Targeted mutation of secretogranin-2 disrupts sexual behavior and reproduction in zebrafish.

The luteinizing hormone surge is essential for fertility as it triggers ovulation in females and sperm release in males. We previously reported that secretoneurin-a, a neuropeptide derived from the processing of secretogranin-2a (Scg2a), stimulates luteinizing hormone release, suggesting a role in reproduction. Here we provide evidence that mutation of the scg2a and scg2b genes using TALENs in zebrafish reduces sexual behavior, ovulation, oviposition, and fertility. Large-scale spawning within-line crossings (n = 82 to 101) were conducted. Wild-type (WT) males paired with WT females successfully spawned in 62% of the breeding trials. Spawning success was reduced to 37% (P = 0.006), 44% (P = 0.0169), and 6% (P < 0.0001) for scg2a-/- , scg2b-/- , and scg2a-/-;scg2b-/- mutants, respectively. Comprehensive video analysis indicates that scg2a-/-;scg2b-/- mutation reduces all male courtship behaviors. Spawning success was 47% in saline-injected WT controls compared to 11% in saline-injected scg2a-/-;scg2b-/- double mutants. For these mutants, spawning success increased 3-fold following a single intraperitoneal (i.p.) injection of synthetic secretoneurin-a (P = 0.0403) and increased 3.5-fold with injection of human chorionic gonadotropin (hCG). Embryonic survival at 24 h remained on average lower in scg2a-/-;scg2b-/- fish compared to WT injected with secretoneurin-a (P < 0.001). Significant reductions in the expression of gonadotropin-releasing hormone 3 in the hypothalamus, and luteinizing hormone beta and glycoprotein alpha subunits in the pituitary provide evidence for disrupted hypothalamo-pituitary function in scg2a and scg2b mutant fish. Our results indicate that secretogranin-2 is required for optimal reproductive function and support the hypothesis that secretoneurin is a reproductive hormone.

Petroleum-derived naphthenic acids disrupt hormone-dependent sexual behaviours in male Western clawed frogs.

Naphthenic acids (NAs), the carboxylic acids found in petroleum, are of emerging concern as they contaminate coastlines after oil spills, leech into freshwater ecosystems of oil sands areas and have wide industrial applications. They are acutely toxic in fish and tadpoles and may be endocrine disruptors at sublethal levels. We characterized androgen-dependent courtship behaviours and their disruption by NAs in male Western clawed frogs, Silurana tropicalis. Courtship primarily consists of males producing low trills and achieving amplexus, a mating position where a male clasps a female. Adult males were exposed for 5 days to 20 mg/l NA and injected with human chorionic gonadotropin to induce calling. The duration of calling activity was significantly reduced by NA exposure. Other acoustic parameters such as dominant frequency, click rate and trill length were not affected. Vocalization and amplexus were both inhibited after NA exposure and restored after 2 weeks of recovery in clean water. To determine possible disruption at the level of the testes, the effects of NA exposure on gene expression of key players in steroidogenesis was determined. Exposure to NAs decreased srd5a on average by ~ 25%. The enzyme 5α-reductase, encoded by srd5a, converts testosterone to its more bioactive form 5α-dihydrotestosterone (DHT), so NAs may be affecting this steroidogenic step. However, the observed upregulation of lhr, star and cyp17a1 suggests that NA-exposed males may be attempting to counteract the reduced potential to produce DHT. Yet, these NA-exposed frogs have dramatically reduced calling duration, so the observed upregulation of star and cyp17a1 is decoupled from the vocalizations. Calling duration and the ability of males to amplex females is reversibly disrupted by NA exposure, implying that environmental reduction and removal of NAs may help improve habitability of contaminated ecosystems.

Agrochemicals disrupt multiple endocrine axes in amphibians.

Concern over global amphibian declines and possible links to agrochemical use has led to research on the endocrine disrupting actions of agrochemicals, such as fertilizers, fungicides, insecticides, acaricides, herbicides, metals, and mixtures. Amphibians, like other species, have to partition resources for body maintenance, growth, and reproduction. Recent studies suggest that metabolic impairments induced by endocrine disrupting chemicals, and more particularly agrichemicals, may disrupt physiological constraints associated with these limited resources and could cause deleterious effects on growth and reproduction. Metabolic disruption has hardly been considered for amphibian species following agrichemical exposure. As for metamorphosis, the key thyroid hormone-dependent developmental phase for amphibians, it can either be advanced or delayed by agrichemicals with consequences for juvenile and adult health and survival. While numerous agrichemicals affect anuran sexual development, including sex reversal and intersex in several species, little is known about the mechanisms involved in dysregulation of the sex differentiation processes. Adult anurans display stereotypical male mating calls and female phonotaxis responses leading to successful amplexus and spawning. These are hormone-dependent behaviours at the foundation of reproductive success. Therefore, male vocalizations are highly ecologically-relevant and may be a non-invasive low-cost method for the assessment of endocrine disruption at the population level. While it is clear that agrochemicals disrupt multiple endocrine systems in frogs, very little has been uncovered regarding the molecular and cellular mechanisms at the basis of these actions. This is surprising, given the importance of the frog models to our deep understanding of developmental biology and thyroid hormone action to understand human health. Several agrochemicals were found to have multiple endocrine effects at once (e.g., targeting both the thyroid and gonadal axes); therefore, the assessment of agrochemicals that alter cross-talk between hormonal systems must be further addressed. Given the diversity of life-history traits in Anura, Caudata, and the Gymnophiona, it is essential that studies on endocrine disruption expand to include the lesser known taxa. Research under ecologically-relevant conditions will also be paramount. Closer collaboration between molecular and cellular endocrinologists and ecotoxicologists and ecologists is thus recommended.