Full term development of rabbit oocytes fertilized by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has been applied successfully in the treatment of male infertility in humans and in fertilization research in mice. However, the technique has had limited success in producing offspring in other species including the rabbit. The aim of this research was to test the in vitro and in vivo developmental of rabbit oocytes after ICSI. Sperm used for ICSI were collected from mature Dutch Belted buck and washed 2-3 times with PBS +0.1% polyvinyl alcohol (PVA) and then mixed with 10% polyvinyl pyrrolidone (PVP) prior to microinjection. Oocytes were collected from superovulated does 14-15 hr after hCG injection and were fertilized by microinjection of a single sperm into the ooplasm of each oocyte without additional activation treatment. After ICSI, the presumed zygotes were either cultured in KSOM +0.3% BSA for 4 days or transferred into oviducts of recipient does at the pronuclear or 2-cell stage. A high percentage of fertilization (78%, n = 114) and blastocyst development (39%) was obtained after ICSI. Control oocytes, receiving a sham injection, exhibited a lower activation rate (31%, n = 51) and were unable to develop to the blastocyst stage, suggesting that the blastocysts developed following ICSI were derived from successful fertilization rather than parthenogenetic development. A total of 113 embryos were transferred to six recipient does. Two recipients became pregnant and delivered seven live young. Our results demonstrated that rabbit oocytes can be successfully fertilized and activated by ICSI and can result in the birth of live offspring.     

Thermostability of sperm nuclei assessed by microinjection into hamster oocytes

Nuclei isolated from spermatozoa of various species (golden hamster, mouse, human, rooster, and the fish tilapia) were heated at 60 degrees-125 degrees C for 20-120 min and then microinjected into hamster oocytes to determine whether they could decondense and develop into pronuclei. Mature, mammalian sperm nuclei, which are stabilized by protamine disulfide bonds, were moderately heat resistant. For example, they remained capable of pronucleus formation even after pretreatment for 30 min at 90 degrees C. Indeed, a temperature of 125 degrees C (steam) was required to inactivate hamster sperm nuclei completely. On the other hand, nuclei of rooster and tilapia spermatozoa and those of immature hamster and mouse spermatozoa, which are not stabilized by protamine disulfide bonds, were sensitive to heating; although some of them decondensed after exposure to 90 degrees C, none formed male pronuclei. Furthermore, nuclei of mature hamster sperm became heat labile when they were pretreated with dithiothreitol to reduce their protamine disulfide bonds. These observations suggest that the thermostability shown by the nuclei of mature spermatozoa of eutherian mammals is related to disulfide cross-linking of sperm protamines.     

小鼠精子注入兔卵母细胞受精研究

The methods of intracytoplasmic sperm injection (ICSI) and subzonal injection (SUZI) were used to study heterologous fertilization and embryonic development between the mouse and the rabbit. Results were as follows: 1. The mouse sperm nuclei decondensed and formed pronuclei following microinjection into cytoplasm and perivitelline space (PVS) of rabbit oocytes; 2. The hybrid embryos developed to the stage of 8-cell when cultured in vitro; 3. The karyotype analysis showed a normal complement of rabbit oocyte and mouse sperm chromosomes in the 4-cell hybrid embryos; 4. The ultrastructure of 4-cell hybrid embryos was similar to that of normal 4-cell rabbit embryos; 5. The fertilization rate (32.4%) and cleavage rate (22.2%) when 5-10 mouse spermatozoa were injected were higher than those of injection of a single spermatozoon into PVS of the rabbit oocyte, but the difference was not significant (P > 0.05). The fertilization rate (42.3%) and cleavage rate (30.8%) in rabbit oocytes in vitro matured for 11-12 h were higher than those in the oocytes which were in vitro matured for 24-25 h following microinjection of 1-2 mouse spermatozoa into PVS, but the difference was not significant (P > 0.05).     

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.     

Human sperm injection into the perivitelline space (SI-PVS) of hamster oocytes: Effect of sperm pretreatment by calcium-ionophore A23187 and freezing-thawing on the penetration rate and polyspermy

Calcium-ionophore A23187 and freezing-thawing were used as sperm treatments before human sperm injection into the perivitelline space (SI-PVS) of hamster oocytes. The penetration rate (PR) was higher when SI-PVS was performed with calcium-ionophore-treated (28%) or frozen-thawed (51%) sperm than with untreated sperm (8%). Optimal PR occurred when five calcium-ionophore-treated (57%) or frozen-thawed (71%) sperm were injected under the zona pellucida. When the sperm:egg ratio was 1:1, PR was higher for calcium-ionophore-treated (18.5%) or frozen-thawed (27.8%) sperm than for untreated sperm (0.0%). Calcium-ionophore sperm treatment had no effect on the polyspermic oocyte rate (POR) or the mean number of swollen sperm nuclei per penetrated oocyte (Pd) or per injected sperm (SR). This may result from premature oocyte activation induced by Ca-ionophore. However, POR was higher with frozen-thawed (74%) than with untreated (50%) or Ca-ionophore-treated (50%) sperm. Whatever the sperm treatment, there was a trend toward a lower SR as the number of injected sperm increased. Cytoplasmic regulation of polyspermy in the hamster oocyte is discussed.     

Immunolocalization of proacrosin/acrosin in rabbit sperm during acrosome reaction and in spermatozoa recovered from the perivitelline space

The participation of acrosin in mammalian sperm penetration through the zona pellucida has been amply debated. In this paper we report the immunolocalization—by silver enhanced immunogold technique using ACRO-8C10 monoclonal antibody to human acrosin—of proacrosin/acrosin on ejaculated rabbit spermatozoa incubated in vitro in a capacitating medium and on spermatozoa recovered from the perivitelline space. After incubation in a capacitating medium, four different patterns were observed: (1) no labeling on acrosome intact spermatozoa; (2) labeling on the rim of the head; (3) labeling on the whole acrosome area; and (4) no labeling on acrosome reacted spermatozoa. At the start of incubation, spermatozoa with pattern 1 were the most abundant, whereas at the end of the 32 h incubation period, patterns 2 and 3 were the most frequent. On the other hand, 625 perivitelline spermatozoa were recovered from 17 fertilized rabbit eggs, of which 26% were labeled with the anti-acrosin monoclonal antibody ACRO-8C10 in two different areas: (1) only on the equatorial region; and (2) only on the postacrosomal area. These results are consistent with the idea that proacrosin/acrosin remains associated to the acrosome reacted spermatozoa for long periods of time, and that proacrosin/acrosin associated to perivitelline spermatozoa could be responsible for the second penetration of fresh rabbit eggs by perivitelline spermatozoa. © 1994 Wiley-Liss, Inc.     

production of transgenic rodents by the microinjection of cloned DNA into fertilized one-cell eggs

Transgenic technologies that enable rapid movement between genotype and phenotype through specific loss-of-function, overexpression, or misexpression phenotypes will be crucial in the elucidation of gene sequences emerging from genome projects. This article describes detailed procedures for the generation of transgenic mice and rats by the injection of cloned DNA into the pronuclei of fertilized one-cell eggs.     

In vitro fertilization of hamster and human oocytes by microinjection of human sperm

In this study, swollen sperm heads were obtained after the injection of human sperm into the perivitelline space of hamster oocytes. The number of injected sperm and the sperm concentration in the preincubation medium were found to have an influcnce on the rate of penetrated hamster oocytes. The optimal injected sperm number was always between five to 12 to obtain 8, 37, and 36% penetration for donors A, B, and C, respectively. The optimal sperm concentration in preincubation medium was between 6 and 22 × 10⁶ sperm/ ml to obtain 16, 47, and 43% penetration for donors A, B, and C, respectively. The rate of polyspermic oocytes was related to the injected sperm number (0, 55, and 100% for one to four, five to 12, and more than 12 injected sperm respectively). Ten human mature oocytes were injected with the sperm from six normal donors. Five fertilized eggs were obtained, and of these four cleaved in in vitro culture.     

Developmental potential of oocytes fertilized by conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) after cryopreservation and mesometrial autotransplantation of rabbit ovarian tissue

Objective: To evaluate mesometrial transplantation of frozen-thawed ovarian tissue in rabbit and to choose the optimized fertilization method for oocytes retrieved from grafts by investigating the capability of oocyte fertilization and further development. Forty rabbits were divided into three groups randomly: control group, fresh tissues transplantation group and frozen-thawed tissues transplantation group. Three months after the transplantation, rabbits were stimulated with FSH and oocytes were retrieved 13 h after human chorionic gonadotropin (HCG) injection. Oocytes matured in vivo or in vitro were then fertilized by conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), followed by observation and evaluation of fertilization rate and blastocyst formation rate. Blastocytes embryos were transferred to pseudopregnancy rabbits to observe pregnancy rate and birth rate. There were no significant differences in the percentage of oocytes matured either in vivo or in vitro among the three groups. The fertilization rate, cleavage rate and blastocyst formation rate of in vivo-matured oocytes had no difference among the three groups, whether they were fertilized by IVF or ICSI. Significantly higher fertilization rates of in vitro-matured oocytes were observed with ICSI compared with IVF in each group. The blastocyst formation rate of in vitro-matured oocytes was significantly lower than that of in vivo-matured oocytes in each group. The birth rate of in vivo-matured oocytes was significantly higher than that of in vitro-matured oocytes, although the pregnancy rate was similar between them. Mesometrial transplantation of frozen-thawed ovarian tissue may provide favorable conditions for follicle development. Oocytes retrieved from mesometrial grafts can develop to the blastocyst stage and produce live offspring. ICSI can optimize the fertilization rate of in vitro-matured oocytes retrieved from grafts.     

Activation of mammalian oocytes by a factor obtained from rabbit sperm

In this study a fraction was prepared from rabbit sperm that activated rabbit and mouse oocytes following injection into the cytoplasm. The sperm factor activated oocytes exhibited cortical granule exocytosis, pronuclear formation, and cleavage. The sperm factor was soluble in aqueous solution and was not active extracellularly. Unlike most artificial activation methods that are only effective with aged oocytes, the sperm factor activated recently ovulated oocytes. The factor appears to be a protein or associated with a protein but not an acrosomal protein. Fractions from both mouse and bull sperm did not activate rabbit or mouse oocytes. Their inactivity may be owing to the techniques used to recover the fractions or differences between species in sperm morphology and fertilization processes. These observations support the hypothesis that oocyte activation is induced by a factor within sperm that is released into the cytoplasm of the oocyte at the time of sperm-oocyte fusion.     

Selective sequestration of an oviductal fluid glycoprotein in the perivitelline space of mouse oocytes and embryos

Previously, we identified a 215 kd glycoprotein, GP215, which is associated with postovulatory oocytes and embryos, but not with preovulatory oocytes (Kapur and Johnson, '85). In this paper a polyclonal antibody that specifically recognizes GP215 has been used to study the distribution of the molecule in association with ova and preimplantation embryos and in the female reproductive tract. GP215 is present in epithelial cells lining the cranial portions of the oviduct and in oviductal fluid, ovarian bursal fluid, and medium conditioned by oviductal tissue in vitro. Immunofluorescence assays of the ovum and early embryo show that GP215 is sequestered in the perivitelline space. Since preovulatory oocytes exposed to bursal fluid in vitro acquire GP215, we hypothesize that GP215 is synthesized and secreted by the oviductal epithelium and secondarily associates with the ovulated oocyte. Sequestration of GP215 within the perivitelline space is relatively specific since mouse serum albumin, a major constituent of oviductal fluid, and other high molecular weight proteins are not similarly retained. These observations indicate that the composition of the perivitelline space may be significantly different from the greater environment external to the zona pellucida such that fertilization and early development of mammalian ova potentially take place in a distinct perivitelline microenvironment.     

Maternal restraint stress diminishes the developmental potential of oocytes

Although studies of both humans and animals suggest detrimental effects of psychological (restraint) stress on reproduction, reports concerning the direct effect of psychological (restraint) stress on the oocyte are few and conflicting. In the present study, a restraint system that allows mice free intake of feed and water while restraining their movement was established, and effects of maternal restraint on oocyte competence were examined by observing embryo development in vitro and in vivo. The results indicated that restraint stress applied to both gonadotropin-stimulated and unstimulated females during oocyte growth and maturation increased their plasma cortisol level but impaired ovulation and oocyte developmental potential. Injection of cortisol also decreased oocyte developmental potential in both stimulated and unstimulated mice. However, whereas restraint stress reduced the plasma follicle-stimulating hormone (FSH) level of unstimulated mice, injection of cortisol did not. Because the stimulated mice had received very high doses of FSH and luteinizing hormone from injection with equine chorionic gonadotropin injection, the results suggested that whereas cortisol acts directly on the ovary to damage the oocyte, restraint stress impairs oocyte competence by actions on both the hypothalamic-pituitary-gonadal and the hypothalamic-pituitary-adrenal axes. However, exposing the cumulus-oocyte complexes (COCs) to physiological levels of cortisol did not affect oocyte nuclear and cytoplasmic maturation in vitro. Thus, cortisol might have impaired ovulation and oocyte potential by an indirect effect on ovarian tissues other than the COCs.     

Resveratrol improved the developmental potential of oocytes after vitrification by modifying the epigenetics

Resveratrol (Res) has been reported to be able to improve oocyte vitrification because of its antioxidative properties. The objective of this study was to further assess the positive effect of Res addition on the developmental potential of vitrified mouse oocytes from the perspective of epigenetic alterations. First, 2 μM Res was chosen as the optimal concentration on the basis of its effects on survival and its antioxidative properties. We found that Res addition significantly promoted fertilization (63.8% vs. 42.9%) and blastocyst formation (68.3% vs. 50.2%) after oocyte vitrification. The quality of the derived blastocysts was also higher after Res treatment. Regarding epigenetic aspects, the expression of the important deacetylase SIRT1 was found to decrease significantly upon vitrification, but it was rescued by Res. The abnormal levels of H3K9 acetylation and DNA methylation in vitrified oocytes were restored by Res addition. Moreover, the expression of several imprinted genes was affected by oocyte vitrification. Among them, abnormal Gtl2 and Peg3 expression levels were restored by Res addition. Therefore, the methylation of their imprinted control regions (ICRs) was examined. Surprisingly, the abnormal patterns of Gtl2 and Peg3 methylation in blastocysts developed from vitrified oocytes were both restored by Res addition. Finally, the full‐term embryonic development showed that the birth rate was improved significantly by Res addition (56.2% vs. 38.1%). Collectively, Res was beneficial for the pre‐ and postimplantation embryonic development. Except for the antioxidative activity, Res also played a role in the correction of some abnormal epigenetic modifications caused by oocyte vitrification.     

Production of mouse chimeras by injection of embryonic stem cells into the perivitelline space of one-cell stage embryos

Generation of mouse chimeras is useful for the elucidation of gene function. In the present report, we describe a new technique for the production of chimeras by injection of R1 embryonic stem (ES) cells into the perivitelline space of one-cell stage mouse embryos. One-cell embryos are injected with 2–6 ES cells into the perivitelline space under the zona pellucida without laser-assistance. Our embryo culture experiments reveal that ES cells injected at the one-cell stage embryo start to be incorporated into the blastomeres beginning at the 8-cell stage and form a chimeric blastocyst after 4 days. We have used this approach to successfully produce a high rate of mouse chimeras in two different mouse genetic backgrounds permitting the establishment of germ line transmitters. This method allows for the earlier introduction of ES cells into mouse embryos, and should free up the possibility of using frozen one-cell embryos for this purpose.     

Morphological differences in nuclear materials released from hamster sperm heads at an early stage of incorporation into immature oocytes,mature oocytes,or fertilized eggs

To elucidate the effects of ooplasmic factors on the early morphological changes in hamster sperm heads within the ooplasm, immature ovarian oocytes at the germinal vesicle stage (GV oocytes), ovulated fully mature oocytes, and fertilized eggs at anaphase II or the pronuclear stage (PN eggs) were examined in detail 15–30 min after insemination or reinsemination. Thin-sectioning studies demonstrated distinct materials released from the sperm nucleus over the entire postacrosomal nuclear surface immediately after disappearance of the sperm nuclear envelope. The release occurred in all of the oocytes and eggs prior to or even in the absence of subsequent chromatin decondensation. Depending upon the stage of the penetrated oocyte or egg, however, the materials varied in morphology: several hemispherical projections of amorphous material within mature oocytes; a number of electron-dense globules within GV oocytes and PN eggs; and both forms within eggs at anaphase II-telophase II. These observations and the fact that only the release of the amorphous material was accompanied by sperm chromatin decondensation indicate that this release was the initial process of chromatin decondensation, whereas the release of the globules resulted from a deficiency or lack of ooplasmic factors affecting the sperm nucleus. Restriction of the release in both forms of material to the late meiotic phase suggests changes in the factors associated with progression of meiosis. To approach an understanding of the mechanism of successful decondensation of sperm chromatin, the ooplasmic factors considered responsible for the stage-dependent release of nuclear materials are discussed. © 1996 Wiley-Liss, Inc.     

Blastocyst formation rates in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection

This study was conducted to evaluate in vivo and in vitro development of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. Oocytes were collected from slaughterhouse-derived ovaries, matured in vitro, and injected with frozen-thawed stallion sperm. In vivo development was assessed after transfer of injected oocytes to the oviducts of recipient mares. Mares were killed 7.5-8.5 days after transfer and the uterus and oviducts flushed for embryo recovery. Of 132 injected oocytes transferred, 69 (52%) were recovered; of these, 25 (36%) were blastocysts with a blastocoele and capsule. In vitro development was assessed in three culture systems. Culture of zygotes in modified Chatot, Ziomek, Bavister medium with BSA containing either 5.5 mM glucose for 7.5 days or 0.55 mM glucose for 3 days, followed by 3 mM glucose for 2 days, then 4.3 mM glucose for 2.5 days, did not result in blastocyst formation. Culture of zygotes in Dulbecco modified Eagle medium (DMEM)/F-12 with 10% fetal bovine serum with and without coculture with equine oviductal epithelial explants yielded 16% and 15% blastocyst development, respectively. Development to blastocyst was significantly lower in G1.3/2.3/BSA than in DMEM/F-12/BSA or in either medium with 10% added serum (2% vs. 18%, 18% or 20%; P < 0.05), suggesting that requirements for equine embryo development differ from those for other species. These results indicate that in vitro-matured equine oocytes are sufficiently competent to form 36% blastocysts in an optimal environment (in vivo). While we identified an in vitro culture system that provided repeatable blastocyst development without coculture, this yielded only half the rate of development achieved in vivo.     

Analysis of the intracellular localization and assembly of Ro ribonucleoprotein particles by microinjection into Xenopus laevis oocytes

Xenopus laevis oocytes have been used to determine the intracellular localization of components of Ro ribonucleoprotein particles (Ro RNPs) and to study the assembly of these RNA-protein complexes. Microinjection of the protein components of human Ro RNPs, i.e., La, Ro60, and Ro52, in X. laevis oocytes showed that all three proteins are able to enter the nucleus, albeit with different efficiencies. In contrast, the RNA components of human Ro RNPs (the Y RNAs) accumulate in the X. laevis cytoplasm upon injection. Localization studies performed at low temperatures indicated that both nuclear import of Ro RNP proteins and nuclear export of Y RNAs are mediated by active transport mechanisms. Immunoprecipitation experiments using monospecific anti-La and anti-Ro60 antibodies showed that the X. laevis La and Ro60 homologues were cross-reactive with the respective antibodies and that both X. laevis proteins were able to interact with human Y1 RNA. Further analyses indicated that: (a) association of X. laevis La and Ro60 with Y RNAs most likely takes place in the nucleus; (b) once formed, Ro RNPs are rapidly exported out of the nucleus; and (c) the association with La is lost during or shortly after nuclear export.     

Maturity at collection and the developmental potential of rhesus monkey oocytes

The purpose of this study was to evaluate the in vitro fertilizability of rhesus monkey oocytes and the developmental capacity of the resulting embryos as they relate to oocyte maturation at the time of follicular aspiration. Animals were hyperstimulated with human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH), with follicular aspiration performed 27 h after administration of an ovulatory stimulus (1000 IU human chorionic gonadotropin [hCG] or 3 x 100 micrograms gonadotropin-releasing hormone [GnRH]). In 7 animals exhibiting a continuously rising pattern of serum estradiol through Day 10 of hyperstimulation, 45 germinal vesicle-intact (GV), 106 metaphase I (MI), and 24 metaphase II (MII) oocytes were collected and cultured in vitro. Upon reaching MII, oocytes were inseminated with 5 x 10(4) motile sperm/ml. Twenty-four percent of GV oocytes cultured in vitro matured to MII with 11 inseminated and none fertilized. Seventy-three percent of MI oocytes matured to MII in vitro with 50% inseminated and 32% fertilized. Oocytes collected at MII stage and inseminated underwent fertilization at a high rate of efficiency (93%). Pronuclear to 8-cell stage embryos were frozen and, upon thawing, 67% (10/15) survived with all blastomeres intact. Frozen-thawed embryos (2- to 6-cell) were transferred to the oviducts of 4 recipients (2 embryos/recipient) during the early luteal phase (1-3 days post LH surge) of natural menstrual cycles. Three twin pregnancies resulted. Thus, a positive correlation exists between the degree of nuclear maturation of rhesus monkey oocytes at collection and their potential for fertilization in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)