Whitten's Medium Research Articles

Selected Metabolites Found in Equine Oviductal Fluid do not Modify the Parameters Associated to Capacitation of the Frozen-thawed Equine Spermatozoa In Vitro

In the horse, a repeatable protocol for in vitro fertilization has not been developed, possibly due to incomplete sperm capacitation. We have previously identified the metabolites present in equine oviductal fluid (OF). We aimed to test the effects of different metabolites found in equine oviductal fluid on quality parameters of frozen-thawed spermatozoa. Different concentrations of myoinositol (5–25 mM), lactate (6–60 mM), glycine (0.1–5 mM), β-alanine (1–6 mM), and histamine (0.05–0.4 mM) were added independently to modified Whitten's medium (pH = 7.25). Thawed equine spermatozoa (three stallions, one ejaculate per stallion, n = 3) were incubated for 2 hours at 37˚C in presence of the selected metabolites. After sperm incubation, total motility (TM), and progressive motility (PM) were evaluated by computer-assisted sperm analysis. Viability (SYBR-14+/PI−), mitochondrial membrane potential (ΔΨm) (JC-1), acrosome reaction (PNA+/PI−) and reactive oxygen species (ROS) production (CellRox+/PI−), were evaluated by flow cytometry. Protein tyrosine phosphorylation (PY) was evaluated by indirect immunofluorescence. Our results show that the addition of the metabolites at the dosages tested does not exert any effect on the sperm parameters analyzed. More research is needed to ascertain if metabolite addition at the dosages found in the equine OF exerts any remarkable effect on in vitro equine sperm capacitation.

Effect of culture conditions and method of conception on mouse live birth rate

To understand in a mouse model whether there are differences in the decidua and live birth rate after transfer of blastocysts generated by invitro fertilization (IVF) or by superovulation with spontaneous mating into unstimulated recipients. Animal experiment. University-affiliated tertiary hospital. Mice. IVF embryos were generated and cultured in either Whitten medium (WM, suboptimal conditions) and 20% O2 or KSOM medium with amino acids (KAA, optimal conditions) and 5% O2. The control blastocysts from superovulated mice were flushed out of the uterus 3.5 days (E3.5) after mating (FB group). The resulting blastocysts were transferred to nonsuperovulated CF1 recipients mated to vasectomized males. To understand whether anomalies of decidua were present, the expression of genes involved in decidual development and inflammation was analyzed at E7.5 and E18.5. Similarly, immunostaining was used to evaluate whether the pathways involved in activation of mTORC1 (p-S6) and Cox2 signaling (Cox 2 staining) were altered. Live birth rate, gene expression, and immunostaining of decidua. Implantation rates at E7.5 were similar, but invivo embryos (FB groups) were predicted to result in live births 3.3 times higher (2.2-5.1) and 6.6 times higher (4.7-9.3) compared with optimal and suboptimal cultures, respectively. Expression of genes involved in decidual development and inflammation or localization and intensity of staining for p-S6 (mTOR pathway), or inflammation (Cox 2 pathway) were not different among the groups. The predicted live birth rate was decreased in mouse embryos generated by IVF compared with embryos generated by mating, whereas the implantation rate was not different. Suboptimal culture conditions resulted in lower birth rate. We did not find evidence of abnormalities in decidualization that could explain these findings. These data indicate that blastocysts cultured in stressful conditions are less competent, suggesting that decreasing the number of embryonic manipulations may result in higher live birth rates.

116 Effect of different energy sources on motility and calcium ionophore-induced acrosome reaction in equine sperm

Equine sperm appears to differ from sperm of other species, preferring oxidative phosphorylation over glycolysis for energy production. However, there is little information about the effects of different energy sources on capacitation-related events in equine sperm. We evaluated stallion sperm incubated in the presence of different energy sources at a pH (~8.3) known to support some aspects of capacitation. Fresh stallion semen (n=9 ejaculates) was washed, then diluted to 30×106 spermmL−1 with modified Whitten's medium containing 25mM bicarbonate, 7mgmL−1 bovine serum albumin (BSA), and only one energy source (5mM glucose, 10mM lactate, or 10mM pyruvate). The diluted sperm were incubated in air for 0.5, 2, or 4h, then exposed to 5 µM calcium ionophore A23187 (CaI) or vehicle (V) for 10min. Sperm were washed and incubated in the assigned medium for 30min, then evaluated by computer-assisted sperm motility analysis and by live/dead staining (Fixable Live/Dead Red, Molecular Probes) followed by fixation (2% v/v paraformaldehyde) and staining for acrosome reaction, using Pisum sativum agglutinin (FITC-PSA, Sigma Aldrich). Stained sperm were evaluated via flow cytometry to detect live acrosome-reacted sperm. Data were analysed using ANOVA with Tukey post hoc comparison (SAS 9.4, SAS Institute). Medium pH at 4h was 8.37 to 8.48. There were no significant differences between CaI and V treatments for any experimental endpoint (P>0.05). At 4h, pyruvate values for total motile sperm (46±5%) and curvilinear velocity (113±8µm s−1) were significantly higher than those for lactate (32±5% and 86±5µm s−1) or glucose (27±4% and 90±5µm s−1). The percentage of live sperm that were acrosome-reacted (%L-AR) did not increase significantly until 4 h; at this time,%L-AR was higher for lactate (46±3%) than for pyruvate (21±3%) or glucose (18±3%; P<0.05). To the best of our knowledge, this is the first critical evaluation of the effect of different energy sources on motility and acrosome reactivity of equine sperm. The higher motility in pyruvate supports the concept that stallion sperm utilises oxidative phosphorylation more effectively than glycolysis. The apparent lack of effect of CaI on%L-AR may be related to the 1- to 2-h delay typically seen after its use in equine sperm; the present data show that incubation even for 4h in potentially capacitating conditions does not prepare equine sperm to respond quickly to CaI. Surprisingly, a high rate of spontaneous acrosome reaction was achieved in the lactate treatment. The notably higher%L-AR with lactate than with pyruvate suggests that this effect is not due to utilisation of substrate for oxidative phosphorylation but may be associated with conversion of lactate to pyruvate via lactate dehydrogenase, or possibly with effects of lactate through other pathways. Although pyruvate was associated with greater motility, this could be related to higher oxidative stress, which could have influenced the%L-AR. Further work is needed to determine whether the acrosomal changes observed are related to functional capacitation.

Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation.

Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (∆Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P-tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30- and 120-min incubation. Membrane fluidity (MC540) increased in both media at 30- and 120-min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2- and 4-hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation-related parameters in stallion sperm.

Extracellular calcium regulates protein tyrosine phosphorylation through calcium-sensing receptor (CaSR) in stallion sperm.

Protein tyrosine phosphorylation (PY), a hallmark of sperm capacitation, is inhibited by extracellular calcium in stallion sperm. The objective of this study was to determine the presence and influence of the calcium-sensing receptor (CaSR) in this phenomenon. First, the presence of the CaSR was demonstrated in stallion sperm. We then tested its function in these gametes using its inhibitor NPS2143 or its agonist AC34356. Sperm were capacitated for 4 hr in modified Whitten's medium with 25 mM bicarbonate plus NPS2143 and 2.4 mM calcium or AC34356 alone, followed by analysis of PY. Inhibition of CaSR with NPS2143 prevented the calcium-dependent PY inhibition in a dose-dependent manner (5, 10, and 15 μM) whereas AC34356 (100 μM) inhibited PY similarly to calcium. Stallion sperm motility and viability significantly decreased in presence of 15 μM of NPS2143 whereas only sperm motility decreased with 100 μM of AC34356. CaSR function was also studied in the complete absence of calcium by including 2 mM ethylene glycol tetraacetic acid (EGTA); under these conditions, AC34356 again inhibited PY, but this time induced a significant increase in sperm motility. Inhibition of calmodulin by W-7 did not recover the AC34356-mediated PY inhibition. When stallion sperm were incubated under capacitating conditions (calcium, bicarbonate, plus bovine serum albumin) at elevated pH (7.9 or 8.5) AC34356 did not block PY. These results thus elucidate the effect of extracellular conditions on the regulation of CaSR, and point to its modulatory role on stallion sperm PY, motility, and viability. Mol. Reprod. Dev. 83: 236-245, 2016. © 2016 Wiley Periodicals, Inc.

Effect of Different Media and Protein Source on Equine Gametes: Potential Impact During In Vitro Fertilization.

Equine in vitro fertilization (IVF) is still inconsistent. In the present work, we studied how modified Whitten's (MW) medium and Tissue Culture Medium 199 (TCM) added with Foetal Bovine Serum (FBS; 10% v/v) or Bovine Serum Albumin (BSA; 7 mg/ml) affected equine gametes to subsequently run IVF trials. Compact (Cp) and expanded (Ex) cumuli equine oocytes were matured and placed in TCM or MW supplemented with BSA or FBS for 18-20 h (no sperm added). In Ex oocytes, TCM-199 added with FBS or BSA resulted in higher metaphase II (MII) rates (75.7% and 62.7%, respectively) than MW added with BSA (54%) or FBS (52.2%; p < 0.05); this was not observed for Cp oocytes. Equine sperm were capacitated in the same media at 10 × 10(6) sperm/ml for 4 h at 37°C; total motility and protein tyrosine phosphorylation (PY) were evaluated. While motility remained unchanged, TCM or MW added with FBS enhanced the number of sperm showing PY-stained tails (25 ± 4.8% and 31 ± 6.6%; mean ± SEM, respectively) over BSA supplemented media (3 ± 1.2% and 11.7 ± 1.1%) for TCM and MW (p < 0.05). In view of the previous results, sperm were capacitated in TCM + FBS and MW + BSA (control); IVF trials were run in the same media supplemented with 200 ng/ml of progesterone, but no fertilization occurred. Our results show that TCM + FBS enhances Ex equine oocyte's meiotic competence over MW + BSA and TCM or MW added with FBS successfully induce equine PY over media supplemented with BSA.

In Vitro Fertilization Affects Growth and Glucose Metabolism in a Sex-Specific Manner in an Outbred Mouse Model1

The preimplantation period is a time of reprogramming that may be vulnerable to disruption. This question has wide clinical relevance since the number of children conceived by in vitro fertilization (IVF) is rising. To examine this question, outbred mice (CF1 × B6D2F1) conceived by IVF and cultured using Whitten medium and 20% O2 (IVFWM group, less optimal) or K simplex optimized medium with amino acids and 5% O2 (IVFKAA group, more optimal and similar to conditions used in human IVF) were studied postnatally. We found that flushed blastocysts transferred to recipient mice provided the best control group (FB group), as this accounted for the effects of superovulation, embryo transfer, and litter size. We observed that many physiological parameters were normal. Reassuringly, IVFKAA offspring did not differ significantly from FB offspring. However, male IVFWM mice (but not females) were larger during the first 19 wk of life and exhibited glucose intolerance. Male IVFWM mice also showed enlarged left heart despite normal blood pressure. Expression of candidate imprinted genes (H19, Igf2, and Slc38a4) in multiple adult tissues did not show differences among the groups; only Slc38a4 was down-regulated following IVF (in both culture conditions) in female adipose tissue. These studies demonstrate that adult metabolism is affected by the type of conditions encountered during the preimplantation stage. Further, the postnatal growth trajectory and glucose homeostasis following ex vivo manipulation may be sexual dimorphic. Future work on the long-term effects of IVF offspring should focus on glucose metabolism and the cardiovascular system.

Effects of embryo culture media do not persist after implantation: a histological study in mice

Is post-implantation embryonic development after blastocyst transfer affected by exposure to different assisted reproduction technology (ART) culture media? Fetal development and placental histology of ART embryos cultured in vitro in different ART media was not impaired compared with embryos grown in vivo. The application of different in vitro culture (IVC) media for human ART has an effect on birthweight of newborns. In the mouse model, differences in blastocyst formation were reported after culture in different ART media. Moreover, abnormalities in the liver and heart have been detected as a result of suboptimal IVC conditions. Fertilized oocytes from inbred and outbred breeding schemes were retrieved and either immediately transferred to foster mothers or incubated in control or human ART culture media up to the blastocyst stage prior to transfer. Placental and fetal anatomy and particularly bone development were evaluated. B6C3F1 female mice were used as oocyte donors after ovulation induction. C57Bl/6 and CD1 males were used for mating and CD1 females as foster mothers for embryo transfer. Fertilized oocytes were recovered from mated females and incubated in sequential human ART media (ISM1/ISM2 and HTF/Multiblast), in control media [KSOM(aa) and Whitten's medium] or grown in utero without IVC (zygote control). As in vivo, control B6C3F1 females were superovulated and left untreated. Fetuses and placentae were isolated by Caesarean section and analysed at 18.5 days post-coitum (dpc) for placenta composition and at 15.5 dpc for body weight, crown-rump length (CRL), fetal organ development, morphological development, total bone length and extent of bone ossification. No major differences in the number of implantation sites or in histological appearance of the placentae were detected. CRL of KSOM(aa) fetuses was higher compared with zygote control and Whitten's medium. Histological analysis of tissue sections revealed no gross morphological differences compared with the in vitro groups or in vivo controls. Furthermore, no changes in skeletal development and degree of ossification were observed. However, fibula and tibia of ISM1/ISM2 fetuses were longer than the respective ones from in vivo fetuses. Findings in the mouse embryo and fetus may not be fully transferable to humans. In addition to skeletal development and placentation, there may be other parameters, e.g. on the molecular level which respond to IVC in ART media. Some comparisons have limited statistical power. Our data suggest that once implantation is achieved, subsequent post-implantation development unfolds normally, resulting in healthy fetuses. With mouse models, we gather information for the safety of human ART culture media. Our mouse study is reassuring for the safety of ART conditions on human embryonic development, given the lack of bold detrimental effects observed in the mouse model. This work was supported by the Deutsche Forschungsgemeinschaft (BO 2540/4-1 and SCHL 394/9-1) and by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (S.L.G.); Bilateral grant NWO-DFG 63-258. None of the authors has any conflict of interest to declare. Not applicable.

Effects of the use of assisted reproduction and high-caloric diet consumption on body weight and cardiovascular health of juvenile mouse offspring

Maternal obesity and the use of assisted reproductive technologies (ART) are two suboptimal developmental environments that can lead to offspring obesity and cardiovascular disease. We hypothesized that these environments independently and synergistically adversely affect the offspring's weight and cardiovascular performance at ~7 weeks of age. Mice were fed either 24% fat and 17.5% high-fructose (HF) corn syrup or maintenance chow (5% fat; low-fat, no-fructose (LF)). Dams were subdivided into no ART and ART groups. ART embryos were cultured in Whitten's medium and transferred into pseudopregnant recipients consuming the same diet as the donor. Offspring were fed the same diet as the mother. Body weights (BW) were measured weekly and mean arterial pressure (MAP) was collected through carotid artery catheterization at killing (55±0.5 days old). Expression of genes involved in cardiovascular remodeling was measured in thoracic aorta using qRT-PCR, and levels of reactive oxygen species (ROS) were measured intracellularly and extracellularly in mesenteric resistance arteries. ART resulted in increased BW at weaning. This effect decreased over time and diet was the predominant determinant of BW by killing. Males had greater MAP than females (P=0.002) and HF consumption was associated with greater MAP regardless of sex (P<0.05). Gene expression was affected by sex (P<0.05) and diet (P<0.1). Lastly, the use of ART resulted in offspring with increased intracellular ROS (P=0.05). In summary, exposure to an obesogenic diet pre- and/or post-natally affects weight, MAP, and gene expression while ART increases oxidative stress in mesenteric resistance arteries of juvenile offspring, no synergistic effects were observed.

Determination of Allelic Expression of H19 in Pre- and Peri-Implantation Mouse Embryos1

H19 is a maternally expressed, imprinted, noncoding RNA with tumor-suppressor activity. During mouse preimplantation development, H19 is primarily expressed in the trophectoderm cells. The purpose of this project was to determine allelic expression of H19 in pre- and peri-implantation mouse embryos. We were further interested in determining if loss of imprinted H19 expression during blastocyst development occurred as a result of superovulation and/or culture. Our last goal was to ascertain if differential H19 allelic expression occurred between the inner cell mass (ICM)-containing half and the primary trophoblast giant cell (PTGC)-containing half of the embryo. C57BL/6J((Cast-7))xC57BL/6J F1 embryos were collected from the uterus at 84, 96, and 108 h following natural ovulation or superovulation. In vitro-cultured F1 embryos were harvested from the oviduct at the 2-cell stage and cultured in KSOM + aa supplemented with amino acids or Whitten media and collected at the above-mentioned times. Allele-specific H19 expression in single embryos was determined by qRT-PCR followed by fluorescence resonance electron transfer or RT-PCR followed by restriction fragment length polymorphism and polyacrylamide gel electrophoresis (RFLP-PAGE). Peri-implantation embryos were microdissected into two sections, one containing the ICM and the other containing the PTGC. TaqMan probes for Dek, Pou5f1, Itga7, H19, and Igf2 were used to ascertain gene expression enrichment in each section. Allele-specific H19 expression in embryo sections was determined by RFLP-PAGE. We found that as embryos advance through preimplantation development they start expressing H19 in a biallelic manner and this phenomenon was observed in the cultured and the in vivo-developed embryos. The PTGC-containing half of the embryo had greater expression of H19 when compared to the ICM-containing half of the embryo, as determined by qRT-PCR. In conclusion, loss of imprinting of H19 occurs in the PTGC-containing section of peri-implantation mouse embryos. We speculate that this is part of a physiologic event at the time of implantation in the mouse.

The Effect of Maternal High-Fat Diet and ART on Cardiovascular Outcome in Offspring.

The Barker Hypothesis states that the maternal environment a fetus is exposed to can affect its subsequent development. Two examples of suboptimal maternal environments found in women today are those created by obesity and Assisted Reproductive Technologies (ART). Obesity affects approximately 300 million people world-wide, and over one-third of women of reproductive age in the United States are obese. ART is used to overcome infertility; a problem that nearly ten percent of women in the United States face. These suboptimal maternal environments have been associated with unfavorable outcomes in the offspring such as cardiovascular disease and insulin resistance. Therefore, for the present study we hypothesized that obesity and ART independently and synergistically can adversely affect the cardiovascular health of the offspring. We examined the effects that maternal consumption of a high (HF) or low (LF) fat diet prior to and during pregnancy in combination with the use of ART (i.e. superovulation, embryo culture, and embryo transfer) or no ART would have on the cardiovascular performance of juvenile offspring in mice. Embryo cultures were performed in either KSOM supplemented with amino acids or in Whitten's medium. Blastocysts were transferred into pseudopregnant females fed either a HF or LF diet. In addition, we challenged the offspring generated from that scheme to either a HF or LF diet after weaning to assess if offspring diet would exacerbate or ameliorate cardiovascular problems resulting from being gestated in a HF environment and/or conceived by ART (in all 40 permutations were attempted). The offspring were sacrificed on average at 7 weeks, and the measurements taken were body weight, blood pressure (by carotid artery catheterization), and resistance vessel vasoconstriction- to norepinephrine [10-8 to 10-5M]- or vasodilation- to acetylcholine or sodium nitroprusside [10−8 to 10−5 or 10−7 to 10−4M; respectively]. Results indicate that mice that developed in Whitten's medium during preimplantation development had greater body weight at seven weeks of age when compared to mice cultured in KSOM or those conceived without the use of ART (control female = 21.40 ± 1.1, KSOM female = 24.10 ± 1.3, Whitten's female = 27.80 ± 1.6, control male = 29.30 ± 1.2, KSOM male = 30.00 ± 1.6, Whitten's male = 36.30 ± 1.6 g; P < 0.01). These results were not confounded by consumption of HF diet at any stage during the experiment. Further, we observed that a group of ART males in the HF diet had higher mean arterial pressure when compared to controls (102.7 ± 6.17 and 74.8 ± 5.24 and mmHg, respectively; P < 0.02). We found that when offspring of obese dams were fed a HF diet, they had increased body weight (P<0.05) and their resistance arteries had greater (P<0.05) vasconstrictive responses to norepinephrine than those from control mice. Differences were observed between males and females that had been exposed to a high fat maternal and offspring diet for body weight (P<0.05) and response to acetylcholine (endothelium dependent response; P<0.05). However, diet did not differently affect the response to sodium nitroprusside (endothelium independent response) between sexes. Together, these data suggest an interaction between the uterine environment and the health of the offspring.

Determination of Allelic Expression of H19 in Peri-Implantation Mouse Embryos.

H19 is a maternally-expressed imprinted non-coding RNA with tumor suppressor activity. During mouse preimplantation development, H19 is primarily expressed in the trophectoderm cells. It has been documented that preimplantation embryo manipulation affects the allelic expression of this gene causing it to become biallelically expressed. We have observed biallelic expression of H19 in embryos that developed in vivo leading us to hypothesize that H19 biallelic expression might be a normal occurrence in mouse embryos immediately prior to implantation. Expression of H19 was determined in C7xB6 F1 embryos derived from superovulated C57BL/6J(Cast7) (C7) females mated to C57BL/6J (B6) males. In vivo-produced control embryos were collected from the uterus. In vitro-cultured embryos were collected from the oviduct at the 2-cell stage and cultured in KSOM supplemented with amino acids or Whitten's media. The embryo collection times chosen were 84, 96, and 108 hours post presumed ovulation to obtain embryos at the early, mid, and late blastocyst stages of development. QPCR with subsequent FRET or RFLP followed by PAGE allowed us to determine allele-specific gene expression in single embryos. For an embryo to be considered to have biallelic H19 expression, =10% of the total H19 RNA must be paternal in origin. So far we have analyzed H19 expression in 21, 32, 47 in vivo produced embryos (for 84, 96, 108 h; respectively), 18, 52, 58 KSOM cultured embryos, and 11, 44, 31 Whitten's cultured embryos. Those results show that the paternal allele is expressed in 24,31, 77 % of in vivo produced embryos (for 84, 96, 108 h respectively), 28, 31, 60 % of KSOM cultured embryos and 18, 15, 55 % of Whitten's cultured embryos. As recent reports suggest a possible effect of superovulation on imprinting, we are repeating the experiment with embryos produced after natural ovulation. Thus far we have analyzed H19 expression in 5, 25, 15 in vivo produced embryos (for 84, 96, 108 h, respectively), 1, 19, 25 KSOM cultured embryos and 0, 8, 20 Whitten's cultured embryos. Those results show that the paternal allele is expressed in 20, 32, 80 % of in vivo produced embryos (for 84, 96, 108 h respectively), 0, 32, 60 % of KSOM culture embryos and not detected, 25, 30 % of Whitten's cultured embryos. We are currently performing DNA methylation assays to ascertain if the observed loss of imprinting correlates with a concomitant decrease in DNA methylation at the H19 regulatory region. Peri-implantation mouse embryos are composed of at least five cell types, namely, inner cell mass, primitive endoderm, mural trophectoderm, polar trophectoderm, and trophoblast giant cells. We speculate that biallelic H19 expression is the result of the differentiation of the trophectoderm trophoblast giant cells and that this may be relevant for implantation. We are currently performing microdissection studies to divide the embryo in two-three parts, one containing the inner cell mass cells, primitive endoderm cells and/or adjacent polar trophectoderm and the other containing the mural trophectoderm and primary trophoblast giant cells. Using a combination of TaqMan probes for H19, Igf2, Pou5f1, Cdx2, Rdx, Dek we intend to ascertain the enrichment of giant cells in our mural trophectoderm sample. Initial work in this area demonstrates our ability to have mural trophectoderm cells free or with minimal inner cell mass contamination based on lack of expression of Pou5f1 in this embryo section.

Calcium–calmodulin and pH regulate protein tyrosine phosphorylation in stallion sperm

The mechanisms leading to capacitation in stallion sperm are poorly understood. The objective of our study was to define factors associated with regulation of protein tyrosine phosphorylation in stallion sperm. Stallion sperm were incubated for 4 h in modified Whitten's media with or without bicarbonate, calcium, or BSA. When sperm were incubated in air at 30×10⁶/ml at initial pH 7.25, protein tyrosine phosphorylation was detected only in medium containing 25 mM bicarbonate alone; calcium and BSA inhibited phosphorylation. Surprisingly, this inhibition did not occur when sperm were incubated at 10×10⁶/ml. The final pH values after incubation at 30×10⁶ and 10×10⁶ sperm/ml were 7.43 ± 0.04 and 7.83 ± 0.07 (mean ± s.e.m.) respectively. Sperm were then incubated at initial pH values of 7.25, 7.90, or 8.50 in either air or 5% CO₂. Protein tyrosine phosphorylation increased with increasing final medium pH, regardless of the addition of bicarbonate or BSA. An increase in environmental pH was observed when raw semen was instilled into the uteri of estrous mares and retrieved after 30 min (from 7.47 ± 0.10 to 7.85 ± 0.08), demonstrating a potential physiological role for pH regulation of capacitation. Sperm incubated in the presence of the calmodulin (CaM) inhibitor W-7 exhibited a dose-dependent increase in protein tyrosine phosphorylation, suggesting that the inhibitory effect of calcium was CaM mediated. These results show for the first time a major regulatory role of external pH, calcium, and CaM in stallion sperm protein tyrosine phosphorylation.

163 HYPERACTIVATION OF STALLION SPERM IN FOLLICULAR FLUID FOR IN VITRO FERTILIZATION OF EQUINE OOCYTES

In vitro fertilization has remained elusive in the horse, as evidenced by low sperm penetration rates when IVF has been attempted with in vivo- or in vitro-matured oocytes. It is likely that the low sperm penetration rates observed in IVF studies are due to the inability to appropriately capacitate or hyperactivate, or both, stallion sperm in the laboratory. The acquisition of hyperactivated sperm motility has been observed within the oviducts of mammals at the time of fertilization and is required for zona pellucida penetration in conjunction with the acrosome reaction (AR). Although the zona pellucida is considered the prime physiological inducer of AR, previous studies have shown a low incidence of AR in zona pellucida-bound stallion spermatozoa after 1 h of in vitro binding. This low incidence suggests that, besides the zona pellucida glycoproteins, another major factor might be responsible for AR. Protein-bound progesterone, present in equine follicular fluid (FF), has been demonstrated to induce AR in stallion spermatozoa. In this context, the aims of this study were (1) to hyperactivate stallion sperm in FF and (2) to verify whether this hyperactivation supports equine IVF. Pooled FF, aspirated from the preovulatory follicles of oestrous mares, was used and its progesterone concentration was determined by immune enzymatic assay. Spermatozoa from fertile stallions selected by a swim-up procedure were pre-incubated for 6 h in capacitating medium (modifed Whittens's medium (WM) supplemented with 25 mM NaHCO3 and 7 mg mL–1 of BSA) and then incubated for 6 h at 37°C in either FF or capacitating WM. Sperm motility was assayed by computer-assisted semen analysis, rates of AR were assessed by fluorescein isothiocyanate-PNA staining and rate of apoptosis was assessed by an annexin V test. For IVF, spermatozoa were incubated at 10 × 106 sperm mL–1 in capacitating WM for 6 h and then diluted to 1 × 106 sperm mL–1 in capacitating WM with or without 10% of FF. Five mature mare oocytes were transferred into droplets (100 μL) of the sperm suspensions covered with mineral oil and then incubated for 18 h at 38.5°C in 5% CO2 in humidified air. After that, oocytes were transferred to an embryo culture medium (DMEM/F-12) for an additional 3 days. Data were analysed by ANOVA. Treatment of sperm with FF resulted in a significant (P ≤ 0.05) decrease of 3 motility variables indicative of hyperactivation: straight line velocity, straightness and linearity. The highest rate of AR (29.44%) and a lower rate of apoptosis (16.93%) were obtained after 4 h of incubation in follicular fluid. By coupling capacitating conditions with the induction of hyperactivation using follicular fluid, we have obtained reproducible percentages of 8-cell-stage embryos (18.56%) in our IVF experiments. Conversely, sperm incubated in capacitating conditions but not treated with FF did not fertilize (0%). It is concluded that mare FF does not impair sperm viability, stimulates equine sperm hyperactivation in vitro, induces the AR and supports equine IVF.

Culture medium, gas atmosphere and MAPK inhibition affect regulation of RNA-binding protein targets during mouse preimplantation development

During oogenesis, mammalian oocytes accumulate maternal mRNAs that support the embryo until embryonic genome activation. RNA-binding proteins (RBP) may regulate the stability and turnover of maternal and embryonic mRNAs. We hypothesised that varying embryo culture conditions, such as culture medium, oxygen tension and MAPK inhibition, affects regulation of RBPs and their targets during preimplantation development. STAU1, ELAVL1, KHSRP and ZFP36 proteins and mRNAs were detected throughout mouse preimplantation development, whereas Elavl2 mRNA decreased after the two-cell stage. Potential target mRNAs of RBP regulation, Gclc, Slc2a1 and Slc7a1 were detected during mouse preimplantation development. Gclc mRNA was significantly elevated in embryos cultured in Whitten's medium compared with embryos cultured in KSOMaa, and Gclc mRNA was elevated under high-oxygen conditions. Inhibition of the p38 MAPK pathway reduced Slc7a1 mRNA expression while inhibition of ERK increased Slc2a1 mRNA expression. The half-lives of the potential RBP mRNA targets are not regulated in parallel; Slc2a1 mRNA displayed the longest half-life. Our results indicate that mRNAs and proteins encoding five RBPs are present during preimplantation development and more importantly, demonstrate that expression of RBP target mRNAs are regulated by culture medium, gas atmosphere and MAPK pathways.

Effect of procaine, pentoxifylline and trolox on capacitation and hyperactivation of stallion spermatozoa

Reasons for low in vitro fertilisation rates in the horse include the difficulties in inducing capacitation and/or hyperactivation of stallion spermatozoa. The aim of this study was to analyse the effect of noncapacitating and capacitating modified Whitten's (MW) and modified Tyrode's medium (MT) and treatment with procaine (5 mmol), pentoxifylline (3.5 mmol) and trolox (120 mmol) on motility (CASA), capacitation, acrosomal status, viability and mitochondrial membrane potential of stallion spermatozoa (n = 4). While there was no influence of MW and MT on sperm motility, a significant increase in the percentage of viable-capacitated spermatozoa was observed after incubation in capacitating MW (P < 0.05). Pentoxifylline showed no significant effect on the motility pattern but increased the proportion of live-capacitated spermatozoa (P < 0.05). Trolox had no detectable effect on either capacitation or hyperactivation. Procaine was the only agent that induced hyperactivation in terms of a reduced proportion of progressively motile spermatozoa, straight line velocity, straightness, linearity and beat-cross frequency and an increase in the amplitude of lateral head displacement (P < 0.05). The combination of capacitating Whitten's medium and procaine showed the best results for the induction of capacitation and hyperactivation in stallion spermatozoa; this was possible even after short-term incubation.

Lack of Tyrosylprotein Sulfotransferase-2 Activity Results in Altered Sperm-Egg Interactions and Loss of ADAM3 and ADAM6 in Epididymal Sperm

Tyrosine O-sulfation is a post-translational modification catalyzed by two tyrosylprotein sulfotransferases (TPST-1 and TPST-2) in the trans-Golgi network. Tpst2-deficient mice have male infertility, sperm motility defects, and possible abnormalities in sperm-egg membrane interactions. Studies here show that compared with wild-type sperm, fewer Tpst2-null sperm bind to the egg membrane, but more of these bound sperm progress to membrane fusion. Similar outcomes were observed with wild-type sperm treated with the anti-sulfotyrosine antibody PSG2. The increased extent of sperm-egg fusion is not due to a failure of Tpst2-null sperm to trigger establishment of the egg membrane block to polyspermy. Anti-sulfotyrosine staining of sperm showed localization similar to that of IZUMO1, a sperm protein that is essential for gamete fusion, but we detected little to no tyrosine sulfation of IZUMO1 and found that IZUMO1 expression and localization were normal in Tpst2-null sperm. Turning to a discovery-driven approach, we used mass spectrometry to characterize sperm proteins that associated with PSG2. This identified ADAM6, a member of the A disintegrin and A metalloprotease (ADAM) family; members of this protein family are associated with multiple sperm functions. Subsequent studies revealed that Tpst2-null sperm lack ADAM6 and ADAM3. Loss of ADAM3 is strongly associated with male infertility and is observed in knockouts of male germ line-specific endoplasmic reticulum-resident chaperones, raising the possibility that TPST-2 may function in quality control in the secretory pathway. These data suggest that TPST-2-mediated tyrosine O-sulfation participates in regulating the sperm surface proteome or membrane order, ultimately affecting male fertility.

Is Biallelic Expression of the Imprinted Gene H19 in Preimplantation Mouse Embryos the Result of a Molecular Clock Rather than Embryo Manipulation?

H19 is an imprinted non-coding RNA known to be transcribed from the maternally inherited allele. Genomic imprinting is defined as parent-specific gene expression. Previous research has shown maternal-specific expression of H19 in mouse blastocysts developed in vivo and biallelic expression in embryos cultured in vitro. In those studies embryos were collected at a particular stage of embryonic development (i.e. late blastocyst stage) despite the time disparity for embryos in different treatment groups to reach the desired developmental stage. For this study, we determined H19's allelic expression in embryos produced after ovarian hyperstimulation at specific times (84 hours, 96 hours, or 108 hours) following presumed ovulation, as opposed to specific stages of development. In addition, we determined the effects of two culture conditions (KSOM augmented with amino acids or Whitten's medium) on imprinted H19 expression in embryos cultured from the 2-cell stage, collected at 32 h post presumed ovulation, until 84, 96, or 108 h post-ovulation. RNA was collected and cDNA was synthesized from single embryos. Results showed that approximately 25% of embryos in all groups expressed biallelic H19 at 84 and 96 hours post-ovulation. Surprisingly, 50-77% of blastocysts expressed H19 from the normally silent paternal allele in all three treatment groups (i.e. in vivo produced, KSOM and Whitten's) at 108 hours post-ovulation. From these results we concluded that biallelic H19 expression is a normal event around the time of implantation in mouse embryos. However, recent data from our laboratory shows that superovulation can affect the level of global DNA methylation in growing oocytes. Therefore, we set out to determine if the high level of biallelic H19 expression was really the result of a developmental cue or simply an adverse outcome of the superovulation scheme. For the second study, we followed the same design as that described for embryos from superovulated females but this time the embryo donors did not receive hormonal stimulation. Briefly, females were observed for the presence of a copulatory plug to establish approximate time of ovulation. Embryos were either collected at the 2-cell stage and cultured as above or flushed from the uterus at 84 hours, 96 hours, or 108 hours. As in the previous experiment, cultured embryos were collected at equivalent times post ovulation. Again, RNA was collected and cDNA was synthesized from single embryos. Following PCR, products were restricted with an enzyme unable to cut the paternal allele allowing us to differentiate expression of each parental allele. Preliminary results show a similar pattern of biallelic expression in embryos produced after natural ovulation as those produced after superovulation. We suggest that during the peri-implantation stage, biallelic expression of H19 may be attributed to a molecular clock and not to be the result of embryo culture as has been previously concluded. (poster)

Effect of ICSI on gene expression and development of mouse preimplantation embryos

In vitro culture (IVC) and IVF of preimplantation mouse embryos are associated with changes in gene expression. It is however not known whether ICSI has additional effects on the transcriptome of mouse blastocysts. We compared gene expression and development of mouse blastocysts produced by ICSI and cultured in Whitten's medium (ICSI(WM)) or KSOM medium with amino acids (ICSI(KSOMaa)) with control blastocysts flushed out of the uterus on post coital Day 3.5 (in vivo). In addition, we compared gene expression in embryos generated by IVF or ICSI using WM. Global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. Blastocysts from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared with embryos generated in vivo. Approximately 1000 genes are differentially expressed between ICSI blastocyst and in vivo blastocysts; proliferation, apoptosis and morphogenetic pathways are the most common pathways altered after IVC. Unexpectedly, expression of only 41 genes was significantly different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOM(aa)). Our results suggest that fertilization by ICSI may play a more important role in shaping the transcriptome of the developing mouse embryo than the culture media used.

Side-by-Side Comparison of Five Commercial Media Systems in a Mouse Model: Suboptimal In Vitro Culture Interferes with Imprint Maintenance1

Assisted reproductive technologies (ARTs) are becoming increasingly prevalent and are generally considered to be safe medical procedures. However, evidence indicates that embryo culture may adversely affect the developmental potential and overall health of the embryo. One of the least studied but most important areas in this regard is the effects of embryo culture on epigenetic phenomena, and on genomic imprinting in particular, because assisted reproduction has been linked to development of the human imprinting disorders Angelman and Beckwith-Wiedemann syndromes. In this study, we performed side-by-side comparisons of five commercial embryo culture systems (KSOMaa, Global, Human Tubal Fluid, Preimplantation 1/Multiblast, and G1v5PLUS/G2v5PLUS) in relation to a best-case (in vivo-derived embryos) and a worst-case (Whitten culture) scenario. Imprinted DNA methylation and expression were examined at three well-studied loci, H19, Peg3, and Snrpn, in mouse embryos cultured from the 2-cell to the blastocyst stage. We show that embryo culture in all commercial media systems resulted in imprinted methylation loss compared to in vivo-derived embryos, although some media systems were able to maintain imprinted methylation levels more similar to those of in vivo-derived embryos in comparison to embryos cultured in Whitten medium. However, all media systems exhibited loss of imprinted H19 expression comparable to that using Whitten medium. Combined treatment of superovulation and embryo culture resulted in increased perturbation of genomic imprinting, above that from culture alone, indicating that multiple ART procedures further disrupt genomic imprinting. These results suggest that time in culture and number of ART procedures should be minimized to ensure fidelity of genomic imprinting during preimplantation development.