Abstract Background Changes in the abundance of ovarian proteins play a key role in the regulation of reproduction. However, to date, no studies have investigated such changes in pubescent goats. Herein we applied isobaric tags for relative and absolute quantitation (iTRAQ) and liquid chromatography–tandem mass spectrometry to analyze the expression levels of ovarian proteins in pre-pubertal (n = 3) and pubertal (n = 3) goats. Results Overall, 7,550 proteins were recognized; 301 (176 up- and 125 downregulated) were identified as differentially abundant proteins (DAPs). Five DAPs were randomly selected for expression level validation by Western blotting; the results of Western blotting and iTRAQ analysis were consistent. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that DAPs were enriched in olfactory transduction, glutathione metabolism, and calcium signaling pathways. Besides, gene ontology functional enrichment analysis revealed that several DAPs enriched in biological processes were associated with cellular process, biological regulation, metabolic process, and response to stimulus. Protein–protein interaction network showed that proteins interacting with CDK1, HSPA1A, and UCK2 were the most abundant. Conclusions We identified 301 DAPs, which were enriched in olfactory transduction, glutathione metabolism, and calcium signaling pathways, suggesting the involvement of these processes in the onset of puberty. Further studies are warranted to more comprehensively explore the function of the identified DAPs and aforementioned signaling pathways to gain novel, deeper insights into the mechanisms underlying the onset of puberty. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-022-08699-y. Keywords: Proteomics, iTRAQ, LC–MS/MS, Pubertal onset, Differentially abundant proteins, PPI network, Anhui white goats Background Puberty is the transitional period between the juvenile state and adulthood; during this phase, animals gain reproductive capacity, which is crucial for their growth and development [[55]1]. In female animals, the hypothalamic–pituitary–ovarian axis regulates puberty and reproductive function [[56]2, [57]3], and the ovary is the final target of this axis. The physiological activity of the ovary plays a crucial role in regulating the growth and development of animals [[58]4]. Gonadotropin-releasing hormone (GnRH) neurons in the arcuate nucleus of the hypothalamus synthesize and secrete GnRH, and during the onset of puberty, their activity and consequently GnRH secretion are increased, which is characterized by pulsed GnRH release [[59]5]. GnRH stimulates gonadotropic cells in the anterior pituitary gland to secrete follicle-stimulating hormone and luteinizing hormone. These hormones act on the ovaries via blood circulation to facilitate the secretion of sex hormones, thereby promoting the rapid development of reproductive organs as well as sexual characteristics and playing a pivotal role in the onset of animal puberty [[60]6, [61]7]. Besides, the ovaries in turn regulate the hypothalamic–pituitary–ovarian axis by positive and negative feedback via the secreted sex hormones, further affecting puberty [[62]8]. The mechanism underlying the onset of puberty is complex and reportedly involves several factors, including neuroendocrinological, genetic, and environmental factors [[63]9]. The specific regulatory mechanism nevertheless remains unclear. Nguyen et al. performed adipose tissue proteomic analyses to study puberty in Brahman heifers; 51 significantly differentially abundant proteins (DAPs) were identified between pre- and post-pubertal heifers. These DAPs were enriched in estrogen signaling and PI3K–Akt signaling pathways, which are known integrators of metabolism and reproduction [[64]10]. Further, Fortes et al. investigated how the uterine tissue and its secretion changes in relation to puberty in Brahman heifers. Using a combination of proteomics and transcriptomics, they identified 258 DAPs in the uterine fluid of pre- and post-pubertal cows [[65]11]. Similarly, Ye et al. applied proteomics to examine the hypothalamus of pre-pubertal and pubertal female goats, which led to the identification of 69 DAPs. These proteins were enriched in the MAPK, RAS, and PI3K–Akt–mTOR signaling pathways, indicating that the identified DAPs and their related signaling pathways are crucial in regulating puberty in goats [[66]12]. Collectively, the results of such studies indicate that proteomics can be effectively used to assess the hypothalamus as well as other tissues to explore the mechanisms underlying puberty. Although the expression of retinol-binding protein 4 in the ovaries of female mice has been found to remain unchanged until puberty, a significant increase was noted during puberty [[67]13]; moreover, retinol-binding protein 4 evidently plays a chief role in the transportation and storage of cells in follicular fluid [[68]14]. Tahir et al. used whole ovaries of six pre- and six post-pubertal Brahman heifers to perform differential abundance analyses of protein profiles between the two physiological states, identifying 32 steroidogenesis-associated DAPs [[69]15]. An increase in steroidogenesis is observed during puberty [[70]16]. A study identified 36 proteins in ovarian follicular fluid of goats at different developmental stages [[71]17]. Overall, these findings suggest that ovarian proteins play a fundamental role in pubertal development in animals. Goats play an important socioeconomic role in the lives of people considering that they are a source of, for example, milk and meat. Puberty is a pivotal stage in female animal development; it marks the first occurrence of ovulation and the onset of reproductive capability [[72]18]. A study reported that lncRNA is related to ovarian steroid hormone synthesis, oogenesis, and oocyte maturation during the growth and development of goats [[73]19]; moreover, the levels of hormones in goats have been found to significantly change during puberty [[74]20]. However, to date, no studies have used proteomics to assess changes in the expression level of proteins in the ovaries of pre-pubertal and pubertal goats. Herein we thus aimed to identify proteins and pathways associated with the onset of puberty by measuring protein abundance levels in the ovaries of prepubertal and pubertal goats. Figure [75]1 shows the experimental design and workflow. We believe our results should enhance our understanding of the key factors and mechanisms that regulate puberty in goats. Fig. 1. [76]Fig. 1 [77]Open in a new tab Experimental design and workflow to identify differentially abundant proteins (DAPs) in pre-pubertal (n = 3) and pubertal (n = 3) goat ovaries Results Protein Identification Overall, 7,550 proteins were identified and quantified by isobaric tags for relative and absolute quantitation (iTRAQ) proteomics, of which 2,623 were uncharacterized proteins and 4,927 were proteins with known functions. In total, 301 DAPs were identified: 176 of them were up- and 125 were downregulated (Fig. [78]2a, b). On comparing data pertaining to pubertal with pre-pubertal goat ovaries, MHC class II antigen showed the highest relative upregulation, while DUF4537 domain-containing protein showed the highest relative downregulation. Tables [79]1 and [80]2 show the top 15 up- and downregulated proteins, respectively, in pubertal ovaries compared with pre-pubertal ones. Fig. 2. [81]Fig. 2 [82]Open in a new tab DAP screening. a Volcano map. Red and green dots indicate up- and downregulation of protein expression levels, respectively. Gray dots indicate DAPs with insignificant changes in expression. b Number of DAPs. In total, 301 DAPs were identified. Orange and green columns represent the number of up- (n = 176) and downregulated (n = 125) proteins, respectively Table 1. Top 15 upregulated proteins in pubertal compared to pre-pubertal goat ovaries Accession Gene Name Description Coverage [%] Unique Peptides P Mean_Ratio [83]Q6BCN2 Cahi-DQA1 MHC class II antigen (Fragment) 0.207 1 0.0010 6.3115 A0A068B4V9 GSTA3 Glutathione S-transferase 0.414 1 0.0034 5.8213 A0A452F8N3 TET3 Methylcytosine dioxygenase TET 0.018 1 0.0050 5.6453 A0A452FL66 Ig-like domain-containing protein 0.155 2 0.0189 5.6192 A0A452F0V7 USP42 USP domain-containing protein 0.006 1 0.0049 5.4207 A0A452FRG5 LMNTD2 LTD domain-containing protein 0.021 1 0.0081 5.3130 [84]P0CH26 HBAII II alpha globin 0.627 1 0.0086 5.2103 A0A452EDD0 NR5A2 Uncharacterized protein 0.018 1 0.0440 4.8056 A0A452DRV6 ZNF529 Uncharacterized protein 0.014 1 0.0022 4.7709 I6TE27 KIAA1239 (Fragment) 0.017 1 0.0027 4.7686 A0A452F5F2 SERPINI2 SERPIN domain-containing protein 0.044 1 0.0145 4.7063 A0A452DS42 IGv domain-containing protein 0.065 1 0.0003 4.4793 A0A452EDE4 KMT2C [Histone H3]-lysine (4) N-trimethyltransferase 0.002 1 0.0009 4.2239 A0A452FX60 LOC102185917 Ig-like domain-containing protein 0.147 1 0.0234 3.8227 A0A452FZ42 CCNE1 Cyclin N-terminal domain-containing protein 0.019 1 0.0081 3.7908 [85]Open in a new tab Table 2. Top 15 downregulated proteins in pubertal compared to pre-pubertal goat ovaries Accession Gene Name Description Coverage [%] Unique Peptides P Mean_Ratio A0A452GA12 C11orf16 DUF4537 domain-containing protein 0.015 1 0.0006 0.3087 A0A452FKP1 EFCAB6 Uncharacterized protein 0.004 1 0.0001 0.3770 A0A452E9S9 CNMD BRICHOS domain-containing protein 0.015 1 0.0030 0.3841 [86]P02082 Hemoglobin fetal subunit beta 0.855 10 0.0032 0.4100 A0A452FUY5 PER2 Uncharacterized protein 0.006 1 0.0027 0.4138 A0A452E681 ZP2 ZP domain-containing protein 0.049 4 0.0002 0.4267 A0A452G903 Metallothionein 0.328 1 0.0000 0.4319 A0A452FKU4 ZP3 Zona pellucida sperm-binding protein 3 0.229 9 0.0007 0.4391 A0A452FFM6 G_PROTEIN_RECEP_F1_2 domain-containing protein 0.025 1 0.0002 0.4455 A0A452DYI9 FLYWCH family member 2 0.052 1 0.0000 0.4803 A0A452FEE2 Ig-like domain-containing protein 0.215 2 0.0022 0.4892 A0A452G9F8 Uncharacterized protein 0.189 3 0.0003 0.4930 A0A452EAM2 Uncharacterized protein 0.037 1 0.0095 0.4947 A0A452FCF1 LOC108635821 AIG1-type G domain-containing protein 0.196 2 0.0026 0.4983 A0A0H4PMF2 Esco2 0.01 1 0.0096 0.5003 [87]Open in a new tab Validation of protein expression levels by Western Blotting (WB) To confirm the results of iTRAQ–liquid chromatography mass spectrometry (LC–MS)/MS analysis and facilitate subsequent functional study, the abundance of five reproduction-related proteins [upregulated: calcipressin-1 (RCAN1), insulin-like growth factor I (IGF-1), and delta (24)-sterol reductase (DHCR24); downregulated: stathmin (STMN1) and cyclin-dependent kinase 1 (CDK1)] was verified in pubertal and pre-pubertal goat ovaries by WB. WB and iTRAQ–LC–MS/MS analysis data were consistent (Fig. [88]3a, b), indicating that our proteomics data were highly reliable. Fig. 3. [89]Fig. 3 [90]Open in a new tab iTRAQ–LC–MS/MS data validation by Western blotting (WB). a Abundance of three upregulated [IGF-1 (full-length blots/gels are presented in Additional file [91]5), RCAN1 (full-length blots/gels are presented in Additional file [92]6), and DHCR24 (full-length blots/gels are presented in Additional file [93]7)] and two downregulated [CDK1 (full-length blots/gels are presented in Additional file [94]8) and STMN1 (full-length blots/gels are presented in Additional file [95]9)] proteins were analyzed by WB. β-Tubulin (full-length blots/gels are presented in Additional file [96]8) served as the internal reference. (Some positions of marker were in the middle and some were on the edge of the gels. To display the images more aesthetically, we have cropped them.) b Quantitative results for proteins. Values represent mean ± SEM (n = 3/group). Statistical significance was assessed using Student’s t-test; P < 0.05 Cluster analysis of DAPs Euclidean distance and hierarchical clustering were used to cluster DAPs, and dynamic changes in DAPs were compared (Fig. [97]4). We found significant differences in protein abundance intensity in the ovaries of pubertal and pre-pubertal goats, indicative of differences in protein abundance levels between pubertal and pre-pubertal goat ovaries. Fig. 4. [98]Fig. 4 [99]Open in a new tab Cluster heatmap showing expression intensity of DAPs. Euclidean distance and hierarchical clustering were used to cluster protein expression patterns. Colors represent relative expression levels of proteins in the ovaries of pre- and pubertal goats, with red and blue representing up- and downregulation, respectively Gene Ontology (GO) Functional Enrichment Analysis. To further analyze the functions of the 301 DAPs, they were classified into the following major categories based on their GO annotations: biological process, cellular component, and molecular function. DAPs enriched in biological processes were mainly associated with cellular process, metabolic process, biological regulation, regulation of biological process, and response to stimulus (Fig. [100]5). Further, the five most abundant terms in the cellular component category were cell part, cell, organelle, membrane, and extracellular region, and those in the molecular function category were binding, catalytic activity, molecular function regulator, transcription regulator activity, and transporter activity. Fig. 5. [101]Fig. 5 [102]Open in a new tab Gene ontology functional analysis of DAPs. P < 0.05 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis To further explore the signaling pathways involved in regulating puberty onset in goats, DAPs were subjected to KEGG [[103]21] pathway enrichment analysis (Fig. [104]6). The 301 DAPs were mapped to 260 KEGG pathways, including glutathione metabolism, olfactory transduction, and calcium signaling pathways, indicating their involvement with pubertal onset in goats. Fig. 6. [105]Fig. 6 [106]Open in a new tab Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of DAPs. The larger the bubble, the more the number of DAPs. Bubble color indicates the P value; the smaller the P value, the higher the level of significance Protein–Protein Interaction (PPI) network analysis The interaction relationships among DAPs in the enriched signaling pathways (KEGG analysis) were found in the STRING database [[107]22], and a PPI network was constructed (Fig. [108]7), which revealed some key interactions among DAPs. In the PPI network, nodes represent proteins and edges denote predicted functional associations; 222 nodes and 192 edges were found to be interconnected. CDK1, recombinant heat shock 70 kDa protein 1A (HSPA1A), and uridine cytidine kinase 2 (UCK2) occupied the central position in the PPI network and were observed to interact with other DAPs as a hub. Fig. 7. [109]Fig. 7 [110]Open in a new tab Protein–protein interaction network analysis Discussion Puberty is a phase of anatomical and physiological development, leading to sexual maturity and reproductive capacity [[111]23]; however, the specific mechanisms underlying puberty remain unclear. Proteins are the material basis of life [[112]24], so changes in the abundance of ovarian proteins are bound to affect pubertal onset. Therefore, we herein performed iTRAQ–LC–MS/MS analysis to compare the expression levels of ovarian proteins in pre-pubertal (n = 3) and pubertal (n = 3) goats so as to identify core proteins involved in puberty regulation. This led to the identification of 7,550 proteins, of which 301 DAPs met the screening standard. Among the DAPs identified in this study, MHC class II antigen (fragment) was the most upregulated one in the ovaries of pubertal goats. MHC class II molecules are encoded by the MHC gene locus, which is located on chromosome 6 in humans and chromosome 17 in mice [[113]25]. These molecules play a key regulatory role in several immune responses and are involved in most autoimmune diseases [[114]26]. In addition, MHC genes reportedly influence mating preferences and odor in