Providing recombinant gonadotropin-based therapies that induce oogenesis from previtellogenic oocytes to produce viable larvae in a teleost, the flathead grey mullet (Mugil cephalus)
Introduction
The flathead grey mullet (Mugil cephalus) is a catadromous teleost with a worldwide distribution (between latitudes 40° North and South) (McDonough et al., 2005) that has been cultured for several centuries principally in some Asian countries and around the Mediterranean basin. Many positive attributes of flathead grey mullet culture have made this species a suitable option for aquaculture. Flathead grey mullet has fast growth (approximately 1 kg per year) (FAO, 2019), does not require dietary fish meal and oil and can be reared in a wide range of salinities and culture systems (González-Castro and Minos, 2016). In addition, the final product marketed in various forms has good texture, taste (Yousif et al., 2010) and is an excellent source of omega-3 essential fatty acids (Khemis et al., 2019).
Flathead grey mullet show a wide variation in reproductive strategies and characteristics across the species worldwide distribution. The species is gonochoristic and generally matures at the age of 3 years. In the Mediterranean, the spawning period has been observed from July to October when breeders spawn externally fertilised pelagic eggs in the sea (Whitfield et al., 2012). Females appear to spawn one set of ova a year (Rao and Babu, 2016). However, in captive conditions flathead grey mullet exhibit different degrees of reproductive dysfunctions in both genders. Despite of the species long history of culture, these dysfunctions that have not been overcome, have limited the possibility to close the life cycle and, thus, culture is still based on the capture of wild juveniles (González-Castro and Minos, 2016; Yousif et al., 2010) or the induction of oocyte maturation and spawning of wild breeders captured at advanced stages of gonadal development (Abraham et al., 1999; Das et al., 2014; El-Gharabawy and Assem, 2006; Karim et al., 2016; Vazirzadeh and Ezhdehakoshpour, 2014). However, the use of wild caught mature fish is unsustainable as it relies on fisheries that are in decline (González-Castro and Minos, 2016) and offers no possibility to close the life cycle in captivity and make genetic improvements of cultured stocks. Flathead grey mullet held in aquaculture facilities present two types of reproductive dysfunction: arrest in late or early stages of gametogenesis. Arrest in late stages of gametogenesis (maturation and ovulation) has been observed in recently caught wild flathead grey mullet or wild flathead grey mullet that were acclimated to ponds or large tanks (El-Greisy and Shaheen, 2007; Kuo et al., 1973; Yousif et al., 2010). This is the most commonly observed dysfunction in fish and can be controlled by hormonally inducing spawning (Mañanós et al., 2009; Zohar and Mylonas, 2001) as has been achieved for flathead grey mullet with therapies that combine different substances such as: carp pituitary homogenates, steroids, human Chorionic Gonadotropin (hCG), gonadotropin releasing hormone synthetic analogues (GnRHa) and drugs that inhibit dopamine (see review by González-Castro and Minos, 2016). In comparison, in wild and hatchery-reared fish held in intensive culture conditions in the Mediterranean region, a more severe reproductive dysfunction has been observed where development was arrested in the early stages of gametogenesis. The artificial propagation of these fish in intensive culture systems would be a sustainable solution for a consistent supply of juveniles (Yousif et al., 2010). In these intensive conditions, females did not initiate vitellogenesis; remained at the primary growth stage or cortical alveoli stage (present study), or were arrested at early stages of vitellogenesis (Aizen et al., 2005). Males failed to initiate spermiation (De Monbrison et al., 1997; Yashouv, 1969) or produced highly viscous milt that could not fertilize the eggs (Shehadeh et al., 1973). These reproductive dysfunctions may be related to alterations in the endocrine control in the brain-pituitary-gonadal (BPG) axis.
In vertebrates, the pituitary gonadotropins (Gths), the follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), are generally accepted to be the central components of the BPG axis in the control of gonad development. Current knowledge in teleost suggest that the major role of Fsh is to promote gametogenesis from early stages through to late stages (vitellogenesis in females and spermatogenesis in males), while Lh is involved in gamete final maturation and release (ovulation and spermiation, in females and males, respectively) (Lubzens et al., 2010; Mañanós et al., 2009). The mechanism underlying the reproductive dysfunctions in Mediterranean captive flathead grey mullet has been described as an inhibition by dopamine (DA) on the action of gonadotropin releasing hormone (GnRH) to release Gths in both females (Aizen et al., 2005) and males (Glubokov et al., 1994). Therefore, methods based on the mechanisms controlling gametogenesis are required to induce complete gonadal development, from early stages (i.e. previtellogenesis) through to the late stages. In the case of males, 17α-methyltestosterone (MT) implants enhanced spermatogenesis and spermiation (Aizen et al., 2005; De Monbrison et al., 1997). In females, treatment with GnRH agonist (GnRHa) in combination with a DA antagonist (Aizen et al., 2005) or a single injection of recombinant Fsh produced in the yeast Pichia pastoris (Meiri-Ashkenazi et al., 2018) increased the number of vitellogenic females by promoting the release of Gths from the pituitary. However, hormonal therapies to enhance endogenous Lh release have been observed to be less effective when the pituitary Lh content was low (Yaron et al., 2009), indicating that alternative therapies may be required in these situations.
A strategy to control gametogenesis in flathead grey mullet as in other teleost, which would not require the availability of endogenous Gths from the pituitary, is the long-term use of recombinant Fsh and Lh (rFsh and rLh, respectively). This approach is nowadays possible through the production of large amounts of species-specific rGths in heterologous expression systems, such as the Drosophila S2 cell line (Kazeto et al., 2008; Zmora et al., 2007), the yeast Pichia pastoris (Aizen et al., 2007; Chen et al., 2012; Kamei et al., 2003; Kasuto and Levavi-Sivan, 2005; Palma et al., 2018; Sanchís-Benlloch et al., 2017), baculovirus silkworm larvae (Cui et al., 2007; Glubokov et al., 1994; Ko et al., 2007; Kobayashi et al., 2010; Kobayashi et al., 2003; Meri et al., 2000), HEK293 cells (Kazeto et al., 2019) and Chinese hamster ovary (CHO) cells (Chauvigné et al., 2017; Choi et al., 2005; Giménez et al., 2015; Molés et al., 2011; Peñaranda et al., 2018; So et al., 2005). The application of rGths based therapies has shown promise to control gametogenesis in different teleost (Chauvigné et al., 2018; Chauvigné et al., 2017; Giménez et al., 2015; Kamei et al., 2006; Peñaranda et al., 2018) and, therefore, could be an effective method to induce gametogenesis in cultured flathead grey mullet arrested in the early stages of sexual maturation.
The present study aimed to use homologous single-chain rGths produced in CHO cells as the basis of a long-term hormone therapy to obtain viable offspring from flathead grey mullet females that were arrested in previtellogenesis and males that did not have flowing sperm. For this purpose, Mugil cephalus rFsh was administered to induce gametogenesis followed by treatments to induce oocyte maturation and ovulation, which were either (a) therapies previously employed in this species such hCG and GnRHa with a DA agonist, or (b) Mugil cephalus rLh.
Section snippets
Study animals and maintenance
Flathead grey mullet were used in different experiments to examine the effect of rGth hormone therapies. An in vivo dose-response test was carried out for rFsh. Experiment 1 examined the long-term effect of rFsh on vitellogenesis and the use of hormone therapies (hCG or GnRHa with DA agonist) previously used in female flathead grey mullet to induce final oocyte maturation and ovulation. Experiment 2 examined the effect of a combined rFsh and rLh therapy in females. In order to obtain sperm,
In vivo dose-response of rFsh on female steroid production
There were no significant increases from the E2 basal values after the application of doses of 0 (Control), 3, 6 and 9 μg kg−1 of rFsh (Fig. 3). A great individual variation in magnitude of response was observed when a dose of 9 μg kg−1 was administered. The administration of 12 μg kg−1 of rFsh produced significant increases in E2 levels on 3 to 6 days after the injection with respect to basal levels. The administration of 15 μg kg−1 of rFsh produced a significant increase in E2 levels on day 3
Discussion
The present study shows that rFsh drives oogenesis from early to late gonad developmental stages in female flathead grey mullet, that rLh is influential to achieve oocyte maturation and ovulation and that rGths can be used to produce milt from male flathead grey mullet. These findings are significant to both demonstrate the accepted roles of the Gths in teleost reproductive development and to provide advances for the control of reproduction in teleost species that experience reproductive
Author statement
Ramos-Júdez, S: Conceptualization, Methodology, Investigation, Formal analysis, Writing- Original draft preparation, Visualization. Chauvigné, F: Investigation, Writing- Reviewing and Editing. González-López, WA: Investigation, Writing- Reviewing and Editing. Rosenfeld, H: Conceptualization, Writing- Reviewing and Editing, Funding acquisition. Cerdà, J: Conceptualization, Investigation, Writing- Reviewing and Editing. Giménez, I: Conceptualization, Methodology, Writing- Reviewing and Editing,
Declaration of Competing Interest
Ignacio Giménez is associated with the biotech company Rara Avis Biotec, S. L., which produced the recombinant gonadotropins employed in this study. Other than this, there are no patents or products in development to declare.
Acknowledgments
The authors would like to thank Josep Lluis Celades and the IRTA staff for technical help. Special thanks to Narciso Mazuelos from Finca Veta La Palma (Isla Mayor, Sevilla) for providing us with flathead grey mullet broodstock. This study has been supported with funding from the European Union's Seventh Framework Programme for research, technological development and demonstration (GA 603121, DIVERSIFY to H.R. and N.D.), the Spanish National Institute for Agronomic Research (Instituto Nacional
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2022, AquacultureCitation Excerpt :Moreover, rFSH and rLH therapy have been used in cases of reproductive dysfunctions to induce gonadal development. Grey mullet (Mugil cephalus) females that remained arrested in previtellogenic stages of gonadal development were treated intramuscularly with specific homogenous rFsh in a dose/time-response study as well as a combined rFSH and rLH therapy was applied by Ramos-Júdez et al. (Ramos-Júdez et al., 2021). Both rGTHs are effective in inducing oogenesis from previtellogenesis to oocyst maturation to obtain eggs, larvae, and high-quality sperm (Ramos-Júdez et al., 2021).
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2022, Animal Reproduction ScienceCitation Excerpt :Therefore, research efforts are currently underway to optimize juvenile production techniques from broodstock management to larval and juveniles rearing. Recent advances in our knowledge of sex determination mechanisms (Dor et al., 2020; Ferraresso et al., 2021), the development of hormonal therapies to induce sexual maturation (Ramos-Júdez et al., 2021, 2022), as well as the identification of suitable compounds for spawning induction and synchronization (Mousa, 2010; Besbes et al., 2020; Vallainc et al., 2021), are paving the way for a more knowledge based approach to Mugilids broodstock management. If implemented at commercial scale, these new protocols could contribute to overcome the current bottlenecks in the commercial scale-up and intensification of mullet aquaculture.