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. 2013 Jun;28(6):1635-46.
doi: 10.1093/humrep/det043. Epub 2013 Mar 12.

The RHOX Homeobox Gene Cluster Is Selectively Expressed in Human Oocytes and Male Germ Cells

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Free PMC article

The RHOX Homeobox Gene Cluster Is Selectively Expressed in Human Oocytes and Male Germ Cells

H W Song et al. Hum Reprod. .
Free PMC article

Abstract

Study question: What human tissues and cell types express the X-linked reproductive homeobox (RHOX) gene cluster?

Summary answer: The RHOX homeobox genes and proteins are selectively expressed in germ cells in both the ovary and testis.

What is known already: The RHOX homeobox transcription factors are encoded by an X-linked gene cluster whose members are selectively expressed in the male and female reproductive tract of mice and rats. The Rhox genes have undergone strong selection pressure to rapidly evolve, making it uncertain whether they maintain their reproductive tissue-centric expression pattern in humans, an issue we address in this report.

Study design, size, duration: We examined the expression of all members of the human RHOX gene cluster in 11 fetal and 8 adult tissues. The focus of our analysis was on fetal testes, where we evaluated 16 different samples from 8 to 20 weeks gestation. We also analyzed fixed sections from fetal testes, adult testes and adult ovaries to determine the cell type-specific expression pattern of the proteins encoded by RHOX genes.

Participants/materials, setting, methods: We used quantitative reverse transcription-polymerase chain reaction analysis to assay human RHOX gene expression. We generated antisera against RHOX proteins and used them for western blotting, immunohistochemical and immunofluorescence analyses of RHOXF1 and RHOXF2/2B protein expression.

Main results and the role of chance: We found that the RHOXF1 and RHOXF2/2B genes are highly expressed in the testis and exhibit low or undetectable expression in most other organs. Using RHOXF1- and RHOXF2/2B-specific antiserum, we found that both RHOXF1 and RHOXF2/2B are primarily expressed in germ cells in the adult testis. Early stage germ cells (spermatogonia and early spermatocytes) express RHOXF2/2B, while later stage germ cells (pachytene spermatocytes and round spermatids) express RHOXF1. Both RHOXF1 and RHOXF2/2B are expressed in prespermatogonia in human fetal testes. Consistent with this, RHOXF1 and RHOXF2/2B mRNA expression increases in the second trimester during fetal testes development when gonocytes differentiate into prespermatogonia. In the human adult ovary, we found that RHOXF1 and RHOXF2/2B are primarily expressed in oocytes.

Limitations, reasons for caution: While the average level of expression of RHOX genes was low or undetectable in all 19 human tissues other than testes, it is still possible that RHOX genes are highly expressed in a small subset of cells in some of these non-testicular tissues. As a case in point, we found that RHOX proteins are highly expressed in oocytes within the human ovary, despite low levels of RHOX mRNA in the whole ovary.

Wider implications of the findings: The cell type-specific and developmentally regulated expression pattern of the RHOX transcription factors suggests that they perform regulatory functions during human fetal germ cell development, spermatogenesis and oogenesis. Our results also raise the possibility that modulation of RHOX gene levels could correct some cases of human infertility and that their encoded proteins are candidate targets for contraceptive drug design.

Keywords: homeobox gene cluster; oogenesis; ovary; spermatogenesis; testis.

Figures

Figure 1
Figure 1
(A) The human RHOX cluster. The map position on the X chromosome is in accordance with the high coverage assembly GRCh37 from the Genome Reference Consortium (Ensembl.org). (B) The exon–intron structure of the human RHOX genes. The numbers indicate the nucleotide length of the exons and introns. The colors denote protein-coding regions. (C) Alignment of the predicted amino-acid sequences in the homeodomain region of the human RHOX proteins. The four amino acids that mediate base-specific binding to DNA (Gehring et al., 1994) are indicated in green. The single amino-acid difference between RHOXF2 and RHOXF2B in the homeodomain region is indicated in red. The yellow-shaded residues are the most conserved amino acids in all known homeodomains; they serve as crucial anchor points to appropriately fold the homeodomain structure (Duboule, 1994). (D) qRT–PCR analysis of total cellular RNA from the indicated human tissues. Values were normalized to RPL32 mRNA level and denote the mean fold change ± standard error of the mean (SEM).
Figure 2
Figure 2
Cell type-specific expression of RHOX transcription factors in adult human testes. (A) Western blot analysis to validate the specificity of the antiserum we generated against RHOXF1 and RHOXF2/2B. Representative figures out of three replicates are shown. Loaded are extracts from human testicular tissue from two individuals (lanes 1, 3, 5 and 7 are from one individual, while lanes 2, 4, 6 and 8 are from another individual). These extracts were incubated with purified RHOXF1 antiserum (lanes 1 and 2), purified RHOXF1 antiserum preincubated with recombinant GST-RHOXF1 protein for 2 h (lanes 3 and 4), purified RHOXF2/2B antiserum (lanes 5 and 6), or purified RHOXF2/2B antiserum preincubated with recombinant GST-RHOXF2 protein for 2 h (lanes 7 and 8). The arrows and asterisks indicate specific and non-specific bands, respectively. Red rectangles denote the position of bands largely abolished by pre-incubation with recombinant RHOX proteins. β-actin serves as a loading control. (BE) Immunohistochemical analysis performed on adult human testis sections with purified RHOXF1 antiserum (B left and D), purified RHOXF1 antiserum preincubated with GST-RHOXF1 protein (B right), purified RHOXF2/2B antiserum (C left and E) and purified RHOXF2/2B antiserum preincubated with GST-RHOXF2 protein (C right). Nuclei were counterstained with hematoxylin. Representative figures out of three replicates are shown. Arrowheads denote selected cells stained with the RHOXF1 or RHOXF2/2B antiserum. Inset boxes show enlarged views of RHOXF1- and RHOXF2/2B antisera stained cells. P, pachytene spermatocytes; R, round spermatids; B, type B spermatogonia; PL, preleptotene spermatocytes; L, leptotene spermatocytes. Scale bar: 20 µm (C, E) and 100 µm (B, D).
Figure 3
Figure 3
RHOX expression in human fetal testes. (A, B) qRT–PCR analysis of total cellular RNA from human fetal testes of the indicated gestation weeks (from 6, 8 and 5 pooled fetal testes derived from 8–9-, 13–16- and 17–20-week-old fetuses, respectively). Values were normalized to RPL32 mRNA level and denote the mean fold change ± SEM. (C, D) Immunohistochemical analysis performed on 18–19 week fetal testes sections with purified antiserum against the indicated protein or preimmune IgG at low magnification (C) or high magnification (D). Representative figures out of three replicates are shown. Nuclei were counterstained with hematoxylin. Arrows denote selected cells expressing the protein recognized by the antisera. Inset boxes show enlarged views of RHOXF1- and RHOXF2/2B antisera stained cells. Scale bar: 20 µm (C) and 100 µm (D).
Figure 4
Figure 4
RHOX proteins are specifically expressed in prespermatogonia in human fetal testes. Double immunofluorescence analysis performed on 18-week human fetal testes sections first incubated with preimmune IgG (red, A), purified RHOXF1 antiserum (red, B) or purified RHOXF2/2B antiserum (red, C), followed by incubation with purified MAGE-A4 antiserum (green, A–C). Representative figures out of three replicates are shown. Enlarged views of MAGE-A4-stained and RHOXF1- or RHOXF2/2B-co-stained cells are shown in the inset boxes. F1, RHOXF1; F2/2B, RHOXF2/2B.
Figure 5
Figure 5
RHOX proteins are expressed in the oocytes in adult human ovaries. Representative figures are shown from immunohistochemical analysis of human adult ovary sections from nine individuals using purified RHOXF1 antiserum, purified RHOXF2/2B antiserum or preimmune IgG. Representative staining of primordial follicles (A), primary follicles (B), secondary follicles (C) and antral follicles (D). The red dotted lines outline oocytes and the yellow arrows demarcate granulosa cells. Scale bar: 20 µm.

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