No abstract

Oocytes and embryos are typically exposed sequentially to varying culture media in standard in-vitro protocols. Expenditures of energy may be required following each medium change to adjust to the changing environment. Therefore, a single base medium was evaluated for its ability to support in-vitro maturation, fertilization and pre-implantation development (IVM/F/C) of bovine oocytes and embryos. Four treatments were examined: a standard maturation [tissue culture medium (TCM) 199 with bovine calf serum (BCS)], fertilization (modified Tyrode's medium with albumin, lactate and pyruvate) and culture (hamster embryo culture medium/TCM with BCS) system (control) and three synthetic oviductal fluid (SOF) treatments; maturation in SOF with bovine serum albumin (SOFBSA), SOF with bovine calf serum (SOFBCS) or the control maturation medium (TCM199 with BCS; SOF199), followed by fertilization and culture in SOF medium. The percentage of total inseminated oocytes successfully developing to the morula and blastocyst stage did not differ (P > 0. 05) between treatments (control, 30.5 +/- 3.5; SOFBSA, 24.6 +/- 3.2; SOFBCS, 22.4 +/- 4.7; SOF199, 27.3 +/- 3.2). Embryos cultured in SOFBCS (92.1 +/- 6.4) had significantly higher cell numbers (P < 0. 05) than those cultured in control (74.8 +/- 4.8) and SOFBSA (71.6 +/- 6.6) but not SOF199 (81.2 +/- 6.8). In conclusion, a single medium can be used successfully throughout maturation, fertilization and pre-implantation embryo development. Moreover, inclusion of serum during maturation in the single medium system resulted in significantly greater cell numbers, possibly reflecting increased quality of the embryos produced.

No abstract

Embryo metabolism is an indicator of viability and, therefore, efficiency of the culture medium. Currently, little is known regarding porcine embryo metabolism. The objective of our study was to evaluate glucose and pyruvate uptake and lactate production in porcine embryos cultured in two different media systems. Oocytes were matured and fertilized according to standard protocols. Embryos were allocated randomly into two culture treatments, NCSU23 medium or G1.2/G2.2 sequential culture media 6–8 h post‐insemination (hpi). Embryo substrate utilization was measured at the two‐cell (24–30 hpi), 8‐cell (80 hpi), morula (120 hpi), and blastocyst (144 hpi) stages using ultramicrofluorimetry. Glucose uptake was higher (P < 0.05) in two‐cell embryos cultured in G1.2 than in NCSU23 medium (4.54 ± 0.71, 2.16 ± 0.87 pmol/embryo/h, respectively). Embryos cultured in G1.2/G2.2 produced significantly more lactate than those in NCSU23 at the eight‐cell stage (9.41 ± 0.71, 4.42 ± 0.95 pmol/embryo/hr, respectively) as well as the morula stage (11.03 ± 2.31, 6.29 ± 0.77 pmol/embryo/hr, respectively). Pyruvate uptake was higher (P < 0.05) in morula cultured in G1.2/G2.2 versus NCSU23 (22.59 ± 3.92, 11.29 ± 1.57 pmol/embryo/h, respectively). Lactate production was greater (P < 0.05) in blastocysts cultured in G1.2/G2.2 (38.13 ± 15.94 pmol/embryo/h) than blastocysts cultured in NCSU23 (8.46 ± 2.38 pmol/embryo/h). Pyruvate uptake was also greater in blastocysts cultured in G1.2/G2.2 (24.3 ± 11.04) than those in NCSU23 (11.30 ± 2.70). When cultured in NCSU23 medium, two‐ and eight‐cell embryos utilized less glucose than morulae and blastocysts, and two‐cell embryos produced less lactate than blastocysts (P < 0.05). In G1.2/G2.2 media, two‐cells took up less pyruvate than morulae or blastocysts, while blastocysts produced more lactate and utilized more glucose than two‐cell, eight‐cell and morula stage embryos (P < 0.05). As in other species, glycolysis appears to be the primary metabolic pathway in post‐compaction stage porcine embryos. Culture medium composition affects not only substrate uptake, but also metabolic pathways by which these substrates are utilized in porcine embryos at several developmental stages. Mol. Reprod. Dev. 58:269–275, 2001. © 2001 Wiley‐Liss, Inc.

High-Grade Serous Carcinoma (HGSC) is the most common and lethal ovarian cancer subtype. PARP-inhibitors (PARPi) have become the mainstay of HGSC targeted therapy, given that these tumors are driven by a high degree of genomic instability and Homologous Recombination (HR) defects. Nonetheless, ~30% of patients initially respond to treatment, ultimately relapsing with resistant disease. Thus, despite recent advances in drug development and an increased understanding of genetic alterations driving HGSC progression, mortality has not declined, highlighting the need for novel therapies. Using a Small Molecule Activator of Protein Phosphatase 2A (PP2A) (SMAP-061), we investigated the mechanism by which PP2A stabilization induces apoptosis in Patient-Derived HGSC cells and Xenograft (PDX) models alone or in combination with PARPi. We uncovered that PP2A genes essential for cellular transformation (B56,B56 and PR72) and basal phosphatase activity (PP2A-A and -C) are heterozygously lost in the majority of HGSC. Moreover, loss of these PP2A genes correlate with worse overall patient survival. We show that SMAP-061 stabilization of PP2A inhibits the homologous recombination (HR) output by targeting RAD51, leading to chronic accumulation of DNA damage and ultimately apoptosis. Furthermore, combination of SMAP-061 and PARPi leads to enhanced apoptosis in both HR-proficient and HR-deficient cells and in PDX models. Our studies identifies PP2A as a novel regulator of HR and indicates PP2A modulators as a therapeutic therapy for HGSC. In sum, our findings further emphasize the potential of PP2A modulators to overcome PARPi insensitivity, given that targeting RAD51 presents benefits in overcoming PARPi-resistance driven by BRCA1/2 mutation reversions.