Nutritional Regulation Of Mouse Germ Cell Development In Vitro

The development of mammalian germ cells requires complex regulation of multiplicate hormones, growth factors and extrinsic nutritional substances. Ginsenosides (GS) and daidzein (DAI) have multiple biological functions, including antioxidative activity, estrogenic activity and reproductive regulation. However, the actions of GS and DAI on reproduction remain unclear. Therefore, the present study adopted type A spermatogonia and oocytes of mouse to evaluate effects of GS or DAI on spermatogonial proliferation and oocyte meiotic maturation and provided evidences for nutritional regulation of vegetal biological active substances on development of germ cells.1. Regulations of hormones on mouse type A spermatogonial proliferation.The serum-free germ-somatic cell coculture model was established to evaluate effects of hormones and growth factors on type A spermatogonial proliferation. Testicular cells of 6-day-old ICR mouse were dispersed by collagenase and c-kit immunocytochemistry identified type A spermatogonial cells. Immunocytochemical staining for proliferating cell nuclear antigen (PCNA) showed that ITS medium supplemented with insulin (10μg/ml), transferring (5μg/ml) and selenite (3x10-8 mol/L) could maintain spermatogonial cell survival and proliferation. The present study evaluated the effects of follicle-stimulating hormone (FSH), epidermal growth factor (EGF), testosterone (T) and 17β-estradiol (E2) on mouse type A spermatogonial proliferation. EGF and epidermal growth factor receptor (EGFR) were mainly expressed on type A spermatogonial cells. FSH (1～100 ng/ml) or EGF (1～10 ng/ml) treatment alone significantly stimulated spermatogonial cell proliferation. Furthermore, FSH (10 ng/ml) could synergize with EGF (0.1 ng/ml) to promote cell proliferation, but higher EGF at 1～10 ng/ml inhibited FSH-induced stimulating effect. T (10-8～10-6 mol/L) and E2 (10-8～10-6 mol/L) promoted proliferation of type A spermatogonia. Flutamide (FLU, an testosterone receptor antagonist) inhibited T-stimulated spermatogonial proliferation, and tamoxifen (TMX, an estrogen receptor a antagonist) inhibited the action of E2. These results showed that the germ-somatic cell coculture model allowed evaluating regulation of gonadotropin, sex steroids and growth factors on spermatogonial proliferation. FSH, T and E2 exerted stimulating actions through a paracine pathway involving indirect effects on somatic cells and EGF promoted spermatogonial proliferation through direct effects on spermatogonia. The above results indicated that the serum-free germ-somatic cell coculture model was appropriate to evaluate effects of extrinsic factors on type A spermatogonial proliferation. Furthermore, the model would be available to study the regulating mechanism of spermatogonial development.2. Effect of ginsenosides on proliferation of type A spermatogoniaThe serum-free germ-somatic cell coculture model was used to evaluate the effect of GS on proliferation of type A spermatogonia. After 72 h culture, Ginsenosides (1.0～10μg/ml) significantly stimulated proliferation of spermatogonia assessed by increased the number and area of spermatogonial colonies. Activation of protein kinase C (PKC) by Phorbol 12-myristate 13-acetate (PMA) elicited proliferation on spermatogonia at 10'8 to 10-7mol/l and PKC inhibitor H7 inhibited this effect. Likewise, forskolin (FSK), an activator of protein kinase A (PKA) exerted proliferating action on type A spermatogonia at concentration of 10-6-10-5 mol/1 and PKA inhibitor H89 inhibited this effect, suggesting activation of PKC and PKA involved type A spermatogonial proliferation. Furthermore, GS (1.0μg/ml) could synergize with PMA to promote spermatogonial proliferation. GS-stimulated spermatogonial proliferation was suppressed by combined treatment with H7. However, H89 did not influence GS-stimulated spermatogonial proliferation, suggesting the effect was not mediated by PKA pathway. These results indicated that activation of protein kinases A and C promoted proliferation of mouse type A spermatogonia and the proliferating effect of GS might be mediated by a mechanism involving the PKC signaling transduction pathway.3. Attenuating effect of daidzein on polychlorinated biphenyls-induced toxicity in mouse testicular germ cellsType A spermatogonial cells form colonies in culture. However, germ cells of 3-week-old mouse grow dispersively in vitro, which accord facilities for observation of cell morphological change. Therefore, the present study adopted mouse testicular germ cells to evaluate the attenuating effects of DAI on A1254-induced oxidative damage in testicular germ cells, with emphasis on the protective mechanism. DAI belongs to the most common isoflavones existed in leguminous plants and possesses multiple pharmacological effects. The most important biological action of DAI contributes to the antioxidant activity. Polychlorinated Biphenyls (PCBs) are members of environmental contaminations, which belong to the halogenated hydrocarbon class and impose toxic effects on normal development and reproductive system of human and animal. PCBs were ubiquitous in the environment due to the persistent and lipophilic characters. Cells were exposed to the reactive oxygen species (ROS)-producing system hypoxanthine/xanthine oxidase (HX/XO) to determine the antioxidant action of DAI. Results showed that A1254 (20～30μg/ml) and HX/XO (10-5M; 2.5×10-3IU/ml) exerted toxic effects on germ cells, induced oxidative damage and cell death. DAI obviously attenuated the oxidative damage induced by PCB and HX/XO system, increased the cell number, and restored A1254-induced decrease in superoxide dismutase (SOD) activity and glutathione (GSH) level and A1254-induced increase in malondialdehyde (MDA) formation. These results indicated DAI exerted antioxidant activity and attenuated A1254-induced oxidative damage in mouse testicle germ cells. Therefore, DAI would play critical role in preventing PCBs-induced generative toxicity.4. Effect of ginsenosides on mouse oocyte maturationMouse oocyte culture model was established to determine the role of GS on meiotic maturation of murine oocytes and further evaluate our understanding of effects of GS on generative health. Cumulus oocyte complexes (COC) and denuded oocytes (DO) in germinal vesicle (GV) stage were isolated from pregnant mare serum gonadotropin (PMSG)-primed immature mice. Germinal vesicle breakdown (GVBD) and first polar body (PB1) extrusion were used as the mark of oocyte meiotic maturation. Oocytes in GV stage could undergo spontaneous maturation after they cultured for 24 h in 10% fetal calf serum (FCS) medium. Hypoxanthine (HX, 4 mmol/L) maintained meiotic arrest and inhibited mouse oocytes from resuming meiosis in vitro maturation. Follicle-stimulating hormone (FSH) induced mouse oocytes to overcome the HX-maintained and resume meiosis in COC. The result showed this model was appropriate to evaluate effects of extrinsic factors on oocyte development. GS (1.0-10μg/ml) stimulated meiotic maturation in COC assessed by increased GVBD and PB1 extrusion. No effect of GS was observed on DO. In addition, GS (1.0μg/ml) could synergize with FSH (0.1 IU/ml) to promote oocyte meiotic maturation in COC. TMX did not influence GS-induced meiotic maturation, suggesting the effect was not mediated by estrogen. However,1 mmol/L Nω-nitro-L-arginine methyl ester (L-NAME, a nitric oxide (NO) synthase inhibitor) reduced the GS-induced oocyte maturation in COC. Immunocytochemical analysis demonstrated that GS enhanced expression of inducible nitric oxide synthase (iNOS) in the cumulus cells. L-NAME reduced the immunocytochemically detected increase in iNOS following ginsenoside exposure. These results suggested that GS might stimulate murine oocytes meiotic maturation through a paracine pathway involving the NO/iNOS system.Conclusion:Mouse type A spermatogonial cell and oocyte culture models were established to determine the roles of vegetal biological active substances on development of germ cells. These results showed ITS medium supplemented with insulin, transferrin and selenite could maintain spermatogonial cell survival. FSH, T, E2, EGF and activation of PKA and PKC could promote type A spermatogonial proliferation. GS promoted type A spermatogonial proliferation by a mechanism involving the PKC signaling transduction pathway. In addition, DAI exerted antioxidant activity and attenuated A1254-induced oxidative damage in mouse testicle germ cells. GS might stimulate murine oocytes meiotic maturation through a paracine pathway involving the NO/iNOS system. These results will provide theoretic guidance for nutritional regulation of germ cell development and improvement of reproductive performance.