Telling the story of XX sex reversal in the goat: highlighting the sex-crossroad in domestic mammals.

The conditions for sex reversal in vertebrate species have been studied extensively and have highlighted numerous key factors involved in sex differentiation. We review here the history of the development of knowledge, referring to one example of complete female-to-male XX sex reversal associated with a polled phenotype in the goat. The results and hypotheses concerning this polled intersex syndrome (PIS) are then presented, firstly with respect to the transcriptional regulatory effects of the PIS mutation, and secondly regarding the role of the main ovarian-differentiating factor in this PIS locus, the FOXL2 gene.

Loss of R-spondin1 and Foxl2 amplifies female-to-male sex reversal in XX mice.

In vertebrates, 2 main genetic pathways have been shown to regulate ovarian development. Indeed, a loss of function mutations in Rspo1 and Foxl2 promote partial female-to-male sex reversal. In mice, it has been shown that the secreted protein RSPO1 is involved in ovarian differentiation and the transcription factor FOXL2 is required for follicular formation. Here, we analysed the potential interactions between these 2 genetic pathways and have shown that while Rspo1 expression seems to be independent of Foxl2 up-regulation, Foxl2 expression partly depends of Rspo1 signalisation. This suggests that different Foxl2-positive somatic cell lineages exist within the ovaries. In addition, a combination of both mutated genes in XX Foxl2(-/-)/Rspo1(-/-) gonads promotes sex reversal, detectable at earlier stages than in XX Rspo1(-/-) mutants. Ectopic development of the steroidogenic lineage is more pronounced in XX Foxl2(-/-)/Rspo1(-/-) gonads than in XX Rspo1(-/-) embryos, suggesting that Foxl2 is involved in preventing ectopic steroidogenesis in foetal ovaries.

Attempt to rescue sex-reversal by transgenic expression of the PISRT1 gene in XX PIS-/- goats.

The Polled Intersex Syndrome (PIS mutation) in goats leads to an absence of horn and to an early sex-reversal of the XX gonads. This mutation is a deletion of an 11.7-kb DNA fragment showing a tissue-specific regulatory activity. Indeed, in XX PIS(-/-) gonads the deletion of PIS leads to the transcriptional extinction of at least 3 neighboring genes, FOXL2, PFOXic and PISRT1. Among them, only FOXL2 is a 'classical' gene, encoding a highly conserved transcription factor. On the other hand, knock-out of Foxl2 in mice results in an early blocking of follicle formation without sex-reversal. This phenotype discrepancy leads to two hypotheses, either FOXL2 is responsible for XX sex-reversal in goat assuming distinct functions of its protein during ovarian differentiation in different mammals, or other PIS-regulated genes are involved. To assess the second possibility, PISRT1 expression was constitutively restored in XX PIS(-/-) gonads. Six transgenic fetuses were obtained by nuclear transfer and studied at 2 developmental stages, 41 and 46 days post-reconstruction. The gonads of these fetuses appear phenotypically identical to those of cloned non-transgenic controls. Conclusively, this result argues for FOXL2 being responsible for the PIS gonad-associated phenotype. Its invalidation in goat will help to better understand this complex syndrome.

Expression studies of the PIS-regulated genes suggest different mechanisms of sex determination within mammals.

In mammals, the Y-located SRY gene is known to induce testis formation from the indifferent gonad. A related gene, SOX9, also plays a critical role in testis differentiation in mammals, in birds and reptiles. It is now assumed that SRY acts upstream of SOX9 in the sex determination cascade, but the regulatory link which should exist between these two genes remains unknown. Studies on XX sex reversal in polled goats (PIS mutation: Polled Intersex Syndrome) have led to the discovery of a female-specific locus crucial for ovarian differentiation. This genomic region is composed of at least two genes, FOXL2 and PISRT1, which share a common transcriptional regulatory region, PIS. In this review, we present the expression pattern of these PIS-regulated genes in mice. The FOXL2 expression profile of mice is similar to that described in goats in accordance with a conserved role of this ovarian differentiating gene in mammals. On the contrary, the PISRT1 expression profile is different between mice and goats, suggesting different mechanisms of the primary switch in the testis determination process within mammals. A model based on two different modes of SOX9 regulation in mice and other mammals is proposed in order to integrate our results into the current scheme of gonad differentiation.

Structure, evolution and expression of the FOXL2 transcription unit.

FOXL2 is a putative transcription factor involved in ovarian development and function. Its mutations in humans are responsible for the blepharophimosis syndrome, characterized by eyelid malformations and premature ovarian failure (POF). Here we have performed a comparative sequence analysis of FOXL2 sequences of ten vertebrate species. We demonstrate that the entire open reading frame (ORF) is under purifying selection leading to strong protein conservation. We also review recent data on FOXL2 transcript and protein expression. FOXL2 has been shown 1) to be the earliest known sex dimorphic marker of ovarian determination/differentiation in vertebrates, 2) to have, at least in mammals, an ovarian expression persisting until adulthood. The conservation of its sequence and pattern of expression suggests that FOXL2 might be a key factor in the early development of the vertebrate female gonad and involved later in adult ovarian function. Finally, we provide arguments for the existence of an alternative transcript in rodents, that may arise from a differential polyadenylation. Although it has only been demonstrated in rodents, its presence/absence in other species deserves further investigation.

Evaluation of the EYTEX(TM) system in the COLIPA eye irritation program.

In the context of the 6th Amendment of the European Directive on Cosmetics, several cosmetic companies concentrate their basic research on the development of the best adapted battery of in vitro tests able to be incorporated in the ocular risk assessment process. Consequently, the European Cosmetic Toiletry and Perfumery Association (COLIPA) has initiated an international multicentric study with the main purpose to validate available alternatives in vitro methods for assessing the eye irritation potential of cosmetic raw materials and formulations. The alternative methods assessed in this validation study were chosen since all of these tests had already been used and continue to be conducted in the risk assessment process. The different endpoints of these assays are mainly biological parameters except for the biochemical assay named EYTEX(TM). In this article, the defined prediction models and the different protocols used in the COLIPA study are described. Then, the EYTEX assay results are presented and discussed in details in order to understand the failure of this assay during this validation study. The relevance and the reliability of the EYTEX assay were particularly low in two laboratories, whereas one laboratory presented acceptable data with a low compatibility with tested samples. These results underline the problem of the complex qualification process of this assay, since sometimes the same sample has been qualified with different protocols in the three laboratories. This validation study also demonstrates that, in the case of EYTEX assay, the criteria used to establish a prediction model have not been rigorous enough. For instance, the mixture of all the EYTEX protocols is not suitable for the establishment of a well-adapted prediction model. Furthermore, a clearer definition of limitations of the EYTEX assay seems to be necessary to better harmonize the qualification procedure in the three laboratories. The COLIPA validation process clearly demonstrated that the EYTEX assay was first, not suitable for the assessment of the eye irritation potential of surfactants and formulations based on surfactants, and secondly not ready for a validation study requiring the establishment of adequate and well defined mathematical prediction models. However, internal comparative studies with specific benchmarks on emulsions containing a low percentage of surfactants may be more adaptable to this type of assay.

Effects of endothelin on the lethality induced by platelet-activating factor or endotoxin in the mouse.

Platelet-activating factor (PAF) is a mediator that decreases cardiac output and total peripheral resistances leading to profound hypotension. It seems to be involved in shock states and in the deleterious effects of endotoxin. As the natural peptide endothelin (ET) shows potent vasoconstrictor properties, we evaluate its activity towards PAF and endotoxin-induced lethality in mice. ET, at doses which per se did not exert any effects on mice vitality, produced a dose-dependent decrease in the PAF-induced lethality with a total protection at 5 micrograms/kg. But conversely to these results, ET potentiated the mortality induced by endotoxin. These results suggest that ET or endothelin analogs could be worthy for therapeutic use in some types of shock, at least when endotoxin is not involved. Complementary studies are necessary to strengthen these preliminary results.

[Value of cumulative electrodermal responses in subliminal auditory perception. A preliminary study]. / Intérêt des réponses électrodermales cumulées dans la perception subliminale auditive. Une étude préliminaire.

This exploratory study examined the usefulness of averaging electrodermal potential responses for research on subliminal auditory perception. Eighteen female subjects were exposed to three kinds (emotional, neutral and 1000 Hz tone) of auditory stimulation which were repeated six times at three intensities (detection threshold, 10 dB under this threshold and 10 dB above identification threshold). Analysis of electrodermal potential responses showed that the number of responses was related to the emotionality of subliminal stimuli presented at detection threshold but not at 10 dB under it. The data interpretation proposed refers to perceptual defence theory. This study indicates that electrodermal response count constitutes a useful measure for subliminal auditory perception research, but averaging those responses was not shown to bring additional information.