The proteins in the seminal plasma and on the sperm surface play important roles in sperm function and numerous reproductive processes. The cysteine-rich secretory proteins (CRISPs) are enriched biasedly in the male reproductive tract of mammals, and CRISP2 is the sole member of CRISPs produced during spermatogenesis; whereas the role of CRISP2 in fertilization and its association with fertility of boars are still unclear. This study aimed to investigate the relationship between the sperm CRISP2 and boar fertility, and explore its impact sperm fertilizing ability. The levels of CRISP2 protein in sperm were quantified by ELISA; correlation analysis was performed to evaluate the association between CRISP2 protein levels and boar reproductive parameters. Meanwhile, the expression of CRISP2 in boar reproductive organs and sperm, and the effects of CRISP2 on in vitro fertilization (IVF) were examined. The results showed that boars with high sperm levels of CRISP2 had high fertility. The protein levels of CRISP2 in sperm were positively correlated with the litter size ( r = 0.412, p = 0.026), the number of live-born piglets ( r = 0.421, p = 0.023) and the qualified piglets per litter ( r = 0.381, p = 0.042). CRISP2 is specifically expressed in the testis and sperm of adult boars, and its location on sperm changed mainly from the post-acrosomal region to the apical segment of acrosome during capacitation. The cleavage rate was significantly decreased by adding the anti-CRISP2 antibody to the IVF medium, which indicates CRISP2 plays a critical role in fertilization. In conclusion, CRISP2 protein is specifically expressed in the adult testis and sperm and is associated with sperm fertilizing ability and boar fertility. Further mechanistic studies are warranted, in order to fully decipher the role of CRISP2 in the boar reproduction.

In this study, we performed the isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis in the spermatozoa of Landrace boars with different fertility potentials and investigated the ability of sperm acrosome associated 4 (SPACA4) and IZUMO family member 2 (IZUMO2) to predict the reproductive perform of boars. The iTRAQ results revealed that 202 proteins were up-regulated and 43 proteins were down-regulated in the spermatozoa from high fertility boars. SPACA4 and IZUMO2 protein levels were significantly up-regulated in the spermatozoa from high fertility boars. SPACA4 and IZUMO2 expression were specifically detected in the adult boar testis. SPACA4 levels were positively correlated with Sow's farrowing rate and reproductive efficiency, but not litter size. IZUMO2 were positively correlated with litter size, Sow's farrowing rate and reproductive efficiency. Treating the boar semen with SPACA4 or IZUMO2 antibodies for 30 min and 60 min failed to affect the sperm motility; while treating the semen with SPACA4 antibody significantly reduced the fertilization and cleavage rates. Similar results for fertilization and cleavage rates were found in IZUMO2 antibody-treated semen. Collectively, our results indicated that protein levels of SPACA4 and IZUMO2 in the spermatozoa were positively related to the reproductive performance of Landrace boars.

文章从禽鸟免疫球蛋白重链基因的结构、类型、多样性机制出发,总结了目前禽鸟中免疫球蛋白重链基因的研究情况,发现目前禽鸟免疫球蛋白重链基因的研究主要集中在鸡形目、雁形目、雀形目和平胸总目在内的部分代表性物种,禽鸟中:普遍存在IgM、IgA、IgY 3种免疫球蛋白重链基因类型,分别由μ、α、γ基因所编码;基因座为典型的易位子结构Vn-Dn-Jn-Cμ-Cα-Qγ;禽鸟中普遍缺失编码IgD的δ基因;α基因在基因座上反向插入;免疫球蛋白的多样性主要通过基因转换进行.雁形目中额外高表达IgY(△Fc),该转录本缺失γ基因的第三、第四个外显子,可能导致雁形目特殊的免疫机制.IgY(△Fc)的产生机制及功能的全面研究将有助于开辟禽病治疗的新机制.

Previous studies on the immunoglobulin (Ig) genes in avian species are limited (mainly to galliformes and anseriformes) but have revealed several interesting features, including the absence of the IgD and Igκ encoding genes, inversion of the IgA encoding gene and the use of gene conversion as the primary mechanism to generate an antibody repertoire. To better understand the Ig genes and their evolutionary development in birds, we analyzed the Ig genes in the ostrich (Struthio camelus), which is one of the most primitive birds. Similar to the chicken and duck, the ostrich expressed only three IgH chain isotypes (IgM, IgA and IgY) and λ light chains. The IgM and IgY constant domains are similar to their counterparts described in other vertebrates. Although conventional IgM, IgA and IgY cDNAs were identified in the ostrich, we also detected a transcript encoding a short membrane-bound form of IgA (lacking the last two C(H) exons) that was undetectable at the protein level. No IgD or κ encoding genes were identified. The presence of a single leader peptide in the expressed heavy chain and light chain V regions indicates that gene conversion also plays a major role in the generation of antibody diversity in the ostrich. Because the ostrich is one of the most primitive living aves, this study suggests that the distinct features of the bird Ig genes appeared very early during the divergence of the avian species and are thus shared by most, if not all, avian species.

IgY(ΔFc), containing only CH1 and CH2 domains, is expressed in the serum of some birds and reptiles, such as ducks and turtles. The duck IgY(ΔFc) is produced by the same υ gene that expresses the intact IgY form (CH1–4) using different transcriptional termination sites. In this study, we show that intact IgY and IgY(ΔFc) are encoded by distinct genes in the red-eared turtle (Trachemys scripta elegans). At least eight IgY and five IgY(ΔFc) transcripts were found in a single turtle. Together with Southern blotting, our data suggest that multiple genes encoding both IgY forms are present in the turtle genome. Both of the IgY forms were detected in the serum using rabbit polyclonal Abs. In addition, we show that multiple copies of the turtle δ gene are present in the genome and that alternative splicing is extensively involved in the generation of both the secretory and membrane-bound forms of the IgD H chain transcripts. Although a single μ gene was identified, the α gene was not identified in this species.

现有的遗传学研究认为高IgD综合征（HIDS）与甲羟戊酸激酶（MVK）基因发生突变有关,但具体的发病机制还不清楚,血清中IgD的升高与HIDS临床表征之间的关系还不知道.本研究运用融合PCR的方法构建肝脏特异性高表达嵌合分泌型IgD（csIgD）载体,并通过显微共注射,基因组PCR和Southern blotting鉴定出7只转基因F0代阳性小鼠.对F1代阳性小鼠在RNA和蛋白质水平分别进行了RT-PCR,Q-PCR和Western blotting,ELISA检测.结果显示,转基因csIgD能够在肝脏特异性表达,并具有遗传稳定性.对阳性小鼠骨髓和脾脏细胞进行的流式分析发现,转基因csIgD的表达并未影响到B细胞的发育.形态解剖学观察,发现一些小鼠出现了皮肤破损和肝脾肾肿大的现象;病理切片显示,肝脾肾组织出现大量的细胞坏死和纤维样蛋白沉淀.TAIL-PCR（thermal asymmetric in terlaced PCR）检测结果表明,目的基因片段没有破坏位于小鼠5号染色体的MVK基因.本转基因小鼠模型的构建,有利于进一步对HIDS发病机制进行探讨.

For decades, it has remained unknown whether artiodactyls, such as cattle, pigs, and sheep, express immunoglobulin D (IgD), although the δ gene was identified in these species nearly 10 years ago. By developing a mouse anti-bovine IgD heavy chain monoclonal antibody (13C2), we show that secreted bovine IgD was present mainly as a monomer in serum and was heavily glycosylated by N-linked saccharides. Nonetheless, IgD was detectable in some but not all of the Holstein cattle examined. Membrane-bound IgD was detected in the spleen by western blotting. Flow cytometric analysis demonstrated that IgD-positive B cells constituted a much lower percentage of B cells in the bovine spleen (∼6.8% of total B cells), jejunal Peyer's patches (∼0.8%), and peripheral blood leukocytes (∼1.2%) than in humans and mice. Furthermore, IgD-positive B cells were almost undetectable in bovine bone marrow and ileal Peyer's patches. We also demonstrated that the bovine δ gene can be expressed via class switch recombination. Accordingly, bovine δ germline transcription, which involves an Iδ exon and is highly homologous to Iμ, was confirmed. However, we could not identify an Iδ promoter, despite bovine Eμ demonstrating both enhancer and promoter activity. This study has answered a long-standing question in cattle B cell biology and significantly contributes to our understanding of B cell development in this species.

Mutation of mevalonate kinase (MVK) is thought to account for most cases of hyperimmunoglobulinemia D syndrome (HIDS) with recurrent fever. However, its mechanism and the relationship between elevated serum immunoglobulin D (IgD) and the clinical features of HIDS are unclear. In this study, we generated by fusion PCR a vector to express high levels of chimeric secretory IgD (csIgD) specifically in the liver. We then generated seven founder lines of transgenic mice by co-microinjection, and verified them using genomic PCR and Southern blotting. We detected the expression of csIgD by reverse transcription PCR, quantitative PCR, western blotting, and enzyme-linked immunosorbent assays. We demonstrated that csIgD could be specifically and stably expressed in the liver. We used flow cytometry to show that overexpression of csIgD in the bone marrow and spleen cells had no effect on B cell development. Morphologic and anatomical observation of the transgenic mice revealed skin damage, hepatosplenomegaly, and nephromegaly in some transgenic mice; in these mice, pathological sections showed high levels of cell necrosis and protein-like sediments in the liver, spleen, and kidney. We demonstrated that the genomic insertion sites of the transgenes did not disrupt the MVK gene on mouse chromosome 5. This transgenic mouse will be useful to explore the pathogenesis of HIDS.