Molecules involved in sperm-zona pellucida interaction in mammals. Role in human fertility

Fertilization in mammals requires an initial interaction of sperm with the oocyte envelope, the zona pellucida (ZP), before it reaches the oocyte. ZP is a highly glycosylated structure, composed of three (mouse) or four (rabbit, boar, bovine, humans...) glycoproteins. The presence of ZP around the oocyte does not allow heterospecific fertilization. This barrier is principally due to the presence of species-specific glycosylations on ZP proteins. Sperm bind ZP by means of membrane receptors which recognize carbohydrate moieties on ZP glycoproteins according to a well-precised sequential process. Upon initial attachment, spermatozoa bind ZP3/ZP4 which induces the sperm acrosome exocytosis followed by a secondary binding of acrosome reacted spermatozoa to ZP2 and by ZP penetration. The sperm receptors are adhesive proteins or integral plasma membrane proteins linked to intraspermatic signalling pathways activating the acrosome reaction. Over the last twenty years, numerous studies have been carried out to identify sperm receptors to ZP in several species, but the data in humans are still incomplete. Work initiated in our research group has identified several proteins interacting with recombinant human ZP2, ZP3 and ZP4, among which are glycolytic enzymes. These enzymes are involved in the gamete interaction by means of their affinity to sugars and not by their catalytic properties. From a clinical point of view, an observed lack or weak expression of some sperm receptors to ZP3 in cases of idiopathic infertility associated with in vitro fertilization failure suggests that knowing the molecular mechanism driving the gamete recognition can be important at the diagnostic level. Furthermore, it has been shown that proteins that mediate gamete recognition diverge rapidly, as a result of positive darwinian selection. A sexual conflict can drive co-evolution of reproductive molecules in both sexes resulting in reproductive isolation and species emergence.     

Biosynthesis of hamster zona pellucida is restricted to the oocyte

The zona pellucida (ZP) is an extracellular coat that surrounds the mammalian oocyte and the early embryo until implantation. This coat mediates several critical aspects of fertilization, including species-selective sperm recognition, the blocking of polyspermy and protection of the oocyte and the preimplantation embryo. Depending on the species, the ZP is composed of three to four different glycoproteins encoded by three or four genes. These genes have been cloned and sequenced for different species. However, controversy exists about the cell type specificity of the ZP glycoproteins, for which several models have been proposed. Different groups have reported that ZP is produced only by the oocytes, by the granulosa cells or by both cell types, depending on the species under study. We recently described the expression of four ZP proteins in the hamster ovary. By means of the complete set of the hamster ZP cDNAs, we undertook the study of the origin and expression pattern of the four ZP genes. In the present work, the expression of ZP1, ZP2, ZP3 and ZP4 is carefully analyzed by in situ hybridization (ISH) in hamster ovaries. Our data suggest that the four hamster ZP genes are expressed in a coordinate and oocyte-specific manner during folliculogenesis. Furthermore, this expression is maximal during the first stages of the oocyte development and declines in oocytes from later development stages, particularly within large antral follicles.     

Cluster of genes encoding the major egg envelope protein of zebrafish

The proteins of fish egg envelopes are encoded by genes that are closely related to the genes for human zona pellucida proteins. A cluster of three genes coding for an egg envelope protein was isolated from the zebrafish, Danio rerio. The three genes, zp2a, zp2b, and zp2c, are located within an 11 kb region and are each comprised of eight exons spanning 1.85 kb. The exon-intron structures of the genes are nearly identical; however, their deduced amino acid sequences diverge at exon 7 (zp2b and zp2c from zp2a) and exon 8 (zp2c from zp2b). Exons 2-7 have a structural organization similar to exons in the carboxy-terminal half of the human zona pellucida ZP1, ZP2, and ZPB genes, suggesting they arose from a common ancestral gene. Sequence comparisons indicate that the deduced zebrafish proteins are most closely related to human ZPB. Zebrafish mRNAs coding for each of the three ZP2 variants have been found either as full-length cDNAs or expressed sequence tags. Distinct from the wf(female) gene of winter flounder which we first reported (Lyons et al., 1993: J Biol Chem 268:21351-21358), expression of the zebrafish zp2 genes was found to be ovary-specific, instead of liver-specific, and the promoter regions of zp2a and zp2b, while different, both contained E-box sequences (CANNTG) that have been demonstrated to be essential for coordination of zona pellucida gene expression in mammalian oocytes. Mixed peptide sequence analysis was used to identify the major polypeptide component of isolated zebrafish egg envelopes as the zp2 gene product.     

Immunohistochemical localization of zona pellucida proteins ZPA, ZPB and ZPC in human, cynomolgus monkey and mouse ovaries

The zona pellucida of mammalian oocytes plays an important role in binding and activation of sperm cells during the molecular events leading to fertilization. The genes coding for the three zona pellucida glycoproteins ZPA, ZPB, and ZPC of various species including mouse, dog, and human have been cloned and sequenced by several groups. However, it has remained a matter of debate as to whether the oocytes alone or in conjunction with the surrounding granulosa cells express and deposit these proteins to form the zona pellucida matrix. Addressing this unresolved issue, we assessed the expression and localization of all three zona pellucida proteins in ovaries of human, cynomolgus monkey and mice using immunohistochemical methods. In addition, oocyte-specific expression of ZPC from the primordial stage onward was confirmed by in situ hybridization. In sections of human ovaries, ZPA, ZPB, and ZPC proteins were immunohistochemically detected in the cytoplasms of primordial oocytes and during later stages of folliculogenesis in the zona pellucida matrices of oocytes. In sections fixed with formalin, a clear homogeneous ring was visible around the oocyte and no staining of granulosa cells was observed. In contrast, staining of ZP proteins was also observed between granulosa cells when Bouin's reagent had been used for tissue fixation. Thus, the original zona pellucida architecture was better preserved by formalin fixation. We further demonstrated that dissolution of the zona pellucida of isolated bovine oocytes occurred after they were exposed to Bouin's reagent. In summary, these results demonstrate that in mice, monkeys and humans, zona proteins are expressed and assembled exclusively by the oocyte and not by the granulosa cells. Previously observed results of ZP expression by an involvement of granulosa cells might therefore be the result of an improper fixation of the tissues leading to the disruption of the zona pellucida. Additionally this study highlights the importance of choosing the correct fixative for immunohistochemistry, not only for the usual reason of retaining antigenicity, but rather to retain the entire architectural structure.     

Molecular analysis of the antigenicity and immunogenicity of recombinant zona pellucida antigens in a primate model.

The studies reported here are the first to demonstrate that recombinant zona pellucida (ZP) proteins will elicit a humoral immune response that recognizes native ZP proteins. Three cDNAs encoding rabbit ZP protein antigens expressed in bacteria were used to immunize cynomolgus monkeys. Four groups of six monkeys each were immunized with bacterially expressed cro-beta-galactosidase recombinant proteins encoded by a full-length cDNA (rc55) encoding the 55-kDa rabbit ZP recombinant protein (rec55), two partial cDNAs (rc75a and rc75b) encoding two recombinant peptides (rec75a and rec75b) of the 75-kDa rabbit ZP protein, and the plasmid-encoded cro-beta-galactosidase control protein. Initial immunizations with these fusion proteins using the muramyl dipeptide adjuvant did not elicit significant levels of antibodies to native or recombinant ZP proteins. Further immunizations were therefore carried out using recombinant ZP proteins conjugated to either protein A or keyhole limpet hemocyanin. Antibodies were detected in the groups immunized with the rec55 and rec75a; however, no antibodies were generated against the rec75b protein. These antibodies have been characterized by two-dimensional PAGE immunoblotting and shown to recognize antigenic domains associated with two of the native rabbit ZP proteins. Reprobes of these immunoblots with sheep anti-total native rabbit ZP proteins, affinity-purified on pig ZP, further demonstrate that a fourth distinct rabbit ZP antigen may be present. The characterization of species-conserved antigenic domains of mammalian ZP proteins is important for studies of the functional regions of ZP proteins and is critical for the design of safe and effective contraceptive vaccines.     

Expressed sequence tag analysis of expression profiles of zebrafish testis and ovary

In the present study, two gonad cDNA libraries from zebrafish testes and ovaries were constructed and a total of 1025 expressed sequence tag (EST) clones were generated from the two libraries: 501 from the testis library and 524 from the ovary library. A total of 641 of the EST clones were identified to share significant sequence identity with known sequences in GenBank, representing at least 478 different zebrafish genes. In order to understand the molecular compositions of the two gonad organs, the expression profiles of the identified clones in these two gonad cDNA libraries were analyzed. Both gonad libraries have a higher portion of clones for nuclear proteins and a lower portion for proteins in translational machinery, cytoskeleton and mitochondria than our previously characterized whole-adult cDNA library. Most abundant cDNA clones in the two gonad libraries were identified and over 10% of ovary clones were found to encode egg membrane proteins (zona pellucida or ZP proteins). Furthermore, the testis library showed a more even distribution of cDNA clones with relatively fewer abundant clones that tend to contribute redundant clones in EST projects; thus, the testis library can supply more unique and novel cDNA sequences in a zebrafish EST project. Another aim of this study is to identify cDNA clones that can be used as molecular markers for the analysis of the gonad development in zebrafish. Eleven potential clones were selected to analyze their expression patterns by Northern blot hybridization. Most of them showed a specific or predominant expression in the expected testis or ovary tissue. At last, four of the clones were found, by section in situ hybridization, to be expressed specifically in the germ cells of the testis or ovary and thus they are suitable molecular markers for analyses of spermatogenesis and oogenesis.     

Isoforms of alanine aminotransferases in human tissues and serum--differential tissue expression using novel antibodies

Serum alanine aminotransferase (ALT) is used as a clinical marker of hepatotoxicity. Two forms of ALT have been identified, ALT1 and ALT2, encoded by separate genes. The cellular and tissue distribution of the different ALT proteins has not been characterized in humans, and their relative contribution to serum is unknown. Here, we describe the development of novel isoenzyme specific ALT1 and ALT2 antibodies and the expression of the enzymes in human cells and organs. In normal human tissue, high expression of ALT1 was found in liver, skeletal muscle and kidney and low levels in heart muscle and not detectable in pancreas. High ALT2 reactivity was detected in heart and skeletal muscle, while no ALT2 expression was found in liver or kidney. Using immunohistochemistry, strong ALT1 reactivity was found in hepatocytes, renaltubular epithelial cells and in salivary gland epithelial cells, while ALT2 was expressed in adrenal gland cortex, neuronal cell bodies, cardiac myocytes, skeletal muscle fibers and endocrine pancreas. Immunoprecipitation using ALT antibodies on normal human serums showed ALT1 to be mainly responsible for basal ALT activity. Together, the results points to a differential expression of ALT1 and ALT2 in human organs and substantiate a need for investigations regarding the possible impacts on ALT measurements.     

Lipid-transfer proteins: Tools for manipulating membrane lipids

Like other eukaryotic cells, plant cells contain proteins able to bind or to transfer lipids. Since they are able to facilitate movements of various phospholipids between membranes and are also capable of binding fatty acids or acyl-CoAs, they have been termed lipid-transfer proteins (LTP). LTPs are basic proteins containing 90 to 95 residues (molecular mass 9 kDa), eight of them being cysteines found in conserved locations. These proteins have been used to manipulate in vitro the lipid composition of isolated membranes either from plant or mammalian sources. In addition to purified LTPs, recombinant LTPs produced by genes expressed in microorganisms can be used for this purpose. Several genes coding for these proteins have been characterized in various plants with different patterns of expression. However, it remains to be investigated whether these recombinant proteins behave functionally as LTPs. The use of purified or recombinant LTPs is promising for the study of the effect of lipid composition on membrane functional properties.     

利用阵列分析水稻AP2/EREBP家族基因的表达特性

AP2／EREBP蛋白是广泛存在于高等植物中的且包含AP2／EREBP功能域的重要转录因子家族,通常可分为包含单功能域的EREBP类蛋白和包含两个功能域的AP2类蛋白,它们的功能涉及植物生长发育调控和对逆境应答等许多方面。据预测．水稻基因组编码150个左右的AP2／EREBP家族成员,但目前绝大多数蛋白的功能仍不清楚。为了解这些基因在水稻不同器官中的表达特性,我们以AP2／EREBP功能域的氨基酸序列为基础,从水稻基因组数据库中搜索到12个AP2类以及20个EREBP类预测基因,利用PCR扩增的编码区序列制备了这些预测基因的macro—array。以幼芽、幼根、幼叶、颖花和灌浆期成熟叶的cDNA为探针,杂交分析结果显示：不同AP2类预测基因之间的表达量差别较大,但同一个基因在不同器官中表达量基本一致：与此不同的是,大部分EREBP类预测基因在幼根和成熟叶片中表达量较高,而在幼芽和幼叶中表达量较低。这些预测基因的表达模式可能与它们的功能密切相关。     

利用阵列分析水稻AP2/EREBP 家族基因的表达特性

AP2/EREBP蛋白是广泛存在于高等植物中的且包含AP2/EREBP功能域的重要转录因子家族, 通常可分为包含单功能域的EREBP类蛋白和包含两个功能域的AP2类蛋白, 它们的功能涉及植物生长发育调控和对逆境应答等许多方面。据预测,水稻基因组编码150个左右的AP2/EREBP 家族成员, 但目前绝大多数蛋白的功能仍不清楚。为了解这些基因在水稻不同器官中的表达特性, 我们以AP2/EREBP功能域的氨基酸序列为基础, 从水稻基因组数据库中搜索到12个AP2类以及20个EREBP类预测基因, 利用PCR扩增的编码区序列制备了这些预测基因的macro-array。 以幼芽、幼根、幼叶、颖花和灌浆期成熟叶的 cDNA为探针, 杂交分析结果显示: 不同AP2类预测基因之间的表达量差别较大, 但同一个基因在不同器官中表达量基本一致; 与此不同的是, 大部分EREBP类预测基因在幼根和成熟叶片中表达量较高, 而在幼芽和幼叶中表达量较低。这些预测基因的表达模式可能与它们的功能密切相关。     

Expression analysis of Arabidopsis thaliana small secreted protein genes

Small proteins secreted to the extracellular matrix in plants regulate many physiological activities, including pathogen response, material transport, and morphogenesis, but the functions of most small secreted proteins have not been elucidated except for some well-known small secreted proteins. To predict the functions and physiological roles of unidentified small secreted proteins, information on their expression patterns is valuable. Here, we report expression analysis of Arabidopsis thaliana small secreted protein (ATSP) genes that encode proteins possessing a signal peptide at N-terminal, and protein sizes were less than 100 amino acid residues. By promoter:reporter experiments, we examined the expression of 122 ATSPs, including 47 unannotated ATSPs that do not have any discernable motifs, in tissues and at the cellular level in Arabidopsis seedlings, and floral organs. As a result, 79 ATSP genes were expressed in various regions of the seedlings, and 37 ATSP genes were specifically expressed.     

The SAC domain-containing protein gene family in Arabidopsis

The SAC domain was first identified in the yeast (Saccharomyces cerevisiae) Sac1p phosphoinositide phosphatase protein and subsequently found in a number of proteins from yeast and animals. The SAC domain is approximately 400 amino acids in length and is characterized by seven conserved motifs. The SAC domains of several proteins have been recently demonstrated to possess phosphoinositide phosphatase activities. Sac1p has been shown to regulate the levels of various phosphoinositides in the phosphoinositide pool and affect diverse cellular functions such as actin cytoskeleton organization, Golgi function, and maintenance of vacuole morphology. The Arabidopsis genome contains a total of nine genes encoding SAC domain-containing proteins (AtSACs). The SAC domains of the AtSACs possess the conserved amino acid motifs that are believed to be important for the phosphoinositide phosphatase activities of yeast and animal SAC domain proteins. AtSACs can be divided into three subgroups based on their sequence similarities, hydropathy profiles, and phylogenetic relationship. Gene expression analysis demonstrated that the AtSAC genes exhibited differential expression patterns in different organs and, in particular, the AtSAC6 gene was predominantly expressed in flowers. Moreover, the expression of the AtSAC6 gene was highly induced by salinity. These results provide a foundation for future studies on the elucidation of the cellular functions of SAC domain-containing proteins in Arabidopsis.     

Differential screening and characterization analysis of the egg envelope glycoprotein ZP3 cDNAs between gynogenetic and gonochoristic crucian carp

INTRODUCTIONFertilization in animals is the trigger that ini-tiates development, and results in a series of well-choreographed interactions between molecules lo-cated on the surfaces of egg and sperm[1, 2]. Somecell surface proteins, such as zolla pellucida gly-coprotein ZPI, ZPZ and ZP3, acrosomal proteinbindin, acrosin and lysin, and sperm plasma mem-brane protein a- and p--fertilin, have been identi-fied to mediate the interaction process[2-8]. Al-though studies on the regulative fa…