Abstract are unidirectional and the cleavage reactions facilitated by

Abstract

Steroidogenesis is a process which governs
the conversion of cholesterol to active steroid hormones. The de novo biosynthesis of active steroid
hormones from the precursor, cholesterol, involves the mobilization of
cholesterol from the cellular stores to the inner mitochondrial membrane in
order to produce the first steroid hormone, pregnenolone. This trafficking of
cholesterol to the inner mitochondrial membrane is accomplished by the actions
of steroidogenic acute regulatory protein (StAR) and thereby initiating the
conversion of cholesterol into several steroid hormones via a series of
enzymatic steps. This review article summarizes the current knowledge of the
enzymatic regulation of steroidogenesis with special reference to males. Most
of the steroidogenic enzymes are either cytochrome P450(s) or hydroxysteroid dehydrogenase(s).
The cytochrome P450 enzymes are unidirectional and the cleavage reactions
facilitated by these enzymes are irreversible due to which the accumulation of
the products does not drive flux backto the precursor unlike the reactions
mediated by the hydroxysteroid dehydrogenase(s) that are mostly reversible. Significant
advances have arisen over past three decades in the understanding of the molecular
characterization of these steroidogenic enzymes that have prompted several
scientists to look upon the aspects of steroidogenic regulation in detail. Some
of the recent findings include identification, molecular cloning and
characterization of different steroidogenic enzymes genes and associated
transcription factors in lower vertebrates. This review attempts to impart an
integrative view on the regulation of testicular development and
spermatogenesis together with the characteristics of few candidate
proteins/factors that are involved in the processes with an emphasis on teleost
fish. Highlights on the crosstalk of factors governing these two processes will
be discussed in terms of sex-reversal and artificial induction to provide an
outlook to understand male reproduction in teleosts.

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Keywords:                                                                                                                                   

Steroidogenesis;
steroid hormones; sex reversal; spermatogenesis; steroidogenic enzyme genes.

I.          Introduction

Steroid
hormones biosynthesis or steroidogenesis regulates an array of developmental
and physiological processes in the life span of an organism. The gonadal
development is preliminary controlled by steroidogenesis followed by the
hormonal regulation by the hypothalamic-pituitary axis. Steroidogenesis is a
complex process that involves a number of enzymatic processes by which
cholesterol is converted to biologically active steroid hormones, the potential
end products being the estrogens and the androgens. The biochemistry of the
enzymes involved in steroidogenesis has been reviewed in detail concerning on human steroidogenesis and its disorders
(1). Steroidogenesis, in other words, can be marked as the beginning
of cholesterol trafficking from the sub cellular stores to the mitochondria,
where by the first enzymatic conversion occurs, i.e., pregnenolone is formed by
the action of cholesterol side chain cleavage cytochrome P450 (P450scc). Steroid
hormone synthesis there by requires a number of essential enzymes namely,
steroidogenic acute regulatory protein (StAR), cholesterol side chain cleavage
cytochrome P450 (P450scc), cytochrome P450 17-hydroxylase/ C17–20 lyase
(CYP17), and 3?-hydroxysteroid dehydrogenase/isomerase (3?-HSD) and further
more. The upstream in the steroid hormone biosynthesis is taken care of by
Steroidogenic Acute Regulatory protein (StAR), its function being the
translocation of cholesterol to the inner mitochondrial membrane. This review
summarizes the pertinent literature focussing on the enzymatic regulation of
androgen biosynthesis with special reference to teleostean models.  

In teleosts,
two biologically important steroid mediators have been identified with reference
to spermatogenesis and sperm maturation namely, 11-ketotestosterone (11-KT) and
17?,20?-dihydroxy-4-pregnen-3-one (17?,20?-DP). Biosynthesis of these steroids
and gonadal development is controlled by the expression of steroidogenic enzyme
genes and their related transcription factors. In fishes, testosterone (T) and
11-KT are the principal androgens (2). Several studies in teleosts (Hippoglossus hippoglossus, Cyprinus carpio) depicts high levels of 11-KT during
spermatogenesis as compared to T implicating former’s potential role in
spermatogenesis progression (3, 4) and inducing male sex phenotype,
secondary sexual characteristics and female-to-male sex-reversal (5).
This prompted researchers to speculate whether 11-KT could pilot testis
formation from bipotential gonad during critical period of sex differentiation,
similar to estradiol-17?, whose presence in juvenile fish favours ovarian
development (6, 7, 8). However, the role of 11-KT seems to have no
role testis determination so also T (9). Nevertheless, testicular
growth, spermiation and recrudescence are essentially regulated by 11-KT and T
in teleosts. Factors responsible for sperm maturation are largely unknown
except for the identification of activin and inhibin playing critical role in
spermatogenesis using eel and zebrafish models. In lower vertebrates including
catfish, multiple forms of dmrt1, sox9 and wt1 have been identified that play crucial role in
gonadal differentiation wherein dmrt1
influences female to male sex reversal (10, 11). Previous study also
suggested that 11?-hsd promoter is
controlled dominantly by the binding of Sox3 with a moderate influence of Wt1
that regulates testicular development and recrudescence (12). In
spite of these, distinct information pertaining to factors or genes and their
interactions during testicular growth and recrudescence are not clear. Thus,
understanding the regulation of steroidogenic enzyme genes directly or indirectly
involved in T and 11-KT production seems critical to address the molecular
mechanisms of spermatogenesis with special reference to fish reproduction.

II.        Steroidogenic
Enzymes Genes: Overview of the Steroidogenesis Cascade

There
have been tremendous developments in understanding the mechanisms of
cholesterol trafficking as well as in the identification and characterisation
of several steroidogenic enzyme genes in the last two decades. These research
and findings have further substantiated the current knowledge of the molecular
steroidogenesis. As steroidogenesis is predominantly controlled by the
expression of steroidogenic enzyme genes, several studies have been made
towards the characterization of different steroidogenic enzymes genes and the
associated transcription factors in lower vertebrates.

 (a)       Cytochrome P450 Enzymes

There
are six cytochrome P450 enzymes whose existence is familiar to the researchers.
P450scc otherwise known as CYP11A1 coordinates a rate limiting enzymatic step
in steroidogenesis i.e., the conversion of cholesterol to pregnenolone. Steroid
11?- hydroxylase, commonly known as CYP11B1catalyses the conversion of
glucocorticoids i.e., 11-deoxy- cortisol to cortisol and deoxy-corticosterone
to corticosterone and androgens i.e., T to 11? – hydroxytestosterone. CYP11B1
is an isozyme for aldosterone synthase, otherwise known as, CYP11B2. CYP11B2
converts deoxy-corticosterone to aldosterone. CYP17 is essential for the
synthesis of both glucocorticoids (17?-hydroxylase activity) and sex steroids
(17,20 lyase activity). P450c21, commonly called as, CYP21A2 is helps in the
21-hydroxylation of both glucocorticoids and mineralocorticoids. P450aromastase
or CYP19A1 is required for the conversion of androgens to estrogens namely, T
to estradiol and androstenedione to estrone.

(b)       Hydroxysteroid
dehydrogenases

The cleavage reactions facilitated by cytochrome P450
enzymes are unidirectional and thus, irreversible. However, the reactions
mediated by hydroxysteroid dehydrogenases are most reversible resulting in the
accumulation of the products that drive flux back to the precursor. Four
hydroxysteroid dehydrogenases are known to the endocrinologists till date
namely 3?-HSD, 20?-hydroxysteroid dehydrogenase (20?-HSD), 17?-hydroxysteroid
dehydrogenase (17?-HSD) and 11?-hydroxysteroid dehydrogenase (11?-HSD). 3?-HSD is responsible for the biosynthesis of progesterone
from pregnenolone, 17?-hydroxyprogesterone from 17?-hydroxypregnenolone, and
androstenedione from dehydroepiandrosterone (DHEA) in the adrenal gland. 20?-HSD
catalyzes the formation of (17?,20?-DP) from 17?-hydroxyprogesterone.
17?-HSD converts androstenedione to T where as 11?-HSD facilitates the conversion of inert 11 keto-products to active steroid
hormones and vice versa.

III.       Regulation
of Steroidogenic Enzymes Genes

The crucial genes that are known to be
involved in sexual development till date, accounts mainly from mammalian
studies. For example, Wilm’s tumour suppressor-1 (WT1), steroidogenic factor-1
(SF-1), and GATA-binding protein 4 (GATA-4) are known to be crucial for the
bipotential gonad development. Genes, namely, double sex and mab-3 related
transcription factor 1 (DMRT1), SRY-related box 9 (SOX9), dosage-sensitive
sex-reversal-adrenal hypoplasia congenital critical region of X chromosome,
gene 1 (DAX1), fibroblast growth factor 9 (FGF9) and desert hedgehog (DHH) are
involved in male sex determination where as anti-mullerian hoemone (AMH) and
androgen receptor (AR) is involved in male sex differentiation. Furthermore,
the genes and factors namely, Wingless-type MMTV integration site family member
4 (WNT4), R-spondin-1 (RSPO-1), forkhead box transcription factor L2 (FOXL2),
and follistatin (FST) are involved in female sex determination where as  CYP19A1 or the ovarian aromatase in  essential for female sex differentiation
(13,14). Similar reports have been documents in lower vertebrate models (15,
16, 17) including teleost (1,18, 19, 20, 21). Inspite of these, the findings
have never been satisfying and truly conclusive with reference to testicular
development and spermatogenesis in contrast to ovarian development that has
been studied upon well where in aromatase plays a very crucial role.

IV. Steroids in Testicular development

Similar
to other vertebrates, fish reproduction is also controlled by the
brain-pituitary-gonadal axis (22). However, the median eminence that connects
the hypothalamus with the pituitary lacks in teleosts. Thereby, the
neuro-endocrine hormones stimulate or inhibit the biosynthesis of the
gonadotrophins directly by reaching the cells of the anterior lobe of the
adenohypophysis. Further, gametogenesis and gonadal steroidogenesis is
regulated. Steroids contribute to the regulation of gametogenesis in teleosts,
similar to mammals. Sex steroids play a crucial role in relaying the sexual
status to the brain-pituitary axis, thereby initiating the neuro-endrocrine
regulation cascade in gonadal development. 
Testis, being the primary target organ for the gonadotropin action, facilitates
gamete maturation, spawning and spermatogenesis. Follicular stimulating hormone
(FSH) accounts for spermatogenesis where as Luteinizing hormone (LH) stimulates
androgen release taking care of spawning and sperm maturation. T and 11- KT,
being the potent fish androgens are regulated in multiple levels during
steroidogenesis. 17?, 20?-DP id known to induce spermiation, increase milt
production and stimulate sperm motility (23).

V.        Hormonal
Regulation of Spermatogenesis

Tropic
hormones regulate The function of steroidogenic cells is regulated by the binding
of the tropic hormones to their specific receptors (mostly G-protien coupled receptors)
on the cell surface, there by activating intracellular signal transduction cascade.
This is followed by a series of reactions that causes a short-lived increase in
the levels of the secondary messengers. Protein kinases (A and C) and phospholipases
are thus activated and the hormonal signal is transmitted inside the cell. However,
if the Protien kinase A pathway coincides with that of protein kinase C, it is known
to suppress the expression of the steroidogenic enzyme genes (24). Two such
tropic hormones are FSH (that acts specifically on Sertoli cells) and LH (which
acts on Leydig cells) whose release is controlled by Gonadotropin releasing
hormone (GnRH) there by controlling the reproductive axis.

VI.       The
crosstalk: Comparison of various teleostean species

a.     
Sex reversal

b.     
Induction

Artificial
induction is one of the most widely used techniques that can boost the sexual
maturation in seasonal breeders during the off-breeding season. Several
induction methods using GnRH and its agonists have been well demonstrated, that
are known to be quite effective (25,26). In Japanese eel, it has been stated that human chorionic
gonadotropin (hCG) can induce all stages of spermatogenesis (27).
The activity of hCG is similar to that of LH considering the fact that they
share the same receptor and hCG is known to possess a longer half-life and a
very high affinity to its receptors (28). Although
the procedures entailing administration (by intraperitoneal injection) of the hormone
followed by in vitro fertilisation
are effective enough to advance the gonadal maturation in teleost (29),
researchers have been investigating for a better way of sustained
administration that may result in higher yield. Induction through hCG in
vitro/in vivo evokes
modulation in sex steroid levels (T, 11-KT, estradiol 17-? and  17?,20?-DP) and up regulation of various steroidogenic enzyme genes (P450c17, 20?-hsd,
cyp11a1, 3?-hsd, 17?-hsd1 and 12, 11?-hsd2, 11?-h and cyp19a1a)
and factors (dmrt1, sox9a,
sox9b, ad4bp/sf-1wt1,  pax2,
gata4 and  foxl2), thereby,
facilitating successful fertilization. Several studies in fishes (for example, Salmo
salar and Pagrus major) stated different methods
of LH-releasing hormone administration i.e., using  cholesterol pellet
and  copolymer respectively (30, 31). However,
the sustained release of hCG have been made possible lately by the use of
osmotic pump in order to induce ovulation and spermiation respectively in
female and male japanese eels (32, 33).

VII.     Discussion
/ Future Perspective

In reference to hormonal
regulation of spermatogenesis,  gonadotropins are
known to regulate gametogenesis by controlling gonadal steroidogenesis. Few
genes and crucial factors  that are known
to have a central role with respect to steroidogenesis have been identified in
teleost, out of which many are found to be regulated by gonadotropins. Advancement
of sexual maturation, in other word, artificial induction of reproduction in teleost
may benefit tremendously for teleosts fish which is failed due to rainfall
delay and other environmental factors mostly. Considering the plight of the
average spawning time for the teleostean fish being short lived, induced
breeding may be proven extensively significant in promoting aquaculture.

 

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