A cell cycle-associated change in Ca2+ releasing activity leads to the generation of Ca2+ transients in mouse embryos during the first mitotic division.

We have used Ca2+-sensitive fluorescent dyes to monitor intracellular Ca2+ during mitosis in one-cell mouse embryos. We find that fertilized embryos generate Ca2+ transients at nuclear envelope breakdown (NEBD) and during mitosis. In addition, fertilized embryos arrested in metaphase using colcemid continue to generate Ca2+ transients. In contrast, parthenogenetic embryos produced by a 2-h exposure to strontium containing medium do not generate detectable Ca2+ transients at NEBD or in mitosis. However, when parthenogenetic embryos are cultured continuously in strontium containing medium Ca2+ transients are detected in mitosis but not in interphase. This suggests that mitotic Ca2+ transients are detected in the presence of an appropriate stimulus such as fertilization or strontium. The Ca2+ transient detected in fertilized embryos is not necessary for inducing NEBD since parthenogenetic embryos undergo nuclear envelope breakdown (NEBD). Also the first sign that NEBD is imminent occurs several minutes before the Ca2+ transient. The Ca2+ transient at NEBD appears to be associated with the nucleus since nuclear transfer experiments show that the presence of a karyoplast from a fertilized embryo is essential. Finally, we show that the intracellular Ca2+ chelator Bapta inhibits NEBD in fertilized and parthenogenetic embryos in a dose-dependent manner. These studies show that during mitosis there is an endogenous increase in Ca2+ releasing activity that leads to the generation of Ca2+ transients specifically during mitosis. The ability of Ca2+ buffers to inhibit NEBD regardless of the presence of global Ca2+ transients suggests that the underlying cell cycle-associated Ca2+ releasing activity may take the form of localized Ca2+ transients.

Survival of mouse embryos frozen to -196 degrees and -269 degrees C.

Mouse embryos survived freezing to -196 degrees C. Survival required slow cooling (0.3 degrees to 2 degrees C per minute) and slow warming (4 degrees to 25 degrees C per minute). Depending on the specific rates used, 50 to 70 percent of more than 2500 frozen and thawed early embryos developed into blastocysts in culture after storage at -196 degrees C for up to 8 days. When approximately 1000 of the survivors, including some frozen to -269 degrees C (4 degrees K), were transferred into foster mothers, 65 percent of the recipients became pregnant. More than 40 percent of the embryos in these pregnant mice gave rise to normal, living full-term fetuses or newborn mice.

Mycoplasmas and in vitro fertilization.

Semen samples taken from 135 patients attending an in vitro fertilization clinic were shown to be colonized, 53 with Ureaplasma urealyticum (39%) and 16 with Mycoplasma hominis (12%). An unidentified mycoplasma species was isolated from the sperm of two patients. M. hominis was recovered from all the washed sperm samples taken from colonized semen, whereas washing the sperm eradicated U. urealyticum from 71% of colonized semen. The presence of mycoplasmas in semen made no significant difference to the sperm count, sperm motility, sperm abnormalities, or fertilization of eggs.

Growth in vitro and acquisition of meiotic competence after the cryopreservation of isolated mouse primary ovarian follicles.

The growth and acquisition of meiotic competence of oocytes from fresh and frozen-thawed primary follicles collected from 10-day-old mice was compared during culture in collagen gels for 12 days. The oocytes contained in primary follicles have a mean diameter of about 48 microns and do not resume meiosis without further growth and development. During the 12-day culture period the mean diameter of the oocytes increased to over 60 microns. The oocytes were capable of resuming meiosis when isolated from the gel and cultured in the absence of follicular cells in a manner similar to that observed in vivo. Freezing and thawing did not affect oocyte growth or the ability to resume meiosis; this demonstrates the possibility of storing large numbers of female gametes for subsequent development.

Alterations in the cell cycle of mouse cumulus granulosa cells during expansion and mucification in vivo and in vitro.

The cell cycle characteristics of mouse cumulus granulosa cells were determined before, during and following their expansion and mucification in vivo and in vitro. Cumulus-oocyte complexes (COC) were recovered from ovarian follicles or oviducts of prepubertal mice previously injected with pregnant mare serum gonadotrophin (PMSG) or a mixture of PMSG and human chorionic gonadotrophin (PMSG+hCG) to synchronize follicle differentiation and ovulation. Cell cycle parameters were determined by monitoring DNA content of cumulus cell nuclei, collected under rigorously controlled conditions, by flow cytometry. The proportion of cumulus cells in three cell cycle-related populations (G0/G1; S; G2/M) was calculated before and after exposure to various experimental conditions in vivo or in vitro. About 30% of cumulus cells recovered from undifferentiated (compact) COC isolated 43-45 h after PMSG injections were in S phase and 63% were in G0/G1 (2C DNA content). Less than 10% of the cells were in the G2/M population. Cell cycle profiles of cumulus cells recovered from mucified COC (oviducal) after PMSG+hCG-induced ovulation varied markedly from those collected before hCG injection and were characterized by the relative absence of S-phase cells and an increased proportion of cells in G0/G1. Cell cycle profiles of cumulus cells collected from mucified COC recovered from mouse ovarian follicles before ovulation (9-10 h after hCG) were also characterized by loss of S-phase cells and an increased G0/G1 population. Results suggest that changes in cell cycle parameters in vivo are primarily mediated in response to physiological changes that occur in the intrafollicular environment initiated by the ovulatory stimulus. A similar lack of S-phase cells was observed in mucified cumulus cells collected 24 h after exposure in vitro of compact COC to dibutyryl cyclic adenosine monophosphate (DBcAMP), follicle-stimulating hormone or epidermal growth factor (EGF). Additionally, the proportion of cumulus cells in G2/M was enhanced in COC exposed to DBcAMP, suggesting that cell division was inhibited under these conditions. Thus, both the G1-->S-phase and G2-->M-phase transitions in the cell cycle appear to be amenable to physiological regulation. Time course studies revealed dose-dependent changes in morphology occurred within 6 h of exposure in vitro of COC to EGF or DBcAMP. Results suggest that the disappearance of the S-phase population is a consequence of a decline in the number of cells beginning DNA synthesis and exit of cells from the S phase following completion of DNA synthesis. Furthermore, loss of proliferative activity in cumulus cells appears to be closely associated with COC expansion and mucification, whether induced under physiological conditions in vivo or in response to a range of hormonal stimuli in vitro. The observations indicate that several signal-transducing pathways mediate changes in cell cycle parameters during cumulus cell differentiation.

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.

Some factors affecting embryo storage in laboratory animals.

Various factors affect the viability of embryos after freezing and thawing. When mouse embryos are supercooled below -6 degrees C before ice induction (seeding), survival is markedly reduced, apparently by inadequate dehydration of the embryos during subsequent cooling. Immediately after thawing, eight-cell mouse embryos and blastocysts experience a delay before normal embryonic development is resumed. A restorative period in culture combined with a modification of the synchrony between embryo and recipient helps to maximize survival following transfer. No loss of viability was observed in eight-cell mouse embryos after storage at -196 degrees C for four years. The preservation of the embryos of laboratory species other than the mouse and rabbit is limited by our lack of knowledge of the culture requirements for the development of such embryos.

Role of facilitative glucose uptake in the glucose-inorganic phosphate-mediated retardation and inhibition of development in different strains of mouse embryos.

Mouse embryos from different strains develop differently in vitro depending on the composition of the culture medium, and in particular on the presence or absence of glucose and inorganic phosphate. Glucose is both stimulatory and inhibitory in certain conditions. Glucose uptake by cells can be passive, down concentration gradients, or active, through sodium driven pumps, or can occur through facilitative transport. This study investigated the effects of inhibition of facilitative glucose transport on the glucose-inorganic phosphate-mediated blocks in development in three different strains of mouse embryo, CF-1, CD-1 and an F2 hybrid. Development of CF-1 and CD-1 embryos is blocked in medium containing glucose and inorganic phosphate but not in medium containing glucose alone, and F2 embryos are not affected. Inhibition of facilitated glucose transport to the eight-cell-morula stage in CF-1 and CD-1 embryos resulted in development in medium containing both glucose and inorganic phosphate, indicating that the prevention of facilitative glucose uptake can overcome the developmental block. Removal of inhibition before the eight-cell-morula stage resulted in total arrest of CF-1 embryos and minimum development of CD-1 embryos. F2 embryos are not affected by inorganic phosphate and glucose and showed no response to the transporter inhibitor at any stage. These data support the contention that facilitated glucose transport is active in embryos, is phosphate-dependent and that its inhibition can overcome the glucose-inorganic phosphate-mediated developmental blocks in mouse embryos.

The roles of pyruvate, lactate and glucose during preimplantation development of embryos from F1 hybrid mice in vitro.

Embryos of certain inbred mouse strains, and their F1 hybrids, are able to develop from the 1-cell to blastocyst stage in simple chemically defined media containing lactate (L), pyruvate (P) and glucose (G). The individual roles of these substrates in supporting complete preimplantation development in vitro was examined with 1-cell F2 embryos from B6CBF1 hybrid mice. Embryos collected between 26 and 27 h post hCG were cultured in medium containing L, P, LP or LPG. After 50 h in culture, the proportions developing to the morula stage were 1%, 83%, 94% and 100%, respectively. In combination, lactate and pyruvate appeared to act synergistically and both the rate and level of development to the morula stage were unaffected by the absence of glucose. After a further 46 h in culture, only the embryos grown in the presence of glucose developed into blastocysts. In LP medium, embryos arrested at the compacted morula stage late on day 3 of development. As culture continued in the absence of glucose, embryos decompacted (approximately 82 h post hCG) and subsequently degenerated. Exposure to medium containing glucose for the first, second or third 24 h period in culture was sufficient to support the morula-to-blastocyst transition. Glucose still supported this transition when embryos were transferred to LPG medium 3 h after the completion of compaction (76 h post hCG), but was ineffective 6 h later (82 h post hCG) once decompaction had commenced. We conclude that lactate and pyruvate together are able to support normal development of 1-cell F2 embryos to the morula stage in vitro, but that glucose is an essential component of the culture medium for development to the blastocyst stage.

Prenatal diagnosis in the human pre-implantation period. Meeting held at the Ciba Foundation on the 13th November 1986.

There is a clinical need for prenatal diagnosis before implantation for patients at high genetic risk who are unable to face the uncertainty of a pregnancy associated with conventional diagnostic methods, as well as for IVF patients who are at high risk for genetic or chromosomal anomalies. Research on human pre-embryos is required to establish: biopsy techniques during cleavage and at the blastocyst stage, in-vitro viability after biopsy, the culture potential of cells removed at these stages, cryopreservation of biopsied pre-embryos, and diagnostic procedures on biopsy material. The consensus view was that blastocyst biopsy is the most likely to succeed. The possibility should be considered of obtaining blastocysts by uterine lavage for diagnostic purposes in fertile women at high genetic risk. However it would be unethical to use uterine lavage to obtain blastocysts for research purposes. Rapid methods of typing the embryos the avoid the need for cryostorage, but there was a consensus (among those on the laboratory diagnostic side) that cryostorage would relieve some of the pressure of the ongoing diagnosis, and allow unhurried detailed analysis. All those present agreed that there is a need for further scientific research to establish pre-implantation diagnosis as a clinical reality. Close collaboration between workers in the fields represented will be necessary to achieve this goal.

The dynamic provision of different energy substrates improves development of one-cell random-bred mouse embryos in vitro.

Preliminary observations showed that one-cell embryos from random-bred MF1 mice avoid cleavage arrest at the two-cell stage ('in vitro two-cell block') when cultured in modified M16 culture medium containing lactate and pyruvate but lacking glucose. The roles of lactate, pyruvate and glucose during preimplantation development of embryos from random-bred mice in vitro were therefore examined. When all three substrates were present continuously during culture, one-cell embryos arrested at the two- to four-cell stages. Improved development to the morula stage after 96 h in culture was obtained in media containing pyruvate alone, lactate and pyruvate, pyruvate and glucose, lactate pyruvate and glucose for the first 24 h, and medium containing lactate and pyruvate for the remaining 72 h. In a second experiment, embryos were cultured in medium containing pyruvate alone, lactate and pyruvate or pyruvate and glucose for the first 24 h, and lactate plus pyruvate medium for the second 24 h. Subsequent transfer to medium containing lactate, pyruvate and glucose supported the morula to blastocyst transition. These results show that developmental arrest in vitro can be overcome by changing the combination of energy substrates at different stages of preimplantation development.

Imprinting of phosphoribosyltransferases during preimplantation development of the mouse mutant, Hprtb-m3.

The measurement of the activity of the X-linked enzyme HPRT has been widely used as an indicator of X-chromosome activity during preimplantation development in the mouse. More recently, the concomitant measurement of the activity of the autosomally-encoded enzyme APRT has been used in an attempt to decrease the variability inherent in the measurement of enzyme activity from minute samples such as preimplantation embryos. In this study the use of the HPRT-deficient mouse mutant, Hprtb-m3, allowed the unequivocal identification of the parental origin of HPRT activity measured in embryos derived from crosses between wild-type mice, and mice which were homozygous or hemizygous for the Hprtb-m3 allele. Results were similar to those of a previous study, where oocyte-encoded HPRT activity accounted for about 10% of total HPRT activity at 76 hours post human chorionic gonadotrophin injection and the paternally-derived Hprt allele was shown to be transcriptionally active by the late 2-cell stage. In contrast to other studies, differential expression of the two Hprt alleles was detected during the preimplantation period, in embryos derived from crosses between wild-type and HPRT-deficient mice. Evidence was also found for the existence of an X-linked locus which influences the amount of APRT activity in the unfertilized oocyte. We propose that the expression pattern of this locus may be influenced by its parental origin.

Capacitation-like changes occur in mouse spermatozoa cooled to low temperatures.

Previously we showed that >70% of mouse spermatozoa cooled slowly from 37 degrees C to 4 degrees C and warmed have undergone capacitation-like changes as examined by a chlortetracycline staining assay. These membrane changes are reflected in the ability of cooled spermatozoa to achieve fertilization rates in vitro similar to those of uncooled controls when added to oocytes immediately upon warming. The aim of this study was to determine the nature of these membrane changes. We found they were not dependent upon the rate of cooling to 4 degrees C and similar changes were observed when spermatozoa were cooled to higher temperatures (10 degrees and 20 degrees C), but it took longer for 50% of the spermatozoa to undergo such changes (3, 18, and 27 min for spermatozoa held at 4 degrees, 10 degrees, and 20 degrees C, respectively). Mixing cooled spermatozoa with oocytes immediately upon warming produced fertilization rates similar to fresh spermatozoa capacitated in vitro for 90 min before the oocytes were added. The rate of sperm penetration as determined by the fluorescent DNA stain Hoescht 33258 was also similar. However, the penetration time for cooled spermatozoa was significantly shortened when they were preincubated for 90 min before being added to oocytes. We conclude that membrane changes resembling capacitation (1) occur during cooling to temperatures above freezing, (2) are independent of cooling rate, (3) proceed faster at lower temperatures, and (4) obviate the need for prior capacitation in vitro before mixing with oocytes.

Analysis of the chromosome complement of frozen-thawed mouse oocytes after parthenogenetic activation.

Frozen-thawed mouse oocytes were artificially activated with Sr2+ and analyzed cytogenetically at the first cleavage division to examine the behavior of the maternal chromosomes independently of the paternal complement. There was no significant difference in the rate of activation between frozen-thawed and freshly collected oocytes and the majority of oocytes (> 90%) had a normal haploid chromosome constitution. The incidence of second polar body retention in frozen-thawed oocytes was low and did not differ significantly from that observed in fresh oocytes and oocytes exposed to dimethylsulfoxide (DMSO) at 0 degree C or 37 degrees C for extended periods beyond those required for protection. The frequency of aneuploidy was similar for frozen-thawed and fresh oocytes but oocytes held at 0 degree C without DMSO or held at 37 degrees C with DMSO for 1 hr showed a 2.5 and 12-fold increase in the frequency of aneuploidy compared with oocytes subjected to a conventional oocyte/embryo freezing regime. It is concluded that the procedures used in successful oocyte cryopreservation do not increase the incidence of chromosomal abnormalities of maternal origin in the resulting embryos.

A comparison of sperm- and IP3-induced Ca2+ release in activated and aging mouse oocytes.

Ca2+ release mechanisms in oocytes are highly sensitive and a number of agents including sperm and inositol trisphosphate (IP3) generate Ca2+ transients. Recently it was shown that this sensitivity decreases after fertilization and subsequent entry into the first mitotic cell cycle (Jones et al., Development 121, 3259-3266, 1995). In this study a similar decrease in the ability of IP3 to cause repetitive Ca2+ transients was observed in parthenogenetic embryos following activation with Sr2+, ethanol, or cycloheximide. This indicates that the decline in sensitivity of the Ca2+ releasing mechanism after oocyte activation is not associated with the fertilizing sperm. A similar decline in IP3-induced Ca2+ release was observed in metaphase II oocytes at 24 hr post hCG or later, although repetitive Ca2+ transients were induced in the aged oocytes after in vitro fertilization. Sperm-induced Ca2+ transients in aged oocytes were similar in duration and peak amplitude to younger oocytes, 15-18 hr post hCG. However, they showed a much reduced rate of rise which was also observed in younger oocytes after the intracellular stores had been depleted by thapsigargin. The results suggest that factors within the oocyte, such as store size, are important in enabling sperm to generate repetitive Ca2+ transients. Also, the Ca2+ release processes decline as the oocyte ages as well as after activation.

Influence of genetic background and media components on the development of mouse embryos in vitro.

One-cell embryos from some inbred and random-bred mice, but not those derived from certain F1 hybrids, suffer from a block during in vitro development known as the two-cell block. This two-cell block can be overcome by removing glucose or inorganic phosphate from the culture system or by altering the ratio of other medium components such as sodium, potassium, or bicarbonate. This issue is made more complex by the fact that the rate of development is different for each strain of mouse and this rate of development is invariably slowed under in vitro culture conditions. This study investigated the role of glucose and inorganic phosphate, individually or in combination, in relation to the two-cell block, and rate of development in vitro of two random-bred strains (CF-1 and CD-1) and an F2 hybrid derived from a nonblocking F1 hybrid cross (C57Bl/6NCr x C3H/HeNCr). Results were compared with in vivo data for each strain, and between media. There was a significant difference in the rate of preimplantation development in vivo of the three strains chosen, which was mirrored in vitro, regardless of the medium. The two random-bred strains suffered from a glucose-related two-cell block which was primarily mediated by inorganic phosphate. Inorganic phosphate was detrimental to embryo development regardless of strain or the presence of glucose. Although glucose, in the absence of inorganic phosphate, resulted in some blocking in development in the inbred strains initially, its presence in media was associated with increased rates of development at later stages in embryos that did not block. Glucose, but not inorganic phosphate, was beneficial but not essential to the development of the F2 embryos. The results of this study demonstrated that mouse embryos from different strains have differential rates of development in vivo and in vitro, and different sensitivities to glucose and inorganic phosphate. The two-cell block was primarily induced in the combined presence of glucose and inorganic phosphate. Glucose was beneficial in the absence of inorganic phosphate, and inorganic phosphate was detrimental to the rate of in vitro development.

Effect of cooling mouse spermatozoa to 4 degrees C on fertilization and embryonic development.

Attempts to freeze mouse spermatozoa in liquid nitrogen (-196 degrees C) have met with limited success. In an attempt to identify the factor(s) that damage mouse spermatozoa during cryopreservation, the effect of slow cooling to 4 degrees C was examined. Epididymal spermatozoa were collected into a variety of media at 37 degrees C, cooled slowly to 4 degrees C over 4 h and warmed in a water bath at 37 degrees C for 5 min. Survival of spermatozoa was assessed by motility, membrane integrity and acrosome status. Labelling with chlortetracycline showed that > 80% of spermatozoa were capacitated and had intact acrosomes immediately after warming compared with < 20% of freshly collected (control) spermatozoa. The rate of fertilization in vitro was similar using spermatozoa cooled in Dulbecco's phosphate-buffered saline and then mixed with oocytes immediately after warming and with control spermatozoa incubated for 2 h before mixing with oocytes (85%). Fewer oocytes were fertilized with spermatozoa cooled in either a modified HEPES-buffered Tyrode's medium or a simple HEPES-buffered medium with a high osmolarity (D3), 63% and 58%, respectively. Two-cell embryos were transferred to the oviducts of pseudopregnant recipients. Implantation was similar in all groups (81-88%) and 54-74% of embryos formed normal late stage fetuses.

Metabolism of [14C]glucose by postimplantation mouse embryos in vitro.

Carbon dioxide and lactate production from [14C]glucose were measured for post-implantation mouse embryos aged 6 to 9 1/2 days post coitum in static cultures with a defined medium. The rate of metabolism increased rapidly and paralleled the increase in protein content indicating a fairly uniform rate of metabolism throughout the period. At all stages studied more than 90% of the glucose utilized was converted to lactate. Over a quarter of carbon dioxide produced was derived from the C-1 position resulting in high C-1:C-6 ratios, indicating that the Pentose Phosphate Shunt is a major oxidative pathway. The influence of various culture condition on CO2 production showed that high concentrations of glucose did not affect glucose utilization whilst high lactate concentrations had a significant inhibitory effect. Pyruvate had no discernible effect.