Impaired Fertility In Female Mice Lacking Crybb2

Now days, infertility has become an important issue affecting our reproductive health,and infertility caused by ovarian dysplasia is also increasing in young people. Ovarianfollicles development includes four stages: primordial follicles stage, primary folliclesstage, secondary follicles stage and mature follicle stage. Only a small part of the folliclesare able to reach maturity for ovulation because follicles atresia happening to most of themduring the development. There are two major influencing factors of ovarian development:one is the hypothalamus-pituitary-gonadal axis regulation and the other is the regulationfrom some inside factors of ovary.Crystallins contain α-, β-and γ-crystallins, which are the major components ofvertebrate eye lens. βB2crystallin is a major component of the β crystallins family, and isessential for maintenance of lens transparency and refractive index. In recent years, thefunction of βB2crystallin in some extralenticμlar tissues has become the new hot spot ofresearch, especially in reproductive function. It is reported that βB2crystallin has animportant impact on the development and function of testis, but its impact on ovary israrely reported.Previous studies have shown that the fertility of male mice with targeted disruption ofβB2crystallin is decreased. This leads to the below questions: whether βB2crystallin alsohas some impact on the fertility of female mice. Whether it will affect the development ofthe ovary of female mice, and how does it affect ovarian development? Our research is toanswer these questions.Having generated mice with targeted deletion of the Crybb2gene provides the base forresearching the effects of Crybb2gene on reproductive function of female mice. Base onprevious study, this paper does a further research on the function of Crybb2on female micereproduction, and explores the underlying mechanism of βB2crystallin affecting femalereproduction. We hope to provide a new idea for the clinical study of infertility.Purpose:To observe the reproductive and estrous cycle changes of female Crybb2deficient mice,analysis the changes of structure and function in female Crybb2deficient mice ovary,identify Crybb2localization in ovarian cells, and initially explore the mechanism ofCrybb2in the regulation of female reproductive function in related cells of mouse ovary, Hope to provide the new ideas on the study of female clinical infertility patients.Methods:1. Observe the influence of Crybb2deficient on mouse fertilityWT and Crybb2-/-female mice in the same age mated with WT male mice, Observedfive month to record the total number of litters and pups, litter size and litters per female.2. Observe the influence of Crybb2deficient on mouse estrous cycleSelected adult WT and Crybb2-/-female mice, and estrous cycle was determined byvaginal smears. Observed for21days and recorded (proestrus, estrus, metestrus, diestrusperiod) percentage.3. Observe the change of structure and function of ovary in WT and Crybb2-/-femalemiceWeight and size of adult WT and Crybb2-/-mice ovary were measured. Follicle numberwas measured by HE staining, and hormone secretion were measured bychemiluminescence.4. Clear the expression of Crybb2in mice ovaryUse western blot method to detect the expression of Crybb2in normal WT andCrybb2-/-mice ovary. Immunohistochemistry was used to analysize the position of Crybb2within the WT mouse ovary.5. Detection of the proliferation and apoptosis of granulosa cellsFlow cytometry was uesd to detect the apoptosis and proliferation of the Crybb2-/-mouse granulosa cells. Real time PCR method was uesd to detect the level of related geneof apoptosis (bcl-2) and cell cycle (cyclinD2, cdk4).6. Detection of estrogen receptor mRNA levelsReal time PCR was used to detect the mRNA level of ERα and ERβ expression in WTand Crybb2-/-mice granulosa cells.Result:1. The influence of Crybb2deficient on female mice fertilityCompared with WT female mice, total number of pups, litter number, litter size andlitters per female were reduced significantly in Crybb2-/-female mice in the samereproductive cycle.2. The influence of Crybb2deficient on mice estrous cycleThere was no difference in proestrus and metestrus rate between Crybb2-/-and WT mice, but estrus rate was significantly lower than WT mice, diestrus significantly higherthan WT mice, within21days the number of the estrous cycle was significant lower thanWT mice.3. The change of structure and function of ovary in WT and Crybb2-/-female miceThe ovarian weight and size of Crybb2-/-mice were significantly reduced comparedwith WT mice, HE staining appeared that mature follicle was reduced in Crybb2-/-miceovaries, no significant corpus luteum, atretic follicles was increased, the number of newlyformed follicles was rarely. The number of Crybb2-/-mice ovarian primordial follicles,secondary follicles, and mature follicles was significantly reduced.Crybb2-/-mice estradiol secretion was significantly higher than WT mice in metestrusand diestrus, and secretion of progesterone in diestrus was lower than WT mice, butandrogen secretion was no statistic differences.4. The expression of Crybb2in mice ovaryCrybb2was expressed in the WT mice ovary by the methods of Western blot, whileno expression in the Crybb2-/-mice ovary. Immunofluorescence showed that Crybb2wasmainly expressed in the cytoplasm of granulosa cells of WT mice ovary.5. The results of the proliferation and apoptosis detectionIn granulosa cells cultured from Crybb2-/-mice, G0/G1phase percentage was morethan WT mice, S phase and G2/M phase percentage were less than WT mice. Earlyapoptosis ratio increased significantly in Crybb2-/-mice ovarian granulosa cells. RT-PCRshowed in Crybb2-/-mice granulosa cells, bcl-2, cyclinD2and cdk4mRNA level weresignificantly reduced.6. Detection of estrogen receptor mRNA levelsRT-PCR results show the estrogen receptor ERα mRNA level was decreased whileERβ mRNAlevel was not statistically significant.Conclusion:1. The fertility of female mice was significantly reduced in Crybb2-/-mice.2. Estrous cycle was disorder with the disruptive of Crybb2. Reduction of estrous ratio andthe number of the estrous cycle will affect the mating of male and female mice, therebyreduced the pregnancy rate.3. Crybb2knockout mice were ovarian dysplasia, the number of development follicles wasreduced, and ovulation was decreased, further affecting reproduction. Disorder of secretionof estrogen and progesterone also affect ovarian function. 4. Crybb2was expressed in ovarian granulosa cell cytoplasm, and may through relatedgene to affect apoptosis, proliferation and hormone secretion, causing granulosa cellproliferation, apoptosis and hormone secretion abmormal, then lead to ovarian structureand function changing, further affected the reproductive function.