Integrative omics analysis reveals differentially distributed proteins in dimorphic euspermatozoa of the squid, Loligo bleekeri

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Highlights

  • The squid Loligo bleekeri produce two types of males and two types of spermatozoa.

  • We undertook integrative omics approach to understand molecular bases of sperm dimorphism.

  • Expression of dynein heavy chain is biased to sneaker sperm.

  • Enzymes involving energy metabolism are largely biased to consort sperm.

Abstract

In the coastal squid Loligo bleekeri, each male produces one of two types of fertilization-competent spermatozoa (eusperm) that exhibit morphological and behavioral differences. Large “consort” males produce short-tailed spermatozoa that display free-swimming behavior when ejaculated into seawater. Small “sneaker” males, on the other hand, produce long-tailed spermatozoa that exhibit a self-swarming trait after ejaculation. To understand the molecular basis for adaptive traits employed by alternative male mating tactics, we performed the transcriptome deep sequencing (RNA-seq) and proteome analyses to search for differences in testicular mRNAs and sperm proteins, respectively. From mature male testes we identified a total of 236,455 contigs (FPKM ≧1) where 3789 and 2789 were preferentially (≧10-fold) expressed in consort and sneaker testes, respectively. A proteomic analysis detected 4302 proteins in the mature sperm as post-translational products. A strongly biased (≧10-fold) distribution occurred in 55 consort proteins and 61 sneaker proteins. There was no clear mRNA–protein correlation, making a ballpark estimate impossible for not only overall protein abundance but also the degree of biased sperm type expressed in the spermatozoa. A family encoding dynein heavy chain gene, however, was found to be biased towards sneakers, whereas many enzymes involving energy metabolism were heavily biased towards consort spermatozoa. The difference in flagellar length matched exactly the different amount of tubulins. From these results we hypothesize that discrete differential traits in dimorphic eusperm arose from a series of innovative alterations in the intracellular components of spermatozoa.

Introduction

In essence, most cells can respond and adapt to their environments by means of transcriptional, post-transcriptional and post-translational events [1]. In particular, a specific set of gene expression facilitates a cell’s optimal reactions in favorable or unfavorable conditions. Spermatozoa are highly specialized, differentiated haploid cells that largely lack transcriptional and translational activities, although some exceptional cases have been reported [2]. Basically, sperm design and their behavioral traits are endowed during spermatogenesis and spermiogenesis. Therefore, in the same species, each sperm cell is expected to behave equally in response to the same environmental cue. In this context, the squid Loligo bleekeri offers a very unique feature regarding two separate sperm traits in two separate male types; (1) large males called “consort” produce small spermatozoa and small “sneaker” males produce ∼50% much larger sperm [3], (2) for the sneakers only, spermatozoa form a cluster in response to carbon dioxide [4], [5]. Such sperm dimorphism is tightly linked to male mating behaviors. Large males have an advantage in copulation, placing sperm inside the oviduct. Small males, however, avoid male-male competition and undertake “sneaky copulation” placing their spermatozoa on the buccal membrane of the female [6], [3], [7]. Fitness consequences of these different sperm phenotypes remain unknown. Moreover, the mode of inheritance as well as the ontogenetic question of how this male dimorphism arose remains unanswered.

Apart from trying to gather some insight into these intriguing evolutionary questions, we attempted to understand the developmental processes that drive spermatogenesis into different types of sperm cells. Because sperm are in general translationally silent, the differences should be evident at the levels of transcription and/or translation in the testis. Using this assumption, we used a comparative “omics” approach to identify differentially expressed transcripts and proteins that can potentially contribute to consort- or sneaker-specific traits.

Section snippets

Handling of animals and spermatozoa

Mature L. bleekeri were collected at Miura (Sagami-bay, Kanagawa) or Matsumae (southeast Hokkaido Island), Japan, and transported in chilled containers (4 °C) to the laboratory within two days. Adult males produce spermatophores, which are cylindrical capsules containing mature sperm. Consort males and sneaker males were distinguished by spermatophore lengths and spermatangium morphology [6], [3]. Spermatozoa were released from spermatophores by mechanically stimulating the spermatophoric

Results and discussion

The lifespan of L. bleekeri is estimated to be ∼1 year [16] and during the last few months (Dec–Apr) of their lifetime individuals conduct the mass spawning in shallow water. Sneaker and consort males were collected simultaneously at the same place to obtain comparative gene/protein expression profiles (Fig. 1). We undertook this in the middle of the spawning season (Feb in Sagami-bay) and relatively smaller mature individuals for both male types were chosen to collect testicular mRNAs, because

Acknowledgments

The authors thank NIG Cell Innovation program (http://cell-innovation.nig.ac.jp/g). Supported by MEXT (NH), Yamada Science Foundation (NH), and NRF (WHHS).

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    1

    These authors equally contributed to this work.

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