Cytogenetic Characterization and Fluorescence in situ Hybridization of (GATA)₁₀ Repeats on Established Primary Cell Cultures from Indian Water Snake <i>(Natrix piscator)</i> and Indian Mugger <i>(Crocodylus palustris)</i> Embryos

Abstract

Sex determination among reptiles has continued to draw the attention of geneticists and the mechanisms involved have been extensively studied and documented in the past 3 decades. The setting up of primary cell lines of reptilian tissues is an important tool in the present study which is a unique aspect not applied in earlier studies. Establishing the cell lines from various species of reptiles would help in our understanding of the mechanisms of evolution and differentiation of sex chromosomes. Therefore, in the present study, we have established for the first time primary cell cultures from Indian water snake (Natrix piscator) and Indian mugger (Crocodylus palustris) embryos. In the preliminary growth stage, 2 types of cells, fibroblast- and epithelial-like, were found to be attached and proliferating in vitro. These fibroblast-like cell cultures were later overtaken by epithelial cells. The cell lines were grown in minimal essential medium supplemented with fetal bovine serum and subcultured for a period of 8–10 months. The morphology of cell types was kept under constant observation microscopically. Interestingly, at a subsequent passage of the cells sporadically scattered neuronal-like and beating cells were observed. The suitable temperature for growth of these cell cultures was 28–30°C. Chromosome analysis was performed from the actively proliferating cells, which revealed 5 pairs of macrochromosomes and 15 pairs of microchromosomes in Natrix piscator, and 15 pairs of only macrochromosomes in Crocodylus palustris. (GATA)_n repeats are well known to be associated with sex chromosomes. Fluorescence in situ hybridization performed with (GATA)₁₀ repeats delineated the W chromosome in the cells of Natrix piscator which has so far not been reported. This cell culture method has presently only been applied to water snakes and crocodile embryos in the current study, but it will be employed in other reptilian species and could go a long way to being a sustainable source of primary cells. This would eventually serve as an important tool for molecular studies in reptiles and other species in the future.

Cytogenetics of the Javan file snake (Acrochordus javanicus) and the evolution of snake sex chromosomes

“…The size of the Z chromosome corresponds to the fourth pair of the complement in the majority of the advanced snakes (Matsubara et al., ; Mengden & Stock, ; Oguiura et al., ; Olmo & Signorino, ), and less often to the fifth pair (Olmo & Signorino, ; Rao et al., ; Rovatsos, Johnson Pokorná, et al., ), although it was demonstrated that the Z chromosomes across all caenophidian families share partial gene content (Rovatsos, Vukić, et al., ). The heterochromatic W chromosomes can vary significantly from small‐to‐medium sized among species of advanced snakes (Matsubara et al., ; Mengden & Stock, ; Oguiura et al., ; Olmo & Signorino, ; Rao et al., ; Rovatsos, Johnson Pokorná, et al., ). Non‐caenophidian snakes mostly have homomorphic sex chromosomes without accumulated heterochromatin (Matsubara et al., ; O'Meally et al., ; Oguiura et al., ; Vicoso et al., ).…”

“…Well‐differentiated and mostly heteromorphic sex chromosomes with a heterochromatic W chromosome have been described in many species of Colubroidea (Beçak & Beçak, ; Matsubara et al., ; O'Meally et al., ; Oguiura et al., ; Ohno, ; Vicoso et al., ) including X. javanicus (Rovatsos, Johnson Pokorná, et al., ). The size of the Z chromosome corresponds to the fourth pair of the complement in the majority of the advanced snakes (Matsubara et al., ; Mengden & Stock, ; Oguiura et al., ; Olmo & Signorino, ), and less often to the fifth pair (Olmo & Signorino, ; Rao et al., ; Rovatsos, Johnson Pokorná, et al., ), although it was demonstrated that the Z chromosomes across all caenophidian families share partial gene content (Rovatsos, Vukić, et al., ). The heterochromatic W chromosomes can vary significantly from small‐to‐medium sized among species of advanced snakes (Matsubara et al., ; Mengden & Stock, ; Oguiura et al., ; Olmo & Signorino, ; Rao et al., ; Rovatsos, Johnson Pokorná, et al., ).…”

Karyomorphological delineation, and the NOR loci on the sex chromosome in three species of Chrysopeleinid (Chrysopeleinae: Colubridae) from Thailand

“…This situation is normal in an early stage of ZZ/ZW sex chromosomes and differs in Colubroidea snakes (Matsubara et al 2006;Oguiura et al 2009). The position of the Z chromosome was usually presented on the fourth pair in almost all of the advanced snakes and is often found in pair 5 (Mengden and Stock 1980;Rao et al 2009;Rovatsos et al 2015b). Falcione et al (2016) proposed the degree of heteromorphy of the sex chromosome (ZW) is caused by the heterochromatin distribution that leads to similar or differs morphologically in shape and/or size.…”

Evidence for Sex Chromosome Turnover in Proteid Salamanders

“…Among amphibians, Y-autosome fusions occur in some 12 species of terraranan frogs, genera Strabomantis , Pristimantis , and Eleutherodactylus [Schmid et al, 2002[Schmid et al, , 2003[Schmid et al, , 2010. Both Y-autosome and W-autosome fusions are known to occur in cypriniform, cyprinodontiform, perciform, salmoniform, and tetraodontiform fishes [de Oliviera et al, 2008;Rao et al, 2009] and, among reptiles, in gekkonid, iguanid, teiid, and scincid lizards, and elapid snakes 310 [Leaché and Sites, 2009;Oguiura et al, 2009]. Such translocations are also known among various mammals [Schmid et al, 2010].…”