Technical note: Mice produced by intracytoplasmic sperm injection using a modified conventional method.

A piezo-driven pipette that includes a small amount of mercury to enhance efficiency is widely used for mouse intracytoplasmic sperm injection (ICSI). Unfortunately, the use of toxic mercury is not permitted in hospital facilities and alternatives to mercury that enhance performance of the device do not work as well in the mouse. We have eliminated mercury toxicity and obtained acceptable ICSI efficiency using a modified conventional method. With this technique, oocyte survival, fertilization (number of 2-cell) and blastocyst rates were 77/126 (61.1%), 65/77 (84.4%), and 45/65 (69.2%), respectively. Eleven live pups were born from the transfer of thirty-two 2- to 4-cell embryos to 2 surrogate mothers. This conventional method is efficient, simple, and does not need the assistance of piezo-driven devices.

One-step versus two-step culture of mouse preimplantation embryos: is there a difference?

BACKGROUND: A comparison has been made of the development of mouse zygotes in either one-step or two-step culture systems. METHODS: Embryo culture, blastocyst cell counts and embryo transfer were done. RESULTS: No significant differences were observed in the proportions of blastocysts, rates of hatching, numbers of cells in the inner cell mass (ICM) and trophectoderm (TE) that developed in protocols: one-step culture in potassium-enriched simplex optimized medium supplemented with glucose and amino acids (KSOMg(AA)), two-step culture in KSOMg(AA)/KSOMg(AA), and two-step culture in G1.2/G2.2. No gross abnormalities were observed in the fetuses that developed from zygotes in the one-step protocol using KSOMg(AA) and a two-step protocol using G1.2/G2.2. The body weights of these two groups of fetuses were not significantly different and no developmental abnormalities were observed. No significant differences were observed in the proportions of blastocysts, rates of hatching, numbers of cells in the ICM and TE that developed in protocols: one-step culture in KSOMg(AA), two-step culture in KSOMg(AA)/KSOMg(AA), and two-step culture in DM2/DM1. EDTA is not toxic to the initial cleavage stages of development at a concentration of 0.01 mmol/l in KSOMg(AA). CONCLUSIONS: Two-step culture protocols are sufficient for the support of preimplantation mouse development in vitro but they are not necessary.

Mouse embryo development following IVF in media containing either L-glutamine or glycyl-L-glutamine.

BACKGROUND: The development of the mouse zygote following fertilization in vitro in a KSOM-type medium containing either L-glutamine or glycyl-L-glutamine has been examined, and compared with the development of mouse zygotes produced by natural fertilization. METHODS: Mouse IVF, embryo culture and embryo transfer. RESULTS: Fertilization rates, development to the blastocyst stage, implantation rate, gross fetal development and fetal body weight are not different in a KSOM-type medium containing either L-glutamine or glycyl-L-glutamine. No evidence of abnormal fetal development, such as exencephaly, was observed. The replacement of L-glutamine with glycyl-L-glutamine favoured the development of relatively more inner cell mass cells than trophectoderm cells, and reduced the numbers of pyknotic and fragmented nuclei in the blastocysts that developed in vitro. CONCLUSIONS: There is no evidence that the presence of glutamine in the medium used for IVF influences significantly the subsequent development of the zygote. Replacing glutamine with glycyl-L-glutamine may be advantageous.

When to avoid creating surplus human embryos.

The advice that should be given to a couple considering assisted reproductive technologies for the treatment of their infertility, when they are completely opposed to the destruction of surplus embryos, is discussed. It is urged that they do not use treatments that generate surplus embryos. They should be given the options of declining IVF and considering adoption, or less efficient treatments, namely limited ovarian stimulation, limited insemination of available ova or natural cycle IVF where no surplus embryos are generated.

Research in the canine block.

This article, in honor of Dr. Anne McLaren, describes research done during 1955-1959 in the Canine Block at the Royal Veterinary College, London. During that period, Anne in collaboration with the author demonstrated that cultured mouse preimplantation embryos could develop into normal mice after transfer to surrogate mothers. We also studied in depth the control of variability of experimental animals and reproductive aging. In recalling this period, I reminisce about Anne and the scientific environment in which the research was done.

Evidence that glucose is not always an inhibitor of mouse preimplantation development in vitro.

A factorial experimental design was used to examine the effects of 16 combinations of four concentrations of glucose (0.20, 0.60, 1.8, 5.4 mmol/l) and four concentrations of potassium dihydrogen phosphate (KH(2)PO(4); 0.05, 0.15, 0.45, 1.35 mmol/l) on the development in vitro of outbred CF1 mouse zygotes. Three responses were measured: (i) the number of zona-enclosed blastocysts; (ii) the number of blastocysts that started to hatch; and (iii) the total cell counts in the blastocysts. General linear modelling was used to estimate the most parsimonious two-dimensional concentration-response surfaces that represent the three responses to the different concentrations of glucose and KH(2)PO(4). There were no significant interactions between the effects of glucose and KH(2)PO(4) in all cases. Thus, the effects of glucose and phosphate are independent. No significant effects of glucose on blastocyst formation and the initiation of hatching were observed. Increasing the concentration of KH(2)PO(4) inhibited slightly (

IVF of mouse ova in a simplex optimized medium supplemented with amino acids.

The addition of amino acids to a modified simplex optimized medium (mKSOM) did not increase the percentage of blastocysts that develop from CF1 mouse ova fertilized in vitro. In contrast, the percentage of blastocysts that began to hatch and the number of cells in these blastocysts, particularly in the inner cell mass, was increased. The added amino acids also supported the development of a more organized extracellular matrix in the same blastocysts. The results suggest that zygotes produced in amino acid-supplemented mKSOM have a greater developmental potential, perhaps developing at a faster rate, than zygotes produced in mKSOM. This enhanced developmental potential may be caused by the alleviation of osmotic stress on the ova and zygotes by the amino acids that are osmolytes. The fertilization of human ova in vitro may benefit from the inclusion of free amino acids in the fertilizing medium. The availability of a medium that can be used to support both IVF and preimplantation development in the mouse is likely to benefit the recovery of mouse strains from cryopreserved spermatozoa.

Amino acids and preimplantation development of the mouse in protein-free potassium simplex optimized medium.

Development of outbred CF1 mouse zygotes in vitro was studied in a chemically defined, protein-free medium both with and without amino acids. The addition of amino acids to protein-free potassium simplex optimized medium (KSOM) had little effect on the proportion of embryos that developed at least to the zona-enclosed blastocyst stage. In contrast, amino acids stimulated very significantly, in a dilution-dependent way, the proportion of blastocysts that at least partially or completely hatched. Amino acids also stimulated cell proliferation in both the trophectoderm and inner cell mass (ICM) cells, at rates that favored proliferation of cells in the ICM; had no effect on the incidence of cell death (oncosis or apoptosis); and improved development of the basement membranes, which form on the blastocoelic surface of the trophectoderm and between the primitive endoderm and the primitive ectoderm. Thus, KSOM, supplemented with amino acids but containing no protein supplements, supports development of a newly fertilized ovum to the late blastocyst stage, in which its normal, three-dimensional structure is preserved and in which the ICM has been partitioned into the primitive ectoderm and primitive endoderm.

Effects of cryoprotectants and ice-seeding temperature on intracellular freezing and survival of human oocytes.

The accurate determination of the freezing conditions that promote intracellular ice formation (IIF) is crucial for designing cryopreservation protocols for cells. In this paper, the range of temperatures at which IIF occurs in human oocytes was determined. Fresh oocytes with a germinal vesicle, failed-to-fertilize (metaphase I and metaphase II stages) and polyspermic eggs were used for this study. The occurrence of IIF was first visualized at a cooling rate of 120 degrees C/min using a programmable thermal microscope stage connected to a videomicroscope. Then, with a cooling rate of 0.2 degrees C/min, the seeding temperature of the extracellular ice was modified to decrease the incidence of IIF and increase the survival rate of frozen-thawed human oocytes. After adding different cryoprotectants, the median temperature of IIF (TMED) was decreased by approximately 23 degrees C in mouse and only by approximately 6.5 degrees C in human oocytes. Using 1.5 M propylene glycol and seeding temperatures of -8.0, -6.0 and -4.5 degrees C, the incidence of IIF was 22/28 (78%), 8/24 (33%) and 0/33 (0%) and the 24 h post-thaw survival rate was 10/31(32%), 19/34 (56%) and 52/56 (93%) respectively. The results show that IIF occurs more readily in human oocytes, and that ice seeding between -6 degrees C and -8 degrees C triggers IIF in a large number of human oocytes. Undesirable IIF can be prevented and survival rates maximized by raising the seeding temperature as close as possible to the melting point of the solution, which in our instrument was -4.5 degrees C.

Coordinate action of Wt1 and a modifier gene supports embryonic survival in the oviduct.

The Wt1 gene, originally identified as a tumor suppressor gene associated with Wilms' tumors, encodes a zinc finger containing transcription factor expressed during gonadal and kidney development. Although Wt1 appears to be required for gonadal and kidney development, no reproductive defects were observed in outbred females heterozygous for a targeted mutation in Wt1. In contrast, no litters were obtained from Wt1 +/- females on a strain 129/Sv inbred genetic background. Ovaries were smaller in Wt1 +/- 129/Sv mice and produced fewer ova, but transplanted Wt1 +/- ovaries from 129/Sv females were able to support successful pregnancies. The inability of Wt1 +/- 129/Sv females to produce successful implantations after ovulation and fertilization appeared to be due to the failure of one-cell embryos to undergo mitosis, such that they were lost in the oviduct before reaching the uterus. Approximately 50% of Wt1 +/- females generated from a backcross of Wt1 +/- 129/Sv:C57BI/6 F1 hybrids to 129/Sv were fertile, indicating the presence of a Wt1 modifier gene that affects survival of the preimplantation embryo. Neither levels of WT1 protein nor the ratio of WT1 spice forms were significantly altered in Wt1 +/- reproductive organs, suggesting that this modifier effect acts downstream of WT1. Wt1 is therefore among a small subset of genes required for survival of the pre-implantation embryo, and appears to function non-autonomously.

Transforming growth factor-beta stimulates mouse blastocyst outgrowth through a mechanism involving parathyroid hormone-related protein.

The goals of this study were 1) to compare the effects of transforming growth factor-beta (TGF-beta) and parathyroid hormone-related protein (PTHrP) on mouse blastocyst attachment and outgrowth in vitro, 2) to determine whether TGF-beta acts through a mechanism involving PTHrP, 3) to examine effects of PTHrP on preimplantation mouse embryo development, and 4) to determine the pattern of expression of PTHrP protein in the uterus of the mouse during early gestation. In the first set of experiments, hatched blastocysts were placed in fibronectin-coated wells. Cultures were treated with PTHrP or TGF-beta1 and assessed at 24, 48, and 72 h for attachment and surface area of blastocyst outgrowth. Results showed that both PTHrP and TGF-beta1 increased blastocyst outgrowth significantly. A PTHrP-neutralizing antibody blocked the stimulatory effect of both PTHrP and TGF-beta1, suggesting that TGF-beta1 acts to increase endogenous production of PTHrP by the blastocyst. Immunoassay of conditioned medium from blastocysts treated with either TGF-beta1 or PTHrP 1-34 confirmed a 3- to 4-fold increase in levels of PTHrP 1-141. In the second series of experiments, pronuclear zygotes were cultured in various concentrations of PTHrP for 96 h. Blastocysts then were subjected to differential fluorescent staining of inner cell mass and trophectoderm cells. Treatment of mouse embryos with the various concentrations of PTHrP altered neither the number developing to the blastocyst stage nor the number of inner cell mass or trophectoderm cells in the resulting blastocysts. In the third experiment, pregnant mice were killed at Days 3, 4, 5, 6, and 7 of gestation, and uterine horns were processed for immunohistochemistry. Uterine sections were stained with antibodies to PTHrP, desmin, and laminin. On Days 3, 4, and 5, uterine luminal and glandular epithelial cells stained intensely for PTHrP, while stromal cells were negative. By Days 6 and 7, decidualized stromal cells stained positively for PTHrP, desmin, and laminin. These results support the hypothesis that TGF-beta and PTHrP play an important role in the process of implantation.

Reflections on the culture of the preimplantation embryo.

There has been a considerable improvement in the media available for the culture of preimplantation mouse embryos during the 40 years since mouse embryos were first cultured and successfully transferred to uterine foster mothers. Two new media, KSOM and mMTF, are becoming more commonly used. The history of the development of these media, including recent work on KSOM and mMTF, is reviewed. A major artefact in the earlier work was the two-cell block. The causes of the two-cell block and the methods by which it has been overcome are reviewed. It is concluded that even the best available media inevitably cause imbalances in the environment in which the embryos are forced to develop, because they consist of only a small subset of the compounds present in the natural environments. As a result, the embryos must adapt to these abnormal conditions if they are to survive. The implications of these conclusions on the choice of media for specific purposes are discussed.

Polyvinyl alcohol and amino acids as substitutes for bovine serum albumin in culture media for mouse preimplantation embryos.

The effect of replacing bovine serum albumin (BSA) in a simple defined medium (KSOM) with polyvinyl alcohol (PVA) and/or amino acids on the percentages of mouse zygotes that develop to at least the blastocyst stage and that hatch at least partially or completely is reported. Blastocysts could form when BSA was replaced with only PVA, but at a moderately reduced rate; however, partial hatching, and hence complete hatching, were severely impaired when BSA was replaced with only PVA. The substitution of BSA with amino acids alone resulted in a high rate of blastocyst formation and moderate impairment of hatching. The addition of PVA to BSA-free KSOM supplemented with amino acids had no extra effect. BSA had significant effects when added to BSA-free KSOM supplemented with amino acids. The BSA caused a significant increase in the rate of partial hatching, and may even have had a small effect on the rate of blastocyst formation. The results also showed that glucose, at a high concentration of 5.56 mM, does not inhibit the development of mouse zygotes to hatched blastocysts when cultured in KSOM supplemented with amino acids.

Intracellular ion concentrations and their maintenance by Na+/K(+)-ATPase in preimplantation mouse embryos.

We have measured the amounts of Na+, K+ and C- in preimplantation mouse embryos (1-cell, 2-cell and morula) using electron probe X-ray microanalysis. The levels of these ions do not vary much over this period, and are approximately the same as those found in other mammalian cells, contrary to previous reports. We have confirmed that preimplantation embryos exhibit Na+/K(+)-ATPase activity at all stages examined, and have shown that the ATPase maintains high K+/Na+ ratios (12-16) in all these embryonic stages, comparable to those seen in other healthy cells; this is in contrast to the low ratios reported in earlier work. Inhibition of the Na+/K(+)-ATPase results in the slow exchange of intracellular K+ for extracellular Na+ (half-time approximately 5 h), indicating that Na+/K(+)-ATPase activity maintains steep Na+ and K+ gradients in preimplantation mouse embryos as it does in most other cells.

Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis.

THE D-type cyclins (D1, D2 and D3) are critical governors of the cell-cycle clock apparatus during the G1 phase of the mammalian cell cycle. These three D-type cyclins are expressed in overlapping, apparently redundant fashion in the proliferating tissues. To investigate why mammalian cells need three distinct D-type cyclins, we have generated mice bearing a disrupted cyclin D2 gene by using gene targeting in embryonic stem cells. Cyclin D2-deficient females are sterile owing to the inability of ovarian granulosa cells to proliferate normally in response to follicle-stimulating hormone (FSH), whereas mutant males display hypoplastic testes. In ovarian granulosa cells, cyclin D2 is specifically induced by FSH via a cyclic-AMP-dependent pathway, indicating that expression of the various D-type cyclins is under control of distinct intracellular signalling pathways. The hypoplasia seen in cyclin D2(-/-) ovaries and testes prompted us to examine human cancers deriving from corresponding tissues. We find that some human ovarian and testicular tumours contain high levels of cyclin D2 messenger RNA.

Cryobiology of non-human primate oocytes.

The responses to various stresses involved with cryopreservation protocols were investigated using non-human primate oocytes. Fluorescence microscopy was used to assess the status of the F-actin microfilament system of rhesus monkey oocytes after exposure to different concentrations of glycerol. The F-actin organization around the cortex and in the transzonal processes was modified by exposure to 1.0 ot 2.0 M glycerol at ambient temperature. These effects were reduced significantly when exposure to glycerol was combined with cooling to O degrees C. Cynomolgus monkey oocytes were also subjected to hyperosmotic stress and observed for morphological changes. An irregular shrinkage phenomenon was observed with germinal vesicle or metaphase I but not metaphase II (MII) oocytes. The irregular shrinkage became uniform and spherical when the oocytes were pretreated with ethyleneglycol-bis-(beta-aminoethyl ether)N,N,N'N' tetraacetic acid (EGTA) before exposure to hypertonic solution. Also, in-vitro-matured MII oocytes from cynomolgus monkeys were used to determine crucial biophysical parameters for freezing primate oocytes. The permeability of oocyte plasma membrane to water, Lpg, and its activation energy, ELp, were determined between 0 and -12 degrees C in the absence of cryoprotective additives. The Lpg was found to be 3.8x10(-14) m3N/s and the ELp was 141.5 kJ/mol. the pre-exponential kinetic and exponential thermodynamic parameters of intracellular ice formation were determined to be 8x108 m2/S and 2. 2x10(9) K5 respectively. By combining models of water transport and intracellular ice formation, the cumulative fraction of oocytes with intracellular ice as a function of the cooling rate was also predicted, and it was shown to correlate reasonably with experimental observations.

Zinc is a possible toxic contaminant of silicone oil in microdrop cultures of preimplantation mouse embryos.

A batch of silicone oil (dimethylpolysiloxane) is described which had differential effects on the development of 1- and 2-cell preimplantation mouse embryos in vitro when used as a microdrop overlay over two culture media: CZB and KSOM. A high rate of development into blastocysts was observed when using CZB medium; in contrast, development was strongly inhibited when KSOM was used. Other batches of silicone oil or paraffin oil permitted development from the zygote to the blastocyst of an outbred strain of mouse without arrest at the 2-cell stage. Our results show that the higher concentrations of ethylenediaminetetraacetic acid (EDTA) and bovine serum albumin (BSA) in CZB medium, in comparison with KSOM, protect against the toxic component in the oil. Observations also gave circumstantial evidence that the toxic component in the oil is zinc. The beneficial effect of including EDTA in a medium is usually attributed to its chelating toxic metals introduced as impurities in other components of the medium. Our results now show that EDTA also protects against impurities in the oil overlay.

Fertilization in vitro of mouse ova from inbred and outbred strains: complete preimplantation embryo development in glucose-supplemented KSOM.

A new medium derived from the use of sequential simplex optimization methods (SOM) that overcomes the block to development beyond two cells in vitro in embryos of the CF1-cultured strain of mouse has recently been described. Contrary to previous reports, glucose was shown to have no significant inhibiting effect on embryo development to the blastocyst stage in SOM. A modification of SOM, designated KSOM, with an increased concentration of Na+ (95 mM) and K+ (2.5 mM), which has been described elsewhere, also supports growth beyond the two-cell block. KSOM produces a higher rate of compaction, a larger yield of blastocysts, and an increased rate of cell division of the trophoblast cells. We have reexamined the glucose effect by varying the concentration of glucose (either 0.2 mM or 5.56 mM) in KSOM and determined the ability of these media to support preimplantation development of CF1 female x B6D2F1 male zygotes through the blastocyst stage. Glucose is shown to have no significant inhibiting effect on development to the blastocyst stage. The yield of blastocysts is typically 85%-90%. A modification of KSOM derived from this study, designated modified KSOM, with an increased concentration of glucose (5.56 mM) and supplemented with 4 mg ml-1 BSA is now shown to support high rates of fertilization in vitro of CF1 ova with hybrid B6D2F1/CrlBR sperm and subsequent development of zygotes beyond the two-cell stage to blastocysts in high yield.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatin organization, meiotic status and meiotic competence acquisition in mouse oocytes from cultured ovarian follicles.

Changes in chromatin organization, meiotic status and the development of meiotic competence in oocytes retained within mouse ovarian follicles from day 0 to day 6 in culture were examined. The effects of exposure for 24 h to human luteinizing hormone (hLH) during the last day in culture was also determined. Preantral follicles from 22- to 24-day-old (prepubertal) mice develop antra and undergo significant growth from day 0 to day 4 in culture, after which the growth rates slow. The statistical significance of meiotic progression was examined using exact logistical regression analysis, which is particularly useful when the data are sparse and unbalanced. The transition from rimmed to unrimmed germinal vesicle stages was found to occur between day 2 and day 4 of follicle culture and was not influenced by exposure to hLH. Treatment with hLH caused a significant increase in the proportion of intrafollicular oocytes resuming meiosis. Assays of meiotic competence performed in vitro in oocytes retrieved from cultured follicles demonstrated that the transition from an unrimmed to a rimmed state is closely coincident with the acquisition and expression of meiotic competence. Forty-six per cent of competent oocytes from follicle cultures at day 3 progressed to metaphase II. These results indicate that the follicle culture system used in these studies supports the transformation of enclosed oocytes from a precompetent to a competent state and can maintain meiotic arrest for up to 6 days in culture. However, an increasing proportion of oocytes exhibit abnormal meiotic progression with continued follicle culture beyond 4 days.