Expression of a male-specific factor on various stages of preimplantation bovine embryos

Four-cell to blastocyst stage bovine embryos were collected from superovulated donors and cultured for 90 min in Ham's F-10 medium (HF-10) containing 10% (V/V) absorbed anti-histocompatibility (H)-Y antiserum. Embryos were then washed 3 times and placed in HF-10 supplemented with 10% (V/V) fluorescein isothiocynate (FITC)-conjugated goat anti-mouse gamma globulin. After an additional wash, embryos were placed in fresh drops of HF-10, individually evaluated at 200 X magnification, and classified as either fluorescent (H-Y-positive) or nonfluorescent (H-Y-negative). Embryos were then placed in drops of HF-10 containing 14% vinblastin and cultured for 4-6 h. Embryos were coded and individually karotyped, and the sex chromosomes were identified. H-Y antigen was detected as early as the eight-cell stage, but not at the four-cell stage. Seventy-nine percent of fluorescent embryos and 89% of nonfluorescent embryos were XY and XX, respectively. Another experiment was carried out in which H-Y antigen was detected on intact inner cell masses (ICM) isolated by immunosurgery from expanded blastocysts that also had been assayed for H-Y antigen. Eighty-eight and 92%, respectively, of ICM classified as fluorescent or nonfluorescent had been scored the same as intact blastocysts. It is concluded from these data that H-Y antigen can be detected on eight-cell to blastocyst stage bovine embryos. There appears to be a localization of detectable antigen in the area of the ICM at the expanded blastocyst stage. Detection of H-Y antigen is an effective, noninvasive method for identification of the sex of preimplantation bovine embryos.     

Identification of a male-specific histocompatibility protein on preimplantation porcine embryos

An indirect immunofluorescence assay was used to detect the presence of male-specific protein(s) on various stages of preimplantation porcine embryos. Embryos were collected at slaughter from the reproductive tracts of day?2.5, ?4, ?5, ?6, and ?8 (day 0 = first day of estrus) sows and gilts. Embryos were placed in medium containing an anti-male primary antibody, washed, and transferred to culture drops containing a fluorescein isothiocyanate (FITC)-labeled secondary antibody. Embryos were classified as either fluorescent (H-Y positive) or nonfluorescent (H-Y negative), transferred to coded drops, and karyotyped to examine sex chromosomes. A total of 91 eight-cell to blastocyst stage embryos were evaluated; of these, 46% were classified as fluorescent and 54% as nonfluorescent. Of readable metaphase spreads (65%) from these embryos, 81% (48 of 59, P < 0.005) were correctly sexed by immunological detection of the male-specific antigen. Although 13 % (2/15)of four-cell embryos evaluated were classified as fluorescent, the accuracy with which embryos at this stage were sexed by detection of H-Y antigen was not different from 50%. Fifty percent of eight-cell embryos were classified as H-Y positive with 78% of embryos correctly sexed. It was concluded that the eight-cell embryo is the earliest stage of development for which there is evidence for expression of H-Y antigen. Detection of the male-specific protein was difficult at the expanded blastocyst stage.     

Embryo sex selection by a rat male-specific antibody and the cytogenetic and developmental confirmation in cattle embryos

Embryos of mouse, rabbit, goat, sheep, and cattle were separated into 2 groups on the basis of their morphology when incubated with a male-specific antibody (qualified here as the H-Y antibody) prepared from newborn rat testis. When morula-stage embryos were cultured in the presence of this H-Y antibody, the development of roughly one half of the embryos was arrested at that stage, whereas the other half continued to develop to the blastocyst stage. The developmentaly arrested group of embryos resumed their development into blastocysts when cultured in antibody-free medium. Eighty to 90% of cattle embryos whose development was unaffected by the antibody were shown to possess a female karyotype (XX), and close to 80% of those embryos whose development was arrested possessed a male karyotype (XY). Cattle embryos whose sex had been presumptively identified by development in the presence of the H-Y antibody were cryopreserved and transferred, and the sex of the calves was examined. The overt sex of the young born from sexed embryos was found to be the same as that determined by chromosomal analysis. © 1993 Wiley-Liss, Inc.     

Investigation of sperm cytotoxicity as an indicator of ability of antisera to detect male-specific antigen on preimplantation mouse embryos

H-Y antisera were produced in C57BL/6 female mice by repeated intraperitoneal injections of syngeneic male spleen cells. Epididymal spermatozoa were incubated in the presence of H-Y antisera and guinea-pig serum as a complement source. Levels of ATP remaining after treatment were used to calculate the amount of specific killing. Sera of different cytotoxic titres were used in an indirect immunofluorescent assay with a fluorescein isothiocyanate-conjugated IgG fraction of goat anti-mouse IgG (Fc fragment specific) as second antibody. Embryos were classified as fluorescent or nonfluorescent, transferred to pseudopregnant recipients, and allowed to develop to term. Of 12 sera tested for sperm cytotoxicity, 5 were different from a nonimmunized control serum (P less than 0.05). Percentage specific killing in each of these sera was 7.8 +/- 4.2, 11.7 +/- 3.0, 26.0 +/- 2.2, 27.7 +/- 3.7 and 39.2 +/- 4.8, respectively (mean +/- s.e.m. with three replicates). The 5 sera and an additional one (4.9 +/- 1.3% specific killing) were used in the embryo sexing experiment. The accuracy with which these sera correctly identified sex of preimplantation embryos was 60, 46, 74, 73, 74 and 48%, respectively. Correlation coefficients were 0.86 (P less than 0.05) for specific sperm cytotoxicity and percentage of nonfluorescent embryos that were female and 0.78 (n.s.) for specific sperm cytotoxicity and percentage of fluorescent embryos that were male. Therefore, although the sperm cytotoxicity test is useful for screening antisera for the study of H-Y antigen expression on preimplantation embryos, nonfluorescent embryos are more accurately classified as females than are fluorescent embryos as male.     

Effects of conditioned media on porcine embryos at different stages of development

The objective of our study was to determine the effect of conditioning media with homologous porcine uterine cells on the developmental rate of porcine embryos. Cell monolayers were prepared by selective dissection and digestion of sections from the uterus of prepuberal gilts that were primed with PMSG and hCG. Conditioned media were used with 2 type of embryos: 4-cell stage (Experiment 1) or blastocyst stage (Experiment 2). In Experiment 1, embryos were collected surgically by flushing the oviducts, 36 to 48 h following the first of 2 inseminations. Embryos were cultured in Whitten's medium containing 1.5% BSA as a protein source until they attained the 4-cell stage. Embryos at the 4-cell stage were cultured randomly in either Whitten's medium with 1.5% BSA or Whitten's medium with 1.5% BSA that was previously conditioned for 24 h with an endometrial epithelial cell monolayer. Embryos were cultured in 50-microl drops covered with oil in a 38.5 degrees C, 5% CO(2) in air incubator. There was no advantage to using the conditioned media with the 4-cell stage embryos. The embryos were less developed than those cultured in nonconditioned Whitten's medium (P <0.001). In Experiment 2, embryos were cultured at the blastocyst stage. They were recovered the same way as in Experiment 1 and then cultured in Whitten's medium containing 1.5% BSA until they reached the blastocyst stage. At the blastocyst stage (Day 6), embryos were randomly assigned to 1 of the 6 following treatments: Whitten's with 1.5% BSA or Whitten's plus 1.5% BSA that was previously conditioned with endometrial epithelial cell monolayer, TCM-199 containing 0.4% BSA or TCM-199 plus 0.4% BSA that was previously conditioned with endometrial epithelial cell monolayer, finally, TCM-199 containing 10% serum or TCM-199 plus 10% serum that was previously conditioned with endometrial epithelial cell monolayer. Results show that initiation of hatching was significantly enhanced by conditioning the Whitten's media.     

Sexing in vitro produced bovine embryos, at different stages of development, using rat H-Y antiserum

The male-specific H-Y antigen is present on mammalian cell membranes and has been identified by various methods, including antiserum cytotoxicity. The objective of the present study was to determine the sex of in vitro produced (IVP) bovine embryos, at varying stages of development, by culturing in the presence of rat monoclonal H-Y antibodies. Embryos derived from IVM/IVF were classified according to the interval after IVF (48, 96 or 120 h) as Category 1, 2 or 3 if they had 4 to 8, <32, and >32 cells, respectively. Embryos of each category were cultured for 24h in TCM-199 supplemented with bovine oviductal epithelial cells, fetal calf serum (FCS), and antibiotics (Control group), to which the following had been added: guinea pig serum (GPS; C' group); H-Y antiserum (HY group); or GPS and H-Y antiserum (C' + HY group). After culture, embryos were designated as "affected" when development was arrested or one or more blastomeres was degenerate; embryos lacking these changes were designated "unaffected." The sex of each embryo was subsequently determined by chromosome analysis. After 48h of IVF (Category 1), within each of the four treatments, the proportion of unaffected embryos was higher than the proportion of unaffected embryos (81% versus 19%, P < 0.05). Similarly, the Control, C' and HY groups of Categories 2 and 3 embryos had different proportions of unaffected versus affected embryos (75% versus 25%, P < 0.05). In all these groups, the male:female ratio did not significantly differ from 1:1. In contrast, in the C' + HY group of Categories 2 and 3 embryos, the ratio of unaffected versus affected embryos was 41% versus 59% (P < 0.05) and the male:female ratio differed (P < 0.05) from the expected 1:1 ratio (approximately 0.3:1 and 4.5:1 for unaffected versus affected, respectively). In conclusion, when bovine embryos were cultured in the presence of rat monoclonal H-Y antibodies and compliment, alterations occurred in embryos that were beyond the 8-cell stage; we inferred that the antibodies cross-reacted with H-Y antigens.     

Cytolytic and fluorescent detection of H-Y antigen on preimplantation mouse embryos

Monoclonal antibodies to histocompatibility (H-Y) antigen, of IgM subclass, were used to immunologically determine the sex of mouse embryos prior to transfer to pseudopregnant recipients. Two experiments were performed, one using cytolysis of H-Y positive embryos and the other using binding of a Fluorescein Isothiocyanate-(FITC) labeled second antibody. Eight- to 16-cell embryos used in the cytolytic assay were cultured in Whitten's Medium without bovine serum albumin (WM), to which monoclonal antibody and normal guinea pig serum were added. Embryos were classified as affected or unaffected, based on morphology of the embryo and its blastomeres. A total of 550 embryos were cultured; 294 (53.5%) were scored as unaffected and 263 of these were transferred to recipients. Forty-three (81.1%) of 53 pups born were female. Morulae and early blastocysts were used in the FITC-labeled second antibody assay. Embryos were cultured in WM containing monoclonal antibody, washed and placed in drops of WM containing FITC-labeled anti-IgM. Following another wash embryos were individually evaluated at 200X for fluorescence. Fifty-five percent (169 of 305) of the embryos displayed cell-specific fluorescence. A total of twenty-three pups, 18 males (78.3%) and five females (21.7%), were born following transfer of 156 fluorescing embryos. Four male (17.4%) and nineteen female (82.6%) pups resulted from embryos classified as non-fluorescing.     

The infection of mouse preimplantation embryos to Sendai virus (parainfluenza I)

To provide information on the susceptibility of mouse embryos to Sendai virus, it was investigated if viral replication occurs in the preimplantation embryo at different stages of development, with or without the zona pellucida (ZP). Mice were induced to superovulate, and embryos were collected on Days 2, 3 and 4 after mating. The ZP was removed by digestion with 0.5% pronase. Embryos were exposed to Sendai virus, washed, and allowed to develop in fresh culture medium. The presence of viral antigen in the embryonic cells was examined by the fluorescent antibody test (FAT). Specific immunofluorescence was demonstrated in the ZP-free morula and ZP-intact blastocyst. However, viral antigen was not detected in the ZP-intact two-cell, four-cell, eight-cell or morula stage embryos. Infected embryos developed normally to expanded blastocysts. These findings show that mouse embryonic cells are permissive hosts to Sendai virus replication and that the ZP played the role of a barrier against the virus.     

Comparison of hybrid and purebred in vitro-derived cattle embryos during in vitro culture

Frozen-thawed spermatozoa collected from a beef bull (Japanese Black) were used for in vitro fertilization (IVF) of matured oocytes obtained from dairy (Holstein) and beef (Japanese Black) females. Embryos were examined for fertilization, cleavage rate, interval between insemination and blastocyst production (experiment I), total cell number per embryo and sex ratio during blastocyst formation (experiment II), and blastocyst production rate of zygotes that developed to 2-, 4-, and 8-cell stages at 48h post-fertilization (experiment III). Fertilized oocytes were cultured in vitro on a cumulus cell co-culture system. The fertilization and cleavage rate of oocytes groups were similar, however, the blastocyst production rate was greater (P<0.05) in hybrid than from purebred embryos (27% versus 20%). Development of blastocysts produced from hybrid embryos developed at a faster rate than blastocysts produced from the straightbred embryos. In hybrid embryos, blastocyst production was significantly greater on day 7 (56%) and gradually decreased from 20% on day 8 to 17% on day 9. In contrast, blastocyst production rate from the purebred embryos was lower on day 7 (17%), increasing on day 8 to 59% and then decreased on day 9 to 24%. The total number of cells per embryo and sex ratio of in vitro-produced blastocysts were not different between hybrid and purebred embryos. The number of blastocysts obtained from embryos at the 8-cell stage of development by 48h post-fertilization (94%) was greater (P<0.01) than the number of zygotes producing blastocysts that had developed to the 4-cell stage (4%) and the 2-cell stage (2%) during the same interval. These results show that the blastocyst production rate and developmental rate to the blastocyst stage were different between hybrid and purebred embryos, and that almost all of the in vitro-produced blastocysts were obtained from zygotes that had developed to the 8-cell stage 48h post-fertilization.     

An in vitro model of gonad differentiation in the chicken. Estradiol-induced sex-inversion results in the occurrence of serological H-Y antigen

Dissociated cells from the gonads and mesonephros of 8-day-old chicken embryos were reorganized in rotation culture. The aggregates obtained from gonadal cells exhibited specific morphologic and histologic sex differences. In the presence of estradiol, aggregates from testicular cells showed characteristics similar to control ovarian aggregates, while in ovarian aggregates under estradiol treatment the female organization became more pronounced. Determination of serological H-Y antigen revealed that male aggregates of gonads and mesonephros were negative for H-Y and those of female embryos were positive for H-Y. Administration of estradiol did not change the H-Y findings in female aggregates. In contrast, in the male, gonadal cultures became H-Y positive while mesonephros cultures remained negative. It is assumed that estradiol induces the occurrence of H-Y antigen in the gonads.     

Co-culture of day-5 to day-7 equine embryos in medium with oviductal tissue

Oviductal and uterine embryos were collected from mares at 5 to 7 days following ovulation 1) to evaluate the effects of oviductal tissue explants on in vitro growth and development of equine embryos and 2) to study the morphologic development of equine embryos in culture. Embryos were incubated for 5 days in a medium (control group) or in medium supplemented with oviductal tissue explants (co-culture group). Embryos were evaluated and the media changed daily. Following 5 days in culture, 10 10 (100%) control embryos and 27 29 (93%) co-cultured embryos had doubled in diameter. All embryos that were recovered as morulae developed to the blastocyst stage in culture. By 5 days in culture, 6 10 (60%) control embryos and 19 29 (66%) co-cultured embryos had reached the hatching blastocyst stage of development. By 3 days in culture, significantly more (P<0.05) control embryos versus co-cultured embryos had degenerated (4 10 vs 2 29 , respectively). By 5 days in culture, significantly more (P<0.01) control embryos versus co-cultured embryos had degenerated (6 10 vs. 3 29 , respectively). Embryos cultured with oviductal tissue were sustained longer than embryos cultured in medium alone. Hatching was characterized by the blastocyst squeezing through a small opening in the zona pellucida or by the zona pellucida thinning over approximately half of the blastocyst surface and subsequently disappearing entirely.     

In vitro development of day-2 equine embryos co-cultured with oviductal explants or trophoblastic vesicles

This study compared the in vitro development of Day-2 equine embryos co-cultured with either trophoblastic vesicles or oviductal explants. Embryos were collected surgically from the oviducts of pony mares 2 d after ovulation and assessed for stage of development. Culture medium was Ham's F12 and Dulbecco's Modified Eagle's Medium (50:50 v/v) in a humidified atmosphere of 5% CO(2) in air at 38.5 degrees C with either trophoblastic vesicles or oviductal explants. The quality score of embryos was assessed daily. After 4 d in culture, embryos were stained (Hoechst 33342) and evaluated with epifluorescence to determine the number of nuclei present. Six of seven embryos co-cultured with oviductal exmplants developed to the morula/blastocyst stage, while four of seven embryos co-cultured with trophoblastic vesicles developed to the morula stage. More (P = 0.1) embryos co-cultured with oviductal explants reached the blastocyst stage than embryos co-cultured with trophoblastic vesicles (3 7 vs 0 7 , respectively). The number of cells was higher (P = 0.1) for embryos co-cultured with oviductal explants than for embryos co-cultured with trophoblastic vesicles (162.6 +/- 32 vs 87.3 +/- 28, respectively). The number of cells for embryos co-cultured with either oviductal explants or trophoblastic vesicles appeared to be lower than for embryos matured in vivo that were recovered from the uterus at Day 6 (378, 399, >1000). The co-culture of early equine embryos in a completely defined medium with either trophoblastic vesicles or oviductal explants can support development to at least the morula stage. The co-culture of embryos with oviductal explants resulted in superior development of four-to eight-cell embryos, as indicated by the proportion that reached the blastocyst stage and by the number of cells.     

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.     

H-Y antigen in the study of sex determination and control of sex ratio

It has been proposed, on the basis of widespread phylogenetic conservation, that H-Y antigen is the inducer of primary sex, causing the undifferentiated XY gonad to become a testis in male heterogametic species such as the human and bovine. That proposition has withstood extensive testing in vivo and in vitro. Freemartin gonads are H-Y⁺, for example, and masculinization of the freemartin gonad has been attributed to soluble H-Y, borne and transmitted in the serum of the bull twin, and bound in ovarian receptors of the female. We have applied monoclonal H-Y antibodies to the identification of gender in embryos of the bovine. Our preliminary results imply presence of H-Y in bovine embryos of the morula and blastocyst stages recovered at about 6–12 days of gestation. Assignment of H-Y phenotype -- positive in males and negative in females -- allows selective implantation of male and female during embryo transfer. Thus in an early study, we correctly identified gender in 6 of 7 calves born healthy at term, after transfer of 8 blastocysts.     

Cryopreservation of embryos as a means of germ plasm conservation in sheep

Embryos were collected from 4 lines of Targhee sheep between 1986 and 1990. The lines were selected for preweaning growth rate (Lines DH and HW) or for multiple births (Line HT); Line C served as an unselected control group. Estrus was synchronized using fluorogestone acetate-impregnated vaginal pessaries, and ewes were superovulated with FSH. Embryos at the morula or blastocyst stage were surgically recovered from mature ewes at Days 5 to 6 and were frozen following morphological evaluation. The overall average number of freezable embryos per collection was 2.9, and did not differ significantly among years or among lines. Of the embryos collected between 1986 and 1988, 92 were transferred to 53 recipients in 1989, producing 53 lambs. Survival rates were 60.9 and 47.8%, respectively, for embryos evaluated as good and fair after thawing. Good-quality blastocysts yielded the highest survival rate (64.4%). Analyses indicated no significant effects of line, developmental stage or embryo evaluation on the incidence of lambing. It was concluded that embryos of morula or blastocyst stage can be successfully frozen for extended periods. The data on embryo yield and survival following cryopreservation were used to calculate numbers of donors needed to preserve and reconstitute a population of specified size.     

Sexing of murine and bovine embryos by developmental arrest induced by high-titer H-Y antisera

Murine and bovine embryos at the late morula stage were cultured in medium containing high-titer rat H-Y antisera. After 12h of incubation, embryos blocked at the late morulae stage were classified as males and those at the blastocyst stage were classified as females. Sexing of murine embryos by PCR and cytogenetics revealed that 83% of the embryos classified as males and 82% of those classified as females had their sex correctly predicted (P < 0.05). Bovine embryos were transferred to recipient females. Pregnancy rates were 71.4% (10/14) for embryos classified as males and 68.8% (11/16) for embryos classified as females. The sex was correctly predicted for 80% (8/10) of the embryos classified as males and for 81.8% (9/11) of those classified as females (overall accuracy, 80.9%, P < 0.05). Therefore, the induction of developmental arrest by high-titer male-specific antisera was an efficient strategy for non-invasive embryo sexing. The procedure was straightforward and has considerable commercial potential for sexing bovine embryos.     

Effects of Nu-Serum® on in vitro development of goat (Capra hircus) embryos

To evaluate the effects of a commercially produced serum substitute on the in vitro development of caprine embryos, registered Nubian doelings were synchronized with norgestomet-impregnated implants (Synchromate-B®: CEVA) and superovulated with descending doses of FSH-p® (Schering). A total of 246 embryos was collected and placed in Tissue Culture Medium 199 (TCM 199, Gibco Laboratories) containing Nu-Serum® (NuS) at concentrations of 2.5%, 5.0%, 10%, or 20%. Control treatments consisted of TCM 199 alone or TCM 199 plus 10% heat-inactivated fetal bovine serum (FBS). Embryos developed in all concentrations of NuS to the morula, blastocyst, and expanded blastocyst stages. The TCM 199 plus 10% NuS had significantly higher percentages of embryos developing to the expanded blastocyst stage than TCM 199 plus 10% FBS. Time to expanded blastocyst development in NuS was shorter than in the TCM 199 plus FBS. No stage-specific block to development was observed with embryos collected and cultured in vitro for any of the treatments. These results demonstrate that NuS, when compared to FBS, allowed a higher percentage (P < 0.05) of caprine embryos to develop to the expanded blastocyst stage, thus providing a valuable substitute for FBS.     

Analysis of apoptosis in the preimplantation bovine embryo using TUNEL

The occurrence of cell death by apoptosis was examined in blastocyst and preblastocyst stage bovine embryos. Zygotes were obtained by in vitro maturation and in vitro fertilization of oocytes from abattoir derived ovaries. Two-cell to hatched blastocyst stage embryos were stained with propidium iodide to label all nuclei and by terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP nick end-labelling (TUNEL) to label apoptotic nuclei, and were analysed by epifluorescent and confocal microscopy. Apoptosis was first observed at the 9-16-cell stage of development, decreasing at the morula stage before increasing at the blastocyst stage. Apoptotic dead cell index in day 7 blastocysts was negatively correlated with the total number of cells; the percentage of dead cells ranged from approximately 1 to 10% and occurred predominantly within the inner cell mass. In addition, apoptotic dead cell index was significantly higher (P < 0.05) in blastocysts cultured (from the two-cell stage) in the presence of 10% fetal bovine serum compared with those developed in serum-free medium. Embryos selected for early cleavage at < 29 h after fertilization and cultured together until the blastocyst stage showed a significantly lower rate of apoptosis (P < 0.01) compared with slower cleaving embryos.     

Na+ / H+ exchanger-3 is involved in mouse blastocyst formation

The mouse blastocyst consists of the trophectoderm, the inner cell mass, and a fluid-filled cavity, the blastocoel. Formation and subsequent expansion of this cavity is important for further differentiation of the inner cell mass and successful implantation. Previous work provided evidence that vectorial transport of Na+ and CL- ions through the trophectoderm into the blastocoel generates an osmotic gradient that drives fluid across this epithelium. As the activity of the Na+ / H+ exchanger (NHE) has been implicated as the exchanger responsible for facilitating the transtrophectodermal Na+ flux, the functional role of NHE in mouse blastocoel development was determined. Embryos were cultured in the presence of subtype-specific NHE inhibitors to examine the role of NHEs in blastocoel development. When 2-cell stage embryos were treated continuously with a specific inhibitor of NHE-1, cariporide, the embryos passed beyond the 8-cell stage and became blastocysts. However, in the presence of a specific inhibitor of NHE-3, S3226, the 2-cell stage embryos developed to the morula stage but formation of the blastocyst were inhibited in a dose-dependent manner. Cariporide did not inhibit the formation of the blastocoel cavity from the morula stage whereas S3226 did inhibit that process. S3226 also reduced the rate of re-expansion of blastocysts collapsed by cytochalasin D upon transfer to the control medium. An immunofluorescence study showed that NHE-3 was detected in the vicinity of the cell membrane of the trophectoderm, especially in the apical cell margins of the trophectoderm. These results suggest that NHE-3 is likely involved in blastocyst formation.     

Establishment and maintenance of a regionalized glycoprotein distribution during early mouse development

The regionalization of the cell membranes of the mouse embryo into apical and basolateral zones has been studied using antibodies to a pair of glycoproteins expressed during the two-cell to early blastocyst stage. These antigens are found on the outer, free surface and in the underlying cortical cytoplasm, but are not detectable at areas of cell contact. In the early blastocyst stage, antigen also appears at the free surfaces of cells bordering the blastocoel. Antigen regionalization is also reestablished after experimental manipulation and appears to be a direct consequence of cell contact. Thus, blastomeres examined 4 hr after dissociation from four- and eight-cell stage embryos express antigen in cortical areas underlying newly exposed surfaces and new sites of contact between embryos in multiple-embryo aggregates lose detectable antigen within 2 to 4 hr of the formation of the contacts. Microfilaments are involved in controlling the regional expression of these glycoproteins. Incubation of embryos from the two-cell stage in medium containing cytochalasin B interferes with antigen targeting, resulting in abnormal expression of the antigens both on the surface and in the cytoplasm of the embryos. Cytochalasin B treatment of later stage embryos results in an uneven distribution of the antigen in cortical cytoplasm and prevents the complete removal of antigen from new sites of cell contact in multiple-embryo aggregates. The presence of nocodozole, which inhibits the polymerization of microtubules, had no detectable effect on the expression of the antigens. Interference with the glycosylation of these proteins, by incubation of embryos in the presence of tunicamycin, did not alter the regionalized pattern of expression.