Epidermal growth factor stimulates hydrolysis of phosphatidylinositol4,5-bisphosphate, generation of diacylglycerol and exocytosis in mouse spermatozoa

Mouse spermatozoa stimulated with epidermal growth factor (EGF) or zona pellucida (ZP) experienced phosphatidylinositol 4,5-bisphosphate hydrolysis, diacylglycerol (DAG) generation and acrosomal exocytosis. The agonists showed additive effects but the action of EGF is likely to be mediated by a distinct receptor because maximal stimulation achieved with EGF was enhanced further by ZP. Generation of DAG and exocytosis stimulated by EGF were inhibited by tyrphostin A48, indicating that tyrosine kinase activity mediates EGF action. On the other hand, pertussis toxin did not affect the EGF-induced formation of DAG or exocytosis, ruling out the involvement of sperm G_i-like proteins. These results indicate that EGF could be an important co-factor in the initiation of exocytosis in spermatozoa.     

Cumulus cell function during bovine oocyte maturation,fertilization, and embryo development in vitro

Several contemporary micromanipulation techniques, such as sperm microinjection, nuclear transfer, and gene transfer by pronuclear injection, require removal of cumulus cells from oocytes or zygotes at various stages. In humans, the cumulus cells are often removed after 15–18 hr of sperm-oocyte coincubation to assist the identification of the fertilization status. This study was designed to evaluate the function of cumulus cells during oocyte maturation, fertilization, and in vitro development in cattle. Cumulus cells were removed before and after maturation and after fertilization for 0,7,20, and 48 hr. The cumulus-free oocytes or embryos were cultured either alone or on cumulus cell monolayers prepared on the day of maturation culture. Percentages of oocyte maturation, fertilization, and development to cleavage, morula, and blastocyst stages and to expanding or hatched blastocysts were recorded for statistical analysis by categorical data modeling (CATMOD) procedures. Cumulus cells removed before maturation significantly reduced the rate of oocyte maturation (4–26% vs. 93–96%), fertilization (0–9% vs. 91–92%), and in vitro development at all stages evaluated. Cumulus cells removed immediately prior to in vitro fertilization (IVF) or 7 hr after IVF reduced the rates of fertilization (58–60% and 71%, respectively, vs. 91–92% for controls), cleavage development (40–47% and 53–54% vs. 74–78% for controls), and morula plus blastocyst development (15% and 24% vs. 45%, P < 0.05). Cumulus cell co-culture started at various stages had no effect on fertilization and cleavage development but significantly improved rates of embryo development to morula or blastocyst stages (P < 0.05). Cumulus cell removal at 20 hr after IVF resulted in similar development to controls (P > 0.05) at all stages tested in this study. The intact state of surrounding cumulus cells of oocytes or embryos appears to be beneficial before or shortly after insemination (at or before 7 hr of IVF) but not essential at 20 hr after IVF. © 1995 Wiley-Liss, Inc.     

猪精子凝集素在精卵结合中的作用机制

猪精子凝集素（ＢＳＬ）位于精子头部,既与精子蛋白结合,亦与透明带糖蛋白ＺＰ３结合。并且ＢＳＬ自身分子间亦会聚合。与ＺＰ３结合的片段亦结合于岩藻素－Ｓｅｐｈａｒｏｓｅ亲和柱,但不与胎球蛋白－Ｓｅｐｈａｒｏｓｅ柱结合。该片段具有较强的抑制精卵结合的活性。ＢＳＬ经ＮＢＳ修饰后,血凝活力大大下降,同时推动与精子结合的活性,但不影响它与ＺＰ３的结合。修饰后的ＢＳＬ亦显著抑制精卵结合。表明ＢＳＬ以两个不同的部     

EARLY STEPS OF SPERM—EGG INTERACTIONS DURING MAMMALIAN FERTILIZATION

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, inin vitrofertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.     

SILAC‐based quantitative proteomic analysis of secretome of Marc‐145 cells infected with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which causes severe reproductive failure in sows, respiratory disease in young and growing pigs, and enormous economic losses to the global swine industry. In this study, SILAC combined with MS/MS was used to quantitatively identify the secretory proteins differentially expressed in PRRSV‐infected Marc‐145 cells compared with mock‐infected controls. In total, we identified 204 secretory proteins showing significant differences in infected cells (163 upregulated, 41 downregulated). Intensive bioinformatic analysis of secretome data revealed that PRRSV infection strongly activated nonclassical protein secretion, especially vesicle‐mediated release of exosomal proteins, including different danger‐associated molecular pattern molecules and the majority of secreted proteins involved in protein binding and transport, regulation of response to stimulus, metabolic processes, and immune responses. According to the functional proteins analysis, we speculate that proteins functioning in binding, transport, and the immune response are exploited by PRRSV to facilitate virus replication and immune evasion. Our study for the first time analyzes the secretory protein profile of PRRSV‐infected Marc‐145 cells and provides valuable insight into the host response to PRRSV infection.     

Skeletal muscle mitochondrial phospholipase A2 and the interaction of mitochondria and sarcoplasmic reticulum in porcine malignant hyperthermia

Comparative studies were carried out on the Ca²⁺-transport systems of mitochondria and sarcoplasmic reticulum from longissimus dorsi muscle of genetically selected malignant hyperthermia-prone and normal pigs in order to identify the biochemical lesion responsible for the enhanced release of Ca²⁺ in the sarcoplasm occurring in porcine malignant hyperthermia. Mitochondria isolated from longissimus dorsi muscle of malignant hyperthermia-prone pigs contained a significantly (P < 0.001) higher amount of endogenous long-chain fatty acids. Similar amounts of endogenous mitochondrial phospholipase A₂ were observed in both types of pigs, but the total activity in malignant hyperthermia-prone pigs was at least twice that of normal. Spermine, a phospholipase A₂ inhibitor, lowered the activity in both types of mitochondria to a similar final level. Mitochondria of malignant hyperthermia-prone pigs showed a significantly (P < 0.001) higher oligomycin-insensitive (Ca²⁺ + Mg²⁺)-ATPase activity, but the Mg²⁺-ATPase and the (Ca²⁺ + Mg²⁺)-ATPase activities were similar in both types of pigs. Sarcoplasmic reticulum isolated from longissimus dorsi muscle of malignant hyperthermia-prone pigs showed a significantly higher (Ca²⁺ + Mg²⁺)-ATPase activity and a lower rate of Ca²⁺ uptake; the maximal amount and the rate of Ca²⁺ uptake by sarcoplasmic reticulum of malignant hyperthermia-prone pigs were half that of normal. Mitochondria from longissimus dorsi muscle of malignant hyperthermia-prone pigs inhibited the Ca²⁺-transport system of the sarcoplasmic reticulum of longissimus dorsi from both normal and malignant hyperthermia-prone pigs, but mitochondria from normal pigs had no influence on the sarcoplasmic reticulum from either type. Experimental evidence favours the concept that long-chain fatty acids released from skeletal muscle mitochondria by endogenous mitochondrial phospholipase A₂ are responsible for the enhanced release of Ca²⁺ from mitochondria (Cheah, K.S. and Cheah, A.M. (1981) Biochim. Biophys. Acta 634, 70–84), and also additional release of Ca²⁺ from sarcoplasmic reticulum into the sarcoplasm during porcine malignant hyperthermia syndrome.     

Gestational changes in electrolyte transport,electrical activity,and permeability of the porcine placenta

Summary 1. The electrolyte transport capacities of the porcine placenta and fetal membranes (amnion, chorion, and allantois) during gestation (47–112 days) were assessed in vitro and in the absence of electrochemical, osmotic, or hydrostatic driving forces. Net transmural transport of ²²Na and ³⁶Cl across sections of porcine allantochorion-endometrium (ALCE), amniochorionendometrium (AMCE), and allantoamnion (AA) mounted in Ussing chambers could not be detected at any stage of gestation. 2. These tissues were characterised throughout gestation by low electrical potential difference (PD; <5 mV, fetal side positive with respect to maternal side) and short circuit current (SCC; <16 A · cm^-1) which were amiloride and iodoacetate sensitive. 3. A periparturitent reversal in the polarity of PD to fetal side negative and a change in the direction of the SCC was observed in tissues obtained from sows (n=3) at 111–112 day gestation. These changes were accompanied by a 73% increase in electrical resistance (R) and a 2–10-fold decrease in the permeability of tissues to Na and Cl. 4. Morphological studies revealed a decrease in the thickness of allantoamnion and a decrease in the height of epithelia from ALCE, AMCE, and AA during gestation, indicating a decrease in activity. These morphological changes only partly explained the pre-term electrophysiological changes in these tissues.Abbreviations AA allantoamnion - ALCE allantochorionendometrium - AMCE Amniochorion-endometrium - fm feto-maternal - mf materno-fetal - PD electrical potential difference - PVP polyvinyl pyrrolidone - R resistance - SCC short circuit current     

A mammalian sperm lectin related to rat hepatocyte lectin-2/3

In rat liver the asialoglycoprotein receptor is composed of three polypeptides, RHL-1, RHL-2 and RHL-3 [6]. In rat testis and spermatozoa a galactosyl receptor (RTG-r) which is immunologically related to RHL-2/3 has been described [7]. We now report that in addition to its presence in the rat, an antigenic species of 54 kDa related to RHL-2/3 is present on rabbit, human, pig and mouse spermatozoa. Purified rabbit testis galactosyl receptor (RbTG-r) consists of two major proteins of 54 and 49 kDa, while purified rabbit liver galactose lectin consists of two major proteins of 43 and 40 kDa. In an ELISA the purified rabbit testis galactosyl receptor was shown to bind biotinylated heat solubilized rabbit zonae, while the purified liver galactose lectin did not. We conclude that one of the mammalian sperm's zona binding proteins is a galactose lectin of 54 kDa related to rat liver RHL-2/3.