Efficient injection of bull spermatozoa into oocytes using a Piezo-driven pipette

Recently, mouse and human offspring have been successfully obtained from embryos developed after intracytoplasmic sperm injection(ICSI), using a Piezo micromanipulator. In this study, the Piezo-ICSI procedure was used with in vitro matured bovine oocytes known to be difficult to fertilize microsurgically. The efficacy of Piezo-ICSI versus conventional ICSI was examined after oocytes were activated and fertilized with or without calcium ionophore (A23187) exposure. In conventional ICSI, the rate of fertilization was 19% (11/59) with A23187 and 5% (2/38) without it. However, when the Piezo-ICSI procedure was performed, the fertilization rate was 72% (47/65) with A23187 and 72% (28/39) without it. The rate of oocyte survival after microinjection was nearly similar for both methods. We suggest that the bovine oocyte is successfully activated and fertilized when an immobilized spermatozoon is injected exactly into the ooplasm through the oolemma, perforated easily by the pulsation of the Piezo. Moreover, an activating procedure such as exposure of oocytes to A23187 is not necessary, because the so-called sperm factor (oocyte activating substances) is incorporated into the ooplasm along with a spermatozoon. In this respect, the Piezo-ICSI was more efficient than the conventional ICSI method for fertilizing and thus obtaining more bovine embryos.     

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.     

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.     

Recovery of mare oocytes on a fixed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection

Oocytes may be collected from live mares from either the stimulated preovulatory follicle or from all visible immature follicles. We evaluated the yield of mature oocytes, and of blastocysts after intracytoplasmic sperm injection (ICSI), for both follicle types. In Experiment 1, mares were assigned to Progesterone (1.2 g biorelease progesterone weekly) or Control treatments. Transvaginal aspiration of all follicles was performed every 14 d. Overall, 596 follicles were aspirated, with a 54% oocyte recovery rate. There was no difference between treatments in number of follicles punctured (9.0 to 9.1) or oocytes recovered (4.8 to 5.0) per mare per aspiration session. Of 314 oocytes recovered, 180 (57%) matured in culture. Thirty-six mature oocytes were subjected to ICSI; 33% formed blastocysts (63% per mare per aspiration session). In Experiment 2, the preovulatory follicle was aspirated every 14 d for three to four cycles. Prostaglandin F_2α was given on Days 6 and 7 after aspiration. A follicle ≥25 mm in diameter was present on Day 13, the day of deslorelin administration, in 23 of 24 cycles, and ovulatory response (granulosa expansion) was seen in 24 of 25 follicles aspirated. Blastocyst development after ICSI was 41% per injected oocyte, or an estimated 33% per mare per aspiration session. We concluded that both aspiration of immature follicles and aspiration of the preovulatory follicle can be performed effectively every 14 d without monitoring ovarian follicular growth. As performed in these separate experiments, aspiration of immature follicles provided more blastocysts per aspiration session.     

Sperm movement in the ooplasm, dithiothreitol pretreatment and sperm freezing are not required for the development of porcine embryos derived from injection of head membrane-damaged sperm

The present study investigated the correlation of sperm movement in the ooplasm, pretreatment of sperm with dithiothreitol (DTT) and sperm freezing with the development of porcine embryos derived from modified intracytoplasmic sperm injection (ICSI). In vitro, matured gilt oocytes without centrifugation were injected with head membrane-damaged spermatozoa aspirated tail-first. In Exp. 1, frozen-thawed sperm were categorized into three groups: impaired, immotile or motile. Oocytes injected with motile sperm (43.6%) showed a higher (P < 0.05) fertilization rate compared to oocytes injected with impaired or immotile sperm (34.5 or 37.2%). The survival rate was significantly higher (P < 0.05) in oocytes injected with impaired sperm (92.9%) than in oocytes injected with immotile or motile sperm (84.8 or 86.7%). No differences were observed in the rates of cleavage or blastocyst formation, and in total cell number of blastocysts among three groups of oocytes. In Exp. 2, motile frozen-thawed sperm were pretreated with DTT before injection and non-treated sperm served as controls. Higher rates (P < 0.05) of fertilization, male pronucleus (MPN) and decondensed sperm head (DSH) formation were observed in oocytes injected with control sperm (41.1, 50.0 and 91.1%, respectively) than in oocytes injected with DTT-treated sperm (22.1, 30.2 and 72.1%, respectively). No differences in embryo development and total cell number of blastocysts were observed between two groups of oocytes. In Exp. 3, motile frozen-thawed or fresh sperm without DTT pretreatment were injected into oocytes. The rates of fertilization and MPN formation were significantly higher (P < 0.05) in oocytes injected with fresh sperm (59.8 and 73.5%) than in oocytes injected with frozen-thawed sperm (36.7 and 59.2%). No differences in embryo development and total cell number of blastocysts were observed between two groups of oocytes. In conclusion, the present study clearly demonstrated that sperm movement in the ooplasm, use of DTT and fresh spermatozoa did not significantly affect on embryo development in porcine modified ICSI.     

Effect of sperm immobilisation and demembranation on the oocyte activation rate in the mouse.

To analyse the effect of the state of the sperm plasma membrane on oocyte activation rate following intracytoplasmic sperm injection (ICSI), three types of human and mouse spermatozoa (intact, immobilised and Triton X-100 treated) were individually injected into mouse oocytes. At 30, 60 and 120 min after injection, maternal chromosomes and sperm nuclei within oocytes were examined. Following human sperm injection, the fastest and the most efficient oocyte activation and sperm head decondensation occurred when the spermatozoa were treated with Triton X-100. Intact spermatozoa were the least effective in activating oocytes. Thus, the rate of mouse oocyte activation following human sperm injection is greatly influenced by the state of the sperm plasma membrane during injection. When mouse spermatozoa were injected into mouse oocytes, the rates of oocyte activation and sperm head decondensation within activated oocytes were the same irrespective of the type of sperm treatment prior to injection. We witnessed that live human spermatozoa injected into moue oocytes often kept moving very actively within the ooplasm for more than 60 min, whereas motile mouse spermatozoa usually became immotile within 20 min after injection into the ooplasm. In 0.002% Triton X-100 solution, mouse spermatozoa are immobilised faster than human spermatozoa. These facts seem to suggest that human sperm plasma membranes are physically and biochemically more stable than those of mouse spermatozoa. Perhaps the physical and chemical properties of the sperm plasma membrane vary from species to species. For those species whose spermatozoa have 'stable' plasma membranes, prior removal or 'damage' of sperm plasma membranes would increase the success rate of ICSI.     

Patterns of intracellular calcium oscillations in horse oocytes fertilized by intracytoplasmic sperm injection: possible explanations for the low success of this assisted reproduction technique in the horse

In all species studied, fertilization induces intracellular Ca2+ ([Ca2+]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca2+]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single motile stallion sperm. Few oocytes displayed [Ca2+]i responses regardless of oocyte source and we hypothesized that this may result from insufficient release of the sperm-borne active molecule (sperm factor) into the oocyte. However, permeabilization of sperm membranes with Triton-X or by sonication did not alleviate the deficient [Ca2+]i responses in mare oocytes. Thus, we hypothesized that a step downstream of release, possibly required for sperm factor function, is not appropriately accomplished in horse oocytes. To test this, ICSI-fertilized horse oocytes were fused to unfertilized mouse oocytes, which are known to respond with [Ca2+]i oscillations to injection of stallion sperm, and [Ca2+]i monitoring was performed. Such pairs consistently displayed [Ca2+]i responses demonstrating that the sperm factor is appropriately released into the ooplasm of horse oocytes, but that these are unable to activate and/or provide the appropriate substrate that is required for the sperm factor delivered by ICSI to initiate oscillations. These findings may have implications to improve the success of ICSI in the equine and other livestock species.     

Oocyte activation and parthenogenetic development of bovine oocytes following intracytoplasmic sperm injection.

Development of bovine oocytes after intracytoplasmic sperm injection (ICSI) was investigated. Oocytes were matured for 24-26 h in vitro and injected with isolated sperm heads. When treated with 7% ethanol (v/v) for 5 min, 71.7% of ICSI oocytes were activated as shown by the resumption of meiosis and the formation of female pronuclei. However, 41.5% of injected sperm heads remained condensed at 18-20 h after injection into the ooplasm. The incidence of decondensing sperm and that of male pronuclei at this stage were 15.1% and 26.4%, respectively. A total of 55.5% of oocytes reached the 2-cell stage following sperm head injection and 54.7% after sham-ICSI; these percentages were not significantly different from those following in vitro fertilisation (IVF) (73.1%). The percentage of 2-cell embryos reaching the 8-cell stage following ICSI was 37.5%, and 27.6% after sham-ICSI, which were significantly lower (p < 0.01) than the equivalent percentage following IVF (62.4%). The percentages of parthenogenetic embryos reaching the 2-cell, 4-cell and 8-cell stages following ICSI were 56.4%, 48.9% and 30.0%, respectively. These results indicate that the low rate of normal embryonic development of bovine oocytes following ICSI is largely due to the parthenogenetic activation of the oocytes.     

Intracytoplasmic sperm injection of bovine oocytes with stallion spermatozoa

Five experiments were designed to study the fertilizability and development of bovine oocytes fertilized by intracytoplasmic sperm injection (ICSI) with stallion spermatozoa. Experiment 1 determined the time required for pronuclear formation after ICSI. Equine sperm head decondensation began 3 h after ICSI; 42% were decondensed 6 h after ICSI. Male pronuclei (MPN) began to form 12 h after ICSI. Female pronuclei (FPN), however, formed as early as 6 h after ICSI. In Experiment 2, ionomycin, ionomycin plus 6-dimethylaminopurine (DMAP), and thimerosal were used to activate ICSI ova. None of the ICSI ova cleaved after treatment with thimerosal. Ionomycin activation after 24 and 30 h of oocyte maturation resulted in 29 and 48% cleavage rates, respectively. Ionomycin combined with DMAP resulted in 49, 6 and 3% cleavage, morula and blastocyst rates, respectively, when oocytes were activated after 24 h maturation. In Experiment 3, rates of cleavage (45-60%) and development to morulae (4-13%) and blastocysts (1-5%) stages following ICSI were not different (P>0.05) among three stallions. Treatment of stallion spermatozoa with ionomycin did not affect cleavage or development of ova fertilized by ICSI. The chromosomal constitution of blastocysts derived from ICSI was bovine, not bovine and equine hybrids. In Experiment 4, to make male and FPN form synchronously, colchicine and DMAP were used for 4 h to inhibit oocytes at metaphase during activation; 63% of oocytes were still at metaphase 8h after ICSI when treated with colchicine, and 50% of sperm nuclei were decondensed. About 18 h after ICSI, 21 and 50% male and FPN had formed, respectively, but cleavage rates were low, and only 1% developed to morulae. In Experiment 5, to test if capacitated equine sperm could fuse with the bovine oolemma, capacitated spermatozoa were injected subzonally (SUZI). Of the 182 SUZI oocytes, 49 (27%) contained extruded second polar bodies. After activation of oocytes with second polar bodies, 44, 22 and 15% developed to 2-, 4- and 8-cell stages, respectively, but development stopped at the 8-cell stage. None of the unactivated oocytes cleaved. In conclusion, equine spermatozoa can decondense and form MPN in bovine oocytes after ICSI, but subsequent embryonic development is parthenogenetic with only bovine chromosomes being found.     

Generation of porcine diploid blastocysts after injection of spermatozoa grown in nude mice

It is anticipated that the utilization of spermatogonia through testicular xenografting will open new avenues for the conservation of male gametes. With the aim of establishing this new technique for genetic preservation of pigs, we used it in combination with intracytoplasmic sperm injection (ICSI). Testicular tissues derived from neonatal piglets, which contained seminiferous cords consisting of only gonocytes/spermatogonia, were transplanted under the back skin of castrated nude mice. Between 125 and 192 d after xenografting, sperm (morphologically similar to epididymal sperm) were recovered from 41 of the 65 host mice (63.1%). Testicular spermatozoa from adult boars were used as a positive control. A single spermatozoon was injected into an in vitro matured porcine oocyte, and the oocytes were electro-stimulated and cultured (graft-ICSI and testis-ICSI, respectively). Blastocyst rates in both ICSI groups (24.9% and 37.4%, respectively) were higher (P < 0.05) than those without the injection procedure (parthenogenetic; 12.7%) and after injection of a small amount of injection buffer (sham; 13.0%). Rates of diploid blastocysts in both graft-ICSI and testis-ICSI groups (48.9% and 60.6%) were higher (P < 0.05) than those in the parthenogenetic and sham groups (13.5% and 28.0%). Therefore, we demonstrated that porcine oocytes injected with xenogeneic sperm have in vitro developmental ability to the blastocyst stage.     

Fertilisability of ovine, bovine or minke whale (Balaenoptera acutorostrata) spermatozoa intracytoplasmically injected into bovine oocytes

This study was conducted to investigate the possibility of using bovine oocytes for a heterologous fertility test by intracytoplasmic sperm injection (ICSI) and to compare the pronuclear formation of ram, bull and minke whale spermatozoa after injection into bovine oocytes. Bovine oocytes were cultured in vitro for 24 h and those with a polar body were selected for ICSI. Frozen-thawed semen from the three species were treated with 5 mM dithiothreitol for 1 h and spermatozoa were killed by storing them in a -20 degrees C refrigerator before use. ICSI was performed using a Piezo system. Three experiments were designed. In experiment 1, a higher (p < 0.05) male pronuclear formation rate was found in the oocytes injected with ram (52.6%) or bull (53.4%) spermatozoa than with minke whale spermatozoa (39.1%). In experiment 2, sperm head decondensation was detected at 2 h after ICSI in the oocytes injected with a spermatozoon of each species. Male pronuclei were first observed at 4 h in the oocytes injected with ram or bull spermatozoa and at 6 h in oocytes injected with minke whale spermatozoa. The mean diameters of male pronuclei derived from both whale and bull spermatozoa were larger than those from ram spermatozoa (30.4 microm and 28.3 microm vs 22.4 microm, p < 0.005). The mean diameter of female pronuclei in the oocytes injected with whale spermatozoa was also larger than with ram spermatozoa (29.3 microm vs 24.7 microm, p < 0.05). The development of male and female pronuclei was synchronous. In experiment 3, ethanol-activated oocytes injected with a spermatozoon from any of the three species achieved significantly higher (p < 0.05-0.001) cleavage rates than control oocytes. Blastocyst formation was only observed when bull spermatozoa were used. The results of this study indicate that dead foreign spermatozoa can participate in fertilisation activities in bovine oocytes after ICSI.     

Cleavage development of bovine oocytes fertilized by sperm injection

Whole in vitro capacitated bovine spermatozoa were microinjected directly into the ooplasm of in vitro matured bovine oocytes in order to determine whether oocytes fertilized by sperm injection could undergo normal pronuclear formation and cleavage development. Immature oocytes recovered from follicles (2-5 mm) of unstimulated ovaries were cultured for 24-25 h in modified TCM 199 medium supplemented with heat-treated day 20 cow serum, luteinizing hormone (LH), and estradiol 17-B. In vitro capacitated, frozen-thawed spermatozoa were injected into the ooplasm, and the injected oocytes were cultured for an additional 24-28 h. Twenty-one percent (21/101) of the sperm-injected oocytes contained a sperm within the ooplasm; however, only 2% (2/101) cleaved. The remaining oocytes either did not contain a sperm or had degenerated. After oocyte activation induced by a 5 min incubation in 1 microM A23187, sperm nuclear decondensation occurred in the A23187-activated, injected oocytes but not in the unactivated, injected controls (37% vs. 0% after 3 h). Those injected, activated oocytes that contained a male pronucleus also exhibited a female pronucleus and second polar body. Furthermore, a significantly higher number (28%, 6/21) of the injected, activated oocytes cleaved to a two- to four-cell stage after 48 h than did the injected, unactivated oocytes (4%). These results indicate that, unlike hamster and rabbit oocytes, bovine oocytes are not sufficiently stimulated by the injection procedure to complete meiosis, but, upon activation by calcium ionophore, they will undergo normal-appearing cleavage development following fertilization by sperm injection.     

Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI)

In the human and the mouse, intracytoplasmic sperm injection (ICSI) apparently triggers normal fertilization and may result in offspring. In the bovine, injection of spermatozoa must be accompanied by artificial methods of oocyte activation in order to achieve normal fertilization events (e.g., pronuclear formation). In this study, different methods of oocyte activation were tested following ICSI of in vitro-matured bovine oocytes. Bovine oocytes were centrifuged to facilitate sperm injection, and spermatozoa were pretreated with 5 mM dithiothreitol (DTT) to promote decondensation. Sperm-injected or sham-injected oocytes were activated with 5 microM ionomycin (A23187). Three hours after activation, oocytes with second polar bodies were selected and treated with 1.9 mM 6-dimethylaminopurine (DMAP). The cleavage rate of sperm-injected oocytes treated with ionomycin and DMAP was higher than with ionomycin alone (62 vs 27%, P < or = 0.05). Blastocysts (2 of 41 cleaved) were obtained only from the sperm-injected, ionomycin + DMAP-treated oocytes. Upon examination 16 h after ICSI, pronuclear formation was observed in 33 of 47 (70%) DMAP-treated oocytes. Two pronuclei were present in 18 of 33 (55%), while 1 and 3 pronuclei were seen in 8 of 33 (24%) and 7 of 33 (21%) oocytes, respectively. In sham-injected oocytes, pronuclear formation was observed in 15 of 38 (39%) with 9 (60%) having 2 pronuclei. Asa single calcium stimulation was insufficient and DMAP treatment could result in triploidy, activation by multiple calcium stimulations was tested. Three calcium stimulations (5 microM ionomycin) were given at 30-min intervals following ICSI. Two pronuclei were found in 12 of 41 (29%) injected oocytes. Increasing the concentration of ionomycin from 5 to 50 microM resulted in a higher rate of activation (41 vs 26%). The rate of metaphase III arrest was lower while the rate of pronuclear formation and cleavage development was higher in sperm-injected than sham-injected oocytes, suggesting that spermatozoa contribute to the activation process. Further improvements in oocyte activation following ICSI in the bovine are necessary.     

Sperm membrane incorporation into oolemma contributes to the oolemma block to sperm penetration: Evidence based on intracytoplasmic sperm injection experiments in the mouse

Mouse oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and reinseminated after the removal of zonae pellucidae at pronuclear stage or at the 2-cell stage. Although these oocytes were activated normally by ICSI, as evidenced by resumption of meiosis and cortical granule exocytosis, they did not develop oolemma block to sperm penetration. They could be penetrated by spermatozoa at pronuclear stage and even at the 2-cell stage. This supports the notion that incorporation of sperm plasma membrane into oolemma contributes to the changes in oolemma that block sperm penetration. © 1996 Wiley-Liss, Inc.     

In vitro production of llama (Lama glama) embryos by intracytoplasmic sperm injection: effect of chemical activation treatments and culture conditions

Assisted reproductive technologies in the llama (Lama glama) are needed to provide alternative methods for the propagation, selection and genetic improvement; however, recovery of adequate quantity and quality of spermatozoa for conventional IVF is problematic. Therefore, an effort was made to adapt the intracytoplasmic sperm injection (ICSI) procedure for the in vitro production of llama embryos. The specific objectives of this study were: (1) to determine in vitro maturation rates of oocytes recovered by transvaginal ultrasound-guided oocyte aspiration (TUGA) or flank laparotomy; (2) to evaluate the effects of activation treatments following ICSI; (3) to evaluate the development of llama ICSI embryos in CR1aa medium or in an oviduct cell co-culture system. Llamas were superstimulated by double dominant follicle reduction followed by oFSH administered in daily descending doses over a 3-day interval. Oocytes were harvested by flank laparotomy or TUGA and matured in vitro for 30 h. Mature oocytes were subjected to ICSI followed by no chemical activation (Treatment A), ionomycin only (Treatment B) or ionomycin/DMAP activation (Treatment C). More oocytes were recovered by flank laparotomy procedure compared with TUGA (94% versus 61%, P<0.05) and a greater number of oocytes harvested by flank laparotomy reached the metaphase-II stage (77% versus 44%, P<0.05). After ICSI, the proportion of cleaved and 4-8-cell stages embryos was significantly greater when injected oocytes were activated with ionomycin/DMAP combination (63% and 38%, respectively, P<0.05). The co-culture of ICSI embryos with llama oviduct epithelial cells resulted in progression to morula (25%) and blastocyst (12%) stages; whereas, all embryos cultured in CR1aa medium arrested at the 8-16-cell developmental stage.     

Sperm-oocyte membrane fusion in the human during monospermic fertilization

Sperm-oocyte membrane fusion has been observed during monospermic fertilization of a human oocyte in vitro. Women were stimulated with both clomiphene citrate and human menopausal gonadotropin and were given human chorionic gonadotropin before a LH-surge. Twelve oocytes, collected at laparoscopy from six women who became pregnant by IVF, were allowed to mature for 7–14 hours in vitro and inseminated with preincubated sperm, fixed between 1–3 hours after insemination, and examined by transmission electron microscopy. Membrane fusion had occurred in one ovum 2 hours after insemination, and the oocyte had resumed maturation and was at anaphase II of meiosis. Cortical granules had been exocytosed, and some of their contents were visible at the surface close to the oolemma all around the oocyte. The sperm that fused with this oocyte was acrosome-reacted and had been partly incorporated into the ooplasm, while the anterior two-thirds of its head was phagocytosed by a tongue of cortical ooplasm. Membrane fusion had occurred between the oolemma and the plasma membrane overlying the postacrosomal segment of the sperm head, posterior to the equatorial vestige. Sperm chromatin had not decondensed, and serial sections revealed a midpiece attached to the basal plate and a tail located deeper in the ooplasm, all devoid of plasma membrane. Supplementary sperm penetrating the inner zona, approaching the perivitelline space, had undergone the acrosome reaction but had a persistent vestige of the equatorial segment of the acrosome with intact plasma membrane. Evidence of sperm chromatin decondensation was seen in other oocytes, 3 hours after insemination, which were at telophase II of meiosis. Eight oocytes penetrated by sperm were monospermic, while four were unfertilized. The general pattern of sperm fusion and incorporation appears to conform to that seen in most other mammals. The study also reveals that sperm have to complete the acrosome reaction before fusing with the egg.     

Requirement of sperm‐oocyte plasma membrane fusion for establishment of the plasma membrane block to polyspermy in human pronuclear oocytes

We investigated whether the incorporation of the sperm membrane into the oolemma contributes to the human plasma membrane block to polyspermy. We used zona pellucida–free oocytes fertilized by intracytoplasmic sperm injection (ICSI) or activated by parthenogenetic activation. Only two of the 35 pronuclear oocytes fertilized by spermatozoa (control) demonstrated one single penetrating spermatozoa. In contrast, the majority of ICSI and parthenogenetically activated pronuclear oocytes were penetrated with an average of three spermatozoa per oocyte. The number of fused and binding spermatozoa of ICSI and parthenogenetically activated oocytes were significantly higher than in control oocytes (3.5 ± 0.6 and 4.3 ± 0.6 for ICSI; 3.0 ± 0.3 and 3.8 ± 0.4 for activated and 0.2 ± 0.1 and 0.6 ± 0.2 for controls, respectively, P < 0.01). Furthermore, the cortical granules were released from the cortex of ICSI and calcium ionophore‐puromycin‐activated pronuclear oocytes to the same extent as that of pronuclear oocytes fertilized by spermatozoa. These results suggest that the establishment of the plasma membrane block to sperm penetration in the human oocyte may require a fusion process between sperm and oocyte plasma membranes. Mol. Reprod. Dev. 52:183–188, 1999. © 1999 Wiley‐Liss, Inc.     

Fertilization and development of quail oocytes after intracytoplasmic sperm injection

The present study was conducted to establish the intracytoplasmic sperm injection (ICSI) method for in vitro fertilization and development in quail. The efficiency of fertilization of oocytes was compared 1) between spontaneous and premature ovulation and 2) among testicular round spermatids, elongated spermatids, and immature and mature spermatozoa. The oocytes were injected with a single spermatozoon or spermatid and cultured for 24 h. Cell division was histologically observed with hematoxylin-eosin (HE) and a nucleus-specific fluorescent dye (DAPI). Five of 30 (16.6%) and 4 of 30 (13.3%) oocytes injected with mature sperm were fertilized in the spontaneous and induced ovulation group, respectively. Those embryos showed development at stages II-VII. Half the number (three of six) of the oocytes injected with testicular spermatozoa were fertilized and developed to stages IV-VII, and two of five oocytes injected with elongated spermatids were fertilized and developed to stage VI. All ooocytes injected with round spermatids were unfertilized. The results demonstrate that intracytoplasmic injection of a single sperm into quail oocyte can activate the oocyte and lead to fertilization. Oocytes prematurely ovulated are capable of fertilizing with mature sperm as are those spontaneously ovulated. In addition, the results suggest that the testicular round spermatids may not possess sufficient oocyte-activating potency but that the elongated spermatids and immature spermatozoa are competent to participate in fertilization and early embryonic development in quail.     

Equine blastocyst development after intracytoplasmic injection of sperm subjected to two freeze-thaw cycles

This study was conducted to evaluate the effects of thawing, division into aliquots and refreezing on fertilizing capacity (ability to support embryo development after intracytoplasmic sperm injection; ICSI) of frozen stallion semen. Frozen semen from a fertile stallion was thawed, diluted 1:100 with freezing extender, and refrozen (2F treatment). Control semen was frozen only once. In vitro matured equine oocytes were injected with: (1) motile control spermatozoa; (2) motile 2F spermatozoa; (3) non-motile 2F spermatozoa; or (4) non-motile 2F spermatozoa, followed by injection of sperm extract. Blastocyst development after ICSI was equivalent between control spermatozoa and motile 2F spermatozoa (27 and 23%, respectively). Blastocyst development after injection of non-motile 2F spermatozoa (13%) tended (P=0.07) to be lower than that for control spermatozoa. Injection of sperm extract into oocytes that received non-motile 2F spermatozoa resulted in a significant decrease in blastocyst development (to 2%) compared with injection of non-motile 2F spermatozoa alone. Spermatozoa from a subfertile stallion was similarly processed and used for ICSI; blastocyst development for both motile control (once frozen) spermatozoa and motile 2F spermatozoa was 9%. In conclusion, frozen stallion semen may be thawed, diluted, and refrozen without effect on the ability of motile spermatozoa to initiate embryo development after ICSI. Non-motile spermatozoa from reprocessed semen may also achieve embryo development after ICSI. To our knowledge, this is the first report evaluating the ability of refrozen spermatozoa to produce embryos by ICSI in any species.     

Influence of oocyte collection technique on initial chromatin configuration, meiotic competence, and male pronucleus formation after intracytoplasmic sperm injection (ICSI) of equine oocytes

There is a great variability in the success of horse oocyte maturation and fertilization among laboratories. This study was conducted to determine if the meiotic and developmental competence of horse oocytes could be dependent on the method of oocyte collection, i.e., aspiration of follicular fluid with a vacuum apparatus, or opening follicles and scraping the granulosa layer. Horse oocytes were recovered from abattoir ovaries by aspiration or scraping and classified as having compact (Cp), expanded (Ex), or partial (P) cumuli. In Experiment 1 (Part A in May and Part B in October), oocytes were fixed immediately after collection to assess whether the collection method influenced the initial chromatin configuration of oocytes. In Experiment 2, in vitro maturation rates of oocytes recovered by aspiration or scraping were compared. In Experiment 3, oocytes were matured in vitro and submitted to intracytoplasmic sperm injection (ICSI). Initial chromatin configuration differed according to collection method in that there was a significantly higher prevalence of diffuse chromatin within the germinal vesicle in oocytes recovered by scraping than in oocytes recovered by aspiration (29/87, 33% and 28/166, 17%, respectively; P < 0.01). Maturation of oocytes to metaphase II did not significantly differ between scraped and aspirated oocytes (56/101, 55.4 % vs. 65/106, 61.4%, respectively). The overall pronucleus formation rate after ICSI of oocytes recovered by scraping was not significantly different than that of oocytes recovered by aspiration (50/99, 52.6% vs. 50/85, 68.5 %, respectively); however, the rate of abnormal fertilization was significantly higher for oocytes collected by aspiration (14/73, 19% vs. 6/94, 6%, respectively; P <0.05). These results demonstrate that the collection method affects the population of recovered oocytes and may contribute to differences in results observed among laboratories working with horse oocytes.