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.     

Sexing of rat embryos with antisera specific for male rats.

Male-specific antigenicity (H-Y antigen) of rat embryos has been examined, and the feasibility of sexing rat embryos by use of H-Y antibodies has been studied. Rat H-Y antisera were produced by immunization of female Wistar rats with a homogenate of testes from male Wistar neonates. Male specificity of the antiserum (H-Y antibody) was determined by retention of cytotoxicity to male epidermal cells after absorption with female cells. After cultivation of rat embryos for 5 to 6 hr in the presence of antibody, half of the embryos were arrested at the morula stage. However, these embryos developed into blastocysts after removal of the antiserum, and then they grew into male young in recipient foster mothers. Eighty percent of the embryos that developed to blastocysts in the presence of the antiserum grew into female young.     

Evaluation of the presence of a specific histocompatibility protein on equine embryonic cells

An indirect immunofluorescence assay was used to detect the presence of H-Y antigen on equine blastocysts. A total of 33 blastocyst stage horse embryos were collected 6 to 7 days post-ovulation by trans-cervical flush and were immediately evaluated for the presence of H-Y antigen. Additionally, 17 embryos, were collected and cultured for 72 h to the expanded blastocyst stage and similarly evaluated. Embryos were placed in medium containing monoclonal antibodies to H-Y antigen followed by incubation in medium containing 1/10 (v/v) fluorescein isothiocyanate conjugated goat anti-mouse IgM Fc specific antiserum. Embryos were individually evaluated at 400X to identify cell specific fluorescence. Following evaluation, embryonic sex was independently verified with karyotypes to identify sex chromosomes. Of the 50 embryos evaluated, 29 were evaluated as non-fluorescent and 21 fluorescent. Expression of H-Y antigen was determined to be uniform in those embryos classified as fluorescent. Twenty-three of 28 (82%) readable karyotypes corresponded to the predicted sex. These results indicate a specific histocompatibility antigen is expressed and maintained at the blastocyst stage of development. In addition, no segregation of this protein on specific cell types occurs in this species.     

Selective passage through the uterotubal junction of sperm from a mixed population produced by chimeras of calmegin-knockout and wild-type male mice

Loss of calmegin, a testis-specific putative chaperone protein of the endoplasmic reticulum, leads to male sterility because the sperm show defects in migration into the oviduct and do not bind to the zona pellucida. To clarify the mechanism of defective migration, XY <--> XY chimeras were produced by aggregating wild-type embryos with embryos of transgenic mice lacking functional calmegin genes and expressing enhanced green fluorescent protein (EGFP) in their acrosomes. Chimeric ejaculates contained wild-type, nonfluorescent sperm as well as sperm with EGFP-tagged acrosomes and the defective calmegin gene. Transgenic, wild-type, and chimeric males were mated to wild-type females; however, only wild-type sperm were ever found within the oviducts. Calmegin-knockout sperm, even when they were combined in chimeric ejaculates with wild-type sperm, remained outside of the uterotubal junction. These findings indicate that the presence of wild-type sperm cannot compensate for the inability of calmegin-knockout sperm to enter the oviduct and that successful ascent into the oviduct depends on the capabilities of individual sperm.     

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.     

Generation of the Y-chromosome linked red fluorescent protein transgenic mouse model and sexing at the preimplantation stage

In mammals, sexual fate is determined by the chromosomes of the male and female gametes during fertilization. Males (XY) or females (XX) are produced when a sperm containing a Y or X-chromosome respectively fertilizes an X-chromosome-containing unfertilized egg. However, sexing of preimplantation stage embryos cannot be conducted visually. To address this, transgenic male mouse models with the ubiquitously expressed green fluorescent protein (GFP) transgene on X- (X-GFP) or Y-chromosomes (Y-GFP) have been established. However, when crossed with wild-type females, sexing of the preimplantation stage embryos by observing the GFP signal is problematic in some cases due to X-inactivation, loss of Y-chromosome (LOY), or loss of transgene fluorescence. In this study, a mouse model with the ubiquitously expressed red fluorescent protein (RFP) transgene on the Y-chromosome was generated since RFP is easily distinguishable from GFP signals. Unfortunately, the ubiquitously expressed tdTomato RFP transgene on the Y-chromosome (Y-RFP) mouse showed the lethal phenotype after birth. No lethal phenotypes were observed when the mitochondrial locating signal N-terminal of tdTomato (mtRFP) was included in the transgene construct. Almost half of the collected fertilized eggs from Y-mtRFP male mice crossed with wild-type females had an RFP signal at the preimplantation stage (E1.5). Therefore, XY eggs were recognized as RFP-positive embryos at the preimplantation stage. Furthermore, 100% sexing was observed at the preimplantation stage using the X-linked GFP/Y-linked RFP male mouse. The established Y-mtRFP mouse models may be used to study sex chromosome related research.     

Survival after transfer of “sexed” mouse embryos exposed to H-Y antisera

Immunological means were used to determine the sex of mouse embryos prior to transfer to pseudopregnant recipients. Antisera to histocompatibility-Y (H-Y) antigen were prepared in adult C57BL/6 female mice by repeated intraperitoneal injections of spleen cells from males of the same strain. Eight-to 16-cell embryos were cultured in BMOC-3 alone or BMOC-3 without bovine serum albumin to which one of the following had been added: H-Y antiserum and normal guinea pig serum (NGPS), NGPS alone, normal mouse serum alone or normal mouse serum and NGPS. After 24 hr of culture, embryos were classified as either affected or unaffected. An embryo was classified as affected if degeneration of the embryo or breakdown of one or more cells was observed. A total of 1000 embryos were cultured in BMOC-3 with H-Y antiserum and NGPS (treated embryos). Two hundred and fifty embryos were cultured in each of the other four media (control embryos). Eighty-seven (9%) of the control embryos and 479 (48%) of the treated embryos were classified as affected after culture. Unaffected embryos, approximately 12 each, were transferred to pseudopregnant recipients. One-hundred forty control embryos (17%) survived to term with 67 females (48%) and 73 males (52%) born. Fifty-eight treated embryos (14%) survived to term, producing 50 females (86%) and 8 males (14%). Percentage of females from embryos cultured in antiserum was greater than for embryos cultured in any other media (P<0.001). These results demonstrate that detection of H-Y antigen on preimplantation embryos may be a useful and effective method of determining sex of an embryo prior to transfer.     

Characterization of antisera against mouse teratocarcinoma OTT6050: Molecular species recognized on embryoid bodies,preimplantation embryos,and sperm

Antisera raised in rabbits by hyperimmunization with small embryoid bodies of the transplantable teratocarcinoma OTT6050 recognize several distinct antigenic protein species on the surfaces of cells of the immunogen. Some of these antigens were found on the cells of preimplantation mouse embryos, on cells from parietal yolk sac carcinoma, and on mouse sperm. These antigens have been distinguished by polyacrylamide electrophoresis of the immune precipitates from detergent extracts of lactoperoxidase-iodinated cells. The intact embryoid bodies from the ascitic form of the OTT6050 teratocarcinoma exhibited five major protein bands (approximate MW 150K, 115K, 82K, 48K, and 12K), one band that ran at the dye front of the gels (R_f ≥1) and one minor band (approximate MW 22K). Two different rabbit antisera recognized an essentially identical pattern of antigens which, however, varied on the different cell types tested. Antisera were also elicited in syngeneic male mice using glutaraldehyde-fixed or irradiated OTT6050 embryoid bodies. The isoantisera had very poor titers in comparison to the absorbed xenoantisera, as assessed by complement-mediated cytotoxic activity against the immunizing cell types. Complement-mediated cytotoxicity could also be demonstrated using parietal yolk sac carcinoma cells, preimplantation mouse embryos from all cleavage stages, blastocysts, and immunosurgically isolated inner cell masses, as targets. The complexity of the antisera generated by intact embryoid bodies described here indicates that these structures bear multiple antigenic specificities not present on adult somatic cells, some of which are stage-specific embryonic polypeptides.     

Clq enhancement of IgG-dependent eosinophil-mediated killing of schistosomula in vitro

Antibody-dependent eosinophil-mediated cytotoxicity plays a role in host protection against metazoan parasite invasion. We examined a possible role for Clq in eosinophil-mediated cytotoxicity by using a Schistosoma mansoni schistosomula killing system in vitro. The addition of monomeric purified human Clq enhanced IgG-dependent human eosinophil-mediated killing from 1.4-fold to 2.3-fold (mean percent killing 12% +/- 4 vs 21% +/- 4, p less than 0.005) when the immune IgG concentration was low. In contrast, there was no significant enhancement of neutrophil-mediated killing. When the IgG concentration was increased fourfold Clq did not cause enhancement of eosinophil-mediated killing (35% +/- 9 vs 37% +/- 5). Preincubation of eosinophils with type 1 collagen abrogated Clq enhancement of killing, raising the possibility of a receptor-mediated process, which depends upon cellular binding of Clq via the collagenous portion of the molecule. Eosinophils and neutrophils were examined for the presence of Clq receptors by using 125I labeled Clq. Clq binding to both cell types was saturable, reversible, and specific, indicating that binding is through specific receptors. Type 1 collagen inhibited binding of Clq to cells, suggesting that Clq binding is via the collagenous stalk of Clq. The number of receptors was approximately twice as high for eosinophils as compared with neutrophils (1.9 X 10(7) vs 1.1 X 10(7), p less than 0.025). Affinity constants for the two cell types were similar (1.5 X 10(7) vs 1.3 X 10(7). These findings suggest that Clq and receptors for Clq on eosinophils may be important for eosinophil-mediated schistosomula killing.     

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.     

PCR sexing and developmental rate differences in preimplantation mouse embryos fertilized and cultured in vitro

A two-step polymerase chain reaction (PCR) assay was used to determine the sex of mouse preimplantation embryos obtained from oocytes fertilized and cultured in vitro, to investigate the differences in the developmental rates of mouse embryos according to the sex. All the in vitro developed embryos could be analyzed by this method. When the embryos were classified according to the time of morula to blastocyst transition as fast-intermediate- and slow-growing embryos, a significantly high percentage (78.0%) of the fast-developing embryos were identified as males; while a significantly lower percentage (42.5%) of slow-developing embryos were identified as males. The intermediate-developing embryos presented a sex ratio not significantly different from the total (57.5%). The deviation of sex ratio was further confirmed by embryo transfer experiment, where fast- and slow-developing embryos gave 76.2% and 25.7% male fetuses, respectively. We concluded that male mouse embryos fertilized and cultured in vitro develop faster than female embryos. © 1993 Wiley-Liss, Inc.     

Developmental potential and transgene expression of porcine nuclear transfer embryos using somatic cells

We examined whether porcine nuclear transfer (NT) embryos carrying somatic cells have a developmental potential and NT embryos carrying transformed fibroblasts express transgenes in the preimplantation stages. In Experiment 1, different activation methods were applied to NT embryos and the development rates were examined. Relative to A23187 only or A23187/6-DMAP, electrical pulse made a significant increase in both cleavage rate (58.1+/-13.9 or 60.7+/-6.3 vs. 74.9+/-7.5%) and development rate of NT embryos to the blastocyst stage (2.2+/-2.8 or 2.2+/-1.5 vs. 11.0+/-4.1%). In Experiment 2, in vitro developmental competence of NT embryos was investigated. The developmental rate to the blastocyst stage of NT embryos (9.9+/- 2.4% for cumulus cells and 9.8+/-1.6% for fibroblast cells) was significantly lower than that (22.9+/-3.5%) of IVF-derived embryos (P<0.01). NT blastocysts derived from either cumulus (28.9+/-11.4, n = 26) or fibroblast cells (30.2+/-9.9, n = 27) showed smaller mean nuclei numbers than IVF-derived blastocysts (38.6+/-10.4, n = 62) (P<0.05). In Experiment 3, nuclear transfer of porcine fibroblasts expressing the GFP (green fluorescent protein) gene resulted in green blastocysts without losing developmental potential. These results suggest that porcine embryos reconstructed by somatic cell nuclear transfer are capable of developing to preimplantation stage. We conclude that somatic cells expressing exogenous genes can be used as nuclei donors in the production of NT-mediated transgenic pig.     

Serology of H-Y antigen

Summary Anti-H-Y antiserum is generally obtained from female inbred mice or rats that have been hyperimmunized with syngeneic male cells. The specificity of such antiserum is defined by its reactivity for male but not female cells. A number of conventional serological assays have been used to measure that reactivity. However, H-Y is a weak antigen, evidently represented sparingly on the surfaces of cells other than sperm, epidermal cells and brain cells; thus the srological assays for H-Y are technically difficult. Yet H-Y serology has enabled significant progress toward the understanding of primary sex differentiation.A recent advance in H-Y serology is the establishment of monoclonal anti-H-Y antisera which promise to facilitate analysis and clarification of the H-Y system.     

Dual fluorescent in situ hybridisation for simultaneous detection of X and Y chromosome-specific probes for the sexing of human preimplantation embryonic nuclei

Summary Dual fluorescent in situ hybridisation has been used for the simultaneous detection of X and Y chromosome-specific probes in single cleavage nuclei from disaggregated 4- to 7-cell human embryos. Based on the presence of a Y signal or 2 X signals in the absence of a Y, 89% of poor quality metaphases and 72% of interphase nuclei could be classified as male or female. With further refinements, this technique will offer a credible alternative to the polymerase chain reaction for the diagnosis of sex in human preimplantation embryos in families segregating for X-linked genetic disease.     

Antisperm antibodies in human immunodeficiency virus infection: effects on fertilization and embryonic development

Sera from human immunodeficiency virus (HIV)-infected males (n = 10) and females (n = 5) were analyzed for the presence of antisperm antibodies reacting against sperm-specific antigens. Of the HIV-positive males tested, sera of 40% were positive for human sperm extract (HSE), 70% for protamine, and 70% for fertilization antigen (FA-1) for at least one class of antibodies, compared to sera from HIV-negative males. Of the HIV-positive females tested, sera of 40% were positive for HSE, 30% for protamine, and 30% for FA-1 compared to sera from HIV-negative females. The majority of the sperm antigen-reactive antibodies belonged to the IgG class. The reactions observed with FA-1 were weaker than those with other antigens. Ninety percent of HIV-positive male sera and 80% of the HIV-negative female sera were found to contain immune complexes, 20% of which showed the presence of FA-1. HIV-positive male or female sera did not bind to any specific protein on the Western blot of HSE. The minimal amount of free anti-FA-1 antibodies present in sera did not bind to live sperm in the sperm immobilization technique, sperm agglutination technique, or immunobead binding technique and thus were incapable of affecting human sperm penetration of zona-free hamster ova (SPA). Nor did HIV-positive sera induce any apparent abnormality in the development of 2-cell embryos to blastocysts in vitro in murine bioassay. In conclusion, these results indicate that HIV-infected patients have sperm-specific antibodies in their sera that do not adversely affect SPA and murine embryo bioassay. There was a high incidence of immune complex formation after HIV infection. These data will provide the basis for exploring further the role of sperm antigens in altering the immunoregulatory mechanisms after HIV infection.     

The role of autophagy during the oocyte-to-embryo transition

After fertilization, the maternal proteins stored in oocytes are degraded and new proteins encoded by the zygotic genome are synthesized. Although several proteins are degraded by the ubiquitin-proteasome system, the mechanism underlying the dynamic protein turnover during this process remains largely unknown. We recently reported that autophagy plays a critical role during preimplantation embryonic development. We found that the level of autophagy was low in unfertilized oocytes; however, autophagy was activated shortly after fertilization. The function of autophagy was further analyzed using oocyte-specific Atg5 (autophagy-related 5) knockout mice. Atg5-null oocytes could develop if they were fertilized with wild-type sperm, but could not develop beyond the four- and eight-cell stages if they were fertilized with Atg5-null sperm. Furthermore, protein synthesis rates were reduced in the autophagy-deficient embryos. We have previously reported that Atg5-null oocytes derived from Atg5+/- mice, which should contain maternally inherited Atg5 protein in the oocyte, were able to produce Atg5-/- neonates, emphasizing the specific importance of autophagy during very early embryogenesis. Thus, the degradation of maternal factors by autophagy is essential for preimplantation development in mammals.

Addendum to: Tsukamoto S, Kuma A, Murakami M, Kishi C, Yamamoto A, Mizushima N. Autophagy is essential for preimplantation development of mouse embryos. Science 2008; 321:117-20.     

Divalent antibodies to mouse embryonal carcinoma cells inhibit compaction in the mouse embryo

Divalent antibodies from two antisera to embryonal carcinoma (EC) cells, F9 line, inhibited compaction in the preimplantation mouse embryo. One of these antisera, AF9-2, completely inhibited compaction at the 8-cell stage when the embryos were continuously incubated in a $\text{1}\text{100}$ dilution of the antiserum in culture medium from the 4-cell stage. Cell divisions continued and no cellular degeneration occurred. When cultured control embryos (preimmune and nonimmune sera) were normal blastocysts, many of the AF9-2-treated embryos were characterized by vacuolated cells and rounded rather than squamous, trophoectodermal cells. Anti-mouse spleen serum ($\text{1}\text{10}$, $\text{1}\text{100}$) had no effect on development. Purified divalent IgGs from AF9-2 (ammonium sulfate precipitation and DEAE chromatography) also were inhibitory at 30 μg/ml. Inhibition of compaction by AF9-2 was reversible. When uncompacted midmorula-stage embryos in AF9-2 ($\text{1}\text{10}$) were transferred to medium without serum, there was a threefold increase in the percentage (70%) of normal blastocysts at the end of culture. Fluorescence microscopy demonstrated that AF9-2 antibodies, unlike preimmune and nonimmune sera, were bound to the surface of 8-cell and early morula-stage embryos. Inhibition by AF9-2 does not occur merely by nonspecifically coating cell surfaces so that they no longer recognize each other, since antispleen antibodies show similar binding by immunofluorescence but no inhibition. This study provides strong evidence that AF9-2 has specific divalent antibodies which block morphogenesis. Since newly compacted embryos lost their compacted appearance in AF9-2, these divalent antibodies cause a loss of cell adhesion, do not extensively cross-link adjacent cell surfaces, and cannot cause the compacted phenotype by agglutination.     

Strain variations in anti-sperm antibody responses and anti-fertility effects in inbred mice

This study provides evidence for polygenic controls of antisperm antibody levels in inbred male mice immunized with syngenic testis and epididymis. H2-linked and non-H2-linked genes were involved. Mice of H-2d haplotype were high responders, whereas those with H-2k haplotype were nonresponders; however, B10.D2/nSnJ mice (H-2d) were also nonresponders. In vitro fertilization inhibition by antisera correlated positively with the serum antisperm antibody levels, particularly with antibody of the immunoglobulin (Ig) G class. Inheritance of antibody response that inhibited in vitro fertilization (IVF) was an autosomal dominant trait, but this was not apparent for the control of antibody levels per se. Since IVF was inhibited by both IgG and fragment antigen-binding (Fab) isolated from immune sera, but not by immune IgG previously absorbed by sperm or testis, the biologic effect is antigen-specific and probably involved blockade of functional antigenic epitopes. Antisera to testis, caput sperm or cauda sperm were found to inhibit IVF to a similar degree. Inbred strains of mice that produced the highest levels of serum antisperm antibodies that inhibited IVF were A/J, SJL/J, DBA/1J and BALB/cByJ mice, and their antisera immunoprecipitated a common sperm antigen molecule of 35,000 to 40,000 Mr. In contrast, C57BL/6 and C57BL/10 mice produced significant antibody levels that had no effect on IVF, and their sera did not react with the 35,000- to 40,000-Mr peak. Moreover, among BALB/c H-2 congenic mice, only antiserum of responder BALB/cByJ (H-2d) mice immunoprecipitated the 35,000- to 40,000 Mr peak. Thus the 35,000- to 40,000-Mr protein may be of functional significance in the fertilization process.     

Chromosome breakage in human preimplantation embryos from carriers of structural chromosomal abnormalities in relation to fragile sites, maternal age, and poor sperm factors

Chromosome breakage is a fairly widespread phenomenon in preimplantation embryos affecting at least 10% of day 3 cleavage stage embryos. It may be detected during preimplantation genetic diagnosis (PGD). For carriers of structural chromosomal abnormalities, PGD involves the removal and testing of single blastomeres from cleavage stage embryos, aiming towards an unaffected pregnancy. Twenty-two such couples were referred for PGD, and biopsied blastomeres on day 3 and untransferred embryos (day 5/6) were tested using fluorescence in situ hybridisation (FISH) with appropriate probes. This study investigated whether chromosome breakage (a) was detected more frequently in cases where the breakpoint of the aberration was in the same chromosomal band as a fragile site and (b) was influenced by maternal age, sperm parameters, reproductive history, or the sex of the carrier parent. The frequency of breakage seemed to be independent of fragile sites, maternal age, reproductive history, and sex of the carrier parent. However, chromosome breakage was very significantly higher in embryos from male carriers with poor sperm parameters versus embryos from male carriers with normal sperm parameters. Consequently, embryos from certain couples were more prone to chromosome breakage, fragment loss, and hence chromosomally unbalanced embryos, independently of meiotic segregation.