Effects Of RAD6B Deficit On Growth And Female Reproduction In Mice

Methods:1)RAD6B^-/-mice were obtained by crossbreeding with RAD6B^+/-mice in the same litter.The offspring of backcrossed parents increased RAD6B^-/-rate,and wild-type mice in the same litter were used as control.2)Weight changes of mice and related indicators of female reproduction were monitored from birth.3)Pituitary sections of mice were frozen and stained with HE to compare the difference between RAD6B^+/+and RAD6B^-/-mice in pituitary volume and quality.The secretion of growth and reproductive hormones in peripheral serum of mice was measured by ELISA;5)RNA was extracted from pituitary tissue,and the expression of five specific pituitary cells was measured by RT-PCR;6)CT was performed on RAD6B^+/+and RAD6B^-/-mice to explore the difference of growth and development;7)primary pituitary cells were isolated and cultured,and CCK8 cells were measured.Growth curve and hormone secretion;DNA damage of primary pituitary cells induced by 500uM H₂O₂,growth trend and hormone secretion after induction were determined by CCK8;8)the number of beta-galactosidase positive cells in pituitary cells was detected by frozen section beta-galactosidase?beta-Gal?staining;9)immunofluorescence?IF?staining was used to observe the distribution density of growth hormone?GH?cells and P16positive cells.The expression levels of GH?growth hormone?,P21 and PRLR?prolactin receptor?were measured by Western Blot.Results:The body weight of RAD6B knockout mice at birth was not significantly different from that of wild type mice,but the body weight difference increased gradually from 1 week old,and the most significant difference was between 3 and 4weeks old?P<0.01?;however,the body weight difference decreased gradually from7 weeks old until disappeared?about 10 weeks old?.Pituitary plays an important role in growth and development,metabolism,reproduction and so on.Therefore,we took mouse pituitary for histological and morphological differences.The results of pituitary weighing and HE showed that RAD6B^-/-mouse pituitary tissue was light in weight,the total volume of pituitary was small,and the volume of neurohypophysis and pituitary lobe was not significantly different from WT,so the difference of pituitary volume was mainly in the anterior pituitary.In addition,the pituitary GH?growth hormone?gene expression was down-regulated?P<0.01?,and the pituitary GH?growth hormone?immunofluorescence staining?IF?results showed that the number of pituitary GH cells decreased significantly?P<0.001?,and the secretion of growth hormone in peripheral blood also decreased significantly?P<0.05?.Hepatogenic IGF-1?insulin-like factor-1?is the downstream protein of GH?growth hormone?.The effect of GH on the growth of somatic cells is achieved through the direct and indirect effects of GH.The level of IGF-1 mRNA measured by RT-PCR showed that pituitary growth hormone?GH?and hepatic IGF-1 mRNA expression decreased by about 50%in 3-week-old mice?GH,P<0.01,IGF-1,P<0.05?.GH/IGF-1 axis is a key component of GH?growth hormone?/IGF-1?insulin-like factor?axis.GH/IGF-1 axis plays a key regulatory role in growth and reproduction,and has an important impact on the growth of spine and tibia.CT results showed that:1)Compared with RAD6B^-/-mice,the longitudinal length of bone?portal teeth to caudal end of sacrum?was significantly shorter at 4 weeks of age than RAD6B^+/+?P<0.01?,and the difference disappeared at 10 weeks of age.The femoral and tibial lengths of 24-week-old?6-month-old?and 4-week-old RAD6B^-/-mice were also shorter than those of RAD6B^+/+mice?P<0.01?,and the differences of femoral and tibial lengths of 10-week-old mice were gradually reduced and tended to be equal?P<0.05?;3)The results of spinal curvature measurement showed that the spinal curvature of KO mice at 4-week-old was slightly higher than that of WT?P<0.05?,and that at 10-week-old?P<0.05?.The curvature gradually disappeared,but the curvature of RAD6B KO mice at 24-week-old?6-month-old?increased gradually?P<0.01?.In addition,we also found that the reproductive organs of 4-week-old RAD6B-/-mice were hypoplasia,and the mammary glands were small and not prominent,and the adjacent mammary glands were discontinuous.Pituitary prolactin plays an important role in the development,stimulation and maintenance of mammary gland.So we measured the expression of pituitary prolactin?PRL?mrna.The results showed that the expression of PRL mRNA increased slightly?P>0.05?.Then we extracted the protein and RNA of mammary gland tissue.We measured the expression of PRL receptor?Prlr?protein and RNA by WB and ELISA.The results showed that the expression of PRL mRNA increased slightly?P>0.05?.The results showed that the expression of prolactin receptor protein and mRNA in RAD6B^-/-mice decreased?P<0.01?,and the reproductive interval cycle of RAD6B^-/-female mice prolonged and the litter yield decreased.Primary pituitary cells were cultured and DNA damage was induced by ROS?H₂O₂?.We found that the proliferation of RAD6B^-/-pituitary cells without any treatment was weaker than that of RAD6B^+/+?P<0.05?.Cell growth curve was measured after DNA damage was induced by 500 uM H2O2.The results showed that the proliferation of RAD6B^-/-pituitary primary cells increased at 2hours?P>0.05?,but at 4 hours the proliferation energy was increased.The secretion of RAD6B^+/+and GH?growth hormone?of RAD6B^-/-pituitary primary cells decreased significantly after adding 500 uM H₂O₂,but the decrease of RAD6B^-/-pituitary primary cells was more obvious?P<0.01?.Then we stained the pituitary tissues of 6-month-old mice with beta-Gal and P16 fluorescence staining and extracted pituitary protein to determine the expression of P21 protein.The results showed that the number of beta-Gal positive cells in RAD6B KO mice was significantly more than that in WT?P<0.0001?,and the number of P16 positive cells was also more than that in WT?P<0.01?,while the expression of KO in P21 was less than that in WT?P<0.01?.Conclusion:1)The weight of RAD6B-/-mice decreased,the volume of anterior pituitary decreased,and the proliferation of primary cells in anterior pituitary decreased;the number of growth promoting hormone cells in anterior pituitary decreased,and the secretion of growth hormone decreased.The above results showed that the deletion of RAD6B gene caused hypoplasia and dysfunction of pituitary in mice.2)The decrease of GH?growth hormone?and the expression of insulin-like factor-1?insulin-like factor-1?in liver may cause GH/IGF-1 axis dysfunction,leading to growth retardation,reproductive organ dysplasia and puberty delay?sexual maturity delay?3,prolactin receptor?PRLR?protein and gene expression decline,leading to immature breast development.4)After adding ROS?H₂O₂?DNA damage inducer,the proliferation of primary pituitary cells in anterior pituitary decreased significantly and GH?growth hormone?secretion decreased significantly,indicating that RAD6B gene defect increased the sensitivity of anterior pituitary cells to DNA damage,and led to the decrease of pituitary growth promoting hormone cell function by 5%,and increased the number of beta-Gal and P16 positive cells in pituitary tissues of RAD6B^-/-mice.These results suggest that the deletion of RAD6B gene results in senescence of anterior pituitary cells.