A Mechanistic Study Of βB2-crystallin In Regulating Reproductive Of Male Mice

In recent years, there is a rising trend of infertility of couples who with thechildbearing age. The USA epidemiological investigations showed that, the prevalencerate of infertility is about10-12%in the couples who with childbearing age, andapproximately10-15%are happened in developing countries, male factor accounting for30%of the causes of infertility, which not only seriously troubled the couples and theirfamilies, also was a heaven burden to the society. However, the occurrence mechanism ofinfertility is unclear. Therefore, deeply investigate the mechanism of the infertility andexploy the new ways to prevent and treat it, not only has important scientific value, butwill have a great prospect and social benefits.Our research found that the reproductive of male mice with targeted distruption ofβB2-crystallin (Crybb2) was decreased, and the influence was much more than thenon-reproductive gene which was knocked out.We speculated that Crybb2may be animportant gene in regulating the reproductive, which has not been reported. Then, itbrings the questions that, how Crybb2regulating the reproductive of male mice? What’sthe influence factor? If can improve the reproductive function through promoting theexpression of Crybb2? Which just is the purpose of this research.Crystallins are the major structural protein components of the vertebrate eyelens.They are built in the same manner and contain α-, β-and γ-crystallins according tomigration in the electric field. α-crystallins are recognized to function both as structuralproteins and as chaperones in the lens[1,2]but little is known about β-crystallins. Crybb2is significantly higher compared with others in the β-crystallin family, and itswater-soluble components increase abnormally with age. The regμlar structure andwater-soluble ingredient point to an important role in maintaining lens high refractiveindex and transparencyPrevious studies showed that, Crybb2has a particular structural and function, inaddition to high express in the lens and is essential for maintenance of lens transparencyand other normal biological characteristics, Crybb2also has been reported to express insome extralenticμlar tissues such as retina, brain, and testis, and has a certain influence on the related function of these tissues. In rescent studies, Crybb2has been implicated in thesubfertility of mice exhibiting mutant Crybb2, but the actual mechanism remains elusive.To understand further the function of Crybb2gene and corresponding protein, wehave generated mice with targeted deletion of the Crybb2gene in previous studies.Surprisingly, fertility reduced markedly in male homozygous knock-out mice comparedwith that of wild-type mice. To investigate the reasons, further investigation wasundertaken to observe and research the function of Crybb2on male reproductive, explorethe underlying mechanism of subfertility in male Crybb2deficient mice. In order toprovide the new ideas and ways on the mechanism study of clinical infertility patients.Purpose:To observe the reproductive changes of male mice with targeted disruption of Crybb2,analysis the function of Crybb2in the maintenance of male normal reproductive function;explore the underlying mechanism of subfertility in male Crybb2deficient mice. In orderto provide the new ideas and ways on the mechanism study of clinical infertility patients.Methods:1. Identification of mouse genotype with targeted disruption of Crybb2Before the experiment, take the organization of mouse tail end and extract thegenomic DNA. Then, identify the genotype of the mice by PCR and agarose gelelectrophoresis analysis.2. Clear the expression of Crybb2in mouse testisUsing Realtime PCR and Western blot detection to detect the expression of Crybb2in normal wild-type mouse testis and no expression in KO mice. Further, using theimmunohistochemistry detection to analysize the position of Crybb2within the normalmouse testis.3. Observe the influence of Crybb2knock out on mouse reproductive functionIn the same period, observe the number of litters, total number of pups and litter sizeof female mice, which paired with the WT and KO male mice respectively, count thesperm number of the KO mice and observe the testicular tissue development of malemice after birth1,2,3,4,6,8weeks.4. Detection of the proliferation and apoptosis of testis germ cellsUsing the TUNEL and BrdU methods to detect the apoptosis and proliferation of theKO mouse testicular tissue germ cells, to analyse the possible causes of the reduced sperm counts and the reproductive.5. Test of the relative proteinsUsing Realtime PCR and Western blot methods to detect the level of CaMKIV,Crem, Tsg23, Bax, Bcl-2in KO mouse testis which proteins are related to thereproductive, proliferation and apoptosis.6. Detect the level of testosterone in mouse serumAdopt enzyme linked immunosorbent assay (ELISA) to examine the testosteronelevel of mouse serum, analysis of whether the sperm count and reproductive changescaused due to the changes of testosterone concentration.Results:1. Identification results of the mouse genotypeTwo genotype was successfμlly amplified by PCR and agarose gel electrophoresisanalysis, the homozygous KO mice and the homozygous WT mice, which provide theguarantee for the following experiment.2. The expression of Crybb2in mouse testisThere was Crybb2gene and its protein expression in the WT mouse testis by themethods of Realtime PCR and Western blot, while no Crybb2expression in the KOmouse testis; immunofluorescence test showed that Crybb2was mainly expressed in thespermatogonia of WT mouse seminiferous tubules.3. The impact of Crybb2knock out on male reproductive functionWithin the same productin cycle, the number of litters, total number of pups andlitter size were reduced significantly by the female mice which paired with KO male mice,while five couples of KO mice gave birth to13litters (mean litter size9.1±1.4) in athree-month period, only7litters (mean litter size4.8±1.5) were yielded from an equalnumber of WT mice; sperm counts was also reduced in KO mature male mice; the testisof4-week-old male mice was hyperplasia apparently, the testis weight was increased theorgan mass of testis was also higher, while there was no significant difference in bodyweight; HE showed that the seminiferous tubules were thinner and scattered.4. The results of the proliferation and apoptosis testsThe results of BrdU and TUNEL assays showed that, compared to the normal mousetestis germ cells, the proliferation and apoptosis of KO mouse spermatogonia in testiswere increased significantly. 5. The expression changes of the protein in mouse testisWe detected the level of CaMKIV, Crem, Tsg23, Cklfsf2b, Bax, Bcl-2in KO mousetestises by Real time PCR and Western blot methods, and the results indicated that bothCaMKIV mRNA and protein were decreased, as well as Bcl-2, in KO mouse testisescompared with those in WT mouse testises, the level of Bax protein was increased whilethere was no changes in Crem,Tsg23and Cklfsf2b levels.6. The level of serum testosterone was decreased in KO miceELISA assay showed that, the serum testosterone level of the KO mice wasdecreased significantly compared with the normal wild-type mice.Conclus`ion:1.`There was a high expression level of Crybb2in the mouse testis.2.`The reproductive of male mice was significantly reduced with the disruption ofCrybb2.3.`The decreased level of CaMKIV in the KO mouse testis may affect the expression ofBcl-2, which further disturbs the proliferation and apoptosis of germ cells in KO mousetestis, with the addition of the decreased testosterone level, contributes to the reducedfertility of KO mice.