In this study, SSH technology was used to construct the differential gene libraries of pituitary tissues in JG and LC goats at different growth stages. Through sequencing, sequence alignment and statistics, the SSH library of pituitary contains 707 ESTs, 37.48% of which were expressed in API group and 30.69% were expressed in EPI group and 31.82% were expressed in BPI group. At the same time, the GO annotation of these libraries showed that most of the genes were mainly involved in biological process, cellular component or molecular function such as metabolic process, cellular process, catalytic activity, binding, etc., and some genes were also involved in the developmental process. This indicates that the molecular mechanism of different growth stages affecting the precocious puberty of goats is comprehensive and complex, which might regulate the growth, development, metabolism and development of goats through multiple signaling pathways.
According to the GO annotation of SSH library, 18 precocious puberty-related differential expression genes were screened from three groups, 44.44% of them in API group, 11.11% in EPI group and 44.44% in BPI group. The results indicated that most precocious puberty related genes were shown in the same growth stages of different goat breeding. The influence of growth stages is more important for the goat precocious puberty.
Some of these precocious puberty related differential genes were differentially expressed in the three groups. Among them, eighteen genes were appeared more than twice in three groups including RPS25, GNB2L1,RPS12, GH1,PET100,PLP1,RPL10A,RPL37,RPLP0,RPS19,PKN1,RPL23A,RPS10, RPS14,RPS20.S,RPS7, HSPA8 and SLC25A5. RPLP0 protein (ribosomal protein lateral stalk subunit P0) encodes one out of approximately 80 ribosomal proteins in human, which are involved in protein synthesis and apoptosis processes [15]. In a previous study, Ragni et al. classified the RPLP0 as the most stable reference gene in expression assays performed in mesenchymal stem cell differentiation, osteoblasts precursor cells [16]. In the study interactions of cytotoxic T lymphocytes with tumor cells, RPLP0 is stably expressed in melanoma cells [17]. RPLP0 gene as reference genes are stably expressed in GCs in both controls and polycystic ovarian syndrome patients and can be used for normalization in gene expression profiling by qRT-PCR [18]. Nikishin et al. confirmed the conclusion of RPLP0 gene as the most stable reference genes in the case of vitrified/thawed human ovarian cortical tissue [19].
PKN1 (protein kinase N1) is a stress-responsive kinase and a member of the protein kinase novel (PKN) family also known as protein kinase C-related kinases (PRKs) [20, 21]. The interaction of Rho with PKN1 induces a conformational change in PKN1 leading to activation loop phosphorylation by 3-Phosphoinositide-Dependent Kinase-1(PDK1) on Thr774 in the case of human PKN1 (Thr778 in mouse/rat) which is necessary for the catalytic activation of PKN1/214 and critical for the stability of the protein [22,23]. PKN1 is shown to provide a basal cardioprotective function in the face of I/R injury [24]. In the currently study found that PKN1 as a novel key player in fine-tuning the balance between axonal outgrowth and presynaptic differentiation in the parallel fiber–forming (PF-forming) cerebellar granule cells (Cgcs) [25]. In neurons PKN1 is the most abundant isoform and has been implicated in a variety of functions including cytoskeletal organization and neuronal differentiation [26-29]. In the precocious puberty related studies found that PKN1 gene modulated TGFβ and EGF dependent regulation of the responses of HEC-A-1 endometrial cancer cells proliferation, migration, and invasiveness, and therefore is a component of the network signaling downstream of TGFβ and EGF [30]. Attarha et al. used proteomics, systems biology, and immunohistochemistry to explore protein expression in human endometrial tumours, identified PKN1 may be considered as predictive biomarkers of endometrial cancer [31].
SLC25A5 (solute carrier family 25 member 5) facilitated the transport of molecules involved in the urea and citric acid cycles, oxidative phosphorylation, DNA maintenance and iron metabolism processes [32, 33]. In the dysregulated expression of androgen metabolism genes and genetic analysis in hypospadias, the data provided direct evidence that SLC25A5 may contribute the genetic etiology of hypospadias [34]. In the bovine reproduction study, SLC25A5 gene were found high expressed in the SSH library of bovine blastocyst [35]. RPL37 (ribosomal protein L37), as a component of the 60S subunit of ribosomes and belongs to the L37E family of ribosomal proteins. In the Zebra Finch Song System study, RPL37 might all influence sexual differentiation, perhaps with the hormone and proteins interacting, such that an appropriate balance is required for normal development [36]. PRL37 protein as a histotype-specific prognostic biomarker was found in the Ovarian Carcinomas Using Immunohistochemistry [37]. These genes above mentioned were potential molecular markers for the study of goat precocious puberty.
In our experiment, 18 DEGs were related to precocious puberty directly or indirectly. Among them, RPL10A, RPL23A, RPL24, RPL34, RPL37, RPLP0, RPS3A , RPS6 and RPS7 are all common protein family ribosomal protein, they play an important role in the development of ovarian and breast cancers related to precocious puberty [38,39]. And EIF5A, HNRNPDL, HSPA8, PKN1, PRKAR2B, SLC40A1, SLC25A5, SNRPD2 and YWHAH, play key roles in oocyte meiosis, steroid biosynthesis and conduction of reproduction process [40–42].
Precocious puberty is regulated via hormones of the hypothalamic-pituitary-gonadal (HPG) axis. In our study, we analysis of the hypothalamic-pituitary-gonadal (HPG) axis related genes by the online tool STRING. We found that most of the DEGs from the three tissues enriched in one pathway, the EIF5A and YWHAH gene play an important role in the network (Fig.9).
eIF5A (eukaryotic translation initiation factor 5A) is a highly conserved 17-kDa protein that is expressed ubiquitously in all cells. eIF5A participates in diverse cellular processes, including protein translation [43-45], nucleocytoplasmic transport of RNA [46], cell proliferation [47], inflammation, and apoptosis[48,49]. Stimulation of follicular growth by follicle-stimulating hormone (FSH) is associated with increased expression of luteinizing hormone/choriogonadotropin receptors (LHCGRs) in granulosa cells [50, 51]. EIF5A was identified as one of the proteins that interact with LRBP and lead to the degradation of LHCGR mRNA by facilitating the transport of LHCGR mRNA-LRBP complex to P bodies for degradation [52-54]. In the currently, a series studies were carried out to determine the mechanism by which inhibition of hypusination of EIF5A causes an increase in LHCGR mRNA expression [55]. The EIF5A gene usually expressed in ovary tissue, in our study the expression of EIF5A gene were detected in the pituitary tissue. The result indicated that this gene might as an important gene expressed in the hypothalamic-pituitary-gonadal (HPG) axis to regulate the goats precocious puberty.
YWHAH (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta) is an isoform of YWHA protein. Numerous studies implicate YWHA as a critical regulator of the cell cycle in meiotic and mitotic cells as well as other cellular processes [56, 57]. The results indicate that oocyte-specific or global elimination of YWHAH protein does not result in abnormal fertility, oocyte maturation or development. Breeding and development of pups was normal in the absence of YWHAH or YWHAE in females with oocyte-specific knockout of these genes. Global inactivation of Ywhah in female mice does not appear to alter oogenesis, oocyte maturation and early development [58]. The previously noted that YWHAH may play an important role in meiotic spindle formation by employing antisense morpholino knockdown approaches [59].
The effect of differential expressed genes from pituitary tissues at different growth stages may be related to the process of precocious puberty. However, the exact pathway remains unknown, and there are few reports on how these differential genes affect the precocious puberty of goats. In the future, more function analysis will be needed to further explore the effects of these genes on precocious puberty in goats.