Regulation of the gonadal transcriptome during sex determination and testis morphogenesis: comparative candidate genes

Abstract

Gene expression profiles during sex determination and gonadal differentiation were investigated to identify new potential regulatory factors. Embryonic day 13 (E13), E14, and E16 rat testes and ovaries were used for microarray analysis, as well as E13 testis organ cultures that undergo testis morphogenesis and develop seminiferous cords in vitro. A list of 109 genes resulted from a selective analysis for genes present in male gonadal development and with a 1.5-fold change in expression between E13 and E16. Characterization of these 109 genes potentially important for testis development revealed that cytoskeletal-associated proteins, extracellular matrix factors, and signaling factors were highly represented. Throughout the developmental period (E13-E16), sex-enriched transcripts were more prevalent in the male with 34 of the 109 genes having testis-enriched expression during sex determination. In ovaries, the total number of transcripts with a 1.5-fold change in expression between E13 and E16 was similar to the testis, but none of those genes were both ovary enriched and regulated during the developmental period. Genes conserved in sex determination were identified by comparing changing transcripts in the rat analysis herein, to transcripts altered in previously published mouse studies of gonadal sex determination. A comparison of changing mouse and rat transcripts identified 43 genes with species conservation in sex determination and testis development. Profiles of gene expression during E13-E16 rat testis and ovary development are presented and candidate genes for involvement in sex determination and testis differentiation are identified. Analysis of cellular pathways did not reveal any specific pathways involving multiple candidate genes. However, the genes and gene network identified influence numerous cellular processes with cellular differentiation, proliferation, focal contact, RNA localization, and development being predominant.

Figure 1

Histology of embryonic rat testis and ovary. Tissue sections from E13 (A), E14 (B), and E16 (C) ovary were analyzed. Tissue sections from E13 (D), E14 (E), and E16 (F) testis, as well as from E13 testis cultured for 3 days (G) were fixed and stained for morphological analysis. Serial sections were stained. Black arrows indicate testis cords in testes or oocyte nests in ovaries.

Figure 2

Dendrogram analysis of microarray data reveals the relative relatedness of gonadal transcriptomes. Dendrograms were produced in GeneSpring 7.2 using an unsupervised cluster analysis. Genes are clustered by pattern of expression. Sample sets or time points are clustered by relatedness of gene expression patterns as indicated by the left margin connective illustrations (i.e. links). (A) Dendrogram of male E13, E14, and E16 testis gene expression above a signal value of 75. (B) Dendrogram of female ovary gene expression above a signal of 75 at E13, E14, and E16. (C) Male gonadal genes for E13, E14, E16, and E13 cultured testis expressed above a signal of 75 and with a 1.5-fold significant change during the developmental period. (D) Female gonadal genes for E13, E14, and E16 expressed above a signal of 75 and with a 1.5-fold significant change.

Figure 3

Expressed gene numbers in male and female gonadal development during sexual differentiation. Venn diagrams comparing numbers of genes expressed above a raw signal of 75 were produced in Genespring 7.2. Genes expressed over 75 in E13, E14, and E16 in the rat testis (A) and ovary (B) are compared.

Figure 4

Numbers of regulated genes from testis and ovary developmental periods. (A) Numbers of genes expressed during testis development that have an expression signal of at least 75 and a statistically significant increase or decrease of 1.5-fold between each time interval are represented in a bar graph. (B) Numbers of ovary development genes with a signal of at least 75 and a statistically significant 1.5-fold increase or decrease. (C) The number of candidate genes for involvement in testis development and cord formation was reduced by comparison with E13 testis culture. Genes that are expressed above a raw signal of 75, have a 1.5-fold increase or decrease in the male time course and have similar patterns of expression changes from E13 to cultured testis as from E13 to E14 or E13 to E16. (D) Candidate testis development genes after subtraction of ovary expressed genes and comparison of testis organ cultures.

Figure 5

Functional categorization of the 109 gene list. The number of genes in each functional category of the 109 gene list of male-enhanced transcripts regulated in testis development.

Figure 6

Functional gene network analysis of testis development genes. The 109 gene list was analyzed by Pathway Assist. Cell processes involved in testis development were determined based on the number of arrows connected to each box (connectivity). Rectangles are the cellular processes, light shaded shapes are a subset of the 109 list, and dark circles represent interconnecting proteins not on the 109 list.

Figure 7

The number of gender-enriched (A) enhanced (B) transcripts between E13 and E16 in the testis and ovary. (A) All genes gender-enriched in the male and female time courses with given signal cut off values. Genes expressed above a signal of 75 in any time point of one sex were removed from lists of genes above a specified signal cut-off in at least one time point of the other sex to obtain the number of gender-enriched genes for a given signal cut off. (B) Numbers of gender-enhanced genes at a single given time point with at least a 1.5-fold change between the sexes were determined.