Zichong granules promote differentiation of ovarian granulosa-like cells from human embryonic stem cells in vitro.

OBJECTIVE: To investigate whether Zichong granules (, ZCKL), a very effective herbal formula for treating infertility, have an impact on the differentiation of ovarian granulosa cells from human embryonic stem cells (hESCs) in vitro, and to explore the cellular mechanisms of its clinical effects. METHODS: Serum from ZCKL-medicated rats was prepared and used to treat mesoderm cells derived from hESCs for 6 d. Normal rat serum and a set of growth factors were used as negative and positive controls, respectively. RESULTS: ZCKL-medicated rat serum, but not normal rat serum, induced hESCs-derived mesoderm cells to differentiate into functional ovarian granulosa-like cells (OGLCs) in a similar manner to defined growth factors. The induced OGLCs resembled the morphology of native human granulosa cells, expressed granulosa cell-specific markers at both the mRNA and protein levels, produced high levels of estradiol and strongly responded to follicle-stimulating hormone stimulation. Furthermore, mRNA levels of follistatin, mothers against decapentaplegic homolog 8 and bone morphogenetic protein 6 were dynamically changed during the process. CONCLUSION: In the ZCKL treatment of infertility, one mechanism by which ZCKL may act is by influencing ovarian granulosa cell differentiation and development, possibly through the follistatin and BMP/SMAD signaling pathways.

Effect of Rat Medicated Serum Containing Zuo Gui Wan and/or You Gui Wan on the Differentiation of Stem Cells Derived from Human First Trimester Umbilical Cord into Oocyte-Like Cells In Vitro.

Zuo Gui Wan (ZGW) and You Gui Wan (YGW) are two classic formulas used in clinical treatment of infertility in traditional Chinese medicine (TCM). However, the actions of the formulas remain to be proven at the cellular and molecular levels. In this study, we investigate whether the two formulas have any effect on germ cell formation and differentiation by culturing rat medicated serums containing YGW or ZGW with stem cells derived from human first trimester umbilical cord. Our results showed that while the normal rat serums had no significant effects, the rat medicated serums had significant effects on the differentiation of the stem cells into oocyte-like cells (OLCs) based on (1) cell morphological changes that resembled purative cumulus-oocyte complexes (COCs); (2) expressions of specific markers that were indicative of germ cell formation and oocyte development; and (3) estradiol production by the COC-like cells. Furthermore, ZGW medicated serums exhibited more obvious effects on specific gene expressions of germ cells, whereas YGW medicated serums showed stronger effects on estradiol production. Accordingly, our study provides evidence demonstrating for the first time that one of molecular and cellular actions of YGW or ZGW in treating human reproductive dysfunctions may be through an enhancement of neooogenesis.

Effect of rat medicated serum containing you gui wan on mouse oocyte in vitro maturation and subsequent fertilization competence.

You Gui Wan (YGW) is a classic herbal formula in traditional Chinese medicine (TCM) used for the clinical treatment of infertility. This study was to explore whether YGW has an impact on mouse oocyte maturation in vitro and subsequent fertilization competence. Rat medicated serum containing YGW was prepared by orally administrating YGW. Mouse immature oocytes were cultured with YGW medicated serum and compared to those cultured with or without normal rat serum or follicle-stimulating hormone (FSH). YGW medicated serum significantly increased the percentages of matured oocytes when compared to the groups with or without normal rat serum (P < 0.01). Furthermore, YGW medicated serum increased the rate of in vitro fertilization (IVF) when compared to the groups treated with FSH and with or without normal rat serum (P < 0.001). YGW medicated serum also had significant effects on the mRNA expressions of PKA, CREB, MAPK, PKC, PKG, and MPF and the concentrations of cAMP, cGMP, and NO in matured oocytes. These results indicate that YGW can promote mouse oocyte maturation and IVF in vitro. Signaling pathways, such as the cAMP/PKA/MAPK, the PKC-MAPK, and the NO-cGMP-PKG pathway, which are similar to those induced by FSH, may be responsible for this action.

Impacts of You Gui Wan on the expression of estrogen receptors and angiogenic factors in OVX­rat vagina: a possible mechanism for the trophic effect of the formula on OVX­induced vaginal atrophy.

The administration of You Gui Wan (YGW) decoction has been observed to improve vaginal atrophy induced by ovariectomy (OVX) in rats. The aim of the current study was to explore the possible mechanisms underlying this effect. Following OVX, 37 Sprague Dawley female rats were randomly divided into three groups which were orally administered with YGW decoction, saline or estrogen for 11 weeks. In parallel with this, 19 normal and 17 rats with sham-surgery were used as controls. The effects of these treatments on estrogen receptors (ER) and various angiogenic factors, including vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-1 (VEGFR-1), angiopoietin (Ang)1 and 2 and basic fibroblast growth factor (bFGF) in the vagina were compared using immunohistochemistry or quantitative polymerase chain reaction (qPCR). OVX was found to induce significant vaginal atrophy and decrease the expression of ER and various angiogenic factors when compared with the normal and sham-surgery animals (all P<0.05). Estrogen replacement and the administration of YGW decoction reversed the vaginal atrophic process. The hormonal replacement and YGW treatment recovered the protein expression of ER-α and -ß, VEGF and VEGFR-1 and the mRNA levels of ER-α, VEGF, VEGFR-1, Ang1 and 2, and bFGF when compared with OVX-rats with saline, normal and sham-surgery treatments (all P<0.05). Thus, it may be concluded that a possible mechanism underlying the effect of YGW on OVX-induced vaginal atrophy may be the upregulated expression of ER and various angiogenic factors in the vaginal tissue.

You Gui Wan can reverse atrophic effect of ovariectomy on rat vaginal fold and blood vessels in the lamina propria.

The aim of this study is to investigate whether or not You Gui Wan (YGW), a classical herbal formula in Traditional Chinese Medicine (TCM), has an impact on rat uterine and vaginal atrophic processes induced by ovariectomy (OVX). Thirty-four OVX Sprague-Dawley (SD) rats were randomly divided into three sets, and orally administrated with YGW decoction, saline or estrogen for 11 weeks, respectively. Histomorphological changes of the uterus and vagina, and serum estradiol levels were then compared. Results showed that OVX caused a dramatic atrophy of the uterus and vagina in the rats. Estrogen replacement reversed the effect of OVX, but with a side effect of endometrial hyperplasia. YGW had no significant effect on blood estradiol concentration or uterine histology, but it significantly overturned the atrophic processes of the vaginal fold and blood vessels in the lamina propria. In order to initially explore the mechanisms underlying these effects, immunostaining of estrogen receptor (ER)-α and -ß in the vagina was performed. It was shown that OVX reduced expressions of ER while YGW and estrogen replacement reversed this reduction. Our findings suggest that YGW can reverse the atrophic effect of OVX on rat vaginal plica and blood vessels in the lamina propria with little adverse effect on endometrial hyperplasia. This indicates the herbal formula as an alternative to hormone replacement therapy in the management of menopausal vaginal atrophy. Recovery of ER expressions in the vagina might be one of mechanisms underlying the effects of YGW.