Studies on the fertilization of mouse eggs in vitro.

The association of abnormal chromosome constitutions and disorders of sex development in domestic animals has been recorded since the beginnings of conventional cytogenetic analysis. Deviated karyotypes consisting of abnormal sex chromosome sets (e.g. aneuploidy) and/or the coexistence of cells with different sex chromosome constitutions (e.g. mosaicism or chimerism) in an individual seem to be the main causes of anomalies of sex determination and sex differentiation. Molecular cytogenetics and genetics have increased our understanding of these pathologies, where human and mouse models have provided a substantial amount of knowledge, leading to the discovery of a number of genes implicated in mammalian sex determination and differentiation. Additionally, other genes, which appeared to be involved in ovary differentiation, have been found by investigations in domestic species such as the goat. In this paper, we present an overview of the biology of mammalian sex development as a scientific background for better understanding the body of knowledge of the clinical cytogenetics of disorders of sex development in domestic animals. An attempt to summarize of what has been described in that particular subject of veterinary medicine for each of the main mammalian domestic species is presented here.

Classical and molecular cytogenetics of disorders of sex development in domestic animals.

Chromosomes of the pig.

To investigate whether cytogenetic risks occur using the mouse intracytoplasmic sperm injection (ICSI) technique, the incidence of chromosome aberrations was compared in one-cell embryos produced by ICSI technique and those by conventional in vitro fertilization (IVF) technique. Spermatozoa were incubated in TYH medium for 1.5-2 h before IVF insemination. For the ICSI technique, spermatozoa were incubated in five different media: TYH, Hepes-buffered TYH (H-TYH), modified CZB (mCZB), Hepes-buffered mCZB (H-mCZB), and PB1 for 0.5 h, 2-2.5 h, and 6 h before injection into metaphase II oocytes. The incidence of IVF embryos with structural chromosome aberrations was 2%, whereas the occurrence of structural chromosome aberrations in ICSI embryos was dependent on the kind of medium and sperm incubation time. When spermatozoa were incubated in TYH medium for 2 h or more, the aberration rates in the resultant ICSI embryos (4%) were not significantly different from that of IVF embryos. However, there was a significant increase in aberration rates in ICSI embryos derived from spermatozoa that were incubated in other culture conditions (6%-28%). In addition, a time-dependent increase in aberration rates was found in ICSI embryos when H-TYH, H-mCZB, and PB1 were used for sperm incubation. There was no significant difference in incidence of aneuploidy between IVF and ICSI embryos. The chromosome analysis results of one-cell embryos were reflected by the performance of postimplantation embryo development. The causal mechanism of chromosome damage in ICSI embryos was discussed in relation to the plasma membrane cholesterol, the acrosome, and in vitro aging of spermatozoa.

https://bioone.org/journals/biology-of-reproduction/volume-77/issue-2/biolreprod.106.057778/Evaluation-of-Chromosomal-Risk-Following-Intracytoplasmic-Sperm-Injection-in-the/10.1095/biolreprod.106.057778.pdf

Evaluation of Chromosomal Risk Following Intracytoplasmic Sperm Injection in the Mouse

The present study was conducted to determine the relationship between embryonic development speed at different stages (the cleaved stage at 52 h and the blastocyst stage at 6 days post insemination) and incidences of chromosome abnormalities in in vitro produced porcine embryos. Porcine oocytes were collected from 3-6-mm ovarian follicles obtained at a slaughterhouse and matured in modified NCSU-37 medium for 44-46 h. Following in vitro fertilization with a final concentration of 1 x 10(5) sperm/ml for 3 h, all oocytes were cultured in vitro for 52 h. Day-2 (52 h after insemination) embryos were classified according to their cleaved stages into 2-cell, 3- to 4-cell, 5- to 8-cell, and >8-cell stages; these were cultured separately for additional 4 days (Day 6). The resultant Day-6 blastocysts were classified according to the morphological diameter into 3 grades: Grade A, expanded blastocysts; Grade B, expanding blastocysts; and Grade C, early blastocysts. They were then analyzed chromosomally. The 3- to 4-cell and 5- to 8-cell embryos had significantly high blastocyst development rates (46.1 and 36.9%, respectively), and these blastocysts contained significantly more cells (40.2 and 42.4 cells, respectively) than those derived from 2-cell embryos and >8-cell embryos (28.6 and 26.5 cells, respectively). The incidence of chromosomal abnormalities was significantly higher in the blastocysts derived from 2-cell and >8-cell stage embryos than in the blastocysts derived from the other stage embryos. Furthermore, the grade A blastocysts had the lowest incidence of chromosomal abnormalities (35.3%) and contained the most cells (48.7 cells). Porcine in vitro production (IVP) yielded a high blastocyst rate and an excellent embryo quality when 3- to 4-cell and 5- to 8-cell stage embryos were selected on Day 2 after insemination. The same criteria yielded a higher quality of expanded blastocysts based on the stage of embryo development and morphology.

The blastocyst production rate and incidence of chromosomal abnormalities by developmental stage in in vitro produced porcine embryos.

Superovulated eggs in (BALB/c×C57BL/6) F1 and ICR (outbred in a closed colony) female mice were fertilized in vitro with spermatozoa obtained from caudal epididymides of ICR males. Air-dried chromosome preparations were made from colcemid-primed 1-cell eggs and stained by the C-banding method. The fertilization rate was lower in F1 eggs than in ICR eggs (88 vs. 92%, P<0.02). The incidence of metaphase ("syngamy") eggs was higher in F1 eggs, and the incidence of pronuclear and late prometaphase ("pre-syngamy") eggs was higher in ICR eggs (P<0.001, P<0.005), showing delayed progress of the first cleavage in the ICR eggs. Developmental rates from 2-cell to 4-cell stage and from morula to blastocyst stage were significantly lower in ICR eggs (P<0.001) than in F1 eggs. These results show that a delay of embryo development had already appeared before male and female genomes fused (pre-syngamy). The incidence of triploidy, which might be caused by dispermy, was higher in F1 eggs, in both the pronuclear and mitotic stages (P<0.001). A little aneuploidy and structural aberration of chromosomes occurred in both F1 and ICR eggs, and no significant difference was observed between F1 and ICR eggs. The sex ratio at the first cleavage stage in both F1 and ICR eggs showed no significant deviation from equality.

/pdf/analyses-of-developmental-ability-of-balb-c-c57bl-6-f1-and-4aune4awsv.pdf

Analyses of Developmental Ability of (BALB/c*C57BL/6) F1 and ICR Mouse Embryos Fertilized In Vitro and Their Chromosome at the First Cleavage Division.

Eggs derived from superovulated F1(BALB/c × C57BL/6) mice were fertilized in a modified Krebs-Ringer bicarbonate medium (TYH) containing 2-mM caffeine. Spermatozoa obtained from the cauda epididymis of ICR male mice were preincubated in the caffeine-containing for 2 h before the mixing of gametes. Gametes preincubated and inseminated in the caffeine-free medium served as controls. Chromosome analysis at the first cleavage showed a significantly (P<0.05) higher incidence of triploidy in eggs fertilized in the caffeine-containing medium (22.9%) than in the control (15.8%). The incidence of aneuploidy and the sex ratio were not significantly different between the two groups of eggs. Most of the triploid eggs observed were considered to be dispermic in origin.

Chromosomal Analysis of First-Cleavage Mouse Eggs Fertilized in Caffeine-Containing Medium

One-celled eggs were recovered from mated, colchicine-primed dd female mice, and chromosome preparations were made by the air-drying and C-staining methods. Their mitotic stages were mostly at late prometaphase with two haploid groups of chromosomes and at early metaphase, a stage of syngamy, and partly at the chromatid stage of metaphase. The Y, a small chromosome with no C-band, was distinguished not only at the chromatid stage of metaphase but also at the other stages above-mentioned. Out of 2, 053 eggs examined 767 eggs could be sexed, sexing rate being 37.4%. Of these, 334 eggs were identified as males and 433 females, with a sex ratio of 43.5% males. The sex ratio was further classified according to the stages above-mentioned. Sex ratio before syngamy was significantly higher than that at syngamy and the chromatid stage of metaphase.

https://www.jstage.jst.go.jp/article/jrd1977/31/2/31_2_78/_pdf

Sexing of mouse eggs at the first cleavage division by the use of C-staining method.

細胞分裂阻止剤の一種であるビンブラスチンをマウス桑実胚の染色体標本作製に用いる場合の至適濃度と処理時間について検討した.とくに,供試胚のすべてにおいて,できるだけ多くの分析可能な中期像を得ることを目的とした.17時間処理で濃度差について検討したところ,処理胚に対する中期像を有する胚の割合は,5-50ng/mlの濃度で96-100%であった.胚の割球数に対する中期像の頻度は,30ng/ml以上の濃度で約90%またはそれ以上であったが,10-20ng/mlでは75～77%台に低下した.このことから,中期像の最大出現率を得るための限界濃度は0.03μg/mlであると考えられた.30ng/mlにおける処理時間の検討では,割球数に対する中期像の出現率は8時間処理では有意に低く,10および17時間処理では高かった.しかし,各処理時間におけるNo.1染色体の平均長を比較したところ,各群間で有意な差が認められ,17時間処理では染色体の収縮が顕著であった.結論として,マウス桑実胚におけるビンブラスチンの至適濃度と処理時間は30ng/ml,10時間であると考えられた.中期像をもつ胚の98.4%において染色体分析が可能であり,そのうち正常な2倍体98.8%,高2倍体0.7%,3倍体0.5%であった.

Adequate Concentration and Duration of Vinblastine Treatment for Chromosome Preparation in Mouse Embryos

1間性豚の生殖器管および染色体構成について調査し,次の所見を得た.1. 表現型は正常で,外部生殖器は雌様で陰核が異常に発達していた.生殖腺は左右とも精巣で,精巣上体,精管,精のうおよび前立腺を認めた.子宮は比較的良く発達し,膣部も認められた.2. 白血球,左右の肩部の皮膚および生殖腺の培養細胞について染色体構成をしらべた結果,性染色体はXXで雌型であったが,常染色体中に正常では見られない大型の次中部着糸型染色体を認めた.この染色体は,G-バンド染色によって,端部着糸型のNo.13と17の染色体がセントリックな融合(centric fusion)をして形成されたものであることが証明された.この転座は調査細胞のすべてに認められた.

Akira Okamoto

Papers

Analyses of Developmental Ability of (BALB/c*C57BL/6) F1 and ICR Mouse Embryos Fertilized In Vitro and Their Chromosome at the First Cleavage Division.

Chromosomal Analysis of First-Cleavage Mouse Eggs Fertilized in Caffeine-Containing Medium

Sexing of mouse eggs at the first cleavage division by the use of C-staining method.

Adequate Concentration and Duration of Vinblastine Treatment for Chromosome Preparation in Mouse Embryos

utosomal Abnormality in a Swine