Expression of Cell Proliferation Regulatory Factors Bricd5, Tnfrsf21, Cdk1 Correlates With Expression of Clock Gene Cry1 in Testis of Hu Rams During Puberty


 The cry1 gene plays an important role in mammalian ontogeny and widely exists in various tissues. Studies showed that cry1 gene was expressed in testis and participated in the regulation of mammalian reproductive activities. To identify the genes that are positively correlated with cry1 gene expression in the testis of rams during the first estrus, and to explore the relationship between cry1 gene and the number of spermatogenic cells. qRT-PCR was used to detect the mRNA transcription levels of cry1, bricd5, tnfrsf21, cdk1 and tcfl5 in testicular tissues of Hu sheep at 0, 30, 60, 90, 120, 150 and 180 days postpartum (dpp) . In testicular tissue, the expression of cry1 mRNA increased with age in testis showed an upward trend, and increased significantly in the estrous phase. The cry1 mRNA at 180dpp was significantly higher than that of 90-day-old testis (p < 0.05). The expression of cell proliferation related genes bricd5, tnfrsf21, cdk1, cry1 upstream specific transcription factor tcfl5 was identified, and the mRNA expression at 180 dpp was significantly higher than that of 3-month-old testis (p < 0.05) The expression level of cry1 mRNA was similar to that of cry1. cry1 gene is highly expressed in the testis of sheep during puberty, and has a significant correlation with the proliferation of spermatogenic cells.

Introduction 49 The first estrus period of male animals refers to the first estrus of male animals and 50 the release of fertilized sperm, which shows a complete behavioral sequence. It is a sign 51 of animals from birth to reproductive capacity. The external performance of estrus is that 52 the body develops most rapidly and produces sexual impulse; the internal performance is 53 that the release of hypothalamic gonadotropin-releasing hormone and the secretion of 54 anterior pituitary hormone begin to strengthen, and the production of androgen in testis 55 increases, which promotes the production of mature germ cells [1]. The occurrence of 56 estrus affects the life-long reproductive performance of livestock and poultry [2], in which 57 spermatogenesis is an important part of the occurrence of estrus. 58 So far, 14 clock genes have been found, namely clock, bmal1, per1, per2, per3, dec1, 59 dec2, cry1, cry2, tim, ckiε, rorα, rev-erbα and npas2. The clock gene including cry1 gene 60 is expressed in the suprachiasmatic nucleus of hypothalamus and almost all peripheral 61 tissues of mammals [3]. Different clock genes can affect the physiological and 62 biochemical functions of cells by regulating different downstream clock genes. In recent 63 years, the research on the function of clock gene in mammalian reproductive organs such 64 as ovary, uterus and testis has become a hot spot [4][5][6][7], indicating that clock gene is related 65 to mammalian reproductive activities [8]. The change of clock gene expression in 66 reproductive organs can affect the level of steroid reproductive hormones [9-10], and may 67 affect sperm motility and spermatogenesis. In recent years, there have been similar reports 68 that the Clock gene can affect the activity acrosin activity [11]; CLOCK combines with 69 SF3B3 and RANBP9 proteins to form a complex, and participates in the alternative 70 splicing of genes related to spermatogenesis [12]. 71 The cry1 gene is one of the important negative feedback genes in the biological 72 clock system. In addition to participating in the regulation of circadian rhythm, it may 73 also be related to DNA damage repair, adipogenesis, follicular development and 74 spermatogenesis. For example, the cry1 gene regulates the classical Wnt/β -catenin 75 signaling pathway regulates adipogenic differentiation; after knockout of mouse cry1 76 gene, the number of spermatogenic cell apoptosis increased and sperm count decreased 77 significantly [13]; And, cry1 gene, as a negative regulator of hypoxia inducible factor 78 HIF1-α, participates in carcinogenesis [14]. 79 It's found that cry1 gene was mainly expressed in Leydig cells, Sertoli cells and 80 spermatogenic cells in mouse testis [13]; The similar result is also observed in other 81 species, such as camels [15]. These results suggest that cry1 gene may play an important 82 role in spermatogenesis. Therefore, in this study, we detected the expression levels of cry1 83 upstream transcription factor and related genes before and after puberty, and analyzed 84 their expression patterns, and observed the number of spermatogenic cells at all levels of 85 testicular immune tissue in Hu sheep, and analyzed the relationship between them. In 86 order to explore the effect of testicular core clock gene on the development of 87 spermatogenic cells in male sheep, the expression level of cry1, a core clock gene, was 88 studied. The results are of great significance to clarify the function of cry1 gene in the 89 initiation of estrus. 90

Materials and methods 91
Sampling 92 For this study, 21 male Hu sheep with average ages of 0, 30, 60, 90, 120, 150 and 93 180 days old were randomly selected and purchased from the Zhiqinghe agriculture and 94 animal husbandry Co., Ltd. (Yichang, Hubei, China). Each age group comprised three 95 sheep (Unpublished). The sheep were raised in the same environmental conditions. The 96 sheep were sedated after intramuscular injection by using 0.1ml/kg su mian xin also 97 known as xylazine hydrochloride (Shengda, Changchun, Jilin, China), and the testicles 98 were surgically collected and weighed using an electronic balance. One testis was fixed 99 with 4% paraformaldehyde, and the other was frozen immediately in liquid nitrogen and 100 then stored at −80°C. 101

HE staining of testicular tissue 102
The testis was washed with 0.9% saline, fixed with 4% paraformaldehyde for 48h at primer3plus.com/cgi-bin/dev/primer3plus.cgi). The relative gene expression levels 124 were calculated using the 2 −∆∆Ct methods using β-actin as the reference genes. 125

Sequence Characterization of TF binding sites of cry1 in sheep 126
The promoter sequences prediction, a 5.00 kb genomic region upstream of the sheep 127 cry1 gene, was carried out using Jaspar online tools(http://jaspar.genereg.net/ ).The

Statistical analysis 139
The statistical significance of the results was analyzed through one-way ANOVA 140 followed by Duncan's multiple comparison. Statistical significance was defined at the 141 level of p < 0.05. Analyses were performed by applying SAS® OnDemand for Academics 142 (SAS Institute, Inc., Cary, NC, USA). Results were presented as means ± standard 143 deviation (SD). 144

Histological analysis of testis during different postnatal stages 146
The results of HE staining of testicular tissue from 1 to 180 days postpartum (dpp) 147 were shown in Figure 1. From the first day to the 60th day, there were only spermatogonia 148 and Sertoli cells in the seminiferous tubules. At the 90th day, spermatogonia began to 149 proliferate in some seminiferous tubules, and a few primary spermatocytes appeared. At 150 the 120th day, there were cavities in the seminiferous tubules, and the number of 151 spermatocytes, early spermatocytes and a few spermatozoa were significantly higher than 152 those at the 90th day. The number of layers of spermatogenic epithelial cells increased 153 and the number of spermatogenic cells at all levels increased. 154 We measured the expression of cry1, bricd5, tnfrsf21 and cdk1 by real time PCR for 157 the testis tissues of rams at 1-180 dpp. It can be seen that the expression level of cry1 158 gene in Hu sheep testis showed an upward trend from 0-180 dpp, and the relative 159 expression level of cry1 gene in Hu sheep testis at 6 months old was significantly higher 160 than that at 90 dpp (p < 0.05); the relative expression level of bricd5 gene in Hu sheep 161 testis showed an upward trend after 90 dpp, and the relative expression level of bricd5 162 gene at 120-180 dpp was significantly higher than that at 90 dpp. The relative expression 163 of tnfrsf21 gene in the testis of Hu sheep aged 0-180 dpp showed an upward trend, and 164 the relative expression of tnfrsf21 gene in the testis of Hu sheep aged 180 dpp was 165 significantly higher than that in the testis of Hu sheep aged 90 dpp (p < 0.05); the relative 166 expression of cdk1 gene in the testis of Hu sheep aged 90 dpp showed an upward trend, 167 and the relative expression of cdk1 gene in the testis of Hu sheep aged 150 dpp was 168 significantly higher than that in the testis of Hu sheep aged 90 dpp. The expression level 169 was significantly increased (p < 0.05). At each time point, the same letters indicated no 170 significant difference, while different letters indicated significant difference (p < 0.05). 171  Table 1). 191 There was a significant regression relationship between the expression level of cry1 192 gene and the number of germ cells in testis of Hu sheep from birth to 180 dpp (p < 0.05). 193 In the process of spermatogenesis, the fitting degree of regression model between cry1 194 gene expression level and spermatogonia number, primary spermatocyte number, 195 secondary spermatocyte number, spermatocyte and sperm decreased in turn (p < 0.05, 196 Rspt 2 = 0.4441, Rpri 2 = 0.4232, Rsec 2 = 0.4433) ( Table 1). 197

Classification of transcription factors of the 5' upstream region expressed in testis 198
A total of 144 transcription factors were screened within 5000 bp upstream of the 199 start codon of cry1 gene (Fig. 3a). According to the transcriptome sequencing data, the The function loss of cry1 inhibited the (MAPK) -Erk signaling pathway, 257 which affected the proliferation of osteoblasts [22]. After the interfe rence of 258 cry1 gene expression in testis supporting cells, the expression of MT1 protein 259 also decreased obviously [23]. After overexpression of cry1 in ovarian