Effects of Rho-associated coiled-coil-forming kinase inhibitor Y27632 on mouse preantral follicles development in vitro / 解剖学报

Objective To investigate the effects of Rho-associated coiled-coil-forming kinase (ROCK) inhibitor Y27632 on the development of mouse preantral follicles in vitro. Methods The ovaries of 12.5-day-old mice were collected and single preantral follicle was isolated by mechanical method to culture Nunclon Sphera 96-well U-bottom 3D cell culture plate in vitro. Set up a control group and treated group containing Y27632, and evaluate the overall development of the follicles through follicular morphology, follicle diameter, the number of mature follicles, and changes in oocyte spindles. The expression of oocytes [bone morphogenetic protein 15(BMP 15), growth differentiation factor 9(GDF9)], granulosa cells [follicle stimulating hormone receptor (FSHR)] and apoptosis related genes (Bad, Bax and Caspase-3) were also assessed by the Real-time PCR technique. Meanwhile, the follicle viability was detected by follicular activity test during follicular development. Results Rock inhibitor Y27632 had no significant effect on the growth diameter and basic development indicators (follicle survival rate, cavity formation rate and maturation rate) (P>0.05). And abnormal spindle assembly occurred during follicle development in the control group. After 8 days of culture, compared with the control group, the granulosa cell-specific gene FSHR was up-regulated in the experimental group; As well as the oocyte-specific genes BMP 15 and GDF9 were up-regulated, while forkhead box 03(Fox03) was down-regulated; The apoptosis genes Bax, Bad and Caspase-3 were also showed down-regulated. And the granulosa cell apoptosis in the experimental group was significantly less than that in the control group. Conclusion ROCK inhibitor Y27632 can inhibit the apoptosis of granulosa cells and prevents the abnormal assembly of oocyte spindles to improve the quality of follicle development in vitro.

In vitro maturation of human oocytes: Its role in infertility treatment and new possibilities / 대한생식의학회지

IVM refers to the maturation of immature oocytes in culture after their recovery from small antral follicles at the stage prior to selection and dominance. IVM requires little or no FSH in vivo and has been proposed as an alternative to conventional IVF, since it reduces the primary adverse effects caused by controlled ovarian stimulation, including the ovarian hyperstimulation syndrome. Moreover, IVM is a promising option for cases for which no standard protocol is suitable, such as FSH resistance, contraindications for ovarian stimulatory drugs, and the need for urgent fertility preservation. Recently, IVM has been used in women with regular cycles and normal ovaries. However, the pregnancy rate following IVM is suboptimal compared with that of conventional IVF, indicating that further studies to optimize the protocol and the culture conditions are warranted.

Regulation and 3 dimensional culture of tertiary follicle growth / 대한생식의학회지

It has been revealed that multiple cohorts of tertiary follicles develop during some animal estrous cycle and the human menstrual cycle. To reach developmental competence, oocytes need the support of somatic cells. During embryogenesis, the primordial germ cells appear, travel to the gonadal rudiments, and form follicles. The female germ cells develop within the somatic cells of the ovary, granulosa cells, and theca cells. How the oocyte and follicle cells support each other has been seriously studied. The latest technologies in genes and proteins and genetic engineering have allowed us to collect a great deal of information about folliculogenesis. For example, a few web pages (http://www.ncbi.nlm.nih.gov; http://mrg.genetics.washington.edu) provide access to databases of genomes, sequences of transcriptomes, and various tools for analyzing and discovering genes important in ovarian development. Formation of the antrum (tertiary follicle) is the final phase of folliculogenesis and the transition from intraovarian to extraovian regulation. This final step coordinates with the hypothalamic-pituitary-ovarian axis. On the other hand, currently, follicle physiology is under intense investigation, as little is known about how to overcome women's ovarian problems or how to develop competent oocytes from in vitro follicle culture or transplantation. In this review, some of the known roles of hormones and some of the genes involved in tertiary follicle growth and the general characteristics of tertiary follicles are summarized. In addition, in vitro culture of tertiary follicles is also discussed as a study model and an assisted reproductive technology model.

Effects of Substance P on the Expression of Various Factors to control Hair Growth in Human Hair Follicle Culture / 대한피부과학회지

BACKGROUND: Among constituents of the skin, hair follicle is an organ where nerve fibers with the most highest density are distributed. Recently, it has been reported that neuropeptides, which are secreted by nerve fibers, have important roles in the hair growth and hair cycle change, and that, the expression of various growth factors and apoptosis-related molecules are important in the hair growth and hair cycle change. Therefore, it was thought of import to analyse the relationship between the effect of neuropeptides and the expression of various factors to control hair growth in the hair follicle and hair follicle cells. OBJECTIVE: The purpose of this study was to investigate the relationship between the effect of neuropeptides and the expression of various hair growth-related factors at the level of hair follicle after pretreatment of cultured hair follicles and dermal papilla cells with SP. METHODS: Normal human scalp samples were obtained, and anagen hair follicles and dermal papilla cells were isolated and were cultured in Dulbeco's Modified Eagle's Medium (DMEM) with several combination of supplements in an atmosphere of 5% CO2/95% air incubator. We divided the culture plates into two groups; i.e. control group (DMEM only) and SP group (10-6M SP dissolved in DMEM). The results were evaluated by measuring linear hair growth and hair follicle morphology under a light microscope. Also, after pretreatment of cultured hair follicle and dermal papilla cells with SP, we examined changes of expression of hair growth factors (FGF-7, IGF-1, VEGF), hair growth-inhibitory factors (IL-1alpha, IL-1beta), and apoptosis-related molecules (p53, caspase-3). RESULTS: The following results were obtained. 1. SP did not have any statistically significant effect on the rate of linear hair growth in cultured hair follicles. However, it prolonged the anagen stage of hair cycle. 2. In hair follicles, the expression of FGF-7, a hair growth factor, was increased more than control, while the expression of caspase-3, an apoptosis-related molecule, was decreased more than control. Also, morphological changes as well as the changes of expression of hair growth factors and apoptosis-related molecules were not found in dermal papilla cells. However, the expression of IL-1beta, a hair growth-inhibitory factor, was decreased more than control. CONCLUSION: We can conclude from the results that SP has growth-stimulatory effect and especially prolongs the duration of anagen phase without affecting the rate of linear hair growth. Also, in hair follicles and dermal papilla cells, SP shows hair growth-stimulatory effect at the molecular levels.

Effects of TNF-alpha and Minoxidil on Human Hair Growth in Vitro / 대한피부과학회지

BACKGROUND: The factors that regulate hair follicle growth are still poorly understood. In vitro models may be useful in elucidating some aspects of hair follicle biology. TNF-alpha is potent inhibitor of hair follicle growth. TNF-alpha induces the formation of a club-like hair follicle, similar to catagen morphology of the hair bulb. Minoxidil induces generalized hypertrichosis when administered systemically, or localized hair regrowth when applied topically to sites of alopecia. OBJECTIVE: The purpose of this study is to investigate the effect of TNF-alpha and minoxidil on human hair growth using in vitro model. METHODS: Healthy human hair follicles without any visible damage were collected, they were cultured in Williams E medium with several combinations of supplements and TNF-alpha and/or minoxidil were added to the media. The results were evaluated by measuring linear hair fiber growth and hair follicle morphology on light microsocopy and by measuring radioisotope uptake of (methyl-3H)thymidine of hair follicle. RESULTS: The following results were obtained from this study. TNF-alpha have inhibitory effect on the rate of linear hair growth in cultured hair follicles. Minoxidil has no stimulatory effect on the rate of linear hair growth and no protective effect on the TNF-alpha induced growth inhibition in cultured hair follicles. CONCLUSION: TNF-alpha has growth-inhibitory effect and minoxidil has no protective effect on the TNF-alpha induced hair change.