Abnormal Ocular Pigmentation And Reduced Fertility In Pale Ear And Pearl Mice

The eye has pigmented cells of two different embryonic origins and therefore it is a good model for studying melanosome biogenesis and melanin production/deposition.Specifically,melanocytes in the choroid and stroma of the iris and ciliary body develop from the neural crest,while pigmented cells in the RPE and pigment epithelium of the iris and ciliary body migrate from the neuroepithelium of optic cup.The Hermansky-Pudlak syndrome(HPS)is an autosomal recessive disorder characterized by oculocutaneous albinism,prolonged bleeding,ceroid/lipofuscin deposition and lung fibrosis.These symptoms were caused by defective lysosome-related organelles(LROs),including lysosomes,melanosomes,and platelet-dense granules.The mouse genes for HPS,pale ear(ep)and pearl(pe),are homologous to the human HPS1 and HPS2 genes.To analyze the melanin deposition of different embryonic-originated tissues in HPS1 and HPS2 mutants,we ivestigated the pigmentation in eyes of pale ear and pearl mice.Here,we reported the delayed and reduced pigmentation in eyes of pale ear and pearl mice in early postnatal stages and adulthood.In eyes of pearl mice,the hypopigmentation was more severe than pale ear mice after postnatal day 7,especially in the neural crest-derived tissues,including the choroid and the stroma of iris and ciliary body.Tyrosinase assay and L-3,4-dihydroxyphenylalanine(L-DOPA)gel staining assay revealed that tyrosinase activity in eyes of pale ear mutants was greatly reduced in early postnatal stages and increased gradually after postnatal day 7(P7).However,the tyrosinase activity in eyes of pearl mice increased slower than that in pale ear mice after P7.Further histological examination revealed that hypopigmentation in the retinal pigment epithelium(RPE)and pigment epithelium of the iris and ciliary body,which are derived from the optic cup,was more severe than that in neural crest-derived tissues.In addition,macromelanosomes were exclusively present in neural crest-derived melanocytes of pale ear adults,but absent at early postnatal stages.Taken together,the mutation in the HPS1 gene could cause two distinct phenotypes in pigmented cells of different embryonic origins.Although the pigmentation was weaker in eyes of pearl mice than pale ear mice,the tyrosinase activity was stronger in pearl mice,suggesting that the degradation of aberrantly transported tyrosinase maybe weaker in pear1 mice than pale ear mice.Furthermore,the pigmentation in eyes of mice doubly heterozygous for HPS1 and HPS2 genes was similar to the wild-type,while the hypopigmentation in iris of double mutant was more severe than either single mutant.Different from the pale ear mice,macromelanosomes were absent in the iris stroma of pearl miceBeside the severe hypopigmentation,our research also revealed that there was a melanin-degradation during the period from postnatal day 28 to 3 months in the iris of pearl mice.This observation was further confirmed by the TEM analysis.In the iris of pearl mice,melanosome counts increased slower,and the melanosome maturity decreased faster than that in pale ear mice.Based on the melanin-degradation in the iris of pearl mice and the observation that macromelanosomes were absent in the iris stroma of pale ear mice at early postnatal stages,we considered that the effects of HPS1 and HPS2 mutations on melanosome biogenesis may gradually exhibited with age.Further Western Blotting analysis revealed that the protein Cathepsin-D in the eyes of pearl mice expressed more than that in pale ear and wild-type mice at early postnatal days.This observation suggested that the immature melanosomes in eyes of pearl mice was degraded by Cathepsin-D,leading to reduced melanosome numbers in eyes of pearl mice.The dominant pigment in melanosomes is melanin that protects the ocular cells in many ways.Melanin in eyes not only absorbs excessive visible light and UV radiation,but also acts as an antioxidant against oxygen species and a weak scavenger of free radicals,and thus protects the retina against photo-oxidative stress.Furthermore,melanin can protect ocular cells against chemical stress,largely by binding to various toxic chemicals.Lipofuscin is another pigment in ocular pigmented cells.It is a non-degradable end product that results from degradation of photoreceptor outer segments,and appears in an inverse relationship to the amount of melanin in RPE cells.Lipofuscin accumulates with age in the RPE and has significant photo-toxic potential.In eyes of pale ear mice,an increased accumulation of lipofuscin in RPE was observed.Furthermore,the wavelength of lipofuscin autofluorescence in the pale ear RPE was shorter than that in wild-type RPE.However,the intensity and wavelength of autofluorescence signals in the RPE of pearl mice were similar to that of wild types at all analyzed stages.We investigated the pigmentation and tyrosinase distribution in pale ear and pearl mice,and found that a single gene mutation could cause two distinct phenotypes in pigmented cells of different embryonic origins.Our study also indicated that HPS1 and HPS2 play distinct roles in the production of melanosomes and accumulation of lipofuscin.The investigation could deepen our knowledge of protain trafficking and organelle formation.Our study may be helpful to find new treatments inoculocutaneous albnisim.The Hermansky-Pudlak syndrome is a form of oculocutaneous albinism characterized by albinism,prolonged bleeding and lung fibrosis.The syndrome is single gene inheritance disease,but different subtypes of the syndrome were due to distinct mutant genes.These mutations affect the protein traffiking,causing defects in lysosomes,melanosomes,and platelet-dense granules.The mouse genes for HPS,pale ear(ep)and pearl(pe),are homologous to the human HPS1 and HPS2 genes.The mutation of these genes could cause defects in lysosomes.The acrosome of sperm is a secretory lysosome,and autophagy in the lysosome is essential for degradation of cytoplasmic contents in spermatid.Therefore,defects in the lysosome-endosome traffic system may affect the spermgenesis and acrosome reaction.However,very little studies has mentioned the abnormities in spermgenesis and acrosome of in pale ear and pearl mice.Herein,we investigated the development of reproductive organs and spermgenesis in pale ear and pearl mice.Herein,we reported that the pale ear mice have a lower ability to impregnate the female mice and smaller litter size.While in the pearl male,the main phenotype of subfertility was a lower ability to impregnate the female mice,and the litter size of pearl mouse was closed to that of wild-type.Although the shape and size in pale ear mice were similar to the wild-type mice,a delayed development of testis and dysplasia of epididymis were observed in the pearl mice.The spermgenesis in pearl mice was extremely lower than wild-type mice at 6 weeks.The spermgenesis in pearl mice gradually increased as testis developed,but the sperm count was still smaller than that in wild-type and pale ear mice(sperm counts:B6>ep>pe).Furthermore,the ability of testis in secreting testosterone was also affected in testis of the pearl mouse.Beside the reduced number of the sperms,the sperm motility was also decreased in the pale ear and pearl mouse(sperm motility:B6>pe>ep).The CTC staining assay also revealed a mild abnormity in acrosin release of pale ear mice.Constitutive WNT/β-catenin signaling in murine sertoli cells disrupts their differentiation and ability to support spermatogenesis and in mutant mice thatβ-catanin is stabilized in Sertoli cells,the testis size was much smaller than that of control littermates.PCP-2,a protein tyrosine phosphates(PTPs)transmembrane receptor,dephosphorylate β-catanin to regulate its intracellular distribution.It has been reported that AP-3 is involved in the PCP-trafficking.We found that the expression of cathepsin-D was enhanced in cytoplasmic extract of pearl mice,indicating that the disruption of the AP-3 pathway may result in abnormal cellular location of PCP-2,which in turn remove the inhibition in the beta-catenin signaling.The abnormity of acrosome of pearl mouse were mainly detected in round spermatids at cap phase.In C57BL-6J,the electron density of the electron-lucent acrosomal vesicles was lower than the electron density of the adjacent nucleus.While in the pearl mouse,the electron density of the electron-lucent acrosomal vesicles was higher than that of the nucleus.Metal ions play enssital roles in the maintenance of mammalian fertility.Moreover,the transporters of metal-ion may be transported though the lysosome-endosome traffic system.We found that the magnesium content in the whole blood sample of pearl mice was significant lower than that of wild-type mice.Besides,we revealed that the zinc distribution in sperm head in pale ear and pearl mice were different from that in the wild-type mice.These results indicated that the AP3B1 and HPS1 genes may affect the development of reproductive organs and spermgenesis by regulating the trafficking of metal-ions.We investigated the fertility of pale ear and pearl mice in multiple levels.Although the ep and pe male mice have the ability to breed,the mutations still affect the fertility of these mice by many direct or indirect pathways.Investigations in related pathways could improve our knowledge in protein trafficking and organelle formation,and may further provide new research earas in clinical infertility study.