Prolonged storage of epididymal spermatozoa does not affect their capacity to fertilise in vitro-matured domestic cat (Felis catus) oocytes when using ICSI.

The impact of different storage conditions of epididymal spermatozoa (including prolonged storage, cryopreservation and freeze-drying) on their fertilisation capacity was tested using intracytoplasmic sperm injection (ICSI). This kind of information is urgently needed when applying assisted reproductive technology to endangered felids in zoos. In particular, the utilisation of epididymal spermatozoa of castrated or deceased felids often requires time-consuming transportation and is therefore susceptible to loss of gamete quality. Sperm cells were stored at 4 °C for up to 72 h followed by cryopreservation or freeze-drying. Thawed motile and immotile spermatozoa were used for ICSI and the embryo cleavage rate was assessed 36 h after injection. A significant impact on the fertilisation rate of oocytes could only be detected when using immotile thawed or rehydrated spermatozoa. Cryopreservation or storage at 4 °C showed no influence. The simulation of transport conditions using domestic cat spermatozoa revealed that in vitro production of felid embryos with gametes from euthanised individuals is possible if testes are stored cool and arrive at the laboratory within 72 h. An essential prerequisite is the application of ICSI to achieve fertilisation even with single motile spermatozoa. Additional cryopreservation of spermatozoa after transportation is possible and will allow the establishment of a sperm bank for felids.

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

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 α 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.

Functional role of feline zona pellucida protein 4 trefoil domain: a sperm receptor or structural component of the domestic cat zona pellucida?

The trefoil domain (TFD) is a protein structure characterized by six cysteines, which form a typical three-loop structure by three disulphide bridges. It is assumed that two of these loops generate a hydrophobic groove, which could be a binding site for carbohydrate residues or proteins. The zona pellucida (ZP) protein, ZP4, contains such a TFD. The carbohydrate-/protein-binding property of TFD allows us to assume a potential sperm receptor function of this domain. Additionally, gastrointestinal trefoil peptides are stable against proteases; therefore, a structural role of TFD within the ZP might also be possible. We were able to show that the synthesized and natural folded feline TFD (fTFD) expresses the typical protease resistance that vanished under reducing conditions and after substitution of cysteine residues within the peptide. Furthermore, an antibody directed against the first loop of fTFD was almost unable to bind to intact in vitro mature cat oocytes. Pre-incubation of oocytes in the reducing agent (DDT), however, improved antibody binding substantially. Therefore, we suggest structural masking of the fTFD domain within the intact ZP. An interaction between fTFD and feline sperm cells was examined using several methods, including immunocytochemistry, immunoelectron microscopy, co-immunoprecipitation and far western blot, but we found no indication for an involvement of TFD in the primary sperm binding to the ZP. To summarize, there is increasing evidence that the TFD of fZP4 has a structural rather than a sperm-binding function.