Abstract

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
Wnt, Sonic hedgehog, 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 proteins that are
encoded by amh, dax1 and cyp19a1 which play important roles 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 the absolute requirement for development,
maintenance, homeostasis and reproduction. In adult vertebrates, these steroids
are produced at appropriate times in specialized steroid producing cells called
gonads. These cells express an group of steroidogenic enzyme genes whose
products modify its cholesterol and derivatives 1. 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. Although many
steroids are chemically identical in all major vertebrate classes, the role of
these steroids may differ. However, unique steroid hormones have evolved in
some vertebrate classes, especially amongst fishes, to fulfil particular
functions.

 

Sex
differentiation is initiated and controlled by gonadal steroid hormones. These
hormones performed different functions to permanently differentiate into sex
organs during development.Steroid hormones are synthesized in 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 usually occur on
order of minutes and hours, respectively. Chronic regulation of steroidogenesis
by LH or ACTH occurs at the level of gene transcription 3. The acute response is initiated by the
mobilization and delivery of the substrate for all steroid hormone biosynthesis. Cholesterol, from the outer to
the inner mitochondrial membrane, where, it is metabolized to the pregnenolone
by the cytochrome P450 cholesterol side chain cleavage enzyme (p450scc).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
observationsby robust steroid hormone synthesis followed co-transfection of
StAR and the cholesterol side-chain cleavage system into nonsteroidogenic COS-1
cells 4, 5. Second, patients with StAR mutations have
congenital lipoid adrenal hyperplasia, in which all adrenal and gonadal
steroidogenesis was disrupted 5, 6. The expression of this protein is
predominantly regulated by cAMP-dependent mechanism in the adrenal and gonads.

Gonadal development

Vertebrate reproduction depends on the function of
two distinct gametes, sperm and eggs, which develop into different organs, the
testis and the ovary. These are composed of germ cells, supporting cells and
interstitial cells.The ovary and the testis are essential for gametogenesis. A
mature ovary consists of an ovarian cavity, the germinal or surface epithelium,
and the stromal compartment. In fishes such as teleosts, germ line stem cells
and mitotically active oogonia reside in the germinal epithelium. This
structure is similar to the surface epithelium in mammals. Follicles are
present in the stromal compartment where oocytes grow and steroid hormones are
produced. In the testis, spermatogenesis from germ line stem cells to sperm
occurs in tubules or lobules, and the interstitial tissue that produce steroid
hormones resides between these structures. The two reproductive organs are
grossly different, but they both are composed of developmentally common cell
lineages, supporting cells, interstitial cells and germ cells. Germ cells,
critical for conveying the genetic information to the next generation, are very
special in that they are segregated from other cells at a very early
developmental stage when major positional information is being established, and
migrate into the future gonadal area.

 Early stage
germ cells that have not reached the gonad are called primordial germ cells
(PGCs). PGCs in teleosts are morphologically identified and functionally
specified by the allocation of cytoplasmic determinants that includes
RNA-binding proteins NANOS, VASA and TUDOR which are localized on granule-like
structures or nuage 7. This finding was similar to which has been
observed in other lower vertebrates and Drosophila. In some fish such as
medaka, nanos3 was found to be the earliest marker so far examined for germ
cells, and using this marker, PGCs was first identified at an early
gastrulation stage 8. The migration of PGCs consists of at least three
mechanically distinct modes 8, 9. First, at an early gastrulation stage, PGCs
actively migrate towards the marginal zone, a process which depends on the
chemokine receptor CXCR4 and its ligand, SDF1A. Second, at the late
gastrulation and early somitogenesis stages, PGC movement depends on the
convergent movement of somatic cells. Third, 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 were
suggested to share a common origin, namely, the supporting cells expressing the
sox9b gene in the bipotential gonadal primordia. Interestingly, sox9b
expression is found in both XX and XY supporting cells 11,
an observation that is in sharp contrast to the situation in mammals, where
sox9 is only expressed in Sertoli cells and is both required and sufficient for
testicular development 12, 13.
The sox9b expressing cells begin to express dmrt1, an indication of
differentiation into Sertoli cells, and are mainly located inside the lobules
of the adult testis as gonadal primordia develop into ovarian structures, the
sox9b -expressing cells constitute germline stem cell niches, or germinal
cradles. As mentioned above, early oogenesis, from germ line stem cells to
early diplotene oocytes, proceeds in the cradles. Subsequently the diplotene
oocytes and the surrounding somatic cells exit from the germinal cradle and recruit
theca cells to form follicles. As a result, the follicles in the stromal
compartment have 2 layers of somatic cells, outer theca cells and inner
granulosa cells. During this step, the granulosa cells lose sox9b expression
while foxl2, a marker of granulosa cells, is activated 11, 14,
suggesting 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.
Histological analysis of other teleost fish also supports this observation 15, 16.
Interestingly, in the testis, sox9b expression is very intense in the Sertoli
cells located most distally in the lobules. The distal region is predominately
occupied by the most undifferentiated type of germ cells, type Aspermatogonia.
In the ovary, sox9b is expressed in the germinal cradles representing niche
regions. Collectively, these observations suggest that the common function of
sox9b -expressing cells may be the maintenance of stem-type germ cells during
early gametogenesis.

During testicular development, steroidogenic genes
required for the production of steroid hormone(s), e.g. p450scc/cyp11a1 and
hsd3b, begin to be expressed in
presumptive Leydig cells located in the marginal regions of the lobule
Steroidogenic genes are expressed in ftz-f1 -expressing cells during testicular
development 17.
This 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, P450scc/ CYP11A1, HSD3B, and P450c17/CYP17A1 were all
co-localized in interstitial Leydig cells 18.
By contrast, during ovarian development, at least 2 types of theca cells seem
to be present in medaka. Some fishes express only aromatase. Expression
analysis using aromatase -reporter transgenic medaka fish has revealed p450c17
and aromatase were exclusively expressed 19.
These results suggest that theca cells may be derived from at least two distinct
populations in medaka. 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 by24, 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 gonodogenesis are GATA4 and FOG2 26.
Mammalian gonads arise in both sexes from bilateral genital ridge which 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 emergingmodels that
suggest complex interactions amongthese genes in gonadal development (Keith
Parker). SRY isthe critical initiator of
testis development is a gene located immediately adjacent to the
pseudoautosomal 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 
was necessary and sufficient to initiate the male development cascade 40.  In the absence of Sry, or if it is expressed at
insufficient levels, the support cell precursors differentiate intogranulosa
cells, thus initiating the ovarian pathway 26.  The molecular mechanisms and the regulations of Sry were
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 (for
review, see28, 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 programme that leads to the male phenotypic sexual
differentiation21,
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.
Müllerian-inhibiting substance (MIS), also named Müllerian-inhibiting factor
(MIF) or anti-Müllerian hormone (AMH), produced by fetal Sertoli cells induces
regression of the Müllerian ducts.

 

 

Sex
differentiation

Sex is usually defined by
the presence or absence of the sex specific chromosome ass
in case of mammals. 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 in zebra fish. The genes FTZ-F1 were
crucial as they were involved in regulating interrenal development thereby
steroid biosynthesis, as well as they also showed expression patterns similar
with reproductive tissue differentiation and function. It was observed that it
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 it
inhibition leads to male to female sex reversal. FTZ-F1 genes are strongly
linked to steroid biosynthesis and also regulatory region of Cyp19 that contain
binding sites for FTZ-F1 genes, further linking FTZ-F1 in sex differentiation
process 44.Since now no
sex-linked genes have been found fish or lower vertebrates, allelic variants
and dosage effects of autosomal genes, such as the Fushi Tarazu factor-1
(FTZ-F1) genes, WT-1, SRY HMG box related gene 9 (Sox9), Anti-Mullerian Hormone (AMH), GATA4 (a zinc
finger transcription factor) and double sex-mab 3 related gene (Dmrt1) might be
involved in determining sex and directing gonad development. 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 fish46-48. It was found that HMG-Box
-cis element has been identified in gene promoter of fushitarazu factor
1a (ff1a) 49. Sox9a was also able to
bind specifically at this site in vitro (von Hofsten et
al., unpublished) indicating that a regulatory connection between Sox9a
and ff1a is present. It was also found that Sox9 alone does not direct sex
determination and differentiation in zebrafish.  Fish usually lack Mullerian
ducts, but other AMH functions may be important for gonad development. AMH
inhibits the expression of aromatase in developing gonads50 therefore negatively
modulates the differentiation and function of Leydigcells by down regulating
several enzymes involved in the steroidogenic pathway51. WT1 has been shown
to be expressed in the intermediate mesoderm prior to and during renal tissue
differentiation52. 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 expression56, 57.

Signal
Transduction pathways

Steroid hormone biosynthesis 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 PKAin mouse58, 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 wasevidence 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
systems62.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 levels63-65. It was observed that EGF and IGF-1 uses the
MAPK/ERK pathways for steroid synthesis and StARexpression65, 66. IGF-1 phosphorylated CREB/activating transcription
factor-1 and activator protein-1 family member cJun/JunDwere also found to be
involved in steroidogenesis.

Role of Gonadotropins and
Gonadaotropin releasing hormone

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 cells67-70. 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 is 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. Gonads are originated from thickening of the ventrolateral surface
of the embryonic mesonephros called the genital ridge. 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 signaling pathway involving ERK1/2, JNK/SAPK, and ERK5 MAPKs in regulation
of StAR expression during steroidogenesis in different steroidogenic tissues.
This emphasize various genetic events are involved at the early and late
development of the process of gonadal development in steroidogenic tissues.