Abstract in chordates and arthropods. Fishes, for

AbstractIn general adrenal glands and gonads play a veryimportant role in sex differentiation and steroidogenesis. These two aresystems are closely related as they share a common region of origin i.e.mesoderm and both are involved in steroidogenesis.

Various biological eventsoccur during adrenal and gonadal steroidogenesis. In this article important signaling pathways and transcription factorsinvolved in regulation of steroidogenesis and adrenal growth have beensummarized. Present review illustrates various novel signaling pathways such Sonichedgehog ,Wnt, Notch, ?-catenin involved in adrenal gland morphology and itsfunctions that are deeply interconnected. Certain nuclear receptor such asSteroidogenic Factor-1 acts as critical regulator of development andhomeostasis of the adrenal cortex and gonads. SF-1 is a nuclear receptoralmost exclusively expressed in the steroidogenic tissues of the hypothalamicpituitary-adrenal/gonadal axis. Mitogen-activated protein kinases areserine/threonine kinases involved in the expression of the Steroidogenic acuteregulatory protein and steroidogenesis. Characterization  of certain proteins that are encoded by dax1, amh, and cyp19a1 whichplays very important role in gonad differentiation and to evaluate the relationbetween gonadal expression of Fushitarazu factor-1, StAR and cytochrome P450-11A in reproductive maturation process.

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This article aimed to describe the varioussignaling mechanisms and novel transcription factors involved at genomic levelin 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 proteinkinases;       Introduction Steroidogenesis involves the synthesis of steroidhormones that are derivatives of cholesterol which are synthesized by varioustissues, most prominently the adrenalgland andgonads. These are usually found in chordates and arthropods. Fishes, for example teleosts, produce several types ofbioactive gonadal steroids, including progestogens, estrogens, androgens andvarious derivatives of steroids. Steroids are required for development,maintenance, homeostasis and reproduction.

Steroids direct the development of germ cellsand 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 areproduced at appropriate times in specialized steroid producing cells called gonads. Thesecells express a group of steroidogenic enzyme genes whose products modify itscholesterol and derivatives 1. Although many steroids areidentical chemically in all major vertebrate classes, the role of thesesteroids may differ. However steroid hormones have evolved in some vertebrateclasses, especially amongst fishes, to fulfil particular functions. Steroidhormones 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 totrophic hormones and other external stimuli with rapid surge in steroid hormoneproduction 2. Theacute and chronic regulation of steroidogenesis is controlled by trophichormones that normally occur in  order of minutes and hours, respectively.

Chronic regulation of steroidogenesis by LH or ACTH occurs at the level of genetranscription 3. Cholesterolis metabolized to pregnenolone by  cytochromeP450 cholesterol side chain cleavageenzyme (p450scc) and transferred from the outer to the innermitochondrial membrane. The Steroidogenic acute regulatory (StAR) protein isthe one which regulates the truerate-limiting step in steroid biosynthesis, i.

e. the delivery of cholesterolfrom the outer to the inner mitochondrial membrane 4. The central role of StAR was proven by twoobservations by robust steroid hormone synthesis followed co-transfection ofStAR and the cholesterol side-chain cleavage system into nonsteroidogenic COS-1cells 4, 5. In other patients with StAR mutations havecongenital lipoid adrenal hyperplasia, whereby all adrenal and gonadalsteroidogenesis was distrupted 5, 6. Sex differentiation is initiated and controlled by gonadal steroidhormones. These hormones performdifferent functions and permanently differentiated into sex organs duringdevelopment. The expression of this protein is predominantly regulatedby cAMP-dependent mechanism in the adrenal and gonads.Gonadal developmentReproduction in vertebrates depends on function  two distinct gametes, sperm and eggs thatdevelop 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, supportingcells, interstitial cells and germ cells. A mature ovary consists an ovarian cavity, germinalepithelium and stromal compartment. In fishes such as teleosts, the germ linestem cells are mitotically active oogonia that reside in germinal epithelium.Their structure was similar to surface epithelium in mammals. Steroid hormonesare produced by the follicles that are present in the stromal compartment wherethe oocytes grow.

In the testis,spermatogenesis starting from the germ line stem cells to sperm productionoccurs in tubules or lobules, and the interstitial tissue that produce steroidhormones resides between these structures. The germ cells at their early stages that have notreached the gonad are termed as primordial germ cells(PGC). These PGCs wereidentified morphologically and functionally specified by the distribution ofcytoplasmic determinants that includes RNA-binding proteins NANOS, TUDOR andVASA which were localized on granule-like structures or nuage 7. This similarity have been previously observed inother lower vertebrates and Drosophila.

Nanos3 was found to be the earliestmarker in some fish such as medaka, and using this marker, PGCs were firstidentified at an early gastrulation stage 8. Three mechanically distinct modes were observed for migration of PGCs 8, 9. In the early gastrulation stage migration towardsmarginal zone depends on the chemokine receptor CXCR4 and its ligand, SDF1A.Second, at the late gastrulation and early somitogenesis stages, it depends onthe convergent movement of somatic cells. After aligning bilaterally, PGCs,governed by interactions between CXCR4 and SDF1B, resume active and directionalmigration towards the posterior end of the lateral plate mesoderm, wheregonadal somatic precursors arise 10.In teleosts,Sertoli and granulosa cells  share acommon origin, namely, the supporting cells expressing the sox9b gene in thebipotential gonadal primordia. Normallysox9b was found to be express inboth supporting cells 11, an observation that is contrast to thesituation in mammals, where sox9 is only expressed in Sertoli cells and is bothrequired testicular development 12, 13. Sox9 along with  steroidogenic factor1, regulates transcription of the anti-Mullerianhormone (AMH) gene.

SOX-9 alsoplays an important role in male sexual development. The sox9b expressingcells begin to express dmrt1, indicating the differentiation into Sertolicells. In the early oogenesis, from germ line stem cells to early diploteneoocytes it proceeds in the cradles. Subsequently the diplotene oocytessurrounding somatic cells exit from the germinal cradle and recruit theca cellsto form follicles. Theca cell layer formation isan important physiological event that occur during early follicular development.Finally the follicles in the stromal compartment have two layers of somaticcells, 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 granulosacells lose sox9b expression while foxl2, a marker of granulosa cells, isactivated 11, 14. This suggests that granulosa cellsoriginate from the sox9b -expressing cells. Both follicular formation andoocyte exit from germinal cradles appears to depend upon a series of successiveprocesses 14 which is also observed in other teleost fishby histological analysis 15, 16. In somestudies it indicated that sox9b and amh,that are involved in testicular differentiation in vertebrates, were implicatedin testicular formation and spermatogenesis during the sex change as well.Interestingly, in the testis, sox9b expression is very intense in the Sertolicells located most distally in the lobules. In the ovary, sox9b is expressed inthe germinal cradles representing niche regions.The common function of sox9b-expressing cells is for the maintenance of stem-type germ cells during earlygametogenesis.

In some studies, they examinedthe roles of amh and dmrt1 in male germcell development by generating theirmutants with Crispr/Cas9 technology in zebrafish.  Amh mutantzebrafish displayed a female-biased sex ratio, and both male and female amh mutantsdeveloped hypertrophic gonads due to uncontrolled proliferation and impaireddifferentiation of germ cells. It was also found that amh  acts asa guardian to control the balance between proliferation and differentiation ofmale 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 steroidhormone(s), e.g. p450scc/cyp11a1 and hsd3b,begin to be express in presumptive Leydig cells located in the marginal regionsof the lobule. These genes are expressed in  ftz-f1 -expressing cells during testiculardevelopment 17.

It suggests that ftzf1 regulates a set of steroidogenic genes and that androgenproduction may occur in a single cell lineage of ftz-f1 – expressing cells. Inrainbow trout, immunohistochemical analysis also revealed that P45011B/CYP11B,HSD3B, P450scc/ CYP11A1 and P450c17/CYP17A1 were all co-localized ininterstitial Leydig cells 18.In  contrast, during ovarian development,at least two types of theca cells seem to be present in medaka.

Some fishesexpress only aromatase. It has also revealed that p450c17 and aromatase wereexclusively expressed in transgenic medaka fish by expression analysis usingaromatase-reporter 19.Alternatively, the two types of theca cells may share a common precursor thatexpresses the ftz-f1 gene, and that generates offspring capable of eithermaintaining or down regulating ftz-f1 expression and initiating aromataseexpression 20.

AdrenaldevelopmentIn vertebrates, adrenal glands composed of twodistinct parts, outer adrenal cortex and inner adrenal medulla. Adrenal cortexsecretes three major hormones glucocorticoids, mineralocarticoids and adrenalandrogens. Adrenal androgens involved in the gender differentiation in humanbeings mainly dehydroepiandrosterone (DHEA) and testosterone. Cellularorganization of gonads is similar in all vertebrates, based on differentprogression can trigger bipotential gonads, forms either ovaries or testis.Gonads are originated from thickening of the ventrolateral surface of theembryonic mesonephros called the genital ridge. The classic experiment of Jost 21demonstrated that female differentiation occurs irrespective of the genetic sexin the absence of testicular hormones. Previous expression data suggestedthat GATA4 was involved in sex determination 22, 23and in vitro data suggested a role for GATA4 in the regulation of genesexpressed 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 differentadaptive responses in the internal and external environment stress ofvertebrates.

The sex determination region of Y chromosome (SRY) gene requiredto initiate signaling for male gonadal differentiation. Many other genesinvolved in gonoadogenesis are GATA4 and FOG2 26.Mammalian gonads arise in both sexes from bilateral genital ridge that have thepotential to develop as ovaries or testes 27, 28.

In humans gonadal differentiation occurs from the 10th through 12thembryonic week.  Steroidogenic factor 1 (SF-1) transcription factorcritical for adrenocortical development and homeostasis. SF1 is also known asadrenal four-binding protein or nuclear hormone receptor Ad4BP, encoded by thegene NR5A1.

All cells that belong to steroidogenic lineages of the adrenal andgonads express SF1, including subpopulations of long-term retained progenitorcells in each organ 29, 30. Therefore, SF1 expression defines the identity ofthese cells and commitment to steroidogenic differentiation 31-33. The expression of SF1 is detectable early in fetallife, between the AGP formation and the ultimate establishment of the adrenal primordium30. Genetic loss of Nr5a1 or its upstreamtranscriptional regulators Pbx1, Wt1, and Cited2, interferes with AGP formationleading to various degrees of adrenal hypoplasia in mice 34-36. While Nr5a1 is continuously expressed from thetime of adrenal primordium formation throughout the adult life, duringembryonic stages and early fetal life in mice, the Nr5a1 expression is drivenby the fetal adrenal-specific enhancer (FAdE), which becomes inactive when thedefinitive cortex forms, suggesting that distinct mechanisms sustain Nr5a1expression in the fetal and in the definitive cortex .

Genes essential forearly gonadal development: Acquisition of sexual dimorphic phenotype conditionis an important role in mammalian gonadal development. In this absence orpresence of Y chromosome at fertilization embryonic gonads differentiate in toeither ovaries or testis. Major four genes are known to be required for development ofbipotential gonads (a) the orphan nuclear receptor Steroidogenic factor-1 (SF1 orFtz-F1) 37 (b) Wilms tumor associated gene  (WT1; 38,  a zinc fingerDNA–binding protein, (c) Lhx1 (also known as Lim1), and (d) Lhx9, two LIM classhomeobox proteins 39.

SRY,SF-1, Wilms’ tumor related 1(WT1), GATA4, and SOX9, were emerging models thatsuggest complex interactions among these genes in gonadal development (KeithParker). SRY is the critical initiator oftestis development is a gene located immediately adjacent to the pseudo autosomalregion of the short arm of the Y chromosome, designated SRY for Sex-determiningRegion-Y chromosome. SRY has been identified as the testis-determining factor(TDF), the key gene responsible for testis development in XY embryos. Once thegonads are formed, the pivotal event in male sexual differentiation isexpression of the SRY gene.

SRY is necessary andsufficient to initiate the male development cascade 40.  In the absence of Sry, or if Sry isexpressed at insufficient levels, the support cell precursors differentiate asgranulosa cells, thus initiating the ovarian pathway 26.  The molecular mechanisms upstream and downstreamregulations of Sry are not well understood. Trivosian et al.

26, demonstrated that the transcription factor GATA4 and itsco-factor FOG2 are required for gonadal differentiation.The physiological target genes forSRY/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 WT1play key roles in both sexes in the development of the indifferent gonad. SF-1and WT1, together with SOX9, and GATA4, cooperate to regulate the expression oftarget genes (e.

g., AMH, Insl3, and the steroid hydroxylases) that mediate malesexual differentiation. DAX-1, a negative regulator of the male developmentalpathway, inhibits the activation of critical target genes by SF-1, WT1, SOX9,and GATA4.GATA4 and FOG2 and their physical interaction arerequired for normal gonadal development, WT1 and SF1, which are expressedprior to SRY and necessary for gonad development in both sexes.The tissue distribution of DAX-1 (adrenal cortex, gonads, hypothalamus, andpituitary) is the same as that of another orphan nuclear receptor,steroidogenic factor 1 (SF-1) that is required for development of the adrenalglands and gonads.

Dmrt is also one ofthe gene involved in testes differentiation in higher and lower vertebrates, DMRT1 geneencodes a zinc finger–like DNA-binding protein and is expressed very early in asex-specific manner in male gonads of all the classes of vertebrates,regardless of the sex-determining mechanism (chromosomal or environmental). Inmice, Dmrt1 is expressed in genital ridges of both sexes andthen becomes testis specific at the end of the sex-determining stage. In testis, Dmrt1 isexpressed in germ cells and Sertoli cells 42, 43have recently shown that Dmrt1 is required for testis but notovarian differentiation.Hormones produced by the testis trigger thedevelopmental process that leads to the male phenotypic sexual differentiation 21,independence of gonads and gonadal hormones in normal female birth. There arethree essential hormones secreted by the testes, androgens, MIS, and Insl3. Thesehormones secreted by testes called testicular hormones in male-specific developmentof the bipotential reproductive system.

Mullerian-inhibiting substance (MIS),also named Mullerian-inhibiting factor (MIF) or anti-Mulerian hormone (AMH), produced by fetal Sertoli cells inducesregression of the Mullerian ducts.SexdifferentiationAmong all vertebratesfishes exhibit  greatest diversity inspecies and act as a relevant model for genetic and evolutionary studies. Hermaphroditismis also a common feature of several fish species. It was observed that fewgenes have been linked in the process of sex determination or differentiation asin zebra fish. The genes Fushi Tarazu factor-1 (FTZ-F1) play crucial role as they were involved inregulating interrenal development  and  alsoshowed expression patterns similar with reproductive tissue differentiation andfunction.  For most of the fish speciesthe differentiation of gonad into a testis or an ovary is strongly influencedby hormonal, environmentalor social factors. In the studies it was observed that they can be sexreversed by exposure to estrogens, suggesting that the estrogen levels play acrucial role during sex differentiation. The Cyp19 gene product aromataseusually converts testosterone into 17 beta-estradiol but when inhibited leadsto male to female sex reversal.

FTZ-F1 genes are strongly linked to steroidbiosynthesis as well as regulatory region of Cyp19 contains binding sites forFTZ-F1 genes, further linking FTZ-F1 in this sex differentiation process 44. The Dax-1 gene has howeverbeen identified in the Nile Tilapia 18, 45, suggesting that otherfish species may also have Dax-1 homologues that play a role in sexdifferentiation. Several HMG-box containing genes, Sox-genes, have beenidentified in fish 46-48. It was found that HMG-Boxcis element has been identified in gene promoter of fushi tarazu factor1a (ff1a) 49. Sox9a was also able tobind specifically at this site in vitro indicating that aregulatory connection between Sox9a and ff1a is present. It was also observedthat Sox9 alone does not direct sex determination and differentiation in zebra fish.

AMH inhibits the expression of aromatase in developing gonads 50 therefore negativelymodulates the differentiation and function of Leydig cells by down regulatingseveral enzymes involved in the steroidogenic pathway 51. WT1 has been shownto be expressed in the intermediate mesoderm prior to and during renal tissuedifferentiation 52. It is also essential forthe steroidogenic interrenal development together with ff1b 53, 54. WT1 is thereby animportant factor in the early events during development of gonadal primordium.Dmrt1 also play an important role in testis determination in teleosts, sincealteration of aromatase levels during sex differentiation can cause sexreversals 55.

These Dmrt is usuallyregulated by GATA factors. GATA factors processes the binding sites of cyp19gene promoter that indicates its role in regulating aromatase expression 56, 57. SignalTransduction pathwaysSteroid hormone biosynthesis normally occurring insteroidogenic cells is regulated by trophic hormone activation of proteinkinase A (PKA) signaling pathways. It was observed that this trophic hormonestimulation results in the activation of G proteins which stimulateadenylatecyclase activity that produces increased intracellular levels of cAMPand PKA in mouse 58, 59. During this signaling, many proteins such ascholesteryl ester hydrolase gets phosphorylated along with transcriptionfactors such as steroidogenic factor 1, GATA-4 and cAMP response-elementbinding protein(CREB)/cAMP response element modulator that activates the genessuch as StAR involved in steroidogenesis.

60, 61. However, there was evidence that regulation ofsteroidogenesis can also modulated by signaling pathways without involving cAMP.These include growth factors, steroidogenic inducing protein (SIP), macrophagederived factors, chloride ions and calcium messenger systems 62. Several evidences show that growth factors such asepidermal 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 theMAPK/ERK pathways for steroid synthesis and StAR expression 65, 66. IGF-1 phosphorylated CREB/activating transcriptionfactor-1 and activator protein-1 family member c Jun/Jun D were also found tobe involved in steroidogenesis.Role of Gonadotropins Gonadotropins are released from the pituitary glandand play an important role in steroidogenesis. They have shown to activate bothp58 and ERK1/2 MAPKs that result in varying effects on StAR expression andsteroidogenesis in ovarian granulosa cells 67-70.

Apart from this it was also observed thatinhibition of p38 decreases both P450arom and estradiol synthesis, and theseevents were tightly correlated with the liver receptor homolog-1 and DAX-11expression demonstrating that p38 targets these transcription factors inregulating steroidogenesis. Other signaling pathways such as ERK/BMK1, JNK/SAPKalso regulate in steroidogenesis. Gonadotropinreleasing hormone (GnRH) is widely expressed outside of the classical brain inareas of the olfactory brain, telencephalon, preoptic area and midbrain. GnRH, is best known in vertebrates for itsexpression in neurons and its role in stimulating the release of gonadotropinsfrom the pituitary gland.

In some earlier studies analysis of the genomeconfirmed showing that many teleosts have three forms of GnRH each encoding byseparate gene. Ovary and testis are major sites of interest because they expressboth GnRH and GnRH receptors.  It wasalso revealed  that peripheral GnRHproduction is important in the early development and maturation of the gonadsof fish, but are not required at least in large quantities when the fish havereached maturity even though the GnRH genes continue to be expressed. Conclusion: Adrenal developmentand gonadal development are two most fundamental biological processes.

Various signaling mechanisms andtranscription factors and several pathways are involved at genomic level incommon to adrenal and gonadal development in fishes and lower vertebratesduring 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 therole of signalling pathway involving ERK1/2, JNK/SAPK, and ERK5 MAPKs inregulation of StAR expression during steroidogenesis in different steroidogenictissues. This emphazise various genetic events are involved at the early andlate development of the process of gonadal development in steroidogenic tissues


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