(381) Post-Thaw Treatment with Astaxanthin Enhances Sperm Function Following Cryopreservation

Amey K. Hughes;1 Daniel Harrington;2 Sarah Szelecki1; Simon F.R. Hinkley;2 Janet L. Pitman1 

1School of Biological Sciences and 2Ferrier Research Institute, Te Herenga Waka Victoria University of Wellington, Wellington, New Zealand 

 


Abstract Text: Kākāpō (Strigops habroptilus) are a critically endangered parrot that is endemic to Aotearoa, New Zealand (NZ).  The Kākāpō Recovery Team, Department of Conservation and local iwi have brought this species back from the brink of extinction to ~250 individuals which reside in predator-free off-shore sanctuaries. Kākāpō breed intermittently every 2-5 years, coinciding with the fruiting of Dacrydium cupressinum (NZ rimu) but the mechanistic link remains a mystery. One hypothesis is that phytoestrogens in rimu fruit bolster endogenous oestrogen levels, which act upon oestrogen receptors (ESR1) in the liver to stimulate egg yolk protein production leading to ovarian follicle maturation1. In support of this hypothesis, we have identified an amino acid indel in the ligand-binding domain of ESR1 of kākāpō̄ that may increase their sensitivity to phytoestrogens2 and have obtained preliminary data of oestrogenic activity in crude rimu fruit extracts3.The aim of this study was to develop a yeast-based bioassay to compare the sensitivity of human and kākāpō ESR1 to phytoestrogens present in rimu fruit.To construct sensitive yeast bioassays, the cell wall mannoprotein genes (CWP1 and CWP2) and pleiotropic drug response genes (PDR5 and PDR10) were first deleted from W303a yeast using homologous recombination of the loxP–kanMX–loxP target gene cassette and a lithium acetate procedure4. The Cyc1 terminator sequence, Ura3-1 gene, and Gal1/10 dual directional promoter were amplified by PCR and inserted into the pUC19 plasmid5. The kākāpō (k)ESR1 or human (h)ESR1 were amplified by PCR and inserted into this modified pUC19 construct. The oestrogen response element (ERE) was transferred from the pRR-ERα-5z plasmid into the p</span>TXRE5z plasmid. The steroid receptor coactivator gene, SRC-1e (human) or Ncoa1 (kākāpō), was inserted into the pESC-LEU plasmid. All cloning were performed using either traditional methods or NEBuilder HiFi DNA Assembly. The modified pUC19, pTXRE5z and pESC-LEU plasmids were incorporated into the modified W303a yeast using lithium acetate methods. Rimu fruit, seeds and unfertilised ovules extracted in a range of solvents for testing in the yeast ESR1 bioassays. Preliminary results from the human ESR1 yeast bioassay revealed that all rimu specimens, i.e. foliage, seeds, unfertilised ovules and berries, showed oestrogenic activity in some, but not all extractions. Interestingly, all specimens extracted using water-based, compared to methanol, acetone and chloroform, methodologies exhibited higher estrogenic activity. The results from the kākāpō ESR1 yeast bioassay are in progress and will be compared to that of the human ESR1 yeast bioassay. In summary, the presence of phytoestrogens in aqueous extracts of rimu fruit mirrors the birds eating habit of crushing the berries and discarding the fibrous chews, supporting a physiological role of phytoestrogens in kākāpō.