Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin beta

We produced mice lacking the sperm surface protein cyritestin (ADAM 3) and found mutant males are infertile. Similar to fertilin beta (ADAM 2) null sperm (C. Cho et al., 1998, Science 281, 1857-1859), cyritestin null sperm are drastically deficient in adhesion to the egg zona pellucida (0.3% of wild type) and to the egg plasma membrane (9% of wild type). Thus sperm from male mice with a gene deletion of either ADAM have a loss of adhesive function in at least two steps of fertilization. We found deletion of either ADAM gene resulted in the loss of multiple gene products. This loss of multiple gene products (sperm membrane proteins) appears to result from a novel, developmental mechanism during sperm differentiation. Because the altered sperm protein expression must be responsible for the fertilization defects, our data suggest new models for the molecular basis of the affected steps in fertilization.     

Male mice deficient for germ-cell cyritestin are infertile

Cyritestin is a membrane-anchored sperm protein belonging to the ADAM (f1.gif" BORDER="0"> f2.gif" BORDER="0">isintegrin and f1.gif" BORDER="0"> f3.gif" BORDER="0">etalloprotease) family of proteins, which are proposed to be involved in cell-cell adhesion through binding to integrin receptors. Several lines of evidence support a role of cyritestin and other members of this protein family in the fusion of sperm and the egg plasma membrane. In an effort to elucidate the physiological function of cyritestin, we have disrupted its locus by homologous recombination. Male homozygous null mutants are infertile, even though spermatogenesis, mating, and migration of sperm from the uterus into the oviduct are normal. In vitro experiments showed that infertility is due to the inability of the cyritestin-deficient sperm to bind to the zona pellucida. However, after removal of the zona pellucida, sperm-egg membrane fusion monitored by the presence of pronuclei and generation of 2- and 4-cell embryos did not reveal any differences from the wild-type situation. These results demonstrate that cyritestin is crucial in the fertilization process at the level of the sperm-zona pellucida interaction.     

Defects in secretory pathway trafficking during sperm development in Adam2 knockout mice

Adam2-null and Adam3-null male mice exhibit reduced levels of one or more ADAM proteins on mature sperm, in addition to the loss of the genetically targeted protein. ADAM protein loss was believed to occur posttranslationally, although the timing of loss and the mechanism by which the loss occurred were not explored. In this study we have found that in Adam3-null mice, fertilin beta (also known as ADAM2) is lost during the formation of testicular sperm. In Adam2-null males, most cyritestin (ADAM3) protein is also lost at this stage, but 25% of cyritestin is lost later, during sperm passage through the epididymis. Although normal levels of cyritestin are synthesized and acquire Endoglycosidase H resistance, indicating transit through the Golgi, the protein does not reach the cell surface. We also discovered that the majority of both fertilin beta and cyritestin are found in a Triton X-100 insoluble compartment on testicular sperm, when most of the cyritestin was observed on the cell surface. This insoluble compartment may represent a sorting platform, because in Adam2-knockout cells, only a small fraction of the cyritestin becomes Triton X-100 insoluble. Thus, it appears that cyritestin loss in Adam2-knockout mice may result, at least in part, from a disruption in protein trafficking.     

The molecular players of sperm-egg fusion in mammals

Fertilization includes a series of cellular interactions culminating with the fusion of gamete membranes, creating a zygote. Two ADAM proteins present on sperm, fertilin beta and cyritestin, drew much attention. However, gene deletion in mice showed that fusion can happen in their absence. The presence of the integrin alpha6beta1 on egg, a putative fertilin beta receptor, is also dispensable. In contrast, sperm lacking Izumo, a molecule with a single Ig domain, are unable to fuse. On the egg side, a role for GPI-anchored molecules has been shown, and in mice lacking both tetraspanins CD9 and CD81 fertilization is completely blocked.     

A Role for the Disintegrin Domain of Cyritestin, a Sperm Surface Protein Belonging to the ADAM Family, in Mouse Sperm–Egg Plasma Membrane Adhesion and Fusion

Sperm–egg plasma membrane fusion is preceded by sperm adhesion to the egg plasma membrane. Cell–cell adhesion frequently involves multiple adhesion molecules on the adhering cells. One sperm surface protein with a role in sperm–egg plasma membrane adhesion is fertilin, a transmembrane heterodimer (α and β subunits). Fertilin α and β are the first identified members of a new family of membrane proteins that each has the following domains: pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domain. This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain. Previous studies indicate that the disintegrin domain of fertilin β functions in sperm–egg adhesion leading to fusion. Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin α, fertilin β, cyritestin, ADAM 4, and ADAM 5. The presence of the disintegrin domain, a known integrin ligand, suggests that like fertilin β, other testis ADAMs could be involved in sperm adhesion to the egg membrane. We tested peptide mimetics from the predicted binding sites in the disintegrin domains of the five testis-expressed ADAMs in a sperm–egg plasma membrane adhesion and fusion assay. The active site peptide from cyritestin strongly inhibited (80–90%) sperm adhesion and fusion and was a more potent inhibitor than the fertilin β active site peptide. Antibodies generated against the active site region of either cyritestin or fertilin β also strongly inhibited (80–90%) both sperm–egg adhesion and fusion. Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm. Indirect immunofluorescence on live, acrosome-reacted sperm using antibodies against either cyritestin or fertilin β showed staining of the equatorial region, a region of the sperm membrane that participates in the early steps of membrane fusion. Collectively, these data indicate that a second ADAM family member, cyritestin, functions with fertilin β in sperm–egg plasma membrane adhesion leading to fusion.     

Male Germ Cell-Expressed Mouse Gene TAZ83 Encodes a Putative, Cysteine-rich Transmembrane Protein (cyritestin) Sharing Homologies with Snake Toxins and Sperm-Egg Fusion Proteins

Data obtained by cloning of a mouse cDNA ( TAZ83 ) are presented. Its corresponding gene is expressed in meiotic and haploid testicular germ cells. The gene encodes a putative, cysteine-rich transmembrane protein with a deduced molecular weight of 90 kilodaltons and an isoelectric point of 5.4. Cysteine patterns within the predicted amino acid sequence of the TAZ83 gene product ( cyritestin , cysteine-rich, testicular) are highly conserved when compared to various snake toxins of the disintegrin metalloproteinase type. The cysteine pattern conservation between cyritestin and a guinea pig sperm-egg fusion protein suggests that TAZ83 codes for a mouse protein with comparable properties or function.     

Sperm defects in mice lacking a functional Niemann-Pick C1 protein

The Niemann-Pick C1 (NPC1) gene encodes for a multiple membrane spanning protein, which regulates the trafficking of low-density lipoprotein-mediated endocytosed cholesterol. Mutation of the human NPC1 gene causes Niemann-Pick type C (NPC) disease. The Npc1(NIH) mice, a model of human NPC disease, bear a spontaneous mutation of the Npc1 gene, and are infertile. In this study, we have performed sperm analysis to search for the cause of male infertility in the Npc1(NIH) mouse. The number of cauda sperms in Npc1(-/-) mice was decreased roughly three-and-half-fold of that in wild-type mice. The decreased sperm number in Npc1(-/-) mice is due, at least in part, to partial arrest of spermatogenesis in the testes, as revealed by histological analysis. Compared to wild-type sperm, Npc1(-/-) sperm displayed a high frequency of morphological abnormalities, including tailless heads and aberrant heads. In the in vitro fertilization (IVF) assay using cumulus-intact eggs, Npc1(-/-) sperm failed to produce two-cell embryos. In the IVF assay where zona-free eggs were used, Npc1(-/-) sperm bound normally but could not fuse with the egg. Further analysis indicated that Npc1(-/-) sperms are drastically impaired in the binding to the egg zona pellucida, only 14% of the level of wild-type sperm. Moreover, on Npc1(-/-) cauda sperm, one-third of the total cyritestin protein was not proteolytically processed, while fertilin beta was processed normally. Taken together, these results demonstrate that there are multiple defects in sperms from mice lacking a functional NPC1 protein, and these observed sperm defects may result in sterility.     

影响山羊体外受精的因素

以屠宰山羊卵母细胞为材料研究了公羊个体、附睾不同部位精子、成熟培养和受精时卵丘存在与否、卵丘扩展程度及卵龄对山羊体外受精的影响。结果表明 :1)不同公羊精液在受精、卵裂和桑椹 /囊胚率上都有显著差异 ;2 )附睾尾精子和鲜精的受精、卵裂和桑椹 /囊胚率无显著差异 ,但显著高于附睾体和附睾头精子 ;3)成熟培养 2 4和 2 7h卵母细胞的的桑椹胚 /囊胚率显著高于培养 2 1和 30h卵母细胞 ;4 )卵丘扩展 3和 4级卵母细胞受精和桑椹胚 /囊胚率显著高于扩展 0和 1级卵母细胞 ;5 )成熟培养前机械去卵丘严重影响卵母细胞体外受精和桑椹胚 /囊胚率 ;6 )受精前完全去掉卵丘显著影响桑椹胚 /囊胚率     

Complete fertilization failure in ICSI

Abstract  Fertilization failure is one of the causes of infertility that becomes evident only after in vitro fertilization (TVF) and intracytoplasmic sperm injection (ICSI) have been attempted. Although the frequency of incidence of fertilization failure is low, if fertilization failure is encountered, medical treatment is usually stopped and serious psychological damage may occur to the patient.
While fertilization failure in IVF can be dealt with using ICSI, there is no treatment for fertilization failure in ICSI. At present, clinical investigations are being conducted to evaluate oocyte activation in combination with ICSI to cope with fertilization failure of ICSI.     

小鼠体内外受精植入前胚胎全基因组甲基化模式比较

为考察体外受精、操作及培养环境对体外受精的小鼠植入前胚胎全基因组DNA甲基化模式的影响,本研究以体内受精的植入前胚胎作为对照,采用间接免疫荧光法检测小鼠体内外受精植入前胚胎基因组DNA甲基化模式.实验结果表明,体外受精各期植入前胚胎呈现出与之相应时期的体内受精植入前胚胎不同的DNA甲基化模式和水平,原核期甲基化水平较高,2-4-、8-细胞期明显降低,而桑葚胚和囊胚期又略有升高.各期体外受精植入前胚胎的基因组DNA甲基化水平都比同时期体内受精胚胎的甲基化水平低.本实验结果部分显示了体外受精、操作及培养环境可能对正常的DNA甲基化模式产生影响,造成体外受精植入前胚胎甲基化模式异常.     

氮、磷、钾对豫麦50旗叶蔗糖和籽粒淀粉积累的影响

以豫麦50为对象,探讨了氮、磷、钾对小麦旗叶中蔗糖的积累及相关酶活性以及籽粒中淀粉含量和组分的影响.结果表明,施氮可以增加灌浆前期旗叶中的糖含量,施钾则提高了灌浆后期旗叶中的糖含量,而施磷则对旗叶中的糖含量影响不大.施氮、磷、钾均能增加蔗糖合成酶活性,但它们的作用时间不同：施氮活性增加在籽粒灌浆中期,施磷在灌浆前期,而施钾在灌浆前、中期.但氮亦可增加花后24 d的磷酸蔗糖合成酶活性,施磷增加了灌浆前、中期磷酸蔗糖合成酶活性,施钾则增加了灌浆后期旗叶磷酸蔗糖酶活性.施氮、磷、钾都提高了籽粒中总糖含量,增加籽粒中淀粉含量,其中施钾效果最为明显.施磷提高籽粒中直链淀粉的积累,而施钾则显著提高了籽粒中支链淀粉的含量.     

Effect of methyl-beta-cyclodextrin treatment of pig spermatozoa on in vitro fertilization and embryo development in the absence or presence of caffeine

A series of experiments were carried out to develop a new method to reduce pig polyspermic fertilization and produce more normal embryos, in vitro. Experiment 1 determined the effect of methyl-beta-cyclodextrin (MCD) treatment during cryopreservation on sperm acrosome reaction and sperm fertilization. Compared to the non-MCD-treated control, MCD treatment increased the percentage of acrosome-reacted spermatozoa at thawing and 2h after incubation in fertilization medium (P<0.01). Treatment with MCD also increased (P<0.05) sperm-penetration rate, number of spermatozoa in oocytes, and fertilization efficiency in the caffeine-free fertilization medium. Experiment 2 was designed to examine the effect of withdrawal of caffeine (caffeine-free) from fertilization medium on fertilization parameters and early embryo development. Using MCD-treated spermatozoa, there was no difference in sperm-penetration rate, oocyte cleavage rate, and blastocyst formation rate between the caffeine-free and caffeine-supplemented groups. However, polyspermic fertilization rate was lower, and fertilization efficiency and blastocyst cell number were higher in the caffeine-free group compared to the caffeine-supplemented group (P<0.05). Experiment 3 studied the effect of caffeine and different concentrations of spermatozoa on fertilization parameters. Sperm-penetration rate did not differ between the caffeine-free and the caffeine-supplemented groups at different sperm concentrations. Caffeine and sperm concentration had an effect on the number of spermatozoa in oocytes and on the polyspermic fertilization rate (P<0.002). Caffeine also affected fertilization efficiency (P<0.05). In conclusion, treating spermatozoa with MCD and withdrawing caffeine from fertilization medium may provide a new method to produce a large number of normal embryos, in vitro.     

改变施肥管理后不同肥力稻田土壤CO2排放特征

利用一个长达30a水稻土长期定位试验,在保证原有定位试验继续正常开展的前提下,将原化肥处理改施有机肥,原有机肥处理改施化肥或者增施有机肥。通过观测田间试验2012—2013年双季稻轮作周期内不同肥力水平稻田土壤施肥管理改变后的土体CO2排放通量(FCO2),研究不同后续施肥管理对不同肥力红壤性水稻土CO2排放的影响。结果表明:变更施肥能明显改变CO2排放动态变化,其中长期施用有机肥处理改施化肥后其FCO2明显减小,长期施用化肥或有机肥处理增施有机肥后其FCO2显著增大。有机肥和土壤有机碳均可促进土体CO2排放,有机肥处理有机物料碳添加量与CO2-C年排放量呈极显著的正相关关系(r=0.9015**,n=21),单施化肥处理土壤有机碳含量与土体CO2-C年排放量符合线性方程:y=10.962x-68.86(R2=0.7507,n=9,P0.01)。长期施用有机肥土壤改施化肥会导致其有机碳矿化损失,土壤有机碳含量越高,矿化损失量越多,最终其有机碳水平将与长期施用化肥的土壤有机碳平衡值一致;长期施用化肥或有机肥土壤改施或增施有机肥可促进土壤有机碳积累,外源添加碳越多,土壤积累碳越多;相同有机肥施用量下土壤有机碳含量越高,有机物料表观分解率越大,积累于土壤中的有机碳越少,不同有机碳水平土壤在相同有机肥管理下其有机碳最终会达到相同的平衡值。在有机碳水平较低(20.46 g/kg)红壤稻田上增施有机肥是提升已培肥水稻土有机碳含量的可持续发展措施,而在有机碳水平较高(14.45 g/kg)红壤稻田上应避免改施化肥。总之,在有机碳含量较高或者较低的中国南方红壤性水稻土上,持续的有机肥施用是保持或者提高其有机碳水平的必要措施。     

根区水肥空间耦合对冬小麦生长及产量的影响

利用管栽试验研究了根区不同湿润方式（整体湿润、上湿下干、上干下湿）、施肥方式（整体施肥、上层施肥、下层施肥）及其耦合对冬小麦不同生育期生长及产量的影响.结果表明：下层施肥方式显著降低了分蘖期冬小麦的株高和叶面积,而不同湿润方式对分蘖期株高和叶面积的影响不显著,拔节期水肥同区方式的株高大于水肥异区方式,表现出协同耦合效应.上干下湿方式和下层施肥方式显著降低了根系干物质量、地上部干物质量和总干物质量,上层施肥方式有利于增加冬小麦生物量,而上湿下干方式与施肥处理对地上部干物质量和总干物质量的耦合效应明显.水肥同区处理的根冠比高于水肥异区处理;上干下湿方式的水分利用效率显著高于整体湿润和上湿下干方式,水肥同区处理的水分利用效率高于水肥异区处理,但下层施肥方式的水分利用效率较低.与上干下湿方式相比,上湿下干和整体湿润方式的冬小麦单穗粒数分别增加了41.7%和61.9%,上层施肥和整体施肥方式的单穗粒数高于下层施肥方式,上湿下干方式与施肥处理对小麦产量及产量构成因素（除千粒重外）的水肥耦合效应明显.不同水肥处理主要通过影响单穗粒数来影响冬小麦产量.     

不同覆盖施肥措施对黄土旱塬冬小麦土壤水分的影响

于2007年9月—2008年7月在位于黄土高原渭北旱塬的王东沟试验区进行冬小麦不同覆盖施肥措施（包括不施肥、农民习惯施肥、推荐施肥、推荐施肥+有机肥、推荐施肥+垄上覆膜、推荐施肥+垄上覆膜+沟内覆草、推荐施肥+全区覆草7个处理）田间试验,并采用水分中子仪定期观测土壤含水量,研究黄土高原旱塬区不同栽培措施下土壤水分的变化特征.结果表明：在干旱季节（春季）,推荐施肥+垄上覆膜+沟内覆草措施有利于贮存更多的土壤水分,其土壤储水量约比最低值（推荐施肥+有机肥）高48.2 mm,并可将土壤水分保持到冬小麦需水的关键期,而且推荐施肥+垄上覆膜措施仅次于推荐施肥+垄上覆膜+沟内覆草,表明这两种措施能够在田间蓄积较多天然降水,有利于黄土高原旱区雨养农业的发展.     

Fertilization in echinoderms

For more than 150years, echinoderm eggs have served as overly favored experimental model systems in which to study fertilization. Sea urchin and starfish belong to the same phylum and thus share many similarities in their fertilization patterns. However, several subtle but fundamental differences do exist in the fertilization of sea urchin and starfish, reflecting their phylogenetic bifurcation approximately 500 million years ago. In this article we review some of the seminal and recent findings that feature similarities and differences in sea urchin and starfish at fertilization.     

Fertilization and the cytoskeleton in the red alga Bostrychia moritziana (Rhodomelaceae,Rhodophyta)

Time-lapse videomicroscopy was used to observe the effects of various cytoskeletal inhibitors on three important fertilization events in Bostrychia moritziana: spermatial mitosis, gamete fusion (formation of a fertilization pore) and nuclear migration along the trichogyne. The microtubule inhibitor oryzalin disrupted spermatial mitosis but had no other effect on fertilization. The actin inhibitors, jasplakinolide, cytochalasin B, latrunculin A and B and mycalolide B inhibited gamete fusion while BDM, a myosin-disrupting drug, inhibited all three major fertilization events. FL-Phallacidin was used to stain actin filaments in spermatia and trichogynes while microtubules were labelled with antibodies at appropriate stages of fertilization. Microtubules were only evident during spermatial nuclear division. Actin filaments were present in both trichogynes and spermatia throughout fertilization; they formed a discrete ring around the fertilization pore and ensheathed male nuclei as the latter migrated into and along the trichogyne. These results suggest that the actin/myosin system plays a role in the events of fertilization.     

Inhibition of in vivo and in vitro fertilization in rodents by gonadotropin-releasing hormone antagonists

We have examined the effect of two GnRH antagonists, Ac-D-Nal(1)-Cl-D-Phe(2)-3-Pyr-D-Ala(3)-Arg(5)-D-Glu(AA)(6)-GnRH (Nal-Glu) and Ac(3,4)-dehydro-Pro(1),-p-fluoro-D-Phe(2),D-Trp(3,6)-GnRH (4pF), on in vivo and in vitro fertilization in rodents. Female rats were treated in the afternoon of proestrus with 2 micro l of Nal-Glu or 4pF (0.5 and 5 mM) injected directly into one oviductal horn (experimental); saline was injected into the contralateral horn (control). Females were then mated and the oviducts were perfused for egg and sperm recovery. The results indicate that both antagonists inhibited in vivo fertilization. Thus, the percentage of fertilized eggs in control oviducts ranged from 92% +/- 5% to 100% +/- 0%, whereas in treated oviducts, fertilization ranged from 25% +/- 6% to 73% +/- 5%. GnRH antagonists did not interfere with the process of ovulation, sperm migration to the site of fertilization, or early embryo development. In additional experiments with mice, GnRH antagonists inhibited in vitro fertilization. One fertilization event that was specifically inhibited by GnRH antagonists was the process of sperm binding to the zona pellucida. This step was precisely monitored using the hemizona assay. GnRH antagonists did not affect sperm movement or acrosomal status. These observations indicated that local treatment with GnRH antagonists inhibit in vivo fertilization and give additional support to the idea that endogenous GnRH may play an important role during fertilization by increasing the efficiency of sperm-zona binding.     

Evidence that the voltage-dependent component in the fertilization process is contributed by the sperm

To investigate the mechanisms that account for the voltage dependence of fertilization and provide an electrical block to polyspermy, we studied cross-fertilizations between three species of amphibians having different degrees of voltage dependence. Anurans, such as the toad Bufo japonicus, as well as the primitive urodele Hynobius nebulosus, have voltage-dependent fertilization; other urodeles, such as Cynops pyrrhogaster, have voltage-independent fertilization (Y. Iwao, 1989, Dev. Biol. 134, 438-445). Entry of Hynobius sperm into Cynops eggs was blocked by clamping the egg's membrane potential at +40 mV, as is the case for fertilization of Hynobius eggs with Hynobius sperm, but not for fertilization of Cynops eggs with Cynops sperm. Therefore, fertilization was voltage dependent in an experimental condition where only the sperm could be contributing this characteristic. The voltage-dependent properties of fertilization between Bufo eggs and Hynobius sperm were also characteristic of the sperm species; fertilization was blocked at +50 mV as in Hynobius fertilization, but not at +20 mV as in Bufo fertilization. These results support the conclusion that the voltage dependence of fertilization results from a component contributed by the sperm.