Introduction
Goats (
Capra hircus) are an important type of domestic and commercial livestock in China and are an important source of meat, skin, fur and fibre for humans. However, the low fecundity of goats has seriously constrained the development of the goat industry [
1]. In recent years, the Jining Grey goat has been used as an ideal model to study the growth and development process of sexually mature ovaries in domestic animals due to its precocious puberty, higher fertility and year-round oestrus [
2]. Puberty (first ovulation) occurs at 2 months of age in Jining Grey goats, and sexual maturity occurs significantly earlier (3–4 months) in these goats than in other goats [
3,
4]. This suggests that key reproductive traits, such as ovarian function and hormone regulation, are manifested in Jining Grey goats in the early growth stages. Their excellent reproductive characteristics provide more possibilities for the study of sexual maturity.
Sexual maturation refers to the period from birth to full sexual maturity (capable of normal reproduction), and past studies have reported that sexual maturity is directly related to the maturation of the hypothalamic–pituitary–ovarian (HPO) axis. The ovary plays a crucial role during sexual maturation by directly mediating the secretion of oestrogen and progesterone and maintaining reproductive capability through follicular development and ovulation [
5]. With the onset of sexual maturity, the endocrine function of the animal changes, and ovarian tissues show different physiological states and functional manifestations. For example, the HPO axis is activated at the onset of puberty. The levels of gonadotropin-releasing hormone released by the hypothalamus and gonadotropins (follicle-stimulating hormone and luteinizing hormone) secreted by the pituitary gland change. This results in the development of the ovaries of the goat, the secretion of hormones such as oestrogen and progesterone, and ultimately, oestrous and ovulation [
6]. During sexual maturation, follicles develop rapidly, ovulation frequency increases, and granulosa cells secrete more oestrogen and progesterone. During this stage, the ovaries are most active in terms of their reproductive and endocrine functions to support the mature oestrous cycles and normal ovulation [
2]. Therefore, sexual maturation is the main stage of ovarian development. However, the biological mechanisms underlying the developmental changes in ovarian tissue during sexual maturation are still poorly understood.
MicroRNAs (miRNAs) are single-stranded and noncoding RNAs that are key posttranscriptional regulators. In animals, miRNAs control gene expression by interacting with the 3’ untranslated region (3’UTR) of the target mRNA and then degrading or inhibiting mRNA translation [
7]. These molecules play a role in a variety of biological processes, including cell proliferation [
8], cell differentiation [
9], apoptosis [
10], tumorigenesis [
11], hormone secretion [
12], and metabolism [
13]. There is growing evidence that miRNAs play a widely recognized role in almost all ovarian biological processes, including ovary maturation, ovarian cell development, luteal development and regression [
14‐
16]. For example, miR-17-5p regulates the expression of the antiangiogenic factor tissue inhibitor of metalloproteinase 1, which is involved in corpus luteum angiogenesis, the lack of which can lead to infertility [
17]. miR-34a regulates the proliferation and function of luteal cells during the transition of the corpus luteum from the developmental to the functional stages [
18]. There is a correlation and temporal relationship between the overexpression of miR-21 and the posttranscriptional regulation of programmed cell death 4 (PDCD4) during oocyte maturation [
19]. Let-7 family members play critical roles in cell fate determination and have been implicated in the regulation of housekeeping genes during ovarian development [
20]. Several studies have shown that miR-133 family members are involved in the regulation of a variety of cellular processes, including cell proliferation, migration, and invasion [
21]. miR-133b binds to the FOXL2-3’UTR in epithelial-like granulosa cells (GC) and reduces the expression of FOXL2. Reduced binding of FOXL2 to the promoters of STAR and CYP19A1 promotes their expression, thereby promoting oestrogen secretion by GCs [
22]. Therefore, studying the expression patterns and potential functions of miRNAs can help us better understand the molecular regulatory mechanisms of ovarian development during sexual maturation.
In the present study, we measured serum levels of reproductive hormones to study their temporal changes at four specific time points (1 day after birth (D1), 2 months after birth (M2), 4 months after birth (M4), and 6 months after birth (M6)) in Jining Grey goats and carried out small-RNA sequencing of ovarian tissue samples to detect the differentially expressed miRNAs and their potential functions during sexual maturation in goats. These results can help to better understand the molecular regulatory mechanisms of ovarian development during the biological process and provide an effective basis for further exploring the improvement of the reproductive regulatory network of the Jining Grey goat, thus promoting progress in the field of goat breeding.
Discussion
To study the serum levels of reproductive hormones during sexual maturation in goats, we measured the levels of FSH, LH, E2 and PROG. Puberty is a critical stage of life in the physiological changes associated with sexual maturation, representing a complex process subject to multiple endocrine and genetic controls [
38]. Pubertal onset is considered to begin with an increase in GnRH, which activates the HPO axis [
39]. Hypothalamic activation and secretion of GnRH are key upstream regulators that initiate the reproductive cascade to stimulate pulsatile release of FSH and LH [
40]. During prepubertal stages, the levels of LH, FSH and E2 have been reported to increase gradually [
41]. LH and FSH stimulate the secretion of steroid hormones in the gonads, while high levels of E2 exert negative feedback effect on the hypothalamus and anterior pituitary, thereby reducing the secretion of gonadotropins [
42]. From mid-puberty onwards, E2 also produces positive feedback, which is a necessary process for ovulation. This requires sufficient LH release from the pituitary and for the follicles to reach a sufficient size to produce enough E2. Oestradiol increases pituitary sensitivity to GnRH, thereby increasing LH secretion [
43]. The rise in E2 inhibits the secretion of FSH within the ovaries, causing the dominant follicle to undergo luteinization under the influence of LH, followed by ovulation. In the present study, we observed that the average levels of LH and FSH increased with the developmental process of puberty, but the rise in LH was more substantial than that of FSH. These elevations may be a result of higher baseline levels of both LH and FSH, as well as an increase in the frequency of LH surges [
44]. Under the influence of high levels of E2, the levels of FSH and LH may exhibit a declining trend at the M6 stage. In addition, exogenous PROG has been demonstrated to increase gonadotropin levels [
45]. PROG secretion at early prepuberty is regulated and influenced by LH, FSH and E2 [
46]. We observed that at the M2 stage, PROG levels were significantly higher than those at the D1 stage, possibly because of the cooperative promotion of follicle development and ovulation by PROG and E2 and subsequent ovarian luteinization and an increase in PROG levels [
47]. A delicate balance between different reproductive hormone signals is needed for proper development during sexual maturation.
The main function of the ovary is to affect the oestrous cycle and fertility of mammals, making it an important reproductive organ [
48]. miRNAs are important regulators of gene expression and play key roles in ovarian disease prevention, ageing, cell proliferation, apoptosis and metabolism [
49]. Many miRNAs have been identified from ovarian tissues or germ cells of goats [
50‐
52], sheep [
53‐
55], cows [
56,
57], and humans [
58,
59]; however, a complete analysis of the transcriptome of intact ovaries during the sexual maturity period of goats, especially Jining Grey goats, has rarely been reported thus far. Our analysis of the four developmental stage libraries identified a total of 667 miRNAs; only 474 miRNAs were commonly expressed in all four stages (Fig.
2A), suggesting that the expression of many miRNAs is time-specific [
60]. The predominant length of the miRNAs we identified was 22 nt (Fig.
2B, C). This finding is consistent with previous findings in sheep [
61], goats [
1], pigs [
62], and mice [
63] but differed in bovine ovaries, where miRNAs of 20 nt size were most abundant [
64], which may reflect species differences. According to the family analysis, the let-7 family had the highest number of annotated miRNAs (Fig.
2E). The family is one of the earliest miRNA groups discovered, and its family members are highly conserved throughout the species [
65]. The let-7 family plays an important role in a variety of biological processes, such as cell proliferation, differentiation, tissue development, and tumour suppression [
66]. Studies have shown that the expression of let-7 family members varies during follicular atresia. For example, downregulation of let-7c is associated with premature ovarian failure, suggesting that let-7c plays a key role in promoting healthy follicular development. Interestingly, the expression pattern of let-7 g in follicles appears to be different from that of other members of the family, as it is significantly upregulated during the atresia period [
67]. The antiapoptotic gene MAP3K1 (mitogen-activated protein kinase kinase kinase 1) was found to be a direct target of let-7 g. Downregulation of MAP3K1 by let-7 g leads to the expression and dephosphorylation of the transcription factor FoxO1, which accumulates in the nucleus and triggers granulosa cell apoptosis [
68]. These data suggest that the let-7 miRNA family has great potential in regulating follicular atresia, but further studies are needed to fully elucidate its underlying mechanisms.
Gene expression patterns can be inferred from differences in expression levels [
69]. In this study, we analysed the expression levels of miRNAs and found 254 differentially expressed miRNAs across the four developmental stages. Among them, the majority of miRNAs exhibited differential expression exclusively in the D1 vs. M2, D1 vs. M4, and D1 vs. M6 comparison groups, with the number of DE miRNAs exceeding 170 in each of these groups. Therefore, we hypothesized that the period between D1 and M2 is a critical period for ovarian development, whereas the expression patterns of miRNAs and their main functions were similar in stages M2, M4 and M6, which may be due to these miRNAs stabilizing follicular development and maturation in goats after the M2 stage. We identified 15 miRNAs with high expression levels at all stages (Fig.
4). miR-148a had the highest average expression level and was reported to be the most highly expressed miRNA in follicular fluid extracellular vesicles of large and small follicles in goats [
70]. miR-10a plays a key role in inducing apoptosis and inhibiting cell proliferation in ovarian GCs by inhibiting the brain-derived neurotrophic factor (BDNF) and TGF-β pathways [
71]. In addition, miR-10a also regulates lipid metabolism and steroid hormone synthesis in sheep GCs by targeting the PTEN-AKT/WNT pathway [
72]. Let-7a-5p, let-7c-5p, let-7b-5p, and let-7e-5p belong to the conserved let-7 family. As key regulators of gene expression, let-7 miRNAs are highly expressed in mammalian gonadal tissues [
67,
73], and have been shown to regulate sperm formation and oocyte maturation [
74,
75], as well as participate in the initial regulation of mammalian sexual maturation [
76‐
78]. This study showed that the expression levels of let-7a/b were significantly higher than those of other let-7 miRNAs in tissues associated with ovarian development, suggesting that let-7a/b may play a more important role in ovarian development, which is in line with reports that a large number of miRNAs have been identified in mammalian ovaries. This is consistent with previous findings that let-7a and let-7b are two of the ten most abundant miRNAs in human, cow, and sheep ovaries [
79‐
81]. These findings suggest that different members of let-7 miRNAs may have distinct roles during animal ovarian development and that their expression is stage dependent. Moreover, miR-125 is produced from the polycistronic let-7 complex gene region, and its expression disrupts the Notch-activated positive feedback loop, shifting the cell from receiving Notch signals to sending Notch signals [
82]. Angiogenesis plays a crucial role in ovarian development and the restoration of ovarian function [
83]. miR-126-3p has been shown to be important for vascular integrity and angiogenesis [
84,
85].
To investigate the functions of DE miRNAs with distinct expression profiles, we analysed the expression patterns of DE miRNAs and identified three clusters (Fig.
5). The results revealed that the expression profiles of DE miRNAs were correlated with the developmental stages of goats. The miRNAs in Cluster 1 were upregulated from stage D1 to stage M2, with higher expression levels in the M2 group. Their target genes were significantly enriched in the MAPK signaling pathway, calcium signaling pathway, Ras signaling pathway, oestrogen signaling pathway, and glycerophospholipid metabolism. An increase in Ca2 + /CaMKII may activate the ERK and MAPK pathways in cumulus cells, promoting amphiregulin and epiregulin expression as well as progesterone production and, thus influencing cell cycle progression [
86]. EGF induces CREB activity via the MAPK3/1 and Ca
2+/CaMKII signaling pathways, promoting the expression of expansion-related genes and the expansion of cumulus cells [
87]. Impairment of FGF23 function prior to primordial follicle formation leads to activation of the MAPK signaling pathway in mouse oocytes, which induces massive apoptosis of oocytes in the ovary. Previous studies have shown that the above signaling pathway may be involved in gonadal development, ovarian steroidogenesis and oocyte maturation [
88]. The DE miRNAs in Cluster 2 were most highly expressed in the D1 group, and their target genes were mainly enriched in signaling pathways related to cellular processes, such as metabolic pathways, neutrophil extracellular trap formation, amino sugar and nucleotide sugar metabolism, and folate biosynthesis, with the highest number of enriched genes being metabolic pathways. Rapid morphological growth of the ovary and a significant increase in ovarian weight and volume (5 times higher than at birth) have been reported in goats during 0–30 days after birth [
2]. This is consistent with our experimental results that the expression of genes related to organ and tissue growth and metabolic processes is higher in the D1 group after goat birth in goats. Cluster 3 showed that the expression levels of DE miRNAs increased from the D1 to M4 stages, with higher expression levels in the M4 group. Their target genes were significantly enriched in the RIG-I-like receptor signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, Notch signaling pathway and Rap1 signaling pathway, as well as other metabolic-related pathways. The PI3k-Akt signaling pathway induces the transcription of target genes and mediates angiogenesis, cell invasion, metastasis, proliferation and apoptosis [
89]. During follicle development, the PI3K signaling pathway controls primordial follicle activation through FOXO3. During the assembly of primordial follicles, FOXO3 is nonphosphorylated and localized in the nucleus, where it acts as an inhibitor of primordial follicle activation. Activation of PI3K/AKT leads to the phosphorylation of FOXO3 and its export from the nucleus, which triggers the activation of primordial follicles [
90]. The Notch signaling pathway is an evolutionarily conserved pathway that plays a major role in cell proliferation, differentiation, migration, adhesion, and apoptosis, among many other cellular processes. It has been shown that constitutive Notch signaling in adult transgenic mice inhibits bFGF-induced angiogenesis and follicular development [
91] and that granulosa cell proliferation is also dependent on Notch signaling [
92]. The Rap1 signaling pathway regulates cell adhesion, cell‒cell junction formation, and cell polarity by cycling between inactive GDP-bound and active GTP-bound conformations [
93]. Therefore, our experimental results suggest that in the early postnatal period of Jining Grey goats, ovarian development may be related to cell growth, which is mainly manifested by the rapid growth of ovarian morphology. During sexual maturation, ovarian function and performance were enhanced, as evidenced by the development and maturation of follicles and ovaries. This is similar to the observations reported by Shi [
2]. Overall, spatially and temporally specific expression of miRNAs in the ovaries of Jining Grey goats plays a critical role in ovarian development and early maturation.
We constructed a regulatory network of miRNAs and target genes associated with ovarian development. Chi-miR-1343 had the highest number of target genes, and its targets were significantly enriched in the PI3K-Akt signaling pathway. miR-1343 has been reported to play an important functional role in bovine follicular development, as its expression level increases during the transition from secondary follicles to preovulation [
94]. Moreover, TGFBR1, a type I receptor in the TGF-β signaling pathway, impedes GC apoptosis of GCs in the ovary, and miR-1343 silences the expression of TGFBR1. Interestingly, miR-1343 was found to downregulate TGFBR1 expression, which promotes the apoptosis pathway and inhibits cell proliferation [
95]. Our results showed that miR-1343 was expressed at a lower level in the D1 group and at higher levels in the M2, M4 and M6 groups, which further indicates that its function may be related to the maintenance of ovarian physiological function, but more experimental evidence is still needed. miR-331-3p is a member of the miR-331 family and has been shown to be a tumour suppressor miRNA [
96]. It has been reported that miR-331-3p targets HER2 through the PI3K/Akt and extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) pathways, thereby inhibiting the proliferation of colorectal cancer cells [
97]. In gastric cancer, miR-331-3p directly targeted E2F1 and induced cell growth defects [
98]. It was found that miR-331-3p could inhibit tumour cell invasion and metastasis by regulating ErbB2 and VAV2 to attenuate the epithelial–mesenchymal transition in non-small cell lung cancer [
99]. These findings indicate that miR-331-3p plays an important role in tumour suppression during cancer development. However, the role of miR-331-3p in ovarian development is still unclear and requires further research and exploration. miR-342-5p is an intron hosted in the Ena/Vasodilator-Stimulated Phosphoprotein-Like (Ena/VASP-like, EVL) gene miRNA, belonging to the Ena/VASP family, is involved in actin cytoskeleton remodelling and has been reported to enhance ERK-maintained cell proliferation [
100]. As a downstream effector of Notch signaling, miR-342-5p regulates the proliferation and differentiation of mouse neural stem cells [
101]. It was previously reported that miR-296-3p is expressed in mouse ovaries [
102], inhibits cell plasticity in different tumour lines [
103], and promotes apoptosis in liver [
104] and mammalian pancreatic α cells [
105]. Furthermore, miR-296 was observed to be epigenetically regulated as part of the imprinted Gnas/GNAS clusters [
106]. miR-296-3p, miR-328-3p, miR-128-5p and miR-34a all target NOTCH4, which was significantly enriched in the Notch signaling pathway. The Notch pathway comprises a conserved family of transmembrane receptors that interact with a number of specific ligands to regulate cell fate [
107]. Notch signaling plays a key role in many developmental processes, affecting cellular differentiation, proliferation, and apoptosis [
108,
109]. In addition, NOTCH4 has been identified as a novel factor involved in the regulation of angiogenesis [
110]. Overall, we predicted that core miRNAs and potential target genes may be involved in the regulation of ovarian development.