Effects Of Oviductus Ranae On Follicle Development

Oviductus Ranae(OR),which was included in the “Chinese Pharmacopoeia” under the name of “toad oil”,is said to be sweet,salty,flat in nature,and belongs to the kidney and lung meridians;it is effective in tonifying the kidney and benefiting the essence,nourishing the yin and moistening the lung.Modern studies have shown that OR has a variety of bioactive functions and high medicinal value.It has been used clinically to treat chronic nephritis,coronary heart disease,perimenopausal estrogen deficiency in women and osteoporosis,etc.Previous studies by our group found that the effect of OR on the improvement of estrogen deficiency in vivo was different from the effect of exogenous hormones,in this paper,we explored the mechanism of action of OR from the perspective of its effect on the development of developmentally restricted ovaries,and investigated the specific mechanism of action of OR on the enhancement of estrogen content in rats from both in vivo and in vitro perspectives,providing a theoretical basis for the estrogen-like effects of OR.The effect of OR on the model of follicular developmental dysfunction caused by triptolian polyglycosides.Sixty female WISTAR rats with normal estrous cycle were randomly divided into control group(CG),model group(MG),positive drug group(PG),OR high dose group(ORHG)and OR low dose group(ORLG),12 rats in each group.Follicular developmental dysfunction model was prepared by continuous administration of triptolide solution for 8weeks to rats in all groups except CG rats,while rats in the other groups were administered the test drugs for 8 weeks.The rats in estrus were treated at week 8.Eight rats were treated in each group,The rats were anesthetized by intraperitoneal injection of 10% sodium pentobarbital solution one hour after the last gavage,and blood was collected from the abdominal aorta to determine serum estradiol(E2),progesterone(P),luteinizing hormone(LH),follicle stimulating hormone(FSH),and testosterone(T)contents;Records of weighing of ovaries and uteri were enucleated and the organ index was calculated;One ovary was made serial pathological sections,the ovary was subjected to HE staining and TUNEL staining.The other side of the ovary was preserved in liquid nitrogen,and the relative amounts of PI3 K,Akt,PTEN and m TOR m RNA were determined by RT-PCR,and the PI3 K,Akt,and p-Akt protein contents in the ovary were determined by Western blotting.The remaining rats in each group were subjected to a litter experiment,and the number of litters produced by the rats was counted.Our results showed that OR could promote the development of secondary follicles,decrease the rate of ovarian follicle atresia,elevate serum E2 and T contents,reduce the serum FSH content,promote the PI3 K protein expression and Akt protein phosphorylation levels in the ovaries of follicle development disorder model,down-regulate the PTEN m RNA expression,up-regulate the PI3 K protein expression and promote the Akt protein phosphorylation levels in the ovaries of follicle development disorder rats caused by triptolide.There was a non positive correlation between ovarian effect and dose,and overdose of OR did not enhance the effect of linfrog oil on follicle development disorder model.The effect of OR-containing serum on follicle culture in vitro.Bilateral ovaries of SPF grade16-day-old female Wistar rats were enucleated in a sterile environment and 110-130 μm anterior sinus follicles were collected by mechanical +enzymatic methods.Healthy follicles selected by inverted microscope were randomly divided into blank control group(CG),normal rat serum group(NG),OR high-dose drug containing serum group(ORHG),and OR low-dose drug containing serum group(ORLG).Follicles were cultured for semi-quantitative fluid replacement on days 1,3,5 and 7,and the culture medium was collected to detect E2 content.Follicles were collected on day 7and the relative expression of m RNA for PI3 K,Akt,PTEN and m TOR was determined by RT-PCR.The results indicated that the medicated serum of OR could promote the growth and development of preantral follicles in vitro and promote follicular secretion of E2.It promoted granulosa cell proliferation in preantral follicles,inhibitted the expression of PTEN m RNA in follicles,and upregulated the expression of PI3K/Akt signaling pathway genes.