Progress 07/15/05 to 08/14/08
Outputs
OUTPUTS: Progress Report: 2007/07/15 to 2008/07/14 Analysis of samples from a full time series to determine changes in gene expression and in oocyte development that occur after unilateral ovariectomy (ULO) of rainbow trout just after commencement of vitellogenesis was completed. Sampling occurred at 1, 2, 4, 6, 9 and 12 weeks after ULO. Radioimmunoassay for estradiol showed a significant decrease in levels after ULO only at week 2. However, plasma FSH levels were significantly (3-5X) elevated between week 4 and week 9. Since E2 levels were back to normal by week 4, it is unlikely that decreased negative feedback of E2 on FSH secretion was the cause of the prolonged elevation in plasma FSH levels. Since several classes of follicles are present in the ULO fish, we ran mRNA analyses on five separate samples of follicles: samples with all follicles present, samples of the most advanced follicles, a sample of the second crop of follicles recruited into vitellogenesis and identifiable at 9 and 12 weeks, previtellogenic follicles, and a sample in which the most advanced follicles were removed. Steroidogenic acute regulatory protein (StAR) transcripts were significantly higher in the all follicle and most advanced follicle sample from ULO fish at weeks 4 and 6, and were also elevated in the second crop at weeks 9 and 12. Transcripts for 3beta-hydroxysteroid dehydrogenase were elevated in ULO fish but this also varied with follicle type sampled: all follicle samples were elevated from at weeks 4 and 6, previtellogenic follicles from ULO fish displayed elevated 3beta-hydroxysteroid dehydrogenase transcripts at weeks 6 and 9, transcripts in ULO second crop follicles were elevated at weeks 9 and 12, as were levels at weeks 9 and 12 in samples from which the most advanced follicles had been removed. Several other steroidogenic enzymes showed no change. FSH receptor transcripts were only elevated in the previtellogenic follicles sampled from ULO fish at weeks 6 and 9. Together, the ULO results suggest that increased expression of FSH, StAR and 3beta-hydroxysteroid dehydrogenase are key events in the recruitment of new follicles into vitellogenesis. Expected Impact: 2007/07/15 to 2008/07/14 We are focusing on the events associated with the recruitment of salmonid oocytes into the primary growth pathway, an area that is relatively unexplored in teleosts, but which is of fundamental and applied importance because fecundity is largely determined during the primary oocyte growth phase, and aspects of egg quality are probably also determined at this time. Results of this project will be of use in the development of methods to manipulate fecundity and the timing of puberty onset, improving reproductive efficiency in cultured species. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Expected Impact: 2007/07/15 to 2008/07/14 We are focusing on the events associated with the recruitment of salmonid oocytes into the primary growth pathway, an area that is relatively unexplored in teleosts, but which is of fundamental and applied importance because fecundity is largely determined during the primary oocyte growth phase, and aspects of egg quality are probably also determined at this time. Results of this project will be of use in the development of methods to manipulate fecundity and the timing of puberty onset, improving reproductive efficiency in cultured species.
Publications
- Publications: 2007/07/15 to 2008/07/14
- Esther Lubzens, E. , Young, G, Bobe, J. and Cerda, Joan. Oogenesis in teleosts: how fish eggs are formed. General and Comparative Endocrinology (in press, 2009).
- Luckenbach, J.A., Kusakabe, M., Swanson, P. and Young, G. Unilateral ovariectomy induces compensatory hypertrophy of existing vitellogenic follicles and reduced follicular atresia in the semelparous coho salmon (Oncorhynchus kisutch). Journal of Experimental Zoology 309A: 1-9 (2008)
- Nakamura, I., Kusakabe, M. and Young, G. Differential suppressive effects of low physiological doses of estradiol-17b in vivo on levels of mRNAs encoding steroidogenic acute regulatory protein and three steroidogenic enzymes in pre-vitellogenic follicles of rainbow trout. General and Comparative Endocrinology (in press, 2009).
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Progress 07/15/06 to 07/14/07
Outputs
The primary objectives this year was to continue and complete analysis of samples from a full time series to determine changes in gene expression and in oocyte development that occur after unilateral ovariectomy (ULO) of rainbow trout just after commencement of vitellogenesis and continue in vitro studies. Radioimmunoassay for estradiol showed a significant decrease in levels after ULO that were restored to those of controls within 4-6 weeks. Preliminary assays indicate short-term increases in FSH during recruitment. Recruitment of new follicles into vitellogenesis was associated with marked increases in transcripts for steroidogenic acute regulatory protein (StAR), and 3beta-hydroxysteroid dehydrogenase, but several other steroidogenic enzymes showed no change. Interestingly, our in vitro studies have indicated that expression of StAR and 3beta-hydroxysteroid dehydrogenase is highly regulated by gonadotropins, which is not the case for most other steroidogenic enzymes
we have measured. Together, the ULO results suggest that increased expression of FSH, StAR and 3beta-hydroxysteroid dehydrogenase are key events in the recruitment of new follicles into vitellogenesis. Measurement of transcripts for IGF-I, IGF-I receptor, and gonadotropin receptors are underway. In vitro studies are focused on determining when the follicle first becomes responsive to IGF-1 and gonadotropins, and to determine the interaction between these factors. A collaborative study with Drs. Luckenbach and Swanson has shown that unlike trout, ULO does not induce oocyte recruitment in coho but it results in hypertrophy of existing follicles such that the remaining ovary of the ULO coho has the same mass as the two ovaries of controls. A publication on the in vitro work is in preparation and a manuscript describing the response of coho to ULO has been submitted.
Impacts
We are focusing on the events associated with the recruitment of salmonid oocytes into the primary growth pathway, an area that is relatively unexplored in teleosts, but which is of fundamental and applied importance because fecundity is largely determined during the primary oocyte growth phase, and aspects of egg quality are probably also determined at this time. Results of this project will be of use in the development of methods to manipulate fecundity and the timing of puberty onset, improving reproductive efficiency in cultured species.
Publications
- No publications reported this period
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Progress 07/15/05 to 07/15/06
Outputs
Our main objective this year was to complete a full time series to determine changes in gene expression and in oocyte development that occur after unilateral ovariectomy (ULO) of rainbow trout just after commencement of vitellogenesis. Female rainbow trout of around 1 kg were subject to ULO or sham operation and blood, ovary and pituitary samples taken at regular 1-2 week intervals for 12 weeks. At the time of sampling, fish were treated with injected with 5-bromo-2-deoxyuridine (BrdU) and samples after 24-h to label proliferating cells. Sample analysis is ongoing but we have measured ovarian transcripts for follicle stimulating hormone receptor (FSH-R) and insulin-like growth factor I (IGF-I) cDNAs and for steroidogenic acute regulatory protein (StAR), and several steroidogenic enzymes. Growing primary follicles in the remaining ovary were observed within two weeks of ULO, and between 4-6 weeks, they appeared to be entering vitellogenesis. This coincided with a
marked increase in transcripts (deyolked ovarian tissue) for StAR and for 3beta-hydroxysteroid dehydrogenase; most of the other target transcripts we have measured so far showed some increase during this time period but not always significantly so. However, during sampling we also separated existing vitellogenic follicles from ovarian tissue, and it is likely that measuring transcripts in the latter will reveal robust changes in expression associated with growth of primary follicles and recruitment into vitellogenesis. Analysis of plasma for steroids and plasma and pituitary for FSH and LH will be completed shortly. Studies to assess the in vitro effects of GTHs, steroids and IGF-I on components of the ovarian endocrine system are underway. We have found that ovarian StAR expression is inhibited by low physiological doses of estradiol, and that IGF-I is consistently a strong regulator of aromatase expression, even at times when gonadotropins are without effect. Collaborative studies
with Drs. T. and C. Miura have commenced to develop a long-term explant culture system for trout ovarian tissue for further in vitro analysis of the endocrine control of oogenesis. Two papers s detailing changes in steroidogenic protein expression in male and female trout during gametogenesis have been published, and another publication comes from collaborative work using salmon that reports how growth at critical periods affects ovarian development, especially critical transitions from early to late cortical alveoli stages and from late cortical alveoli to vitellogenesis.
Impacts
We are focusing on the events associated with the recruitment of salmonid oocytes into the primary growth pathway, an area that is relatively unexplored in teleosts, but which is of fundamental and applied importance because fecundity is largely determined during the primary oocyte growth phase, and aspects of egg quality are probably also determined at this time. Results of this project will be of use in the development of methods to manipulate fecundity and the timing of puberty onset, improving reproductive efficiency in cultured species.
Publications
- Nakamura, I., Evans, J.C., Kusakabe, M., Nagahama, Y. and Young, G. Changes in steroidogenic enzyme and steroidogenic acute regulatory protein messenger RNAs in ovarian follicles during ovarian development of rainbow trout (Oncorhynchus mykiss). General and Comparative Endocrinology 144: 224-231 (2005).
- Young, G., Lokman, P.M., Kusakabe, M., Nakamura, I. and Goetz F.W. Gonadal steroidogenesis in teleost fish. In Molecular Aspects of Fish and Marine Biology (C. Hew series ed.), volume 2: Hormones and their receptors in fish reproduction (N. Sherwood and P. Melamed eds), pp. 155-223. World Scientific Press, Singapore (2005).
- Campbell, B., Dickey, J., Beckman, B., Young, G., Pierce, A., Fukada, H. and Swanson, P. Previtellogenic oocyte growth in salmon: relationships among body growth, plasma insulin-like growth factor-1, estradiol-17beta, follicle-stimulating hormone and expression of ovarian genes for insulin-like growth factors, steroidogenic acute regulatory protein and receptors for gonadotropins, growth hormone,and somatolactin. Biology of Reproduction 75: 34-44 (2006).
- Kusakabe, M., Nakamura, I., Evans, J., Swanson, P and Young, G. Changes in mRNAs encoding steroidogenic acute regulatory protein, steroidogenic enzymes and receptors for gonadotropins during spermatogenesis in rainbow trout testes. Journal of Endocrinology189: 541-554 (2006).
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