In vitro and in vivo effects of ulipristal acetate on fertilization and early embryo development in mice.

STUDY QUESTION: Does ulipristal acetate (UPA), a selective progesterone receptor modulator used for emergency contraception (EC), interfere with fertilization or early embryo development in vitro and in vivo? SUMMARY ANSWER: At doses similar to those used for EC, UPA does not affect mouse gamete transport, fertilization or embryo development. WHAT IS KNOWN ALREADY: UPA acts as an emergency contraceptive mainly by inhibiting or delaying ovulation. However, there is little information regarding its effects on post-ovulatory events preceding implantation. STUDY DESIGN, SIZE, DURATION: This was an in vitro and in vivo experimental study involving the use of mouse gametes and embryos from at least three animals in each set of experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: For in vitro fertilization experiments, mouse epididymal spermatozoa capacitated in the presence of different concentrations of UPA (0-1000 ng/ml) were used to inseminate cumulus-intact or cumulus-free eggs in the presence or absence of UPA during gamete co-incubation, and the percentage of fertilized eggs was determined. For in vivo fertilization experiments, superovulated females caged with proven fertile males were injected with UPA (40 mg/kg) or vehicle just before or just after mating and the percentage of fertilized eggs recovered from the ampulla was determined. To investigate the effect of UPA on embryo development, zygotes were recovered from mated females, cultured in the presence of UPA (1000 ng/ml) for 4 days and the progression of embryo development was monitored daily. MAIN RESULTS AND THE ROLE OF CHANCE: In vitro studies revealed that the presence of UPA during capacitation and/or gamete co-incubation does not affect fertilization. Whereas the in vivo administration of UPA at the same time as hCG injection produced a decrease in the number of eggs ovulated compared with controls (vehicle injected animals, P < 0.05), no effects on fertilization were observed when UPA was administered shortly before or after mating. No differences were observed in either the percentage of cleaved embryos or the cleavage speed when UPA was present during in vitro embryo culture. LIMITATIONS, REASONS FOR CAUTION: Considering the ethical and technical limitations inherent to the use of human gametes for fertilization studies, the mouse model was used as an approach for exploring the potential effects of UPA on in vivo sperm transport and fertilization. Nevertheless, the extrapolation of these results to humans requires further investigation. WIDER IMPLICATIONS OF THE FINDINGS: This study presents new evidence on the lack of effect of UPA on gamete interaction and embryo development, providing new insights into the mechanism of action of UPA as an emergency contraceptive method with potential clinical implications. These new findings could contribute to increase the acceptability and proper use of UPA as an emergency contraceptive method. STUDY FUNDING/COMPETING INTERESTS: This study was partially supported by a National Agency of Scientific and Technological Promotion (ANPCyT), Argentina grants PICT 2011-061 to D.J.C. and PICT 2011-2023 to P.S.C. None of the authors has any competing interests to declare.

Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression, Functionality and Involvement in Motility Regulation.

Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility.

Glucose-regulated protein 78 (Grp78/BiP) is secreted by human oviduct epithelial cells and the recombinant protein modulates sperm-zona pellucida binding.

OBJECTIVE: To determine the secretion of Grp78 by human oviduct epithelial cells, its association to spermatozoa, and its involvement in gamete interaction. DESIGN: Prospective study. SETTING: Basic research laboratory. SUBJECT(S): Semen samples obtained from normozoospermic volunteers. Tubal tissue provided by patients undergoing hysterectomies. Oocytes collected from women undergoing IVF-ET. INTERVENTION(S): Analysis of Grp78 expression and secretion by oviductal tissue. Gamete incubation with recombinant Grp78 (rec-Grp78). MAIN OUTCOME MEASURE(S): Assessment of protein expression and secretion by immunohistochemistry and Western immunoblotting, respectively. Evaluation of rec-Grp78 binding to human spermatozoa by immunocytochemistry, and analysis of its effect upon gamete interaction using the hemizona assay. RESULT(S): Grp78 was found in the surface of oviduct epithelial cells. Soluble Grp78 was detected in oviductal fluids from women in the periovulatory period and in oviductal tissue conditioned medium. Rec-Grp78 was able to bind to the sperm acrosomal cap, and its presence during gamete interaction led to a decrease in the number of spermatozoa bound to the zona pellucida (ZP). When calcium ions from the incubation medium were replaced by strontium, rec-Grp78 enhanced sperm-ZP interaction. CONCLUSION(S): Grp78 is expressed and secreted by oviduct epithelial cells. The protein would bind to the gametes and may modulate their interaction in a calcium-dependent manner.

Bicarbonate is required for migration of sperm epididymal protein DE (CRISP-1) to the equatorial segment and expression of rat sperm fusion ability.

Numerous studies have demonstrated that sperm capacitation is a bicarbonate-dependent process. In the rat, capacitation has not been studied as much as in other species, mainly because of the difficulties in carrying out functional assays with this animal model. In the present study, we have examined the influence of bicarbonate in the overall rat sperm capacitation process by analyzing involvement of the anion in 1) protein tyrosine phosphorylation, 2) migration of epididymal protein DE (also known as CRISP-1) from the dorsal region to the equatorial segment of the sperm head that occurs during capacitation, and 3) ability of sperm to fuse with the egg. Incubation of sperm under capacitating conditions produced a time-dependent increase in protein tyrosine phosphorylation. This phosphorylation did not occur in the absence of HCO3- and rapidly increased by either exposure of sperm to HCO3- or replacement of the anion by a cAMP analog (dibutyryl-cAMP) and a phosphodiesterase inhibitor (pentoxifylline). The absence of HCO3- also produced a significant decrease in the percentage of cells showing migration of DE to the equatorial segment. This parameter was completely restored by addition of the anion, but dibutyryl-cAMP and pentoxifylline were not sufficient to overcome the decrease in DE migration. Sperm capacitated in the absence of HCO3- were unable to penetrate zona-free eggs independent of the presence of the anion during gamete coincubation. Exposure of these sperm to bicarbonate, or replacement of the anion by dibutyryl-cAMP and pentoxifylline, only partially restored the sperm fusion ability. Altogether, these results indicate that, in addition to its influence on protein tyrosine phosphorylation, bicarbonate is required to support other rat sperm capacitation- associated events, such as migration of DE to the equatorial segment, and expression of the ability of sperm to fuse with the egg.