Establishment Of Isl1 Pituitary Specific Deletion Mouse Model And The Effects Of Pituitary Isl1 Knockout On The Mouse Fertility

Gonadotropins,including follicle stimulating hormone(FSH)and luteining hormone(LH),are the key hormones to regulate the animal reproductive activities.It remains unclear about the molecular and cellular mechanisms of FSH and LH synthesis and secretion,although our previous studies in vitro have shown that LIM homeodomain transcription factor Isl1 is a key factor regulating FSH and LH synthesis and secretion in LPT2 cells.In addition,it is difficult to prove the physiological function of Isl1 because the Isl1 mutant mice die at early embryonic development.The present study was thus designed to establish inducible and pituitary special Isl1 knockout mouse models,and study the effects of Isl1 on the synthesis and secretion of FSH and LH.The most important results included the following points:We firstly detected the expression pattern of Isl1 in mouse developing pituitary gland using Real-Time quantitative PCR(RT-qPCR)and immunohistochemistry(IHC).The results showed that Isl1 was persistently expressed from E11.5 to adult pituitary.The Isl1 and ?GSU co-localization results showed that about 80%?GSU positive cells expressed Isl1.These data suggest that Isl1 is involved in the earlier development of pituitary gland and the differentiation of ?GSU progenitor cells.Secondly,we established the inducible and pituitary special Isl1 knockout mouse models using Cre-LoxP system.Isl1LoxP/LoxPmice mated with Isl1MCM/+ mice,in which DNA recombinase(Cre)MCM cassettes is activated under Isl1 promoter,to generate Isl1MCM/LoxP offspring.Tamoxifen was administrated to the Isl1MCM/LoxP mice or their primary pituitary cells.The efficiency of Isl1 ablation was detected by RT-qPCR.The results showed that tamoxifen could induce the knockdown of Isl1 gene effectively in pituitary cells in vitro,but failed to induce the knockout of Isl1 in mice pituitary in vivo.Furher,Isl1LoP/LoxP mice mated with ?GSUCre/+ mice,in which DNA recombinase(Cre)is activated under ?GSU promoter,to produce ?GSUCre/+Isl1LoxP/LoxP(Isl1 CKO)offspring.The efficiency of Isl1 ablation was detected by RT-qPCR,Western blot and immunofluorescence labling.The results showed that pituitary Isl1 was efficiently knockdown in the Isl1 CKO mice.The aGSUCre/+Isl1LoxP/LoxP mice model,in which Isl1 is truncated after Cre expression in gonadotropes,was used to investigate effects of Isll on the FSH and LH synthesis and secretion.The results demonstrated that Isll CKO mice exhibited a reduction in gonadotropes in pituitary,and the expression levels of LH? and FSH? mRNA decreased significantly.It is unexpected that the concentration of FSH in the serum reduced obviously,while LH in the serum showed no obvious changes in Isl1 CKO mice.Finally,we detected the effects of pituitary Isl1 specific deletion on mouse fertility.The results of the mating assays showed that both male and female mice were fertile.The relative fecundity of female mice was significantly impaired,while there were no obvious changes in male mice.The follicle development and estrous cyclicity were abnormal in Isl1 CKO female mice.The number of ovulation in Isll CKO female mice was decreased by about 87%,but which was recovered by supplying gonadotropin.These indicate that Isll is involved in the regulation of FSH and LH synthesis and secretion.Taken together,the present study demonstrated that Isll was expressed persistently in the mouse developing pituitary.Then we established inducible and pituitary special Isll knockout mice,and found that the FSH synthesis and secretion were decreased in pituitary Isll special knockout mice which resulted in the impaired female fertility.These results are important to further understanding the pituitary development and the regulations and related mechanisms of gonadotropin and reproduction.