Detection of the mechanism of immunotoxicity of cyclosporine A in murine in vitro and in vivo models.

Transcriptomics in combination with in vitro cell systems is a powerful approach to unravel modes of action of toxicants. An important question is to which extent the modes of action as revealed by transcriptomics depend on cell type, species and study type (in vitro or in vivo). To acquire more insight into this, we assessed the transcriptomic effects of the immunosuppressive drug cyclosporine A (CsA) upon 6 h of exposure of the mouse cytotoxic T cell line CTLL-2, the thymoma EL-4 and primary splenocytes and compared these to the effects in spleens of mice orally treated with CsA for 7 days. EL-4 and CTLL-2 cells showed the highest similarities in response. CsA affected many genes in primary splenocytes that were not affected in EL-4 or CTLL-2. Pathway analysis demonstrated that CsA upregulated the unfolded protein response, endoplasmic reticulum stress and NRF2 activation in EL-4 cells, CTLL-2 cells and primary mouse splenocytes but not in mouse spleen in vivo. As expected, CsA downregulated cell cycle and immune response in splenocytes in vitro, spleens in vivo as well as CTLL-2 in vitro. Genes up- and downregulated in human Jurkat, HepG2 and renal proximal tubular cells were similarly affected in CTLL-2, EL-4 and primary splenocytes in vitro. In conclusion, of the models tested in this study, the known mechanism of immunotoxicity of CsA is best represented in the mouse cytotoxic T cell line CTLL-2. This is likely due to the fact that this cell line is cultured in the presence of a T cell activation stimulant (IL-2) making it more suitable to detect inhibitory effects on T cell activation.

Tailored microarray platform for the detection of marine toxins.

Currently, there are no fast in vitro broad spectrum screening bioassays for the detection of marine toxins. The aim of this study was to develop such an assay. In gene expression profiling experiments 17 marker genes were provisionally selected that were differentially regulated in human intestinal Caco-2 cells upon exposure to the lipophilic shellfish poisons azaspiracid-1 (AZA1) or dinophysis toxin-1 (DTX1). These 17 genes together with two control genes were the basis for the design of a tailored microarray platform for the detection of these marine toxins and potentially others. Five out of the 17 selected marker genes on this dedicated DNA microarray gave clear signals, whereby the resulting fingerprints could be used to detect these toxins. CEACAM1, DDIT4, and TUBB3 were up-regulated by both AZA1 and DTX1, TRIB3 was up-regulated by AZA1 only, and OSR2 by DTX1 only. Analysis by singleplex qRT-PCR revealed the up- and down-regulation of the selected RGS16 and NPPB marker genes by DTX1, that were not envisioned by the new developed dedicated array. The qRT-PCR targeting the DDIT4, RSG16 and NPPB genes thus already resulted in a specific pattern for AZA1 and DTX1 indicating that for this specific case qRT-PCR might a be more suitable approach than a dedicated array.

Effect of different stages of the follicular wave on in vitro developmental competence of bovine oocytes.

This study aimed to investigate the developmental competence of ovum pick-up collected oocytes on three stages of the follicular wave: Days 2, 5 and 8. A group of 11 cows was used in successive cycles to perform ovum pick-up on either Day 2, 5 or 8 of an induced follicular wave (three sessions per stage). Follicular waves were initiated by puncturing the dominant follicle and all other follicles sized > or = 5 mm at Days 5-7 of the cycle. The plasma progesterone concentrations did not differ between the days of ovum pick-up: 4.0 +/- 1.8, 5.1 +/- 1.6 and 5.2 +/- 1.7 ng/ml for Days 2, 5 and 8, respectively. The proportion of oocytes with three or more layers of non-expanded cumulus cells was higher for Day 5 than Day 8, while Days 2 and 5 did not significantly differ from each other (85, 96 and 68% of 113, 60 and 101 oocytes for Days 2, 5 and 8, respectively). The proportion of oocytes competent to develop a blastocyst in an in vitro production system was higher for Days 2 and 5 than for Day 8: 27, 29 and 15% for the oocytes with fair to good cumulus investment and 23, 27 and 11%, respectively, when all oocytes were taken in account. This indicates that the dominant follicle reduces the developmental competence of oocytes from subordinate follicles at a relatively late stage of dominance. This finding has practical consequences for the handling of cows that undergo ovum pick-up only once or very irregularly. The embryo yield can then be improved by performing the ovum pick-up at Days 2-5 of the cycle or 2-5 days after ablation of the large follicles.

Follicular dynamics around the recruitment of the first follicular wave in the cow.

The present study aimed to test the generally accepted view that a follicular wave starts with follicles newly recruited from the population smaller than 3 mm, which later compete for dominance. According to this view, subordinate follicles are expected to be too atretic to join the next follicular wave. Ten cows were ovariectomized shortly prior to the LH surge, thus around the start of the first follicular wave of the cycle. Per cow, on average, 14.4 follicles of >/=3 mm were dissected. Follicular health was determined on the basis of four parameters: 1) judgment of the degree of atresia by stereomicroscope, 2) incidence of apoptotic nuclei among the granulosa cells, 3) estradiol and progesterone concentrations, and 4) insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs)-2, -4, and -5 concentrations in the follicular fluid. In addition to the preovulatory follicle, 3.1 other follicles, mainly sized 3-4.5 mm, were found to be healthy based on the proportion of apoptotic nuclei, and concentrations of estradiol/progesterone, and IGFBPs. The ability of these follicles to respond with growth on the preovulatory and periovulatory FSH surges was supported by a comparison to the follicular population of four cows 31-68 h after the LH surge. The present results point to an alteration of the view on the follicular wave. The larger follicles during the first days of the follicular wave are, in general, derived from follicles that also joined the previous wave. A portion of these growing follicles are estradiol active and compete for dominance. Other growing follicles lack estradiol production and are probably derived from rather atretic follicles. The first newly recruited follicles do not reach the size of 3 mm before 31 h after the preovulatory FSH surge. At that time, the larger follicles are already competing for dominance.

Effects of oocyte maturation regimen on the relative abundance of gene transcripts in bovine blastocysts derived in vitro or in vivo.

Bovine embryos produced in vitro differ substantially from embryos produced in vivo in the mRNA expression patterns of genes important for development. Several factors in the in vitro production systems have profound effects on embryonic mRNA expression patterns. The effects of the type of maturation on the expression pattern of genes important for development in blastocysts produced in vitro have not yet been investigated. The aim of the present study was to investigate the effects of various maturational protocols on the relative abundance of a panel of six marker genes, indicative of compaction and cavitation, metabolism, stress susceptibility and RNA processing, in bovine blastocysts produced in vitro. Four groups of blastocysts were analysed by a sensitive semi-quantitative RT-PCR assay. Blastocysts were produced in vitro from oocytes of different origin from: (1) 3-8 mm follicles; (2) preovulatory follicles before the LH surge; and (3) preovulatory follicles 24 h after the LH surge. The first two groups were matured in vitro, whereas the third group had undergone maturation in vivo. A fourth group comprised blastocysts developed entirely in vivo. Expression of glucose transporter 1 was significantly (P < 0.05) higher, and expression of desmocollin 2 and plakophilin tended to be higher (P < 0.1) for in vivo (group 4) compared with in vitro blastocysts (group 1), whereas no differences were found for heat shock protein 70.1, E-cadherin and poly(A) polymerase. Expression of the six transcripts did not differ among blastocysts produced in vitro from oocytes of groups 1, 2 and 3. Results indicate that alterations in the relative abundance of these transcripts in blastocysts produced in vitro cannot primarily be attributed to the origin of the oocyte, but are likely to have been induced by post-maturation or fertilization culture conditions.

Effects of in vivo prematuration and in vivo final maturation on developmental capacity and quality of pre-implantation embryos.

In current in vitro production (IVP) systems, oocytes lack in vivo dominant and preovulatory follicular development, which may compromise pregnancy and viability of calves born. When an oocyte sets off in vivo on the road toward fertilization, it contains numerous transcripts and proteins necessary to survive the first few cell cycles of embryonic development. It is not yet known during which period of development the oocyte builds up the store, possibly primarily during the major growth phase of the oocyte, which is completed at the time a follicle reaches the size of 3 mm. Here, we investigated to what extent the later phases of follicular development, such as prematuration in the dominant follicle before the LH surge and ensuing final maturation in the preovulatory follicle, contribute to oocyte competence and development into viable biastocysts. Recent studies on in vivo vs in vitro oocyte maturation employed oocytes from an identical preovulatory development by applying ovum pick-up (OPU) twice (before and 24 h after the LH surge) in each cow treated for superovulation with a controlled LH surge. The embryo recovery rates at Day 7 of IVC after IVF were similar: 44% (97/219) for in vivo- vs 41% (87/213) for in vitro-matured oocytes, which shows that the natural environment during final maturation is not essential for the mere in vitro development of the prematured oocyte beyond the 8- to 16-cell stage. However, in vivo maturation appeared to contribute to the oocyte's quality in a more subtle way, as indicated by a significant increase in the proportion of expanded blastocysts and a more physiological degree of chromosome aberrations of the embryos. In blastocysts derived from in vivo-matured oocytes, 21% of the embryos were mixoploid vs 50% from in vitro-matured oocytes, concomitant with a higher number of cells (96 vs 54 per normal blastocyst). The expression pattern of a set of six developmentally important genes was, however, not significantly altered in blastocysts derived from in vivo-matured oocytes. Certain deviations were observed compared with the levels of entirely in vivo-developed control blastocysts, which suggests that the beneficial effects of in vivo maturation are possibly exerted at initial stages of embryonic development. Prematuration in vivo, occurring in a dominant follicle developing from about 8 mm into the preovulatory follicle, is accompanied by changes in protein synthesis of the cumulus oocyte complex (COC). Presumably, the differentially expressed proteins are involved in equipping the oocyte with further developmental competence. Although we have unraveled some important biochemical and cellular biological features of the oocyte, further research on in vivo processes is essential to improve in vitro embryo production in practice.

Ribosomal RNA gene expression and chromosome aberrations in bovine oocytes and preimplantation embryos.

This review focuses on the key features of development of the bovine oocyte and embryo, with comparisons of the developmental characteristics of embryos produced in vivo and in vitro. The oocyte is transcriptionally quiescent in the primordial and primary follicle. In the secondary follicle transcription is initiated in the oocyte and a ribosome-synthesizing nucleolus is established in this cell. Transcription and nucleolar activity are enhanced in the tertiary follicle during oocyte growth. When the oocyte reaches approximately 110 microm in diameter, corresponding to a follicle of about 3 mm in diameter, transcription ceases and the nucleolus is inactivated, forming a dense spherical remnant. During the final phase of follicular dominance this remnant becomes vacuolated and, in conjunction with resumption of meiosis, it disperses. The rRNA genes are apparently re-activated during the four-cell stage, that is, the third cell cycle after fertilization, but a nucleolus is not formed. During the subsequent cell cycle, that is, during the eight-cell stage, ribosome-synthesizing nucleoli are again established. Bovine embryos produced in vitro apparently display the same pattern of nucleolus development as that in embryos developed in vivo. Examination of the ploidy of embryonic cells using fluorescence in situ hybridization has revealed that the production of bovine embryos in vitro is associated with increased chromosome aberrations in the embryos. Blastocysts produced in vitro display a significantly higher rate of mixoploidy, that is, when the embryo consists of both normal diploid and abnormal polyploid cells, than that in embryos developed in vivo. The rate of mixoploidy among embryos produced in vitro increases with increasing developmental stage. Moreover, after fertilization in vitro, initially there is a high rate of 'true' polyploidy, that is, when all cells of the embryos are polyploid. However, the polyploid embryos are eliminated before they cleave beyond the eight-cell stage, the stage at which major activation of the embryonic genome occurs in cattle.

Chromosomal abnormalities and developmental kinetics in in vivo-developed cattle embryos at days 2 to 5 after ovulation.

The frequency of chromosome abnormalities was investigated in cattle embryos (n = 256) derived from superovulated heifers (n = 35) on Days 2, 3, 4, and 5 postovulation (PO). Interphase nuclei (n = 4358) were analyzed for chromosome abnormalities using fluorescent in situ hybridization with chromosome 6- and chromosome 7-specific probes and the developmental rate was described by scoring cell numbers. We found that 93%, 85%, 84%, and 69% of the embryos from Days 2, 3, 4, and 5 PO, respectively, displayed a normal diploid chromosome number in all cells. Of the embryos containing abnormal cells, mixoploidy was significantly more frequent than polyploidy. The percentage of mixoploidy at Days 2, 3, 4, and 5 PO was 5%, 13%, 16%, and 31%, respectively, whereas the percentages of polyploidy were 2%, 2%, 0%, and 0%, respectively. The mean number of cells per embryo was 4.7, 8, 11.5, and 48.3, respectively, at Days 2, 3, 4, and 5 PO. Thus, in vivo-developed embryos were significantly more advanced than the in vitro-produced (IVP) embryos except for Day 2. In conclusion, a significantly lower frequency of chromosomally abnormal embryos, in particular displaying polyploidy early after fertilization, was seen in in vivo versus IVP embryos, and these chromosomal abnormalities may be inherent to the process of IVP in cattle.

Impaired final follicular maturation in heifers after superovulation with recombinant human FSH.

The aim of this study was to investigate whether human FSH without contaminating LH can exert a normal superovulation response in cows. One group of heifers (n = 9) was stimulated with recombinant human FSH (rhFSH), an FSH source without any LH activity, and another group (n = 9) was treated with equine chorionic gonadotrophin (eCG), an FSH source with high LH activity. Daily transrectal ultrasonography showed that eCG- and rhFSH-stimulated heifers (n = 9 per group) had the same follicular growth characteristics and equal numbers of follicles > 8 mm in diameter after 3 days of stimulation. The treatment groups differed considerably in steroid production: rhFSH-treated heifers produced much lower oestradiol concentrations than did eCG-stimulated heifers during the first days of stimulation and much lower progesterone concentrations in the period after the LH surge. During the 27-35 h after prostaglandin injection, rhFSH-treated heifers had fewer LH pulses than did eCG-treated heifers (0.3 versus 3.0 per heifer, respectively; n = 3 per group). All rhFSH-treated heifers (n = 6) underwent a preovulatory LH surge, but this occurred significantly later than in the eCG-treated heifers (n = 4; 39.4 +/- 1.9 h versus 47.1 +/- 1.5 h in rhFSH- and eCG-treated heifers, respectively). Multiple ovulations occurred in only three of six rhFSH-treated heifers, but in all four eCG-treated heifers with an LH surge. At 24 h after the LH surge, the percentage of metaphase II stage oocytes with cortical granules distributed close to the oolemma was significantly lower in the rhFSH group (7.3%) than in the eCG group (55.9%). In conclusion, final follicular maturation is impaired in heifers treated with rhFSH, which might be due to the combination of a lack of LH activity in the gonadotrophin preparation and the severe suppression of LH pulsatility.

Bovine follicular development and its effect on the in vitro competence of oocytes.

The current knowledge is reviewed concerning correlations between follicular development in the cow and the competence of matured oocytes to develop into an embryo following IVF and IVC. At the follicular size of 3 mm, some oocytes become competent and the proportion of competent oocytes does not increase during development up to 7 mm. The proportion of competent oocytes increases greatly in follicles > 8 mm in both untreated and gonadotropin-stimulated cows. The competence of in vitro-matured oocytes from these large follicles is lower than the competence of in vivo-matured oocytes. These observations lead to the following concept. Oocytes have acquired an intrinsic capacity to develop into an embryo after IVM-IVF-IVC at the follicular stage of 3 mm, but require an additional "prematuration" to express this competence. In vivo, this prematuration occurs during preovulatory development before the occurrence of the LH surge. In follicles of 3-7 mm, a low level of atresia appears to improve the in vitro competence of oocytes which may act via a prematuration-like effect. A thorough understanding, however, of the effect of atresia and other factors on the competence of this highly heterogeneous oocyte population is still missing. Two routes to improve the embryo yield in ovum pick-up (OPU) practice are discussed.

Effect of exogenous glutathione on the in vitro fertilization of bovine oocytes.

Increased amounts of reactive oxygen species (ROS) during in vitro culture may cause cytotoxic damage to gametes and embryos. The main purpose of this study was to investigate the effect of glutathione (GSH), a ROS scavenger, supplemented during IVF of bovine oocytes on embryo development using spermatozoa from different bulls. The following experiments were performed: 1) matured COCs were fertilized in the absence or presence of 1 mM GSH using semen from 4 bulls (Bulls A, B, C and D); 2) matured COCs were fertilized in the absence or presence of 1 mM GSH using semen from Bull C to examine sperm penetration, pronuclear formation and apposition; 3) COCs were fertilized with in the presence of either 0, 0.1, 1.0 or 10 mM GSH to examine the effect of GSH concentration using sperm from Bull C; 4) concentrations of GSH were measured both in the medium and in the oocytes during IVF. Glutathione at 1 mM in IVF medium affected the blastocyst formation, but not the cleavage rate. The effect on blastocyst formation was bull dependent: semen from Bull B and D had a negative, that from Bull C a positive and the one from Bull A no effect. The positive effect of Bull C semen increased the rate of blastocyst formation from 20.1 to 27.3% in control and GSH-treated samples, respectively. The increased rate was due to more zygotes reaching the 8-cell or greater stage by Day 4 after IVF. There was no change in the fertilization or cleavage rates. The GSH was still stable after 18 h incubation in IVF medium, and there was a dose-dependent increase in the GSH concentration in the oocytes. It is concluded that the effect of GSH during IVF on the proportion of blastocysts is dependent on both bull and GSH concentration.

Do X and Y spermatozoa differ in proteins?

This article reviews the current knowledge about X- and Y-chromosomal gene expression during spermatogenesis and possible differences between X- and Y-chromosome-bearing spermatozoa (X and Y sperm) in relation to whether an immunological method of separation of X and Y spermatozoa might some day be feasible. Recent studies demonstrated that X- and Y-chromosome-bearing spermatids do express X- and Y-chromosomal genes that might theoretically result in protein differences between X and Y sperm. Most, if not all, of these gene products, however, are expected to be shared among X and Y spermatids via intercellular bridges. Studies on aberrant mouse strains indicate that complete sharing might not occur for all gene products. This keeps open the possibility that X and Y sperm may differ in proteins, but until now, this has not been confirmed by comparative studies between flow-cytometrically sorted X and Y sperm for H-Y antigen or other membrane proteins.

Testis-specific expression of a functional retroposon encoding glucose-6-phosphate dehydrogenase in the mouse.

The X-chromosomal gene glucose-6-phosphate dehydrogenase (G6pd) is known to be expressed in most cell types of mammalian species. In the mouse, we have detected a novel gene, designated G6pd-2, encoding a G6PD isoenzyme. G6pd-2 does not contain introns and appears to represent a retroposed gene. This gene is uniquely transcribed in postmeiotic spermatogenic cells in which the X-encoded G6pd gene is not transcribed. Expression of the G6pd-2 sequence in a bacterial system showed that the encoded product is an active enzyme. Zymogramic analysis demonstrated that recombinant G6PD-2, but not recombinant G6PD-1 (the X-chromosome-encoded G6PD), formed tetramers under reducing conditions. Under the same conditions, G6PD tetramers were also found in extracts of spermatids and spermatozoa, indicating the presence of G6pd-2-encoded isoenzyme in these cell types. G6pd-2 is one of the very few known expressed retroposons encoding a functional protein, and the presence of this gene is probably related to X chromosome inactivation during spermatogenesis.

Comparison of detergent-solubilized membrane and soluble proteins from flow cytometrically sorted X- and Y-chromosome bearing porcine spermatozoa by high resolution 2-D electrophoresis.

The only known and measurable difference between X- and Y-chromosome bearing spermatozoa is the small difference in their DNA content. The X sperm in the human carry 2.8% more DNA than the Y sperm, while in domestic livestock this difference ranges from 3.0 to 4.2%. The only successful sperm separation method, flow cytometric sorting, is based on this difference in DNA content. Using this technique, X and Y sperm populations with purities greater than 90% can be obtained. The number of spermatozoa that can be sorted in a given time period, however, is too low for application of this technique in routine artificial insemination. Therefore, the search for a marker other than DNA to differentiate between X and Y sperm remains of interest in order to develop a method for large scale X and Y sperm separation. The aim of the present study was to investigate whether porcine X and Y sperm contain some difference in their plasma membrane proteins. The flow cytometric sorting of sperm enabled a direct comparison of the proteins of the X and Y sperm populations. High resolution two-dimensional (2-D) electrophoresis was used; however, adaptations were needed to enable its use for analysis of proteins of flow cytometrically sorted sperm, both in the sorting procedure, membrane protein solubilization, and in the 2-D electrophoresis. Up to 1000 protein spots per gel could be detected and quantified. Comparison of the 2-D protein patterns revealed differences in protein spots between sperm of two individual boars. However, no differences in protein spots between the X and Y sperm fractions were found. These results provide additional support for the view that X- and Y-chromosome bearing spermatozoa are phenotypically identical, and cast doubt on the likelihood that a surface marker can provide a base for X and Y sperm separation.

Postmeiotic transcription of X and Y chromosomal genes during spermatogenesis in the mouse.

During the meiotic prophase of spermatogenesis, the X and Y chromosomes form the heterochromatic sex body, showing little transcriptional activity. It has been suggested that transcription of the Xist gene is involved in this inactivation. After completion of the meiotic divisions, at least two Y chromosomal genes, Zfy and Sry, are transcribed in haploid spermatids. In contrast, postmeiotic transcription of X chromosomal genes has not been demonstrated. Using highly purified preparations of mouse pachytene spermatocytes, round spermatids, and cytoplasmic fragments from elongated spermatids, the present experiments show differential postmeiotic expression of the Y chromosomal genes Ubely and Sry, with highest mRNA levels in round spermatids and cytoplasmic fragments, respectively. Postmeiotic transcription of the X chromosomal gene Ube1x is indicated by an increased level of Ube1x mRNA in round spermatids and cytoplasmic fragments. The X chromosomal gene MHR6A shows a marked temporary postmeiotic expression in round spermatids. This postmeiotic activity of the X chromosome is a novel finding, which may have implications for our understanding of X chromosome inactivation during spermatogenesis and paternal genome imprinting.

The production of anti-H-Y monoclonal antibodies: their potential use in a sex test for bovine embryos.

In an attempt to improve the accuracy of sexing bovine embryos, new anti-H-Y monoclonal antibodies were produced and selected, using an extended screening procedure. In addition to the commonly used screening of soluble H-Y antigen sources, such as testis supernatant and Daudi supernatant, the binding specificity to cell surface H-Y antigen was tested also. A radioimmunoassay (RIA) employing male and, as a control, female bovine lymphocytes, and enzyme-linked immunosorbent assays (ELISAs) on solubilized membrane fractions resulted in the selection of a number of clones producing monoclonal antibody (mAb) with male-enhanced binding. Four of the anti-H-Y mAb were assessed for binding to Day 7 or 8 bovine embryos. The accuracy of sexing bovine embryos ranged from 58% to 71%. Two of the four antibodies did not react with presumed soluble H-Y antigen-containing sources in an ELISA. These results raise doubts about the suitability of the presumed soluble H-Y antigen sources, Daudi, TM4 and testis supernatant, to be used in screening tests for anti-H-Y antibodies.

Daudi supernatant, unlike other H-Y antigen sources, exerts a sex-reversing effect on embryonic chick gonad differentiation.

In vitro cultures of intact chick gonads (organ cultures) and reaggregation cultures of dispersed gonad cells (roller cultures) were made. Gonads or gonad cells from 7-day-old chick embryos, at the stage when sex-specific differentiation begins, were cultured in the presence of presumed H-Y antigen-containing supernatants, or co-cultured in the presence of H-Y antigen-producing cell lines. The H-Y antigen-producing cells tested were of human, mouse, bovine and chicken origin. During organ culture, addition of supernatant of the human lymphoma cell line Daudi, or co-culture with Daudi cells, stimulated a clear proliferation of the germinal epithelium in male gonads, indicating feminization. A similar effect was obtained by treatment with estradiol. In reaggregation culture, the increase in nuclear size of germ cells was chosen as a parameter for feminization. A significant increase of germ cell nuclear size was observed in gonads cultured in the presence of Daudi supernatant. In both organ cultures and reaggregation cultures, other tested H-Y antigen sources and semi-purified H-Y antigen fractions did not exert significant effects on differentiation of the gonads or on the average area of the germ cell nuclei. These findings suggest that it is not H-Y antigen, but another protein produced by Daudi cells, that might be responsible for the sex-reversing effects.

Binding of anti-H-Y monoclonal antibodies to separated X and Y chromosome-bearing porcine and bovine sperm.

Studies designed to answer the question whether or not H-Y antigen is preferentially expressed on Y chromosome bearing sperm have resulted in conflicting results. This is probably due to the absence of reliable methods for estimating the percentage of X and Y chromosome bearing sperm in fractions, enriched or depleted for H-Y antigen positive sperm. In recent years a reliable method for separating X and Y chromosome bearing sperm has been published. With this method, separation is achieved by using a flow cytometer/cell sorter, which detects differences in DNA content. This technique provided the first opportunity for testing anti-H-Y antibody binding to fractions enriched for X and Y chromosome bearing sperm, directly. A total of 7 anti-H-Y monoclonal antibodies were tested using sorted porcine sperm and in one experiment also sorted bovine sperm. All monoclonal antibodies bound only a fraction of the sperm (20 to 50%). However, no difference in binding to the X and Y sperm enriched fractions was found. Therefore, the present experiments do not yield evidence that H-Y antigen is preferentially expressed in Y chromosome bearing sperm.