Crybb2 deficiency impairs fertility in female mice.

Beta-B2-crystallin (CRYBB2), encoded by Crybb2 gene, is a major protein in the mammalian eye lens that plays an important role in maintaining the transparency of the ocular lens. However, CRYBB2 also plays important roles in many extra-lenticular tissues and organs such as the retina, brain and testis. Our previous studies demonstrated that male Crybb2 deficient (Crybb2(-/-)) mice have reduced fertility compared with wild-type (WT) mice, while female Crybb2(-/-) mice exhibited reduced ovary weights and shorter estrous cycle percentages. Here we specifically investigated the role of CRYBB2 in the female reproductive system. Our studies revealed that ovaries from female Crybb2(-/-) mice exhibited significantly reduced numbers of primordial, secondary and pre-ovulatory follicles when compared with WT mice, while the rate of atretic follicles was also increased. Additionally, fewer eggs were collected from the oviduct of Crybb2(-/-) female mice after superovulation. Estrogen levels were higher in the metestrus and diestrus cycles of female Crybb2(-/-) mice, while progesterone levels were lower in diestrus cycles. Furthermore, the expression of survival and cell cycle genes, Bcl-2, Cdk4 and Ccnd2, were significantly decreased in granulosa cells isolated from female Crybb2(-/-) mice, consistent with the predominant expression of CRYBB2 in ovarian granulosa cells. Our results reveal a critical role for CRYBB2 in female fertility and specific effects on the proliferation and survival status of ovarian granulosa cells.

Aquaporin 4 knockdown exacerbates streptozotocin-induced diabetic retinopathy through aggravating inflammatory response.

Diabetic retinopathy is a leading cause of reduced visual acuity and acquired blindness. Diabetes is known to alter the amount of retinal expression of the water-selective channels aquaporin 4 (AQP4). However, the function and impact of AQP4 in diabetic retinopathy is not well understood. In the present work, diabetes was induced by intraperitoneal injection of streptozotocin in Sprague-Dawley rats. Two weeks later, AQP4 shRNA (r) lentiviral particles or negative lentiviral particles were delivered by intravitreal injection to the eyes. Gene delivery was confirmed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blotting analysis. Eight weeks later, BRB breakdown was measured using Evans blue dye. Images of retinal sections were obtained and the thicknesses of the retinas were determined. Retinal leukostasis measurement was performed using acridine orange leukocyte fluorography. The mRNA levels of IL-1ß, IL-6, intercellular adhesion molecule 1 (ICAM-1), glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) were determined using qRT-PCR method. AQP4 shRNA (r) lentiviral particles or negative lentiviral particles were transfected into rMC-1 cells to investigate its effect on inflammation induced by high glucose. Incubation with IL-1ß or IL-6 was performed to test their effect on AQP4 expression in rMC-1 cells. In the current work, it was found that AQP4 expression was enhanced in the retina of diabetic rats. AQP4 knockdown led to exacerbation of retinopathy including enhancing retinal vascular permeability, retinal thickness, pro-inflammatory factors expression, and VEGF and GFAP expression in retinas of diabetic rats. AQP4 knockdown enhanced the expression of pro-inflammatory cytokines induced by high glucose in rMC-1 cells. In addition, AQP4 knockdown enhanced the release of IL-6 and VEGF from rMC-1 cells into the medium. Moreover, it was found that incubation with IL-1ß or IL-6 suppressed AQP4 expression in rMC-1 cells. These results suggested that streptozotocin injection induced diabetes resulted in compensatory increases of AQP4 expression, and downregulation of AQP4 exacerbated diabetic retinopathy through aggravating inflammatory response, at last in part. Therefore, regulation of retinal function by AQP4 may attenuate diabetic retinopathy, offering a promising therapeutic strategy for diabetic retinopathy.

Clinical significance of serum biochemistry changes in mice with targeted disruption of ßB2-crystallin gene.

AIM: To explore the pathogenesis, influencing factors, ways of medical intervention and evaluation indicators of cataract by observing changes in serum biochemical indices in mice with targeted disruption of ßB2-crystallin. METHODS: Nine 6-week-old male mice with targeted knockout of ßB2-crystallin were used as the study group, and nine age- and sex-matched normal wild-type mice as the control group. The genetype of the modeled mice was identified by PCR technique. Tropicamide and phenylephrine eye drops were used as the cycloplegic agents to observe changes in lens opacity with a slit-lamp. The lens was then removed and blood was collected for biochemical evaluation in the serum. RESULTS: Two genotypes were successfully identified by PCR technique. Slit-lamp observation showed that the lens cortex was opaque and GSH level in the lens cortex was remarkably decreased in mice with ßB2-crystallin deficiency compared with the control group (P<0.01). Serum Na(+), Cl(-), Ca(2+), Mg(2+) and Fe(2+) levels, ALT and AST activities, and TP, ALP, Cr, TC, GLU content were decreased significantly compared with the control group (P<0.05). There was no difference in LDH, P, Cu(2+), K(+) levels between the two groups (P>0.05). CONCLUSION: Compared with the wild-type mice, serum biochemical indices underwent significant changes in mice with targeted disruption of ßB2-crystallin gene, especially with abnormal distribution of Na(+)&Ca(2+), which induced the formation of cataract.