M. J. Izquierdo-Rico

The Oviduct: Functional Genomic and Proteomic Approach

The mammalian oviduct is an anatomical part of the female reproductive tract, which plays several important roles in the events related to fertilization and embryo development. This review examines and compares several studies related to the proteomic and transcriptomic profile of the oviduct in different domestic animals. This information could be important for clarifying the role of oviductal factors in different events regulating fertilization and early embryo development, as well as for improving synthetic media for in vitro maturation/in vitro fertilization/embryo culture techniques (IVM/IVF/EC).

Rabbit zona pellucida composition: A molecular, proteomic and phylogenetic approach

The zona pellucida (ZP) participates in sperm-egg interactions during the first steps of fertilization. Recent studies have shown that the ZP matrix of oocytes in several species is composed of four glycoproteins, designated as ZP1, ZP2, ZP3 and ZP4, rather than the three described in mouse, pig and cow. In this study, investigations were carried out to unveil a fourth glycoprotein in the rabbit (Oryctolagus cuniculus) ZP. Using total RNA isolated from rabbit ovaries, the complementary deoxyribonucleic acid (cDNA) encoding rabbit ZP1 was amplified by reverse transcribed polymerase chain reaction (RT-PCR). The ZP1 cDNA contains an open reading frame of 1825 nucleotides encoding a polypeptide of 608 amino acid residues. The deduced amino acid sequence of rabbit ZP1 showed high identity with other species: 70% identity with human and horse ZP1, and 67% identity with mouse and rat ZP1. At the proteomic level, peptides corresponding to the four proteins were detected by mass spectrometry. In addition, a molecular phylogenetic analysis of ZP1 showed that pseudogenization of this gene has occurred at least four times during the evolution of mammals. The data presented in this manuscript provide evidence, for the first time, that the rabbit ZP is composed of four glycoproteins.

Four glycoproteins are expressed in the cat zona pellucida.

The mammalian oocyte is surrounded by a matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding and may be involved in speciation. In cat (Felis catus), this matrix is composed of at least three glycoproteins called ZP2, ZP3, and ZP4. However, recent studies have pointed to the presence of a fourth protein in several mammals (rat, human, hamster or rabbit), meaning that a reevaluation of cat ZP is needed. For this reason, the objective of this research was to analyze the protein composition of cat ZP by means of proteomic analysis. Using ZP from ovaries and oocytes, several peptides corresponding to four proteins were detected, yielding a coverage of 33.17%, 71.50%, 50.23%, and 49.64% for ZP1, ZP2, ZP3, and ZP4, respectively. Moreover, the expression of four genes was confirmed by molecular analysis. Using total RNA isolated from cat ovaries, the complementary deoxyribonucleic acids encoding cat ZP were partially amplified by reverse-transcribed polymerase chain reaction. Furthermore, ZP1 was totally amplified for the first time in this species. As far as we are aware, this is the first study that confirms the presence of four proteins in cat ZP.

Differential gene expression in porcine oviduct during the oestrous cycle

The oviduct undergoes changes under the influence of steroid hormones during the oestrous cycle. However, the molecular mechanisms underlying oviductal regulation are not fully understood. The aim of the present study was to identify the gene expression profile of the porcine oviduct in different stages of the cycle using microarray technology. A systematic study was performed on animals at four different stage: prepubertal gilts, and sows in the preovulatory, postovulatory and luteal phase of the oestrous cycle. The porcine oviduct expressed a total of 4929 genes. Moreover, significant differences in the expression of several genes were detected as the oestrous cycle progressed. Analysis of the differentially expressed genes indicated that a total of 86, 89 and 15 genes were upregulated in prepubertal gilts, preovulatory and luteal sows respectively compared with levels observed in postovulatory sows. Moreover, 80, 51 and 64 genes were downregulated in prepubertal, preovulatory and luteal animals respectively compared with the postovulatory sows. The concentrations of 10 selected transcripts were quantified by real-time reverse transcription-polymerase chain reaction to validate the cDNA array hybridisation data. Conversely, for some genes, localisation of corresponding protein expression in the oviduct was analysed by immunohistochemistry (i.e. cholecystokinin, glutathione peroxidase 2, mucin 1, phosphatidylethanolamine binding protein 4 and tachykinin 3) and mass spectrometry analysis of oviductal fluid allowed identification of peptides from all five proteins. The results of the present study demonstrate that gene expression in the porcine oviduct is clearly regulated during the oestrous cycle, with some oviductal proteins that could be related to several reproductive processes described here for the first time.

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.

Expression of the vascular endothelial growth factor system (VEGF) in the porcine oviduct during the estrous cycle

The expression and localization of VEGFA and its main receptors Flt-1 and KDR is characterized in the oviduct throughout the porcine estrous cycle. Oviducts were sampled from sows at early follicular, late follicular, early luteal and late luteal stages of the estrous cycle and a segment from the mid portion of the ampulla and isthmus studied by real time RT-PCR and quantitative immunohistochemistry. The expression of the three components of the VEGF system was continuous, although differences were observed depending on the oviduct portion, the stage of the estrous cycle and the histological layer. VEGFA and KDR mRNA expressions were higher in ampulla, while Flt-1 mRNA was higher in isthmus. VEGFA protein was also higher in ampulla but Flt-1 and KDR did not show regional differences between ampulla and isthmus. While the mRNA expression of VEGFA, Flt-1 and KDR increased progressively during the luteal period, the amount of VEGFA and Flt-1 protein decreased in the same period (in isthmus only). Contrastinly, KDR protein peaked in ampulla and isthmus just before ovulation. The VEGF system was majorly located in both the secretory and ciliated cells of the oviduct epithelium, but also in the endothelium and fibroblasts of the lamina propia and the muscle fibres and vessels of the tunica muscularis. Our results are consistent with a participation of VEFG in the regulation of the dynamics of oviductal fluid secretion and the oviduct contractibility.

Morphological study of boar sperm during their passage through the female genital tract

Once deposited in the female tract, sperm face a series of challenges that must be overcome to ensure the presence of an adequate normal sperm population close to the site of fertilization. Our aim was to evaluate the influence of the uterine milieu on boar sperm morphology. In experiment 1, sperm morphology was evaluated in the backflow (60 min after insemination) and within the uterotubal junction (UTJ) (collected ~24 h after insemination) following intrauterine sperm deposition (n = 6) and compared with the morphology of the sperm in the insemination dose. In experiment 2, the influence of the uterine fluid (UF) on sperm morphological modifications was evaluated. For this purpose, ejaculated (n = 4) and epididymal (n = 4) sperm were in vitro incubated with or without UF for 2 and 24 h. In both experiments, sperm were classified as normal, having a cytoplasmic droplet (proximal or distal) or having tail defects. The results of experiment 1 pointed to an increase in morphologically abnormal sperm collected in the backflow (27.70%) and a reduction of the same in the UTJ (2.12%) compared with the insemination dose (17.75%) (P < 0.05). In experiment 2, incubation of ejaculated sperm with UF did not provoke any morphological modifications; however, when epididymal sperm were incubated with UF, a pronounced increase in the percentage of normal sperm was evident after 24 h compared with the initial dose (from 25.77% to 53.58%, P < 0.05), mainly due to distal cytoplasmatic droplet shedding (53.22 vs. 20.20%). In conclusion, almost all the sperm that colonize the UTJ had a normal morphology, with part of the abnormal sperm having been discarded in the backflow and part selected/modified on their way to the oviduct. UF seems to influence cytoplasmic distal droplet removal, as demonstrated previously in seminal plasma.

Metabolites involved in cellular communication among human cumulus-oocyte-complex and sperm during in vitro fertilization.

BackgroundFertilization is a key physiological process for the preservation of the species. Consequently, different mechanisms affecting the sperm and the oocyte have been developed to ensure a successful fertilization. Thus, sperm acrosome reaction is necessary for the egg coat penetration and sperm-oolema fusion. Several molecules are able to induce the sperm acrosome reaction; however, this process should be produced coordinately in time and in the space to allow the success of fertilization between gametes.The goal of this study was to analyze the metabolites secreted by cumulus-oocyte-complex (COC) to find out new components that could contribute to the induction of the human sperm acrosome reaction and other physiological processes at the time of gamete interaction and fertilization.MethodsFor the metabolomic analysis, eighteen aliquots of medium were used in each group, containing: a) only COC before insemination and after 3 h of incubation; b) COC and capacitated spermatozoa after insemination and incubated for 16–20 hours; c) only capacitated sperm after 16–20 h in culture and d) only fertilization medium as control. Six patients undergoing assisted reproduction whose male partners provided normozoospermic samples were included in the study. Seventy-two COC were inseminated.ResultsThe metabolites identified were monoacylglycerol (MAG), lysophosphatidylcholine (LPC) and phytosphingosine (PHS). Analysis by PCR and in silico of the gene expression strongly suggests that the cumulus cells contribute to the formation of the PHS and LPC.ConclusionsLPC and PHS are secreted by cumulus cells during in vitro fertilization and they could be involved in the induction of human acrosome reaction (AR). The identification of new molecules with a paracrine effect on oocytes, cumulus cells and spermatozoa will provide a better understanding of gamete interaction.

Composition of marsupial zona pellucida: a molecular and phylogenetic approach

The zona pellucida (ZP) is an extracellular matrix that surrounds mammalian oocytes. In eutherians it is formed from three or four proteins (ZP1, ZP2, ZP3, ZP4). In the few marsupials that have been studied, however, only three of these have been characterised (ZP2, ZP3, ZP4). Nevertheless, the composition in marsupials may be more complex, since a duplication of the ZP3 gene was recently described in one species. The aim of this work was to elucidate the ZP composition in marsupials and relate it to the evolution of the ZP gene family. For that, an in silico and molecular analysis was undertaken, focusing on two South American species (gray short-tailed opossum and common opossum) and five Australian species (brushtail possum, koala, Bennetts wallaby, Tammar wallaby and Tasmanian devil). This analysis identified the presence of ZP1 mRNA and mRNA from two or three paralogues of ZP3 in marsupials. Furthermore, evidence for ZP1 and ZP4 pseudogenes in the South American subfamily Didelphinae and for ZP3 pseudogenes in two marsupials is provided. In conclusion, two different composition models are proposed for marsupials: a model with four proteins (ZP1, ZP2 and ZP3 (two copies)) for the South American species and a model with six proteins (ZP1, ZP2, ZP3 (three copies) and ZP4) for the Australasian species.

Analysis of ZP1 gene reveals differences in zona pellucida composition in carnivores

The zona pellucida (ZP) is an extracellular envelope that surrounds mammalian oocytes. This coat participates in the interaction between gametes, induction of the acrosome reaction, block of polyspermy and protection of the oviductal embryo. Previous studies suggested that carnivore ZP was formed by three glycoproteins (ZP2, ZP3 and ZP4), with ZP1 being a pseudogene. However, a recent study in the cat found that all four proteins were expressed. In the present study, in silico and molecular analyses were performed in several carnivores to clarify the ZP composition in this order of mammals. The in silico analysis demonstrated the presence of the ZP1 gene in five carnivores: cheetah, panda, polar bear, tiger and walrus, whereas in the Antarctic fur seal and the Weddell seal there was evidence of pseudogenisation. Molecular analysis showed the presence of four ZP transcripts in ferret ovaries (ZP1, ZP2, ZP3 and ZP4) and three in fox ovaries (ZP2, ZP3 and ZP4). Analysis of the fox ZP1 gene showed the presence of a stop codon. The results strongly suggest that all four ZP genes are expressed in most carnivores, whereas ZP1 pseudogenisation seems to have independently affected three families (Canidae, Otariidae and Phocidae) of the carnivore tree.