Types of oocytes from guinea pigs and their meiotic progression during IVM.

The arrow indicates the first polar body of the matured oocytes, indicating the oocytes have entered the MII phase. (A) Matured oocytes stained with Cell Truck Bule CMF2HC under the microscope after treatment with ROS for 12 hours. (B) Immature oocytes stained with Cell Truck Bule CMF2HC under the microscope after treatment with ROS for 12 hours. (C) After the removal of IMBX, the fluorescence intensity of matured oocytes treated with IBMX was significantly stronger than that treated with ROS. (D) The fluorescence intensity of GSH in MII oocytes cultured in the maturation medium was relatively weaker, and the first polar body was not obvious as well, compared with that cultured in the maturation medium with ROS. (E) The fluorescence intensity of GSH in MII oocytes cultured in MTA medium supplemented with L-cysteine and cystine was stronger than that of GSH in MII oocytes cultured in basic medium. (F) The fluorescence intensity of GSH cultured in the basic medium was much lower than other groups.</p

The microtubule morphology of MII oocytes from guinea pigs was divided into two types.

For MII oocytes with normal spindles and aggregated chromosomes (A, B and C in the top panel), the chromosomes were distributed and aggregated on the equatorial plate, while for MII oocytes with abnormal spindles and dispersed chromosomes (D, E and F in the bottom panel), the chromosomes could not be neatly distributed on the equatorial plate and are in a diffusion state. A and D are for Hoechst 33342 staining, B and E are for β-tubulin staining, and C and F are merged for A and B, and D and E, respectively. Bar in 50 μm.</p

S1 Dataset -

Guinea pigs are a valuable animal model for studying various diseases, including reproductive diseases. However, techniques for generating embryos via embryo engineering in guinea pigs are limited; for instance, in vitro maturation (IVM) technique is preliminary for guinea pig oocytes. In this study, we aimed to establish and optimize an IVM method for guinea pig oocytes by investigating various factors, such as superovulation induced by different hormones, culture supplementation (e.g., amino acids, hormone, and inhibitors), culture conditions (e.g., oocyte type, culture medium type, and treatment time), and in vivo hCG stimulation. We found that oocytes collected from guinea pigs with superovulation induced by hMG have a higher IVM rate compared to those collected from natural cycling individuals. Moreover, we found that addition of L-cysteine, cystine, and ROS in the culture medium can increase the IVM rate. In addition, we demonstrated that in vivo stimulation with hCG for 3–8 h can further increase the IVM rate. As a result, the overall IVM rate of guinea pig oocytes under our optimized conditions can reach ~69%, and the mature oocytes have high GSH levels and normal morphology. In summary, we established an effective IVM method for guinea pig oocytes by optimizing various factors and conditions, which provides a basis for embryo engineering using guinea pigs as a model.</div

Effects of different hormones on superovulation and on the IVM rate of oocytes from these superovulated guinea pigs.

Effects of different hormones on superovulation and on the IVM rate of oocytes from these superovulated guinea pigs.</p

Effects of different doses of hCG on the IVM rate of oocytes from superovulated guinea pigs.

Effects of different doses of hCG on the IVM rate of oocytes from superovulated guinea pigs.</p

Effects of different culture media on intracellular levels of GSH and morphology of microtubules in MII oocytes.

Effects of different culture media on intracellular levels of GSH and morphology of microtubules in MII oocytes.</p

Effects of different amino acids and inhibitors on the IVM rate of guinea pig oocytes.

Effects of different amino acids and inhibitors on the IVM rate of guinea pig oocytes.</p

Effects of culture duration on the IVM rate of oocytes from superovulated guinea pigs.

Effects of culture duration on the IVM rate of oocytes from superovulated guinea pigs.</p

Effects of PMSG, hCG, FSH, and LH in the culture medium on the IVM rate of oocytes from natural cycling guinea pigs.

Effects of PMSG, hCG, FSH, and LH in the culture medium on the IVM rate of oocytes from natural cycling guinea pigs.</p

Types of guinea pig oocytes and their meiotic progression during IVM.

(A,B,C) Compact COCs collected from the ovaries of guinea pigs. Type A oocytes have three or more layers of cumulus cells (A). Type B oocytes have 1–3 layers of cumulus cells (B). Type C oocytes have few or no cumulus cells (i.e., naked oocytes) (C). (D) Oocytes at the MII stage (matured oocytes) induced from Type A oocytes showing the first polar body (arrow). Scale bar = 200 μm in (A); 100 μm in (B, C); 50 μm in (D).</p