Sperm motility is vital to human reproduction. Malformations of sperm flagella can cause male infertility. Men with multiple morphological abnormalities of the flagella (MMAF) have abnormal spermatozoa with absent, short, coiled, bent, and/or irregular-caliber flagella, which impair sperm motility. The known human MMAF-associated genes, such as DNAH1, only account for fewer than 45% of affected individuals. Pathogenic mechanisms in the genetically unexplained MMAF remain to be elucidated. Here, we conducted genetic analyses by using whole-exome sequencing and genome-wide comparative genomic hybridization microarrays in a multi-center cohort of 30 Han Chinese men affected by MMAF. Among them, 12 subjects could not be genetically explained by any known MMAFassociated genes. Intriguingly, we identified compound-heterozygous mutations in CFAP43 in three subjects and a homozygous frameshift mutation in CFAP44 in one subject. All of these recessive mutations were parentally inherited from heterozygous carriers but were absent in 984 individuals from three Han Chinese control populations. CFAP43 and CFAP44, encoding two cilia-and flagella-associated proteins (CFAPs), are specifically or preferentially expressed in the testis. Using CRISPR/Cas9 technology, we generated two knockout models each deficient in mouse ortholog Cfap43 or Cfap44. Notably, both Cfap43-and Cfap44-deficient male mice presented with MMAF phenotypes, whereas the corresponding female mice were fertile. Our experimental observations on human subjects and animal models strongly suggest that biallelic mutations in either CFAP43 or CFAP44 can cause sperm flagellar abnormalities and impair sperm motility. Further investigations on other CFAP-encoding genes in more genetically unexplained MMAF-affected individuals could uncover novel mechanisms underlying sperm flagellar formation.

Proteolytic processing and degradation of enamel matrix proteins appears to be an essential feature of dental enamel formation. The source and character of proteolytic activity in the enamel matrix of developing teeth changes as enamel formation progresses. Two proteinases have been isolated from the extracellular enamel matrix of developing teeth: enamelysin (MMP-20), a matrix metalloproteinase. and kallikrein-4 (KLK4), a serine proteinase. Here, we ask if the expression of MMP-20 and KLK4 correlate with the stage-associated changes in the digestion of enamel proteins. Using in situ hybridization, we localized MMP-20 and KLK4 mRNA in mouse maxillary first molars on postnatal days 1, 2, 3, 5, 6, 7, 9, 11, and 14. Enamelysin signal was first detected in preameloblasts, ameloblasts, and odontoblasts on day 2, but not in ameloblasts covering the enamel-free zone. Enamelysin signal declined in ameloblasts on day 6 but persisted in the dental pulp. In contrast, KLK4 transcripts were first observed on day 3 in pulp and on day 6 in ameloblasts covering the enamel-free zone. the KLK4 signal was present in maturation-stage ameloblasts on days 9, 11, and 14. The expression patterns of MMP-20 and KLK4 by ameloblasts in mouse molars are stage-specific and complementary.

Amelogenin and enamelin are structural proteins in the enamel matrix of developing teeth. The temporal and spatial patterns of enamelin expression in developing mouse molars have not been characterized, while controversy remains with respect to amelogenin expression by odontoblasts and cementoblasts. Here we report the results of in situ hybridization analyses of amelogenin and enamelin expression in mouse molars from postnatal days 1, 2, 3, 7, 9, 14, and 21. Amelogenin and enamelin mRNA in maxillary first molars was first observed in pre-ameloblasts on the cusp slopes at day 2. The onsets of amelogenin and enamelin expression were approximately synchronous with the initial accumulation of predentin matrix. Both proteins were expressed by ameloblasts throughout the secretory, transition, and early maturation stages. Enamelin expression terminated in maturation stage ameloblasts on day 9, while amelogenin expression is still detected in maturation stage ameloblasts on day 14. No amelogenin expression was observed in day 21 mouse molars. Amelogenin and enamelin RNA messages were restricted to ameloblasts. No expression was observed in pulp, bone, or along the developing root. We conclude that amelogenin and enamelin are enamel-specific and do not directly participate in the formation of dentin or cementum in developing mouse molars.

The oxylipin pathway is of central importance for plant defensive responses. Yet, the first step of the pathway, the liberation of linolenic acid following induction, is poorly understood. Phospholipases D (PLDs) have been hypothesized to mediate this process, but data from Arabidopsis (Arabidopsis thaliana) regarding the role of PLDs in plant resistance have remained controversial. Here, we cloned two chloroplast-localized PLD genes from rice (Oryza sativa), OsPLDa4 and OsPLDa5, both of which were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis, mechanical wounding, and treatment with jasmonic acid (JA). Antisense expression of OsPLDa4 and -a5 (as-pld), which resulted in a 50% reduction of the expression of the two genes, reduced elicited levels of linolenic acid, JA, green leaf volatiles, and ethylene and attenuated the SSB-induced expression of a mitogen-activated protein kinase (OsMPK3), a lipoxygenase (OsHI-LOX), a hydroperoxide lyase (OsHPL3), as well as a 1-aminocyclopropane-1-carboxylic acid synthase (OsACS2). The impaired oxylipin and ethylene signaling in as-pld plants decreased the levels of herbivore-induced trypsin protease inhibitors and volatiles, improved the performance of SSB and the rice brown planthopper Nilaparvata lugens, and reduced the attractiveness of plants to a larval parasitoid of SSB, Apanteles chilonis. The production of trypsin protease inhibitors in as-pld plants could be partially restored by JA, while the resistance to rice brown planthopper and SSB was restored by green leaf volatile application. Our results show that phospholipases function as important components of herbivore-induced direct and indirect defenses in rice.