In mechanisms and novel transcription factors involved at

In general adrenal glands and gonads play a very
important role in sex differentiation and steroidogenesis. These two are
systems are closely related as they share a common region of origin i.e.
mesoderm and both are involved in steroidogenesis. Various biological events
occur during adrenal and gonadal steroidogenesis. In this article important signaling pathways and transcription factors
involved in regulation of steroidogenesis and adrenal growth have been
summarized. Present review illustrates various novel signaling pathways such Sonic
hedgehog ,Wnt, Notch, ?-catenin involved in adrenal gland morphology and its
functions that are deeply interconnected. Certain nuclear receptor such as
Steroidogenic Factor-1 acts as critical regulator of development and
homeostasis of the adrenal cortex and gonads. SF-1 is a nuclear receptor
almost exclusively expressed in the steroidogenic tissues of the hypothalamic
pituitary-adrenal/gonadal axis. Mitogen-activated protein kinases are
serine/threonine kinases involved in the expression of the Steroidogenic acute
regulatory protein and steroidogenesis. Characterization  of certain proteins that are encoded by dax1, amh, and cyp19a1 which
plays very important role in gonad differentiation and to evaluate the relation
between gonadal expression of Fushi
tarazu factor-1, StAR and cytochrome P450-11A in reproductive maturation
process. This article aimed to describe
the various signaling mechanisms and novel transcription factors involved at
genomic level in common to adrenal and gonadal development in fishes and lower
vertebrates.

 

Keywords: Gonadal development; Sex differentiation;
Adrenal growth; Steroidogenic Factor-1;
Steroidogenic acute regulatory protein; Mitogen-activated protein
kinases;

 

 

 

 

 

 

 

Introduction

 

Steroidogenesis involves the synthesis of steroid
hormones that are derivatives of cholesterol which are synthesized by various
tissues, most prominently the adrenal
gland and
gonads. These are usually found in chordates and arthropods. Fishes, for example teleosts, produce several types of
bioactive gonadal steroids, including progestogens, estrogens, androgens and
various derivatives of steroids. Steroids are required for development,
maintenance, homeostasis and reproduction. Steroids direct the development of germ cells
and accessory glands and organs, as well as the modification of the behaviour,
to ensure that sexual reproduction can take place. In adult vertebrates, these steroids are
produced at appropriate times in specialized steroid producing cells called gonads. These
cells express a group of steroidogenic enzyme genes whose products modify its
cholesterol and derivatives 1. Although many steroids are
identical chemically in all major vertebrate classes, the role of these
steroids may differ. However steroid hormones have evolved in some vertebrate
classes, especially amongst fishes, to fulfil particular functions.

 

Steroid
hormones are produced by steroidogenic cells of the ovary testis and brain that are required for normal reproductive function and bodily homeostasis.
Steroidogenic endocrine tissues such as the adrenal and the gonads respond to trophic
hormones and other external stimuli with rapid surge in steroid hormone production 2. The
acute and chronic regulation of steroidogenesis is controlled by trophic
hormones that normally occur in  order of minutes and hours, respectively.
Chronic regulation of steroidogenesis by LH or ACTH occurs at the level of gene
transcription 3. Cholesterol
is metabolized to pregnenolone by  cytochrome
P450 cholesterol side chain cleavage
enzyme (p450scc) and transferred from the outer to the inner
mitochondrial membrane. The Steroidogenic acute regulatory (StAR) protein is
the one which regulates the true
rate-limiting step in steroid biosynthesis, i.e. the delivery of cholesterol
from the outer to the inner mitochondrial membrane 4. The central role of StAR was proven by two
observations by robust steroid hormone synthesis followed co-transfection of
StAR and the cholesterol side-chain cleavage system into nonsteroidogenic COS-1
cells 4, 5. In other patients with StAR mutations have
congenital lipoid adrenal hyperplasia, whereby all adrenal and gonadal
steroidogenesis was distrupted 5, 6. Sex differentiation is initiated and controlled by gonadal steroid
hormones. These hormones perform
different functions and permanently differentiated into sex organs during
development. The expression of this protein is predominantly regulated
by cAMP-dependent mechanism in the adrenal and gonads.

Gonadal development

Reproduction in vertebrates depends on function  two distinct gametes, sperm and eggs that
develop into different organs, the testis and the ovary. The two reproductive organs are grossly different,
but they both are composed of developmentally common cell lineages, supporting
cells, interstitial cells and germ cells. A mature ovary consists an ovarian cavity, germinal
epithelium and stromal compartment. In fishes such as teleosts, the germ line
stem cells are mitotically active oogonia that reside in germinal epithelium.
Their structure was similar to surface epithelium in mammals. Steroid hormones
are produced by the follicles that are present in the stromal compartment where
the oocytes grow. In the testis,
spermatogenesis starting from the germ line stem cells to sperm production
occurs in tubules or lobules, and the interstitial tissue that produce steroid
hormones resides between these structures.

The germ cells at their early stages that have not
reached the gonad are termed as primordial germ cells(PGC). These PGCs were
identified morphologically and functionally specified by the distribution of
cytoplasmic determinants that includes RNA-binding proteins NANOS, TUDOR and
VASA which were localized on granule-like structures or nuage 7. This similarity have been previously observed in
other lower vertebrates and Drosophila. Nanos3 was found to be the earliest
marker in some fish such as medaka, and using this marker, PGCs were first
identified at an early gastrulation stage 8. Three mechanically 
distinct modes were observed for migration of PGCs 8, 9. In the early gastrulation stage migration towards
marginal zone depends on the chemokine receptor CXCR4 and its ligand, SDF1A.
Second, at the late gastrulation and early somitogenesis stages, it depends on
the convergent movement of somatic cells. After aligning bilaterally, PGCs,
governed by interactions between CXCR4 and SDF1B, resume active and directional
migration towards the posterior end of the lateral plate mesoderm, where
gonadal somatic precursors arise 10.

In teleosts,
Sertoli and granulosa cells  share a
common origin, namely, the supporting cells expressing the sox9b gene in the
bipotential gonadal primordia. Normally
sox9b was found to be express in
both supporting cells 11, an observation that is contrast to the
situation in mammals, where sox9 is only expressed in Sertoli cells and is both
required testicular development 12, 13. Sox9 along with  steroidogenic factor 1, regulates transcription of the anti-Mullerian hormone (AMH) gene. SOX-9
also plays an important role in male sexual development. The
sox9b expressing cells begin to express dmrt1, indicating the differentiation
into Sertoli cells. In the early oogenesis, from germ line stem cells to early
diplotene oocytes it proceeds in the cradles. Subsequently the diplotene
oocytes surrounding somatic cells exit from the germinal cradle and recruit
theca cells to form follicles. Theca cell layer
formation is an important physiological event that occur during early
follicular development. Finally the follicles in the stromal
compartment have two layers of somatic cells, outer theca cells and inner
granulosa cells. Granulosa and theca cells of
the ovary act as a support  to germ cells
within the developing follicle.  During this step, the granulosa cells lose
sox9b expression while foxl2, a marker of granulosa cells, is activated 11, 14. This suggests that granulosa cells
originate from the sox9b -expressing cells. Both follicular formation and
oocyte exit from germinal cradles appears to depend upon a series of successive
processes 14 which is also observed in other teleost fish
by histological analysis 15, 16. In some
studies it indicated that sox9b and amh, that
are involved in testicular differentiation in vertebrates, were implicated in
testicular formation and spermatogenesis during the sex change as well.
Interestingly, in the testis, sox9b expression is very intense in the Sertoli
cells located most distally in the lobules. In the ovary, sox9b is expressed in
the germinal cradles representing niche regions.The common function of sox9b
-expressing cells is for the maintenance of stem-type germ cells during early
gametogenesis. In some studies, they examined
the roles of amh and dmrt1 in male germ
cell development by generating their mutants with Crispr/Cas9 technology
in zebrafish.  Amh mutant zebrafish displayed a female-biased sex ratio,
and both male and female amh mutants
developed hypertrophic gonads due to uncontrolled proliferation and impaired
differentiation of germ cells. It was also found that amh  acts as a
guardian to control the balance between proliferation and differentiation of
male germ cells, whereas dmrt1 required for
the maintenance, self-renewal, and differentiation of male germ cells.
During testicular development, genes required for the production of steroid
hormone(s), e.g. p450scc/cyp11a1 and hsd3b,
begin to be express in presumptive Leydig cells located in the marginal regions
of the lobule. These genes are expressed in  ftz-f1 -expressing cells during testicular
development 17.
It suggests that ftzf1 regulates a set of steroidogenic genes and that androgen
production may occur in a single cell lineage of ftz-f1 – expressing cells. In
rainbow trout, immunohistochemical analysis also revealed that P45011B/CYP11B,
HSD3B, P450scc/ CYP11A1 and P450c17/CYP17A1 were all co-localized in
interstitial Leydig cells 18.
In  contrast, during ovarian development,
at least two types of theca cells seem to be present in medaka. Some fishes
express only aromatase. It has also revealed that p450c17 and aromatase were
exclusively expressed in transgenic medaka fish by expression analysis using
aromatase-reporter 19.
Alternatively, the two types of theca cells may share a common precursor that
expresses the ftz-f1 gene, and that generates offspring capable of either
maintaining or down regulating ftz-f1 expression and initiating aromatase
expression 20.

Adrenal
development

In vertebrates, adrenal glands composed of two
distinct parts, outer adrenal cortex and inner adrenal medulla. Adrenal cortex
secretes three major hormones glucocorticoids, mineralocarticoids and adrenal
androgens. Adrenal androgens involved in the gender differentiation in human
beings mainly dehydroepiandrosterone (DHEA) and testosterone. Cellular
organization of gonads is similar in all vertebrates, based on different
progression can trigger bipotential gonads, forms either ovaries or testis.
Gonads are originated from thickening of the ventrolateral surface of the
embryonic mesonephros called the genital ridge. The classic experiment of Jost 21
demonstrated that female differentiation occurs irrespective of the genetic sex
in the absence of testicular hormones. Previous expression data suggested
that GATA4 was involved in sex determination 22, 23
and in vitro data suggested a role for GATA4 in the regulation of genes
expressed in the gonads downstream of Sry, including Mis,
inhibin ?, and steroidogenic acute regulatory protein (StAR) (reviewed by 24, 25.

Adrenal steroid hormones are effective in different
adaptive responses in the internal and external environment stress of
vertebrates. The sex determination region of Y chromosome (SRY) gene required
to initiate signaling for male gonadal differentiation. Many other genes
involved in gonoadogenesis are GATA4 and FOG2 26.
Mammalian gonads arise in both sexes from bilateral genital ridge that have the
potential to develop as ovaries or testes 27, 28.
In humans gonadal differentiation occurs from the 10th through 12th
embryonic week. 

Steroidogenic factor 1 (SF-1) transcription factor
critical for adrenocortical development and homeostasis. SF1 is also known as
adrenal four-binding protein or nuclear hormone receptor Ad4BP, encoded by the
gene NR5A1. All cells that belong to steroidogenic lineages of the adrenal and
gonads express SF1, including subpopulations of long-term retained progenitor
cells in each organ 29, 30. Therefore, SF1 expression defines the identity of
these cells and commitment to steroidogenic differentiation 31-33. The expression of SF1 is detectable early in fetal
life, between the AGP formation and the ultimate establishment of the adrenal
primordium 30. Genetic loss of Nr5a1 or its upstream
transcriptional regulators Pbx1, Wt1, and Cited2, interferes with AGP formation
leading to various degrees of adrenal hypoplasia in mice 34-36. While Nr5a1 is continuously expressed from the
time of adrenal primordium formation throughout the adult life, during
embryonic stages and early fetal life in mice, the Nr5a1 expression is driven
by the fetal adrenal-specific enhancer (FAdE), which becomes inactive when the
definitive cortex forms, suggesting that distinct mechanisms sustain Nr5a1
expression in the fetal and in the definitive cortex .

Genes essential for
early gonadal development:

Acquisition of sexual dimorphic phenotype condition
is an important role in mammalian gonadal development. In this absence or presence
of Y chromosome at fertilization embryonic gonads differentiate in to either
ovaries or testis. Major four genes are known to be required for development of
bipotential gonads (a) the orphan nuclear receptor Steroidogenic factor-1 (SF1 orFtz-F1) 37 (b) Wilms tumor associated gene  (WT1; 38,  a zinc finger
DNA–binding protein, (c) Lhx1 (also known as Lim1), and (d) Lhx9, two LIM class
homeobox proteins 39. SRY,
SF-1, Wilms’ tumor related 1(WT1), GATA4, and SOX9, were emerging models that
suggest complex interactions among these genes in gonadal development (Keith
Parker). SRY is the critical initiator of
testis development is a gene located immediately adjacent to the pseudo autosomal
region of the short arm of the Y chromosome, designated SRY for Sex-determining
Region-Y chromosome. SRY has been identified as the testis-determining factor
(TDF), the key gene responsible for testis development in XY embryos. Once the
gonads are formed, the pivotal event in male sexual differentiation is expression
of the SRY gene. SRY is necessary and sufficient
to initiate the male development cascade 40.  In the absence of Sry, or if Sry is
expressed at insufficient levels, the support cell precursors differentiate as
granulosa cells, thus initiating the ovarian pathway 26.  The molecular mechanisms upstream and downstream
regulations of Sry are not well understood. Trivosian et al. 26, demonstrated that the transcription factor GATA4 and its
co-factor FOG2 are required for gonadal differentiation.

The physiological target genes for
SRY/Sry remain unknown, but potential candidates including Sox9,SF-1, DMRT1, GATA-4, Dhh,
and testatin, are up-regulated during testicular differentiation 28, 41. Genes SF-1 and WT1
play key roles in both sexes in the development of the indifferent gonad. SF-1
and WT1, together with SOX9, and GATA4, cooperate to regulate the expression of
target genes (e.g., AMH, Insl3, and the steroid hydroxylases) that mediate male
sexual differentiation. DAX-1, a negative regulator of the male developmental
pathway, inhibits the activation of critical target genes by SF-1, WT1, SOX9,
and GATA4.

GATA4 and FOG2 and their physical interaction are
required for normal gonadal development, WT1 and SF1, which are expressed
prior to SRY and necessary for gonad development in both sexes.
The tissue distribution of DAX-1 (adrenal cortex, gonads, hypothalamus, and
pituitary) is the same as that of another orphan nuclear receptor,
steroidogenic factor 1 (SF-1) that is required for development of the adrenal
glands and gonads.

Dmrt is also one of
the gene involved in testes differentiation in higher and lower vertebrates, DMRT1 gene
encodes a zinc finger–like DNA-binding protein and is expressed very early in a
sex-specific manner in male gonads of all the classes of vertebrates,
regardless of the sex-determining mechanism (chromosomal or environmental). In
mice, Dmrt1 is expressed in genital ridges of both sexes and
then becomes testis specific at the end of the sex-determining stage. In
testis, Dmrt1 is expressed in germ cells and Sertoli cells 42, 43
have recently shown that Dmrt1 is required for testis but not
ovarian differentiation.

Hormones produced by the testis trigger the
developmental process that leads to the male phenotypic sexual differentiation 21,
independence of gonads and gonadal hormones in normal female birth. There are
three essential hormones secreted by the testes, androgens, MIS, and Insl3. These
hormones secreted by testes called testicular hormones in male-specific
development of the bipotential reproductive system. Mullerian-inhibiting substance
(MIS), also named Mullerian-inhibiting factor (MIF) or anti-Mulerian hormone (AMH), produced by fetal Sertoli cells induces
regression of the Mullerian ducts.

Sex
differentiation

Among all vertebrates
fishes exhibit  greatest diversity in
species and act as a relevant model for genetic and evolutionary studies. Hermaphroditism
is also a common feature of several fish species. It was observed that few
genes have been linked in the process of sex determination or differentiation as
in zebra fish. The genes Fushi Tarazu factor-1 (FTZ-F1) play crucial role as they were involved in
regulating interrenal development  and  also showed expression patterns similar with
reproductive tissue differentiation and function.  For most of the fish species the differentiation of gonad into
a testis or an ovary is strongly influenced by hormonal, environmental or social factors. In the
studies it was observed that they can be sex reversed by exposure to estrogens,
suggesting that the estrogen levels play a crucial role during sex
differentiation. The Cyp19 gene product aromatase usually converts testosterone
into 17 beta-estradiol but when inhibited leads to male to female sex reversal.
FTZ-F1 genes are strongly linked to steroid biosynthesis as well as regulatory
region of Cyp19 contains binding sites for FTZ-F1 genes, further linking FTZ-F1
in this sex differentiation process 44. The Dax-1 gene has however
been identified in the Nile Tilapia 18, 45, suggesting that other
fish species may also have Dax-1 homologues that play a role in sex
differentiation. Several HMG-box containing genes, Sox-genes, have been
identified in fish 46-48. It was found that HMG-Box
cis element has been identified in gene promoter of fushi tarazu factor
1a (ff1a) 49. Sox9a was also able to
bind specifically at this site in vitro indicating that a
regulatory connection between Sox9a and ff1a is present. It was also observed
that Sox9 alone does not direct sex determination and differentiation in zebra fish.
AMH inhibits the expression of aromatase in developing gonads 50 therefore negatively
modulates the differentiation and function of Leydig cells by down regulating
several enzymes involved in the steroidogenic pathway 51. WT1 has been shown
to be expressed in the intermediate mesoderm prior to and during renal tissue
differentiation 52. It is also essential for
the steroidogenic interrenal development together with ff1b 53, 54. WT1 is thereby an
important factor in the early events during development of gonadal primordium.
Dmrt1 also play an important role in testis determination in teleosts, since
alteration of aromatase levels during sex differentiation can cause sex
reversals 55. These Dmrt is usually
regulated by GATA factors. GATA factors processes the binding sites of cyp19
gene promoter that indicates its role in regulating aromatase expression 56, 57.

Signal
Transduction pathways

Steroid hormone biosynthesis normally occurring in
steroidogenic cells is regulated by trophic hormone activation of protein
kinase A (PKA) signaling pathways. It was observed that this trophic hormone stimulation
results in the activation of G proteins which stimulate adenylatecyclase
activity that produces increased intracellular levels of cAMP and PKA in mouse 58, 59. During this signaling, many proteins such as
cholesteryl ester hydrolase gets phosphorylated along with transcription
factors such as steroidogenic factor 1, GATA-4 and cAMP response-element binding
protein(CREB)/cAMP response element modulator that activates the genes such as
StAR involved in steroidogenesis. 60, 61. However, there was evidence that regulation of
steroidogenesis can also modulated by signaling pathways without involving
cAMP. These include growth factors, steroidogenic inducing protein (SIP), macrophage
derived factors, chloride ions and calcium messenger systems 62. Several evidences show that growth factors such as
epidermal growth factor (EGF) and insulin dependent growth factor (IGF-1),
stimulate steroid synthesis without altering cAMP levels 63-65. It was observed that EGF and IGF-1 uses the
MAPK/ERK pathways for steroid synthesis and StAR expression 65, 66. IGF-1 phosphorylated CREB/activating transcription
factor-1 and activator protein-1 family member c Jun/Jun D were also found to
be involved in steroidogenesis.

Role of Gonadotropins

Gonadotropins are released from the pituitary gland
and play an important role in steroidogenesis. They have shown to activate both
p58 and ERK1/2 MAPKs that result in varying effects on StAR expression and
steroidogenesis in ovarian granulosa cells 67-70. Apart from this it was also observed that
inhibition of p38 decreases both P450arom and estradiol synthesis, and these
events were tightly correlated with the liver receptor homolog-1 and DAX-11
expression demonstrating that p38 targets these transcription factors in
regulating steroidogenesis. Other signaling pathways such as ERK/BMK1, JNK/SAPK
also regulate in steroidogenesis. Gonadotropin
releasing hormone (GnRH) is widely expressed outside of the classical brain in
areas of the olfactory brain, telencephalon, preoptic area and midbrain. GnRH, is best known in vertebrates for its
expression in neurons and its role in stimulating the release of gonadotropins
from the pituitary gland. In some earlier studies analysis of the genome
confirmed showing that many teleosts have three forms of GnRH each encoding by
separate gene. Ovary and testis are major sites of interest because they
express both GnRH and GnRH receptors.  It
was also revealed  that peripheral GnRH
production is important in the early development and maturation of the gonads
of fish, but are not required at least in large quantities when the fish have
reached maturity even though the GnRH genes continue to be expressed.

Conclusion:

 

Adrenal
development and gonadal development are two most fundamental biological
processes. Various signaling
mechanisms and transcription factors and several pathways are involved at
genomic level in common to adrenal and gonadal development in fishes and lower
vertebrates during steroid biosynthesis. Sex determinating factors such as FTZ-F1 genes, Sox9a,
GATA4, Dmrt1 and AMH, are involved in the differentiation of gonads. The
studies also summarized the role of signalling pathway involving ERK1/2,
JNK/SAPK, and ERK5 MAPKs in regulation of StAR expression during
steroidogenesis in different steroidogenic tissues. This emphazise various
genetic events are involved at the early and late development of the process of
gonadal development in steroidogenic tissues.