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.

Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa.

Chromosome stability was maintained in mouse spermatozoa after freeze-drying or freezing without cryoprotection in a simple Tris.HCl buffer containing EGTA (50 mM) and NaCl (50 mM). The ability of spermatozoa to activate oocytes spontaneously was not destroyed by freeze-drying or freezing without cryoprotection in this solution. Embryos derived after injecting oocytes with sperm heads from rehydrated freeze-dried and from thawed spermatozoa developed normally. Provided the DNA integrity of the sperm nucleus is maintained, embryos can be generated by the intracytoplasmic sperm injection technique (ICSI) from severely damaged spermatozoa that are no longer capable of normal physiological activity. This procedure was effective for preserving spermatozoa from strains (C57BL/6J, 129/SvJ, and BALB/c) in which the fertility of spermatozoa frozen conventionally is extremely poor. The technique provides an effective means of storing mouse spermatozoa from many different inbred, mutant, and transgenic strains for biomedical research.

Restoration of a normal reproductive lifespan after grafting of cryopreserved mouse ovaries.

Fresh and frozen ovaries from 10 day old C57BL/6J-Gpi-1(a) mice were grafted orthotopically into ovariectomized B6CBF1 (homozygous Gpi-1(b)) recipients. The recipients were mated with B6CBF1 males. The birth and size of each litter was recorded. The electrophoretic variant of glucose phosphate isomerase was determined for each neonate. Twelve of 13 recipients of fresh ovary and 10 of 12 recipients of frozen ovary were fertile. Of these, 10 (fresh) and eight (frozen) had litters derived from the ovarian grafts only, or from the graft and native ovary. The breeding characteristics of recipients of fresh and frozen grafts were similar. The reproductive lifespan of the recipients of fresh (6.2 litters) and frozen (8.4 litters) grafts was similar to that of unoperated C57BL/6-Gpi-1(a) control females mated with B6CBF1 males (6.3 litters). Litter size was similar in recipients of grafted ovaries (fresh, 5.4 pups/litter; frozen, 6.3 pups/litter) and C57BL/6-Gpi-1(a) females (6.3 pups/litter). The results suggest that cryopreservation per se does not affect the long-term viability of ovarian tissue and provides an option for storing female germ cells. This is the first unequivocal demonstration that a normal reproductive lifespan can be restored by orthotopic grafting of frozen ovary.

The effect of follicle stimulating hormone and epidermal growth factor on the developmental capacity of in-vitro matured mouse oocytes.

This study investigates the effects of follicle stimulating hormone (FSH) or epidermal growth factor (EGF) on the development of mouse oocytes matured in vitro. The data show that addition of FSH or EGF does not significantly increase the proportion of oocytes maturing to metaphase II but does increase the ability of these oocytes to cleave to the 2-cell stage after fertilization. After transfer of 2-cell embryos to pseudopregnant recipients, 64-78% of the embryos implanted, which was significantly reduced compared to embryos from in-vivo matured controls (89%). Fewer fetuses at day 14 of gestation were produced from embryos derived from oocytes matured in basal conditions (26%), or in the presence of EGF (32%), compared to oocytes matured in vivo (64%) or in the presence of FSH (58%). Examination of polar bodies and pronuclei of oocytes matured in vitro suggests that an increase in the rate of triploidy may be partly responsible for the increased fetal loss after maturation in the absence of FSH. This study shows that the fertilization rate after in-vitro maturation can be improved by FSH and EGF and that subsequent embryonic development is improved specifically by FSH.

Production of normal offspring from mouse oocytes injected with spermatozoa cryopreserved with or without cryoprotection.

Epididymal mouse spermatozoa were suspended in various physiological solutions (CZB, PBS or isotonic saline) with or without 18% (w/v) raffinose before cooling to -20 degrees, -50 degrees or -196 degrees C and storage for 1-28 days. After thawing, a few spermatozoa frozen with raffinose were partially motile (about 2%) but in all other treatments they were immotile and diagnosed as 'dead' by staining that differentiates between live and dead spermatozoa. Almost all oocytes injected with sperm heads (nuclei) from spermatozoa frozen with and without raffinose were fertilized normally (95-100%) and developed to the two-cell stage (89-100%). No differences were found between the physiological media. The majority of oocytes fertilized with spermatozoa frozen in CZB medium developed to blastocysts (80-94%) but development was significantly reduced after fertilization with spermatozoa frozen in PBS and isotonic saline especially in the absence of raffinose (69 and 70% versus 51 and 50%). Normal fertile offspring were obtained in all treatments but there were significantly fewer offspring with spermatozoa stored at -196 degrees C in isotonic saline with or without raffinose and CZB with raffinose. Testicular spermatozoa were extremely sensitive to cryodamage: about 50% frozen to -196 degrees C in CZB with or without raffinose disintegrated after thawing. Almost 100% of oocytes injected with sperm heads from intact (at light microscope level) testicular spermatozoa developed to the two-cell stage but development to blastocysts was reduced significantly compared with that of controls especially those without raffinose. The data indicate that cryopreservation of sperm nuclei requires less stringent conditions than those for the retention of normal physiological function of intact spermatozoa. Motility and plasma membrane integrity are not essential for fertilization and the production of live offspring when nuclei of nonviable spermatozoa are injected into oocytes.

Effect of cryoprotectants on the survival of follicles in frozen mouse ovaries.

Ovaries from 10-day-old mice were exposed to 1.5 mol l-1 dimethylsulfoxide, 1,2-propanediol, ethanediol or glycerol for 5-60 min at room temperature before freezing. Follicles in fresh and frozen ovaries were counted and scored as normal or damaged in stained serial sections. More primordial follicles survived in ovaries frozen in dimethylsulfoxide, 1,2-propanediol and ethanediol (81-94%) than in those frozen in glycerol (4-28%). Prolonged exposure to ethanediol (60 min) before cooling decreased the survival rate, while increasing the exposure to glycerol (> or = 12 min) increased the survival rate. Fewer than 49% of primary follicles survived freezing. After transfer underneath the kidney capsules of ovariectomized immunodeficient recipients, there was no difference in the establishment of grafts of fresh (92%) and frozen (90%) ovaries, the number of recipients showing vaginal cornification (fresh, 91%, frozen 96%) or the latency of cornification (11 days). Fifteen days after transplantation, similar numbers of follicles remained in grafts of fresh ovaries, in ovaries frozen in dimethylsulfoxide and 1,2-propanediol, and in ovaries frozen after exposure to ethanediol for 5-30 min. Overall, the total number of follicles remaining in grafts of ovaries frozen in dimethylsulfoxide and 1,2-propanediol represented 42-46% of follicles present in ungrafted ovaries. This was not significantly different from grafts of fresh ovaries (63%). Dimethylsulfoxide and 1,2-propanediol are the most effective cryoprotectants for 10-day-old mouse ovaries. The majority of follicles are lost during graft establishment.

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.

Meiotic and mitotic Ca2+ oscillations affect cell composition in resulting blastocysts.

At fertilization periodic Ca2+ oscillations release oocytes from meiotic arrest. The present study examined whether these oscillations have a long-term role in pre- and postimplantation development, independent of their immediate effect. Sr(2+)-containing medium was used to induce oscillations during exit from meiosis and first embryonic mitosis and Sr(2+)-activated parthenotes were compared to ethanol-activated parthenotes and embryos generated by in vitro fertilization. After embryo culture, blastocysts were differentially stained for the inner cell mass and trophectoderm. It was found that oscillations both during exit from meiosis and during mitosis acted to increase the number of inner cell mass cells. In contrast, the trophectoderm cell number was largest in ethanol-activated parthenotes and smallest in fertilized embryos. Postimplantation development was also modestly improved by extending the time of exposure to Sr(2+)-containing medium. Together these data suggest that Ca2+ oscillations have a role in long-term embryonic events and that they provide more than merely a stimulus for meiotic resumption.

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.

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.

Ca2+ release and the development of Ca2+ release mechanisms during oocyte maturation: a prelude to fertilization.

Oogenesis involves the production of an oocyte that can undergo fertilization and support early development. The stimulus that initiates embryogenesis is an increase in the concentration of intracellular Ca2+ in the cytoplasm of the oocyte at the time of fertilization. The development of the ability of the oocyte to release Ca2+ in response to the fertilizing spermatozoon is an essential step in the process of oogenesis. Mammalian oocytes are particularly useful for studying the development of Ca2+ signalling systems, owing to the series of Ca2+ oscillations generated at fertilization, compared with the monotonic Ca2+ increase seen in nonmammalian species. Recent evidence has revealed that Ca2+ release mechanisms are modified during oogenesis. The maximal sensitivity of Ca2+ release is reached in the final stages of oocyte maturation, just before the optimal time for fertilization. In this review, we consider the mechanism underlying Ca2+ release in mammalian oocytes and discuss how the release mechanisms are modified during oocyte maturation. The tight co-ordination of the differentiation of the Ca2+ signalling system with the development of the oocyte provides a means of ensuring successful activation at the time of fertilization. Finally, we consider the consequences for embryo development in circumstances in which the co-ordination is lost.

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.

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.

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.

Repetitive sperm-induced Ca2+ transients in mouse oocytes are cell cycle dependent.

Mature mouse oocytes are arrested at metaphase of the second meiotic division. Completion of meiosis and a block to polyspermy is caused by a series of repetitive Ca2+ transients triggered by the sperm at fertilization. These Ca2+ transients have been widely reported to last for a number of hours but when, or why, they cease is not known. Here we show that Ca2+ transients cease during entry into interphase, at the time when pronuclei are forming. In fertilized oocytes arrested at metaphase using colcemid, Ca2+ transients continued for as long as measurements were made, up to 18 hours after fertilization. Therefore sperm is able to induce Ca2+ transients during metaphase but not during interphase. In addition metaphase II oocytes, but not pronuclear stage 1-cell embryos showed highly repetitive Ca2+ oscillations in response to microinjection of inositol trisphosphate. This was explored further by treating in vitro maturing oocytes at metaphase I for 4-5 hours with cycloheximide, which induced nuclear progression to interphase (nucleus formation) and subsequent re-entry to metaphase (nuclear envelope breakdown). Fertilization of cycloheximide-treated oocytes revealed that continuous Ca2+ oscillations in response to sperm were observed after nuclear envelope breakdown but not during interphase. However interphase oocytes were able to generate Ca2+ transients in response to thimerosal. This data suggests that the ability of the sperm to trigger repetitive Ca2+ transients in oocytes is modulated in a cell cycle-dependent manner.

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.

Cytogenetical analysis and developmental potential of vitrified mouse oocytes.

Mature mouse oocytes were cryopreserved by vitrification in 6 M dimethyl sulfoxide (VS). After warming they were either artificially activated with strontium (Sr2+), and the incidence of chromosome non-disjunction was assessed at first cleavage metaphase; or they were fertilized in vitro, and postimplantation survival was examined at Day 15 of gestation. Similar proportions of vitrified and freshly collected oocytes were activated with Sr2+ (75% and 82%, respectively). The majority of activated oocytes extruded the second polar body and formed a single pronucleus ( > 90%). When the exposure time to VS was increased from 90 to 110 sec without cooling, a significant proportion of activated oocytes arrested at the pronuclear stage (30%), and chromosome condensation did not occur. The frequency of aneuploidy in vitrified and control oocytes was similar, but when exposure to VS without cooling was extended, aneuploidy and second polar body retention were significantly higher than those of controls (p < 0.05). The rates of fertilization of vitrified (85%) and control oocytes (92%) did not differ. After transfer, similar proportions of vitrified and control embryos implanted (68-80%) and formed normal fetuses (38-49%). We conclude that vitrification in 6 M dimethyl sulfoxide is a simple and safe procedure for the preservation of mouse oocytes provided that the time of exposure to the cryoprotectant is carefully controlled.

Follicular development in cryopreserved marmoset ovarian tissue after transplantation.

Pieces of marmoset ovary were frozen by slow cooling in 1.5 M dimethylsulphoxide. The follicles in fresh and frozen tissue were counted and examined for morphological appearance in stained serial sections. The proportion of normal follicles was similar in fresh tissue and frozen tissue examined immediately after thawing. Follicles at all stages of folliculogenesis up to the small antral stage survived freezing and thawing. Fresh and frozen tissue was transplanted underneath the kidney capsules of ovariectomized immunodeficient mice. The establishment of grafts was similar, and oestrogenic activity (cornification of the vaginal epithelium) was observed in the recipients 20 and 16 days after transplantation of fresh and frozen grafts respectively. The total number of follicles and the proportion of normal follicles were similar in fresh and frozen grafts. Grafts of frozen tissue recovered between 7 and 15 days after transfer contained follicles up to the small antral stage of development. Grafts recovered between 21 and 32 days contained follicles at all stages of folliculogenesis, including large antral follicles (1-2 mm diameter). Our results suggest that freezing and thawing do not substantially damage marmoset ovarian tissue, and the cryopreserved tissue retains its ability to support the development of large antral follicles.

Calcium oscillations and protein synthesis inhibition synergistically activate mouse oocytes.

We have examined the ability of the two parthenogenetic agents, strontium (Sr2+) and cycloheximide, to activate mouse oocytes. We demonstrate that Sr2+ and cycloheximide act synergistically to promote parthenogenetic activation up to the pronuclear stage in oocytes collected immediately after ovulation. These two agents appeared to act independently, since incubation in Sr2+ media triggered a series of intracellular Ca2+ rises without affecting protein synthesis and cycloheximide inhibited protein synthesis without causing any intracellular Ca2+ changes. In addition, cycloheximide did not alter the pattern of Ca2+ oscillations induced by Sr2+. In contrast, we show that another commonly used parthenogenetic activation treatment, the Ca2+ ionophore A23187, has dual effects. Exposure of oocytes to the Ca2+ ionophore, A 23187, in Ca(2+)- and Mg(2+)-free medium leads to the activation of young oocytes. However, as well as generating a Ca2+ increase, the treatment of mouse oocytes with A23187 and Ca(2+)- and Mg(2+)-free media led to a marked inhibition of protein synthesis. Our data show that parthenogenetic agents may have two important loci for activating mammalian oocytes and that the combined effect on Ca2+ release and protein synthesis is most effective.

Nuclei from fertilized mouse embryos have calcium-releasing activity.

During mammalian fertilization, the sperm triggers a series of intracellular Ca2+ oscillations which initiate oocyte activation and the formation of pronuclei. Oocyte activation can be induced artificially by a variety of chemical and physical stimuli which elevate intracellular calcium. We show that the transfer of nuclei from 1- and 2-cell-stage fertilized mouse embryos to unfertilized oocytes stimulates the completion of meiosis and the formation of pronuclei. Nuclei from embryos that had developed to the 4-cell stage did not stimulate meiotic resumption. The ability to cause oocyte activation was specific to nuclei transferred from fertilized embryos as nuclei from parthenogenetic embryos or cytoplasts from fertilized or parthenogenetic embryos did not induce activation. Nucleus-induced oocyte activation was associated with the generation of intracellular Ca2+ transients, which were seen after nuclear envelope breakdown of the transferred nuclei. Treatment of the oocyte with the intracellular Ca2+ chelator, BAPTA, prior to nuclear transfer inhibited intracellular Ca2+ transients and oocyte activation. The specific Ca(2+)-releasing activity of the nucleus was not caused by sperm-induced protein synthesis since similar activity was present in nuclei originating from embryos exposed to cycloheximide throughout fertilization. The specific ability of nuclei from fertilized embryos to stimulate Ca2+ transients and oocyte activation was also found in nuclei from embryos parthenogenetically activated by the injection of a partially purified cytosolic sperm factor. The results suggest that the fertilizing sperm introduces Ca(2+)-releasing activity which becomes associated with the nucleus of early mammalian embryos.