Parthenogenetic activation of bovine oocytes using bovine and murine phospholipase C zeta

Background  

During natural fertilization, sperm fusion with the oocyte induces long lasting intracellular calcium oscillations which in turn are responsible for oocyte activation. PLCZ1 has been identified as the factor that the sperm delivers into the egg to induce such a response. We tested the hypothesis that PLCZ1 cRNA injection can be used to activate bovine oocytes.     

Conservation of MAP kinase activity and MSP genes in parthenogenetic nematodes

Background  

MAP (mitogen-activated protein) kinase activation is a prerequisite for oocyte maturation, ovulation and fertilisation in many animals. In the hermaphroditic nematode Caenorhabditis elegans, an MSP (major sperm protein) dependent pathway is utilised for MAP kinase activation and successive oocyte maturation with extracellular MSP released from sperm acting as activator. How oocyte-to-embryo transition is triggered in parthenogenetic nematode species that lack sperm, is not known.     

Slow oocyte freezing and thawing in couples with no sperm or an insufficient number of sperm on the day of <Emphasis Type="Italic">in vitro</Emphasis>fertilization

Background  

The clinical results of in vitro fertilization of slowly frozen-thawed oocytes are known to be significantly worse than those obtained by fresh oocytes. Little is known about the factors affecting the clinical outcome of frozen-thawed oocytes. The aim of this retrospective study was to explore the role of oocyte cryopreservation in the group of patients with no available sperm on the day of in vitro fertilization. Additionally, the effects of the female serum FSH level and sperm quality on the clinical outcome of frozen-thawed oocytes were evaluated.     

Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection

The objective was to determine the effects of various methods of oocyte activation and sperm pretreatment on development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection (ICSI). The second polar body was extruded in the majority (>78.4%) of in vitro-matured (IVM) oocytes 4h after electrical pulse activation. In embryos generated by ICSI and sham-ICSI, a combination of an electrical pulse, with various chemical activators 4 h later, improved (P < 0.05) blastocyst formation rate compared to activation only with a pulse. Treatment with 6-dimethylaminopurine (DMAP) after electrical activation significantly increased the oocyte activation rate. The effects of exposure of sperm to repeated freeze-thaw cycles (without cryoprotectant) on oocyte activation and the effects of sperm pre-incubated with dithiothreitol (DTT) or Triton X-100 on early embryo development were also examined. Blastocyst formation rates after ICSI did not differ between motile sperm and those rendered immotile by one-time freezing and thawing without cryoprotectant. However, sperm rendered immotile by three cycles of freezing/thawing without cryoprotectant had a significantly lower blastocyst formation rate. Although oocytes injected with sperm pre-incubated with Triton X-100 had a higher normal fertilization rate than those pre-incubated with DTT or one-time frozen/thawed sperm, rates of blastocyst formation and cell numbers were similar among the three groups. In conclusion, various methods of oocyte activation and sperm preparation significantly affected the developmental capacity of early porcine embryos derived from IVM and ICSI.     

Expression of the zebrafish intermediate neurofilament Nestin in the developing nervous system and in neural proliferation zones at postembryonic stages

Background

At fertilisation, mammalian oocytes are activated by oscillations of intracellular Ca²⁺ ([Ca²⁺]_i). Phospholipase Cζ, which is introduced by fertilising spermatozoon, triggers [Ca²⁺]_i oscillations through the generation of inositol 1,4,5-triphosphate (IP₃), which causes Ca²⁺ release by binding to IP₃ receptors located on the endoplasmic reticulum (ER) of the oocyte. Ability to respond to this activating stimulus develops during meiotic maturation of the oocyte. Here we examine how the development of this ability is perturbed when a single spermatozoon is introduced into the oocyte prematurely, i.e. during oocyte maturation.

Results

Mouse oocytes during maturation in vitro were fertilised by ICSI (intracytoplasmic sperm injection) 1 – 4 h after germinal vesicle break-down (GVBD) and were subsequently cultured until they reached metaphase II (MII) stage. At MII stage they were fertilised in vitro for the second time (refertilisation). We observed that refertilised oocytes underwent activation with similar frequency as control oocytes, which also went through maturation in vitro, but were fertilised only once at MII stage (87% and 93%, respectively). Refertilised MII oocytes were able to develop [Ca²⁺]_i oscillations in response to penetration by spermatozoa. We found however, that they generated a lower number of transients than control oocytes. We also showed that the oocytes, which were fertilised during maturation had a similar level of MPF activity as control oocytes, which were not subjected to ICSI during maturation, but had reduced level of IP₃ receptors.

Conclusion

Mouse oocytes, which were experimentally fertilised during maturation retain the ability to generate repetitive [Ca²⁺]_i transients, and to be activated after completion of maturation.     

Oocyte diameter in relation to meiotic competence and sperm penetration

This study was conducted to determine the diameter of canine oocytes that are able to attain full meiotic competence and sperm penetration. Oocytes were collected from ovaries of bitches at various stages of the estrous cycle. Only healthy-looking cumulus-oocyte complexes were used for in vitro maturation, and were divided into four groups based on diameter: <100, 100 to <110, 110 to <120 and >120 microm. Following in vitro maturation or fertilization, oocytes were stained to assess nuclear maturation and penetration rates. The mean oocyte diameter was 108.5 +/- 0.4 microm. The oocytes displayed size-related ability to undergo meiotic maturation. After culture for 72 h, the rates of oocytes that remained at the germinal vesicle stage in the <110 microm groups were significantly higher (P<0.01) than in the > or = 110 microm groups. None of the oocytes <110 microm reached metaphase II (MU), but 4.9 and 21.5% of the oocytes that were greater than 110 and 120 microm, respectively, progressed to MII. After in vitro fertilization for 20 h, 10 to 25% of oocytes were penetrated by spermatozoa, but there were no clear relationships between oocyte diameter and penetration rates of the oocyte by sperm. In the <120 microm groups, sperm penetration was mostly found in oocytes arrested at the germinal vesicle stage. However, a total of eight oocytes > or = 120 microm in diameter were penetrated by spermatozoa, of which five oocytes reached MII. These results suggest that there is a clear relationship between oocyte diameter and meiotic competence, but no relationship between oocyte diameter and sperm penetration. Canine oocytes may have acquired meiotic competence once they reach at a diameter of 120 microm, but the oocytes may allow the entry of spermatozoa into the ooplasm irrespective of oocyte diameter.     

Effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection rabbit embryos

This study assessed the effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection (ICSI) rabbit embryos. Oocytes were recovered from female rabbits superovulated with PMSG and hCG, and epididymal sperm were collected from a fertile male rabbit. The oocyte was positioned with the first polar body at 12 o'clock position, and a microinjection needle containing a sperm was inserted into the oocyte at 3 o'clock. Oolemma breakage was achieved by aspirating ooplasm, and the aspirated ooplasm and sperm were re-injected into the oocyte. The injected oocytes were cultured in M199 medium containing 10% fetal calf serum at 38 degrees C with 5% CO2 in air. The results showed that oocytes injected at 1 h post-collection produced a higher (p < 0.05) fertilization rate than those injected at 4 or 7 h post-collection. Blastocyst rate in the 1 h group was higher (p < 0.05) than in the 7 h group. Denuded oocytes (group A) and oocytes with cumulus cells (group B) were injected, respectively. Rates of fertilization and development of ICSI embryos were not significantly different (p > 0.05) between the two groups. Four ICSI methods were applied in this experiment. In methods 1 and 2, the needle tip was pushed across half the diameter of the oocyte, and oolemma breakage was achieved by either a single aspiration (method 1) or repeated aspiration and expulsion (method 2) of ooplasm. In methods 3 and 4, the needle tip was pushed to the oocyte periphery opposite the puncture site, and oolemma breakage was achieved by either a single aspiration (method 3) or repeated aspiration and expulsion (method 4) of ooplasm. Fertilization rate in method 2 was significantly higher (p < 0.05) than in methods 1 and 3. Blastocyst rates were not significantly different (p > 0.05) among methods 1, 3 and 4, but method 2 produced a higher (p < 0.05) blastocyst rate than method 3.     

Brief coincubation of gametes in porcine in vitro fertilization: role of sperm:oocyte ratio and post-coincubation medium

In this study, a short coincubation time of 10 min was used to determine the effect of different sperm:oocyte ratios during in vitro fertilization (IVF), and different periods of post-coincubation in a medium that is not appropriate for IVF, on fertilization parameters. In the first experiment, a total of 1624 in vitro matured oocytes, from 4 replicates, were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000, 1500, 1000 and 500 sperm:oocyte) and coincubated for 10 min or 6 h. The oocytes from 10 min of coincubation were washed in IVF medium to remove spermatozoa not bound to the zona pellucida and transferred to another droplet of the same medium (containing no spermatozoa) for 6h. The oocytes from the other group remained with the spermatozoa for 6h. Oocytes from both groups were then cultured in embryo culture medium (IVC) for 12h to assess fertilization parameters. In the second experiment, 1872 in vitro matured oocytes, in 3 replicates were inseminated with frozen-thawed spermatozoa using the same sperm:oocyte ratios as in the first experiment. The oocytes were coincubated for 10 min and transferred directly to IVC medium for 18 h (group A), to IVF medium (containing no sperm) only for 2h and then to IVC medium for 16 h (group B), or to IVF medium (containing no sperm) for 6h and then to IVC medium for 12 h (group C or control). There was an effect of sperm:oocyte ratio on all fertilization parameters in experiment 1. The efficiency of IVF (number of monospermic oocytes/total number inseminated) was higher (P<0.05) for oocytes coincubated with spermatozoa for 10 min and inseminated with 1500 and 1000 sperm:oocyte (35.8+/-3 and 37.6+/-2.7%, respectively) and for those coincubated for 6h with 500 spermatozoa per oocyte (37.2+/-3.1%). In experiment 2, the penetration and efficiency rates obtained in group A were poor (between 3 and 15%) irrespective of the sperm:oocyte ratio. However, in group B the fertilization parameters were similar to the controls and were also affected by the sperm:oocyte ratio. These results demonstrate that coincubation time may be reduced to 10 min to increase the efficiency of fertilization depending on the sperm:oocyte ratio, and that the spermatozoa bound to the zona pellucida require a maximum of 2h in an appropriate medium to penetrate the oocytes.     

Mitochondrial distribution and microtubule organization in fertilized and cloned porcine embryos: implications for developmental potential

Mitochondrial distribution and microtubule organization were examined in porcine oocytes after parthenogenesis, fertilization and somatic cell nuclear transfer (SCNT). Our results revealed that mitochondria are translocated from the oocyte's cortex to the perinuclear area by microtubules that either constitute the sperm aster in in vitro-fertilized (IVF) oocytes or originate from the donor cell centrosomes in SCNT oocytes. The ability to translocate mitochondria to the perinuclear area was lower in SCNT oocytes than in IVF oocytes. Sperm-induced activation rather than electrical activation of SCNT oocytes as well as the presence of the oocyte spindle enhanced perinuclear mitochondrial association with reconstructed nuclei, while removal of the oocyte spindle prior to sperm penetration decreased mitochondrial association with male pronuclei without having an apparent effect on microtubules. We conclude that factors derived from spermatozoa and oocyte spindles may affect the ability of zygotic microtubules to translocate mitochondria after IVF and SCNT in porcine oocytes. Mitochondrial association with pronuclei was positively related with embryo development after IVF. The reduced mitochondrial association with nuclei in SCNT oocytes may be one of the reasons for the low cloning efficiency which could be corrected by adding yet to be identified, sperm-derived factors that are normally present during physiological fertilization.     

In vitro spontaneous parthenogenetic activation of golden hamster oocytes

Parthenogenetic activation is a major hurdle to be cleared for the examination of the human sperm chromosome after intracytoplasmic injection (ICSI) into golden hamster oocytes. Various factors that affect spontaneous activation of hamster oocytes were, therefore, investigated in this study. We collected cumulus-oocyte complexes (COC) from the oviducts of superovulated females and washed them thoroughly with Ca2+-containing or Ca2+-free TALP-HEPES medium (handling media). We cultured oocytes with intact cumulus or those without cumulus (removed by previous hyaluronidase treatment) in Ca2+-containing or -free m-TALP-3 for 6 or 12 h before examining for their activation. Among the oocytes recovered 17 h post-hCG, 92-94% were parthenogenetically activated by 6 h of in vitro culture. Activation rate in the oocytes collected at 13.5 h post-hCG (53%) was significantly (P < 0.05) lower than that in the oocytes collected 17 h post-hCG (92%), indicating that the spontaneous activation rate increased as the oocytes became older. Both cumulus-intact and cumulus-free oocytes had similar (P > 0.05) activation rates when cultured in vitro, suggesting that hyaluronidase treatment had no effect on the rate of oocyte activation. Omission of Ca2+ from the handling medium also had no effect on the activation of the oocytes. The rate of spontaneous activation of the oocytes cultured in calcium-free medium for 6 (9%) and 12 h (16%) was significantly (P < 0.01) lower than that (94%) of the control oocytes cultured in Ca2+-containing medium, implying a positive influence of Ca2+ on in vitro activation of hamster oocytes. When we cultured the oocytes first in calcium-free medium for 6 h, and then in calcium-containing medium for 6 h, 94% were activated, which is comparable to the rate for oocytes continuously cultured in Ca2+-containing medium. This indicates that the inhibition of hamster oocyte activation in Ca2+-free medium is reversible and can be used to control spontaneous activation of golden hamster oocytes.     

Activation of bovine oocytes matured in vitro by injection of bovine and human spermatozoa or their cytosolic fractions

The aim of this study was to investigate whether bovine spermatozoa possess so-called sperm factor in the cytosolic fraction (CF) which activates bovine oocytes, and whether bovine oocytes matured in vitro are activated by microinjection of CF extracted from spermatozoa of other species. In the first experiment, bovine and human spermatozoa were microinjected into ooplasm of bovine oocytes matured in vitro. Secondly, CF from bovine and human spermatozoa were injected into bovine oocytes. In the third, CF from human spermatozoa was injected into human unfertilised oocytes obtained 18-20 h after clinical intracytoplasmic sperm injection (ICSI). We found that microinjection of bovine spermatozoa into bovine oocytes induced oocyte activation, as shown by resumption of meiosis and formation of a female pronucleus, at a significantly higher rate than the bovine sham injection (63.0% vs 43.0%; p < 0.05). On the other hand, there was no significant difference in activation rate between the human sperm injection (35.9%) and the human sham injection (22.9%). Furthermore, microinjection of bovine sperm CF into bovine oocytes induced oocyte activation at a significantly higher rate than the human CF injection or sham injection (75.9% vs 14.8%, 20.4%; p < 0.01). Formation of a single female pronucleus and second polar body extrusion was observed in 95.1% of activated oocytes after bovine sperm CF injection. When human sperm CF was injected into human unfertilised oocytes, the activation rate was significantly higher than following sham injection (76.9% vs 44.0%; p < 0.05). These results indicate the presence of sperm factor in bovine sperm CF which activate bovine oocytes, and suggest the possibility that sperm factor has species-specificity at least between bovine and human.     

Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs

Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca²⁺ concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca²⁺ concentration contained in the injection medium. In Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3–66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation.     

Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs

Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca²⁺ concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca²⁺ concentration contained in the injection medium. In Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3-66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation.     

Leptin accelerates pronuclear formation following intracytoplasmic sperm injection of porcine oocytes: Possible role for MAP kinase inactivation

Leptin, a multifunctional hormone, is present in mammalian oocytes and follicular fluids and cumulus cells. While leptin modulates oocyte maturation in vitro which seems to result in enhancement of embryo development, it is unclear whether leptin treatment of oocytes affects cytoplasmic maturation and fertilization processes. In order to gain a better understanding of the role of leptin during oocyte maturation, we examined microtubule and microfilament assembly following oocyte maturation and blastocyst formation, mitogen-activated protein kinase (MAPK) activity, and pronuclear formation following parthenogenetic stimuli or intracytoplasmic sperm injection (ICSI) in leptin-treated oocytes. Addition of 10 or 100 ng/ml leptin during oocyte maturation did not increase the proportion of metaphase II oocytes, but enhanced development to blastocyst stage by day 7 (P < 0.01) after parthenogenetic activation (PA), accompanied by increased cell number. However there was no effect on the number of apoptotic cells in blastocysts. Following maturation in the presence of leptin, there were more oocytes with normal spindle formation. MAPK activity decreased more rapidly, and pronuclear formation was accelerated after parthenogenetic activation or ICSI of leptin-treated oocytes. These results suggested that exogeneous leptin enhanced spindle assembly and accelerated pronuclear formation following fertilization, possibly via the MAPK pathway.     

Arachidonic and Linoleic Acid Derivatives Impact Oocyte ICSI Fertilization – A Prospective Analysis of Follicular Fluid and a Matched Oocyte in a ‘One Follicle – One Retrieved Oocyte – One Resulting Embryo’ Investigational Setting

Objective

To evaluate human oocyte ability to undergo fertilization and subsequent preimplantation embryonic development in relation to a wide panel of follicular fluid (FF) arachidonic acid derivatives (AAD) and linoleic acid derivatives (LAD) of prospectively selected patients undergoing intracytoplasmic sperm injection (ICSI).

Methodology

Study was designed as a two center (a university clinic and a private clinic) prospective study. 54 women of 181 consecutive couples undergoing ICSI were prospectively found to be eligible for analysis. ''One follicle – one retrieved oocyte – one resulting embryo'' approach was used. Each individual follicle was aspirated independently and matched to an oocyte growing in this particular follicular milieu. FF samples were assessed for AAD and LAD by high-performance liquid chromatography; additionally, activity of secretory phospholipase A (sPLA₂) was determined by enzyme-linked immunosorbent assay.

Principal Findings

Increased activity of sPLA₂ and significantly higher AAD and LAD levels were found in FF of oocytes that did not show two pronuclei or underwent degeneration after ICSI in comparison to oocytes with the appearance of two pronuclei. Receiver operating characteristics curve analysis identified acids with the highest sensitivity and specificity: 5oxo-hydroxyeicosatetraenoic, 16-hydroxyeicosatetraenoic, 9-hydroxyoctadecadieneoic and 12-hydroxyeicosatetraenoic. No significant differences between AAD and LAD related to embryo quality were found.

Conclusions/Significance

Our study demonstrates for the first time that elevated concentrations of AAD and LAD in FF at the time of oocyte retrieval significantly decrease the ability of oocytes to form pronuclei after ICSI. This may serve as a new tool for non-invasive assessment of oocyte developmental capacity. However, levels of AAD and LAD are not associated with subsequent embryo quality or pregnancy rate, and therefore more studies are needed to determine their usefulness in human IVF procedure.     

Behavior of sperm nuclei in intact and bisected metaphase II mouse oocytes fertilized in the presence of colcemid

Our objective was to examine the developmental fate of sperm nuclei in oocytes fertilized under conditions of meiotic arrest. Therefore zona-free metaphase II oocytes and oocyte fragments (nucleate and anucleate) were fertilized in the presence of colcemid. In anucleate oocyte fragments, normal male pronuclei develop. In contrast, in intact oocytes and nucleate fragments sperm nuclei after initial decondensation undergo secondary condensation. This state is maintained as long as the oocytes are treated with colcemid. When the drug is removed 3 h after insemination, the meiotic spindle(s) is reconstructed, the second polar body(ies) is extruded, and a female pronucleus (or micronuclei) forms. At the same time the sperm nucleus decondenses again and transforms into a male pronucleus. In addition oocytes fertilized in the presence of colcemid could not be refertilized. These observations suggest that oocytes and oocyte fragments fertilized in the presence of colcemid undergo activation despite the failure of pronucleus formation. The inhibitory effect of colcemid on the formation of pronuclei is expressed only in the presence of oocyte chromosomes. We suggest that colcemid stabilizes factors responsible for chromosome condensation that are associated with oocyte chromosomes but not factors (whether the same or different) present in the cytoplasm.     

Intracytoplasmic sperm injection in bovine: effects of oocyte activation, sperm pretreatment and injection technique

Intracytoplasmic sperm injection (ICSI) is a very important technique for treating male subfertility and for basic research. The efficiency of ICSI in bovine is very limited because of the necessity for additional oocyte activation before or after the ICSI procedure. In this study, we compared the effects of seven different protocols on activation and fertilization rates of bovine oocytes after ICSI and on their subsequent development under in vitro conditions. The protocols include 1) different chemical activation of oocytes, 2) pretreated or nonpretreated sperm, and 3) conventional or Piezo-driven injection techniques. In all three groups, ICSI, sham-injected, and noninjected, the highest activation rates were obtained after treatment of oocytes with ionomycin followed by 6-dimethylaminopurine (6-DMAP). Using this treatment for oocyte activation, 59% of oocytes were activated and 31% of oocytes were fertilized using dithiothreitol (DTT) pretreated spermatozoa and Piezo-driven injection. Using the protocols with the same oocyte activation or activation with calcium ionophore (Ca-I) and cycloheximide (CHX), nonpretreated sperm, and conventional injection technique, early cleavage rate (79.6% and 77.6%, respectively) were significantly (P <0.01) higher when compared with all other protocols. The latter protocol resulted in 8% blastocyst and 90% of the obtained blastocysts were found to be diploid. Our results demonstrate that activation of oocytes, sperm treatment, and injection technique separately or together could improve the success of bovine ICSI.     

Sperm chromatin acquires an activity that induces microtubule assembly during residence in the cytoplasm of metaphase oocytes of the mouse

Evidence from several cell types indicates that chromatin can induce microtubule assembly in its vicinity. To determine whether this activity is present in sperm chromatin, whose biochemical composition differs from somatic chromatin, mouse oocytes that were undergoing meiotic maturation were inseminated. Maturing oocytes are not activated by sperm penetration but remain arrested at metaphase. The sperm chromatin within the oocyte cytoplasm initially became dispersed and later, under the influence of oocyte cytoplasmic factors, recondensed into a small mass of individual chromosomes. When inseminated oocytes were processed for immunofluorescence using an anti--tubulin antibody, microtubules were never associated with dispersed sperm chromatin, although the chromosomes of the oocyte were arranged on a spindle. In contrast, microtubules were associated with the majority of sperm nuclei that had become recondensed, and were frequently arranged into a spindle-like structure. When oocytes had been penetrated by more than three sperm, most sperm nuclei remained at the dispersed chromatin stage and these were never associated with microtubules. Exposure of polyspermic oocytes to taxol, which promotes microtubule assembly, failed to induce microtubule assembly around dispersed sperm chromatin. Exposure of monospermic oocytes to nocodazole, which inhibits tubulin polymerization, prevented resolution of the recondensed sperm chromatin into individual chromosomes. These results suggest that sperm chromatin lacks an activity that can induce local microtubule assembly, and that it acquires this activity once modified by oocyte cytoplasmic factors.     

Improved fertilization and embryo development resulting in birth of live piglets after intracytoplasmic sperm injection and in vitro culture in a cysteine-supplemented medium

The effects of cysteine treatment on fertilization rate, intracellular concentration of glutathione, and embryo development in vitro and after embryo transfer were examined following intracytoplasmic sperm injection (ICSI) of in vitro-matured porcine oocytes using a piezo drive unit. Culture of presumed zygotes after ICSI with 1.71-3.71 mM cysteine for 3-12h improved (P<0.05) fertilization rates as compared to treatment with 0.57 mM cysteine or to controls (0mM) (56 to 68%, 48%, 35%, respectively). Extension of treatment time with cysteine beyond 3h did not further increase fertilization rates, suggesting that cysteine promoted early developmental events after ICSI (e.g. decondensation of sperm chromatin). There was no effect of cysteine supplementation on oocyte glutathione levels after ICSI. Pretreatment of spermatozoa for 3h with 1.71 mM cysteine did not improve fertilization rates. The incidence of blastocysts formation when cultured in 1.71 mM cysteine for 3h after ICSI was 31%, which was higher (P<0.05) than controls (18%). Transfer of 20-38 embryos cultured with 1.71 mM cysteine for 3h after ICSI to each of seven recipients yielded three deliveries with an average litter size of 4.0. We concluded that cysteine supplementation for the first 3h after ICSI improved fertilization and embryo development rates, with no influence on glutathione levels in oocytes, and that the cysteine-treated ICSI embryos developed to full term. The study also showed that porcine oocytes matured in a chemically defined medium had the ability for full-term development after piezo-ICSI without additional treatments for oocyte activation.     

The BSA-induced Ca(2+) influx during sperm capacitation is CATSPER channel-dependent

Background  

Serum albumin is a key component in mammalian sperm capacitation, a functional maturation process by which sperm become competent to fertilize oocytes. Capacitation is accompanied by several cellular and molecular changes including an increased tyrosine phosphorylation of sperm proteins and a development of hyperactivated sperm motility. Both of these processes require extracellular calcium, but how calcium enters sperm during capacitation is not well understood.