Abstract
Yolk protein is crucial in oviparous animals because it provides most of the nutrients for developing embryos. The yolk protein precursor in the majority of oviparous animals is vitellogenin (Vtg) which belongs to the large lipid transfer protein (LLTP) superfamily. In the phylum Echinodermata, a transferrin-like protein was identified as the yolk protein precursor in the classes Echinoidea and Holothuroidea and termed major yolk protein (MYP). In the class Asteroidea, a functional Vtg has been recently reported in Parvulastra exigua. However, it is still unknown whether Vtg is the major yolk protein in asteroids or if they have different forms of Vtg. Moreover, is it possible that asteroids synthesise MYP? Do sea stars synthesise vertebrate-like sex steroid hormones (VLSHs), such as estrogen, to control vitellogenesis? To answer these questions, the present study aimed mainly to determine yolk protein precursor transcript abundance and steroidogenic enzyme activity in the New Zealand cushion sea star Patiriella regularis. Furthermore, the present study aimed: i) to describe oocyte formation in P. regularis and categorise the stages of the reproductive cycle (Chapter 2); ii) to identify the yolk protein precursors (Chapter 3); iii) to examine yolk protein precursor mRNA levels and sites of synthesis during oogenesis and iv) to examine steroidogenic enzyme activity throughout the reproductive cycle.
The reproductive cycle of female P. regularis was studied in Chapter 2 using histological analysis and organ index analysis of samples collected on a monthly basis. The annual reproductive cycle of P. regularis was divided into five stages: I) recovery stage, II) growing stage, III) mature stage, IV) partly spawned stage and V) spent stage. P. regularis spawned in the austral summer and oocyte growth occurred mainly between autumn and spring.
Two distinct Vtgs (termed PrVtg1 and PrVtg2) that belong to the LLTP superfamily have been identified and sequenced in Chapter 3. In addition, a partial cDNA encoding MYP (termed PrMYP) was cloned and sequenced. Amino acid sequencing for the prominent proteins in ovulated eggs matched the deduced amino acid sequences of PrVtgs. These results provided the first evidence that at least two Vtgs correspond to yolk proteins in echinoderms. In addition, it is the first report for the expression of MYP in a sea star.
Chapter 4 showed that the expression levels of PrVtg1 differed significantly during the reproductive cycle in both the pyloric caeca and ovary. PrVtg2 mRNA levels changed significantly in the pyloric caeca during the reproductive cycle while it was stable in the ovary. In situ hybridisation analysis showed that PrVtg transcripts were present in the digestive epithelium of the pyloric caeca. In the ovary, PrVtg1 transcripts were localised in the somatic cells rather than follicle cells, while PrVtg2 transcripts were identified in the follicle cells. These results provide evidence that somatic cells in sea star ovaries function in Vtg synthesis and that both the pyloric caeca and the ovary are the main vitellogenic organs in sea stars. Interestingly, PrMYP transcripts were identified in the growing oocyte, which contrasts with findings from other echinoderm species, such as sea urchins.
In Chapter 5, pyloric caeca and ovary were incubated with pregnenolone (P5) and androstenedione (AD). Subsequently, the concentrations of pregnenolone (P4), androstenedione (AD), testosterone (T) and estradiol-17β were measured using liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry. Both organs converted P5 and AD into P4 and T respectively, but the concentration of P4 and T did not change significantly during the reproductive cycle. Analysis of transcriptome data from P. regularis ovary indicated that the transcripts encoding crucial steroidogenic proteins were absent. Thus, it is suggested that VLSHs do not control asteroid reproduction nor are they are synthesised for a particular physiological function.
The present study provided novel results that advance the understanding of vitellogenesis in asteroids. In addition, the present study provided valuable knowledge that will advance the understanding of Vtg evolution in the phylum Echinodermata and the relationship between asteroid Vtgs with Vtgs from other animals.