Prelude to Intersex in Fish: Identifying a Sensitive Period for Feminization

Field studies have shown a high occurrence of intersex (the presence of both male and female characteristics) and ovotestis (the presence of eggs in the testis) in wild populations of a fish known as roach (Rutilus rutilus) in rivers in the United Kingdom that are downstream from wastewater treatment plants. Furthermore, studies have demonstrated that intersex males are less fertile, which may have population-level effects. However, to date, scientists have been unable to induce intersex in male fish with controlled exposures to wastewater effluents. A study conducted at The University of Exeter now shows that the sensitive period for feminization of the reproductive duct—in which the male testis forms an ovary-like cavity—may occur earlier than previously thought, and raises new questions about the conditions that lead to actual germ cell disruption [EHP 113:1299–1307]. 
 
Many questions persist about the causes of and the most vulnerable life stages for various types of sexual effects induced by estrogenic chemicals in wastewater effluent. In this study, the researchers collected two different U.K. wastewater effluents and exposed wild roach at two life stages: during early life and development of the gonads (from fertilization up to 300 days post-hatch) and as adults producing germ cells following annual spawning. These adults included one group of fish that had been raised in clean water and another that had hatched and grown to maturity in the wild. 
 
Both effluents induced synthesis of vitellogenin (an estrogen-dependent yolk precursor and biomarker of estrogen exposure) at both life stages, with the extent of this induction correlating with the steroid estrogen content of the effluent. Previous studies have demonstrated that feminization of the sperm duct to form an ovary-like cavity occurs when exposure to effluent comes during the time of sexual differentiation, which in roach occurs from 50 to 150 days post-hatch. This study showed alteration of the sperm duct with an exposure earlier in life, from fertilization to 60 days post-hatch, before any signs of sexual development appear. The alteration, furthermore, was permanent, persisting even after 240 days’ maintenance in clean water after exposure. 
 
However, no ovotestis was observed in any of the juvenile fish. There was also no evidence of ovotestis in post-spawning adult male roach raised in a clean environment and subsequently exposed to effluent. There was evidence that the wild males had previously been exposed to estrogenic stimuli, as some of males had ovotestis when the study began. The severity of this condition increased slightly during the study period, but the increase occurred across both exposed and control fish and thus appeared unrelated to the study effluent exposure. 
 
The authors suggest possible explanations that need further study—one is that ovotestis is induced only by effluents with greater levels of estrogenic chemicals than those used in the study. The researchers evaluated the effluents for content of two chemicals previously implicated in causing intersex—steroidal estrogens and alkylphenols—and found that these levels were similar to concentrations reported in wastewater effluents in the United Kingdom and worldwide. They emphasize that chemical content and interactions ideally should be taken into account when trying to determine the conditions that lead to sexual effects. 
 
The results of these studies raise the possibility that ovotestis may be a result either of longevity of exposure or of programming in early life that manifests itself as fish mature sexually. Previous findings from the authors support this idea by showing that the severity of intersex increases with age. The authors are further exploring these possibilities now with a laboratory study of roach that includes an environmentally relevant estrogen exposure of two years’ duration.

We wish to report some corrections to our study [1], none of which alters the interpretation of the data or the conclusions drawn. After publication, we noticed that one of the microarray hybridizations (on sample NB11) was performed on the same patient's material as another hybridization (sample NB4; see Table 1; a corrected version of Table 5 [1]). As this error leads to an incorrect subclassification of the patients into the 'favourable' and 'unfavourable' neuroblastoma subgroups, we would like to exclude this data point from the differential expression analysis of favorable versus unfavorable neuroblastoma given under the heading 'Differential expression analysis of favorable and unfavorable neuroblastoma' in the Results section of [1]. Careful reanalysis after exclusion of NB11 did not lead to important changes in the generated gene lists and conclusions; the changes are given in the corrected paragraph and Table 2 (a  corrected version of Table 4 [1]), and the Additional data files 1 and 2 (corrected versions of Additional data files 2 and 3 [1]) available online with this article.
We also noticed that sample NB1 is stage 1 instead of stage 4S and that sample NB2 was not localized to the adrenals (see Table 1).

Differential expression analysis of favorable and unfavorable neuroblastoma
So far, most published microarray studies on neuroblastomas mainly compared favorable with unfavorable neuroblastomas in order to identify prognostic markers or pathways that are involved in these clearly different neuroblastoma tumor types. In order to add value to such an analysis, we contrasted similar differentially expressed gene lists with the normal neuroblast expression profile (Additional data file 1). In a first step, we compared the differentially expressed genes between these two tumor types with published prognostic gene lists. We found that 23 of the 193 genes on our list were previously reported, including the well established markers MYCN, NTRK1, and CD44 (see NBGS analysis in Additional data file 2). This overlap demonstrates the validity of the selected neuroblastoma panel and their expression profile. Subsequently, we looked for the corresponding gene expression levels of the differentially expressed genes in the normal counterpart cells, aiming to select neuroblastoma candidate genes. Of the 100 genes that are more highly expressed in favorable tumors (compared to unfavorable) 41 also have a significant differential expression (either higher or lower) compared to neuroblasts, whereas 43 of the 93 genes that are more highly expressed in unfavorable tumors exhibit differential expression compared to the neuroblasts (Table 2).
From this analysis, a few putative positional tumor suppressor candidates emerge: CDC42 on 1p36, CACNA2D3 on 3p21 and DLK1 on 14q. The latter two genes are of particular interest because they are highly expressed in neuroblasts and favorable neuroblastomas and their expression is significantly lower in unfavorable neuroblastomas. Among the genes that are more highly expressed in unfavorable neuroblastomas than in favorable ones and neuroblasts, the proven oncogenic transcription factor MYCN emerges (and putative downstream genes KIFAP3, OPHN1, RGS7, ASCL1, ODC1, TWIST1 and TYMS, according to NBGS), as well as several other genes that have been identified or studied in the context of neuroblastoma such as ALK and PRAME, and positional candidates on 17q including BIRC5 and RNU2.

Additional data files
Additional data files 1 and 2 containing the corrected data available online with this article. Samples were subdivided into favorable or unfavorable type based on MYCN amplification, ploidy and age at diagnosis. *Neuroblastoma or nodular ganglioneuroblastoma. ND, not determined or unknown.

References
11p -Genes that are differentially expressed compared with neuroblasts among the differentially expressed genes in favorable neuroblastoma (NB) vs unfavorable NB, with an indication of the number of neuroblastoma microarray studies in which these genes were found through NBGS analysis. NBGS, Neuroblastoma Gene Server.