Providing recombinant gonadotropin-based therapies that induce oogenesis from previtellogenic oocytes to produce viable larvae in a teleost, the flathead grey mullet (Mugil cephalus)

Highlights

• Long-term treatment with mullet-specific rGths produced viable gametes and larvae in Mugil cephalus.

• Recombinant Fsh induced oogenesis to advanced stages of vitellogenesis (425 ± 19 μm diameter) in previtellogenic females.

• Recombinant Lh completed oocyte growth (609 ± 5 μm diameter) in rFsh-induced females.

• Recombinant Lh and progesterone induced oocyte maturation and ovulation.

Abstract

Under intensive captive conditions, wild-caught flathead grey mullet (Mugil cephalus) females remained arrested in previtellogenic stages of gonadal development and no sperm could be obtained from males. With the aim to induce oogenesis from previtellogenesis to oocyte maturation, induce the release of sperm and obtain fertilized eggs, female and male flathead grey mullet were treated with Mugil cephalus single-chain recombinant gonadotropins (rGths), follicle-stimulating (rFsh) and luteinizing (rLh) hormones. In Experiment 1, fish were treated with a weekly dose of rFsh (15 μg kg⁻¹), which in females significantly (P < 0.001) increased plasma concentration of 17β-estradiol and induced vitellogenic oocyte growth up to a maximum mean diameter of 425 ± 19 μm after 9 weeks of treatment. In Experiment 2, fish were treated with weekly injections of both rFsh and rLh at different doses (from 2.5 to 12 μg kg⁻¹). Oocyte diameter reached 609 ± 5 μm, from which final oocyte maturation and ovulation was induced with 30 μg kg⁻¹ of rLh and 40 mg kg⁻¹ of progesterone. Good quality sperm (> 75% motile spermatozoa) was obtained from males in both experiments, and in Exp. 2 the addition of rLh induced the production of higher quantities of sperm that were used to fertilise the eggs. Although fertilisation was low (0.4%), these fertilized eggs with embryo development produced viable larvae (71% hatching). In comparison, control females remained arrested at previtellogenesis and control males did not produce sperm. The study demonstrated that both rGths are effective to induce the process of oogenesis in female flathead grey mullet and to obtain flowing sperm from males, adding more data to confirm the roles of the Gths in teleost gametogenesis. This is the first report, in a teleost species, of the use of rGths (rFsh and rLh) to induce oogenesis from previtellogenesis through to maturation to obtain eggs and larvae. This advance provides the bases for the development of therapies for the use in the aquaculture of teleost of commercial interest or the conservation of endangered species.

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.