Puberty onset is crucial for animal reproduction, which could affect on the economics of the animal industry
(Verma et al., 2019). Therefore, puberty onset has become an area of great concern. Recent studies have shown that DNA methylation decreases during the onset of puberty
(Yang et al., 2018).
DNA methylation is an important epigenetic modification that occurs in the CpG islands and the CpG-rich regions (Ma and He. 2014). The methylation status of the promoter region affects the transcription of the corresponding gene
(Lomniczi et al., 2015). In mammals, DNMT1 is a major DNA methyltransferase, which is involved in the process of gene methylation
(Sperlazza et al., 2017). Changes in DNA methylation during puberty might affect specific genes expression in the hypothalamus and thereby regulate the process of puberty onset
(Javed et al., 2015). MeCP2 is a methylated CpG binding protein, which is also the first discovered protein in the MBD family
(Tsuji-Hosokawa et al., 2017). It contains 70 MBD core amino acids, which is characterized as a transcriptional repression domain
(Meehan et al., 1992). Studies have shown that MeCP2 can block the function of RNA polymerase to inhibit the transcription process
(Kinde et al., 2016). At present, the functions of MeCP2 and DNMT1 in the hypothalamus of ewes are unclear and need to be investigated further.
Gonadotropin-releasing hormone (GnRH) is an important regulatory hormone leads to the puberty onset.
KiSS-1 is a key gene involved in puberty onset. Kisspeptin, a product of the
KiSS-1, plays an important role in regulating GnRH secretion in the hypothalamus regulating puberty onset in female animals
(Javed et al., 2015).
In this study, we explore the expression and distribution of DNMT1 and MeCP2 and their relationship with GnRH and kisspeptin, which are the crucial genes for regulating puberty onset. This study will provide a reference for the mechanism of DNA methylation during puberty onset.
Animals and experimental design
This work was conducted at the College of Animal Science and Technology, Jilin Agricultural University, Changchun, China. The protocols of the current study were approved by the Ethical Committee of Jilin Agricultural University, Changchun, China. The ewes from the sheep farm in Changchun City, China were divided into three groups: peri-puberty (90d), pre-puberty (120d) and puberty. Starting from day 120, the teaser rams were used to screen and identify ewes every day. Starting from day 120, the teaser rams were used to screen and identify ewes every day. Three female small-tailed Han sheep in each group were selected. Their progesterone levels in the blood of 90 days (peri-puberty), 120 days (pre-puberty) and puberty sheep were determined using the ELISA Kit (RENJIEBIO, Shanghai, China). The sign of the puberty onset in sheep is that the progesterone concentration reaches 1 ng/mL. Subsequently, all small-tailed Han sheep were slaughtered and the hypothalamus was harvested for further experiments.
Sample collection and preservation
The hypothalamus tissue samples of the small-tailed Han sheep at different stages of puberty onset were harvested. Treating with excessive pentobarbital sodium caused death and subsequently, the hypothalamus was quickly removed after opening the cranium. The collected hypothalamus tissue samples were mainly from the front-end stops at the optic chiasm and the back-end stops at the corpus albicans. One part of the tissue was frozen in liquid nitrogen for total RNA extraction and the other part was used to prepare frozen sections of the hypothalamus for double-labeling immunofluorescence.
RNA isolation and cDNA synthesis
Total RNA was extracted from the hypothalamic tissue samples using RNAiso Plus (Takara, Tokyo, Japan). Total RNA (1 μg) was transcribed into the cDNA using the PrimeScript RT reagent Kit with gDNA Eraser (Takara, Tokyo, Japan).
qRT-PCR
Real-time quantitative PCR was performed using a SYBR® Premix Ex TaqTM II (Takara, Tokyo, Japan) on a Real-Time PCR detection system (Agilent StrataGene Mx3005P, Santa Clara, CA, USA). The primers sequences of
DNMT1,
MeCP2,
GnRH and
KiSS-1 are listed in Table 1. Relative gene expression was calculated using the 2DDCt method.
Double-labeling immunofluorescence
The tissue slices were revived using the Quick Antigen Retrieval Solution for Frozen Sections (Beyotime Institute of Biotechnology, China) and washed with TBST (0.1% Tween20, 1 × TBS) for 5 min. They were then fixed with 4% paraformaldehyde at 4 °C and then Immunol Staining Blocking Buffer (Beyotime Institute of Biotechnology, China) was added to the fixed tissue sections. The sections were incubated for 1 h at 37 °C in the wet box to reduce the non-specific background staining and subsequently incubated for overnight at 4 °C with the first primary antibody. Then, the sections were rinsed in TBST for 3 times at room temperature. Further, the tissue sections were incubated with the corresponding secondary antibody for 1 h at 37°C. Thereafter, the second primary antibody was added directly to the samples and incubated overnight at 4°C. The sections were rinsed in TBST 3 times at room temperature and were further incubated with the corresponding secondary antibody for 1 h at 37°C. Finally, the sections were stained with DAPI (4’, 6-diamidino-2-phenylindole) stain solution for 5 min to induce nuclear counter-staining. The Antifade Mounting Medium (Beyotime Institute of Biotechnology, China) was used to mount the samples. The images were captured and observed under the Laser scanning confocal microscope. All antibodies were purchased from commercial suppliers are indicated in Table 2.
Statistical analysis
The data are presented as mean ± standard error of the mean (SEM). The analysis for significance was performed by one-way analysis of variance (ANOVA) accompanied by Duncan’s Multiple Range Test (DMRT). Different letters of a, b and c indicate a significant difference (
p<0.05). All statistical analyses were performed using SPSS for Windows, version 18.0 (SPSS Inc., Chicago, IL, USA).
The mRNA expression of GnRH, KiSS-1, DNMT1 and MeCP2 in the hypothalamus of ewes
Primer sequences used for qRT-PCR are listed in Table 1. As shown in Fig 1, the expression of
GnRH and
KiSS-1 are significantly increased, while the expression of
DNMT1 and
MeCP2 are significantly decreased during pre-puberty and puberty in the hypothalamus of ewes (
p<0.05).
Expression and distribution of DNMT1 and GnRH in hypothalamic POA and ARC in ewes
Antibodies used for double-labeling immunofluorescence in this study are listed in Table 2. Results of double-labeling immunofluorescence indicate that both DNMT1 and GnRH are uniformly distributed in hypothalamic POA and ARC and exhibit a co-expression pattern (Fig 2A and C). As shown in Fig 2B and D, the rate of co-expressing positive cells is significantly decreased at puberty in hypothalamic POA, while it increased significantly in hypothalamic ARC at puberty (
p<0.05).
Expression and distribution of MeCP2 and GnRH in hypothalamic POA and ARC in ewes
Results of double-labeling immunofluorescence indicate that both MeCP2 and GnRH are also uniformly distributed in hypothalamic POA and ARC and exhibits a co-expression pattern (Fig 3A and C). As shown in Fig 3B, the rate of co-expressing positive cells is significantly decreased at puberty in hypothalamic POA (
p<0.05). Besides, in Fig 3D, it has been shown that the rate of co-expressing positive cells is increased significantly in hypothalamic ARC at puberty (
p<0.05).
Expression and distribution of DNMT1 and kisspeptin in hypothalamic POA and ARC in ewes
As shown in Fig 4, DNMT1 is distributed in hypothalamic POA and ARC. Although kisspeptin is rarely expressed in POA and abundantly expressed in ARC, it is also found to be co-expressed with DNMT1 in the same nucleus. The rate of co-expressing positive cells decreased significantly at puberty in hypothalamic POA, but increased significantly at pre-puberty and puberty in hypothalamic ARC(
p<0.05, Fig 4B and D).
Expression and distribution of MeCP2 and kisspeptin in hypothalamic POA and ARC in ewes
As shown in Fig 5, MeCP2 is distributed in hypothalamic POA and ARC. Although kisspeptin is rarely expressed in POA and abundantly expressed in ARC, it is also found to be co-expressed with MeCP2 in the same nucleus. In addition, the rate of co-expressing positive cells decreased significantly at puberty in hypothalamic POA, but increased significantly at pre-puberty and puberty in hypothalamic ARC (
p<0.05, Fig 5B and D).
Puberty onset can be regulated by hypothalamic DNA methylation
(Yang et al., 2018). However, the mechanism remains unclear. IOur results showed that DNMT1 and MeCP2 were expressed in the hypothalamic preoptic area (POA) and arcuate nucleus (ARC). Besides, DNMT1 and MeCP2 have a co-expression patterns with GnRH and kisspeptin in hypothalamic ARC and POA.
Our results demonstrated that the high expression of
GnRH and
KiSS-1 promotes the puberty onset of the small-tailed Han sheep.
DNMT1 and
MeCP2 were expressed in the hypothalamus and the expression decreased at puberty.
GnRH and kisspeptin expression has been found to increase during puberty in the hypothalamus
(Hellier et al., 2019). DNMT1 expression decreases during puberty onset in female rhesus monkeys
(Roth et al., 2007). Additionally, MeCP2 regulated many genes expression in the hypothalamus
(Chahrour et al., 2008).
In this study, we also found that DNMT1 and MeCP2 are expressed in the hypothalamic POA and ARC. POA and ARC are the key sites for GnRH neurons to receive feedback
(Wankowska and Polkowska. 2009). It has been reported that kisspeptin is abundantly expressed in hypothalamic ARC
(Tomori et al., 2017). Studies have demonstrated that the number of GnRH cell bodies in hypothalamic POA is reduced during puberty in ewes
(Gabel et al., 2015). DNA methylation levels in the GnRH promoter region of rhesus monkeys were found to be reduced during puberty onset
(Kurian and Terasawa. 2013). Kisspeptin is shown to be involved in puberty onset in rats and the distribution in the ventromedial nucleus and ARC is higher than that in the paraventricular nucleus
(Ruiz-Pino et al., 2019). However, there are few reports on the distribution of DNMT1 and MeCP2 in the hypothalamus.
