Differential expression of WNT4 in testicular and ovarian development in a marsupial

Abstract

Background: WNT4 is a key regulator of gonadal differentiation in humans and mice, playing a pivotal role in early embryogenesis. Using a marsupial, the tammar wallaby, in which most gonadal differentiation occurs after birth whilst the young is in the pouch, we show by quantitative PCR during early testicular and ovarian development that WNT4 is differentially expressed in gonads.

Results: Before birth, WNT4 mRNA expression was similar in indifferent gonads of both sexes. After birth, in females WNT4 mRNA dramatically increased during ovarian differentiation, reaching a peak by day 9-13 post partum (pp) when the ovarian cortex and medulla are first distinguishable. WNT4 protein was localised in the ovarian cortex and at the medullary boundary. WNT4 mRNA then steadily decreased to day 49, by which time all the female germ cells have entered meiotic arrest. In males, WNT4 mRNA was down-regulated in testes immediately after birth, coincident with the time that seminiferous cords normally form, and rose gradually after day 8. By day 49, when testicular androgen production normally declines, WNT4 protein was restricted to the Leydig cells.

Conclusion: This is the first localisation of WNT4 protein in developing gonads and is consistent with a role for WNT4 in steroidogenesis. Our data provide strong support for the suggestion that WNT4 not only functions as an anti-testis gene during early development, but is also necessary for later ovarian and testicular function.

Figure 1

Amino acid sequence alignment of WNT4 orthologues. The solid black box indicates the signaling peptide (transmembrane domain) of the WNT4 protein, while the dashed line outlines the WNT gene family domain. Conserved cysteine residues (C) important for secondary structure are marked with an asterisk. Shaded regions indicate residues with shared homology. Numbers indicate residue number.

Figure 2

Similarity and evolution analysis of WNT4 protein. a) Percentage amino acid similarity of WNT4 between the tammar and other species; b) A phylogenetic tree was constructed with WNT4 protein sequence from the tammar wallaby and other species listed in the methods. Phylogenies were performed with PHYLIP3.63 by bootstrap analysis using Maximum-likelihood analysis (1000 replicates, first values), maximum parsimony (1000 replicates, second values) and neighbour-joining (1000 replicates, third values). Numbers along branches indicate reliability of each branch when run 1000 times.

Figure 3

WNT4 gene structure in the tammar. a) The tammar wallaby WNT4 gene consists of 5 exons. The black blocks represent the exons 1–5, of 90bp, 236bp, 132bp, 143bp and 561bp respectively. The size of intron 1 and 2 are unknown while intron 3 and 4 are 154bp and 575bp accordingly; b) Southern blotting of genomic DNA. Male and female genomic DNA was digested with Eco RI, Sal I + Xbal I, respectively, and hybridized with [α-³²P]dCTP-labelled tammar WNT4 cDNA probe.

Figure 4

WNT4 mRNA expression in tammar adult tissues. Expression of the WNT4 gene (209bp) was assessed in a range of adult tissues using the 18S mRNA (100bp) as a reference. There was very strong expression in the testis, ovary and kidney, moderate expression in muscle and liver, weak expression in spleen and lung, and none in the brain, heart and prostate.

Figure 5

Quantitative analysis of WNT4 expression and testosterone in developing gonads. (a) Relative expression of WNT4 mRNA against a reference of 18S mRNA (means ± s.e.m) for female (○) and male (●) gonads from the last 3 days of the 26.5 day gestation to day 40–49 post partum. Ages in parentheses indicate actual range of ages in the samples assayed. In females, WNT4 expression increased sharply between day 4–8 pp and 9–14 pp coinciding with morphological differentiation of the ovary. Relative expression then declined gradually to days 40–49 pp. In males, WNT4 expression fell sharply between fetal stages and day 1–3 pp coinciding with morphological differentiation of the testis. WNT4 expression gradually rose from day 9 to reach levels similar to those seen in females at day 40–49 pp. (b) Testicular (●) and ovarian (○) testosterone concentrations (ng/mg) throughout the first 50 days post partum. From [29]

Figure 6

Western blotting analysis of WNT4. Testis and ovary protein cross-reacted with the human anti-WNT4 antibody (Abcam), giving a 39 KD band as predicated from the tammar WNT4 sequence. The two negative controls, heart and BSA, did not cross-react.

Figure 7

Immunolocalisation of WNT4 protein during gonadal development in the tammar. The relative developmental stage of the fetus at each time point examined is shown in the top panels. In fetal gonads one day before birth (d25 of pregnancy), WNT4 protein was distributed broadly in the somatic cells of both females (a) and males (g); from 1–3 days after birth WNT4 stained strongly in the presumptive ovary (b) while it decreased dramatically in the developing testis (h) as the seminiferous cords form. By 12–13 days post partum, WNT4 staining was more specific and clearer. In testis, there was strong WNT4 staining in the Leydig cells and the tunica albuginea (i); there was strong staining in the somatic cells and medulla of the ovary (c). There was no immunoreactivity in any of the negative controls: d, e, f, j, k and l (IgG, pre-immune serum). Co = cortex; M = mesonephroi; Me = medulla; Ov = ovary; ST = seminiferous tubule; T = testis; TA = tunica albuginea. Scale bars= 100 μm

Figure 8

Immunolocalisation of WNT4 in the testis of the tammar at day 45pp. WNT4 immunostaining was present in the Leydig cells and the tunica at day 45pp, but it was absent from germ cells and Sertoli cells and Wolffian duct (WD). The dashed square on the low-power image (inset) is the area shown in high-power. Germ cells (GC), Sertoli cells (ST), Leydig cells (LC) and tunica albuginea (TA). Scale bars = 100 μmm.