Establishment Of In-vitro Rat And Mouse Follicle Culture Systems And Application For Reproductive Toxicity

Reproductive and developmental dysfunction is one of the major public health problems which imposes a significant impact on human health.The effect of chemicals on female reproductive system will result in ovarian cycle disorder or infertility,which increases significantly the spontaneous abortion rate and the incidence of offspring developmental anomaly and fertility,and so on.Simultaneously,all female gametes have been formed in prenatal,the number of follicles are immovable at birth,if destruction,no supernumerary gametes to retrieve,the consequence is more serious than male.Therefore, it is extremely important to screen and identify chemicals which have female reproductive toxicity and to elucidate the mechanism of their toxic action.Nevertheless,new chemical entities are produced in their thousands each year by combinatorial chemistry and computer-assisted drug design.Efforts are being made to identify and develop alternative methodologies,such as high-throughput and few testing samples.The European Commission and the EU Member States must actively encourage and support the development,validation and acceptance of methods which could reduce, refine or replace the use of laboratory animals.Currently,the guidelines produced by ICH and OECD for reproductive testing are still performed by in-vivo animal studies,mainly in rodents.Evaluation of the femal reproductive function by in vivo studies is currently carried out on the basis of parameters related to fertility outcome,these parameters do not allow elucidation the site of action and the mechanism of toxic damage to the ovary.Since in-vivo animal studies are less sensitivity,require longer testing cycle and more test samples than in-vitro bioassay,so it is necessary to develop in-vitro bioassay for testing femal reproductive toxicity.As yet,no adequate in-vitro models have been validated to identify potentially hazardous products with regard to the reproductive system,including ovarian follicle,oocyte,oogenesis which is the key to evaluate femal reproductive toxicity.In rescent years,in-vitro follicle culture systems have been developed with the aim of growing immature oocytes from early follicle stages to fertilizable oocytes.They are also often used as a tool to study the process of folliculogenesis and oogenesis in vitro.Several research groups have described the conditions for in-vitro culture of mouse ovarian follicles.In-depth study of the culture system demonstrates that the whole in-vitro process mimics normal(in-vivo)physiology from the point of view of morphogenesis, defferentiantion,hormone production.Ovarian follicle function can be analyzed simultaneously on folliculogenesis,hormonogenesis,and oogenesis on variant developmental stages.These systems have been applied succeedly to reproductive biology and physiology studies for the folliculogenesis and follicle regulation.Nevertheless,in-vitro follicle bioassay for reproductive toxicity is are not produced except for individual toxicity parameters.As yet,in-vitro rat preantral follicle culture system were not developed.On basis of the mouse follicle culture system described according to literatures,in-vitro rat preantral follicle culture systems were developed in this study and had never been reported before.The aim of this study is to develop pre-antral mouse/rat follicle culture in-vitro assay in repriductive toxicology testing to provide information regarding the potential mechanism of action and the identification of chemicals which have female reproductive toxicity.1.Establishment of in-vitro pre-antral mouse follicle culture systemThe ovaries were from prepubertal mice(C57B1/6JxCBA/Ca)(aged 12～14 days).Only follicles with 2-3 layers of granulose cells,a centrally located oocyte and a diameter in the range 100～130μm,which were enclosed by an intact basal membrane and had some attached theca cells,were mechanically dissected and randomly allocated to 96-well culture plates.The preantral follicles were subsequently cultured individually for 12 days within culture medium covered by mineral oil.On day 12 of culture,the follicles were induced ovulation for 16h.The in-vitro developmental characteristics of the follicles were observed including hormonogenesis,folliculogenesis and oogenesis,by comparing in vitro growth and maturation of oocytes(IVG) with in vivo growth and in vitro maturation of oocytes(IVM).1.1 Developmental characteristics of mouse preantral follicles in vitroA total of 419 from 7 prepubertal mice,preantral follicles and oocyte with an average diameter of 118.51±10.40μm and 50.75±1.67μm,respectively,376/419(89.74%) of follicles were visibly associated with theca cells and possessed a close follicle(granulosa) cell/oocyte apposition.At day 12 of culture,follicles and oocyte with an average diameter of 118.51±10.40μm and 50.75±1.67μm were measured.31.03%of the cultured follicles reached the pre-ovulatory stage with a large antrum-like cavity,86.87%was survival and 50.60%COCs were released by maturation induction in this study. The morphological observation showed that developmental characteristics of mouse preantral follicles in vitro were similar to those in vivo,including the formation of antral follicle and preovulatory follicle,the proliferation and differentiation of granulosa cell, cumulus cell mucification,releasingof cumulus-oocyte complexes(COCs),oocyte maturation and the formation of first polar body.1.2 Hormone secretion characteristics of in-vitro cultured folliclesEstradiol and progesterone was detected using electrochemiluminescence immunoassay. The results showed that the follicle cultures were characterized by a consistent increase in the secretion of oestradiol as measured in the conditioned medium throughout the in vitro culture period from day 4 onwards.Progesterone secretion increased characteristically only after the in-vitro ovulatory stimulus from day 12 to 13.In a word,the patterns of hormone secretion observed in preantral follicle culture were similar to those characteristic for in-vivo follicle hormone secretion.1.3 The oocyte maturation in vitroThe numbers of oocytes with germinal vesicle(GV) did not differ between IVG and IVM groups,18.89%oocytes form the first polar body in IVG groups and 53.53%oocytes form the first polar body in IVM groups.The results were not found abnormality chromosome segregation in this study.In conclusion,judging from the three aspects of folliculogenesis,hormonogenesis,and oogenesis,the establishment of in-vitro pre-antral mouse follicle culture is successful.Our results were in accordance with that of Wang Yubao.However,the PB formation rate was relatively lower than that reported by Fengyun Sun.2.Establishment of in-vitro pre-antral SD rat follicle culture system2.1 Determination of in-vitro pre-antral SD rat follicle culture conditions(1) The size of follicle for culture:Mimicing in-vitro pre-antral mouse follicle culture system,we studied follicle size for culture.The results displayed that 100～130μm,131～150μm and 151～160μm groups of the follicle diameters increase gradually up to344.29±105.40μm,535.76±105.41μm,587.65±138.69μm,respectively,showing statistical significance comparing to pre-culture(p＜0.05).Antral follicles and surviving follicles in 100～130μm groups were declined significantly comparing to 131～150μm and 151～160μm group.(2) The rat years of age:The ovaries were from prepubertal SD rat aged PND12,13 and 14.The follicles with variant diameter in the range of 100～160μm were mechanically dissected.In PND13 rat,94.87%follicles were 130～150μm in diameters in all dissected follicles,PND12 rat(17.54%) and PND14 rat(about 29.58%).(3) The time of starting to induce ovulation:Observation of the respective releasing of COCs in d10,d11 and d12 of the cultured ovarian follicle showed that there was not significant difference in three groups.Hence,d10,d11 and d12 of culture could be chose as the time of starting to induce ovulation.(4) The time of COCs released from follicles:After inducing ovulation for 16h,18h,20h and 22h respectively,the numbers of COCs released from follicles increased in a time-dependent way.After inducing ovulation for 20h,the rate of COCs released from follicles reached 47.84%.Therefore the best observing time of COCs releasing is 20h.(5) The numbers of ovarian follicle each group:Taking into consideration the fact that the variation of the indexes of rats has are similar to that of mice,16-20 ovarian follicles in each group were chose for examination,according to the report of Val'erie Van Merris.2.2 Developmental characteristics of early rat preantral follicles in vitroOf a total of 296 preantral follicles and oocytes with an average diameter of 141.26±8.82μm and 51.25±1.37μm,respectively.96.28%follicles possessed theca cells and a close follicle(granulosa) cell/oocyte apposition.The size of the follicles increased dramatically up to543.21±131.11μm and 70.48±2.89μm at day11of culture.75.67%of the cultured follicles reached the pre-ovulatory stage with a large antrum-like cavity and 92.23%follicles were survival at day 11 of culture.Mucification with expansion of cumulus cells(44.93%COCs)was observed 20h after the in vitro ovulatory stimulus in this study.The morphological observation showed that developmental characteristics of rat preantral follicles in vitro were similar to those in vivo,including the formation of antral follicle and preovulatory follicle,the proliferation and differentiation of granulosa cell, cumulus cell mucification,releasingof cumulus-oocyte cell complexes(COCs),oocyte maturation and the formation of first polar body.2.3 Hormone secretion characteristics of in vitro cultured folliclesEstradiol and progesterone was measured using electrochemiluminescence immunoassay.The results showed the follicle cultures were characterized by a consistent increase in the secretion of oestradiol as measured in the conditioned medium throughout the in vitro culture period from day 4 onwards.Progesterone secretion increased characteristically only after the in vitro ovulatory stimulus from day 11 to day12.2.4 Oocyte maturation in vitro7.44%(9/121)oocytes was blocked germinal vesicle(GV) stage,57.02%(69/121) oocytes initiated resumption of maturation(GVBD),35.53%(43/121) oocytes formed a first polar bodyin IVG groups,1.86%(6/322)oocytes blocked germinal vesicle(GV) stages,40.06%(129/322)oocytes initiated resumption of maturation(GVBD),56.21% (181/322) oocytes formed a first polar body in IVM groups.The results were not found abnormality chromosome segregation in this study.3.Application of in-vitro SD rat follicle culture system for femal reproductive toxicity testingThe PB formation rate was lower than that reported by Fengyun Sun.On the other hand, rats are the most common experimental animals in reproductive toxicity testing.Therefore, in-vitro rat follicle culture system was applied for femal reproductive toxicity testing in this study.Some chemicals including cadmium chloride(CdCL₂),DEHP,MEHP and Nocodazole were assessed for hormonogenesis,folliculogenesis and oogenesis using the in-vitro SD rat follicle screen system.3.1 Follicle toxicity of cadmium chloride(CdCL₂) in vitroCadmium chloride is a common industrial poison and environment contaminant.As some animal experiments show,cadmium chloride can interrupt the function of ovaries, extend the oestrous cycle of rats,cause histological and structural alteration,inhibit the growth of ovary follicle,and increase the number of atretic follicle.Moreover,it impairs the ovulation and causes infertility.Some in-vitro bioassays show that the progesterone production was significantly decreased by cadmium chloride.3.1.1 Effects of CdCL₂ on follicle development in vitroCdCL₂ with concentrations＞1.2μg/ml had been reduced significantlysurvival follicles rate(66.44±3.95%)compared to the control group(91.57±1.32%) on day11 (p＜0.05),wherea33.57±3.95%degenerated or underwent premature oocyte release This dose dependent decrease showed a coefficient of correlation(R²) of 0.90.CdCL₂ with the concentrations of 1.6μg/ml impaired follicle growth and differentiation,significantly reduced(p＜0.05)the rate of antral follicles(42.51±6.66%)compared to the control(79.68±6.62%).CdCL₂ did not influence the17β-E₂ secretion in the concentration range of 0.8～1.6μg /ml CdCL₂.The basal progesterone production in the treated groups was significantly decreased for＞0.8ug/ml CdCL₂ compared to the control after hCG-administration on days 11 and 12.3.1.2 Effects of CdCL₂ on different developmental stage follicle in vitroTo determine sensitive follicle stage of CdCL₂ exposure,the culture follicles exposed to CdCL₂ on day2(pre-antral follicle),day 6(antral follicle),day11(pre-avulation follicle) for 48h,respecfively.CdCL₂ with concentrations 1.6ug/ml on day 2 had a significantly reduced(p＜0.05) survival rate(52.51±7.84),the rate of antral follicles(42.51±6.66%),compared to follicles exposed to same concentrations CdCL₂ on day 6(81.75±8.13%), (74.24±6.98%),respectively.Anormal follicle rate(47.49±7.48%) increase significantly on day 2,compared to day6(47.49±7.48%).The numbers of GV oocyte(58.34±11.79%) increase significantly on day2,compared to day6(15.66±9.28%),day 11(20.2±2.86%).There are no significant adverse effects of CdCL₂ exposure on day6 upon follicle development and oocyte maturation.In IVM group,COCs treated with concentrations 1.6ug/ml CdCL₂ reduced(p＜0.05) PB-formation rate(34.11±0.34%),compared to control (67.98±6.57%).These results demondated that CdCL₂ would inhibit both follicle development and oocyte maturation from pre-antral follicle and oocyte maturation and PB-formation from pre-ovulation follicle.3.2 Effects of DEHP and MEHP on follicle development in vitro Ai-2-ethylhexyl phttmlate(DEHP) and mono-2-ethylhexyl phthalate(MEHP) belongs to phthalicacidesters(PAEs).Animal experiments showed that the toxicity on reproduction of DEHP result from its metabolite MEHP,which affected granulose cells of ovaries and remarkably inhibited the production of estrogen during preovulatory stage.Some in-vitro bioassays show that MEHP decreased the production of progesterone as well as the FSH-induced cAMP production.Follicles treated with concentrations＞10μg/mLMEHP had a significantly reduced(p＜0.05) survival rate(54.76±3.37%) compared to the control group(91.57±1.32%)on day11. wherea 45.24±3.37%follicles degenerated or underwent premature oocyte release.This dose dependent decrease showed a coefficient of correlation(R²) of 0.92. Concentrations＞20ug/mL MEHP dose dependently impaired follicle growth and differentiation,antral follicles(46.18±1.67%) had a significantly reduced(p＜0.05) compared to the control group(85.91±5.03%).DEHP showed no adverse effects upon follicle development and there were no significant defference between DMSO and control.3.3 Effects of Nocodazole on oocyte maturation in vitroNocodazole,methyl[5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl]carbamate,a synthetic anti-tumour drug interfering with microtubule assembly,competes with colchicine for the same binding site on the Arg-390 of b-tubulin and directly affects microtubule polymerization and microtubuledependent processes.When COCs exposed to Nocodazole concentrations of 100nM for 20h in IVM,there existing oocyte cytotoxicity.The COCs exposed to Nocodazole concentrations of＞5nM, We observed signicant reduced in PB-formation rate compared to its control group. Although the preovulation follicles exposed to Nocodazole concentrations of 10～40nM showed no statistical difference in COCs rate isolated from follicle compared to the controls,there existed a trend.Concentrations of 40nM Nocodazole dose significantly reduced(p＜0.05)PB-formation rate(19.35%) compared to the control group(37.78%)Summary1.This study developed in-vitro mouse pre-antral follicle culture system.2.In-vitro rat preantral follicle culture system were developed and this system had never been reported before 3.Rat preantral follicle culture as a novel in-vitro assay was applied to detect the effects of four chemicals on folliculogenesis,steroidogenesis and oogenesis,and on variant developmental stages,which further revealed that the bioassay is a efficient tool to study ovarian toxicity and their mechanisms of chemical.4.A preliminary protocol of in-vitro rat preantral follicle culture system for the study on female reproductive toxicity was proposed,including the morphological observation, stages of follicle differentiation,hormone secretion profiles,oocyte diameters,the formatiom of PB and the genetoxic of oocyte.