Oviductal environment affects preparation of gametes for fertilization, fertilization itself, and subsequent embryonic development. The aim of this study was to evaluate the effect of oviductal fluid and the possible involvement of deleted in malignant brain tumor 1 (DMBT1) on IVF in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase in the monospermic fertilization rate and that this effect is canceled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase in the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the estrus cycle was shown by western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus-oocyte complexes was shown by western blot analysis, and the localization of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatic and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization.

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.

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Mammalian oocytes are surrounded by an extracellular coat called the zona pellucida (ZP), which, from an evolutionary point of view, is the most ancient of the coats that envelope vertebrate oocytes and conceptuses. This matrix separates the oocyte from cumulus cells and is responsible for species-specific recognition between gametes, preventing polyspermy and protecting the preimplantation embryo. The ZP is a dynamic structure that shows different properties before and after fertilization. Until very recently, mammalian ZP was believed to be composed of only three glycoproteins, ZP1, ZP2 and ZP3, as first described in mouse. However, studies have revealed that this composition is not necessarily applicable to other mammals. Such differences can be explained by an analysis of the molecular evolution of the ZP gene family, during which ZP genes have suffered pseudogenization and duplication events that have resulted in differing models of ZP protein composition. The many discoveries made in recent years related to ZP composition and evolution suggest that a compilation would be useful. Moreover, this review analyses ZP biosynthesis, the role of each ZP protein in different mammalian species and how these proteins may interact among themselves and with other proteins present in the oviductal lumen.

The oviduct plays important roles in reproductive events: sperm reservoir formation, final gamete maturation, fertilization and early embryo development. It is well known that the oviductal environment affects gametes and embryos and, ultimately, the health of offspring, so that in vivo embryos are better in terms of morphology, cryotolerance, pregnancy rates or epigenetic profile than those obtained in vitro. The deciphering of embryo–maternal interaction in the oviduct may provide a better understanding of the embryo needs during the periconception period to improve reproductive efficiency. Here, we perform a comparative analysis among species of oviductal gene expression related to embryonic development during its journey through the oviduct, as described to date. Cross-talk communication between the oviduct environment and embryo will be studied by analyses of the secreted or exosomal proteins of the oviduct and the presence of receptors in the membrane of the embryo blastomeres. Finally, we review the data that are available to date on the expression and characterization of the most abundant protein in the oviduct, oviductin (OVGP1), highlighting its fundamental role in fertilization and embryonic development.

Most wild and domestic donkey breeds are currently endangered or threatened. Their preservation includes the creation of a Genome Resource Bank. Embryo cryopreservation allows preservation of genetics from both male and female and is the fastest method to restore a breed. Embryo production in vivo is limited in equids. We recently established a technique of ovum pick up (OPU) in the donkey. Conditions of in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture of zygotes have been evaluated. Equine abattoirederived oocytes were used as controls. Donkey cumulus-oocyte complexes (COCs) collected by OPU were matured in vitro in TCM199 with fetal calf serum and epidermal growth factor for 24, 30, 34, or 38 hours. Forty-four percent were in metaphase 2 after 34 hours. In our conditions, IVM of donkey oocytes was slower than that of equine oocytes and the optimal duration for donkey oocytes IVM may be 34 hours. Oocytes we co-incubated with frozen-thawed donkey semen treated with procaine for 18 hours and cultured for 30 hours in a Dulbecco Modified Eagle Medium-F12based medium. Only 15% of jennies oocytes contained 2 pronuclei after co-incubation, and none of them developed further after 48 hours after IVF. Treatment of donkey sperm with procaine may not be efficient for IVF. Some parthenogenetic activation occurred. In conclusion, we confirm that our conditions for OPU in jennies yielded high recovery rates that improved with operator experience. Maturation rates of 44% can be achieved using the IVM medium routinely used for equine oocytes in our lab. Further studies are in progress to establish efficient conditions for IVF and development of donkey zygotes.

Mammalian eggs are surrounded by an extracellular matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding, protection of the oviductal embryo, and may be involved in speciation. In eutherian mammals, this coat is formed of three or four glycoproteins (ZP1–ZP4). While Mus musculus has been used as a model to study the ZP for more than 35 years, surprisingly, it is the only eutherian species in which the ZP is formed of three glycoproteins Zp1, Zp2, and Zp3, Zp4 being a pseudogene. Zp4 was lost in the Mus lineage after it diverged from Rattus, although it is not known when precisely this loss occurred. In this work, the status of Zp4 in several murine rodents was tested by phylogenetic, molecular, and proteomic analyses. Additionally, assays of cross in vitro fertilization between three and four ZP rodents were performed to test the effect of the presence of Zp4 in murine ZP and its possible involvement in reproductive isolation. Our results showed that Zp4 pseudogenization is restricted to the subgenus Mus, which diverged around 6 MYA. Heterologous in vitro fertilization assays demonstrate that a ZP formed of four glycoproteins is not a barrier for the spermatozoa of species with a ZP formed of three glycoproteins. This study identifies the existence of several mouse species with four ZPs that can be considered suitable for use as an experimental animal model to understand the structural and functional roles of the four ZP proteins in other species, including human.

Oviductal proteins play an important role in mammalian fertilization, as proteins from seminal fluid. However, in contrast with the latter, their phylogenetic evolution has been poorly studied. Our objective was to study in 16 mammals the evolution of 16 genes that encode oviductal proteins involved in at least one of the following steps: (1) sperm–oviduct interaction, (2) acrosome reaction, and/or (3) sperm–zona pellucida interaction. Most genes were present in all studied mammals. However, some genes were lost along the evolution of mammals and found as pseudogenes: annexin A5 (ANXA5) and deleted in malignant brain tumor 1 (DMBT1) in tarsier; oviductin (OVGP1) in megabat; and probably progestagen-associated endometrial protein (PAEP) in tarsier, mouse, rat, rabbit, dolphin, and megabat; prostaglandin D2 synthase (PTGDS) in microbat; and plasminogen (PLG) in megabat. Four genes [ANXA1, ANXA4, ANXA5, and heat shock 70 kDa protein 5 (HSPA5)] showed branch-site positive selection, whereas for seven genes [ANXA2, lactotransferrin (LTF), OVGP1, PLG, S100 calcium-binding protein A11 (S100A11), Sperm adhesion molecule 1 (SPAM1), and osteopontin (SPP1)] branch-site model and model-site positive selection were observed. These results strongly suggest that genes encoding oviductal proteins that are known to be important for gamete fertilization are subjected to positive selection during evolution, as numerous genes encoding proteins from mammalian seminal fluid. This suggests that such a rapid evolution may have as a consequence that two isolated populations become separate species more rapidly.