Formation Of Female Pronuclei Research Articles

Improvement of Porcine Oocytes with Low Developmental Ability after Fusion of Cytoplasmic Fragments Prepared by Serial Centrifugation

We have shown in pigs that oocytes denuded of cumulus cells at 24 h of in vitro maturation culture and subsequently matured for a total of 46 h (DO24 oocytes) have lower cytoplasmic maturity than those matured with cumulus cells for 46 h and then denuded (DO46 oocytes). In the present study, DO24 zona-free oocytes were fused with one (1C) or two (2C) cytoplasmic fragments produced by serial centrifugation ("centri-fusion") of DO46 oocytes (DO24+1C and DO24+2C oocytes, respectively). Groups of (1) DO46 (a control), (2) DO24, (3) DO24+1C and (4) DO24+2C oocytes were partheno-activated by an electrical pulse or fertilized in vitro and subsequently cultured for 6 days. In the fused groups, female pronucleus (FPN) formation rates were higher than that in the DO24 group after parthenogenetic activation (PA); however, the blastocyst rates were intermediate between those of the control and DO24 groups. After in vitro fertilization, the male pronucleus (MPN) formation rates in the fused groups were similar to that in the control group and higher than that in the DO24 group; the normal fertilization rate in the DO24+2C group was higher than that in the DO24 group and similar to that in the control group, resulting in significantly higher blastocyst rates in the DO24+2C and control groups than that in the DO24 group. These results suggest that centri-fusion using ooplasm from fully matured DO46 oocytes can offer a potentially novel approach for restoration of cytoplasmic maturity to oocytes with low developmental ability and subsequent improvement of fertilization and developmental competence.

DNA synthesis and epigenetic modification during mouse oocyte fertilization by human or hamster sperm injection

To evaluate DNA synthesis and epigenetic modification in mouse oocytes during the first cell cycle following the injection of human or hamster sperm. Mouse oocytes following the injection of human and hamster sperm and cultured in M16 medium. Male and female pronucleus formation, DNA synthesis, histone protein modification, and heterochromatin formation were similar in mouse oocytes injected with human or hamster sperm. However, DNA methylation patterns were altered in mouse oocytes following human sperm injection. Immunocytochemical staining with a histone H3-MeK9 antibody revealed that human and hamster sperm chromatin associated normally with female mouse chromatin, then entered into the metaphase and formed normal, two-cell stage embryos. Although differences in epigenetic modification of DNA were observed, fertilization and cleavage occurred in a species non-specific manner in mouse oocytes.

Progesterone receptor membrane component 1 expression and putative function in bovine oocyte maturation, fertilization, and early embryonic development

Although the mRNA that encodes progesterone receptor membrane component 1 (PGRMC1) is present in mammalian oocytes, nothing is known about either PGRMC1's expression pattern or function in oocytes during maturation, fertilization, and subsequent embryonic development. As PGRMC1 associates with the mitotic spindle in somatic cells, we hypothesized that PGRMC1 is involved in oocyte maturation (meiosis). Western blot analysis confirmed the presence of PGRMC1 in bovine oocytes. This study also shows that PGRMC1 is present at the germinal vesicle (GV)- and MII-stage oocytes and is associated with male and female pronucleus formation of the zygote and is highly expressed in blastocysts. A more detailed examination of PGRMC1 localization using confocal imaging demonstrated that in GV-stage oocytes, PGRMC1 was concentrated throughout the GV but did not localize to the chromatin. With the resumption of meiosis in vitro, PGRMC1 concentrated in the centromeric region of metaphase I chromosomes, while in the anaphase I/telophase I stages the majority of PGRMC1 concentrated between the separating chromosomes. At the metaphase II stage, PGRMC1 re-associated with the centromeric region of the chromosomes. A colocalization study demonstrated that PGRMC1 associated with the phosphorylated form of aurora kinase B, which localizes to the centromeres at metaphase. Finally, PGRMC1 antibody injection significantly lowered the percentage of oocytes that matured and reached the metaphase II stage after 24 h of culture. The majority of the PGRMC1 antibody-injected oocytes arrested in the prometaphase I stage of meiosis. Furthermore, in most of the PGRMC1 antibody-injected oocytes, the chromosomes were disorganized and scattered. Taken together, these data demonstrate that PGRMC1 is expressed in bovine oocytes and its localization changes at specific stages of oocyte maturation. These observations suggest an important role for PGRMC1 in oocyte maturation, which may be specifically related to the mechanism by which chromosomes segregate.

Nuclear Replacement ofIn Vitro-Matured Porcine Oocytes by a Serial Centrifugation and Fusion Method

The objective of the present study was to establish a method for nuclear replacement in metaphase-II (M-II) stage porcine oocytes. Karyoplasts containing M-II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro-matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona-free M-II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2-77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2-32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0-15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri-Fusion) is an effective method for producing M-II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development.

Vitrification solution containing DMSO and EG can induce parthenogenetic activation of in vitro matured ovine oocytes and decrease sperm penetration

This study was designed to examine the reduced incidence of normal fertilization in vitrified ovine oocytes. After in vitro maturation for 24 h, the oocytes were randomly allocated into three groups: (1) untreated (control), (2) exposed to vitrification solution (VS) without being plunged into liquid nitrogen (toxicity), or (3) vitrified by open-pulled straw method (vitrification). In experiment 1, the treated and control oocytes were matured for another 2 h, and the oocytes were then in vitro fertilized for 12 h to examine sperm penetration. The percentage of monospermy in toxicity group (29.3%) and vitrification group (28.2%) dramatically decreased compared to the control group (45.0%) ( P < 0.05). To find the mechanism that the VS decreased the monospermy, some treated and control oocytes were used to test the distribution of CG and the resistance of zona pellucida (ZP) to 0.1% pronase E immediately (IVM 24 h), after another 2 h of maturation (IVM 26 h), and after 12 h of in vitro fertilization (IVF 12 h) respectively. Others were used to examine female pronucleus formation after 12 h of culture in fertilization medium with the absence of sperm. The results showed that the percentage of CG completely release in the oocytes (IVM 24 and 26 h) of toxicity group (41.2% and 39.9%) and vitrification group (41.7% and 51.7%) was significantly higher than that of control group (7.1% and 18.4%) ( P < 0.05). The ZP digestion duration in the oocytes (IVM 26 h) of the toxicity group (435.6 s) and vitrification group (422.3 s) was longer than that of control group (381.6 s) ( P < 0.05). The percentage of female pronucleus formation in toxicity group (58.7%) and vitrification group (63.9%) was higher than that (8.2%) of control group ( P < 0.05). The data above demonstrated that the VS containing DMSO and EG could parthenogenetically activate in vitro matured ovine oocytes, resulting in ZP hardening and decreased sperm penetration.

Effect of centrifugation and electrical activation on male pronucleus formation and embryonic development of porcine oocytes reconstructed with intracytoplasmic sperm injection

Oocyte centrifugation and electrical activation are commonly used in intracytoplasmic sperm injection (ICSI) of bovine and porcine oocytes, to facilitate visual identification of sperm release into the ooplasm and to support oocyte activation following injection with tail membrane-damaged sperm. The present study evaluated the necessity of these steps in porcine modified ICSI. In the first series of experiments, in vitro-matured gilt oocytes with or without centrifugation were injected with head membrane-damaged spermatozoa aspirated tail first. Oocytes without centrifugation exhibited a significantly higher normal fertilisation rate, defined as male pronucleus (MPN) and female pronucleus (FPN) formation and the presence of two polar bodies, than centrifuged oocytes (40% v. 9%, respectively; P < 0.05). The rate of MPN formation was significantly higher in uncentrifuged oocytes compared with centrifuged oocytes (48% v. 17%, respectively; P < 0.05). The rates of survival, cleavage, blastocyst formation and total cell number in blastocysts did not differ between the two groups of oocytes. Next, the effect of electrical activation after ICSI on uncentrifuged oocytes injected with head membrane-damaged spermatozoa was determined. No significant differences were observed in the rate of MPN formation in sperm-injected oocytes regardless of electrical activation. However, the survival rates of sperm-injected or control oocytes without electrical activation were significantly higher than those of sperm-injected or control oocytes with electrical activation (88% and 84% v. 77% and 64%, respectively; P < 0.05). The cleavage rates of sperm-injected oocytes were significantly higher than those of control oocytes, regardless of electrical activation (77% and 81% v. 47% and 61% in sperm-injected and control oocytes with or without electrical activation, respectively; P < 0.05). Although development to blastocysts was similar in all experimental groups, the total cell numbers in blastocysts from control oocytes were significantly higher than those in sperm-injected oocytes, regardless of electrical activation (40 and 44 v. 22 and 26 in control and sperm-injected oocytes with or without electrical activation, respectively; P < 0.05). In conclusion, the present study clearly demonstrated that oocyte centrifugation before sperm injection is not beneficial to normal fertilisation and that electrical activation is not necessary in the modified porcine ICSI.

Low oxygen tension during in vitro maturation of porcine follicular oocytes improves parthenogenetic activation and subsequent development to the blastocyst stage

To establish a reliable in vitro maturation system for activation and subsequent development as nuclear recipients for the effective production of pig clones, we assessed maturation, activation and parthenogenetic development in response to the following: (1) type of immature oocytes (cumulus–oocyte complexes (COCs) or parietal granulosa plus cumulus–oocyte complexes (GCOCs)); (2) oxygen (O 2) tension (5 or 20%); and (3) maturation period (36–60 h). The rate of nuclear maturation to metaphase-II (M-II) in the GCOC group (73.0 ± 3.1%) was higher than that in the COC group ( P < 0.05, 60.6 ± 3.5%), but the rates did not differ between the 5 and 20% O 2 tension groups. M-II rate increased ( P < 0.05) to about 70% after 42 h and then remained constant until 60 h of culture. When oocytes were matured under 5% O 2 tension and stimulated, the rate of normal oocyte activation (a female pronucleus formation and emission of the second polar body) was higher ( P < 0.05, 38.5 ± 3.9%) than when oocytes were matured under 20% O 2 tension (24.5 ± 3.9%). On the other hand, the rate of normal activation was not significantly different between the COC and GCOC groups, and the highest ( P < 0.05) normal activation rate was obtained in oocytes cultured for 48 and 54 h (48.4 ± 5.5% and 47.9 ± 8.2%, respectively). When COC and GCOC matured for 48 h under 5 and 20% O 2 tension were stimulated and subsequently cultured in vitro for 6 days, the rate of blastocyst formation did not differ between the oocyte types nor between the O 2 tension groups. However, blastocyst quality, as measured by mean total cell number, was significantly higher in the 5% O 2 group ( P < 0.05, 34.6 ± 2.0 for COC; 33.8 ± 1.8 for GCOC) compared with the 20% O 2 group (25.9 ± 1.8 for COC; 27.0 ± 2.0 for GCOC). In conclusion, low O 2 tension (5%) during in vitro maturation of porcine oocytes promoted their ability to be activated normally and improved the quality of parthenogenetic blastocysts developed in vitro in modified NCSU-37 solutions. This knowledge may be applicable for preparation of in vitro matured oocytes with good quality as recipient oocytes for generating pig clones.

Effect of sperm preparation method on in vitro fertilization in pigs.

This study was designed to determine the effect of different sperm preparation treatments before IVF on the acrosome reaction, oocyte penetration time, early embryo development and timing of female and male pronucleus formation. Pooled sperm-rich fractions were (i) washed in PBS, (ii) left unwashed, or (iii) layered in a Percoll gradient. In Expt 1, the proportion of acrosome-reacted spermatozoa, determined by staining with fluorescein isothyocyanate-labelled peanut agglutinin lectin and propidium iodide, was highest after treatment with Percoll (P < 0.001). In Expt 2, oocytes matured in vitro were co-cultured with spermatozoa for 2, 4 or 6 h. Attached spermatozoa were then removed and the oocytes were cultured in fresh IVF medium for 16 h. Both sperm treatment and co-culture time were found to affect penetrability and monospermy rates (P < 0.001); spermatozoa treated with Percoll showed fastest oocyte penetration and highest penetrability. In Expt 3, matured oocytes were co-incubated with spermatozoa pretreated by the three above mentioned procedures (i, ii, iii) for 2, 6 and 2 h respectively. Putative zygotes were then washed and transferred to medium NCSU-23 until the blastocyst stage. In this experiment, sperm treatment had a significant effect on the cleavage rate (P < 0.001) and rate of blastocyst formation (P < 0.05); the group treated with Percoll showed the highest rate of blastocyst formation. Finally, in Expt 4, timing of female and male pronucleus formation for each sperm treatment was determined 4, 6 and 8 h after insemination. The time of female and male pronucleus formation was affected by the sperm treatment and was faster for the Percoll group (P < 0.05). The findings of the present study indicate that treatment with Percoll yields the best results in this in vitro pig embryo production system.

Effect of sperm preparation method on in vitro fertilization in pigs

This study was designed to determine the effect of different sperm preparation treatments before IVF on the acrosome reaction, oocyte penetration time, early embryo development and timing of female and male pronucleus formation. Pooled sperm-rich fractions were (i) washed in PBS, (ii) left unwashed, or (iii) layered in a Percoll gradient. In Expt 1, the proportion of acrosome-reacted spermatozoa, determined by staining with fluorescein isothyocyanate-labelled peanut agglutinin lectin and propidium iodide, was highest after treatment with Percoll (P < 0.001). In Expt 2, oocytes matured in vitro were co-cultured with spermatozoa for 2, 4 or 6 h. Attached spermatozoa were then removed and the oocytes were cultured in fresh IVF medium for 16 h. Both sperm treatment and co-culture time were found to affect penetrability and monospermy rates (P < 0.001); spermatozoa treated with Percoll showed fastest oocyte penetration and highest penetrability. In Expt 3, matured oocytes were co-incubated with spermatozoa pretreated by the three above mentioned procedures (i, ii, iii) for 2, 6 and 2 h respectively. Putative zygotes were then washed and transferred to medium NCSU-23 until the blastocyst stage. In this experiment, sperm treatment had a significant effect on the cleavage rate (P < 0.001) and rate of blastocyst formation (P < 0.05); the group treated with Percoll showed the highest rate of blastocyst formation. Finally, in Expt 4, timing of female and male pronucleus formation for each sperm treatment was determined 4, 6 and 8 h after insemination. The time of female and male pronucleus formation was affected by the sperm treatment and was faster for the Percoll group (P < 0.05). The findings of the present study indicate that treatment with Percoll yields the best results in this in vitro pig embryo production system.

Failure of male pronucleus formation is the major cause of lack of fertilization and embryo development in pig oocytes subjected to intracytoplasmic sperm injection.

The objectives of this study were 1) to compare the efficiency of intracytoplasmic sperm injection (ICSI) with and without additional artificial stimulation using frozen-thawed sperm and in vitro-matured porcine oocytes and 2) to determine the nuclear anomalies of ICSI oocytes that failed to fertilize or develop. In experiments 1 and 2, we evaluated the effects of additional activation treatments, e.g., electrical stimulus, Ca ionophore (A23187), and/or cycloheximide, on fertilization and development of ICSI porcine oocytes. Significantly higher fertilization, cleavage, and blastocyst rates were obtained for oocytes treated with a combination of ICSI and electrical activation (EA) (P < 0.05) than for those treated with ICSI alone. However, different combinations of electrical and chemical activation treatments did not further improve the rates of fertilization, cleavage, and blastocyst development for ICSI embryos. To elucidate the association between sperm head decondensation and oocyte activation and to investigate the cause of embryonic development failure, in experiment 3 we evaluated the nuclear morphology of oocytes 16-20 h after ICSI. Nearly 100% of oocytes showed female pronucleus formation after ICSI regardless of activation treatment. However, failure of male pronucleus formation with intact or swelling sperm heads was observed in some ICSI embryos, suggesting that these embryos underwent cell division with the female pronucleus only. Artificial activation (EA and A23187) had a beneficial effect on embryonic development, sperm decondensation was independent of the resumption of meiosis, and the failure of formation of a male pronucleus was the major cause for fertilization failure in porcine ICSI embryos.

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.

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.

Fertilization, embryonic development, and offspring from mouse eggs injected with round spermatids combined with Ca2+ oscillation-inducing sperm factor.

Round spermatids, precursor male gametes, are known to possess the potential to achieve fertilization and embryonic development when injected into eggs. However, injection of spermatids alone seldom activates eggs in the mouse, as spermatids by themselves cannot induce an increase in intracellular Ca2+, a prerequisite for egg activation. We injected a mouse round spermatid into an egg simultaneously with partially purified sperm factor from differentiated hamster spermatozoa. The combined injection produced repetitive Ca2+ increases (Ca2+ oscillations) lasting for at least 4 h as observed at fertilization, and induced activation in 92% of eggs. This method provided 75% fertilization success associated with male and female pronucleus formation and development to 2-cell embryos, while only 7% of eggs were fertilized by injection of a spermatid alone. Of the 2-cell embryos, approximately 50% developed to blastocysts during 5 days of culture in vitro, while no blastocysts were obtained following injection of sperm factor alone. Furthermore, the 2-cell embryos, that were created by spermatids and sperm factor and transplanted into foster mothers, developed into normal offspring, although the percentage was only 22%. All infants grew into healthy adults carrying normal chromosomes. The sperm factor served as a complementary factor for successful fertilization by round spermatid injection.

Protein phosphorylation is essential for formation of male pronucleus in bovine oocytes

This study examined the association between the morphological and protein phosphorylation events following sperm penetration of in vitro matured and in vitro fertilized bovine oocytes. Oocytes were labeled with [32P]-orthophosphate at 3 hr intervals from 3 to 18 hr of following insemination. The phosphorylation of protein complexes of 23 kDa and 18 kDa specifically increased with the formation of male and female pronuclei. In addition, oocytes were treated with 6-Dimethylaminopurine (6-DMAP) or Okadaic acid (OA) at 0, 3, 6, and 9 hr respectively following insemination. Although the formation of female pronucleus was not affected by 6-DMAP, the male pronuclear formation was completely inhibited by the presence of 6-DMAP at 0 and 3 hr of post insemination. The formation of both pronuclei was inhibited by the presence of OA at any time following insemination. These results suggest that the male pronuclear formation is associated with protein phosphorylation and that the formation of the male and the female pronuclei may involve different factors in bovine zygotes since they respond differently to the kinase modulations. Mol. Reprod. Dev. 52:43–49, 1999. © 1999 Wiley-Liss, Inc.

Time course of pronuclear formation and fertilisation after insemination in vitro and intracytoplasmic sperm injection of in vitro matured sheep oocytes.

The time course of sperm decondensation, oocyte activation, pronuclear formation and the possible causes of abnormalities after intracytoplasmic sperm injection (ICSI) and in vitro fertilisation (IVF) were examined. Frozen-thawed and pooled fresh semen from three different rams were washed and capacitated for ICSI or IVF. In vitro matured oocytes were cultured after sperm injection for 0.5, 0.75, 1, 2, 3, 4, 5, 6, 8, 18, 21 and 23 h, and oocytes were cultured after in vitro insemination for the same times other than 18 and 23 h. All oocytes were cultured in bicarbonate-buffered synthetic oviduct fluid medium (BSOF) supplemented with 2% oestrous sheep serum. A total of 746 metaphase II oocytes were injected with a single spermatozoon and 986 oocytes were inseminated for IVF. The earliest oocyte activation after ICSI was observed at 0.5 h, when 14.8% of oocytes were in anaphase II; this was earlier than after IVF, when only 6.4% of the oocytes exhibited anaphase II 1 h after insemination. Decondensing spermatozoa were first observed 1 h after ICSI and 3 h after insemination for IVF. The earliest female and male pronuclei after ICSI were observed at 2 and 3 h respectively, while the female and male pronuclei after IVF were observed at 4 h after insemination. The overall fertilisation rate was lower after ICSI (28.6%) than IVF (70.4%) but the percentage of abnormal fertilisation was not different between ICSI (8.7%) and IVF (15.2%). It was concluded that the fertilisation events were more advanced for ICSI than IVF, using injection and insemination time as reference points. The formation of male and female pronuclei were asynchronous after ICSI, in contrast to IVF when they appeared simultaneously at 4 h. Abnormalities found in fertilisation after ICSI may therefore be induced by the injection technique.

Ultrastructure of gametes after intracytoplasmic sperm injection.

The ultrastructure of 18 metaphase-II oocytes after intracytoplasmic sperm injection (ICSI) was analysed from 30 min to 8 h following the microinjection. Three control metaphase-II oocytes were not injected. The 21 oocytes were embedded individually and examined in serial pole-to-pole sections. Seventeen oocytes had been matured in vitro from the germinal vesicle stage and showed ultrastructural alterations due to long-term culture. In microinjected oocytes, membrane-bound vacuoles and oolemma inclusions were found at the injection site. The ICSI oocytes showed evidence of plasma-membrane damage and increased oocyte exchange processes with residual and multivesiculated bodies. Gamete membrane fusion was not observed. Acrosome reaction was observed within the ooplasm 15 min after ICSI. Sperm elements were not incorporated into any oocyte vacuole. Onset of oocyte activation was associated with sperm chromatin decondensation. Cortical granules were released at 60 min and two pronuclei were formed at 6 h after ICSI. The male and female pronucleus formation was asynchronous. The findings suggest specific ICSI-related morphological features of early fertilization.

Adenophostin, a potent agonist of the inositol 1,4,5-trisphosphate receptor, is useful for fertilization of mouse oocytes injected with round spermatids leading to normal offspring.

Precursor male gametes such as round spermatids and secondary spermatocytes are known to possess the potential to achieve fertilization and embryonic development when injected into mature oocytes, but in previous studies, injected mouse spermatids did not activate the oocytes. In this study, we confirmed that this was the case because spermatids by themselves could not induce an increase in intracellular Ca2+ concentration ([Ca2+]i) of oocytes, the pivotal signal in oocyte activation. Repetitive rises in [Ca2+]i (Ca2+ oscillations), lasting for at least 3 h as observed at fertilization, were produced by a single injection of adenophostin B isolated from fungal products, a novel nonmetabolizable agonist of the inositol 1,4,5-trisphosphate receptor (InsP3r), which mediates Ca2+ release from the endoplasmic reticulum. Ca2+ oscillations were blocked by an antibody against the type 1 InsP3r. About 95% of oocytes were activated by adenophostin (0.3-0.4 microM in the oocyte). Simultaneous injection of a round spermatid and adenophostin resulted in 55% fertilization success in association with male and female pronucleus formation and development to two-cell embryos. Furthermore, 25% of two-cell embryos that were transplanted to foster mothers developed to normal offspring. All infants grew into adults that reproduced healthy second generations. Adenophostin will be useful for parthenogenetic oocyte activation in the biotechnology of animal reproduction. Injection combined with precursor spermatozoa may be applicable to assisted conception therapy for patients with defective spermatogenesis.

Morphological events during in vitro fertilization of prepubertal goat oocytes matured in vitro

Experiments were carried out to study morphological changes temporally associated with in vitro fertilization (IVF) of prepubertal goat oocytes and to elucidate some of the abnormalities occurring during this process. The effects of different intervals of insemination on subsequent embryonic development were also studied. Prepubertal goat oocytes collected at slaughter were matured in TCM199 supplemented with estrous goat serum (20%), FSH (10 μg/ml), LH (10 μg/ml) and estradiol-17 β (1 μg/ml) for 27 h at 38.5 °C. Matured oocytes were inseminated with freshly ejaculated spermatozoa following capacitation as described by Younis et al. (37) but with 100 μg/ml heparin. Representative oocytes were fixed every 2 to 4 h from 2 to 28 h after insemination for a study of sperm penetration, sperm head decondensation, meiotic activation, female chromosome decondensation, and male and female pronuclear formation. At the same intervals after insemination, some of the ova were co-cultured on granulosa cell monolayers for up to 9 d. Sperm penetration into the ooplasm was first observed at 4 h post insemination; decondensation of male and female chromatin and formation of male and female pronuclei occurred at 6 to 8 and 10 to 16 h after insemination, respectively. Highest proportions of oocytes were penetrated after exposure to spermatozoa for 8 h. There were no significant differences in ovum penetration after longer insemination intervals. Cleavage was first observed 24 h after insemination. Three types of abnormalities were observed. These were polyspermy, polygyny and asynchrony in the development of the female and male pronuclei, apparently due to a delay in the decondensation of the male pronucleus. Significantly higher proportions of oocytes cleaved (31.2 to 45.5%) after 20, 24 or 28 h insemination intervals than following shorter intervals of exposure to spermatozoa. However, the sperm exposure interval did not significantly affect subsequent embryonic development to the blastocyst stage. Embryos resulting from oocytes exposed to sperm cells for at least 12 h developed further than the 8-cell stage.

Establishment of the block against sperm penetration in parthenogenetically activated bovine oocytes matured in vitro.

The ability of a single electric pulse to mimic a block against sperm penetration in bovine oocytes matured in vitro was investigated. Confocal laser scanning microscopy detected a global loss of spots, presumed to be cortical granules, stained with Lens culinaris agglutinin, in pulsed oocytes. Transmission electron microscopy revealed that cortical granule exocytosis occurred within 1 min of stimulation and the number of remaining cortical granules was significantly reduced in all pulsed oocytes. The ability of pulsed oocytes to undergo fertilization in vitro was also affected, as only 31% of the pulsed oocytes were penetrated compared with 87% in the control group. Since incidences of penetration in pulsed oocytes (31%), and of polyspermy in control oocytes (18%) did not differ and were highly correlated (P = 0.009) among trials (n = 15), the induced block is considered to be comparable with the natural block triggered by a spermatozoon. The increased resistance of the zona pellucida to pronase E observed in pulsed oocytes suggests that the induced block depends, at least partly, on modifications of zona pellucida glycoproteins. Finally, the majority (66%) of pulsed, penetrated oocytes did not form male pronuclei, probably as a consequence of asynchrony between the formation of female pronucleus and sperm penetration. The reduced ability of the cytoplasm to induce the formation of a male pronucleus was accompanied by a fall in histone H1 kinase activity to basal values by 3 h after stimulation. These results demonstrate that a single electric pulse can induce a block against sperm penetration similar to that of the spermatozoon itself.

Enhancement of the ncdD microtubule motor mutant by mutants of alpha Tub67C.

Ncd is a kinesin-related microtubule motor protein required for chromosome segregation in Drosophila oocytes and early embryos. In tests for interactions with other proteins, we find that mutants of alpha Tub67C, which affect an oocyte- and early embryo-specific alpha-tubulin, enhance meiotic nondisjunction and zygotic loss of ncdD, a partial loss-of-function mutant of ncd. The enhancement is dominant and allele-specific with respect to alpha Tub67C, and depends on the recessive effects of ncdD. Cytologically, embryos of alpha Tub67C/+ show delayed meiotic divisions and defective female pronucleus formation, while meiotic spindle assembly is abnormal in embryos of ncdD/ncdD. Doubly mutant alpha Tub67C ncdD/ncdD embryos are rescued for female pronucleus formation, but show delayed meiotic progression and defective pronuclear conjugation or fusion. Delayed completion of meiosis, together with failure of pronuclear fusion, prevents normal interactions of maternal with paternal chromosomes, enhancing the ncdD mutant phenotype. The genetics and cytology of doubly mutant embryos and the molecular defect of NcdD provide evidence for interaction of Ncd with alpha Tub67C in vivo. These results imply that a specific alpha-tubulin isoform is required for normal cellular function of a kinesin motor protein.