Identification of bovine doppel protein in testis, ovary and ejaculated spermatozoa

Doppel (Dpl) protein is a recently identified prion-like protein. Although Dpl might be expressed in the brain after prion gene deletion, in both human and mice Dpl is normally expressed only in testis and spermatozoa, where it appears to be involved in male fertility. Little information is available so far about the expression pattern of Dpl in bovines, thus, hampering possible research on the role of this protein in bovine infertility. We have thus, designed, produced and validated through Western blotting a polyclonal antibody against bovine Dpl. With this antibody we then screened bovine tissues for Dpl expression by immunohistochemistry. Ejaculated spermatozoa were screened by flow cytometry and immunocytochemistry. Bovine Dpl was expressed in all the developing stages of germinal cells, from spermatogones to ejaculated spermatozoa, in Sertoli cells and in ovarian follicles (granulosa cells and follicular fluid). Dpl immunoreactivity was also found on other tissues, where endothelial cells, peripheral nerves and scattered lymphocytes stained positive. This distribution pattern suggests that Dpl might be involved in sperm maturation/capacitation in bovines, like it might be in mice. This hypothesis needs to be verified by widespread application of the flow cytometric protocol established in this paper on spermatozoa from animals with reduced fertility

Factors involved in vitro maturation of carnivore oocytes

In order to sustain complete maturation of Carnivore oocytes, the modifications of the culture environment by: 1) the addition of gonadotropins that modulate the intracellular levels of regulatory molecules; 2) the addition of gonadotropins and thiol compounds that stimulate endogenous system of cell defense; 3) the use of alternative culture systems, have been investigated in the present thesis. The data showed that the presence of gonadotropins in the maturation medium of feline oocytes caused a moderate increase of the intracellular concentration of cAMP, a significant changes in communications between oocyte and surrounding cumulus cells and an increase of the intracellular concentration of glutathione along with meiotic progression. The use of an alternative culture system for in vitro maturation of canine oocytes, i.e. isolated oviduct, combined with the extension of the culture time (two step: isolated oviduct 24h + drop 48h) positively influenced the oocytes survival and their in vitro maturation rates

Cultural strategies for maturation of carnivore oocytes

Feline and canine oocytes are characterized by unique features and consequently in vitro conditions for maturation differ from other mammalian models. However, the attempt to create a microenvironment similar to that in which oocyte development occurs physiologically is challenging in these species, since little is known on mechanisms regulating oocyte maturation. In vivo, the acquisition of oocyte competence occurs in the follicular and in the oviductal environments and an in vitro maturation system should support the dynamic changes related to nuclear and cytoplasmic maturation occurring to all components of the cumulus-oocyte complex. Modifications of the cultural environment during maturation, by addition of substances that stimulate endogenous systems of cell defence and modulate the intracellular levels of regulatory molecules, or by use of sequential different culture systems, may be interesting strategies for enhancing viability and competence of the Carnivore oocytes

Cultural strategies for maturation of carnivore oocytes

Feline and canine oocytes are characterized by unique features and consequently in vitro conditions for maturation differ from other mammalian models. However, the attempt to create a microenvironment similar to that in which oocyte development occurs physiologically is challenging in these species, since little is known on mechanisms regulating oocyte maturation. In vivo, the acquisition of oocyte competence occurs in the follicular and in the oviductal environments and an in vitro maturation system should support the dynamic changes related to nuclear and cytoplasmic maturation occurring to all components of the cumulus-oocyte complex. Modifications of the cultural environment during maturation, by addition of substances that stimulate endogenous systems of cell defence and modulate the intracellular levels of regulatory molecules, or by use of sequential different culture systems, may be interesting strategies for enhancing viability and competence of the Carnivore oocytes

Culture strategies for maturation of carnivore oocytes

In vitro maturation (IVM) of carnivore oocytes is still under investigation. It is well known that oocytes must accomplish nuclear and cytoplasmic maturation to acquire developmental competence. However, little is known about mechanisms regulating these events in carnivore oocytes. Consequently, IVM rates are still lower than those obtained in other species. To improve results in carnivores, two strategies have to be investigated: one finalized towards preserving in vitro functional integrity and potentiality to accomplish complete maturation of cumulus\u2013oocyte complexes (COCs), the other finalized towards providing culture conditions adequate for sustaining complete maturation of these oocytes. Thus, modifications of the culture environment during IVM, by addition of substances that stimulate endogenous systems of cell defence and modulate the intracellular levels of regulatory molecules, or by use of sequentially different culture systems, are interesting strategies for enhancing viability and competence in terms of complete maturation of carnivore oocytes. This review is focused on recent advances in the study of these aspects developed in feline and/or canine oocytes

In vitro manipulation of cryopreserved gametes in the domestic cat

The availability of preserved gametes would greatly enhance reproductive technologies aimed to the diffusion of valuable cat breeds or to the preservation of biodiversity in wild feline species. In the domestic cat, in vitro fertilization (IVF) or intracytoplasmic injection of frozen spermatozoa resulted in embryo development up to the blastocyst stage. By contrast, cryopreservation of oocytes has met with very little success in animals and humans, due to the unique morphological characteristics of the cells. Various types of cell damage (i.e. cytoskeleton disorganization, DNA abnormality, premature granule exocitosis, etc.) may occur during freezing and meiotic stages vary in their response to cryopreservation. In fact, IVF of frozen cat oocytes resulted in embryo development to the blastocyst stage only when mature (Metaphase II) oocytes were cryopreserved. The cryopreservation of immature oocytes, as donors of germinal vesicles (GVs) for transfer to fresh enucleated oocytes, could be an opportunity to overcome the problem of freezing injuries to the cytoplasm. The transferred GV could respond to meiosis promoting factors present in the recipient cytoplasm, as demonstrated in other species. Our recent results show that high proportions of cat immature oocytes are still viable and their GVs are well preserved after thawing. Removal and transfer of the cryopreserved GVs would allow a better understanding of the physiological mechanisms involved in cat oocyte maturation and a future application of this procedure may aid in the conservation of female valuable germplasm

Preparation of karyoplasts and cytoplasts from feline oocytes at the germinal vesicle stage

Introduction. The homologous transfer of oocyte nucleus stage I or germinal vesicle (GV-karyoplast) into an enucleated mouse oocyte (cytoplast) at the same developmental stage, resulted in resumption of meiosis (1) and embryo development in vitro (2) of the reconstructed GV-oocytes. The transfer of GVs derived from cryopreserved oocytes also resulted in a normal progression to metaphase II (MII) in vitro of the reconstructed mouse and bovine oocytes (3,4). It has been shown that the GV is potentially more resilient to cooling than the spindle of MII, because chromosomes are decondensed and enclosed within the nuclear membrane. However, thawed immature cat oocytes show a poor developmental competence in vitro (5), probably due to the occurrence of chilling-induced damages to the cytoplasm. This compartment has a pivotal role in the resumption and completion of oocyte maturation, which is essential for the developmental competence of the embryo. Since the integrity of the oocyte nucleus may be better preserved than the cytoplasm after cryopreservation, the transfer of cryopreserved GVs into fresh enucleated oocytes could improve the chance of embryo development in culture. In the feline species there are no reports in the literature concerning GV transfer. Hence, the purpose of this work was to make a preliminary evaluation of the feasibility of enucleating immature oocytes in order to produce GV-karyoplasts and cytoplasts for GV transfer in cat oocytes. Materials and methods. A total of 156 immature (GV) cat oocytes collected from anestrous queens after ovariectomy were mechanically deprived of cumulus cells with a small-bore pipette. The oocytes were centrifuged at 14000 rpm for 16 min to obtain the polarization of the cytoplasm and a better visualization of the GV. The nucleus was measured in order to prepare adequate microtools for manipulation. Prior to enucleation, the oocytes were incubated for 30 min at room temperature in a specific medium containing 7.5 \uf06dg/mL of cytochalasin B (Sigma Chemical Co., USA) for inducing an increase of the oolemmal elasticity and 50 \uf06dg/mL of 3-isobutyl-1-methylxanthine (IBMX, Sigma) in order to prevent the GV breakdown (1). Following lancing of the zona pellucida with a sharp-tipped pipette, GV nuclei were extruded using a bevelled glass pipette with a diameter adequate to the size of GV in cat oocytes. The GV was surrounded by a small amount of cytoplasm and encapsulated by a membrane (GV-karyoplast). The enucleated oocytes were considered as cytoplasts. Results. The mean average of GV diameter in oocytes > 120 \uf06dm of diameter was 35.4 + 5.3 \uf06dm. A bevelled glass pipette, with inner diameter of 40-45 \uf06dm, allowed the extrusion of intact and morphologically normal karyoplasts and related cytoplasts in 17.3% (27/156) of micromanipulated oocytes. However, the lancing of the zona pellucida or the extrusion of the karyoplast resulted in rates of 40.4% (63/156) and 42.3% (66/156) of severely damaged oocytes, respectively. Conclusion. These results suggest that is possible to prepare karyoplasts and cytoplasts from feline oocytes, although the efficiency of the technique is low compared to what has been obtained in mouse (around 90%, 1). This is likely due to the thickness and hardness of zona pellucida, and to the larger diameter of the GV of cat oocytes compared to that of mouse (15 \uf06dm) or bovine (25-30 \uf06dm) oocytes. Further experiments based on the partial dissection of the zona pellucida with an acidic solution in order to reduce the oocyte damage and to improve the efficiency of GV transfer in feline oocytes, are in progress in our laboratory. References 1) 1) Liu et al., Human Reprod 1999; 14:2357-61. 2) Takeuchi et al., Hum Reprod 2004;19:975\u201381. 3) Moffa et al., Human Reprod 2002;17:178-83. 4) Luciano et al., Reprod Fertil Dev 2006;18:138. 5) Luvoni and Pellizzari, Theriogenology 2000;53:1529-40

Embryo production in dogs : from in vitro fertilization to cloning

Increased availability of canine embryos would be desirable to develop research and to apply assisted reproductive technologies in the treatment of infertility and in the improvement of reproductive performances in valuable Canids, both domestic and non-domestic. Embryo production through in vitro fertilization and nuclear transfer has been technically achieved in the dog, and the transfer of cloned embryos has recently resulted in the birth of puppies. However, the efficiency of these technologies is still very limited. This is mainly because of the peculiar characteristics of the canine oocyte and the lack of its full acquisition of developmental competence in vitro. This paper discusses the latest results and aspects on which further research should be focused to provide advances in the field