Proteinaceous sperm motility inhibitory factor from the female Indian garden lizard Calotes versicolor

Abstract

Female sperm storage is an intriguing adaptation exhibited by a wide array of both vertebrates and invertebrates. The mechanisms underlying female sperm storage have remained elusive. Using the Indian garden lizard Calotes versicolor as a model organism, we investigated the role of low and high molecular weight factors in this phenomenon. Previously, we demonstrated three distinct phases of the reproductive cycle in this animal with live, motile spermatozoa recovered from the uterovaginal region during the reproductive phase. In the present study, we analysed the uterovaginal contents using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified an abundant protein band corresponding to ~55 kDa regardless of the phase of the reproductive cycle. Analysis of the purified protein by liquid chromatography-tandem mass spectrometry suggested a unique protein without any homology to the National Center for Biotechnology Information database. Exogenous addition of this protein to washed spermatozoa derived from the epididymis reversibly inhibited sperm motility in a concentration- and time-dependent manner, suggesting it plays a key role in sperm storage. These studies are likely to offer new avenues to unravel the secrets of female sperm storage seen across the animal taxa and may have novel applications not only in reproductive biology, but also in general cell storage and preserving endangered animal species.

Sperm storage by females across the animal phyla: A survey on the occurrence and biomolecules involved in sperm storage

“…Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the crude uterovaginal flushings we have found at least 30-50 more proteins (Unpublished data) and this needs further investigation in relation to sperm storage. We also detected low molecular weight factors like lactic acid (Shankar et al, 2018) and citric acid (Unpublished data) in the uterovaginal flushings, and there are likely many other organic acids, which are currently under investigation.…”

“…Based on the available literature (Gwathmey et al, 2003; Kawano et al, 2014; Kumari et al, 1990; Shankar et al, 2015; Srinivas et al, 1995), we infer that, sustaining sperm in a viable condition may probably require substances either secreted from the oviduct (Kumari et al, 1990; Shankar et al, 2015) or deposited into the oviduct with the sperm during mating (Gwathmey et al, 2003; Kawano et al, 2014; Srinivas et al, 1995) which still remains largely an unanswered query. We showed (Shankar et al, 2018) that a unique approximately 55‐kDa protein of the oviductal origin in C. versicolor may be a factor contributing to sperm storage in females. However, another study (Srinivas et al, 1995) suggests that the storage factor may be of male seminal origin as seen in Psammophillus dorsalis , a sympatric species in Southern India (Srinivas et al, 1995).…”

“…In our previous study (Shankar et al, 2018) It is reasonable that sperm require a source of energy for their long-term stay in the female oviduct and in the absence of exogenous carbohydrates, mammalian sperm are known to actively metabolize lipids to supply their energy needs for motility and metabolism (Mann, 1964). In Greater Asiatic yellow bat, Scotophilus heathii increased expression of hormone-sensitive lipase (HSL) and organic cation transporter 2 (OCTN2) is seen in the cytoplasm of luminal epithelial cells in the uterotubal junction, the site of sperm storage (Roy & Krishna, 2012).…”

“…Calotes versicolor as a model organism, we isolated a novel approximately 55-kDa protein from the uterovaginal flushings (lavage) that reversibly inhibited the motility of the sperm in vitro (Shankar et al, 2015;Shankar et al, 2018). However, besides the…”

“…The multiple roles of the reptilian oviduct and the structural specializations for these purposes provide enormous scope for future research (Girling & Girling, 2002). For the past three decades using the Indian garden lizard, Calotes versicolor as a model reptile to study the phenomenon of sperm storage by female (Kumari et al, 1990;Shankar et al, 2015;Shankar et al, 2018), we have documented some of its reproductive characters which are discussed below. A previous study in C. versicolor (Kumari et al, 1990) has shown the histological and histochemical features of the SSTs found at the junction of the uterus and vagina of the oviduct called the uterovaginal junction.…”