Zona-intact Oocytes Research Articles

Lysophosphatidylcholine treatment allows interspecies fertilization of hamster oocytes with zona pellucida by human spermatozoa.

The study of interspecific gamete interactions has been improved by the use of in vitro fertilization techniques. Zona pellucida is at the cellular level the main barrier to cross-fertilization. Moreover, there are several cases in which the heterologous spermatozoa fail to enter the cytoplasm of zona-free oocytes, emphasizing the notion that another important barrier exists to cross-fertilization at the egg plasma membrane level. We report here a simple method to fertilize hamster oocytes with zona pellucida by human spermatozoa. We used lysophosphatidylcholine (LPC), a fusogenic agent, to treat spermatozoa, spermatozoa and zona-intact oocytes, or zona-intact oocytes alone. This molecule allows the penetration of hamster oocytes zona by human spermatozoa. Our results suggest that LPC modifies the zona pellucida of oocytes and the plasma membrane of both gametes in vitro, favouring sperm penetration through the zona pellucida and gamete fusion.

Presence of granulosa cells during oocyte maturation improved in vitro development of IVM-IVF bovine oocytes collected by ultrasound-guided transvaginal aspiration

Frozen-thawed ejaculated stallion spermatozoa were preincubated for 3 h in BO medium containing 5 mM caffeine and then treated with 0.1 μ M calcium ionophore A23187 for 60 sec. Aliquots of the sperm suspension (final concentration 1–2 × 107/ml) were added to the oocytes which had been matured in vitro for 32 h. In Experiment 1, there were 3 groups of oocytes; cumulus intact, denuded zona-intact, and zona-free. Cumulus cells were removed with 0.5% hyaluronidase and the zona pellucida with 0.1% protease. The oocytes were fixed 20 h after insemination with acetic acid:ethanol (1:3) and stained with 1% orcein. The sperm penetration rate of zona-free oocytes was 83%, whereas the sperm penetration rate was very low (1 to 3%) in the cumulus-enclosed or zona-intact oocytes. In Experiment 2, denuded zona-intact oocytes were placed in PBS supplemented with 10% fetal bovine serum 1 h before the end of in vitro maturation. The zona pellucida was micromanipulated with a metal microblade under × 100 magnification within 20 min of treatment with 0.3 M sucrose. For partial zona dissection, a slit in the zona pellucida was made. For partial zona removal, oocytes were transferred to protein-free PBS to fix the oocytes on the bottom of the Petri-dish and to remove a piece of the zona pellucida. Micromanipulated oocytes were subjected to in vitro fertilization as described above. Zona-intact and zona-free oocytes treated with sucrose solution for 20 min were used as controls. The penetration rates were 4 (257), 12 (758), 52 (3160), and 86% (4451) for zona-intact, partially zona dissected, partially zona removed, and zona-free oocytes, respectively. Proportions of oocytes with monospermic penetration were 100 (22), 57 (47), 58 (1831), and 34% (1544), respectively. In Experiment 3, sperm penetration and male pronucleus formation in the partially zona removed oocytes were examined at 2.5 to 20.0 h of insemination. Sperm penetration started 2.5 h post-insemination (22%, 1149), and increased to 38% (2155) at 5 h, to 46% (2350) at 10 h, and to 56% (2748) at 20 h. The transformation of sperm heads into male pronuclei was first observed 10 h post insemination. These results indicate that assisted fertilization techniques may be a useful tool for achieving fertilization and embryo production in vitro in horses.

Infertile couples with normal counts who require subzonal sperm insertion possess a fertility defect that affects zona pellucida penetration.

The results of subzonal sperm insertion (SUZI) have been retrospectively analysed in a subset of patients with normal sperm counts who were found to require SUZI because of poor or absent fertilization of zona-intact oocytes. This patient group is of particular interest because male factor-related infertility cannot be due to insufficient numbers of spermatozoa reaching the oocytes. Thus, failed fertilization can be attributed to deficiencies in one or more steps in the fertilization process, and SUZI provides a method of distinguishing defects of zona pellucida penetration from gamete fusion. A total of 26 such patients were treated identically to and concurrently with a much larger group of SUZI candidates who typically suffered from oligozoospermia, and fertilization results were compared. Fertilization rates after SUZI were higher in patients with normal counts than in oligozoospermic patients (51 and 26% respectively), indicating that the proportion of spermatozoa capable of fusing with the oocyte is the same or higher in the group with normal counts. In addition, nearly all SUZI procedures led to fertilization (23/26), with two out of three failed fertilizations occurring in cases where two or less oocytes were manipulated, results which further indicate that failed fertilization in these patients is not due to a defect at the level of gamete fusion. These findings suggest that infertility in these patients is based upon the inability of the spermatozoa to reach the oolemma and thus, that their fertility defect resides at the step of zona penetration.

In vitro fertilization rate of horse oocytes with partially removed zonae

Frozen-thawed ejaculated stallion spermatozoa were preincubated for 3 h in BO medium containing 5 mM caffeine and then treated with 0.1 micro M calcium ionophore A23187 for 60 sec. Aliquots of the sperm suspension (final concentration 1-2 x 10(7)/ml) were added to the oocytes which had been matured in vitro for 32 h. In Experiment 1, there were 3 groups of oocytes; cumulus intact, denuded zona-intact, and zona-free. Cumulus cells were removed with 0.5% hyaluronidase and the zona pellucida with 0.1% protease. The oocytes were fixed 20 h after insemination with acetic acid:ethanol (1:3) and stained with 1% orcein. The sperm penetration rate of zona-free oocytes was 83%, whereas the sperm penetration rate was very low (1 to 3%) in the cumulus-enclosed or zona-intact oocytes. In Experiment 2, denuded zona-intact oocytes were placed in PBS supplemented with 10% fetal bovine serum 1 h before the end of in vitro maturation. The zona pellucida was micromanipulated with a metal microblade under x 100 magnification within 20 min of treatment with 0.3 M sucrose. For partial zona dissection, a slit in the zona pellucida was made. For partial zona removal, oocytes were transferred to protein-free PBS to fix the oocytes on the bottom of the Petri-dish and to remove a piece of the zona pellucida. Micromanipulated oocytes were subjected to in vitro fertilization as described above. Zona-intact and zona-free oocytes treated with sucrose solution for 20 min were used as controls. The penetration rates were 4 (2/57), 12 (7/58), 52 (31/60), and 86% (44/51) for zona-intact, partially zona dissected, partially zona removed, and zona-free oocytes, respectively. Proportions of oocytes with monospermic penetration were 100 (2/2), 57 (4/7), 58 (18/31), and 34% (15/44), respectively. In Experiment 3, sperm penetration and male pronucleus formation in the partially zona removed oocytes were examined at 2.5 to 20.0 h of insemination. Sperm penetration started 2.5 h post-insemination (22%, 11/49), and increased to 38% (21/55) at 5 h, to 46% (23/50) at 10 h, and to 56% (27/48) at 20 h. The transformation of sperm heads into male pronuclei was first observed 10 h post insemination. These results indicate that assisted fertilization techniques may be a useful tool for achieving fertilization and embryo production in vitro in horses.

Initial pregnancies after intravaginal transport and partial zona dissection of human ova

We report on 72 couples with previous failure of fertilisation during several trials of conventional in-vitro fertilisation (IVF). In the present study part of the aspirated oocytes was subjected to partial zona dissection (PZD) before insemination, thereby achieving 7 pregnancies during 104 cycles. Normal in-vitro insemination of the remaining zona-intact oocytes was again unsuccessful. Since microinsemination techniques, in contrast to conventional IVF, are not yet being offered by many clinics, we developed the following new concept: hormonal stimulation, sonographic control of follicular growth, aspiration of oocytes, and sperm preparation were performed at the usual place (Munich) whereas PZD and control IVF were accomplished in another laboratory (Ulm). In order to transport the gametes between these two institutes, we modified the previously reported intravaginal culture (IVC) of inseminated oocytes by transferring oocytes and spermatozoa into separate plastic capsules. In our view, this procedure offers a suitable opportunity for infertile couples living too far away from a microinsemination centre to afford long daily journeys or even several overnight stays for superovulation treatment. The fertilisation and pregnancy rates in our study are comparable to those reported in the literature.

Removal of the zona pellucida and parthenogenetic activation affect rates of survival of ultrarapidly frozen mouse oocytes

Survival rates on thawing were assessed for ultrarapidly frozen mouse oocytes following the removal of zonae pellucidae using an acid Tyrode's solution, pronase or a mechanical dissection technique. Significantly higher rates of survival were observed for zona-free oocytes than for zona-intact control oocytes (303/684, 44% v. 130/498, 26%; P < 0.001). The rates of survival observed for pronuclear stage embryos (72-76%) were much greater than those observed for oocytes and were not influenced by zona removal techniques. Parthenogenetic activation of oocytes by exposure to a 7% (v/v) ethanol solution was also shown to increase survival rates of ultrarapidly frozen oocytes (155/185, 84% v. 19/102, 19%) indicating that fusion of sperm to the plasma membrane or formation of a male pronucleus are not directly responsible for the increased survival rates of pronuclear stage embryos compared with oocytes. These data support the hypothesis that increased survival of ultrarapidly frozen pronuclear stage embryos is the result of changes to the plasma membrane and/or to the zona pellucida that occur following fertilization.

The effect of partial zona dissection on the fertilizing capacity of chronically obstructed mouse caput epididymal sperm

Having observed that sperm from the chronically obstructed caput epididymis fertilize poorly in vitro, we investigated the effect of partial zona dissection (PZD) on fertilization in a murine model of unilateral proximal epididymal obstruction. Cleavage rates were compared for zona-intact oocytes and PZD oocytes incubated with sperm from the following epididymal segments: the obstructed caput, the contralateral nonobstructed caput, the contralateral cauda, and a sperm-free preparation to control for parthenogenesis. Unilateral epididymal obstruction resulted in significantly higher sperm counts in the obstructed caput compared to the nonobstructed caput, although there was no difference in motility. Cleavage rates for ova incubated with sperm from the obstructed caput and the nonobstructed caput were uniformly poor and did not differ significantly from those for the sperm-free controls. Cauda sperm fertilized significantly better than all other sperm groups (P < 0.05). Partial zona dissection did not improve cleavage rates in any group. We conclude that sperm from the chronically obstructed caput epididymis, like sperm from the normal caput, are unable to fertilize ova, and PZD does not enhance this poor fertilizing capacity. Furthermore, the finding that PZD does not improve the fertilizing capacity of the presumably mature cauda sperm in our mouse model suggests that any beneficial effect of PZD may be strain-specific.

First pregnancies after intravaginal transport and partial zona dissection of human oocytes

We report on 72 couples with previous failure of fertilization during several trials of conventional IVF. For the present study, a part of the aspirated oocytes was subjected to partial zona dissection before insemination, thereby achieving 7 pregnancies during 104 cycles. Normal in vitro insemination of the remaining zona-intact oocytes was unsuccessful again. Because microinsemination techniques, in contrast to routine IVF, are not yet offered by many clinics, we developed the following new concept: hormonal stimulation, sonographic control of follicular growth, aspiration of oocytes, and sperm preparation were performed at the usual place (Munich), whereas partial zona dissection and control IVF were accomplished in another laboratory (Ulm). To transport the gametes between these two institutes, we modified the previously reported intravaginal culture of inseminated oocytes by transferring oocytes and spermatozoa into separate plastic capsules. In our view, this procedure offers an opportunity for infertile couples living too far away from a microinsemination center to afford long daily journeys or even several overnight stays just for superovulation treatment. The fertilization and PRs in our study are comparable with those reported in the literature.

Zona opening with 308 nm XeCl excimer laser improves fertilization by spermatozoa from long-term vasectomized mice.

Spermatozoa from long-term vasectomized mice have greatly reduced fertilizing ability in vivo and in vitro, which makes this a useful animal model for male factor infertility. The purpose of this study was to evaluate the 308 nm XeCl excimer laser for opening the zona pellucida to enhance the fertilization rate with spermatozoa from vasectomized males. Inseminating zona-intact (control) oocytes with 5 x 10(6) spermatozoa/ml resulted in only 6% fertilization and 33.3% development to the blastocyst stage; zona-opened oocytes showed significant improvement with 31.5% fertilization, 90% cleavage to the 2-cell stage, and 72.2% blastocyst formation. Out of the 130 oocytes in the experimental group, zona ablation was performed successfully on 127 and only three were damaged. These results suggest that laser micromanipulation for assisted fertilization potentially offers a simplified and precise method for mechanical zona cutting.

Competition between zonae pellucidae and a proteinase inhibitor for sperm binding.

Murine sperm bind a proteinase inhibitor of seminal vesicle origin at ejaculation. The inhibitor binds in the acrosomal region of the sperm head and is removed during in utero or in vitro incubation. Adding inhibitor to sperm reduces their ability to bind zonae, while adding the purified inhibitor binding site to cumulus-free, zona-intact oocytes reduces the ability of the oocytes to bind sperm. Immuno-aggregation of the inhibitor binding site results in exocytosis of the acrosome. These observations suggest that the inhibitor binding site may participate in zona binding and the acrosome reaction. If the inhibitor binding site binds both the zona and the seminal inhibitor, then these components should compete with each other for that site on the sperm. We show that purified seminal inhibitor, as well as other proteinase inhibitors, block zona-induced acrosome reactions. Likewise, zona glycopeptides block inhibitor/anti-inhibitor-induced acrosome reactions in a concentration-dependent fashion. The inhibitor/anti-inhibitor-induced acrosome reaction is sensitive to pertussis toxin and proteinase inhibitor and thus is similar to zona-induced reactions. These findings support the suggestion that the trypsin inhibitor binding site on the head of the sperm functions to insure sperm-zona binding and induction of the acrosome reaction.

Fertilization, cleavage, and cytogenetics of 48-hour zona-intact and zona-free human unfertilized oocytes reinseminated with donor sperm

To examine the fertilization rates of 48-hour unfertilized oocytes inseminated with fertile donor sperm and to evaluate the cleavage and cytogenetics of ensuing embryos. Prospective. Assisted reproductive technology (ART) program. Four hundred ninety-seven unfertilized oocytes from 97 ART patients were categorized into four groups. A (zona-intact) and B (zona-free) were from patients with partial fertilization failure, whereas C (zona-intact) and D (zona-free) were total fertilization failures. Fertilization rates in groups A and B were significantly higher than C and D (33.2% to 60.9% versus 20.0% to 48.1%; P less than 0.01). Zona-free oocytes had higher fertilization rates than zona-intact oocytes (48.1% to 60.9% versus 20.0% to 32.2%). Multiple pronuclei were high in zona-free oocytes (33.1% to 41.3%). Forty-eight to 54% of embryos generated after donor insemination had chromosome anomalies (mosaicism, aneuploidy, pulverization). One cause of total fertilization failure appears to lie in intrinsic oocyte problems confined to the zona and oolemma. The fertilization of 48-hour unfertilized oocytes may be of some value in diagnosing fertilization failure in ART patients.

Assisted fertilization of mouse oocytes and preliminary results for human oocytes using zona drilling

The zona-drilling procedure was investigated in mouse oocytes prior to a study on human oocytes. The procedure involved the injection of 5-nl volumes of acidic Hepes-buffered medium at pH 2.5 using a microinjection instrument. Zona-drilled mouse oocytes had significantly higher rates of fertilization (60/99; 61%) than zona-intact oocytes (6/103; 6%) at an insemination concentration of 1 x 10(4) sperm/ml (P less than 0.001). The procedure did not induce parthenogenetic activation of oocytes and more than 97% of zygotes developed to the blastocyst stage. A similar rate of live progeny was observed when zona-drilled (38.0%) and control embryos (38.5%) were transferred to pseudopregnant recipients. Chromosome analyses were performed on zona-intact, zona-free, and zona-drilled oocytes inseminated with varying concentrations of sperm and analysed at the first cleavage division. Zona-free oocytes had high rates of polyploidy (greater than or equal to 40%) with varying insemination numbers but the zona-drilled oocytes did not reveal significant increases in the rate of polyploidy or aneuploidy when compared to controls. In the human studies, zona-drilled oocytes achieved higher rates of fertilization than zona-intact oocytes, with sperm numbers as low as 1 x 10(4)/ml (6/8; 75%). Polyspermic fertilization was observed in 1/2 and 2/6 of fertilized oocytes inseminated with 1 x 10(5) and 1 x 10(4) sperm/ml, respectively. With the low sperm concentration 2/4 of those which were normally fertilized developed to healthy blastocysts. These studies suggest that the zona-drilling technique as described can be performed without apparent harm to oocytes and generate normal embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of Calcium-Free Tyrode's Sperm Capacitation Medium for Use in Bovine In Vitro Fertilization

Our objective was to determine if a bovine sperm capacitation technique, developed with zona-free hamster oocytes, could be used for the in vitro fertilization of in vitro matured bovine zona-intact oocytes. Bovine cumulus-enclosed primary oocytes from 2- to 5-mm follicles were matured in tissue culture Medium 199 containing Earle's salts and bicarbonate and supplemented with 10% fetal calf serum, FSH (10µg/ml),and estradiol-17β (1.5µg/ml) for 24h at 37°C under paraffin oil. Ejaculated bovine sperm, washed thrice in bovine serum albumin-saline (pH 7.6) and capacitated for 4h in Ca++-free Tyrode's medium (pH 7.6), were diluted to 2×106 sperm/ml in Medium 199 supplemented with 10% fetal calf serum. Oocytes were added (10/500 µ1 droplet) to this medium containing the capacitated sperm, freeze-thawed killed sperm, or no sperm and incubated for 8h before transfer to fresh medium and then incubated for 40h. At the end of each incubation, a portion of the oocytes were stained and evaluated for development or fertilization. After 24h of culture, 49% of the oocytes had matured (metaphase II). Fertilization rates were 55.6% after exposure of all oocytes to Ca++-free Tyrode's capacitated sperm and 82.5% if only metaphase II oocytes were selected. The parthenogenetic controls were negative (1.4% and 0%). Therefore, the Ca++-free Tyrode's sperm capacitation technique can be used for bovine in vitro fertilization studies.

Effect of cooling temperatures on pre-compacted bovine embryos

This research concerned effects of cooling in vitro matured bovine oocytes on subsequent fertilization and development in vitro. Oocytes were maintained at 39°C (control), 20°C, 10°C or 0°C for 5, 10 or 20 min, then fertilized and cultured in vitro for 7 d. The proportion of fertilized oocytes that cleaved and developed to the morula/blastocyst stage was compared between different treatments. Duration of exposure had no effect on the results. Fertilization rate was higher (P<0.05) for oocytes maintained at 39°C (73.2%) than for oocytes cooled at 20°C (58.6%), 10°C (47.3%), or 0°C (36.9%). Cleavage rates were 58.3, 45.3, 15.7 and 7.0% for 39°C, 20°C, 10°C and 0°C, respectively (P<0.05). The lowest development rate to the blastocyst stage was obtained with oocytes cooled to 10°C (0.0%) or 0°C (0.9%), followed by 20°C (7.1%) and 39°C (16.5%; P< 0.05). In a second experiment, the zona pellucida was removed after cooling but prior to fertilization (zona-free) from a portion of the in vitro- matured bovine oocytes in each treatment. When sperm penetration rates of zona-free oocytes were compared (percentage of oocytes exhibiting ≥ 2 pronuclei), there was no difference (P>0.05) between oocytes cooled at 0°C (59.7%) or 10°C (67.9%). However, penetration rates in these 2 groups were lower (P<0.05) when compared to zona-free oocytes cooled at 20°C (83.1%) or those maintained at 39°C (83.1%). Zona-free oocytes had higher penetration rates (P<0.05) when cooled at 0°C (59.7%) or 10°C (67.9%) than zona-intact oocytes cooled at 0°C (37.3%) or 10°C (47.2%). However, there was no difference in the penetration rate when zona-free and zonaintact oocytes were cooled at 20°C or maintained at 39°C. These data demonstrate that cooling in vitro-matured bovine oocytes decreases the percentage of oocytes that undergo fertilization and subsequently develop in vitro. Moreover, at least part of the decrease in fertilization following oocyte cooling is due to effects on the zona pellucida.