Hybrid Oocytes Research Articles

Kinetochore deterioration concommitant with centromere weakening during aging in mouse oocyte meiosis-I.

Age-related oocyte aneuploidy occurs as a result of chromosome segregation errors in female meiosis-I and meiosis-II, and is caused by a progressive age-related deterioration of the chromosome segregation machinery. Here, we assess the impact of age upon the kinetochore, the multi-protein structure that forms the link between the chromosome and spindle microtubules. We find that in meiosis-I the outer kinetochore assembles at germinal vesicle breakdown, but that a substantially smaller outer kinetochore is assembled in oocytes from aged mice. We show this correlates with a weaker centromere in aged oocytes and, using nuclear transfer approaches to generate young-aged hybrid oocytes, we show that outer kinetochore assembly always mirrors the status of the centromere, regardless of cytoplasmic age. Finally, we show that weaker kinetochores in aged oocytes are associated with thinner microtubule bundles, that are more likely to be mis-attached. We conclude that progressive loss of the centromere with advancing maternal age underpins a loss of the outer kinetochore in meiosis-I, which likely contributes to chromosome segregation fallibility in oocytes from older females.

Genotypic divergence in mouse oocyte transcriptomes: possible pathways to hybrid vigor impacting fertility and embryogenesis.

What causes hybrid vigor phenotypes in mammalian oocytes and preimplantation embryos? Answering this question should provide new insight into determinants of oocyte and embryo quality and infertility. Hybrid vigor could arise through a variety of mechanisms, many of which must operate through posttranscriptional mechanisms affecting oocyte mRNA accumulation, stability, translation, and degradation. The differential regulation of such mRNAs may impact essential pathways and functions within the oocyte. We conducted in-depth transcriptome comparisons of immature and mature oocytes of C57BL/6J and DBA/2J inbred strains and C57BL/6J × DBA/2J F1 (BDF1) hybrid oocytes with RNA sequencing, combined with novel computational methods of analysis. We observed extensive differences in mRNA expression and regulation between parental inbred strains and between inbred and hybrid genotypes, including mRNAs encoding proposed markers of oocyte quality. Unique BDF1 oocyte characteristics arise through a combination of additive dominance and incomplete dominance features in the transcriptome, with a lesser degree of transgressive mRNA expression. Special features of the BDF1 transcriptome most prominently relate to histone expression, mitochondrial function, and oxidative phosphorylation. The study reveals the major underlying mechanisms that contribute to superior properties of hybrid oocytes in a mouse model.

Laser assisted IVF: A simplified technique to improve zygote production rates without the need for ICSI

Background/Aim: Spermatozoa from inbred mice provide a useful model to study male infertility as they produce a low number of zygotes with conventional IVF. This study aimed to assess whether the use of zona laser dissection might improve the number of zygotes developing after conventional IVF with inbred spermatozoa. Materials & Methods: Mouse oocytes from an inbred C57BL/6J strain and a hybrid B6D2F1 strain were allocated to either conventional IVF or laser assisted IVF, using spermatozoa from the inbred strain C57BL/6J. Laser assisted IVF involved use of a 1.48 diode microsurgical laser device to ablate three holes into each zona immediately prior to insemination. Results: Use of a laser significantly improved the zygote production rate compared with zona intact oocytes (88.4% vs. 18.3% for inbred oocytes, and 92.3% vs. 1.6% for hybrid oocytes, P Conclusion: Zona laser dissection is a simple technique that avoids the need for ICSI, significantly reduces operator time and allows numerous oocytes to be treated in a short period. Since high numbers of zygotes can develop using laser assisted IVF for this animal model where the spermatozoa are of poor quality, one might speculate that laser assisted IVF may have a possible role in the treatment male infertility in a clinical setting.

Strain Specific Spontaneous Activation During Mouse Oocyte Maturation.

Objective To examine whether spontaneous oocyte activation is determined by genetic differences and interacts with culture environment. Design Experimental study. Setting Temple University School of Medicine. Animal(s) C57BL/6, DBA/2, C3H/HeJ, and A/J strains, along with reciprocal F1 hybrid female mice (5–6 weeks). Intervention(s) Immature oocytes from different mouse strains collected and cultured in different maturation conditions, including different serum, serum replacement, bovine serum albumin (BSA), and follicle-stimulation hormone (FSH). Main Outcome Measure(s) The emission of first polar body, pronucleus formation, meiotic arrest, spontaneous activation, and expression of maturation regulators. Result(s) Oocytes from C57BL/6 mice display a high rate of delayed first meiotic division and spontaneous activation after the first meiotic division with in vitro maturation (IVM), and the second meiotic division with in vivo maturation (VVM) after superovulation. Spontaneous activation with IVM is sensitive to culture environment. Oocytes that spontaneously activated during the first meiotic division with IVM have unusual replicated sister chromatid pairs with slight connections at centromeres at first mitosis, whereas oocytes that activated in vivo display haploidization from the second meiotic division. Spontaneous activation is also seen in F1 hybrid oocytes, indicating a dominant trait from C57BL/6. Delayed meiosis was associated with reduced cyclin B and securin expression. Conclusion(s) Both mouse strain and culture environment have a statistically significant effect on the incidence of meiotic defects and spontaneous activation. Reduced expression of meiotic regulators may underlie this effect.

Strain-specific spontaneous activation during mouse oocyte maturation

To examine whether spontaneous oocyte activation is determined by genetic differences and interacts with culture environment. Experimental study. Temple University School of Medicine. C57BL/6, DBA/2, C3H/HeJ, and A/J strains, along with reciprocal F1 hybrid female mice (5-6 weeks). Immature oocytes from different mouse strains collected and cultured in different maturation conditions, including different serum, serum replacement, bovine serum albumin (BSA), and follicle-stimulation hormone (FSH). The emission of first polar body, pronucleus formation, meiotic arrest, spontaneous activation, and expression of maturation regulators. Oocytes from C57BL/6 mice display a high rate of delayed first meiotic division and spontaneous activation after the first meiotic division with in vitro maturation (IVM), and the second meiotic division with in vivo maturation (VVM) after superovulation. Spontaneous activation with IVM is sensitive to culture environment. Oocytes that spontaneously activated during the first meiotic division with IVM have unusual replicated sister chromatid pairs with slight connections at centromeres at first mitosis, whereas oocytes that activated in vivo display haploidization from the second meiotic division. Spontaneous activation is also seen in F1 hybrid oocytes, indicating a dominant trait from C57BL/6. Delayed meiosis was associated with reduced cyclin B and securin expression. Both mouse strain and culture environment have a statistically significant effect on the incidence of meiotic defects and spontaneous activation. Reduced expression of meiotic regulators may underlie this effect.

Improving in vitro development of cloned bovine embryos with hybrid (Holstein–Chinese Yellow) recipient oocytes recovered by ovum pick up

In the present study, oocytes from F1 hybrid cattle, as well as their parental lines, were recovered by ovum pick up (OPU) and used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Four F1 hybrid (Holstein dam × Chinese Yellow sire), 10 Holstein and four Chinese Yellow cattle were subjected to OPU once weekly. There were no significant differences among breeds for number of recovered oocytes per session (overall average, 7.8 ± 0.5; mean ± S.E.M.), quality of the recovered oocytes, or oocyte maturation rate (72–73%). Matured oocytes were all used as recipient cytoplasm (without selection) and a single batch of cumulus cells collected from a Holstein cow were used as donor cells. Although reconstructed embryos initiated cleavage sooner when the recipient cytoplasm was from hybrid cattle versus the two parental breeds, the overall cleavage rate was indistinguishable among breeds. At Day 8, the blastocyst rate from the cleaved embryos (51% versus 37% and 27%), the total number of cells per blastocyst (135 ± 4.1 versus 116 ± 3.6 and 101 ± 4.2), and the percentage of Grade-A (excellent quality) blastocysts (54% versus 42% and 29%) in the hybrid group were all higher than that of Holstein and Yellow groups. Furthermore, the proportion of blastocysts obtained at Day 7 (as a percentage of the total number of blastocysts) was greater in the hybrid group than in Holstein and Yellow groups (89% versus 71% and 63%). In conclusion, the use of F1 hybrid oocytes as recipient cytoplasm significantly improved in vitro development of cloned bovine embryos relative to oocytes derived from the parental lines.

Chromosome analysis of human spermatozoa with morphologically abnormal heads by injection into mouse oocytes.

Aim: The risk of chromosome aberrations in human spermatozoa with an abnormal head shape was examined using intracytoplasmic sperm injection into mouse oocytes. Methods: Human spermatozoa with small, large, pointed or elongated head shape from a fertile donor were injected into mouse oocytes, and the hybrid oocytes were cytogenetically analyzed at the first cleavage metaphase. Results: The oocyte activation rate was significantly lower in hybrid oocytes injected with pointed (90.2%) or elongated spermatozoa (94.1%) than with normal spermatozoa (99.0%). However, the frequency of intracytoplasmic sperm injection oocytes at the first cleavage metaphase did not differ among the sperm groups (71.8-77.2%). No difference in the incidences of aneuploidy (1.8, 2.6 and 1.4%), diploidy (0, 0 and 0%) and structural chromosome aberrations (6.3, 10.4 and 8.6%) was observed between small or pointed spermatozoa and normal spermatozoa. Only a small population of large spermatozoa (3.7%) was diploidy. Elongated spermatozoa showed significantly frequent structural chromosome aberrations (33.3%) as compared with normal spermatozoa. Conclusion: The results reveal some interesting details of the mechanism of sperm nuclear condensation: small sperm size is attributed to the problem of nuclear condensation, not a decrease in chromosomes. Diploidy prevents sperm nucleus from condensing, resulting in a large sperm head. Elongation of sperm nucleus causes DNA lesions leading to structural chromosome aberrations. (Reprod Med Biol 2004; 3: 147-152).

Frequent structural chromosome aberrations in immotile human sperm exposed to culture media.

The influence of culture media or centrifugation on chromosomes of immotile human sperm was examined using ICSI into mouse oocytes. In experiment 1, immotile and motile human sperm retrieved directly from ejaculates were injected into mouse oocytes. In experiment 2, immotile human sperm were exposed to seminal plasma or one of four kinds of culture media (HEPES-BWW, modified-BWW, modified-human tubal fluid (HTF) and Dulbecco's phosphate-buffered saline) for 1.5-2.5 h at 18 degrees C in air before microinjection. In experiment 3, immotile human sperm were centrifuged along with HEPES-BWW before microinjection. In experiment 4, frozen-thawed immotile human sperm washed with seminal plasma or HEPES-BWW were injected into mouse oocytes. The hybrid oocytes were prepared for chromosome slides at first cleavage metaphase and were then examined cytogenetically. In experiment 1, there was no significant difference in the incidences of structural chromosome aberrations between motile and immotile sperm (4.3% versus 5.8%). In experiment 2, culture media caused more frequent structural chromosome aberrations (14.3-32.6%) in immotile sperm than did seminal plasma (5.4%). In experiment 3, structural chromosome aberrations were found in 48.1% of the centrifuged immotile sperm, and a live/dead sperm viability test intimated that the aberrant sperm were probably dead. In experiment 4, the incidence of structural chromosome aberrations in frozen-thawed immotile sperm was significantly higher in HEPES-BWW (62.2%) than in seminal plasma (17.2%). The results indicate that immotile sperm do not have significantly more DNA lesions than motile sperm, although DNA of immotile sperm appears to be vulnerable to damage caused by different culture media.

A detailed cytogenetic analysis of large numbers of fresh and frozen-thawed human sperm after ICSI into mouse oocytes.

Since information about chromosome aberrations in micro-manipulated sperm is still inadequate, cytogenetic analysis was performed on large numbers of fresh and frozen-thawed (FT) human sperm after injection into mouse oocytes. The effects of the ICSI procedure on oocytes are also discussed based on analysis of the mouse chromosome complements. After the injection of fresh and FT human sperm into mouse oocytes, chromosomes of the hybrid oocytes were analysed at first cleavage metaphase. Incidences of the hybrid oocytes at the first cleavage metaphase were significantly different between fresh (71.5%) and FT sperm groups (80.1%) (P < 0.05). The chromosome analysis of 477 fresh and 141 FT sperm showed no difference in the incidences of aneuploidy (1.6/0.7%), structural aberrations (8.8/7.8%) or diploidy (0.0/0.0%) between these categories. The cytogenetic result did not differ from our previous result using IVF between human sperm and hamster oocytes. In an additional cytogenetic study on 615 mouse chromosome complements, the incidence of diploidy (5.4%) was significantly higher than those (0.3-2.8%) in the previous mouse cytogenetic studies, and the hybrid oocytes with no mouse chromosomes (2.0%) existed. This result suggests that the ICSI procedure induces no sperm chromosome aberrations but increases numerical aberrations in oocyte chromosome complements.

Evidence that Meishan and large-white hybrid preovulatory follicles may differentially affect oocyte in vitro maturation and fertilization.

Two experiments were carried out to test the hypothesis that follicles recovered from Meishan animals may provide a more favourable environment for oocyte maturation in vitro than follicles recovered from Large-White hybrid animals. In Experiment 1, all follicles > or = 4 mm were recovered from six Meishan and seven Large-White hybrid gilts in the late follicular phase and healthy oocyte cumulus complexes recovered. Cumulus oocyte complexes were randomly divided into two groups, and each group cultured for 27 or 34 h (62 and 64; 56 and 56 for Meishan and Large-White hybrid, respectively) in defined medium in the presence of either of the two largest follicle shells per animal. Subsequent examination of oocyte nuclear maturation showed that although maturation did not differ significantly between the breeds after 27 h, more (P < 0.01) Meishan oocytes co-cultured with Meishan follicles developed to metaphase II stage than Large-White hybrid oocytes co-cultured with Large-White hybrid follicles after 34 h. The next eight largest follicles per animal were cultured for 34 h to produce conditioned media. In Experiment 2, oocytes recovered from the slaughterhouse were matured for 46 h in the presence of conditioned media from Meishan (612 oocytes) or Large-White hybrid (731 oocytes) follicles, or in fresh medium in the presence of a follicle shell from slaughterhouse ovaries. Oocytes were then inseminated and 12 h later examined for penetration and male pronuclear formation. A higher (P < 0.05) percentage of oocytes cultured in Meishan follicle conditioned medium underwent sperm penetration and male pronuclear formation than oocytes cultured in conditioned media from Large-White hybrid animals. Concentration of oestradiol and progesterone in the conditioned media did not differ between the breeds (P > 0.1). In conclusion, these results suggest that (1) Meishan oocytes have advanced maturational capacity when cultured with Meishan preovulatory follicle shells and (2) differences in follicle maturation in the Meishan compared to the Large-White hybrid pig may result in an improved ability of the follicles, via conditioned media, to support oocyte maturation and fertilization in vitro.

Control of germinal vesicle breakdown in bovine x murine hybrid oocytes.

Bovine oocytes cultured in control medium or in medium containing dibutyrylcyclic adenosine monophosphate (dbcAMP) or an inhibitor of cyclic nucleotide phosphodiesterase (3-isobutyl-1-methylxanthine, IBMX) undergo germinal vesicle breakdown (GVBD). On the other hand, mouse oocytes remain arrested at the germinal vesicle (GV) stage when dbcAMP or IBMX is present. When 1 bovine GV stage oocyte is fused to 1 GV stage mouse oocyte, dissolution of both species GV occurred in dbcAMP-supplemented medium. Only when 4 to 5 GV stage mouse oocytes are fused to 1 GV stage bovine oocyte, and these giant cells are cultured in dbcAMP-medium, is maturation arrested with only GVs present in the cytoplasm. The inhibitory effect is more evident in IBMX-supplemented medium. Here nearly 50% of the fused cells exhibit GVs, both mouse and bovine, when 1 cattle GV oocyte is fused to 1 mouse GV oocyte and the fused cells are cultured for 24 h. Moreover, nearly all GVs are well preserved after fusion of 1 bovine oocyte to 2 or more mouse oocytes. When these hybrid cells after 24 h culture in IBMX are then washed and cultured in control medium for a further 24 h, GVBD occurred in all cells. We are of the opinion that this novel approach (ie mixing of sensitive and non-sensitive cytoplasm) may in the future better explain the mechanisms involved in the regulation of mammalian oocyte maturation.

Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at --196 degrees C.

Unfertilized mouse oocytes were frozen and stored at --196 degrees C. Immediately after thawing 331 out of 463 MF1 oocytes (71-5%) and 271 out of 410 F1 (C57BL X CBA) hybrid oocytes (66-1%) were morphologically normal. No significant difference was found between the survival of oocytes frozen and thawed with (70%) or without (66%) the surrounding cumulus cells. Fertilization in vitro of frozen-thawed oocytes was significantly lower than that of freshly collected control oocytes. The overall fertilization rate in vitro for MF1 oocytes was lower than with F1 (C57BL X CBA) hybrid oocytes. The presence or absence of cumulus cells during fertilization in vitro did not affect the fertilization rate. Live 14-day fetuses were obtained after transfer of frozen-thawed unfertilized oocytes directly to the oviducts of females mated with fertile males. However, much higher rates of survival (up to 45%) to 14-day fetuses and live-born were found after the fertilization of frozen-thawed oocytes in vitro and subsequent transfer at the 2-cell or blastocyst stage to pseudopregnant recipients.

H-gene (histocompatibility) mutations induced by triethylenemelamine in the mouse

Based on a simple dermal-graft procedure, the H-test for histo-compatibility mutations screens the mouse “H-system” of not less than 29 and perhaps considerably more than 100 loci that are scattered throughout the genome. Graft-tests with normally heterozygous or hemizygous loci permit scoring mutations as gains, losses, and gains+losses. Tests with normally homozygous loci screen only for gains, but further analysis can detect accompanying losses. Assayed by the H-test, triethylenemelamine (TEM) increased the spontaneous mutation rate per generation by approximately four- to five-fold in the case of BALB/c spermatogonia and F 1 hybrid oocytes (BALB/c females × C57BL/6 males), but had a much smaller effect, if any, on the rate of C57BL/6 spermatogonia. Single intraperitoneal doses of 2.4–4.0 mg/kg were given.