Improving fertilization rate in ICSI cycles by adding myoinositol to the semen preparation procedures: a prospective, bicentric, randomized trial on sibling oocytes.

PURPOSE: To evaluate whether the in vitro incubation of spermatozoa with myoinositol may improve the fertilization rate in ICSI cycles. METHODS: This is a prospective, bicentric, randomized study on 500 MII sibling oocytes injected in 78 ICSI cycles performed between March and October 2013. Randomization of the oocytes into two groups was performed at the time of the denudation. Fertilization rates (per oocyte injected with spermatozoa treated with myoinositol versus per oocyte injected with spermatozoa treated with placebo) were measured as primary outcome and embryo morphology as secondary outcome. Clinical outcomes were also documented. RESULT (S): Fertilization rate (78.9 ± 28.6% vs 63.2 ± 36.7, P = 0.002) and percentage of grade A embryos on day 3 (59.8 ± 35.6% vs 43.5 ± 41.5, P = 0.019) were significantly higher when spermatozoa were treated in vitro with myoinositol versus placebo. No differences were found for the expanded blastocyst formation rate. CONCLUSION (S): In vitro treatment of spermatozoa with myoinositol may optimize ICSI outcomes by improving the fertilization rate and embryo quality on day 3. The improvement of the number and the quality of embryos available in an ICSI cycle may have clinical utility if these findings can be confirmed.

Effect of different cryopreservation protocols on cytoskeleton and gap junction mediated communication integrity in feline germinal vesicle stage oocytes.

Oocyte cryopreservation in carnivores can significantly improve assisted reproductive technologies in animal breeding and preservation programs for endangered species. However, the cooling process severely affects the integrity and the survival of the oocyte after thawing and may irreversibly compromise its subsequent developmental capability. In the present study, two different methods of oocyte cryopreservation, slow freezing and vitrification, were evaluated in order to assess which of them proved more suitable for preserving the functional coupling with cumulus cells as well as nuclear and cytoplasmic competence after warming of immature feline oocytes. From a total of 422 cumulus enclosed oocytes (COCs) obtained from queens after ovariectomy, 137 were stored by vitrification in open pulled straws, 147 by slow freezing and 138 untreated oocytes were used as controls. Immediately after collection and then after warming, functional coupling was assessed by lucifer yellow injection and groups of fresh and cryopreserved oocytes were fixed to analyze tubulin and actin distribution, and chromatin organization. Finally, COCs cryopreserved with both treatments were matured in vitro after warming. In most cases, oocytes cryopreserved by slow freezing showed a cytoskeletal distribution similar to control oocytes, while the process of vitrification induced a loss of organization of cytoskeletal elements. The slow freezing protocol ensured a significantly higher percentage of COCs with functionally open and partially open communications (37.2 vs. 19.0) and higher maturational capability (32.5 vs. 14.1) compared to vitrified oocytes. We conclude that although both protocols impaired intercellular junctions, slow freezing represents a suitable method of GV stage cat oocytes banking since it more efficiently preserves the functional coupling with cumulus cells after thawing as well as nuclear and cytoplasmic competence. Further studies are needed to technically overcome the damage induced by the cryopreservation procedures on immature mammalian 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-oocyte 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.

Effect of gonadotropins during in vitro maturation of feline oocytes on oocyte-cumulus cells functional coupling and intracellular concentration of glutathione.

Information about the mechanisms of meiotic arrest and resumption of meiosis in feline oocytes is still limited. The aim of this study was to investigate the effect of the presence of gonadotropins during IVM, on meiotic progression in relation to the status of gap junction mediated communications between oocyte and cumulus cells, to the cAMP intracellular content, and to the intra-oocyte concentration of glutathione (GSH) in feline oocytes. Our results indicated that about 50% of cumulus-oocyte complexes (COCs) showed functionally open communications at the time of collection, while the remainder were partially or totally closed. After 3h of culture, the percentage of COCs with functional gap junctions was significantly greater in the group matured in the presence of gonadotropins than in those matured without them, where an interruption of communications was observed. Moreover, this precocious uncoupling was associated with a moderate increase of cAMP concentration in the oocyte, lower than in the group exposed to gonadotropins. Intra-oocyte glutathione levels decreased significantly after 24h of IVM, whether gonadotropins were present or absent during the culturing process. The presence of thiol compounds in the IVM medium induced an intra-oocyte GSH concentration significantly higher than that found in oocytes cultured without these compounds, and similar to the GSH content of immature oocytes. Moreover, the intracellular GSH concentration increased as meiosis progressed. The present study suggests that in feline oocytes, gonadotropins affect the dynamic changes in communications between oocyte and cumulus cells during IVM. However, the intracellular concentration of GSH is not influenced by the gonadotropin stimulation. Moreover, the presence of gonadotropins and thiol compounds results in an increase of GSH levels along with meiotic progression of the oocytes.

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 vivo and in vitro maturation of canine oocytes.

The domestic dog could be a valuable model for studying and developing assisted reproduction in taxonomically related endangered Canids. However, the efficiency of in vitro oocyte maturation is very low in this species compared to that of other mammalian species and this limits the development of reproductive biotechnologies, such as in vitro embryo production, cryopreservation, or nucleus transfer. In canine species the female gamete has unique characteristics: the oocyte is exposed to high concentration of progesterone in the follicular environment, it is ovulated in the dictyate state, and resumes and completes meiosis in the oviduct. Therefore, optimum conditions for in vitro maturation of dog oocytes may differ from other mammalian models in which follicles, where estrogens are the dominant hormones, ovulate oocytes at the Metaphase II stage of the first meiotic division. An in vitro culture system needs to be based on in vivo conditions in order to create a microenvironment similar to that in which oocyte development occurs physiologically, but little is known on mechanisms regulating oocyte maturation in the dog. This review analyzes the known factors involved in canine oocyte maturation in vivo and in vitro in order to suggest on which aspects future investigations may be focused.