Studies on Methods and Influencing Factors of Porcine Oocyte Activation

The effects of ionomycin, electrical pulses, dithiothreitol(DTT), 6-DMAP and thimersal, used either alone or in combinations, on the activation rate, pronuclear type and cleavage of porcine oocyte were studied in this paper. The results are as follows:(1)The activation rates of oocytes treated with ionomycin(88.9%)or pulsed(80.0%)alone did not differ significantly from each other and from those in oocytes treated in combination with 6-DMAP(84. 3 and 79. 1%, respectively). While 6-DMAP improved electrical activation(increased the proportion of oocytes with 1 PN), it had no effect on oocyte activation by ionomycin;(2)Post-treatment with DTT significantly enhanced the thimersal activation of porcine oocytes with activation rates increased from 4.6 to 82.6%;(3)When pulsed, the activation rates of oocytes with crimped(85.4%)or fragmented first polar bodies(86. 2%)were significantly higher than those of oocytes with intact polar bodies(58. 8%); the ratio of 1 PN activated oocytes(78. 8%)was significantly higher, but that of 2 PN oocytes(18. 2%)was significantly lower in oocytes with intact polar bodies;(4)The cleavage rate of oocytes activated by electrical stimulus(52.4%)was significantly higher than that in oocytes activated by ionomycin(23.0%)when combined with 6-DMAP, but it was not significantly different from that in oocytes activated by electrical stimulus alone(46.1%).

Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm

Background： In order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine oocytes and sperm selected by swim up（SU） or swim up ＋ zona pellucida（SU ＋ ZP） binding.Results： Experiment 1, 4–20 replicates with total 821 in vitro matured oocytes were chemically activated with ethanol, calcium ionophore or ionomycin, to determine oocyte activation（precense of one PN）. Treatments showed similar results（54, 47, 42 %, respectively） but statistically differents（P 0.05）.Conclusions： Chemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU ＋ ZP increased AR/A and AR/D rates in comparison with SU in fresh and frozen-thawed sperm. According to this, in terms of fertilization rates, chemical activation after ICSI increased oocyte PN formation compared to mechanical activation. Also, fresh sperm treated with SU and SU ＋ ZP were significantly different than frozen-thawed sperm,but between sperm treatments no significant differences were obtained.

Spontaneous activation of hamster oocytes in vitro

Objectives To observe the time of spontaneous activation of hamster oocytes and to find out the early signs of hamster oocytes activation Methods First, 63 hamster oocytes including activated and non activated oocytes (group A) and 51 non activated oocytes (group B) were injected with human sperm and checked for fertilization after 16-18 hours Then, 20 hamster oocytes were cultured in vitro in medium with or without calcium and examined under light microscope every half an hour up to 9 hours Results Under light microscope, hamster oocytes activation were observed to undergo 4 stages: metaphase Ⅱ stage without meiotic spindle, appearance of meiotic spindle near the first polar body, protrusion of oolemma, and complete extrusion of second polar body with pronucleus formation After culture for 1 5 hours in vitro, 30% 40% of the oocytes demonstrated meiotic spindle; but after 9 hours, 50% showed complete activation Culture in medium without calcium showed a little delay in the process of activation The earliest sign of spontaneous activation in hamster oocytes was the appearance of meiotic spindle near the first polar body Although group A and B showed no difference, the damage rate was lower in group B (19 7%±16 6%) than in group A (35 9%±5 7%) ( P =0 08), whereas the fertilization rate (18 3%±10 9%) was higher in group B than in group A (14 2%±8 7%) ( P =0 135) Conclusions Hamster oocytes may be activated spontaneously early after their removal It is important to recognize the earliest sign of spontaneous activation and perform intracytoplasmic sperm injection (ICSI) before it happens

No genetic alterations in infants from intracytoplasmic sperm injection in combination with artificial oocyte activation： a pilot study

The combination of intracytoplasmic sperm injection （ICSI） with artificial oocyte activation has overcome repeated fertilization failure after ICSI due to sperm defects in activating oocyte, resulting in pregnancies and births for many couples. However, in comparison to the growing data illustrating the effectiveness of artificial oocyte activation in reproductive medicine, the safety of this approach has not been well studied. Previously,

Phospholipase C-Zeta Reveals the Underlying Pathological Influence after Density Gradient Centrifugation, Microfluidic Sorting, and Cryopreservation of Human Sperm

Objective:Sperm preparation techniques and cryopreservation are widely used in assisted reproductive techniques(ART).How to improve the quality of sperm management is a matter of great concern.Phospholipase C-zeta(PLCζ)is considered a sperm-specific agent that activates oocyte activation and thus playing a crucial role in male fertility.However,the potential mechanisms by which semen processing and cryopreservation on PLCζcontribute to keyhole have not been addressed.Methods:In this study,semen samples were taken from have not been addressed 10 normozoospermic men.Each semen sample was assigned to the following groups:density gradient centrifugation(DGC)as control,microfluidic sorting,and cryopreservation.Sperm parameters of molity,viability,membrane integrity,and intracellular ROS were evaluated during sperm preparation and cryopreservation.The expression of PLCζin human sperm was determined by immunofluorescence and western blotting.Results:The results showed that molity,viability,and membrane integrity decreased in cryopreservation group.Intracellular ROS were also significantly increased compared to the the control group.There was no significant difference between DGC and microfluidic sorting group.Our investigation revealed that total levels of PLCζwere comparable between DGC and microfluidic sorting,but there were significantly reduced levels of PLCζafter cryopreservation as quantified by both immunofluorescenceand immunoblotting.PLCζimmunofluorescence in sperm revealed different PLCζlocalization patterns around the acrosomal(Ac),equatorial(Eq),post-acrosomal(PA)areas of sperm heads,and their combination.The predominant patterns of PLCζlocalization in DGC were similar to that of microfluidic sorting,with strong,with staining.In contrast,PLCζstaining in freeze-thawed sperm was considerably weaker fluorescence intensity.Conclusion:This study clarified the mechanism of sperm preparation and cryopreservation underlying effect on sperm characteristic,accompanied with PLCζexpresion.We demonstrated that microfluidic sorting provides a highly efficient preparation method for clinical selection of PLCζ-expressing sperm comparable to DGC gene expression.It is suggested that the cryopreservation of sperm has a significant detrimental effect on PLCζ.

A live birth of activated one-day-old unfertilized oocyte for a patient who experienced repeatedly near-total fertilization failure after intracytoplasmic sperm injection

Total or near-total fertilization failure after intracytoplasmic sperm injection （ICSl） is a rare event, but it occurs repeatedly because of sperm defects in activating oocyte. The case presents a successful pregnancy and live birth after calcium ionophore A23187 （A23187） activation on one-day-old unfertilized oocytes in a patient whose husband suffered oligoasthenoteratozoospermia, and who had experienced repeated near-total fertilization failure after ICSI. In the second ICSI cycle, only one oocyte was fertilized while nine were unfertilized. Oocyte activation with A23187 were performed on the one-day-old unfertilized oocytes after ICSI and resulted in fertilization and embryo transfer. A clinical pregnancy was achieved and a healthy baby was born. To our knowledge, this is the first reported case of a healthy birth after oocyte activation on the one-day-old unfertilized oocyte. This indicates that ＂rescue oocyte activation＂ on one-day-old unfertilized oocytes after ICSI may be helpful for preventing total or near-total fertilization failure after ICSI.

Negative biomarker-based male fertility evaluation： sperm phenotypes associated with molecular-level anomalies

Biomarker-based sperm analysis elevates the treatment of human infertility and ameliorates reproductive performance in livestock. The negative biomarker-based approach focuses on proteins and ligands unique to defective spermatozoa, regardless of their morphological phenotype, lending itself to analysis by flow cytometry （FC）. A prime example is the spermatid specific thioredoxin SPTRX3/TXNDC8, retained in the nuclear vacuoles and superfluous cytoplasm of defective human spermatozoa. Infertile couples with high semen SPTRX3 are less likely to conceive by assisted reproductive therapies （ART） and more prone to recurrent miscarriage while low SPTRX3 has been associated with multiple ART births. Ubiquitin, a small, proteolysis-promoting covalent posttranslational protein modifier is found on the surface of defective posttesticular spermatozoa and in the damaged protein aggregates, the aggresomes of spermiogenic origin. Semen ubiquitin content correlates negatively with fertility and conventional semen parameters, and with sperm binding of lectins LCA （Lens culinaris agglutinin; reveals altered sperm surface） and PNA （Arachis hypogaealpeanut agglutinin; reveals acrosomal malformation or damage）. The Postacrosomal Sheath WWl Domain Binding Protein （PAWP）, implicated in oocyte activation during fertilization, is ectopic or absent from defective human and animal spermatozoa. Consequently, FC-parameters of PAWP correlate with ART outcomes in infertile couples and with fertility in bulls. Assays based on the above biomarkers have been combined into multiplex FC semen screening protocols, and the surface expression of lectins and ubiquitin has been utilized to develop nanoparticle-based bull semen purification method validated by field artificial insemination trials. These advances go hand-in-hand with the innovation of FC-technology and genomics/proteomics-based biomarker discovery.

Advancing male age differentially alters levels and localization patterns of PLCzeta in sperm and testes from different mouse strains

Sperm-specific phospholipase C zeta(PLCζ)initiates intracellular calcium(Ca2+)transients which drive a series of concurrent events collectively termed oocyte activation.Numerous investigations have linked abrogation and absence/reduction of PLCζwith forms of male infertility in humans where oocyte activation fails.However,very few studies have examined potential relationships between PLCζand advancing male age,both of which are increasingly considered to be major effectors of male fertility.Initial efforts in humans may be hindered by inherent PLCζvariability within the human population,alongside a lack of sufficient controllable repeats.Herein,utilizing immunoblotting,immunofluorescence,and quantitative reverse transcription PCR(qRT-PCR)we examined for the first time PLCζprotein levels and localization patterns in sperm,and PLCζmRNA levels within testes,from mice at 8 weeks,12 weeks,24 weeks,and 36 weeks of age,from two separate strains of mice,C57BL/6(B6;inbred)and CD1(outbred).Collectively,advancing male age generally diminished levels and variability of PLCζprotein and mRNA in sperm and testes,respectively,when both strains were examined.Furthermore,advancing male age altered the predominant pattern of PLCζlocalization in mouse sperm,with younger mice exhibiting predominantly post-acrosomal,and older mice exhibiting both post-acrosomal and acrosomal populations of PLCζ.However,the specific pattern of such decline in levels of protein and mRNA was strain-specific.Collectively,our results demonstrate a negative relationship between advancing male age and PLCζlevels and localization patterns,indicating that aging male mice from different strains may serve as useful models to investigate PLCζin cases of male infertility and subfertility in humans.