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Vitellogenin and vitellogenesis in greenback flounder Rhombosolea tapirina

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Sun, Biao (2005) Vitellogenin and vitellogenesis in greenback flounder Rhombosolea tapirina. PhD thesis, University of Tasmania.

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Abstract

The overall aim of this study was to obtain knowledge about the yolk precursor,
vitellogenin (Vtg) and the process of vitellogenesis in female greenback flounder
(Rhombosolea tapirina), including purification and characterization of Vtg;
development of a specific Vtg assay; induction of Vtg by l 7P-estradiol (E2) in
vivo and in vitro; relationship between endocrine regulation of vitellogenesis and
oocyte growth in mature females; and the possible role of Vtg feedback on
ovarian steroid production.
Vtg purified from the plasma ofE2-treated male animals by gel filtration
chromatography had an estimated molecular weight (MW) of~ 540 kD, and
resolved into three bands with estimated MW of 152, 102, 80 kD after SDSp
AGE. Western blotting demonstrated that the Vtg consisted of one subunit (152
kD). A polyclonal antibody against the 152 kD band developed from sheep, was
used to establish-an indirect antibody-capture competitive enzyme-linked
immunosorbent assay (ELISA). The validation of the ELISA was confirmed by
the parallelism between the standard Vtg curve and serial dilutions of plasma from
vitellogenic females, but no cross-reaction was found with the plasma of males, or
plasma from a range of other species.
In Ez-treated fish, detectable concentrations of plasma Vtg were first found at 48
h, and reached a peak at 96 h post-injection, and increased as the E2 dose
increased, with a threshold of~ 0.1 mg E2 Kg body weight -1.
Multiple injections of E2 increased the absolute concentrations of Vtg and extended the peak Vtg up
to 288 h, but did not change the threshold dose. The time-course and dose response
for induction of Vtg by E2 in the flounder was similar to that seen in
other species, however, plasma concentrations of Vtg were generally lower than
those found in other teleosts.
Oocyte size-frequency distributions from mature females showed that this species
has multiple clutch group synchronous type of oocyte development. But that
reproductive development is not synchronized within the population. Consistent

with this observation, there were no significant seasonal variations in
gonadosomatic index (IG), hepatosomatic index (IH), or plasma concentrations of
Vtg, E2 and testosterone (T). Significant increases in IG , IH and plasma
concentrations of Vtg, E2 and T were observed in vitellogenic fish, and in fish
undergoing final maturation. Plasma concentrations of Vtg and E2 rose steadily
across oocyte sizes from 100 to 450 μm, and both reached a concentration plateau
at oocyte sizes of around 450 μm. In contrast, plasma concentrations of T showed
no marked increase until oocytes grew beyond 400 μm.
Isolated vitellogenic follicles were incubated with human chorionic gonadotropin
(hCG), dibutyryl cyclic AMP (dbcAMP) and gonadal steroid precursors T, 17-
hydroxyprogesterone (17P) and androstenedione (A) in the presence or absence of
Vtg. High concentrations ofVtg suppressed the production of E2 and T, and the
effect appeared to operate at several levels along the steroidogenic pathways.
These observations combined with findings of other studies indicate that Vtg
might regulate its own production by modulating ovarian steroid synthesis.

Item Type: Thesis (PhD)
Keywords: Flatfishes, Rhombosolea tapirina
Copyright Holders: The Author
Copyright Information:

Copyright 2005 the Author - The University is continuing to endeavour to trace the copyright
owner(s) and in the meantime this item has been reproduced here in good faith. We
would be pleased to hear from the copyright owner(s).

Additional Information:

Author has listed Paper 1 as being in press and an article with the same title as chapter 3 was published, the text of the article does not correspond with chapter 3.

Chapter 5 is the equivalent of the peer reviewed version of the following article: Sun, B. and Pankhurst, N.W. (2004), Patterns of oocyte growth, vitellogenin and gonadal steroid concentrations in greenback flounder. Journal of Fish Biology, 64: 1399–1412. doi: 10.1111/j.0022-1112.2004.00406.x, which has been published in final form at 10.1111/j.0022-1112.2004.00406.x. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.

Parts of chapter 5 were also presented in poster form at the 7th International
Symposium on Reproductive Physiology of Fish, Mie, Japan, May 2003 and also published as:
Sun B. and Pankhurst N.W. (2003). Correlation between oocyte development
and plasma concentrations of steroids and vitellogenin in greenback
flounder Rhombosolea tapirina. Fish Physiology and Biochemistry 28, 367-
368. However similar wording of that in the article does not appear in this thesis.

Chapter 7 is the equivalent of the peer reviewed version of the following article: Sun, B. and Pankhurst, N. W. (2006), In vitro effect of vitellogenin on steroid production by ovarian follicles of greenback flounder Rhombosolea tapirina. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 114 (1): 78-85 doi:10.1016/j.cbpa.2006.02.007

Date Deposited: 04 Feb 2015 23:30
Last Modified: 11 Mar 2016 05:54
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