To investigate the possibility of DHLA-induced cytotoxicity, we treated mouse blastocysts with 25, 50 or 100 μM DHLA at 37 °C for 24 h, and monitored apoptosis using the TUNEL method. Cellular apoptosis was evident in blastocysts treated with 50 μM DHLA (Figure 1A). Quantitative analysis revealed ~2.5 to 7.5-fold more apoptotic cells in 50–100 μM DHLA-treated blastocysts, compared with untreated control cells (Figure 1B). Clearly, DHLA induces apoptosis in mouse blastocysts within the 50–100 μM concentration range.

Differential staining, followed by cell counting, was used to assess cell proliferation in blastocysts either treated with 25, 50 or 100 μM DHLA for 24 h or left untreated. We observed significantly lower cell numbers in 50 μM DHLA-treated blastocysts, compared with control cells (Figure 2A). Annexin V staining revealed markedly higher numbers of Annexin V-positive/PI-negative (apoptotic) cells in the ICM of treated blastocysts versus controls, but no such differences in the trophectoderm (TE) (Figure 2B). Our experiments show that 50–100 μM DHLA induces significant apoptosis in the ICM, but not TE, of mouse blastocysts, further supporting the theory that DHLA impairs the developmental potential of blastocysts.

Untreated control morulae displayed 80% development to blastocysts, whereas only 52% of those treated with 50 μM DHLA developed into blastocysts under our experimental conditions (Figure 3A). To further determine the effects of DHLA on post-implantation events in vitro, we treated blastocysts with or without 25, 50 or 100 μM DHLA, and analyzed subsequent development for 8 days in culture. Importantly, the rate of embryo attachment to fibronectin-coated culture dishes and lack of further development (attachment only group) has no any effects by treatment with DHLA (Figure 3B). However, DHLA-pretreated blastocysts displayed a lower incidence of post-implantation developmental milestones (Figure 3B). Our results clearly indicate that DHLA affects the in vitro potential of blastocysts to develop into post-implantation embryos.

To determine the effects of DHLA on blastocyst development in vivo, we transferred control and DHLA-pretreated mouse blastocysts, and examined the uterine content at 13 days post-transfer (day 18 post-coitus). The implantation ratios in the 50–100 μM DHLA-pretreated groups were significantly lower than that of the untreated control group (Figure 4A). Embryos that implanted but failed to develop were subsequently resorbed. However, the proportion of implanted embryos that failed to develop normally was markedly higher in the group treated with 50–100 μM DHLA (Figure 4A). Interestingly, no significant differences in placental weight were observed between the DHLA-treated and untreated groups (Figure 4B). However, fetal weight was lower in the 100 μM DHLA-treated group compared to the control group (479 ± 61 mg versus 611 ± 68 mg, respectively). Previous studies, including a recent report by our group, showed that 35–40% of fetuses weigh more than 600 mg, and the average weight of total surviving fetuses is about 600 ± 12 mg in the untreated control group at day 18 of pregnancy in a mouse embryo transfer assay [23,27–30]. Fetal weight is an important indicator of developmental status, and the average fetal weight of the untreated control group is used as a key marker of development of blastocysts treated with 100 μM DHLA. Interestingly, only 5.8% of the fetuses in the 100 μM DHLA-pretreated group weighed more than 600 mg (indicative of successful embryonic and fetal development), whereas 43% of control fetuses exceeded this threshold (Figure 4C). These observations collectively indicate that exposure to high concentrations of DHLA (such as 100 μM) at the blastocyst stage reduces embryo implantation and the potential for post-implantation development.

Next, we examined the effects of DHLA on blastocyst development in an animal model. Female mice were fed a standard diet and drinking water supplemented with or without DHLA (25, 50 or 100 μM). DHLA consumption led to significant apoptosis and decreased cell proliferation in mouse blastocysts (Figure 5A,B). In addition, DHLA inhibited embryonic development to the blastocyst stage, causing frequent termination at the 2–16 cell or morula stage or degradation (Figure 5C). These results further validate the theory that exposure to high concentrations of DHLA (50–100 μM) through intake is potentially hazardous for mouse embryonic development.

Pregnant mare’s serum gonadotropin (PMSG), Bovine serum albumin (BSA), sodium pyruvate and dihydrolipoic acid were purchased from Sigma (St. Louis, MO, USA). Human chorionic gonadotropin (hCG) was obtained from Serono (NV Organon Oss, the Netherlands). The TUNEL in situ cell death detection kit was obtained from Roche (Mannheim, Germany) and CMRL-1066 medium was from Gibco Life Technologies (Grand Island, NY, USA).

ICR mice were from National Laboratory Animal Center (Taiwan). This research was also approved by the Animal Research Ethics Board of Chung Yuan Christian University (Taiwan). All animals received humane care, as outlined in the Guidelines for Care and Use of Experimental Animals (Canadian Council on Animal Care, Ottawa, 1984). All mice were maintained on breeder chow (Harlan Teklad chow) with food and water available ad libitum. Housing was in standard 28 cm × 16 cm × 11 cm (height) polypropylene cages with wire-grid tops and kept under a 12 h·day/12 h night regimen. Nulliparous females (6–8 weeks old) were superovulated by injection of 5 IU PMSG followed 48 h later by injection of 5 IU hCG, and then mated overnight with a single fertile male of the same strain. The day a vaginal plug was found was defined as day 0 of gestation. Plug-positive females were separated for experimentation. Morulas were obtained by flushing the uterine tubes on the afternoon of gestation day 3, and blastocysts were obtained by flushing the uterine horn on day 4; in both cases the flushing solution consisted of CMRL-1066 culture medium containing 1 mM glutamine and 1 mM sodium pyruvate. The concentration of glucose in this medium was 5 mM. Expanded blastocysts from different females were pooled and randomly selected for experiments.

Blastocysts were incubated in medium containing the indicated concentrations of DHLA for 24 h. For apoptosis detection, embryos were washed in DHLA-free medium, fixed, permeabilized and subjected to TUNEL labeling using an in situ cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s protocol. Photographic images were taken under bright field illumination using a fluorescence microscope.

Blastocysts were incubated with or without culture medium containing 0, 25, 50 or 100 μM DHLA. After 24 h they were washed with DHLA-free medium and dual differential staining was used to facilitate counting of cell numbers in the inner cell mass (ICM) and trophectoderm (TE) [37]. Blastocysts were incubated in 0.4% pronase in M₂-BSA medium (M₂ medium containing 0.1% bovine serum albumin) for removal of the zona pellucida. The denuded blastocysts were exposed to 1 mM trinitrobenzenesulphonic acid (TNBS) in BSA-free M₂ medium containing 0.1% polyvinylpyrrolidone (PVP) at 4 °C for 30 min, and then washed with M₂ medium [40]. The blastocysts were further treated with 30 μg/mL anti-dinitrophenol-BSA complex antibody in M₂-BSA at 37 °C for 30 min, and then with M₂ medium supplemented with 10% whole guinea-pig serum as a source of complement, along with 20 μg/mL bisbenzimide and 10 μg/mL propidium iodide (PI), at 37 °C for 30 min. The immunolysed blastocysts were gently transferred to slides and protected from light before observation. Under UV light excitation, the ICM cells (which take up bisbenzimidine but exclude PI) appeared blue, whereas the TE cells (which take up both fluorochromes) appeared orange-red. Since multinucleated cells are not common in preimplantation embryos [41], the number of nuclei was considered to represent an accurate measure of the cell number.

Blastocysts were incubated in 25, 50 or 100 μM DHLA for 24 h, washed with DHLA-free culture medium, and then stained using an Annexin V-FLUOS staining kit (Roche), according to the manufacturer's instructions. Briefly, the blastocysts were incubated in M₂-BSA for removal of the zona pellucida, washed with PBS plus 0.3% BSA, and then incubated for 60 min with a mixture of 100 μL binding buffer, 1 μL fluorescein isothiocyanate (FITC)-conjugated Annexin V and 1 μL PI. After incubation, the embryos were washed and photographed using a fluorescence microscope under fluorescent illumination. Cells staining Annexin V+/PI− were considered apoptotic, while those staining Annexin V+/PI+ were considered necrotic.

Blastocysts were cultured according to a modification of the previously reported method [42]. Briefly, embryos were cultured in 4-well multidishes at 37 °C. For group culture, four embryos were cultured per well. The basic medium consisted of CMRL-1066 supplemented with 1 mM glutamine and 1 mM sodium pyruvate plus 50 IU/mL penicillin and 50 mg/mL streptomycin (hereafter called culture medium). For treatments, the embryos were cultured with the indicated concentrations of DHLA for 24 h in serum-free medium. Thereafter, the embryos were cultured for 3 days in culture medium supplemented with 20% fetal calf serum, and for 4 days in culture medium supplemented with 20% heated-inactivated human placental cord serum, for a total culture time of 8 days from the onset of treatment. Embryos were inspected daily under a phase-contrast dissecting microscope, and developmental stages were classified according to established methods [43,44]. Under these culture conditions, each hatched blastocyst attached to the fibronectin and grew to form a cluster of ICM cells over the trophoblastic layer via a process called TE outgrowth. After a total incubation period of 96 h, morphological scores for outgrowth were estimated. Growing embryos were classified as either “attached” or “outgrowth”, with the latter defined by the presence of a cluster of ICM cells over the trophoblastic layer. As described previously [45], ICM clusters were scored according to shape, ranging from compact and rounded ICM (+++) to a few scattered cells (+) over the trophoblastic layer.

To examine the ability of expanded blastocysts to implant and develop in vivo, the generated embryos were transferred to recipient mice. ICR females (white skin color) were mated with vasectomized males (C57BL/6J; black skin color; from National Laboratory Animal Center, Taiwan, ROC) to produce pseudopregnant dams as recipients for embryo transfer. To ensure that all fetuses in the pseudopregnant mice came from embryo transfer (white color) and not from fertilization by C57BL/6J (black color), we examined the skin color of the fetuses at day 18 post-coitus. To assess the impact of DHLA on postimplantation growth in vivo, blastocysts were exposed to 0, 25, 50 and 100 μM DHLA for 24 h, and then 8 embryos were transferred in parallel to the paired uterine horns of day 4 pseudopregnant mice. The surrogate mice were killed on day 18 post-coitus, and the frequency of implantation was calculated as the number of implantation sites per number of embryos transferred. The incidence rates of resorbed and surviving fetuses were calculated as the number of resorptions or surviving fetuses, respectively, per number of implantations. The weights of the surviving fetuses and placentae were measured immediately after dissection.

The data were analyzed using one-way ANOVA and t-tests and are presented as the mean ± SEM, with significance at P < 0.05.