SNAT2 Mediated Regulation Of E₂ And P₄ On Proliferation And Lactation Of Goat Mammary Epithelial Cells

The development of goat mammary gland is strictly controlled by ovarian hormones especially estrogen and progesterone（P₄）.Sodium-dependent neutral amino acid transporter type A System 2（SNAT2）is reported to be essential for tissue development and is found to be highly expressed in mammary tissue of mice during late gestation and lactation.It is found that SNAT2 transcription is regulated by 17β-estradiol（E₂）,however,it is unclear whether SNAT2 mediates the regulation of E₂ and P₄ on mammary gland development and lactation in goats.In this study,we elucidate the role and mechanism of SNAT2-mediated E₂ and P₄ in regulating mammary gland development and lactation at the cellular level in vivo and in vitro.The main contents are as follows:（1）To explore the effects of different concentrations of E₂ and P₄ on proliferation of goat mammary epithelial cells（GMECs）,SNAT2 mRNA and protein expression.Firstly,different concentrations of E₂ and P₄ were used to treat GMECs,CCK-8 and EdU were used to detect cell proliferation of GMECs,and then mRNA and protein expression of SNAT2 were detected by qRT-PCR and Western blot.The results showed that E₂ and P₄ accelerated the proliferation of GMECs in a dose-dependent manner,and the mRNA and protein expression levels of SNAT2 showed the same trend with the proliferation of GMECs under the action of E₂ and P₄.The result suggests that SNAT2 may be involved in E₂ and P₄ regulation of GMECs proliferation;（2）To clarify the role and mechanism of SNAT2 in regulation of GMECs proliferation by E₂ and P₄ through interference or overexpression of SNAT2.Under the optimal concentrations of E₂ and P₄,CCK8 and EdU were used to detect the proliferation of GMECs cells after the interference and overexpression of SNAT2.The results showed that the proliferation of GMECs induced by E₂ and P₄ was inhibited and enhanced When SNAT2 is disturbed and overexpressed.In addition,the expression of mTOR and its downstream signaling molecules decreased or increased significantly after SNAT2 interference or overexpression（P<0.05）.These results indicate that SNAT2 mediates the regulation of E₂ and P₄ on the proliferation of GMECs,and mTOR signaling pathway may be the molecular pathway in SNAT2;（3）Rapamycin was used to block mTOR signaling pathway to clarify that mTOR signaling pathway is the pathway that SNAT2-mediated E₂ and P₄ regulate the proliferation of GMECs.At the optimal concentrations of E₂ and P₄,the promoting effect of overexpression SNAT2 on GMECs was weakened after the mTOR signaling pathway was blocked by addition of rapamycin.These results indicate that SNAT2 regulates GMECs proliferation by influencing the mTOR signaling pathway to mediate E₂ and P₄;（4）Finally,it is revealed whether SNAT2 mediates E₂ and P₄ regulation of lactation through in vitro induction of lactation.In vitro GMECs were given a certain concentration of E₂ and P₄ to simulate the hormone levels on 120th day of pregnancy（Pd120）,Pd135,Pd140,Pd147,and the second day of lactation（Ld2）,Ld30 of goats,the levels of SNAT2 and β-casein in GMECs were detected by Western blot.The result show that except simulated Ld2,SNAT2 during gestation and late lactation showed the same trend withβ-casein,the levels of Pd120～Pd135 and Pd140～Pd147 are significantly increased,and the levels of Pd135～Pd140 are significantly decreased,but the levels of SNAT2 are significantly decreased in Pd147～Ld2,but the levels of β-casein are significantly increased.Through inducing lactation of GMECs in simulated Pd 147 and Ld2 periods,SNAT2 and β-casein were significantly increased after the addition of various lactation related hormones.After overexpression or interference with SNAT2,β-casein levels also increased or decreased（P<0.05）.These results indicate that SNAT2 is also involved in the regulation of GMECs milk protein synthesis by various lactation related hormones.In conclusion,we found that SNAT2 regulates the proliferation of GMECs by influencing mTOR signaling pathway to mediate E₂ and P₄.During pregnancy,SNAT2 may mediate not only cell proliferation but also E₂/P₄ regulation of GMECs milk protein synthesis.In lactation period,SNAT2 may mediate other prolactin hormones rather than E₂ and P₄ to regulate milk protein synthesis.From the perspective of amino acid transport,this study revealed the role and mechanism of reproductive hormones in the regulation of mammary gland development and lactation,providing a new idea for clinical improvement of mammary development and milk synthesis.