Ice easily recrystallizes during warming after vitrification, and antifreeze protein (AFP) can inhibit the re-crystallization. However, no study has evaluated the effect of AFP treatment only thereon during warming. This study sought to compare AFP treatment protocols: a conventional protocol with AFP treatment during vitrification and first-step warming and a new protocol with AFP treatment during the first-step warming only. According to the protocols, 10 mg/mL of LeIBP (a type of AFP) was used. Five-week-old B6D2F1 mouse ovaries were randomly divided into a vitrified-warmed control and two experimental groups, one treated with the conventional AFP treatment protocol (LeIBP-all) and the other with the new AFP treatment protocol (LeIBP-w). For evaluation, ratios of ovarian follicle integrity, apoptosis, and DNA double-strand (DDS) damage/repairing were analyzed. The LeIBP-treated groups showed significantly higher intact follicle ratios than the control, and the results were similar between the LeIBP-treated groups. Apoptotic follicle ratios were significantly lower in both LeIBP-treated groups than the control, and the results were not significantly different between the LeIBP-treated groups. With regard to DDS damage/repairing follicle ratio, significantly lower ratios were recorded in both LeIBP-treated groups, compared to the control, and the results were similar between the LeIBP-treated groups. This study demonstrated that both protocols with LeIBP had a beneficial effect on maintaining follicle integrity and preventing follicle apoptosis and DDS damage. Moreover, the new protocol showed similar results to the conventional protocol. This new protocol could optimize the mouse ovary vitrification-warming procedure using AFP, while minimizing the treatment steps.
Animals ; Antifreeze Proteins/*pharmacology ; Apoptosis/drug effects ; Cryopreservation ; Cryoprotective Agents/pharmacology ; Female ; Mice ; Ovarian Follicle/cytology/drug effects ; Ovary/cytology/drug effects/*physiology ; *Vitrification/drug effects

The effect of high concentrations of testosterone on ovarian follicle development was investigated. Primary follicles and granulosa cells were cultured in vitro in media supplemented with a testosterone concentration gradient. The combined effects of testosterone and follicle-stimulating hormone (FSH) on follicular growth and granulosa cell gonadotropin receptor mRNA expression were also investigated. Follicle growth in the presence of high testosterone concentrations was promoted at early stages (days 1-7), but inhibited at later stage (days 7-14) of in vitro culture. Interestingly, testosterone-induced follicle development arrest was rescued by treatment with high concentrations of FSH (400 mIU/mL). In addition, in cultured granulosa cells, high testosterone concentrations induced cell proliferation, and increased the mRNA expression level of FSH receptor (FSHR), and luteinized hormone/choriogonadotropin receptor. It was concluded that high concentrations of testosterone inhibited follicle development, most likely through regulation of the FSH signaling pathway, although independently from FSHR downregulation. These findings are an important step in further understanding the pathogenesis of polycystic ovary syndrome.
Androgens ; pharmacology ; Animals ; Cell Proliferation ; drug effects ; Female ; Follicle Stimulating Hormone ; genetics ; metabolism ; pharmacology ; Gene Expression Regulation, Developmental ; Granulosa Cells ; cytology ; drug effects ; metabolism ; Mice ; Ovarian Follicle ; cytology ; drug effects ; growth & development ; metabolism ; Primary Cell Culture ; RNA, Messenger ; genetics ; metabolism ; Receptors, FSH ; genetics ; metabolism ; Receptors, Gonadotropin ; genetics ; metabolism ; Receptors, LH ; genetics ; metabolism ; Signal Transduction ; drug effects ; genetics ; Testosterone ; antagonists & inhibitors ; pharmacology

Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200~250 microm) were isolated by micro-dissection and cultured in 0 (control), 10(-3), 10(-5), 10(-7), and 10(-9) M SNP. To examine the reversible effect of SNP, PFs were cultured with 10(-5) M SNP + 1 mM Nomega-nitro-L-arginine methyl ester (L-NAME) or 1.0 microg hemoglobin (Hb). The results showed that greater concentrations of SNP (10(-3), 10(-5), 10(-7) M) inhibited (p < 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10(-9) M) significantly stimulated (p < 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP + L-NAME promoted the inhibitor effect of SNP while a SNP + Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p < 0.05) in a dose-dependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium.
Animals ; *Apoptosis ; Buffaloes/*physiology ; Estradiol/biosynthesis ; Female ; Follicle Stimulating Hormone/metabolism ; NG-Nitroarginine Methyl Ester/pharmacology ; Nitrates/pharmacology ; Nitric Oxide/*metabolism ; Nitric Oxide Donors/pharmacology ; Nitrites/pharmacology ; Nitroprusside/pharmacology ; Oocytes/cytology/drug effects/growth & development/metabolism ; Ovarian Follicle/*cytology/drug effects/growth & development/*metabolism ; Progesterone/biosynthesis

OBJECTIVETo observe the effect of Yangjing Zhongyu Decoction (YZD) on mRNA and protein expression of PCNA, StAR, and FSHR in ovarian granulose cells (GCs) cultured by excess androgen.

METHODSOvarian GCs from porcine follicles were isolated and cultured in vitro. Follicular stimulating hormone (FSH) or YZD was added in the GCs treated by excess testosterone propionate. Totally 48 h later mRNA and protein expression of PCNA, StAR, and FSHR were detected by RT-PCR and Western blot.

RESULTSExcess androgen inhibited mRNA and protein expression of PCNA, StAR, and FSHR of GCs. FSH and YZD could antagonize inhibition of excess androgens, and promote mRNA and protein expression of PCNA, StAR, and FSHR in GCs.

CONCLUSIONYZD could antagonize the inhibition of excess androgen on mRNA and protein expression of PCNA, StAR and FSHR in GCs. Thus, we inferred that YZD could improve the follicle dysplasia by promoting mRNA and protein expression of PCNA, StAR and FSHR in GCs.

Androgens ; pharmacology ; Animals ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Female ; Follicle Stimulating Hormone ; pharmacology ; Granulosa Cells ; cytology ; drug effects ; metabolism ; Membrane Transport Proteins ; genetics ; metabolism ; Ovarian Follicle ; cytology ; drug effects ; Proliferating Cell Nuclear Antigen ; genetics ; metabolism ; RNA, Messenger ; genetics ; Receptors, FSH ; genetics ; metabolism ; Swine

OBJECTIVETo explore the effect of Bushen Tiaojing Recipe (BTR) on the expressions of drosophila mothers against decapentaplegic protein (Smadl), Smad5, Smad8, and Smad4 on human mural granulosa cells.

METHODSSixty-six patients undergoing in vitro fertilization-embryo transfer (IVF-ET) were randomly assigned to two groups in the ratio of 1:2, the treatment group and the control group. Twenty-three patients in the treatment group were treated with BTR and GnRHa/FSH/hCG, while forty-three patients in the control group were treated with GnRHa/FSH/hCG. The mRNA expressions of Smad1, Smad5, Smad8, and Smad4 on mural granulosa cells of the mature follicle were detected by real-time PCR on the ovum retrieval day. The expressions of Smad1, Smad5, Smad8, and Smad4 at the protein level were observed using cell immunofluorescence method.

RESULTSThe mRNA and protein expressions of Smadl in the granulosa cells were significantly higher in the treatment group than in the control group (P <0.05). There was no statistical difference in the mRNA and protein expressions of Smad5, Smad8, and Smad4 between the two groups.

CONCLUSIONThe mechanisms of BTR for improving the pregnancy rate and the ovarian functions might be correlated with up-regulating mRNA and protein expressions of Smadl of human mural granulosa cells.

Adult ; Drugs, Chinese Herbal ; pharmacology ; Female ; Granulosa Cells ; drug effects ; metabolism ; Humans ; Ovarian Follicle ; cytology ; Signal Transduction ; Smad1 Protein ; metabolism ; Smad4 Protein ; metabolism ; Smad5 Protein ; metabolism ; Smad8 Protein ; metabolism

AIM(1) To investigate the mRNA expression of the key angiogenic growth factors in the grafts after transplantation. (2) To investigate the potential impact of danshen (Chinese traditional medicine) administration on grafts angiogenesis.

METHODSThe frozen-thawed ovarian tissue from aborted fetus were xenografted into the renal capsule of the nude mice, recovered 48 h, 7 d and 28 d after respectively. Either danshen or saline (as the control) was administered after transplantation.

RESULTSThe mRNA levels of VEGF showed a temporary raise in 48 h after transplantation, then decreased in one week, and no significant difference was fund between the control group and danshen group. Ang-2 was increased in 48 h after transplantation, when Danshen group was significantly higher than the control group (P < 0.05). The microvessel density significantly increased in all the tissues after transplantation. The control group peaked on day 7 after transplantation, while danshen group peaked in 48 h and kept correspondingly steady after that.

CONCLUSIONEarly angiogenesis began within 48 h after transplantation of the thawed human fetal ovarian tissue, and its microvessel density peaked within the first week after transplantation. Our results also suggested that the use of danshen injection in conjunction with transplantation could facilitate revascularization of the grafts.

Angiopoietin-2 ; genetics ; metabolism ; Animals ; Cryopreservation ; Drugs, Chinese Herbal ; pharmacology ; Female ; Fetal Tissue Transplantation ; methods ; Fetus ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neovascularization, Physiologic ; drug effects ; Ovarian Follicle ; cytology ; growth & development ; transplantation ; RNA, Messenger ; genetics ; metabolism ; Salvia miltiorrhiza ; chemistry ; Transplantation, Heterologous ; methods ; Vascular Endothelial Growth Factor A ; genetics ; metabolism