Transcriptome profiling of laser-captured germ cells and functional characterization of zbtb40 during 17alpha-methyltestosterone-induced spermatogenesis in orange-spotted grouper (Epinephelus coioides)

Xi Wu,Yang Yang,Chaoyue Zhong,Yin Guo,Haoran Lin,Xiaochun Liu,Shuisheng Li

doi:10.1186/s12864-020-6477-4

Abstract

BackgroundSpermatogenesis is an intricate process regulated by a finely organized network. The orange-spotted grouper (Epinephelus coioides) is a protogynous hermaphroditic fish, but the regulatory mechanism of its spermatogenesis is not well-understood. In the present study, transcriptome sequencing of the male germ cells isolated from orange-spotted grouper was performed to explore the molecular mechanism underlying spermatogenesis.ResultsIn this study, the orange-spotted grouper was induced to change sex from female to male by 17alpha-methyltestosterone (MT) implantation. During the spermatogenesis, male germ cells (spermatogonia, spermatocytes, spermatids, and spermatozoa) were isolated by laser capture microdissection. Transcriptomic analysis for the isolated cells was performed. A total of 244,984,338 clean reads were generated from four cDNA libraries. Real-time PCR results of 13 genes related to sex differentiation and hormone metabolism indicated that transcriptome data are reliable. RNA-seq data showed that the female-related genes and genes involved in hormone metabolism were highly expressed in spermatogonia and spermatozoa, suggesting that these genes participate in the spermatogenesis. Interestingly, the expression of zbtb family genes showed significantly changes in the RNA-seq data, and their expression patterns were further examined during spermatogenesis. The analysis of cellular localization of Eczbtb40 and the co-localization of Eczbtb40 and Eccyp17a1 in different gonadal stages suggested that Eczbtb40 might interact with Eccyp17a1 during spermatogenesis.ConclusionsOur study, for the first time, investigated the transcriptome of the male germ cells from orange-spotted grouper, and identified functional genes, GO terms, and KEGG pathways involved in spermatogenesis. Furthermore, Eczbtb40 was first characterized and its role during spermatogenesis was predicted. These data will contribute to future studies on the molecular mechanism of spermatogenesis in teleosts.

Highlights

Spermatogenesis is an intricate process regulated by a finely organized network
Developmental stages of gonads during male by 17alphamethyltestosterone (MT)-induced spermatogenesis Oocytes in the sham group remained in the primarygrowth stage throughout the experimental period (Fig. 1a, b, d and f)
In conclusion, during the spermatogenesis, four different male germ cells were isolated by an optimized protocol of Laser capture microdissection (LCM) and transcriptome analysis was performed

Summary

Introduction

Spermatogenesis is an intricate process regulated by a finely organized network. The orange-spotted grouper (Epinephelus coioides) is a protogynous hermaphroditic fish, but the regulatory mechanism of its spermatogenesis is not well-understood. Transcriptome sequencing of the male germ cells isolated from orange-spotted grouper was performed to explore the molecular mechanism underlying spermatogenesis. Some approaches have already been used to quantify and localize unique genes in the testis during spermatogenesis, such as in situ hybridization [2, 3], immunohistochemistry, and serial analysis of gene expression [4, 5]. These methods can determine the spatial expression of specific gene during spermatogenesis, but cannot provide high throughput gene expression profiles in specific cell populations

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Discussion

Conclusion