, at covering the mushroom body. 71

Figs. 1-11: Photomicrograph (H&E) of a transverse section passing through the olfactory region of different embryonic stages. 72

Fig. 1: At 4 cm snout vents length, showing the vomeronasal placode (VO.P) and the olfactory nerve primordium (OL.N.PR), olfactory sac (OL.SC) and prosencephalon (PRC). Scale bars, 117µm. Fig. 2: At 4 cm snout vents length, showing the vomeronasalpit (VO.PI) and the vomeronasal nerve primordium (VO.N.PR). Scale bars, 117µm. Fig. 3: At 5 cm snout vents length, showing that the opening of the vomeronasal pit into the main olfactory canal (OL.CN), eye (E), oral cavity (OR.CV). Scale bars, 125µm. Fig. 4: At 5 cm snout vents length, showing tubular vomeronasal sac (VO.SC) lies medially to the olfactory canal. Scale bars, 125µm. Fig. 5: At 5.5 cm snout vents length, showing a cresentic shape vomeronasal sac with its corresponding undifferentiated epithelium and mushroom body primordium (M.B.PR). The nasopharyngeal duct (NP.D) of the main olfactory canal also, internal naris (IN.NR), olfactory nerve (OL.N) and vomeronasal nerve (VO.N) appeared. Scale bars, 125µm. Fig. 6: At 5.5 cm snout vents length, showing both olfactory canal and vomeronasal sac with their corresponding canal open into oral cavity by internal naris through a common duct (CO.D). Also, the first appearance of vomeronasal duct primordium (VO.D.PR). Scale bars, 125µm. Fig. 7: At 5.5 cm snout vents length, showing the vomeronasal nerve entering the accessory olfactory bulb (OL.B).Also, internasal septum (IN.S) appeared. Scale bars, 125µm. Fig. 8: A photomicrograph (H&E) of a transverse section passing through the olfactory region at 6.5 cm snout vents length, showing the sensory (V.SE) and non sensory epithelium (V.NSE), of vomeronasal organ.scale bars, 125µm. Fig. 9: At 7 cm snout vents length, showing the vomeronasal organ (VO) with its corresponding vomeronasal duct (VO.D.PR).Scale bars, 125µm. Fig. 10: At 7 cm snout vents length, showing the epithelium of dome shape vomeronasal organ differentiated into an inner supporting layer (SP.LA), intermediate receptor layer (RE.LA) and an outer basal layer (B.LA).And mushroom body (M.B) appeared. Scale bars, 125µm. Fig. 11: At 8 cm snout vents length, showing cartilaginous vomeronasal concha (VO.CO) and septomaxillary bone (SMX) support vomeronasal organ. An opening vomeronasal duct receives from its medial side the nasolacrimal duct primordium (NL.D.PR). Scale bars, 125µm. Stage 8 (SVL: 7 cm (: The developing dome shape cavity through the palate. This duct received from its vomeronasal organ developed a closed vomeronasal duct medial side the nasolacrimal duct (Fig.11, NL.D.PR), for primordium (Fig. 9, VO.D.PR). It loosed its connection the first time. The dorsal dome of vomeronasal organ with the olfactory canal. In this stage (Fig.10), the dorsal formed from sensory epithelium, whereas, the ventral side sensory epithelium of vomeronasal (V.SE) organ of the organ formed from non sensory epithelium (Fig.11). differentiated into 3 layers: an inner supporting layer In this stage, the vomeronasal organ (Fig.11) was (SP.LA), intermediate receptor layer (RE.LA) and an outer encapsulated by a cartilaginous capsule at its anterior basal layer (B.LA). The floor of the developing organ half. Posteriorly, it was supported dorsally and medially (mushroom body) consisting of mesenchymal connective by the septomaxillary bone (SMX) and ventrally by tissue, began to protrude into the lumen forming the prevomer bone. These membrane bones appeared for the vomeronasal concha primordium (VO.CO.PR). This first time in 8 cm SVL stage from the pre-existing covered with a layer of non sensory epithelium which connective tissue. faces a crescentic organ lumen (LU). Stage 9 (SVL: 8 cm (: The vomeronasal organ (VO), DISCUSSION lied ventral to the rostral part of the olfactory chamber. Its dorsal side was bulging and forming the dorsal dome In the current study, the vomeronasal organ while the ventral side invaginated into the organ forming originated from the medial part of the olfactory placode mushroom body (Fig. 11, M.B). The latter was supported after its invagination to form the olfactory sac. The by a developing cartilaginous vomeronasal concha olfactory placode made its appearance as thickened area (VO.CO). The organ had a very narrow opening duct of the ectoderm at 1.2 cm SVL (Snout vent length). Shortly (Fig.11, VO.D.PR) which opened ventrally into the mouth latter on and at 1.5 cm SVL stage, the placode invaginated 73

forming an olfactory pit which soon became more deeper at 1.9 cm SVL stage forming a pouch like structure; the olfactory sac. This was the same results recorded by Parsons [34] in Ckysemyspicta; Thamnophis sp.; Oxybelis sp. and in Alligator mississipensis. The olfactory placodes were specialized areas of the head ectoderm of the vertebrate embryos that typically first become appeared as patches of thickened columnar epithelial cells [32]. Halpern [15] mentioned that, the vomeronasal organ derived from the olfactory placode and was both morphologically and physiologically similar to the main olfactory system. Also, the evolutionary origins of the vomeronasal organ were thought to begin with tetrapods and specific receptor genes that were expressed in the vomeronasal system as compared to the olfactory system proper had been identified [35, 36]. These earlier stages in jacobson's organ development of Malpolon monspessulana were relatively similar to the reptiles; while in the later stage, the members of each species gradually attained their characteristic an adult specialization [15,24, 34]. The obvious difference was the timing sequences [5]. Since discovered, the vomeronasal organ assumed that: it was the organ of secretory nature but suspected that it could also be sensory [37]. It was named as organ on vomeronasal Jacobsoni by Mihalkovics [38]. Today, this organ was recognized as a chemosensory organ for pheromones in mammals and jacobson's organ in reptiles. Concerning reptiles, recent studies suggested that the vomeronasal receptor neurons and not the main olfactory ones were stimulates by a non volatile substances extracted from earth worms which are the favorite food of garter snakes [39]. The vomeronasal organ anatomy shape, position, communication, organization and development vary considerably among vertebrates [40-42]. Phylogenelically, it seemed probably that the vomeronasal organ first evolved in amphibians [11-13, 43, 44]. Although, Hornung and Mozeli [45] and Trotier et al. [46] reported that in frogs, the organ couldn t be considered a true organ as in reptiles and mammals but rather a specialized area of the wall of the inferior chamber of the nose. It attained its greatest development in reptiles but in some of them it was reduced and even absent in others [34, 47-51]. Aves and crocodilians were considered to be sister groups, owing to the absence of this organ in adult stages [12, 40, 52]. Although, there were many studies on the structure of the reptilian vomeronasal organ owing to its variability [15, 53], yet, there was few or scarce information about its ontogenetic development. Among reptiles, the vomeronasal organ was more highly developed in squamates and was largest in snakes [17-18, 54-58]. The developing vomeronasal organ of Malpolon monspessulana remained opened into the main olfactory canal at 5 and 5.5 cm SVL stages. At these stages, the organ repeated its conditions of the adult amphibians. Again, at 5.5 cm SVL stage, the organ lumen acquires a crescentic shape, which was lined with undifferentiated epithelium, with the appearance of the mushroom body primordium. A mushroom body was not developed in Chamaeleon and in the other lizard in which jacobson's organ was reduced [21, 22, 59], although the organ developed normally in early stages. On the other hand, in crocodilian, the organ was transitory and disappeared in later embryonic stages [34]. At 6.5 cm SVL, the vomeronasal organ became completely separated from the main olfactory system and at 7 cm SVL; it started to develop a solid vomeronasal duct into the palate. This duct hollowed out in 8 cm SVL stage to open into the mouth cavity. The disappearance of the connection with the olfactory organ and the appearance of the organ duct that opened into the mouth is atypical adult reptilian character. The epithelium lining the organ started to differentiate into a thick sensory layer on the dorsal side and a thin non sensory epithelium covering the mushroom body at 5.5 cm SVL. This was the same as that mentioned by Taniguchi et al. [60]. The sensory epithelium in stage 7, 8 cm SVL developed into tightly packed polygonal columns interspersed with small amount of connective tissue containing a network of capillaries. These results were mentioned by Wang and Halpern [61] in garter snake. Parsons [34] recorded that, these columns developed only in later embryonic stages. Such columns are not formed clearly in lizards [50, 51]. Again, the capillaries were not present at the earliest stages in the development of the columns of lizards, but they did seem to be there at all stages in Thammopis [34]. CONCLUSION The vomeronasal organ shows the same anatomy and histology as found in other snakes. According to the importance of vomeronasal organ to reptiles and especially to Squamata further studies on vomeronasal organ are required. ACKNOWLEDGEMENT The authors would like to thank the head of Zoology Department and staff members, Faculty of Science, Cairo University, for the help offered me through the preparation of this manuscript. 74

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