Banh, Thi Quyen Quyen (2019) Sex differentiation of barramundi lates calcarifer – understanding male sexual development and its manipulation through exogenous steroids and non-steroidal aromatase inhibitor. PhD thesis, James Cook University.

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Abstract

Barramundi, or Asian seabass (Lates calcarifer), is considered one of the most important commercial tropical aquaculture species in Southeast Asia and Australia. Despite its importance, efforts to improve barramundi production via selective breeding have been challenging, primarily due to difficulties in controlling sex change. Barramundi is a protandrous hermaphrodite, where fish first mature as male and then, a few years later, sex change into female. The gonadal development of barramundi provides a good model to study the genetic mechanisms underlying natural sex change in fish; however, this natural sex change process poses significant challenges for barramundi broodstock management as high quality male broodfish change sex, which requires the constant recruitment of new male broodstock. Alternatively, and relevant to selective breeding programs, the sequential sex change of barramundi results in an age difference between the sexes in spawning groups, where females are one generation older than the males. As the rate of genetic gain is contingent on the generation interval, this inter-generation breeding thereby halves the annualised rate of genetic progress that could otherwise be made through a single-generation selection program. Therefore, sex control of barramundi in hatcheries through either inducing precocious females, or preventing natural sex change from male to female, is of great importance to the industry.

Sex control for fishes (including teleosts) is feasible as the course of gonadal development is a flexible process and may be subject to modification by extrinsic factors, especially during early gonadal development when animals are most sensitive to environmental stimuli (widely referred to as the labile period). Manipulation of environmental factors (e.g. addition of sex steroids, changing water temperature or pH) during early gonadal development may override genetically pre-programmed sex determination mechanisms, thus altering the resultant phenotypic sex of the fish. Yet, the process of testicular development in barramundi is unknown. In particular, there is no information on when and how testicular differentiation commences in the species and what happens when exogenous steroids are subsequently administered. To address these knowledge gaps, the research that is reported in this thesis was conducted in three steps. Firstly, the timing of sexual development and differentiation in the protandrous barramundi was determined through tracking morphological differentiation and gene expression of key sex genes from early embryogenesis through to spermatogenesis. This provided an understanding of the timing of initial gonadal differentiation into the male testis state and an indication of the likely labile period whereby sex could be manipulated through the addition of exogenous factors such as hormones. Once the timing and process of testicular differentiation was established, the research then went on to examine the effect of applying exogenous steroid and non-steroidal hormones for inducing precocious feminisation and/or retaining masculinisation. This was examined through a feeding trial of exogenous steroid to juveniles and use of implants with estrogen and fadrozole in male barramundi.

The labile period of sex determination in barramundi was identified by examining the gonadal morphology of fish from newly hatched larvae till 9 months post-hatch, when barramundi gonads were previously reported as differentiated testes. As primordial germ cells (PGCs) of male fish remain morphologically unchanged for longer periods and are differentiated as the final step of testicular differentiation, early histological indicators for testicular differentiation, such as the slit-like lumen in stromal tissue, appearance of blood vessels, and invagination of the epithelial tissues into lobular structures, are often used. Results showed that the barramundi proto-testes originates as a strip of cells attached to the dorsal coelemic cavity at 4 days post hatch (dph), with the formation of a slit-like lumen by 44 dph (fish total length (TL) 25.8 ± 3.3 mm), which is the first morphological indication of testicular differentiation in the species. This slit-like lumen later develops into the testes efferent duct. Other major indicators of testicular development were invagination among epithelial cells and the formation of capillary vessels at 60 dph, the organization of primary germ cells into lobules at 90 dph and differentiation of the somatic cells into Sertoli cells at 120 dph. Final testicular differentiation was histologically discernible at 140 dph when the somatic cells had differentiated into Sertoli cells and germ cells into spermatocytes. Noticeably, the male-associated gene dmrt1 (double-sex and Mab-2 related transcription factor 1) was detected exclusively to be expressed in Sertoli cells by in situ hybridisation using an mRNA probe. Additionally, gene expression of the important male-related genes, dmrt1 and cyp11b (cytochrome P450 11β-hydroxylase gene), were examined using RT-qPCR. Cyp11b and dmrt1 were expressed highest in the gonad at 90 and 120 dph, respectively, with dmrt1 expression observed to rapidly increase from 70 to 120 dph. The beginning of sex differentiation to the finalisation of testicular development in barramundi was determined in this study to occur from 44 to 140 dph (TL 169.4 ± 40.3 mm).

The next steps in this study were to assess the feminising effect of 17 β-estradiol (E₂) and 17 α-ethinylestradiol (EE₂) on barramundi during their determined labile period. Fish were fed pellets containing 10 mg E₂ kg⁻¹ food, 20 mg E₂ kg⁻¹ food, 5 mg EE₂ kg⁻¹ food and 10 mg EE₂ kg⁻¹ food from 30 to 160 dph, which covers the period of initial male gonad differentiation identified in the first study. The effect of E₂ and EE₂ treatment on gonadal morphology and dmrt1 and cyp19a1a gene expression was analysed by histology and RT-qPCR. Results showed that orally delivered E₂ and EE₂ induced observable changes in gonad morphology. Previtellogenic oocytes (PO) were observed in 33% and 50% of the fish fed with 20 mg E₂ kg⁻¹ food at 160 dph and 12 months post hatch (mph), respectively, while PO were not observed in control fish. The 10 mg E₂ kg⁻¹ food treatment did not induce feminisation, but significantly suppressed testicular development. Treatment with EE₂ resulted in fibrosis within gonad tissues at a dose-dependent rate. E₂ administration resulted in upregulation of the gene cyp19a1a and down-regulation of dmrt1. EE₂ significantly suppressed expression of dmrt1 at 160 dph and 12 mph; while cyp19a1a was not significantly different at 160 dph and was significantly downregulated at 12 mph. The result of this study showed that E₂ is a more suitable hormone than EE₂ to induce precocious feminisation of barramundi, and when delivered through the feed it has a dose-dependent feminising effect.

Whilst feeding of estrogen steroids to juvenile barramundi results in sex change of some individuals in a selective breeding program, from a consumer awareness perspective it may be better to only administer hormone to males selected for use as female broodstock to allay any fears about hormone treatment of fish that may be destined for human consumption. Because of this, it was deemed vital to assess if there is an alternative route to sex change males. The literature shows that sex plasticity of some teleost species remains after sexual differentiation, therefore a trial was conducted to produce precocious female barramundi after fish had differentiated as males. Here, the efficacy of two dosages of E₂ delivered via implants to induce the early feminisation of barramundi were evaluated. Six-month-old male barramundi (405 ± 50 g body weight (BW)) were given a single cholesterol-based pellet implant containing either 0 mg E₂ kg⁻¹ BW (untreated control), 4 mg E₂ kg⁻¹ BW ('low dose'), or 8 mg E₂ kg⁻¹ BW ('high dose'). At 4 and 9 weeks post-implantation, gonads were histologically assessed for morphological changes, liver condition histologically examined and expression profiles of key male (dmrt1, cyp11b and esr1) and female (cyp19a1a and foxl2) sex-related genes examined using RT-qPCR. At 9 weeks post-implantation, significant gonadal morphological changes were observed in E₂-treated fish, while all control fish remained as male. In the 'high dose' E₂ treatment group, 77.8 % (7/9) of fish sex-changed completely to female, signified by gonads containing oocytes (20-30 μm) and no observed residual sperm. Comparably, 44.4 % (4/9) of fish in the 'low dose' E₂ treatment group had sex-changed, while remaining fish showed complete testicular regression with gonads containing only undifferentiated germ cells. In the 'high dose' E₂ treatment, increased expression of female-biased genes (cyp19a1a and foxl2) was observed, while downregulation of male-biased genes (dmrt1, cyp11b and esr1) was induced. The success of artificially-induced sex change in barramundi provides an important tool that is critical to improving selective breeding of this species.

Efforts to control sex of barramundi also means having the ability to maintain high quality broodfish as males. The results from the feeding and implant trials illustrated the important role of the cyp19a1a aromatase gene in barramundi feminisation, suggesting a possible way to block natural sex change in barramundi may be to reduce aromatase activity (estrogens are synthesised from androgens through a reaction catalysed by cytochrome P450 aromatase). Accordingly, a final trial was conducted to assess the impact of a non-steroidal aromatase inhibitor, Fadrozole (FAD), administered solely or with E₂ cholesterol pellet implants, on the gonadal development of mature male barramundi. Fifteen-month-old male barramundi (2102 ± 126 g BW) were divided into four groups of 12 individuals each. Fish in the first group were implanted with a cholesterol pellet without FAD or E₂ as control (n = 12). The three treated groups of fish were implanted with 8 mg E₂ kg⁻¹ (n = 12), 8 mg FAD kg⁻¹ (n = 12), or 8 mg E₂ and 8 mg FAD kg⁻¹ were both implanted into individual fish (n = 12). Survival rates were 100% for all groups during the experiment with no damage observed in liver and kidney of any fish. At the final sampling 9 weeks after treatment, 8 % (1/12) of the untreated control fish were found in early stages of sex reversion (stage T1), whereas 100% of FAD-treated fish were male and 100% of E₂-treated fish were female. Binary treatment with FAD and E₂ resulted in 42 % (5/12) males, 42 % (5/12) females and 16% (2/12) transitional fish. In the FAD-treated fish, expression of male genes (dmrt1 and cyp11b) were significantly upregulated, whilst female-biased gene (foxl2) downregulated. Exposure to E₂ resulted in significantly higher expression of cyp19a1a and foxl2; and lower expression of dmrt1, cyp11b and esr1 than control fish. FAD neutralised the effects of E₂, in terms of gonad morphology and gene expression.

In summary, this PhD research has revealed the important process of sex differentiation of barramundi, a sequential hermaphroditic teleost species. It unveiled the morphological changes at cellular levels of barramundi gonads from undifferentiated to maturity of testes, while revealing the expression patterns of the important sex-related genes during the process. The timing of this process provided the time window for testing the responsiveness of barramundi gonads to the exogenous steroids for sex control purposes. This research has determined a suitable hormone treatment for barramundi feminisation, including hormone types and doses, timing and administration route. It also filled our knowledge gap in gene expression of the important male- and female-biased genes in barramundi gonad exposed to exogenous steroids and non-steroid hormones. Although the effect of masculine fadrozole was investigated in this thesis, it is necessary to optimise the dose for better results. The process of obtaining young females by means of hormonal treatment in this study also require further research of their reproductive potential, as the ultimate goal is to produce dams for selective breeding programs.

Item ID:	64547
Item Type:	Thesis (PhD)
Keywords:	Asian seabass, barramundi, cyp11b, dmrt1, environment, epigenetics, gonad histology, hormonal manipulation, in situ hybridization, Lates calcarifer, primordial germ cells, QTL, reproductive control, Sertoli cells, sex ratio, testicular differentiation, triploidy
Related URLs:	https://researchonline.jcu.edu.au/39217/ https://researchonline.jcu.edu.au/49516/
Copyright Information:	Copyright © 2019 Thi Quyen Quyen Banh.
Additional Information:	Two publications arising from this thesis are stored in ResearchOnline@JCU, at the time of processing. Please see the Related URLs. The publications are: Chapter 1: Budd, Alyssa M., Banh, Quyen Q., Domingos, Jose A., and Jerry, Dean R. (2015) Sex control in fish: approaches, challenges and opportunities for aquaculture. Journal of Marine Science and Engineering, 3 (2). jmse3020329. pp. 329-355. Chapter 2: Banh, Quyen Q., Domingos, Jose A., Zenger, Kyall R., and Jerry, Dean R. (2017) Morphological changes and regulation of the genes dmrt1 and cyp11b during the sex differentiation of barramundi (Lates calcarifer Bloch). Aquaculture, 479. pp. 75-84.
Date Deposited:	08 Oct 2020 02:15
FoR Codes:	07 AGRICULTURAL AND VETERINARY SCIENCES > 0704 Fisheries Sciences > 070405 Fish Physiology and Genetics @ 50% 06 BIOLOGICAL SCIENCES > 0604 Genetics > 060403 Developmental Genetics (incl Sex Determination) @ 50%
SEO Codes:	83 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 8301 Fisheries - Aquaculture > 830102 Aquaculture Fin Fish (excl. Tuna) @ 100%
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