Abstract
It is unknown which factors are involved in regulation of early oocyte growth in oviparous vertebrates; in particular, the involvement of the oocyte-derived growth factors, growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) in teleost reproduction. The regulation of steroidogenic acute regulatory protein (StAR), the true rate- limiting step in steroidogenesis, has also been largely ignored in many fish species. The few studies on early oogenesis have pointed to roles for pituitary hormones, such as follicle stimulating hormone (FSH), luteinizing hormone (LH) and growth hormone (GH) as being possible regulators of GDF-9, BMP-15, StAR gene expression and steroid production. The main aims of this thesis were i) to examine BMP-15 and StAR mRNA levels throughout oogenesis in wild and artificially maturing New Zealand shortfinned eel, Anguilla australis, in vivo ii) to examine whether FSH, LH, GH or the metabolic hormone insulin-like growth factor-I (IGF-I) have any effect on GDF-9, BMP-15 or StAR mRNA levels in the previtellogenic (PV) ovary of A. australis, in vitro iii) to examine the effects of FSH on steroid production in the ovarian tissue of PV A. australis, in vitro.
Both wild and artificially maturing eel cDNA was used to obtain BMP-15 and StAR mRNA levels at all stages of eel oogenesis. BMP-15 mRNA levels were found to significantly increase in wild females in early vitellogenesis (EV) compared with wild PV females (P < 0.01). No difference in BMP-15 mRNA levels was found in artificially maturing eels at different developmental stages. Levels of StAR mRNA throughout shortfinned eel oogenesis were found not to be significantly different in wild females in EV compared with wild PV females. A significant difference in StAR mRNA levels in artificially maturing eels at different developmental stages was found, with a peak at midvitellogenesis (MV) (P < 0.001).
In vitro incubations of PV A. australis ovarian tissue with FSH, LH, GH or IGF-I were conducted and the mRNA levels of GDF-9, BMP-15 and StAR subsequently measured by quantitative real-time PCR (RT-PCR). No effect of FSH, LH or GH on either GDF-9 or BMP-15 mRNA levels was found during PV in A. australis. FSH was however, found to significantly increase StAR mRNA levels (P < 0.05) throughout PV. IGF-I had no effect on GDF-9, BMP-15 or StAR mRNA levels.
To investigate the effect of FSH on steroidogenesis throughout PV, the levels of five steroid hormones; 11-ketotestosterone (11-KT), 11β-hydroxyandrostenedione (11-OHA), estradiol (E2), testosterone (T), 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) were measured in the ovary after 8 h incubation with FSH and tritium labelled pregnenolone (P5) in eel ringer. No change in steroid levels between controls and FSH treatments was found. High levels of 11-OHA and E2 were however, found to be produced in both control and FSH incubations.
In conclusion, neither pituitary hormones nor IGF-I had any effect on GDF-9 or BMP-15 mRNA levels throughout PV in the shortfinned eel in vitro. FSH was found to have an effect on StAR mRNA levels throughout PV in the eel in vitro, which may mean that the gonadotropins may regulate StAR in teleosts. The steroid hormones E2 and 11-OHA were found at elevated levels throughout PV in the eel and may be of great importance to early-stage eel oocytes.