Changes in anandamide levels in mouse uterus are associated with uterine receptivity for embryo implantation

Anandamide (N-arachidonoylethanolamine) is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. This investigation demonstrates that the periimplantation mouse uterus contains the highest levels of anandamide (142-1345 pmol/micromol lipid P; 1-7 microg/g wet weight) yet discovered in a mammalian tissue. The levels fluctuate with the state of pregnancy; down-regulation of anandamide levels is associated with uterine receptivity, while up-regulation is correlated with uterine refractoriness to embryo implantation. Anandamide levels are highest during the nonreceptive phase in the pseudopregnant uterus and in the interimplantation sites, and lowest at the site of embryo implantation. The lower levels of uterine anandamide at the implantation sites may be a mechanism by which implanting embryos protect themselves from the detrimental effects of this endogenous ligand. We also observed a reduced rate of zona-hatching of blastocysts in vitro in the presence of anandamide, and inhibition of implantation by systemic administration of a synthetic cannabinoid agonist CP 55,940. These adverse effects were reversed by SR141716A, a specific CB1-R antagonist. Taken together, the results suggest that an aberrant synthesis of anandamide and/or expression of the cannabinoid receptors in the uterus/embryo may account for early pregnancy failure or female infertility.     

Different expression patterns of MMP-2 and MMP-9 in breast cancer

The role of nitric oxide (NO) in activation of cGMP is well established. It has been proposed that the ratio of cAMP to cGMP may be important in the regulation of preimplantation embryonic growth and differentiation. Therefore, we determined the ability of murine preimplantation embryos to produce NO. In addition, NO as an endogenous smooth muscle relaxant and vasodilator is a candidate for involvement in embryo implantation because this process requires increased vascular permeability and uterine quiescence at the sites of blastocyst apposition. Nitrite assays, an indirect measure of NO production, indicate that preimplantation murine embryos produce NO. This production was reversibly inhibited by culture of embryos in medium containing a nonspecific NO synthase (NOS) inhibitor (NG-nitro-L-arginine). Additionally, inhibition of normal development was observed in embryos cultured with NOS inhibitor. NO levels increased in culture medium when ovariectomized progesterone-treated animals were exposed to estrogen for 1 h in utero. Such hormonal treatment induces implantation. These data indicate that NO levels are regulated by estrogen and may be important in regulation of implantation. In addition, these data demonstrate for the first time that NO production appears to be required for normal embryonic development.     

Nitric oxide synthase regulation during embryonic implantation

It has previously been demonstrated that uterine nitric oxide synthase (NOS) activity increases before embryonic implantation in rats. The aim of the present work was to investigate the regulation and the physiological relevance of the nitric oxide (NO) system in ovoimplantation. The increase in NOS activity in early pregnancy was found to be independent of the presence of embryos in the uterus. Whereas the Ca2+-dependent isoform of NOS increased gradually in the preimplantation days, the Ca2+-independent isoform increased just at the beginning of implantation (Day 5, 1800 hours); then the activity of both isoforms declined. Oestradiol, whose concentration peaks before implantation, might be regulating NOS activity in the uterus, since treatment of rats with tamoxifen, a receptor antagonist, reduces the activity of both isoforms to preimplantation levels. Intraluminal injections of L-NAME (0.5 mg kg[-1]), a competitive inhibitor of NOS, reduced by 50% the number of implanted embryos; this suggests that the NO system plays a role during implantation. The data suggest that oestradiol might be a modulator of NOS activity during nidation and that NO production is necessary to achieve a successful embryo implantation.     

An EORTC international multicenter randomized trial (EORTC number 19923) comparing two dosages of liposomal amphotericin B for treatment of invasive aspergillosis

Experiments were performed to determine the actions of recombinant bovine interleukin-1beta (IL-1beta) on the growth of preimplantation embryos. In the first series of studies, IL-1beta was added at 8-10 h after insemination, and the percentage of oocytes developing to the blastocyst stage was evaluated. IL-1beta increased development to the blastocyst stage when embryos were cultured at high density ( approximately 25-30 embryos/drop) but decreased or had no effect on development when cultured at low density ( approximately 10 embryos/drop). Thus, the positive effect of IL-1beta depends upon some other embryo-derived product. The effect of IL-1beta on embryonic development was maintained in completely denuded embryos, indicating that cumulus cells do not mediate the actions of IL-1beta. Maximum development of embryos cultured at approximately 25-30/drop occurred at 0.1-1 ng/ml; 10 ng/ml was less effective. Addition of IL-1beta to groups of approximately 25-30 embryos/drop at 8-10 h after insemination also increased embryo cell number at Day 5 postinsemination by increasing the proportion of embryos that reached the 9- to 16-cell stage. However, IL-1beta had no effect on the proportion of blastocysts when added at Day 5 postinsemination. Thus, IL-1beta probably acts to increase blastocyst numbers by exerting actions on embryo growth before Day 5. In contrast to its effect on embryos, addition of IL-1beta during oocyte maturation did not affect cumulus expansion, cleavage rate of oocytes, or subsequent development to the blastocyst stage. In conclusion, IL-1beta can modulate growth of bovine embryos at early stages of development in a manner dependent upon embryo density.     

Particle concentration fluorescence as an alternative to radioactivity for cytokine receptor binding assays

OBJECTIVE: To determine the in vitro and in vivo effects of lidocaine on preimplantation mouse embryo development during prolonged exposure. STUDY DESIGN: In vitro experiments: Two-cell mouse embryos were exposed to lidocaine doses of 0, 10, 100, and 1000 micrograms/mL for 72 h. In vivo experiments, female mice were exposed to lidocaine doses of 0, 0.3, 1.7, and 3.3 mg/kg body weight on days 1, 2, 3, and 4 of pregnancy. Early development, cell number, mitotic index, and micronuclei frequency were examined. RESULTS: Development to the blastocyst stage was inhibited by all the doses tested in vitro and in vivo. Most of the affected embryos showed arrest and degeneration. The cell number was decreased, but not the mitotic index. Furthermore, clastogenic damage was observed in vitro. CONCLUSIONS: Lidocaine adversely affects preimplantation mouse embryo development in vitro and in vivo.     

Effects of ammonium dinitramide on preimplantation embryos in Sprague-Dawley rats

Ammonium dinitramide (ADN) is a class 1.1 oxidizer that may be used in rocket propellants and explosives. Previous studies have shown that ADN is a female reproductive toxicant, causing implantation failure in Sprague-Dawley rats when it is administered during the preimplantation period of gestation. The purpose of this follow-up study was to identify the mechanism(s) associated with implantation failure following exposure to ADN. Mated female rats were treated with 2.0 grams per liter (g l-1) ADN in their drinking water for 24, 48, 72, or 96 h before preimplantation embryos were harvested from the oviducts or uterine horns. On gestation day 1 (GD-1), comparable numbers of morphologically normal two-cell embryos were harvested from the oviducts of the treatment and control groups. On GD-2, the development of the embryos harvested from the treated animals was either slowed or halted when compared to the control embryos. By GD-4, 98% of the embryos harvested from the control group had developed to the morula or blastocyst stage; these were collected from the uterine horns. On GD-4 in the treated group, 41% of the harvested embryos remained at the two- to six-cell stage and 59% were degenerate; 82% of these embryos were collected from the oviducts. These data suggest that the implantation failure seen in animals treated with ADN is due to embryolethality.     

Effects of genistein and lavendustin on reproductive processes in domestic animals in vitro

The aim of our experiments was to study the influence of genistein [tyrosine kinase (TK) inhibitor with estrogenic activity] and lavendustin A (TK inhibitor without estrogenic activity) on female reproductive processes in domestic animals in vitro. It was found that genistein (0.001-1 microg/ml) increased IGF-I release by cultured bovine and porcine granulosa cells, but decreased its secretion by rabbit granulosa cells (0.01-10 microg/ml). Genistein stimulated progesterone secretion by bovine and rabbit granulosa cells (at 0.01-10 microg/ml), estradiol output by rabbit granulosa cells (at 1 microg/ml) and porcine ovarian follicles (at 10 microg/ml), as well as cAMP production by bovine (at 0.001-1 microg/ml) and rabbit (at 1 microg/ml) granulosa cells. No effects of genistein (at 10 microg/ml) on PGF-2 alpha and progesterone release by porcine ovarian follicles were observed. Genistein significantly (P < 0.05) stimulated the reinitiation and completion of nuclear maturation of porcine oocytes (at 5 microg/ml), as well as the preimplantation development of rabbit zygotes (at 1 microg/ml). Lavendustin A (0.001-1 microg/ml) increased IGF-I release by bovine (but not by porcine) granulosa cells, cAMP release by bovine granulosa cells, and PGF-2 alpha output by porcine ovarian follicles (at 10 microg/ml). Lavendustin (at 1 microg/ml) had no significant effect on IGF-I release by porcine granulosa cells, on estradiol and cAMP output by rabbit granulosa cells, or on progesterone secretion by porcine follicles (at 10 microg/ml). Inhibitory actions of lavendustin (at 10 microg/ml) on estradiol secretion by porcine follicles were also found. Furthermore, lavendustin, like genistein, promoted the reinitiation and completion of meiosis in porcine oocytes. The present study demonstrates a predominantly stimulatory effect of TK inhibition on endocrine and generative processes in domestic animals. The majority of these effects are similar for both compounds, indirectly suggesting that their action is due to tyrosine kinase inhibition and protein kinase A-stimulation, rather than estrogenic activity.     

Vertebral artery trauma

The developmental consequences of paternal exposure to acrylamide (50 mg/kg i.p. for 5 days) were assessed in preimplantation embryos. There was a significant increase in the proportion of morphologically abnormal embryos after postmeiotic treatment during spermatogenesis (88.7% vs. 14.8% in control). Abnormal embryos had an average of 1.8 +/- 3.5 cells and > 80% had at least one fragmented nucleus. In addition, morphologically normal embryos were significantly delayed (34.3 +/- 12.8 cells per embryo vs. 57.6 +/- 15.7 in control, P < 0.001). Acrylamide caused 10- and 20-fold increases in frequencies of cells with micronuclei (MN) in morphologically normal and abnormal embryos, respectively (41 and 93 MN per 1,000 cells). Both centromere-negative (MN-) and centromere-positive (MN+) were induced. Nuclei of abnormal embryos were significantly larger (900 microm2 vs. 250 microm2) than controls. In addition, MN of abnormal embryos were larger than those of normal embryos (21.2 microm2 vs. 6.5 microm2, P < 0.01). Among control embryos, MN+ were significantly larger than MN- (P < 0.05). These findings suggest that the preimplantation embryo is a sensitive indicator of paternally transmitted effects on early development. Multiple mechanisms appear to be involved, including cytogenetic damage, proliferation arrest/delay, and fertilization failure. Future studies are needed to establish how induced cytological defects in preimplantation embryos contribute to birth defects and other postimplantation abnormalities.     

Preimplantation genetic diagnosis increases the implantation rate in human in vitro fertilization by avoiding the transfer of chromosomally abnormal embryos

OBJECTIVE: To verify the percentage of chromosomally abnormal preimplantation embryos in patients with a poor prognosis and possibly to increase the chance of implantation by selecting chromosomally normal embryos. DESIGN: A prospective, randomized, controlled study. SETTING: In vitro fertilization program at the Reproductive Medicine Unit of the Società Italiana Studi Medicina della Riproduzione, Bologna, Italy. PATIENT(S): In a total of 28 stimulated cycles, the maternal age was > or = 38 years and/or the patient had > or = 3 previous IVF failures, factors that indicated a poor prognosis. After consent, 11 patients underwent preimplantation genetic diagnosis for aneuploidy, whereas 17 controls underwent assisted zona hatching. INTERVENTION(S): Simultaneous analysis of chromosomes X, Y, 13, 18, and 21 in a blastomere biopsied from day-3 embryos. Chromosomal analysis was performed with fluorescence in situ hybridization. Assisted zona hatching was performed on day-3 embryos from the control-group patients. MAIN OUTCOME MEASURE(S): Embryo morphology, results of fluorescence in situ hybridization, clinical pregnancies, and implantation. RESULT(S): In the study group, a total of 61 embryos were analyzed by fluorescence in situ hybridization, and 55% were chromosomally abnormal. Embryo transfer with at least one normal embryo was performed in 10 cycles. Four clinical pregnancies resulted, with a 28.0% implantation rate. In the control group, 41 embryos were transferred in 17 cycles after the assisted zona hatching procedure, yielding four clinical pregnancies and an 11.9% implantation rate. CONCLUSION(S): Infertile patients classified as having a poor prognosis have a high percentage of chromosomally abnormal embryos. The advantage of selecting and transferring embryos with normal fluorescence in situ hybridization results has an immediate impact on implantation.     

The use of early pregnant mares as embryo recipients

Fourteen normal, cyclic mares, treated to synchronise oestrus and ovulation and inseminated artificially with fresh semen, were assigned to a donor or a recipient group after ovulation, with the aim of obtaining a degree of synchrony of > or =2 days. Ten embryos, collected on Day 6 or 7 after ovulation (Day 0), were transferred nonsurgically to inseminated recipient mares (IRM) that had ovulated up to 5 days after the respective donors, or to pregnant recipient mares (PRM) that had ovulated 2-7 days before the donors. Embryonic size and development, as determined by ultrasound examination, were used to distinguish embryos derived from the recipient (recipient embryo = RE) or from the donor (transferred embryo = TE) mare. In cases of twin pregnancy, the RE was manually squeezed on Days 14-16. Abortion was induced in all mares on Day 30. Three of 6 TE developed in IRM. Two of 6 IRM developed a twin pregnancy (RE+TE), while 4 of 6 IRM developed a singleton pregnancy (1 TE and 3 RE). None of 4 TE developed in Day 9-14 PRM and one of these PRM lost her own embryo following ET. The experiment demonstrated that a mare can carry her own embryo and a transferred embryo simultaneously. However, the status of pregnancy does not improve conception rate in recipient mares that ovulate prior to the donor.     

Effect of twenty-four-week treatment with the antiestrogen EM-800 on estrogen-sensitive parameters in intact and ovariectomized mice

Treatment with the antiestrogen EM-800, at the daily oral dose of 3 microg, 10 microg, 30 microg, or 100 microg for 24 weeks, caused a marked inhibition of uterine and vaginal weight in both intact and ovariectomized mice. Maximal 64% and 41% inhibitions of uterine weight were achieved in intact and ovariectomized animals, respectively. Similar inhibitory effects of EM-800 were observed on vaginal weight with maximal inhibitions of 71% and 35%, in intact and ovariectomized animals, respectively. The pure antiestrogenic activity of EM-800 on the hypothalamo-pituitary-ovarian axis is illustrated by the 76-91% increases in ovarian weight observed in intact animals treated with the 10-100 microg doses of the antiestrogen. Serum 17beta-estradiol was 93% increased at the 100 microg daily dose of EM-800, whereas serum androstenedione, testosterone, and dihydrotestosterone were 141-713% increased over control at the same dose of the antiestrogen. Serum LH was increased by treatment with EM-800 in intact animals, whereas no effect was observed on the elevated gonadotropin levels in ovariectomized animals. At all doses used in intact animals, the antiestrogen caused a complete disappearance of the glandular elements of the mammary gland, the atrophy being comparable with that observed in ovariectomized mice. The mammary gland of EM-800-treated animals was exclusively composed of an atrophied ductal system lined by atrophied epithelial cells with an absence of lobulo-glandular elements. No effect of the compound was observed on the histology of the mammary gland in ovariectomized animals, thus showing the pure antiestrogenic effect of EM-800 on the mammary gland, as shown also for the uterus, vagina, and hypothalamo-pituitary axis. At histopathology, all doses of EM-800 in intact animals led to a moderate to severe uterine and vaginal atrophy. The uterine atrophy affected both the myometrium and the endometrium. Interestingly, the uterine atrophy achieved in intact animals treated with EM-800 was greater than that observed after ovariectomy alone, thus clearly demonstrating the pure antiestrogenic activity of EM-800. The present data show the highly potent and pure antiestrogenic activity of EM-800 on all parameters measured after 6 months of treatment in both intact and ovariectomized mice, a maximal effect being reached at the daily 10 microg dose of the antiestrogen in intact animals.     

Postcoital antifertility activity of aminoalcohols

Previously, postcoital antifertility effects of a number of aminoalcohols, including 2-(isopropylamino)-ethanol, have been demonstrated in rodents. In this experiment, we compared the antifertility activity of 2-(isopropylamino)-ethanol to the following analogs: hydroxyethylpiperidine, hydroxyethylpiridine, hydroxyethylpirrolidine, and hydroxyethylpirrolidone. Female rats were gavaged on Days 0 through 5 of gestation with 0.7 mmol/kg/d of these substances. Only 2-(isopropylamino)-ethanol and hydroxyethylpirrolidine showed a strong antifertility activity: females treated with 2-(isopropylamino)-ethanol had no signs of implantation, whereas those treated with hydroxyethylpirrolidine had 100% early resorptions. Treatments with these two substances during the periimplantation period (Days 4 and 5) produced 100% early resorptions. Histologic examination of the implantation sites showed signs of embryonic degeneration starting from Day 6.5 of gestation. The flushing of the uteri of females treated with 2-(isopropylamino)-ethanol on Days 0 through 3 post coitum showed 78% of the embryos at the stage of 1 to 3 blastomeres, whereas the embryos of females treated during the same period with hydroxyethylpirrolidine were normal blastocysts. Therefore, 2-(isopropylamino)-ethanol and hydroxyethylpirrolidine are able to kill embryos during the early implantation stages, whereas 2-(isopropylamino)-ethanol is also able to stop the development of preimplantation embryos.     

Success rates when attempting to nonsurgically collect equine embryos at 144, 156 or 168 hours after ovulation

The purpose of this study was to evaluate the exact age when the equine embryo reaches the uterus. The time of ovulation was determined by hourly ultrasound examinations starting 32 h after an injection of crude equine pituitary gonadotrophin or human chorionic gonadotrophin, or after the first of 4 injections of buserelin. Nonsurgical uterine flushings were carried out 144 h (Day 6), 156 h (Day 6.5) or 168 h (Day 7) after ovulation. Induction of ovulation was attempted in 101 oestrous cycles and 61 of 101 mares (60.4%) ovulated 32-44 h post injection. Sixty embryo collections were performed which yielded: 0/20 embryos at 144 h, 9/17 embryos (53%) at 156 h and 12/23 embryos (52%) at 168 h. The mean (+/- s.e.m.) diameter of the embryo was significantly greater (P<0.01) at Day 7 (244 +/- 15 microm) than at Day 6.5 (186 +/- 9.1 microm), and variability in size was observed among embryos collected from the same mare after synchronous natural multiple ovulations. These results suggest that; i) horse embryos enter the uterus between 144 and 156 h after ovulation, and ii) the time interval between ovulation and fertilisation in mares is inconsistent and/or embryonic development rate may differ between individual embryos.     

A method for endoscopic embryo collection and transfer in the rabbit

Thirty-two rabbits were used for endoscopic embryo collection and transfer. For embryo collection midventral laparoscopy and transcervical endoscopy were combined for orthograd flushing of the oviducts and uterine horns. Transfer was performed transcervically under laparoscopic control. The mean number of corpora lutea counted in nine donors was 13.3 +/- 8.4. A total of 72 morulae/blastocysts were obtained. No embryos were recovered when only the uterine horns were flushed. 291 embryos were transcervically transferred to 23 recipients which resulted in 10 pregnant animals at day 12. Two of three slaughtered recipients showed together 4 implantation sites. Four animals delivered 12 pups.     

Genetic regulation of preimplantation mouse embryo survival

The preimplantation period of mammalian development is characterized by cleavage of a one-cell embryo to a blastocyst stage embryo. During preimplantation development, 15%-50% of the embryos die as a result of factors that are largely unknown. Two parameters of preimplantation development, a fast rate of development and a low degree of fragmentation, are indicative of good embryo quality. There is mounting evidence that genes control both rate of development and degree of fragmentation. We have discovered a gene, Ped (preimplantation embryo development), which controls the rate of preimplantation embryonic cleavage. The Ped gene is encoded by two similar genes, Q7 and Q9, in the Q region of the mouse major histocompatibility complex (MHC). The Ped gene product is an MHC class Ib protein, the Qa-2 antigen. The mechanisms by which the Ped gene controls rate of embryonic cleavage division are being explored. In order to understand genetic mechanisms underlying the second criterion of embryo quality, degree of fragmentation, we have begun to assess expression of the genes that could potentially regulate apoptosis in preimplantation embryos. We have shown that staurosporine can induce apoptosis in mouse blastocysts. By using RT-PCR, we have shown that genes encoding protein in the two major gene families that regulate apoptosis, the Bcl-2 and caspase gene families, are present in preimplantation embryos. We hypothesize that there is a homeostatic mechanism by which genes that regulate cell survival and those that regulate cell death determine the overall viability of preimplantation embryos.     

Embryonic origin of preimplantation factor (PIF): biological activity and partial characterization

Preimplantation factor (PIF) is detected in the serum of women shortly after fertilization; its origin, however, has not been established. In this study, the embryonal origin of PIF was investigated and partial characterization of the factor was carried out. Culture media from viable human 2-8-cell stage embryos and mouse 2-cell-blastocyst stage embryos were analysed using the lymphocyte/platelet binding assay (LPBA). The assay was performed by combining culture media with donor O+ type blood-derived lymphocytes/platelets, complement and an antibody against CD2. Increased autorosette formation between lymphocytes and platelets (> 9%) was an indication for the presence of PIF. In addition, the effect of platelet-activating factor (PAF) and chaperonin 10 on PIF activity was determined. Partial purification of PIF was carried out using gel filtration and reverse-phase high purification liquid chromatography (HPLC), followed by mass spectrometry. Culture media of single human viable fertilized oocytes were negative for PIF; however, the 10-fold concentrated medium was positive for PIF. In medium in which five or more mouse embryos were cultured, PIF activity was observed starting at the morula stage and was higher by the blastocyst stage. Addition of PAF or chaperonin 10 to the PIF assay did not elicit a specific effect on PIF activity. Chromatographic data suggest that PIF activity is due to low molecular weight proteins. PIF appears to be a low molecular weight protein which is derived from viable preimplantation embryos. It is different from PAF or chaperonin 10. Its final characterization will be valuable for better understanding of maternal recognition of pregnancy and implantation.     

Neonatal androgenization potentiates the inhibitory effects of blood withdrawal on ovulation in the rat

Rats treated on Day 5 of life with testosterone propionate (TP) were tested for their ovulatory response to pregnant mare's serum gonadotropin (PMSG) on the 30th day of life with and without cardiac puncture under ketamine/xylazine anesthesia. TP without cardiac puncture in doses of 0.625, 1.25, or 2.5 microg did not inhibit ovulation, but 5.0 microg of TP inhibited ovulation in 15 of 16 rats. When cardiac puncture was performed at 1900 hr in rats not given TP, seven of eight ovulated on Day 33. However, none of the rats that received either 1.25 or 2.5 microg of TP ovulated after cardiac puncture at 1900 hr, and only one of seven given the 0.625-microg dose ovulated. Thus, the stress of bleeding greatly enhanced the inhibitory effect of otherwise ineffective doses of TP. The preovulatory luteinizing hormone (LH) surge was delayed and diminished by neonatal TP in a dose-related manner in animals from which blood was drawn from a previously inserted catheter. When blood drawn from similarly TP-treated animals by cardiac puncture, serum levels of LH were further reduced. Progesterone, given at 1100 hr on Day 32, was capable of partially overcoming the inhibitory effects of the combined treatments on both ovulation and serum LH levels at all but the highest dose of TP tested (5.0 microg). We conclude that neonatal exposure to androgen sensitizes rats to ovulation-inhibiting factors, such as bleeding, in a manner which appears to delay as well as inhibit the preovulatory release of LH. Such early exposure to androgen, therefore, may determine the susceptibility of individuals to reproductive failure in adult life.     

Response of preimplantation murine embryos to heat shock as modified by developmental stage and glutathione status

Objectives were to characterize developmental changes in response to heat shock in the preimplantation mouse embryo and to evaluate whether ability to synthesize glutathione is important for thermal resistance in mouse embryos. Heat shock (41 degrees C for 1 or 2 h) was most effective at disrupting development to the blastocyst stage when applied to embryos at the 2-cell stage that were delayed in development. Effects of heat shock on ability of embryos to undergo hatching were similar for 2-cell, 4-cell, and morula stage embryos. The phenomenon of induced thermotolerance, for which exposure to a mild heat shock increases resistance to a more severe heat shock, depended upon stage of development and whether embryos developed in vitro or in vivo. In particular, induced thermotolerance was observed for morulae derived from development in vivo but not for 2-cell embryos or morulae that developed in culture. Administration of buthionine sulfoximine to inhibit glutathione synthesis did not increase thermal sensitivity of 2-cell embryos or morulae but did reduce subsequent development of 2-cell embryos at both 37 degrees and 41 degrees C. In summary, changes in the ability of 2-cell through morula stages to continue to develop following a single heat shock were generally minimal. However, 2-cell embryos delayed in development had reduced thermal resistance, and therefore, maternal heat stress may be more likely to cause mortality of embryos that are already compromised in development. There were also developmental changes in the capacity of embryos to undergo induced thermotolerance. Glutathione synthesis was important for development of embryos but inhibition of glutathione synthesis did not make embryos more susceptible to heat shock.