Early expression of aromatase and the membrane estrogen receptor GPER in neuromasts reveals a role for estrogens in the development of the frog lateral line system.

Estrogens and their receptors are present at very early stages of vertebrate embryogenesis before gonadal tissues are formed. However, the cellular source and the function of estrogens in embryogenesis remain major questions in developmental endocrinology. We demonstrate the presence of estrogen-synthesizing enzyme aromatase and G protein-coupled estrogen receptor (GPER) proteins throughout early embryogenesis in the model organism, Silurana tropicalis. We provide the first evidence of aromatase in the vertebrate lateral line. High levels of aromatase were detected in the mantle cells of neuromasts, the mechanosensory units of the lateral line, which persisted throughout the course of development (Nieuwkoop and Faber stages 34-47). We show that GPER is expressed in both the accessory and hair cells. Pharmacological activation of GPER with the agonist G-1 disrupted neuromast development and migration. Future study of this novel estrogen system in the amphibian lateral line may shed light on similar systems such as the mammalian inner ear.

Influence of physiological concentrations of androgens on the developmental competence and sex ratio of in vitro produced bovine embryos.

This study was designed to determine if the addition of androgens at ovarian follicular fluid (FF) concentrations to oocyte maturation media would alter the development and sex ratio of bovine embryos. To maximize hormone bioavailability, oil was removed and glass culture dishes were used during in vitro maturation (IVM) phase; this modified system was then used in the present experiment along with the standard IVM system utilizing plastic containers and incubation under oil. Ethanol (0.2%) was the vector for steroid hormone delivery. Oocytes were incubated for 22h in the presence of two doses ("low" and "high") of androstenedione (A4) or testosterone (T); the doses were based on the concentrations of both androgens in preovulatory bovine follicles (A4: 337.5 and 562.5ng/ml; T: 22.2 and 42.6ng/ml). The results of hormone assays indicated that bioavailability of steroid hormones remained relatively constant, regardless of the IVM system used. The plasticware with the addition of T resulted in significantly higher cleavage rates (80.0±2.1%) than any other combination of treatments (plasticware×A4: 71.5±2.6%; glassware×T: 71.2±1.9%; and glassware×A4: 71.4±2.4%). The blastocyst formation rate for the plasticware×T treatment (39.7±2.5%) was significantly greater than for all other combinations (glassware×T: 28.7±2.2%; glassware×A4: 24.0±2.8%; and plasticware×A4: 19.8±3.0%) and the low dose of T (37.1±2.5%) resulted in higher (p<0.05) blastocyst formation rates than all other treatments (T high dose: 29.2±2.5%; A4 high dose: 27.1±2.9%; and A4 low dose: 20.2±3.0%). The proportion of male embryos was greater (p<0.05) in plastic than glass dishes in the low-dose A4 group (59.1±8.7% vs. 38.2±5.5%, plasticware vs. glassware, respectively) and it tended to be greater (p<0.08) in the control groups and high-dose A4 group, but not in the T groups. There was a moderate positive correlation between blastocyst formation rates across all treatment and control groups, and the percentage of male bovine embryos (r=0.38, p<0.05). In summary, specific combinations of androgen and glassware/plasticware treatments did alter early bovine embryo development and sex ratio. The addition of T to IVM media increased the cleavage and blastocyst formation rates in plasticware and may be employed to improve the efficiency of the standard in vitro embryo production systems. Androstenedione appeared to enhance whereas testosterone nullified the deviation in sex ratio (pro-femaleness) associated with the use of glass IVM dishes.

Efficient induction of spawning of northern leopard frogs (Lithobates pipiens) during and outside the natural breeding season.

BACKGROUND: Amphibian declines are now recognized globally. It is also well known that many anurans do not reproduce easily in captivity, especially when held over long periods, or if they require hibernation before breeding. A simple method to induce spawning and subsequent development of large numbers of healthy tadpoles is therefore required to meet research and conservation goals. METHODS: The method is based on simultaneous injection of both female and male leopard frogs, Lithobates pipiens (formerly called Rana pipiens) with a cocktail of a gonadotropin-releasing hormone agonist (GnRH-A) and a dopamine antagonist. We call this the AMPHIPLEX method, which is derived from the combination of the words amphibian and amplexus. Following injection, the animals are thereby induced, and perform amplexus and natural fertilization under captive conditions. RESULTS: We tested combinations of a GnRH agonist with 2 different dopamine antagonists in L. pipiens in the breeding season. The combination of des-Gly(10), D-Ala(6), Pro-NHEt(9)-GnRH (0.4 micrograms/g body weight; GnRH-A) with metoclopramide hydrochloride (10 micrograms/g body weight; MET) or domperidone (DOM) were equally effective, producing 89% and 88% successful spawning, respectively. This yielded more than 44,000 eggs for the 16/18 females that ovulated in the GnRH-A+MET group, and more than 39,000 eggs for the 15/17 females that ovulated in the GnRH-A+DOM group. We further tested the GnRH-A+MET in frogs collected in the wild in late autumn and hibernated for a short period under laboratory conditions, and report a low spawning success (43%). However, GnRH-A priming 24 hours prior to injections of the GnRH-A+MET cocktail in animals hibernated for 5-6 weeks produced out-of-season spawning (89%) and fertilization (85%) comparable to those we observed for in-season spawning. Assessment of age and weight at metamorphosis indicated that L. pipiens tadpoles resulting from out-of-season spawning grew normally and metamorphosed successfully. CONCLUSION: We provide evidence for successful captive breeding of the leopard frog, L. pipiens. This simple protocol can be used to obtain large numbers of eggs in a predictable, timed manner.

A large expansion of the HSFY gene family in cattle shows dispersion across Yq and testis-specific expression.

Heat shock transcription factor, Y-linked (HSFY) is a member of the heat shock transcriptional factor (HSF) family that is found in multiple copies on the Y chromosome and conserved in a number of species. Its function still remains unknown but in humans it is thought to play a role in spermatogenesis. Through real time polymerase chain reaction (PCR) analyses we determined that the HSFY family is largely expanded in cattle (∼70 copies) compared with human (2 functional copies, 4 HSFY-similar copies). Unexpectedly, we found that it does not vary among individual bulls as a copy number variant (CNV). Using fluorescence in situ hybridization (FISH) we found that the copies are dispersed along the long arm of the Y chromosome (Yq). HSFY expression in cattle appears restricted to the testis and its mRNA correlates positively with mRNA markers of spermatogonial and spermatocyte cells (UCHL1 and TRPC2, respectively) which suggests that HSFY is expressed (at least in part) in early germ cells.