A laboratory investigation into features of morphology and physiology for their potential to predict reproductive success in male frogs.

Amphibian populations are declining globally, however, the contribution of reduced reproduction to declines is unknown. We investigated associations between morphological (weight/snout-vent length, nuptial pad colour/size, forelimb width/size) and physiological (nuptial pad/testis histomorphology, plasma hormones, gene expression) features with reproductive success in males as measured by amplexus success and fertility rate (% eggs fertilised) in laboratory maintained Silurana/Xenopus tropicalis. We explored the robustness of these features to predict amplexus success/fertility rate by investigating these associations within a sub-set of frogs exposed to anti-androgens (flutamide (50 µg/L)/linuron (9 or 45 µg/L)). In unexposed males, nuptial pad features (size/colour/number of hooks/androgen receptor mRNA) were positively associated with amplexus success, but not with fertility rate. In exposed males, many of the associations with amplexus success differed from untreated animals (they were either reversed or absent). In the exposed males forelimb width/nuptial pad morphology were also associated with fertility rate. However, a more darkly coloured nuptial pad was positively associated with amplexus success across all groups and was indicative of androgen status. Our findings demonstrate the central role for nuptial pad morphology in reproductive success in S. tropicalis, however, the lack of concordance between unexposed/exposed frogs complicates understanding of the utility of features of nuptial pad morphology as biomarkers in wild populations. In conclusion, our work has indicated that nuptial pad and forelimb morphology have potential for development as biomarkers of reproductive health in wild anurans, however, further research is needed to establish this.

Sex-dependent expression of anti-Müllerian hormone (amh) and amh receptor 2 during sex organ differentiation and characterization of the Müllerian duct development in Xenopus tropicalis.

Amphibian gonadal differentiation involves the action of sex steroids. Recent research indicates that the anti-Müllerian hormone (AMH) is involved in testicular development in some lower vertebrate species. For amphibians there is a lack of data on ontogenetic expression of the AMH receptor AMHR2/amhr2 and of progesterone receptors (PGRS/pgrs). Here we expand the knowledge on amphibian sex differentiation by characterizing ontogenetic mRNA levels of amh, amhr2, intracellular and membrane pgrs (ipgr and mpgr beta) and cytochrome P450 19a1 (cyp19a1) (ovarian marker) in the urogenital complex of the model species Xenopus (Silurana) tropicalis. Furthermore, we characterized the ontogenetic development of the Müllerian ducts (precursors of the female reproductive tract) histologically. The developmental period investigated spanned from beginning of gonadal differentiation, Nieuwkoop and Faber (NF) stage 51, to 4weeks post-metamorphosis. The Müllerian ducts were first observed at NF 64 in both sexes. Male-enhanced amh mRNA levels from NF 53/54 to 6days post-metamorphosis and female-enhanced cyp19a1 levels from NF 53 to 4weeks post-metamorphosis were noted. The sexually dimorphic mRNA level profile was more distinct for amh than for cyp19a1. The pgrs mRNA levels increased over the studied period and showed no sex differences. At later developmental stages, the amhr2 mRNA level was increased in putative females compared with males. Our findings suggest that AMH has a role in gonadal differentiation in X. tropicalis. We propose relative gonadal amh mRNA level as a testicular marker during early gonadal development in amphibians.

Molecular and histological endpoints for developmental reproductive toxicity in Xenopus tropicalis: Levonorgestrel perturbs anti-Müllerian hormone and progesterone receptor expression.

There is an increasing concern regarding the risks associated with developmental exposure to endocrine disrupting chemicals and the consequences for reproductive capability. The present study aimed to refine the Xenopus (Silurana) tropicalis test system for developmental reproductive toxicity by characterising molecular and histological features of sexual development, and to explore effects of exposure to the progestagen levonorgestrel (LNG). Larvae were exposed to LNG (0, 3, 30, 300 ng/L) over the first three weeks of development, encompassing the beginning of gonadal differentiation. mRNA levels of amh (anti-Müllerian hormone), amhr2 (amh receptor 2), ipgr (intracellular progesterone receptor), mpgr beta (membrane progesterone receptor beta), and cyp19a1 (cytochrome p450 19a1) were quantified in larvae and juveniles (4 weeks post-metamorphosis). Relative cyp19a1 and amh expression was used as a molecular marker for phenotypic sex of larvae. Gonadal and Müllerian duct development were characterised histologically in juveniles. Compared to controls, LNG exposure increased the expression of amh and ipgr in male larvae. In juveniles, mpgr beta expression was increased in both sexes and amhr2 expression was decreased in males, implying persistent effects of developmental progestagen exposure on amh and pgr expression signalling. No effects of LNG on the gonadal or Müllerian duct development were found, implying that the exposure window was not critical with regard to these endpoints. In juveniles, folliculogenesis had initiated and the Müllerian ducts were larger in females than in males. This new knowledge on sexual development in X. tropicalis is useful in the development of early life-stage endpoints for developmental reproductive toxicity.

Mixture effects of levonorgestrel and ethinylestradiol: estrogenic biomarkers and hormone receptor mRNA expression during sexual programming.

Synthetic progesterone (progestins) and estrogens are widely used pharmaceuticals. Given that their simultaneous unintentional exposure occurs in wildlife and also in human infants, data on mixture effects of combined exposures to these hormones during development is needed. Using the Xenopus (Silurana) tropicalis test system we investigated mixture effects of levonorgestrel (LNG) and ethinylestradiol (EE2) on hormone sensitive endpoints. After larval exposure to LNG (0.1nM), or EE2 (0.1nM) singly, or in combination with LNG (0.01, 0.1, 1.0nM), the gonadal sex ratio was determined histologically and hepatic mRNA levels of genes encoding vitellogenin (vtg beta1) and the estrogen (esr1, esr2), progesterone (ipgr) and androgen (ar) receptors were quantified using quantitative PCR. All EE2-exposed groups showed female-biased sex ratios and increased vtg beta1 mRNA levels compared with the controls. Compared with the EE2-alone group (positive control) there were no significant alterations in vtg beta1 levels or in sex ratios in the co-exposure groups. Exposure to LNG-alone caused an increase in ar mRNA levels in females, but not in males, compared to the controls and the co-exposed groups, indicating that co-exposure to EE2 counteracted the LNG-induced ar levels. No treatment related impacts on the mRNA expression of esr1, esr2, and ipgr in female tadpoles were found, suggesting that these endpoints are insensitive to long-term exposure to estrogen or progestin. Due to the EE2-induced female-biased sex ratios, the mRNA expression data for the low number of males in the EE2-exposed groups were not statistically analyzed. In conclusion, our results suggest that induced vtg expression is a robust biomarker for estrogenic activity in exposure scenarios involving both estrogens and progestins. Developmental exposure to LNG caused an induction of hepatic ar mRNA expression that was antagonized by combined exposure to EE2 and LNG. To our knowledge this is the first study to report effects of combined exposures to EE2 and LNG during the period of sexual programming.

Presence of alkylresorcinols, potential whole grain biomarkers, in human adipose tissue.

Alkylresorcinols (AR) in plasma samples have been suggested to be short- to medium-term biomarkers of whole grain wheat and rye intake. In the present study, we investigated whether AR are present in human adipose tissues, and if content correlated with long-term whole grain bread intake. Furthermore, we investigated if the relative AR homologue composition reflected what has been found previously in the habitual diet of Swedes. Biopsy samples (10-25 mg) from free-living Swedish women (n 20) were analysed by GC-MS. The mean total AR concentration in the samples was 0.54 (SD 0.35) microg/g, ranging from below limit of quantification ( < 0.08 microg/g) to 1.50 microg/g. Whole grain bread intake was significantly correlated with plasma total AR content (r 0.48, P < 0.05), and the C17 : 0/C21 : 0 ratio was 0.35 (sd 0.24), which is similar to what is found in plasma among free-living subjects consuming a mixed whole grain wheat and rye diet. These results suggest that AR in the adipose tissue should be evaluated as a long-term biomarker of whole grain wheat and rye intake.