Effect of perfluorodecanoic acid on pig oocyte viability, intracellular calcium levels and gap junction intercellular communication during oocyte maturation in vitro.

Perfluorodecanoic acid (PFDA) is a synthetic perfluorinated compound, which has been reported to exert adverse effects on somatic cells. However, its effects on germ cells have not been studied to date. The aim of the present study was to analyze the effects of PFDA on the viability, intracellular calcium levels and gap junction intercellular communication (GJIC) during porcine oocyte maturation in vitro. PFDA negatively impacted oocyte viability (medium lethal concentration, LC50 = 7.8 µM) and maturation (medium inhibition of maturation, IM50 = 3.8 µM). Oocytes exposed to 3.8 µM PFDA showed higher levels of intracellular calcium relative to control oocytes. In addition, GJIC among the cumulus cells and the oocyte was disrupted. The effects of PFDA on oocyte calcium homeostasis and intercellular communication seem to be responsible for the inhibition of oocyte maturation and oocyte death. In addition, since the deleterious effects of PFDA on oocyte viability, maturation and GJIC are significantly stronger than the previously reported effects of another widely used perfluorinated compound (Perfluorooctane sulfonate) in the same model, the use of PFDA in consumer products is questioned.

Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm.

BACKGROUND: In order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine oocytes and sperm selected by swim up (SU) or swim up + zona pellucida (SU + ZP) binding. RESULTS: Experiment 1, 4-20 replicates with total 821 in vitro matured oocytes were chemically activated with ethanol, calcium ionophore or ionomycin, to determine oocyte activation (precense of one PN). Treatments showed similar results (54, 47, 42 %, respectively) but statistically differents (P < 0.05) than mechanical activated oocytes in sham, ICSI and sham injection (13, 25, 32 %, respectively) (10-17 replicates; n = 429). Experiment 2: Twelve ejaculates and 28 straws of semen were used (11-19 replicates). Sperm were selected by SU in BSA-TCM 199-H medium. A total of 2,294 fresh sperm and 2,760 from frozen-thawed semen were analyzed after SU or SU + ZP binding. Fresh sperm selected by SU showed acrosome reaction (AR) of 59 %, the sperm selected by SU + ZP binding increased AR to 91 %. In comparison, the AR of frozen-thawed sperm using SU or SU + ZP binding was 77 and 86 %, respectively (P < 0.05). Experiment 3: fertilization in 200 mechanical activativated oocytes (17 replicates) was 4 %, but fertilization increased in ethanol activated oocytes after ICSI (12-28 %) (5-6 replicates). When fresh sperm only selected by SU were injected to 123 oocytes, a fertilization rate (28 %) was achieved; in sperm selected by SU + ZP was 25 % (73 oocytes). In comparison, in frozen-thawed sperm selected by SU, fertilization was 13 % (70 oocytes), whereas sperm from SU + ZP binding displayed 12 % (51 oocytes) (P > 0.05). CONCLUSIONS: Chemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU + ZP increased AR/A and AR/D rates in comparison with SU in fresh and frozen-thawed sperm. According to this, in terms of fertilization rates, chemical activation after ICSI increased oocyte PN formation compared to mechanical activation. Also, fresh sperm treated with SU and SU + ZP were significantly different than frozen-thawed sperm, but between sperm treatments no significant differences were obtained.

Effect of perfluorooctane sulfonate on viability, maturation and gap junctional intercellular communication of porcine oocytes in vitro.

Perfluorooctane sulfonate (PFOS) is a broadly used man-made surfactant whose long half-life has led to bioaccumulation. This perfluorinated compound is ubiquitous in human body fluids. PFOS concentrations as high as 26µM in plasma have been reported in occupationally exposed populations, and high levels of PFOS in human follicular fluid have been associated with subfertility. However, the effect of PFOS on the maturation of oocytes in mammals has not been reported to date. The aim of this study was to determine the effects of PFOS during oocyte maturation. Results indicate that PFOS inhibits oocyte viability (Lethal Concentration50=32µM) and maturation (inhibition of maturation50=22µM) at physiologically relevant concentrations. In order to evaluate the mechanisms of oocyte maturation inhibition by PFOS, gap junctional intercellular communication (GJIC) between oocytes and granulosa cells was assessed. GJIC between granulosa cells and the oocyte was significantly affected during the first 8h of maturation. However, the inhibitory effect of PFOS on GJIC was not due to an alteration on the expression of connexin genes Cx43, Cx45 and Cx60. These findings suggest that occupationally exposed populations could be at risk, and that PFOS might affect oocyte maturation by interfering the GJIC in the cumulus-oocyte complexes during the first hours of maturation.

Role of Mael in early oogenesis and during germ-cell differentiation from embryonic stem cells in mice in vitro.

In a previous study, we have identified a set of conserved spermatogenic genes whose expression is restricted to testis and ovary and that are developmentally regulated. One of these genes, the transcription factor Mael, has been reported to play an essential role in mouse spermatogenesis. Nevertheless, the role of Mael in mouse oogenesis has not been defined. In order to analyse the role of Mael in mouse oogenesis, the expression of this gene was blocked during early oogenesis in mouse in vitro using RNAi technology. In addition, the role of Mael during differentiation of embryonic stem cells (ESC) into germ cells in vitro was analysed. Results show that downregulation of Mael by a specific short interfering RNA disrupted fetal oocyte growth and differentiation in fetal ovary explants in culture and the expression of several germ-cell markers in ESC during their differentiation. These results suggest that there is an important role for Mael in early oogenesis and during germ-cell differentiation from embryonic stem cells in mouse in vitro.

Interaction of potential porcine sperm ligands with the oocyte plasma membrane.

We previously identified 62, 39, 27 and 7 kDa porcine sperm plasma membrane proteins that demonstrated a predominant affinity for the porcine oocyte plasma membrane by Western ligand blotting. The current experiments were designed to further investigate the potential roles of these molecules in sperm-oocyte plasma membrane interaction. Abilities of these proteins to bind to the oocyte plasma membrane and to inhibit sperm-oocyte interaction were evaluated. Plasma membrane was isolated primarily from the head of ejaculated porcine sperm by nitrogen cavitation and density gradient centrifugation. Fractions containing the 62, 39, 27 and 7 kDa proteins were electroeluted from one dimensional SDS polyacrylamide gels, dialysed and proteins biotinylated. Following incubation with zona-free porcine oocytes, bound protein was visualized with 20 µg TRITC-avidin/ml using confocal microscopy. Fractions of the dialysed, electroeluted proteins were added to porcine in vitro fertilization assays. The 62, 39, 27 and 7 kDa proteins all demonstrated binding to the oocyte plasma membrane in contrast to a biotinylated control protein. Addition of unlabelled sperm plasma membrane proteins to the biotinylated protein visibly reduced binding. Addition of each of these protein fractions to in vitro fertilization assays reduced sperm interaction with the porcine oocyte plasma membrane in a concentration-dependent manner. Binding of these sperm plasma membrane proteins to the oocyte plasma membrane and inhibition of fertilization are consistent with these proteins being involved in sperm-oocyte plasma membrane interaction.

Effect of norethisterone and its A-ring reduced metabolites on the acrosome reaction in porcine spermatozoa.

The synthetic progestin, norethisterone (NET), has been reported as a contragestational postcoital agent in humans, rodents and rabbits. The effect and molecular mechanisms of NET and its A-ring reduced metabolites, 5alpha-NET and 3beta5alpha-NET, on the acrosome reaction (AR) are unknown. The aim of this study was to assess the effect of these compounds on an in vitro progesterone-induced AR in porcine spermatozoa. The spermatozoa were obtained from semen ejaculated by proven fertile adult pigs. Seminal plasma removed and incubated under capacitating conditions was performed in TALP-Hepes medium for 4 h. Progesterone (P4) and three different progestins: norethisterone (NET), 5alpha-norethisterone (5alpha-NET) and 3beta5alpha-NET were then added at equimolar doses, and the spermatozoa were incubated for 15 min. Double-staining with PSA-FITC and Hoechst-33258 assessed the AR and sperm viability. Both P4 and NET induced the AR, while 5alpha-NET not only did not induce this process, but was able to block the effect of P4 on the spermatozoa. 3beta5alpha-NET was not able to inhibit P4 action. These results suggest that NET and its A-ring reduced metabolites act in different ways on the progesterone-induced AR in porcine spermatozoa.

Sperm plasma membrane receptors for the porcine oocyte plasma membrane.

In vitro fertilisation (IVF) was used to assess the ability of solubilised sperm plasma membrane (PM) proteins to inhibit the interaction of intact gametes. This is a first step before evaluating the ability of individual isolated proteins to competitively inhibit sperm-oocyte interaction as part of the process of studying the molecular events of fertilisation. Porcine oocytes were aspirated from ovaries, matured for 48 h in Medium 199, and the zona pellucida (ZP) was removed by exposure to acid Tyrode's solution. ZP-free matured oocytes were exposed to 200-800 micrograms/ml sperm PM protein for 1 h prior to insemination and during gamete co-incubation. Twenty-four hours after insemination with 5 x 10(5) capacitated sperm/ml, the oocytes were fixed, stained and examined. Sperm PM protein clearly inhibited IVF in a concentration-dependent manner (r = -0.87). The inhibition index (I50%), representing the sperm PM protein concentration necessary to inhibit IVF to 50% of the control value, was 310 micrograms/ml. These results demonstrate that solubilised sperm PM protein inhibits the interaction of intact gametes as one might expect for receptor-ligand interactions. Furthermore, the complement of sperm PM proteins appeared maximally effective at a calculated concentration of 690 microns/ml, providing a foundation for further studies with individual proteins.

In vitro fertilizing capacity of frozen-thawed boar semen.

We describe a porcine semen cryopreservation technique and assess the in vitro fertilizing capacity of the frozen-thawed spermatozoa. The thawed spermatozoa did not lose the physiological properties of motility, viability, and acrosome reaction or capacity to fertilize in vitro. Immediately after thawing, the spermatozoa showed 51% mean motility, 60% viability, and 5% induced acrosome reaction. After 2.5 h of incubation in TALP medium, the spermatozoa exhibited 61% motility, 63% viability and 40% induced acrosome reaction. The average in vitro fertilization capacity of thawed spermatozoa was 68% compared with that of spermatozoa from fresh semen (85%). The percentage of polyspermy was highly variable, with frozen-thawed samples ranging from 0 to 28% and fresh samples from 0 to 30%. The results obtained with frozen semen from 5 boars of different breeds did not show considerable variation. This suggests that the freezing-thawing technique is reproducible and adequate for in vitro fertilization.

Inhibition of pig oocyte in vitro fertilization by the action of components of the zona pellucida.

The aim of the present study was to determine whether the previous addition of porcine zona pellucida (ZP) components to spermatozoa of the same species has an inhibitory effect on in vitro fertilization (IVF). Boar spermatozoa were exposed to whole porcine solubilized zona pellucida (SZP), ZP glycoproteins (55 kDa and 90 kDa) and peptides (37 kDa, 40 kDa and 68kDa). Doses tested were 40, 70 and 100 mug/ml. In vitro fertilization was clearly inhibited by each component when the oocytes were compared with those fertilized with untreated spermatozoa. All the components had an effect in a dose dependent manner.