TABLE 2.
Significant results and outcome on effect of UCHs during in vitro maturation and fertilization of mouse oocytes
| Treatment | Replicates | N-oocyte treated | Treatment group | Control group | Main outcome & conclusion | |
|---|---|---|---|---|---|---|
| A | L3i during IVM, then IVF without L3i | 2 | 176 | Fertilization rate in L3i: 56.2 ± 17.6% | Fert. Rate in control: 85.5 ± 9.5 (P = 0.28) | Oocyte matured in the presence of L3i have somewhat reduced fertilizing ability; Conclusion: Inhibition of UCHL3 activity during maturation reduces fertilizing ability of the oocyte. |
| B | L3i during IVF | 3 | 486 | Fertilization rate in L3i: 52.2 ± 3.1% | Fertilization rate in control: 94.7 ± 2.7 P = 0.0005 | Presence of L3i during IVF reduces fertilization rate. Spindle and pronuclear abnormalities were observed in the non-fertilized and fertilized oocyte, respectively. Conclusion: Inhibition of UCHL3 activity during fertilization reduces fertilization rate and causes aberrant pronuclear development. |
| C | UBAL injection in GV oocyte, IVM & IVF | 2 | 164 | Fertilization rate 45.4 ± 14.8% | Fertilization rate 84.8 ± 1.95% (P = 0.12) | Fertilization rate reduced to 54 % of control. Conclusion: Ooplasmic UCHs sensitive to UBAL play a role in fertilization. |
| D | UBAL injection in GV oocyte, IVM & ZF-IVF | 2 | 169 | Fertilization rate: 6.6 ± 0.6% | Fert. rate in PBS- injected control: 82.2 ± 0.6% (P = 0.0001) | Near complete fertilization block in UBAL-injected oocyte (fert. rate in UBAL was 8 % of that in control). Conclusion: The effect of UBAL on fertilization is not due to its effect on ZP. |
| E | UBAL injection in MII-oocyte & IVF | 4 | 301 | Fertilization rate 7.9 ± 5.8% | Fertilization rate 69.9 ± 23.4% (P = 0.04) | Near complete block of fertilization; no sperm accumulation in periviteline space. Conclusion: Ooplasmic UCHs sensitive to UBAL play a role in fertilization. |
| F | UBAL injection in MII-oocyte & ZF-IVF | 3 | 660 | Fertilization rate 47.6 ± 20.7% | Fertilization rate PBS-injected control: 79.2 ± 10.2% (P = 0.56) | Some but not a significant reduction in fertilization rate for UBAL. Conclusion: The effect of UBAL is not immediate; perhaps there is not enough time for injected UBAL to act on its substrates when fertilization is accelerated by ZP removal. |
| G | UBAL injection in MII-oocyte & ICSI | 5 | 733 | Fertilization rate (% 2PN) of UBAL-ICSI oocyte: 80.2 ± 5.3; Fertilization rate of UBAL-IVF oocyte: 1.7 ± 1.7 (P < 0.0001) | Fertilization rate of control ICSI oocyte: 79.8 ± 6.7 | Normal fertilization and PN-development are observed after ICSI of UBAL-injected oocyte; no fertilization after IVF in the same batches of UBAL-injected oocyte. Conclusion: UBAL injection does now harm the oocytes. |
| H | UBAL injection in MII-oocyte & parthenogentic activation | 3 | 412 | All groups including UBAL and PBS injected and non-injected parthenotes, and the IVF control had near 100% fertilization/PN development rates (range 92.1 ± 7.9% or 100%). | Conclusion: UBAL des not affect oocyte viability assessed by their ability to undergo parthenogenetic activation, so UBAL has likely a more specific effect. | |
| I & J | IVF (ZP+ and ZP− free) with UBAL in IVF medium | 4 | 639 | Fert. Rate UBAL-ZP+ IVF: 81.9±7.5%; Fert Rate UBAL ZF-IVF: 60.1±12.8% | Fert rate control ZP+ IVF: 89.5± 5.6% (P=0.47); Fert. Rate control ZF-IVF 84.3 ± 10.3 % (P=0.19) | Addition of UBAL in IVF medium has no significant effect on fertilization rate, regardless of the presence/absence of ZP, though a somewhat reduced fertilization is observed in ZF-IVF of UBAL oocyte. |
| K & K’ | L1-AB injection in GV/MII oocyte & ZF-IV | 4 | 761 | Fertilization rates reduced in oocyte injected at GV stage (P = 0.1) but not in those injected at MII (P = 0.41) | Accelerating fertilization by zona removal reduces the inhibitory effect of AB injected at MII stage, but the inhibition is substantial when AB is injected at GV stage prior to IVM and ZF-IVF. | |
| L | L1-AB injection in MII & IVF | 4 | 544 | Fertilization rate 5.8 ± 5.5% in L1AB injected oocyte (P= 0.0009 between L1AB and NRS) | Fertilization rate 74.8 ± 10.0% in NRS control and 88.0 ± 8.3% in no-injection control. | Fertilization rate in L1AB group reduced to7.8 % of NRS-injected control. Conclusion: UCHL1 is essential for fertilization, may affect sperm-oolemma fusion, sperm incorporation or chemoattractant production by the oocyte |
| TOTAL | 33 | 4559 | N/A | N/A | ||
ABBREVIATIONS
GV-germinal vesicle oocyte
GVBD-germinal vesicle breakdown
IVF-in vitro fertilization
IVM-in vitro maturation
MII-metaphase II-oocyte
L1AB-mouse antibody against UCHL1 (Abcam, #ab20559,)
L3i-inhibitor of UCHL3 (Calbiochem #662069)
L3AB-rabbit antibody against UCHL3 (MBL #A8724)
UBAL-ubiquitin aldehyde
ZP-zona pellucida
ZF-IVF-zona-free IVF (ZP removed before fertilization)
2PN-two pronuclei (male=female) present in an ovum are consistent with normal fertilization by IVF or ICI (one sperm tail must be present in ooplasm in IVF 2PN oocyte for them to be considered fertilized; sperm tail is separated from sperm head prior to murine ICSI)
NRS- non-immune rabbit sera