Previous studies on the pituitary have focused on individual genes and signaling pathways involved in its development [4
], as well as expression and release of pituitary hormones, such as PRL, LH and FSH [20
]. With the advance of the human genome project, a large amount of genomic information is now available for surveying the physiological and pathological processes of humans at a global level. Hormonal genomics has been proposed to develop postgenomic approaches substituting gene-by-gene ones to reveal the subfamilies and pathways for genes involved in the hormones signaling [22
]. The functional subgenomes of secreted extracellular signaling molecules, transmembrane receptors, intracellular signaling molecules, and transcriptional factors can now be analyzed in an integrated manner. Of which, EST sequencing, series analysis of gene expression (SAGE) and cDNA microarray or gene chip analysis are the most efficient strategies for profiling gene expression at the transcriptome level [23
In this study, a total of 3,539 EST clusters were derived from 9,271 ESTs generated from cDNA libraries of human fetal and adult pituitaries. It was not as high as expected because of the limited number of human samples. We did confirm the information by semi-quantitative RT-PCR, in situ
hybridization or reviewing previous studies. Aside from semi-quantitative RT-PCR in this study, microarray analysis and quantitative RT-PCR carried out in our previous study also showed that the expression level of hormones, such as TSHβ, POMC and GH in fetus, were relative lower than that in adult [11
]. On the other hand, some genes such as EDNRA, were highly expressed in fetal pituitary, also in agreement with previous study performed by Dr. Ellestad using microarray [12
]. Although these studies were all to highlight the gene expression profile of pituitary, there is only few genes overlapped between these screens. Most of the variations could be caused by methods different.
The partial transcription maps derived from these ESTs indicated that hormones and hormone-associated genes were predominant in adult pituitary, while development-associated genes were predominant in fetal pituitary, including Sox4 and ST13. Sox4 has been shown to be functionally involved in development of a wide range of tissues and cells, such as heart, B cells, reproductive system and central nervous system [26
]. ST13 may facilitate the chaperone function of Hsc/Hsp70 in protein folding and repair, and in controlling the activity of regulatory proteins such as steroid receptors and regulators of proliferation or apoptosis [27
]. POU1F1, necessary for cell lineage of thyrotrophs, somatotrophs and lactotrophs, was hit once in the cDNA library of fetal pituitary. No significant different of its expression level could be detected between fetal and adult pituitary. It is in agreement with that POU1F1 is necessary but not sufficient for hormones gene activation [29
It was revealed for the first time that Sox4, a member of the SRY-like high-mobility group (HMG) box gene family, was one of the most abundant mRNAs in the fetal pituitary. The HMG box defines a superfamily of eukaryotic DNA-binding proteins of central importance in mammalian gene regulation [30
], and SRY defines the mammalian testis-determining factor encoded by the Y chromosome [31
]. Sox protein is a specific HMG-box factor that is similar to SRY. It is ubiquitous in the animal kingdom and is involved in diverse developmental processes, including germ layer formation, cell type specification, and organogenesis [32
]. Sox4 null mice die during embryogenesis because of failure of endocardial ridge development, and blocking of B-cell lineage progression at the stage of pro-B-lymphocyte expansion [33
Explanted fetal thymic organ cultures (FTOCs) of Sox-4-deficient thymus yielded 10–50-fold fewer CD4/CD8 double-positive and single-positive cells than FTOCs of littermates [34
]. Lioubinski et al
have reported that Sox4 is expressed in insulin-producing pancreas islets, and co-expressed with glucagon during tissue development [16
]. Also, it has been reported that Sox4 is expressed in the mouse uterus under ovarian hormone control. This suggests a developmental role for this gene in the female reproductive system, because its expression always appears to be related to the maturational stage of the cell population [35
]. Furthermore, Cheung et al
] showed that Sox4 might indeed had another functional role in brain development. It has been demonstrated that Sox4 expression counteracts differentiation of radial glia and has to be down-regulated before full maturation can occur in the brain [36
]. Radial glia with prolonged expression of Sox4 fail to migrate to the position normally assumed by Bergmann glia, and do not extend radial fibers toward the pial surface [37
]. The expression pattern of Sox4 in pituitary development suggests that it is involved in pituitary cell differentiation; similar to its role in facilitating thymocyte differentiation, but it normally prevents premature differentiation of human pituitary.
In addition, we investigated the expression of 17 members of the Sox gene family in the fetal human pituitary using RT-PCR. Eleven of these members (Sox 2, 3, 4, 5, 7, 9, 10, 12, 13, 15 and 17) were detected (data not shown). It has recently been reported that Sox2 had a critical role in the development of the hypothalamo-pituitary axis [38
], and deletion of Sox3 can lead to abnormal development of RP and defects in pituitary function [39
]. Sox10 might be necessary for gonadotropin-releasing hormone cell differentiation [41
]. Obviously, members of the Sox gene family might have a predominant role in the regulation of hypothalamus-pituitary axis formation.
Except for the genes known to be associated with pituitary development, our data suggest that some other genes are involved in the differentiation and maturation of human pituitary. They contained genes for cytokines and their receptors, transcription factors, and those involved in the cell cycle, DNA replication and signal transduction. Of which, GA-binding protein transcription factor is a critical and functionally relevant Ets factor that regulates rPRL promoter activity via the BTE site [42
]. HIF-1 exerts an antiapoptotic role in HP75 in hypoxia [43
]. TFDP3 was identified in pituitary for the first time, and only highly espressed in human testis, thymus and fetal pituitary according to our studies (data not show). It was reported as an inhibitor of E2F-induced apoptosis [44
40 novel ESTs were produced in human fetal pituitary. They are all singletons which no EST was matched in a BLAST against the nonredundant EST database at NCBI. It is suggested that they are potential new genes, proteins and microRNAs that need to be investigated in the future. For example, CD239423 was located on Human DNA sequence from clone RP11-123K19 on chromosome 9, jointed by 13118–13292, 26649–26703, 29319–29386, and 39381–39729. The others such as CD239425, CD238497, CD237004 and CD238482 were found to reside in the introns of various genes. And their tissue expression patterns show that some novel ESTs exist particularly in fetal pituitary cells. It is indicated that EST sequencing is an efficient method for gene discovery and expression level analysis.