Abstract plays very important role in gonad


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 transcription
factors and important signaling pathways 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
which acts as critical regulator for the development and homeostasis of the
adrenal cortex and gonads. SF-1 is a nuclear receptor that is expressed
exclusively in the steroidogenic tissues of the hypothalamic
pituitary-adrenal/gonadal axis. Protein kinase such as  Mitogen-activated protein kinases which are
serine/threonine kinases are majorly involved in the expression of the crucial
protein Steroidogenic acute regulatory protein in 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 StAR ,Fushi
tarazu factor-1, and cytochrome P450-11A in reproduction. 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.

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








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, homeostasis ,maintenance, and
reproduction. In adult vertebrates, these steroids are produced at appropriate
times in steroid producing cells termed as gonads. These cells express various  group of steroidogenic enzyme genes that
usually modify cholesterol and its derivatives 1. These steroids play a different role even
though they are chemically identical in major of the vertebrates.



Steroid hormones usually
produced by steroidogenic cells, that include ovary, testis and brain that are required for reproduction  and
bodily homeostasis. 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 through cytochrome P450 cholesterol side chain cleavage enzyme and transferred
from outer to inner mitochondrial membrane. This is regulated by the  Steroidogenic acute regulatory (StAR) protein
the one which regulates the true
rate-limiting step in steroid biosynthesis 4. This central role of StAR protein
regulation was proven by two observations 
5, 6. Sex differentiation is initiated and controlled by gonadal steroid
hormones. These hormones perform different
functions during development  into sex
organs. This is regulated by the expression of proteins  produced by certains signalling pathway such
as  cAMP-dependent mechanism in
the adrenal and gonads.




Gonadal development

in vertebrates depends on function  of
two gametes that is sperm and eggs that develop into different organs i.e , the
testis and the ovary. These two organs are grossly different  and composed of common cell lineages,
interstitial cells, supporting cells and germ cells. Each mature ovary composed
of an ovarian cavity, stromal compartment and germinal epithelium. In fishes
such as teleosts, the germ line stem cells resides in germinal epithelium and
mitotically produce active oogonia . This structure is similar to that of
surface epithelium present in mammals. The steroid hormones are produced by
follicles which helps the oocytes to grow that reside in stromal compartment.
In case of testis,the spermatogenesis starts from the germ line stem cells till
the sperm production that usually occurs in tubules , and the interstitial
tissue producing these hormones resides between these structures.


Factors involved during gonadal development

Germ cells that not have been reached to the gonad
at their early stages are termed as primordial germ cells (PGC). These
primordial germ cells were identified morphologically and specified  functionally by the distribution of
cytoplasmic determinants that includes RNA-binding proteins such as VASA, NANOS
and TUDOR and that were restricted  as granular-like
structures or nuage 7. This similarity have been previously found in some
lower vertebrates and Drosophila. Nanos3 was found to be the earliest marker in
some fish such as medaka, and by using this, PGCs were identified first during early
gastrulation stage 8. Three mechanically 
distinct modes were observed for migration of PGCs 8, 9. In the early gastrulation stage the chemokine
receptor CXCR4 and its ligand, SDF1A were involved in  migration towards the marginal zone. During
the late gastrulation and early somitogenesis stages, it is dependent on the
convergent movement of the somatic cells. Later during the bilateral alignment
the primordial stem cells governs the interactions between CXCR4 and SDF1B,
which resume its activity and migrates towards the posterior end of lateral
plate mesoderm, where the somatic precursors of gonads arise10.


In teleosts, granulosa cells and sertoli
cells shared a common origin, specifically supporting cells that express sox9b
gene in bipotential gonadal primordia. Both supporting cells showed the
expression  of sox9b 11, an observation in contrast to the situation
of mammals, where only sox9 was expressed in Sertoli cells that are required
for testicular development 12, 13. Sox9 along with  (SF-1) steroidogenic factor 1, regulates the transcription of anti-Mullerian hormone (AMH) gene.

The SOX-9 gene
plays a very  important role in male
sexual development. These cells that express sox9b start  to express dmrt1, indicating  the differentiation to Sertoli cells. In the
early stage of oogenesis, from germ line stem cells to an early diplotene
oocytes it proceeds to form cradle structures. Subsequently these diplotene
oocytes that surrounds the somatic cells exit from the germinal cradle and will
recruit theca cells to form the follicles. Theca
cell layer formation is an important physiological event that occurs during
early follicular development.

 Finally theese follicles that are presnt in
stromal compartment possess two layers of somatic cells termed as inner
granulosa cells  and outer theca cells. Granulosa and theca cells of the ovary act as a
support  to germ cells within the
developing follicle.  During this process,
 the granulosa cells will lose the
expression of  sox9b while foxl2, that is
a marker of granulosa cells, gets activated 11, 14. This suggests that granulosa cells which
are originated from the sox9b -expressing cells, both follicular formation and
oocyte will exit from germinal cradles and depends 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.
Sox9 expression was intensively observed in the sertoli cells of testis that
are located more distantly in lobules. In ovary, sox9b was expressed in the
germinal cradles representing niche regions. The most important function of
sox9b-expressing cells is to maintain the stem type germ cells during early
stages of gametogenesis. In some studies, they have observed the functions of dmrt1 and amh during male germ cell development by creating the mutants with
the use of Crispr/cas9 technology in zebrafish. Both female and male amh mutants developed hypertrophic
gonads because of uncontrolled proliferation as well as impaired
differentiation of germ cells whereas amh
mutant zebrafish showed female-based sex ratio.

 It was also found that amh  helps
in controlling the balance between proliferation as well as  differentiation of male germ cells. But
dmrt1 is usually required for self-renewal,  maintenance as well as during differentiation
of male germ cells. During the process of  testicular development, the genes which are required
for the production of steroid hormones such as  p450scc/cyp11a1 and hsd3b, starts to express in Leydig cells that are located in the
marginal regions of lobules. These genes have been expressed in  ftz-f1 -expressing cells during the testicular
development 17. It suggests that ftzf1 regulates various
sets of steroidogenic genes and that are involved in the  androgen production that may normally occur in
a single cell lineage of ftz-f1 expressing cells. In certain fishes such as rainbow
trout, immunohistochemical analysis revealed that HSD3B, P450c17/CYP17A1,
P45011B/CYP11B, , P450scc/ CYP11A1 and P450c17/CYP17A1 were all co-localized in
the  interstitial Leydig cells 18.

two types of theca cells were seem to be present in certain fish such as medaka
during the ovarian development. Some fishes express only aromatase. P450c17 and
aromatase were expressed exclusively in transgenic medaka fish by expression
analysis using aromatase-reporter 19. Alternatively, the two types of
theca cells have been to share a common precursor that normally expressed  the ftz-f1 gene, by which generating an
offspring capable of either by maintaining or down regulating ftz-f1 expression
and initiating the aromatase expression 20.


vertebrates, adrenal glands consists of two distinct parts, outer adrenal
cortex and inner adrenal medulla. The outer adrenal cortex secretes three major
hormones referred as glucocorticoids, mineralocorticoids 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 the embryonic mesonephros with the
thickening of ventrolateral surface which is termed as genital ridge. In the
classic experiment of Jost 21  female differentiation has been observed
irrespective of the genetic sex in the absence of testicular hormones. In
certain expression analysis , it was observed that GATA4 was involved in sex
determination 22,
23. The role of GATA4 in the
regulation of gene expression was also observed by in vitro analysis data in the gonads downstream of  Sry,
including inhibin ? , Mis, and steroidogenic acute regulatory
protein(StAR) 24,

vertebrates, the steroid hormones of the adrenal gland  show a different adaptive responses during
the internal and external environmental stress conditions. Sex determination
region of Y chromosome (SRY) gene required to initiate the signaling for male
gonadal differentiation. Many other genes involved in gonadogenesis are GATA4
and FOG2 26. In mammals, gonads arise from
bilateral genital ridge in both sexes that usually develop as ovaries or testes
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 essential task in mammalian gonadal
development. Absence or presence of Y-chromosome at the embryonic fertilization
level, gonads distinguish in to either ovaries or testis. Up to now there are
four most important genes are known to be essential for progression of bipotential gonads,
orphan nuclear receptor Steroidogenic factor-1 (SF1 or Ftz-F1) 37 (b)
Wilms tumor associated gene (WT1;38 (c)
Lhx1 and (d) Lhx9 39. In
Keith Parker
experiments, SRY (Sex-determining Region-Y chromosome), SF-1, (WT1), GATA4, and
SOX9, were promising genes and interactions along with these genes play major
role in gonadal development. In that SRY
significant initiator of testis development, it is located adjacent to the
pseudoautosomal region of the short arm of the Y chromosome. Usually SRY gene
recognized as the testis-determining factor (TDF), an important gene
accountable for testis development in XY embryos. The critical event in male sexual
differentiation is expression of the SRY gene after that gonad
is formed. SRY  gene is an essential and adequate to start the male
development cascade 40.   Absence or insufficient levels
SRY gene expression, supported cell precursors differentiate into granulosa,
consequently the ovarian pathway in steps forward 26. Molecular level gene expressions and
regulations are still unclear in many ways. Trivosian et al. 26, demonstrated transcription factor GATA4
and its co-factor FOG2 were also play in gonadal development.

Genes like,
SF-1 and WT1 play vital in either sexes
in the gonad differentiation in many vertebrates and lower vertebrates. SF-1
and WT1, jointly with GATA4 and SOX9 assisted in regulation of target genes in
male gonadal development. Genes that are Insl3 and the steroid hydroxylase were
influenced by SF-1 and WT-1. Robert et al., demonstrated that male negative
regulator gene DAX-1 also involves in the differentiation mechanism. DAX-1 down
regulates the key factor genes by WT-1, SF-1, SOX9, and GATA4. The other way target
gene for SRY gene is still unclear. 
Other genes like DMRT1, Dhh
and teststin were up regulated during
gonadal differentiation and testes development 28, 41. 

Dmrt, play a significant role in gonadal development and
testes differentiation in many higher vertebrates as well as lower vertebrates.
Expression of Dmrt1 gene is not independent of sex differentiation and early
expression in testes determination. In mice, Dmrt1 is
expressed in genital ridges of both sexes and then becomes testis specific at
the end of the sex-determining stage. Raymond, C.S., et al., demonstrated that Dmrt1 is up regulated in mice male
germinal and Sertoli cells 42, 43. It showed that that Dmrt1 paly an
important role in testis development but not ovarian.

Hormones also play
significant role in gonadal development and sex differentiation in many
vertebrates and few of lower vertebrates, especially testicular hormones
involved in male phenotypic sexual differentiation 21. Androgens, MIS (Mullerian-inhibiting substance) and
Insl3 are the three important hormones from testes, called testicular hormones
significant role in bipotential reproductive system.

Sex differentiation

all vertebrates’ fishes, exhibit greatest diversity in species and act as a
relevant model for genetic and evolutionary studies. A common feature observed in most of several fish species is
hermaphroditism. In most of the lower invertebrates like fishes, sexual
determination and sexual differentiation extremely influenced by hormones,
biotic and abiotic factors like environmental factors temperarature, quality of
water etc. Many genes that have been linked in the development of sex
determination as observed in like zebra fish model. The genes Fushi-Tarazu factor-1 (FTZ-F1) play crucial role as they were
involved in regulation and expression of interregnal sexual development and
differentiation. FTZ-F1 genes involved in the steroid biosynthesis and
regulatory region of Cyp19 contains binding sites for FTZ-F1 genes that are
involved in sex differentiation process. In certain studies in zebra fish it
was observed gonadal sex differentiation occurred due to the compounds that
interfere the with Cyp19 gene expression. If the of inhibition testosterone
into17 beta-estradiol by Cyp19 gene product aromatase leads to male to female
sex. Kobayashi, T., et al., 18, 44, demonstrated that Dax-1 gene from Nile
Tilapia involved in gonadal development, Dax-1 gene regulation in other fish
species is still unknown.

 Transcription factors like DMRT1 have significant role in gonadal
development especially in testis development 45.
HMG-box (high mobility group box, a DNA binding protein box) containing genes,
like Sox-genes and those transcription factors include dmy, sd4BP/SF1, sox9,
dax1 and wt1 that plays crucial role in sex determination and differentiation 46-48.
Von Hofsten, J., et al was found that HMG-Box, cis element is gene promoter
of fushi tarazu factor 1a (ff1a) 49. It
was observed that Sox9 independently not involved in direct sex determination
in zebra fish. Studies showed that Anti Mullerian Hormone down regulates the
expression of aromatase in the developing gonads 50
therefore negative controlling of Leydig cells by down regulating enzymes that
are implicated in the steroidogenic pathway 51. Other
way the WT-1 gene combination with ff1b factor play crucial role in
steroidogenic interregnal development 52. 53, 54.
Dmrt1 also play significant role in gonadal and testis determination in teleost
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 occurred
by activation of proteinkinase A (PKA) in trophic hormones. It was observed
that this trophic hormone stimulation results in the activation of G proteins
which stimulate adenylatecyclase activity that produces high 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 SF-1, GATA-4, (CREB)/cAMP and 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 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 StAR expression 65, 66. IGF-1 phosphorylated CREB/activating
transcription factor-1 and activator protein-1 family member cJun/JunD 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 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.



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 signaling 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.