Embryonic gene activation in in vitro produced embryos of the domestic cat (Felis catus)

Abstract

Accurate embryonic gene activation (EGA) is essential for the embryo's developmental potency and reflects the quality of in vitro produced embryos. To describe the dynamic and temporal patterns of EGA in the cat, the mRNA expression of developmentally important genes (DNA methyltransferases 1 and 3A, DNMT1 and DNMT3A; gap junction protein a 1, GJA1; transcription factor octamer 4, POU5F1 (OCT4); insulin-like growth factor (IGF) 1 and 2 receptors, IGF1R and IGF2R) was examined by RT-PCR techniques in preimplantation embryos obtained after in vitro maturation and IVF. Furthermore, influences of ICSI and sperm cryopreservation on the relative mRNA abundance in 4-5-days-old morulae were analyzed. Total RNA was obtained from immature and matured oocytes, 2-cell embryos, 4-cell embryos, and 8-16-cell embryos, morulae, and blastocysts. RNA was transcribed into single-stranded cDNA by reverse transcriptase. After amplification, a nonfelid standard RNA was used for semiquantitative analysis. Our results showed an increase in transcript abundance from the matured oocyte to the 2-cell embryo for all examined genes except for IGF2R, indicating that, in vitro, the embryonic genome is activated shortly after fertilization. However, the activation pattern varied markedly between the different genes. We also found different patterns of mRNA expression for the examined genes in morulae produced either by IVF or ICSI, and using fresh or cryopreserved sperm. Owing to high variations within the single groups of compared morulae, we were able to observe only a tendency toward higher relative mRNA expression in embryos derived by IVF with fresh sperm in comparison to all other groups.

In vitro fertilizing potential of urethral and epididymal spermatozoa collected from domestic cats (Felis catus)

“…Freezing extender contained Tris buffer supplemented with 20% (v/v) egg yolk, 6% glycerol (v/v) and 1% Equex STM paste (v/v) (Nova Chemical Sales Inc., Scituate, MA, USA). Washing medium (WM) used for collection and rinsing of oocytes consisted of Medium 199 containing Earle's salts, supplemented with 3 mg/ml BSA, 0.1 mg/ml cysteine, 1.4 mg/ml HEPES, 0.25 mg/ml sodium pyruvate, 0.6 mg/ml sodium lactate, 0.15 mg/ml L-glutamine, and 0.055 mg/ml gentamicin (Waurich 2010). Maturation medium (MM) for in vitro maturation of oocytes consisted of WM supplemented with 0.02 IU/ml of FSH and 0.02 IU/ml LH.…”

“…For each replicate (n=10), cryopreserved CT and EP samples collected from the same male were thawed by immersion in a 37 o C water bath for 30 s. Each sample was centrifuged at 620 x g for 5 min, supernatant was discarded and the pellet were re-sus- pended in HTF+SSS. The concentration of motile spermatozoa was adjusted to 4 x 10 6 /ml (Waurich et al 2010). …”

“…COCs possessing dark, uniform cytoplasm and surrounded by at least several layers of cumulus cells were subjected to in vitro maturation (IVM) in MM at 38.5 o C in 5% CO 2 , for 24 h (Waurich et al 2010). The in vitro matured oocytes were washed twice in WM, randomly divided into two groups (9-11 oocytes per group) and transferred to the separate wells containing 300 μl of HTF+SSS.…”

“…A hundred microliters of CT was added to one well and 100 μl of EP to the second. Co-incubation of gametes lasted for 18h at 38.5 o C in 5% CO 2 (Waurich et al 2010). Next, the presumptive zygotes were washed 2 times in WM and moved to the well containing 400 μl of CSC+SSS.…”

“…For in vitro fertilization in cats several studies were conducted, with the use of different types of semen, miscellaneous media and various culture conditions (Freistedt et al 2001, Herric et al 2007, Waurich et al 2010, Thuwanut et al 2015. The results of these studies show a large variation and it is hard to compare fertilizing potential of spermatozoa obtained by different methods.…”