Reduced oocyte activation and first cleavage rate after ICSI with spermatozoa from a sterile mouse chromosome mutant.

BACKGROUND: Male mice, heterozygous for two semi-identical reciprocal translocations T(1;13)70H and T(1;13)1Wa are usually sterile. We have investigated this oligoasthenoteratozoospermic mouse model using ICSI. METHODS: B6D2F1 oocytes were injected with epididymal or testicular sperm from fertile or sterile translocation carriers and from chromosomally normal fertile controls. ICSI efficiency was determined by pronucleus formation and first cleavage rates. For arrested zygotes, cell cycle progression was evaluated by BrdU incorporation and incubation with okadaic acid. RESULTS: Epididymal sperm from infertile translocation carriers showed a slightly lower fertilization rate (70% vs. 92%, 95% and 95% for fertile translocation carriers and two groups of normal fertile control males, respectively) and a severely reduced cleavage rate (33% vs. 87%, 96% and 89% for the same control groups). However, the use of testicular sperm significantly improved the cleavage rate (62% vs. 83% for normal fertile controls). Development of arrested zygotes was delayed or blocked during S- and G2-phase. CONCLUSIONS: Whereas control testicular and epididymal sperm performed equally well, the use of testicular sperm from oligospermic T/T' males significantly increased first cleavage rates when compared to the low rates with epididymal sperm. Epididymal storage in oligospermics may negatively influence zygote division.

Transmission of gametes with normal or translocation chromosomes in male and female single-sex mouse chimeras.

Three male and four female mouse single-sex chimeras derived from fusions of Rb(11.13)4Bnr T(1;13)70H homozygous embryos with +/+ embryos were caged with T(1;13)70H homozygotes of the opposite sex and followed through their reproductive lifespans. Six animals (three males and three females) were germline chimeras. The fz gene was used as a marker for the T70H reciprocal translocation. The ratio of fz/fz to fz/+ offspring did not change with increasing age in males, but decreased in two of the three female chimeras. Within males, there was generally good agreement between the proportions of translocation and nontranslocation germ cells from spermatogonial mitosis through the first and second meiotic division. In one male, this ratio was also reflected in the offspring. In the other two males, there was significant selection during haplophase, from which both types of spermatozoa could benefit.

Embryo survival in pseudopregnant and in pregnant but genetically semi-sterile recipients after nonsurgical embryo transfer in the mouse.

A new nonsurgical embryo transfer technique was used in the mouse that yielded survival rates of between 40 and 70% depending on embryo stage and, possibly, on the degree of synchrony between the embryo and recipient. Three variables were tested using this embryo transfer technique: a) pseudopregnant recipients vs pregnant but genetically semi-sterile recipients, b) embryos resulting from superovulation vs embryos from natural ovulation, and c) 12-hour vs 24-hour asynchrony between donors and recipients. None of these variables significantly affected the pregnancy rate or the percentage of transferred embryos developing to term. The pregnancy rates were between 77 and 90% in 6 experimental groups of 8 to 13 females. Survival rates were between 41 and 63% when all recipients were considered and between 53 and 68% when only the pregnant recipients were included. The embryo transfer procedure influenced litter size composition of the endogenous conceptuses of the semi-sterile recipients. Too many females were devoid of these. Recipients of expanded blastocysts had significantly better transfer results than recipients that also received morulae and early blastocysts. It was concluded that the transfer success rates were influenced by the recipients and possibly by their preparation for transfer.

Chromosome damage and non-disjunction measured at the first cleavage division in normal and chromosomally mutant female mice irradiated at the diakinesis stage of female meiosis.

We have irradiated primary murine oocytes at the diakinesis stage of the first meiotic division with 0.6 Gy X-rays. Fertilized oocytes were cultured overnight to arrest the first cleavage division and display pronuclear chromosomes. All preparations were preferentially stained for centric constitutive heterochromatin and analyzed for structural and numerical radiation effects. Females of 3 different karyotypes were irradiated (all on a Swiss random-bred genetic background): +/+ (221 female pronuclei analyzed), Rb(11.13)4Bnr T(1;13)70H/Rb(1.13)4Bnr T(1;13)70H with 11.13(1) and 1(13) large and small marker bivalents (RbT/RbT, 242 zygotes analyzed) and the same karyotype but with a 1(13)H;1(13) Wa heteromorphic bivalent (RbT/RbT*, 126 zygotes analyzed). Hyperploid chromosome counts were encountered with frequencies of 11.8% (+/+), 11.9% (RbT/RbT) and 16.6% (RbT/RbT*). In this order of karyotypes, the frequencies of dicentrics per zygote were 0.07, 0.16 and 0.11 and the frequencies of fragments 0.13, 0.18 and 0.31. In about half of the supernumerary chromosome spreads, a dicentric chromosome was included. The long marker bivalent 11.13(1) had a non-disjunction frequency of 2.5 times its control value, partially because it was involved in dicentric formation as well. For the RbT/RbT karyotype, the spontaneous maternal non-disjunction level was 5.4%. For the RbT/RbT* karyotype, it can be assumed to be the same or slightly higher because of the 1(13)H;1(13) Wa heteromorphic bivalent. This increased intrinsic sensitivity for non-disjunction was not expressed as an increased sensitivity for aneuploidy after irradiation. This fact and the numerical association between hyperploidy and dicentric formation, both for normal bivalents and for the 11.13(1) marker bivalent, lead us to suppose that in the female mouse, irradiation-caused aneuploidy is effectuated via chromatid exchange. The data presented do not rule out the existence of another mechanism.

Meiosis in a sterile male mouse with an isoYq marker chromosome.

A male mouse with a metacentric Y chromosome of twice the normal size has been studied chromosomally in bone marrow mitoses, spermatogonial mitoses, and diakinesis-metaphase I primary spermatocytes. A low frequency of nondisjunction for this chromosome (2%) was noted in both bone marrow and spermatogonial mitoses. In spermatogonial mitoses, loss of the Y chromosome had occurred to the extent that 12% of spermatogonia were XO, resulting in 17% XO primary spermatocytes. Hardly any stages beyond the primary spermatocyte stage were encountered, which agrees with testis weights of approximately 30% of normal. Surface-spread pachytene spermatocytes yielded few cells that were analyzable for their total complement of synaptonemal complexes. The Y chromosome showed complete fold-back pairing and was located far away from the X chromosome. X and Y chromosomes were paired in 14.5% of the diakinesis-MI spermatocytes that contained a Y chromosome. The origin of this chromosome is discussed against the background of localization of the gene for the testis-determining factor on the short arm of the mouse Y chromosome.

Embryo loss, blastomere development and chromosome constitution after human chorionic gonadotropin-induced ovulation in mice and rats with regular cycles.

A dose of 7 IU human chorionic gonadotropin (hCG) given 14 h before the expected LH peak on proestrus significantly increased embryonic mortality in Swiss random-bred female mice to 55% of the number of corpora lutea. The use of luteinizing hormone-releasing hormone in a similar injection protocol did not induce embryonic death. The effect found in Swiss random-bred mice resembles that of a dose of 20 IU hCG in the rat. Afternoon-day-4 mouse embryos contained 39.1 +/- 12.6 nuclei after hCG-induced ovulation compared to 46.2 +/- 16.6 nuclei after spontaneous ovulation. For early-day-5 embryos of the rat, these figures were 34.2 +/- 10.1 and 31.7 +/- 8.4, respectively (mating was early on day 1). Numerical chromosome errors were estimated in secondary oocytes of the mouse and early-day-5 embryos of the rat. Compared with data from the literature, hCG seems to induce some extra meiotic nondisjunction in the rat only. Combining all genetic and physiological data, the loss of fecundity after hCG-induced ovulation is a maternal effect.

Lack of correlation between levels of MHC class I antigen and susceptibility to lysis of small cellular lung carcinoma (SCLC) by natural killer cells.

Small cellular lung carcinoma (SCLC) cell lines are susceptible to lysis by NK cells. SCLC, normally negative for MHC class I Ag, were rendered positive for HLA-A and -B Ag by two methods: treatment with IFN-gamma or transfection with HLA class I genes. Exposure to IFN-gamma induced high levels of class I Ag and reduced susceptibility to NK-mediated lysis. However, transfection with either HLA-A2, HLA-B27, or HLA-B27 with beta 2m did not result in reduced susceptibility to NK cells. These transfectants expressed amounts of HLA class I Ag comparable to those in IFN-gamma-treated, untransfected cells. Transfection with the beta 2m gene or plasmid alone neither influenced levels of surface class I Ag nor resulted in reduced susceptibility to lysis by NK cells. Thus, the effects of IFN-gamma on NK susceptibility can be dissociated from the induction of class I Ag.

Male pachytene pairing in single and double translocation heterozygotes and spermatogenic impairment in the mouse.

In order to clarify the relationship between meiotic pairing and progress of spermatogenesis, an analysis of male meiotic pairing was carried out in four reciprocal translocation heterozygotes and two double heterozygotes for two semi-identical reciprocal translocations. The reciprocal translocations were chosen to range from fertility (T70H/+) through almost complete sterility (T31H/+) to complete sterility (T32H/+, T42/H+). If meiotic pairing in the translocation multivalent was incomplete, it concerned terminal or probably more often proximal chromosome segments (Chain IV). If both segments failed to pair the multivalent symbol is Chain III + I. Complete pairing is symbolized by Ring IV. To contrast and complement observations of this type, the double heterozygotes were introduced. Males of this type in theory possess two heteromorphic bivalents with a central area of incomplete meiotic pairing (loop formation). Of the T70H/T1Wa double heterozygotes, 36% of the males are capable of inducing at least one decidual reaction in two females whereas for T26H/T2Wa, 79% of the males can do so. For the reciprocal translocations, it was found that proximity of the multivalent to the sex bivalent during pachytene increased in the order Ring IV, Chain IV, Chain III + I. The degree of spermatogenic impairment as measured from cell counts in histological sections and tubular whole mounts, is positively related to the frequency of proximity between the sex chromosomes and the translocation multivalent and thus to lack of meiotic pairing within the multivalent. The meiotic pairing analysis of the double heterozygotes yielded the following findings. For the long heteromorphic bivalents a true loop was never seen in T70H/T1Wa and only rarely observed in T26H/T2Wa. Small marker bivalents of both types were usually recognizable by the following criteria: pairing confined to distal or proximal segments, both distal and proximal segments pairing and loop formation and pairing covering the entire length of both "homologues" but the longer one often with a "thickened" lateral element. The same positive correlation between the absence of pairing (proximal, distal or central) and the proximity of the small marker bivalent synaptonemal complex to the sex bivalent has been found as for unpaired segments within reciprocal translocation multivalents. One unexpected finding was the occurrence of diploid spermatids and spermatozoa especially in T32H/+ males (70-91%) but also in T31H/+ (3-39%).

Karyotypes of 3- or 4-day-old pig embryos after short in-vitro culture.

Pig embryos (3-4 days old) were grown for approximately 21 h in microdroplets under mineral oil with vinblastin sulphate as the mitotic inhibitor. From 10 gilts, 115 eggs were cultured: 8 proved to be unfertilized oocytes, 9 did not contain mitotic figures, 16 contained fewer than 3 good mitotic figures and 80 were judged to be of normal karyotype. A few polyploid cells were encountered, possibly of trophoblast origin. One embryo was a haploid and another one a 2N/6N mixoploid. In one gilt, in which there was no evidence of fertilization, a tetraploid embryo was discovered, and was assumed to be parthenogenetic.

Meiotic nondisjunction and translocation segregation in dwarf (dw/dw) and control T(1;13)70H heterozygous mice.

The meiotic behavior of translocation heterozygous T70 (1;13)H/+ male mice with a Snell dwarf (dw/dw) genotype was compared with that of nondwarf T70H/+ controls. A four-fold increase in the nondisjunction frequency of the normal bivalents occurred as a consequence of the dwarf genotype. This increase is identical to that seen in karyologically normal dwarf males. No effect of the dwarf condition on the segregation of the translocation multivalent could be noted. Thus, translocation heterozygosity does not enhance the meiotic instability caused by the hypopituitary dwarf condition. From a small sample of oocytes from T70H/+ and chromosomally normal dwarf females it is concluded that nondisjunction in females is not increased by the dwarf condition. In general we conclude that animals with higher spontaneous nondisjunction levels are not necessarily more sensitive to factors increasing nondisjunction.

Chiasma frequency and nondisjunction in heteromorphic bivalents: meiotic behavior in T(1;13)70H/T(1;13)1Wa mice as compared to T(1;13)70H/T(1;13)70H mice.

Two different reciprocal translocations between chromosomes 1 and 13 in the mouse, T(1;13)70H and T(1;13)1Wa, were intercrossed, resulting in T(1;13)70H/T(1;13)1Wa animals, possessing two heteromorphic marker bivalents. The meiotic behavior of these animals was compared to that of T(1;13)70H/T(1;13)70H animals. In females the chiasma frequency in the translocation bivalents was decreased by the presence of the loops following meiotic pairing, but in males chiasma frequencies were not changes. The nondisjunction level of both translocation bivalents was increased in females and males. The nondisjunction level of bivalents not involved in the translocations was increased in the females, but not in the males. Possible causes and relevance of these phenomena are discussed.

The use of translocation-derived "marker-bivalents" for studying the origin of meiotic instability in female mice.

Female mice of two age groups, 3--4 and 11--14 months old, homozygous for the T(1;13)70H reciprocal mouse translocation were used for cytological observations of bivalents (in primary oocytes) and metaphase II chromosomes (in secondary oocytes). Special attention was given to the behavior of the long (131) and short (113) marker chromosomes. In primary oocytes, univalents were considered "true" or "opposite". The aged females showed an eight-folded increase in "true" univalent frequency for chromosomes 113 over the young ones. A nine-fold rise for nondisjunction with regard to this chromosome was observed. For the other chromosomes, these factors were 2 and 1.7, respectively. The absolute levels of nondisjunction remained low at old age (1.42% for chromosome 113, 1.22% for all other chromosomes). The long marker bivalent 131 was used for chiasma counts. No change in chiasma number with age was observed. It is argued that poorer physiological conditions within the maturing oocytes of older females are the major cause for both the increasing frequencies of "true" and "opposite" univalents and the increased incidence for nondisjunction.

Son-sire-regression based heritability estimates of chiasma frequency, using T70H mouse translocation heterozygotes, and the relation between univalence, chiasma frequency and sperm production.

T(1;13)70H/+ translocation heretozygous mice were used for assessing heritability values for chiasma frequencies and the epididymal sperm count. The chiasma frequency estimates were based on 15 son-sire pairs, the translocation heterozygotes being maintained in a Swiss random-bred genetic background. The chiasma frequencies were scored separately for the T70H/+ derived multivalent, specific pairing segments within the multivalent and the remaining bivalents. Chiasma counts within these specified parts of the genome were positively correlated. The heritability estimates, significantly greater than zero, ranged from 0.78-0.98, depending on the chromosome segments included. These results indicate a strong genetic control on a cellular basis for the formation of chiasmata in the mouse. Despite significantly positive correlations and regressions between the various chiasma frequencies and the sperm count (for which 29 pairs of observations were available), no significant heritability estimate for the sperm count was obtained. The relation between the chiasma frequency and the sperm count was weakest when the chiasma count was confined to a region of the translocation-caused multivalent in which the absence of a chiasma almost always resulted in the production of an univalent. This indicates that in the translocation heterozygotes used, the overall chiasma frequency has a greater predictive value for the sperm count than autosomal univalence alone.

Chromosomal radiosensitivity and karyotype in mice using cultured peripheral blood lymphocytes, and comparison with this system in man.

The frequencies were studied X-ray-induced dicentric chromosomes and deletions in peripheral blood lymphocytes of mouse and man, cultured in vitro. After doses of 100 and 200 rad, (a) the mouse was equally sensitive as man to the induction of dicentrics, and (b) the frequency of deletions was higher in the mouse, reaching statistical significance at the 200 rad level only. At the 200 rad level, the mouse with normal karyotype was compared with the T(1;13)70H translocation heterozygote and the Ts(1(13))7OH tertiary trisomic of normal appearance. No differences were found either with respect to dicentrics or to deletions. At the 100 rad level, the normal mouse was compared with the tertiary trisomic mouse of the affected phenotype and with the tobacco mouse. The frequency of dicentrics was significantly higher in the phenotypically abnormal trisomics, whereas the deletion frequency was higher in the tobacco mice. C-banding of the slides enabled the locating of breaks in constitutive hetero-chromatin and euchromatin. When exchanges were classified into three categories, i.e. those between eu- and euchromatin, eu- and hetero-, and hetero- and heterochromatin, there was a preference for the first and the last whereas only few occurred between chromatins of contrasting type. Differences between previous determinations of the chromosomal radiosensitivity of mouse and man, using peripheral blood lymphocytes (i.e. man is twice as sensitive as mouse), and the one presented here could be attributed to differences in harvest time of the mouse peripheral blood lymphocytes. Thus the so-called "arm number" hypothesis of Brewen et al. [5] is not confirmed by the present results.

The production, morphology, karyotypes and transport of spermatozoa from tertiary trisomic mice and the consequences for egg fertilization.

Tertiary trisomic males, carrying the small translocation chromosome from the T(1;13)7OH reciprocal mouse translocation as the extra chromosome, are oligospermic. Uterine and oviductal sperm counts were congruent to 10% of normal. Of the uterine spermatozoa, 77-2% were morphologically abnormal compared with 24-6% in the oviduct. Oligospermy in the tertiary trisomic males leads to delayed fertilization; 34-8% of the 109 eggs scored between 5-5--9-5 hr after mating were fertilized compared with 52-1% (n=343) at Day 2. Of the 179 morulae/blastocysts recovered at Day 4, 46-9% contained the small marker chromosome, which agrees with earlier cytological studies on secondary spermatocytes. These results indicate that euploid and aneuploid spermatozoa are formed in about equal numbers and there is no relationship between sperm morphology and karyotype.

PIP: Studies on sperm function and viability of eggs fertilized by tertiary trisomic male mice carrying the small translocation chromosome t(1; 13)7 OH were conducted. These males are oligospermic: 10 percent of normal sperm numbers were found in uterus and oviduct. 77.2 percent of uterine and 24.6 percent of oviduct spermatozoa were abnormal, usually having aberrant tail attachment and symmetrical heads. Delayed fertilization was suggested since only 34.8 percent of eggs were fertilized by 5.5-9.5 hours after mating, compared with 100 percent in contols; control eggs were in a more advanced stage of development; and on Day 2 50.1 percent of trisomic and 95.5 percent of control eggs were fertilized. Of 179 morulae or blastocysts karyotyped on Day 4, 46.9 percent contained the marker chromosome, indicating 1:1 segregation of meiosis. The sex ratio determined by C-banding was 103.5 males per 100 females. The trisomic males became less fertile with age. Those with abnormal incisors produced smaller litters. The results indicate that eupoloid and aneuploid spermatozoa are formed in equal numbers, and sperm karyotype does not effect sperm morphology or function.