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Endocrine Control of Reproduction, Fish

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DOI: 10.1016/B978-0-12-809633-8.20579-7

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From Biran, J., & Levavi-Sivan, B. (2018). Endocrine Control of Reproduction,

Fish. In M. K. Skinner (Ed.), Encyclopedia of Reproduction. vol. 6, pp. 362–368.
Academic Press: Elsevier. http://dx.doi.org/10.1016/B978-0-12-809633-8.20579-7
ISBN: 9780128118993
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 Author's personal copy

Endocrine Control of Reproduction, Fish

Jakob Biran, Agricultural Research Organization, Rishon Letziyon, Israel
Berta Levavi-Sivan, The Hebrew University of Jerusalem, Rehovot, Israel
© 2018 Elsevier Inc. All rights reserved.

Introduction

Fish comprise more than 40% of all vertebrate species and are extremely diverse. Nevertheless, the general endocrine components
and pathways that regulate fish reproduction are highly conserved and as in other vertebrates, they are generated by the
hypothalamus-pituitary-gonadal (HPG) axis (Levavi-Sivan et al., 2010; Zohar et al., 2010). Sensory information from the internal
and external environments as well as social cues (such as presence of a potential mate, temperature, photoperiod, metabolism,
energy stores, gonadal state etc.), are processed by the fish brain and integrated by the hypothalamus to initiate and regulate
reproductive activity. The hypothalamus reacts to environmental and physiological conditions by synthesizing and releasing
neuropeptides (primarily gonadotropin-releasing hormone; GnRH) and monoamines (mainly dopamine) which regulate the
activity of endocrine cells in the anterior pituitary. That region of the pituitary produces the gonadotropins follicle stimulating
hormone (FSH) and luteinizing hormone (LH). Upon stimulation, gonadotrope cells located in the anterior pituitary produce
and release their hormones into the blood (Levavi-Sivan et al., 2010). Both gonadotropins reach the gonads, where they stimulate
the synthesis of steroidal hormones that, in turn, regulate maturation of gametes, as well as convey feedback to the hypothalamus
and pituitary.
In spite of the diversity of reproductive strategies among the approximately 29,000 recognized fish species, fish share several
distinct endocrine characteristics: (i) The fish neurohypophysis is positioned dorsally to the adenohypophysis, in contrast to the
anterior-posterior organization of the mammalian pituitary. Moreover, unlike the mammalian adenohypophysis, the teleostean
adenohypophysis does not contain a cleft (a remnant of the mammalian Rathke’s pouch), and so there is no clear morphological
separation into a pars distalis and a pars intermedia as in mammals (Pogoda and Hammerschmidt, 2007); (ii) Fish lack both
median eminence and a hypophyseal portal system. Instead, both neurohypophysis and adenohypophysis are directly innervated
by hypothalamic nerve terminals (Ball, 1981) and apply a dual mode of gonadotrope regulation by GnRH, combining both neuro-
glandular and neurovascular components (Golan et al., 2015); (iii) Cells producing distinct gonadotropin are well defined,
spatially arranged, and grouped together in particular regions of the gland. In addition, while a single mammalian gonadotrope
may produce both FSH and LH, fish gonadotropins are produced and secreted by distinct cell types (Levavi-Sivan et al., 2010).
LH cells exhibit close cell-to-cell contacts and form a continuous network throughout the gland while FSH cells are more loosely
distributed, still maintaining some degree of cell-to-cell contact by cytoplasmic processes (Golan et al., 2015); (iv) Due to events of
whole-genome duplications that have occurred during fish evolution, as many as three GnRH-ligand and five GnRH-receptor
(GnRH-R) genes have been identified in certain fish species. By contrast, only one or two such variants occur in mammals. The
gene duplication in GnRH ligands has led to segregation in the expression pattern and central function of each ligand. A
species-specific variant (seabream-type GnRH; GnRH1) that occurs in fish, is mainly located in the preoptic area, and plays a central
role in regulating reproduction. A highly preserved variant (chicken-type cGnRH-II; GnRH2) in the midbrain may act as a neuro-
transmitter and/or neuromodulator. A salmon-type GnRH (sGnRH; GnRH3) that has been primarily identified in the terminal
nerve ganglion and olfactory bulbs, is reported to have a neuromodulatory function (Zohar et al., 2010). GnRH receptors are
G-protein coupled receptors (GPCR). Piscine GnRH-Rs lack their C-terminal tail; therefore, no GnRH desensitization exists
(Levavi-Sivan et al., 2010).
Owing to their commercial value, the reproductive biology of the various fish species has been well studied. Thus, interest
constantly increases in fish as model organisms for basic science, as well as for the agricultural aspects.

Reproductive Strategies

The high variability of fish and the ecological niches they inhabit has led to the adoption of various reproductive strategies including
viviparity (Poecilia), sequential hermaphroditism (the protandrous seabream or the protogenous orange spotted grouper) and even
simultaneous hermaphroditism (Black hamlet). Nevertheless, most fish species are gonochoristic and oviparous, meaning that the
sexes are separate and fertilization and embryonal development are external (Yaron et al., 2003). Fish may have synchronous ovaries
and spawn only once in a season (carp) or even once in their lifetime (river eel and Atlantic salmon); or they can possess asynchro-
nous (or group-synchronous) ovaries, producing several spawning events during the breeding season. During this period, their
ovaries contain several generations of oocytes at various developmental stages. Spawning cycles of asynchronous fish may vary
from 24 h in the gilthead seabream, up to one month in tilapia, (Yaron et al., 2003).
Fish with synchronous ovaries undergo sharp metabolic changes during the vitellogenic period that differ from those
occurring during final oocyte maturation and spawning. Endocrine changes that support each step reflect relatively simple
and well-timed hormonal patterns. In contrast, simultaneous production and maintenance of several oocyte generations in asyn-
chronous ovaries makes the sequential hormone release more complex. These endocrine differences are discussed in the
following sections.

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Endcocrine Control of Reproduction j Endocrine Control of Reproduction, Fish 363

The Hypothalamus-Pituitary-Gonadal (HPG) Axis

The HPG axis consists of three major sites of endocrine regulation. A plethora of neuropeptides are secreted from specific neurons in
the brain (primarily but not only in the hypothalamus) and form an interface between the central nervous system and the endocrine
system. These neuropeptides may directly affect the gonadotropes in the pituitary, or may exert their action via the GnRH neurons.
This action stimulates the production and release of two distinct but chemically related gonadotropins: FSH and LH. FSH and LH
are released into the circulation, and stimulate the gonads to secrete sex-specific steroids that are responsible for gametogenesis,
vitellogenesis and final oocyte maturation in females, as well as spermatogenesis and spermiogenesis in males (Fig. 1).

The Hypothalamus and its Neurohormones

The plethora of neuropeptides and monoamines released by the brain were shown to regulate piscine reproductive functions. These
include: GnRH, pituitary adenylate cyclase-activating peptide (PACAP), kisspeptins, Neurokinin B (NKB), Neurokinin F (NKF),
Neuropeptide Y (NPY), ghrelin, secretoneurin, leptin, gonadotropin-inhibitory hormone (GnIH), dopamine, serotonin, g-amino-
butyric acid (GABA), spexin, dynorphin, and more.
GnRH was first isolated from mammals, and later from other vertebrates (Sherwood et al., 1983). To date, 30 structurally
different forms of GnRH have been identified, while 12 structural variants of the GnRH molecule have been found in different
fish species. The N-terminal amino acid sequence (pGlu-His-Trp-Ser) and carboxyl terminal amino acid sequence (Pro-Gly-NH2)
have been conserved over 400 million years of chordate evolution (Kochman, 2012). As indicated by its name, chicken GnRH II
(GnRH-2) was first isolated from chicken brain and found to be the most ubiquitous form of GnRH molecule. The brain of
most teleosts contains three forms of GnRH, encoded by three distinct genes and differ in their developmental origin, spatial distri-
bution, and function. GnRH1 neurons are hypophysiotropic and are positioned in the preoptic area (POA); GnRH2 neurons reside
in the midbrain tegmentum, and are suggested to be involved in food intake and reproductive behavior; and the GnRH3 neuronal
population is localized in the terminal nerve and has no direct innervation to the pituitary (Zohar et al., 2010). Some teleost species
have only two GnRH forms: the salmon and zebrafish genomes do not contain the GnRH1 form, while catfish and eel lack the
GnRH3. The lack of these forms is compensated by the axonal projections of the remaining forms (Abe and Oka, 2011). For
example, in zebrafish, some of the GnRH3 neurons developing in the terminal nerve migrate into the hypothalamic POA and inner-
vate the pituitary, suggesting that they acquire the role GnRH1 fulfills in “three-form” fish (Zohar et al., 2010).
As in mammals, GnRH1 neurons in the fish POA display irregular and episodic spontaneous electrical activity with circadian
fluctuations in the episodic firing frequency. These irregular activities are in contrast to those of the non-hypothalamic populations

Fig. 1 Schematic diagram displaying HPG axis in fish. Schematic diagram showing potential regulatory signals conveyed by hypothalamic neurons
to pituitary cells, leading to the gonads (HPG axis).

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of GnRH neurons, i.e., GnRH2 and GnRH3, which display regular pacemaker activities, further supporting their functions other
than – hypophysiotropic (Oka, 2010). In contrast to the similarities in the GnRH neurons’ electrical activity between fish and
mammals, a striking difference can be seen in the structure and function of the GnRH-R. In mammals, constant delivery of high
GnRH doses leads to hypoactivity of the gonad and disrupted estrous cycles, due to desensitization of the GnRH-R. In contrast,
chronic administration of high doses of GnRH in fish leads to increased gonadotropin release and even to spawning. (Zohar
et al., 2010).
While hypothalamic GnRH is a positive regulator of the HPG axis, neuroendocrine release of hypothalamic dopamine serves as
a negative effector of HPG activity in many, albeit not all, fish species. Dopamine is a neurotransmitter, synthesized from tyrosine by
tyrosine hydroxylase (the rate-limiting enzyme) and Dopa-decarboxilase. Although dopamine is known to have pleiotropic effects
on both brain and pituitary functions, its involvement in the endocrine regulation of fish reproduction has been demonstrated in
several fish species, including goldfish, zebrafish, carp, African catfish, tilapia, eel, grey mullet, sturgeon, and rainbow trout
[reviewed by Levavi-Sivan et al. (2010); Zohar et al. (2010)]. Contrasting these effects, dopaminergic inhibition was not observed
in several marine species, suggesting that its involvement in reproductive function is mainly important in freshwater fish. Dopamine
receptors are GPCRs and are classically divided into two principal subtypes, according to their ability to activate (D1-like subtype) or
inhibit (D2-like subtype) adenylyl cyclase, the key enzyme in the conversion of adenosine triphosphate to 30 50 -cyclic AMP. Various
agonists and antagonists with high specificity to D1 or D2 receptors were employed to demonstrate that the inhibitory effects of
dopamine on fish reproduction is due to specific activation of D2-like receptors. Fontaine and colleagues have demonstrated the
expression of three D2-like receptors in LH cells in the adenohypophysis of the female zebrafish, and that the dopaminergic inner-
vation of the adenohypophysis originates from the hypothalamic POA (Fontaine et al., 2015). These findings provide neuroanatom-
ical support for the existence of dopaminergic inhibition in piscine reproduction.
During recent years, a large surfeit of novel neuropeptides have been found to regulate the secretion of GnRH in mammals and
other non-mammalian vertebrates. However, it was the identification of kisspeptin as an upstream regulator of the hypophysio-
tropic GnRH neurons that led to a significant change in the way that endocrinologists perceive neuroendocrine regulation of the
HPG axis (Tena-Sempere et al., 2012). The discovery that GnRH neurons have Kiss1-receptors (a.k.a. GPR54) was the first indication
that the kisspeptin system is involved in the control of fish reproduction (Parhar et al., 2004). Unlike most mammals, which possess
only one kisspeptin system (KISS1), the majority of teleosts have two kisspeptin systems, comprised of Kiss1 and Kiss2; and two
cognate receptors, Kiss1r and Kiss2r (Biran et al., 2008). Nevertheless, certain fish species have only one kisspeptin gene and two
Kiss1R genes; in others, two kisspeptin genes were identified with only one Kiss1R gene. This inconsistency can be explained by
the fact that bony fish underwent two complete genome duplications during evolution, and many of these duplicated genes
were subsequently lost (Mechaly et al., 2013). As in the case of GnRH, chronic administration of kisspeptin was found to suppress
reproductive activity in rats and monkeys (Seminara et al., 2006; Thompson et al., 2006), while chronic administration of kisspeptin
in fish leads to increased reproductive functions (Nocillado et al., 2013).
Deletion and site-directed mutagenesis of an estrogen-responsive element (ERE) from the kiss promoters of the protogynous
orange-spotted grouper (Epinephelus coioides), indicated that Kiss1 is regulated by estradiol 17b (E2), via the classical pathway
utilized by Erb1, as well as via an activator protein 1 (Ap1)-dependent, non-classical pathway utilized by Erb2. Kiss2 was also regu-
lated by E2 through the Creb transcription factor, as well as by Erb1 and Erb2 pathways. The effects of gonadal steroids on kisspeptin
in vivo were also demonstrated. In the medaka, Kiss1 neurons drastically change their firing activity according to E2 levels, and in the
European sea bass expression levels of Kiss2 were affected by circulating testosterone levels (Alvarado et al., 2016). Taken together,
accumulating data clearly support the assumption that E2 is involved in the feedback regulation of piscine kisspeptins via various
estrogen receptors.
A relatively new neuropeptide found to be involved in fish reproduction is Neurokinin B (NKB, encoded by the gene tac3). NKB
is a member of the tachykinin peptide family. The mammalian NKB is unique in that its prepro-hormone encodes a single mature
peptide, while other prepro-tachykinins encode two mature peptides. Mammals also have one high affinity receptor for NKB
(NKBR/NKR3). In contrast, fish have up to three NKB receptors and two genes encoding NKB. Importantly, prepro-tachykinin3
of fish matures into two peptides. Because the additional peptide appears only in fish and frogs, it was designated neurokinin F
(NKF; Fig. 1) (Biran et al., 2012). Moreover, while kisspeptin and NKB are co-expressed by the same neurons in the mammalian
hypothalamus, the piscine kisspeptin and NKB are expressed by different neuronal subsets of the fish hypothalamus (Ogawa
et al., 2012). NKB-R are expressed in LH but not FSH cells, suggesting regulation of final oocyte maturation by NKB via LH surge
(Biran et al., 2014). In vivo administration of NKB/NKF peptides increased reproductive functions in zebrafish, tilapia, goldfish and
striped bass (Biran et al., 2012, 2014; Zmora et al., 2017). However, tac3 gene products in carp do not play a role in LH synthesis at
the pituitary level, but may serve as novel stimulators for prolactin and somatolactin synthesis.

The Pituitary and its Gonadotropins

As in other vertebrates, the regulation of fish reproduction involves the synthesis and secretion of hormones from the adenohy-
pophysis, including FSH (GTH-I), LH (GTH-II), growth hormone (GH), thyroid-stimulating hormone (TSH) and adrenocortico-
tropic hormone (ACTH), with FSH and LH serving as the key regulators of gonadal development and function. FSH and LH are
heterodimeric hormones with a distinct b subunit and a common a subunit, which is also common to TSH). Over the years, gonad-
otropin subunit genes were isolated in more than 56 fish species from at least 14 teleost orders, allowing a vast characterization of
fish gonadotropin synthesis and release. Signals from hypothalamic neurohormones and gonadal steroids are integrated in

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Endcocrine Control of Reproduction j Endocrine Control of Reproduction, Fish 365

gonadotrope cells to regulate synthesis and release of both gonadotropins. Nonetheless, GnRH regulation of FSH synthesis and
release is less clear than that of LH (Zohar et al., 2010; Yaron et al., 2003).
As previously described, there are two main modes of gonadal development in fish. Synchronous gonadal development is
accompanied by a clear duality of gonadotropin secretion; while the adenohypophysis of immature salmon contains mainly
FSH cells, the adenohypophysis of sexually mature rainbow trout contains numerus LH-producing cells. In addition, only FSH
is released into the blood of immature coho salmon, with increased levels during the gonadal recruitment step, and reduced levels
towards final oocyte maturation and spawning. While both gonadotropins induce E2 secretion, LH is more potent than FSH in
raising 17a,20b dihydroxy-4-pregnen-3-one (DHP) levels from post vitellogenic oocytes (Swanson, 1991). However, in carp, LH
alone is sufficient to regulate both vitellogenesis and final oocyte maturation while FSH may have another, yet undefined role.
In contrast, both FSH and LH are synthesized and released during all stages of reproduction in multi-spawning fish with asynchro-
nous ovaries like the gilthead seabream, goldfish and tilapia (reviewed by Levavi-Sivan et al. (2010); Yaron et al. (2003)). These
differences are probably due to the need to regulate the development of several oocyte generations in parallel. In this situation,
the differential regulation of each oocyte generation may be attributed to differences in the expression of gonadotropin receptors.
The fish FSH and LH receptors are GPCRs belonging to the Class A of rhodopsin-like receptors. Mammalian FSHR and LHR show
very high selectivity to their cognate ligands, leading to functional specificity. However, ligand-receptor promiscuity was shown in
several fish species. Structural modeling combined with in vitro assays of gonadotropin receptor activation have demonstrated that
in some fish species, FSHR may bind both FSH and LH, while in almost all fish species tested, LHR is highly specific for LH (Aizen
et al., 2012). Concomitant with their importance in induction of ovarian steroidogenesis, gonadotropin receptors are expressed by
follicular cells of the ovary (theca and granulosa cells) and by interstitial and nurse cells of the testis (Leydig and Sertoli cells) (Yaron
and Sivan, 2006).
Gonadal steroids transmit their feedback to regulate the synthesis and release of FSH and LH. This feedback can be of a positive
or negative nature, depending on the reproductive state of the fish. Gonadectomy in sexually mature goldfish, catfish, Atlantic
croaker, bass and salmonids results in increased LH secretion, while treatment with gonadal steroids may reverse this effect. Never-
theless, during earlier stages of gonadal development, gonadal steroids can induce LHb subunit expression and protein levels
(Levavi-Sivan et al., 2010; Yaron et al., 2003). More anatomical work is required to understand whether these effects are direct or
indirect.
LH acts on the ovarian follicle to produce DHP, the maturation-inducing Hormone, (MIH) in most fishes. The dramatic increase
in the capacity of post vitellogenic follicles to produce DHP in response to LH is correlated with a decrease in P450c17 (P450c17-I)
and P450 aromatase (oP450arom) mRNA and increase in the novel form of P450c17 (P450c17-II) and 20b-hydroxysteroid dehy-
drogenase (20b-HSD) mRNA.

The Gonads and Their Steroids

Most fish species used in research and aquaculture are gonochoristic. Therefore, a sexually mature fish would have either a functional
testis (male) or a functional ovary (female). In most gonochoristic fish, FSH and LH stimulate the synthesis of three key sex steroids:
E2 serves as the principal estrogen and stimulates germ-cell proliferation and growth and vitellogenesis; 11 ketotestosterone (11-KT)
serves as the central androgen that regulates spermatogenesis and spermiogenesis; DHP regulates final oocyte maturation and ovula-
tion in females as well as spermatozoa maturation and spermiation in males (Yaron, 1995). In addition, DHP is involved in the
meiosis onset of ogonia in the ovary and of spermatogonia in the testis. Androgens and E2 (under FSH) are involved in the appear-
ance of lipid droplets in previtellogenic oocyes. It is noteworthy that higher vertebrates utilize testosterone and not 11-KT as their
main androgenic hormone. These pathways are regulated in parallel to the above-mentioned feedback regulation of gonadal
steroids on reproductive functions of the hypothalamus and pituitary (Fig. 2).

Male
In spermiogenesis, non-functional sperm undergo the process of sperm maturation to become mature spermatozoa, fully capable of
vigorous motility and fertilization. These processes are mainly controlled by sex steroid hormones. Spermatogonial renewal is
controlled by E2 through the expression of platelet-derived endothelial cell growth factor. The proliferation of spermatogonia
toward meiosis is initiated by 11-KT, which is produced by FSH stimulation. 11-KT prevents the expression of anti-Müllerian
hormone (AMH), which functions to inhibit proliferation of spermatogonia and induce expression of activin B, which functions
in the induction of spermatogonial proliferation. Meiosis is induced by DHP through the action of trypsin. DHP also regulates
sperm maturation through the regulation of seminal plasma pH. Gonadotropic stimulation of Leydig cells in the testis induce
the synthesis and release of 11-KT, which leads to the activation of Sertoli cells, resulting in stimulation of spermatogenesis.
With the advancement of spermatogenesis, 11-KT levels start to decline and DHP levels rise, leading to induction of spermiogenesis.
In some fish species, DHP was shown to further regulate sperm motility (Miura and Miura, 2011; Schulz et al., 2010) (Fig. 2).

Female
In the case of female fish, the endocrine roles of gonadal steroids are more complex; alongside their roles in feedback regulation and
gonadal development, gonadal estrogens induce the synthesis and release of vitellogenin (VTG), the primary storage protein in fish
oocytes, by the female liver (Lubzens et al., 2017). In fish with synchronous ovaries, theca cells of the follicle respond to FSH to
produce testosterone, which is then aromatized into E2 by the granulosa cells. E2 stimulates the production of VTG and in parallel

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366 Endcocrine Control of Reproduction j Endocrine Control of Reproduction, Fish

Fig. 2 Endocrine control of spermatogenesis A schematic diagram summarizing the possible control mechanisms of spermatogenesis in the Japa-
nese eel. FSH, follicle-stimulating hormone; LH, luteinizing hormone; 17a,20b-DHP, 17a,20b-dihydroxy-4-pregnen-3-one; PD-ECGF, platelet-derived
endothelial cell growth factor; AMH, anti-Müllerian hormone; CAll, carbonic anhydrase; eSRS, spermatogenesis related substances. Reproduced from
Miura, C., Miura, T., 2011. Analysis of spermatogenesis using an eel model. Aqua-Biosci. Monogr. 4, 105–129.

regulates its accumulation in the yolk. Following the vitellogenic growth of the oocyte, FSH levels decline concurrently with the rise
in LH levels. This leads to shifts in steroidogenesis that leads the ovary to produce mainly DHP (Yaron, 1995).
In addition to the classic genomic mechanism of steroid action mediated by activation of intracellular nuclear receptors, there is
now extensive evidence that steroids also activate receptors on the cell surface to initiate rapid intracellular signaling and biological
responses that are often non-genomic. Two recently discovered novel proteins with seven-transmembrane domains, GPCR30
(GPR30), and membrane progestin receptors (mPRs), have the ligand binding and signaling characteristics of estrogen and
progestin membrane receptors, respectively. During the first phase – at the end of vitellogenic oocyte growth – the ovarian follicles
produce large amounts of estrogens and minor quantities of progestins, resulting in activation of the inhibitory pathway regulating
oocyte maturation, but not the stimulatory pathway. The estrogens act through GPR30 to activate a stimulatory G protein (Gs),
resulting in stimulation of adenylyl cyclase activity and increases in cAMP production. The high levels of cAMP maintain meiotic
arrest of fish oocytes, possibly through downstream signaling molecules such as protein kinase A. In addition, estrogens upregulate
GPR30 expression to potentiate the inhibitory pathway and down-regulate mPRa expression to block the stimulatory one, through
activation of GPR30 via unknown pathways. As a result, the oocytes remain in meiotic arrest. In the second phase, the steroidogenic
pathway is switched to the production of progestins and estrogen production is declined. At the same time, progestins, through
mPRa, cause down-regulation of GPR30 expression, which together with the reduction in estrogen levels, causes suppression of
the inhibitory pathway. Progestin binding to mPRa results in activation of an inhibitory G protein (Gi) decreasing adenylyl cyclase
activity and cAMP levels, leading to inhibition of protein kinase A, thereby releasing the oocyte from meiotic arrest and allowing
oocyte maturation to proceed (Thomas, 2012).

Genome Editing Era in Fish Reproduction

The emergence of the novel methods of transcription activator-like effector nucleases (TALEN) (Bedell et al., 2012) and clustered
regularly interspaced palindromic repeats-associated (CRISPR)/Cas9 (Gagnon et al., 2014) systems, commenced a technological
revolution in fish genome editing, allowing reverse genetics in fish. These methods are now applicable in several fish species of
commercial importance, including Atlantic salmon (Edvardsen et al., 2014), channel catfish (Khalil et al., 2017), Nile tilapia
(Wu et al., 2016), sea lamprey (Square et al., 2015) and model fish species zebrafish and medaka (Bedell et al., 2012; Gagnon
et al., 2014; Luo et al., 2015). These tools have already provided some exciting findings regarding the endocrine regulation of
fish reproduction.
At the hypothalamic level in zebrafish, no phenotypes were found in fish carrying single or double mutations in genes encoding
kisspeptin ligands or their receptors (Tang et al., 2015). Similarly, no phenotypes were identified in GnRH3 mutant zebrafish, and
even triple knockout fish for both kisspeptin ligands and GnRH3 resulted in normal reproductive development (Spicer et al., 2016;
Liu et al., 2017). These findings strongly challenge the current dogma of neuroendocrine regulation of fish reproduction.
At the pituitary level, zebrafish carrying homozygous mutation in the gene encoding FSHb-subunit, display delayed gonadal
maturation; and mutants in the gene encoding LHb-subunit show normal gonadal development but fail to spawn and hence
are infertile (Zhang et al., 2015). Double mutant zebrafish for both gonadotropins or both gonadotropin receptors develop an

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infertile all-male population phenotype. However, while FSHR mutant females are infertile, FSHR mutant males show no signifi-
cant phenotype (Chu et al., 2015).
At the gonadal level, “loss of function” mutations in the zebrafish aromatase gene resulted in an all-male phenotype (Lau et al.,
2016); and mutating the nuclear progesterone receptor in zebrafish leads to female infertility due to ovulation failure with no repro-
ductive phenotype in males (Tang et al., 2016).
Taken together, these findings demonstrate the high importance and strength of genomic editing in fish. Nevertheless, it also
highlight the need for additional knockout lines in other commercially important fish models.

Concluding Remarks

As in other vertebrates, in fish, endocrine regulation of reproduction is governed by the HPG axis. In the current article, we briefly
reviewed the endocrine components of the axis with emphasis on the unique and different characteristics of these components.
Taken together, the data presented show the high evolutionary importance of fish as model organisms for endocrine research in
the reproductive field; they are an exciting model for studying vertebrate reproduction. Moreover, some features in the piscine
HPG axis may provide explanations to phenomena that cannot be addressed in higher vertebrates. For example, the anatomical
separation between FSH and LH enables the characterization of regulatory pathways for each gonadotropin, while their co-
expression in mammals would hamper such efforts.
Finally, there is no doubt that novel genomic tools will significantly advance endocrine research in fish, which will lead to impor-
tant breakthroughs. Nevertheless, adopting these methodologies should be advanced in concert with development of additional
and more classical endocrine tools, such as specific hormone ELISAs, antibodies and anatomical research.

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