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N Forde and P Lonergan

Establishment of pregnancy in domestic ruminants includes pregnancy recognition signalling by the conceptus, implantation and placentation. Despite the high fertilisation success rate in ruminants, a significant amount of embryo loss occurs, primarily during early gestation. Interferon-tau (IFNT), a type I interferon that is exclusively secreted by the cells of the trophectoderm of the ruminant conceptus, has been recognised as the primary agent for maternal recognition of pregnancy in ruminants. It produces its antiluteolytic effect on the corpus luteum by inhibiting the expression of oxytocin receptors in the uterine epithelial cells, which prevents pulsatile, luteolytic secretion of prostaglandin F2α by the uterine endometrium. While the importance of IFNT in maternal recognition of pregnancy and prevention of luteolysis in ruminants is unequivocal, important questions, for example, relating to the threshold level of IFNT required for pregnancy maintenance, remain unanswered. This paper reviews data linking IFNT with measures of fertility in ruminants.

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H. Khatir, C. Carolan, P. Lonergan and P. Mermillod

The aims of the present study were to characterize the follicular fluid from prepubertal calf follicles of known size and quality and to study the ability of follicular fluid to support cytoplasmic maturation of calf and cow oocytes. Follicular fluid was obtained from 67 calf follicles classified according to size (S: small < 6 mm, M: medium 6–8 mm and L: large > 8 mm in diameter) and quality (HY: healthy, EA: early atretic and A: atretic). Quality was first determined by mitosis:pycnosis ratios in granulosa cell smears and confirmed by insulin-like growth factor binding protein (IGFBP) patterns. There was approximately 90% agreement between the two methods of follicle classification and on this basis the calf follicular fluid was pooled into nine groups. The accuracy of this pooling was confirmed by evaluation of oestradiol concentrations in the nine pools of follicular fluid using radioimmunoassay. Increases in follicle size were characterized by a decreased intensity of bands for IGFBP-2, IGFBP-5 and IGFBP-4, an increase in the proportion of healthy follicles and a decrease in the proportion of follicles in the early stages of atresia. This finding is in agreement with previously published results in cows. All classes of calf follicular fluid contained lower concentrations of oestradiol than previously reported for corresponding classes of cow follicular fluid. Cow oocytes were matured in M199 alone, or supplemented with 10% fetal calf serum (FCS), or 10% calf follicular fluid from one of three pools (LHY, LEA, LA), fertilized, and cultured for 8 days in synthetic oviduct fluid. Addition of FCS or calf follicular fluid to cow oocytes during in vitro maturation increased the yield of blastocysts on day 8 over the control (23%, 21/91), FCS (39%, 37/96, P < 0.05), LA (41% 21/52, P < 0.05), LEA (32%, 28/88), LHY (36%, 32/88), although not significantly in all cases. The rate of hatching of blastocysts was also improved: control (38%, 8/21), FCS (54%, 20/37), LA (62%, 13/21), LEA (75%, 21/28, P < 0.02), LHY (59% 19/32). In contrast, the addition of either FCS, calf follicular fluid or cow follicular fluid did not improve development of calf oocytes compared with the unsupplemented control. In conclusion, it is probable that serum and follicular fluid contain factors that stimulate the acquisition by oocytes, during maturation, of developmental competence and to which prepubertal oocytes are unable to respond. Specific receptors for these factors may develop only around puberty.

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P. Lonergan, H. Khatir, C. Carolan and P. Mermillod

The aims of the present study were to assess the effect of various substances on meiotic resumption and subsequent development to the blastocyst stage of bovine oocytes. Immature cumulus–oocyte complexes were cultured for 24 h in (a) Medium 199 (M199) alone, or M199 supplemented with (b) 10% fetal calf serum (FCS), (c) 1 μg cycloheximide ml−1, (d) 2 mmol 6-dimethylaminopurine (6-DMAP) l−1, or (e) 0.1 mmol vanadate l−1. After 24 h, groups (a) and (b) were inseminated with frozen–thawed spermatozoa and subsequently cultured, while groups (c–e) were washed and cultured for a second 24 h in M199 + FCS, after which they were inseminated and cultured. At all time points a representative sample of oocytes were fixed and stained with orcein to observe the nuclear status, while others were labelled with [35S]methionine to study protein biosynthesis. Incubation with 6-DMAP, cycloheximide or vanadate completely blocked germinal vesicle breakdown with most oocytes remaining at the germinal vesicle stage after 24 h culture (89%, 100% and 85%, respectively). This inhibitory effect was fully reversible in the case of 6-DMAP and cycloheximide; after a second period of incubation, germinal vesicle breakdown occurred in almost all cases (99% and 100%, respectively), and most reached metaphase II (85% and 83%, respectively). In contrast, inhibition with vanadate was only reversible in 56% of oocytes, with only 6% reaching metaphase II. Cleavage rates at 72 h after insemination and blastocyst yields on day 8 of culture were, respectively: (i) M199, 72% and 34%; (ii) M199 + FCS, 80% and 45%; (iii) M199 + cycloheximide, 81% and 19%; (iv) M199 + 6-DMAP, 77% and 14%. 6-DMAP did not modify methionine incorporation. However, cycloheximide completely blocked protein synthesis when present during the period of labelling. Addition of epidermal growth factor to cycloheximide-inhibited oocytes was without effect. In contrast, epidermal growth factor overcame the effect of 6-DMAP in about 50% of oocytes, resulting in lower developmental rates after IVF. These results give an indication of the feasibility of in vitro meiotic inhibition as a tool in the study of the mechanisms involved in acquisition of competence.

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P Bermejo-Alvarez, D Rizos, P Lonergan and A Gutierrez-Adan

Sex chromosome transcripts can lead to a broad transcriptional sexual dimorphism in the absence of concomitant or previous exposure to sex hormones, especially when X-chromosome inactivation (XCI) is not complete. XCI timing has been suggested to differ greatly among species, and in bovine, most of the X-linked transcripts are upregulated in female blastocysts. To determine the timing of XCI, we analyzed in day 14 bovine embryos the sexual dimorphic transcription of seven X-linked genes known to be upregulated in female blastocysts (X24112, brain-expressed X-linked 2 (BEX2), ubiquitin-conjugating enzyme E2A (UBE2A), glucose-6-phosphate dehydrogenase (G6PD), brain-expressed X-linked 1 (BEX1), calpain 6 (CAPN6), and spermidine/spermine N-acetyltransferase 1 (SAT1)). The transcription of five genes whose expression differs between sexes at the blastocyst stage (DNMT3A, interferon tau (IFNT2), glutathione S-transferase mu 3 (GSTM3), progesterone receptor membrane component 1 (PGRMC1), and laminin alpha 1 (LAMA1)) and four genes related with sex determination (Wilms tumor 1 (WT1), gata binding protein 4 (GATA4), zinc finger protein multitype 2 (ZFPM2), and DMRT1) was also analyzed to determine the evolution of transcriptional sexual dimorphism. The expression level of five X-linked transcripts was effectively equalized among sexes suggesting that, in cattle, a substantial XCI occurs during the period between blastocyst hatching and initiation of elongation, although UBE2A and SAT1 displayed significant transcriptional differences. Similarly, sexual dimorphism was also reduced for autosomal genes with only DNMT3A and IFNT2 exhibiting sex-related differences. Among the genes potentially involved in sex determination, Wilms tumor 1 (WT1) was significantly upregulated in males and GATA4 in females, whereas no differences were observed for ZFPM2 and DMRT1. In conclusion, a major XCI occurred between the blastocyst and early elongation stages leading to a reduction in the transcriptional sexual dimorphism of autosomal genes, which makes the period the most susceptible to sex-specific embryo loss.

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P Bermejo-Alvarez, D Rizos, P Lonergan and A Gutierrez-Adan

In adult tissues, sexual dimorphism is largely attributed to sex hormone effects, although there is increasing evidence for a major role of sex chromosome dosage. During preimplantation development, male and female embryos can display phenotypic differences that can only be attributed to the transcriptional differences resulting from their different sex chromosome complements. Thus, all expressed Y-linked genes and those X-linked genes that totally or partially escape X-chromosome inactivation at each specific developmental stage display transcriptional sexual dimorphism. Furthermore, these differentially expressed sex chromosome transcripts can regulate the transcription of autosomal genes, leading to a large transcriptional sexual dimorphism. The sex-dependent transcriptional differences may affect several molecular pathways such as glucose metabolism, DNA methylation and epigenetic regulation, and protein metabolism. These molecular differences may have developmental consequences, including sex-selective embryo loss and sex-specific epigenetic responses to environmental hazards, leading to long-term effects. This review discusses transcriptional sexual dimorphism in preimplantation embryos, its consequences on sex ratio biases and on the developmental origin of health and disease, and its significance for transcriptional studies and adult sexual dimorphism.

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I M Aparicio, M Garcia-Herreros, T Fair and P Lonergan

The aim of this study was to examine the presence and regulation of glycogen synthase kinase-3α (GSK3A) and GSK-3β (GSK3B) in bovine embryos and their possible roles in embryo development. Our results show that GSK3A and GSK3B are present in bovine embryos at the two-cell stage to the hatched blastocyst stage. Bovine embryo development was associated with an increase in the phosphorylation of both isoforms, being statistically significant at blastocyst and hatched blastocyst stages, compared with earlier stages. Inhibition of GSK3 with CT99021 (3 μM) resulted in a significant increase in the percentage and quality of blastocysts, while inhibition of GSK3 with lithium chloride (LiCl; 20 mM) significantly reduced at the proportion of eight-cell embryos on day 3 and inhibited blastocyst formation. The use of LY294002 (10 μM), a specific inhibitor of phosphatidylinositol-3 kinase, also produced a significant decrease in embryo development. In addition, treatment with LiCl and LY294002 produced a significant decrease in the serine phosphorylation of both isoforms of GSK3. Finally, CT99021 and LiCl reduced the phosphorylation of β-catenin on Ser45 in two-cell embryos, while LY294002 increased it. Despite the fact that LiCl inhibited GSK3 activity, as demonstrated by β-catenin phosphorylation, its effects on the bovine embryo could be mediated through other signaling pathways leading finally to a decrease in the phosphorylation of GSK3 and a reduction in embryo development. Therefore, in conclusion, GSK3A/B serine phosphorylation was positively correlated with embryo development, indicating the importance of an accurate regulation of GSK3 activity during developmental stages to achieve normal bovine embryo development.

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JM Lozano, P Lonergan, MP Boland and D O'Callaghan

Two experiments were carried out to study the effect of nutrition on embryo development in two periods in superovulated ewes (Expt 1) and on oocyte developmental capacity during the late follicular phase (Expt 2). In Expt 1, a lower superovulation response in terms of animals ovulating (P < 0.05), ovulation rate per ewe ovulating (P = 0.1) and number of good quality embryos per animal treated (P < 0.07) was noted in ewes fed an ad libitum diet compared with ewes offered control (1.5 times the daily maintenance energy requirements, 1.5 x M) or low energy (0.5 x M) diets. Nutrition also modified the morphological and functional quality of the oocytes and embryos recovered. Thus, 92% of day 4 embryos recovered from ewes offered the control diet were classified as good embryos, compared with 70 and 82% of those recovered from ewes offered the ad libitum and low diets, respectively (P < 0.05). Ewes offered the ad libitum diet had a greater percentage of poorly developed embryos compared with ewes offered the control or low diets (P < 0.05). Ewes fed the low diet tended to have more non-fertilized oocytes than ewes offered the control diet (P = 0.09). Diet of recipient ewes to which good quality embryos were transferred on day 4 did not affect embryo quality, when assessed 12 days later (day 16 of pregnancy). However, recipient diet affected prostaglandin F(2alpha) (PGF(2alpha)) production in vitro, and uterine tissue that originated from recipient ewes on the low diet secreted more PGF(2alpha) relative to uterine tissue that originated from recipients on the control diet (P < 0.05). In Expt 2, fewer total (P < 0.05) and good quality (P < 0.01) oocytes and a lower percentage of good quality oocytes (P < 0.01) were obtained from superovulated ewes offered the ad libitum diet compared with ewes offered the low diet. In addition, cleavage rate tended to be higher (51 versus 35%, P = 0.09) in ewes offered the low diet compared with ewes offered the ad libitum diet. In conclusion, changes in diet can affect the quality of the oocyte and embryo in superovulated sheep. A lower superovulation response and a decrease in the quality of oocytes and embryos indicate that ad libitum diets are highly detrimental for superovulatory programmes when compared with low and control diets. In addition, the results from the present study indicate that a low energy diet during early embryo development increased the uterine production in vitro of PGF(2alpha) which could lead to a poor uterine environment thereby compromising the development of the embryo.

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P. Lonergan, H. Khatir, F. Piumi, D. Rieger, P. Humblot and M. P. Boland

In vitro produced bovine zygotes show substantial variation in the time required to complete the first cell cycle and in their in vitro development potential. A number of reports have highlighted the fact that the fastest developing embryos in vitro are most likely to be comparable with their in vivo counterparts. At 24 h after IVF, presumptive zygotes were cultured in droplets of synthetic oviduct fluid medium. Droplets were examined at regular intervals and all cleaved embryos at each time point were transferred into new droplets and cultured separately for the duration of the experiment. All uncleaved zygotes were returned to the incubator and re-examined at the successive time points until 48 h after insemination, at which time the remaining uncleaved oocytes were retained as a group. A representative number of day 7 blastocysts from zygotes that had cleaved by 30 or 36 h were transferred to synchronized recipients and pregnancy was diagnosed by ultrasonography at day 35. Glucose and glutamine metabolism was examined in zygotes and blastocysts and compared retrospectively with time of first cleavage. A representative number of blastocysts from each of the cleavage groups was sexed using PCR. Data were analysed by chi-squared and regression analysis. Development to the blastocyst stage decreased as the time from insemination to first cleavage increased (r = 0.97, P < 0.03). There was no difference in blastocyst hatching, number of blastocyst cells or pregnancy rate between the 30 and 36 h groups. The overall sex ratio was 62% males (n = 258, P < 0.0001) and was not different in the 30 and 36 h groups (61%, n = 155 versus 63%, n = 95, respectively). These results indicate that although time of first cleavage has a major influence on the probability of an embryo developing to the blastocyst stage, once that stage is attained, subsequent developmental characteristics are unrelated to the time of first cleavage.

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B. Oussaid, J. C. Mariana, N. Poulin, J. Fontaine, P. Lonergan, J. F. Beckers and Y. Cognie

A GnRH antagonist (Antarelix) treatment was used during the breeding season of Romanov ewes, to investigate whether LH pulses are required the day before the preovulatory surge for normal early embryo development in vivo (Expt 1) and in vitro (Expt 2). In Expt 1, at the onset of oestrus after removal of a fluorogestone acetate sponge, group A0.5 (n = 22) received a subcutaneous injection of 0.5 mg Antarelix, and ovulation was induced with an intravenous injection of 3 mg pig LH 24 h later. The control group (group C, n = 20) were untreated. All ewes were mated naturally at 36 and 48 h after oestrus and embryos were recovered 8 days after sponge removal. There were significant differences in the decrease in LH and in the increase in FSH concentration after Antarelix treatment between treated and control groups. The ovulation rate and embryo recovery rate were not significantly different between the two groups but the blastocyst rate was lower (P < 0.0001) in group A0.5 than in group C, with more unfertilized or degenerated oocytes in group A0.5 (69.2%). In Expt 2, 24 h after sponge removal, group A (n = 10) and group B (n = 10) received one subcutaneous injection of 0.5 mg Antarelix. The control group (group C,n = 10) was left untreated. LH pulsatility was re-established in group B with hourly intravenous injections of 5 μg ovine LH for 24 h. Oocytes were collected by flushing the oviducts 28 h after the LH surge, and were fertilized and cultured in vitro for 7 days. Ovulation and cleavage rates were not significantly different among the three groups but a higher rate of blastocysts (P < 0.01) was obtained after Antarelix treatment when LH pulsatility was re-established (group B). Oestradiol concentration was strongly depressed (P < 0.0003) after Antarelix treatment in group A, but was maintained after injection of LH pulses in group B, although at a lower value than before the preovulatory surge in the control group. In conclusion, inhibition of endogenous LH pulses 1 day before the preovulatory surge was not essential for ovulation and in vitro fertilization but was associated with a decrease in plasma oestradiol concentrations and inferior embryo development both in vivo and in vitro. When LH pulsatility was re-established, oestradiol concentrations increased and embryo development was restored.

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B Fernandez-Fuertes, A Blanco-Fernandez, C J Reid, K G Meade, S Fair and P Lonergan

This study tested the hypothesis that sperm sialic acid (Sia) is required to reach the site of fertilization, and that successful fertilization requires recognition of Sia from both the sperm and oocyte to occur. In addition, it has recently been reported that Siglecs (Sia-binding-immunoglobulin-like lectins) are present on the sperm surface. Thus, the possibility that the recognition of oocyte Sia was sperm-Siglec-mediated was also addressed. Sperm exposed to neuraminidase (NMase) exhibited lower overall and progressive motility, which translated to a decreased ability to swim through cervical mucus from cows in oestrus. In addition, when either sperm or cumulus–oocyte complexes (COCs) were treated with NMase, a decrease in cleavage and blastocyst rate was observed. However, incubation of sperm with increasing concentrations of anti-Siglec-2, -5, -6 and -10 antibodies prior to fertilization had no effect on their fertilizing ability. Interestingly, treatment with NMase increased the number of sperm bound to the ZP but also the rate of polyspermic fertilization. Flow cytometry analysis revealed no differences in the percentage of capacitated or acrosome-reacted sperm. These results suggest that Sia are required to reach the site of fertilization but need to be removed for sperm–oocyte interaction. However, fine regulation is needed to avoid abnormal fertilization which can lead to impaired embryo development.