To Investigate The Effect Of Rat Medicated Sera Prepared With Kidney-invigorating Chinese Herbal Formulas On Differentiation Of Human FTM-PVCs Into Oocyte-like Cells In Vitro

Objective:To investigate the effect of rat medicated sera prepared with kidney-invigorating Chinese herbal formulas on differentiation of human first trimester-derived perivascular cells (FTM-PVCs) into oocyte-like cells (OLCs) in vitro.First part:To investigate whether or not the human FTM-PVCs were able to differentiate in vitro into the OLCs and establish an in vitro research platform for the kidney-invigorating Chinese herbs.Methods:1. Methods for inducing human FTM-PVCs into OLCs in vitroThe human FTM-PV cells were first isolated and cultured according to the method published previously. After that, different differentiation protocols were investigated whether or not the cells were able to differentiate into OLCs. The protocols included1)25%human ovarian follicular fluid+FSH+LH,2).25%human ovarian follicular fluid+FSH+LH+E2,3).25%human ovarian follicular fluid+FSH+LH+E2+folic acid,4).25%human ovarian follicular fluid+FSH+LH+E2+IVM culturing medium and5). co-culture with human follicular granulosa cells or culture conditioned medium of human follicular granulosa cells. During the process of differentiation, morphological changes were compared under an inverted microscope among the various differentiation protocols. Expressions of specific markers for oocytes were determined by immunocytochemistry and estradiol concentrations in the culture media were assayed by using an estradiol ELISA.2. Establishment of the in vitro research platform for the kidney-invigorating Chinese herbsAfter comparisons, culture with25%human ovarian follicular fluid+FSH+LH+E2was selected as a standard differentiation protocol. Morphologic observation, detection of specific biomarkers for oocytes such as SCP3, GDF-9, ZP1, ZP2and ZP3at transcription and translation levels, as well as estradiol production were all included into the platform.Results: 1. The OLCs and/or cumulus-oocyte-like complex (COCs) in morphology could be found after24days of culturing the FTM-PVCs in human ovarian follicular fluid plus LH, FSH and E2or co-culturing with human granulosa cells.2. The OLCs could express SCP3, GDF-9, ZP1, ZP2and ZP3at transcription and translation levels.3. The estradiol concentrations in the culture media of the OLCs were significantly higher than that in the culture media of the undifferentiated FTM-PVCs.Conclusions:1. Human FTM-PVCs have a potential ability to differentiate into the OLCs in vitro in the culture with human follicular fluid or co-culture with human granulose cells. To our knowledge, this is the first report of differentiation of OLCs derived from non-embryonic stem cells.2. The characteristics of FTM-PVCs are similar to "essence" as defined by theories of Traditional Chinese Medicine. Therefore, the differentiation process from FTM-PVCs to OLCs could be used as a research platform for investigation of Chinese herbal function.Second part:To investigate the effect of rat medicated sera prepared with kidney-invigorating Chinese herbal formulas on the differentiation process from human FTM-PVCs to OLCs in vitroMethods:1. The preparation of rat medicated sera with invigorating kidney herbsFifty rats were randomly divided into5groups:blank group,1#drug group,2#drug group,3#drug group and5#drug group,10in each group which included5males and5females. The rats in blank group were drenched with distilled water while the rats in1#,2#,3#or5#groups were drenched with corresponding herbal formula decoctions of20ml/kg body weight (equal to5.0g drug/kg body weight)._After90min of drug administrations, rat blood samples were collected from eye-ground veins, and the medicated or blank sera were obtained by centrifugation at3000rpm for10min. The sera were purified with HPLC and filtered to sterile.2. The investigation of the effect of the rat medicated sera on the differentiation process from human FTM-PVCs to OLCs in vitroThe human FTM-PVCs were cultured with2%,5%,10%or20%of the various rat medicated serum. At the same time, the cells were also treated with the rat blank serum as a negative control, while the human follicular fluid as a positive control. Moreover, the rat medicated sera plus human follicular fluid were used to observe whether or not there was a synergistic effect of the rat medicated sera and human follicular fluid. The morphological changes for each group was observed under an inverted microscope. On the24th or31st day after interventions, the expression of GDF-9was examined with immunocytochemistry assay. For the group with2%of the rat medicated serum, mRNA levels of SCP3, GDF-9, ZP1, ZP2and ZP3were evaluated with a semi-quantitative RT-PCR assay. The E2productions in culture medium were measured with an ELISA assay.Results:1. The OLCs in morphology could be found in cultures with the#2,#3and#5rat medicated serum that were similar to the positive controls while no significant changes in cell morphology was found in the negative controls. Moreover, no a synergistic effect of the rat medicated serum and human follicular fluid can be detected.2. After the intervention with the rat medicated sera, the differentiated OLCs could express GDF-9protein, and mRNA levels of SCP3, GDF-9, ZP-1, ZP-2and ZP-3were significantly higher than that in the negative controls.3. E2production in culture media with the rat medicated sera was significantly higher than that in culture media with the rat blank sera.Conclusions:1. The rat medicated sera prepared with invigorating kidney herbs has the ability to induce human FTM-PVCs into OLCs in vitro. The findings may demonstrated the TCM theory of "Kidney controls reproduction and is congenital foundation" from the view of modern Western medicine.2. The differentiation process from the human FTM-PVCs differentiation into the OLCs can be used as a new platform to evaluate the effect of invigorating kidney herbs on the growth and development of germ cells in vitro.Third part:To investigate possible mechanisms underlying the effect of the rats medicated seraMethods:Components of the various rat medicated sera were analyzed with HPLC. Concentrations of various cytokines and hormones in the rat medicated sera or human follicular fluid as well as differentiation culture media with the rat medicated sera or human follicular fluid were compared by using a number of specific ELISA kits, respectively.Results:1. There were different peaks appeared at2-3min or16-17min in1#,3#and5#rat drug serum from the rat blank serum on the HPLC profile.2. In the human ovarian follicular fluid, there were a number of molecules associated with germ cell formation/maturation such as VEGF, SCP1, SCP2, SCP3, MPF and SCF.3. After inducing FTM-PVCs into the OLCs by using human ovarian follicular fluid, the concentrations of VEGF, SCP1, SCP2, MPF in the culture medium of OLCs were significantly increased when compared to the culture medium of undifferentiated cells.4. The levels of P, E2, FSH, LH, F-T, MPF, SCF, EGF, IGF-1, TNF-a, IL-la in the rat medicated sera were not significantly different from that in the rat blank sera. However, the VEGF levels in the2#,3#and5#rat medicated were significantly higher that in rat blank serum.5. Compared to the negative controls, the concentrations of VEGF, SCP1, SCP2, SCP3and MPF in culture medium with the1#rat medicated serum were increased while the levels of E2, SCP2, MPF and F-T in culture medium with2#rat medicated serum and the levels of SCP3and MPF in culture medium with3#and5#rat medicated sera were elevated.Conclusions:1. From the results, it suggests that VEGF, SCP1, SCP2and MPF in human follicular fluid may play an important role in the differentiation of the FTM-PVCs into the OLCs since levels of these molecules in the culture medium of the OLCs were significantly increased compared to that of the undifferentiated cells.2. The possible mechanism underlying the effects of rat medicated sera may be similar to that of human follicular fluid by increase of production of VEGF, SCP1, SCP2, SCP3, MPF and free testosterone from the differentiated cells. However, it may be induced by some unknown active components in the rat medicated sera rather than directly by these molecules since the levels of molecules of rat medicated sera were not different from the blank serum.