MicroRNAs in amniotic fluid and maternal blood plasma associated with sex determination and early gonad differentiation in cattle

MicroRNAs (miRNAs), as gene expression regulators, may play a critical role during the sex determination process. We hypothesised that the expression of miRNAs in amniotic fluid (AF) and maternal blood plasma (MP) during this process would be affected by the sex of the embryo. Amniotic fluid and MP were collected from six pregnant heifers (3 carrying a single male and 3 a single female embryo) following slaughter on Day 39 post insemination, coinciding with the peak of SRY expression. Samples (6 AF and 6 MP) were profiled using a miRNA Serum/Plasma Focus PCR Panel. Differentially expressed (DE) miRNAs were identified in AF (n = 5) and associated MP (n = 56) of male vs female embryos (P < 0.05). Functional analysis showed that inflammatory and immune response were amongst the 13 biological processes enriched by miRNAs DE in MP in the male group (FDR < 0.05), suggesting that these sex-dependent DE miRNAs may be implicated in modulating the receptivity of the dam to a male embryo. Further, we compared the downstream targets of the sex-dependent DE miRNAs detected in MP with genes previously identified as DE in male vs female genital ridges. The analyses revealed potential targets that might be important during this developmental stage such as SHROOM2, DDX3Y, SOX9, SRY, PPP1CB, JARID2, USP9X, KDM6A, and EIF2S3. Results from this study highlight novel aspects of sex determination and embryo-maternal communication in cattle such as the potential role of miRNAs in gonad development as well as in the modulation of the receptivity of the dam to a male embryo.

55 Identification of microRNAs associated with sex determination in bovine amniotic fluid and maternal blood plasma

In most eutherian mammals, sex determination is the process through which a bipotential gonad (also known as genital ridges) develops into a testis or ovary depending on the sex chromosome content of the embryo, specifically by the presence of the SRY/Sry gene (sex-determining region of the Y chromosome). MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression and are involved in diverse functional roles including development, differentiation, apoptosis, and immunity. We hypothesised that the expression of miRNAs in amniotic fluid (AF) and maternal blood plasma (MP) would be affected by the sex of the embryo around the time of sex determination. Amniotic fluid and MP were collected from 6 crossbred beef pregnant heifers (3 carrying a single male and 3 carrying a single female embryo) following slaughter on Day 39 (when the peak of SRY expression occurs in cattle). All heifers had been synchronized and inseminated with semen from the same beef bull. A total of 12 samples (6 AF and 6 MP) were profiled using the miRCURY LNA miRNA Serum/Plasma Focus PCR Panel (Qiagen; 179 assays targeting relevant miRNAs). Data were analysed by GeneGlobe Data Analysis Center (Qiagen). A threshold cycle cut-off of 35 was applied and data were analysed using an unpaired t-test. Gene ontology enrichment analysis was performed using the WebGestaltR package to explore the possible functions of differentially expressed (DE) miRNAs. In this study, DE miRNAs were identified in male vs. female AF (n=5; 3 upregulated and 2 downregulated; P<0.05) and MP (n=57; 54 upregulated and 3 downregulated; P<0.05). Although no enrichment was detected for DE miRNAs in AF (in either sex) or in MP in heifers carrying a female embryo, 37 biological processes were enriched by DE miRNAs in MP of heifers carrying a male embryo (false discovery rate<0.05). Interestingly, the top five most enriched biological processes were male gonad development, development of primary male sexual characteristics, signal transduction in absence of ligand, actomyosin structure organisation, and male sex differentiation, suggesting a potential role of these miRNAs in reproductive traits. Results from this study highlight unique aspects of sex determination in cattle such as the role of miRNAs in gonad development. Moreover, although it is well known that AF provides a protective space around the developing embryo/fetus that allows its movement and growth; here we provide evidence suggesting that its components may play important roles in fetal development. Finally, miRNAs in MP may offer new opportunities to investigate biomarkers for early prediction of embryo/fetal sex in commercial practice. This research was supported by the Science Foundation Ireland (13/IA/1983) and the European Union H2020 Marie Sklodowska-Curie Innovative Training Network project Biology and Technology of Reproductive Health - REP-BIOTECH - 675526.

Effect of heritable symbionts on maternally-derived embryo transcripts

Maternally-transmitted endosymbiotic bacteria are ubiquitous in insects. Among other influential phenotypes, many heritable symbionts of arthropods are notorious for manipulating host reproduction through one of four reproductive syndromes, which are generally exerted during early developmental stages of the host: male feminization; parthenogenesis induction; male killing; and cytoplasmic incompatibility (CI). Major advances have been achieved in understanding mechanisms and identifying symbiont factors involved in reproductive manipulation, particularly male killing and cytoplasmic incompatibility. Nonetheless, whether cytoplasmically-transmitted bacteria influence the maternally-loaded components of the egg or early embryo has not been examined. In the present study, we investigated whether heritable endosymbionts that cause different reproductive phenotypes in Drosophila melanogaster influence the mRNA transcriptome of early embryos. We used mRNA-seq to evaluate differential expression in Drosophila embryos lacking endosymbionts (control) to those harbouring the male-killing Spiroplasma poulsonii strain MSRO-Br, the CI-inducing Wolbachia strain wMel, or Spiroplasma poulsonii strain Hyd1; a strain that lacks a reproductive phenotype and is naturally associated with Drosophila hydei. We found no consistent evidence of influence of symbiont on mRNA composition of early embryos, suggesting that the reproductive manipulation mechanism does not involve alteration of maternally-loaded transcripts. In addition, we capitalized on several available mRNA-seq datasets derived from Spiroplasma-infected Drosophila melanogaster embryos, to search for signals of depurination of rRNA, consistent with the activity of Ribosome Inactivating Proteins (RIPs) encoded by Spiroplasma poulsonii. We found small but statistically significant signals of depurination of Drosophila rRNA in the Spiroplasma treatments (both strains), but not in the symbiont-free control or Wolbachia treatment, consistent with the action of RIPs. The depurination signal was slightly stronger in the treatment with the male-killing strain. This result supports a recent report that RIP-induced damage contributes to male embryo death.

In Vivo Ablation of the Conserved GATA-Binding Motif in the Amh Promoter Impairs Amh Expression in the Male Mouse

GATA4 is an essential transcriptional regulator required for gonadal development, differentiation, and function. In the developing testis, proposed GATA4-regulated genes include steroidogenic factor 1 (Nr5a1), SRY-related HMG box 9 (Sox9), and anti-Müllerian hormone (Amh). Although some of these genes have been validated as genuine GATA4 targets, it remains unclear whether GATA4 is a direct regulator of endogenous Amh transcription. We used a CRISPR/Cas9-based approach to specifically inactivate or delete the sole GATA-binding motif of the proximal mouse Amh promoter. AMH mRNA and protein levels were assessed at developmental time points corresponding to elevated AMH levels: fetal and neonate testes in males and adult ovaries in females. In males, loss of GATA binding to the Amh promoter significantly reduced Amh expression. Although the loss of GATA binding did not block the initiation of Amh transcription, AMH mRNA and protein levels failed to upregulate in the developing fetal and neonate testis. Interestingly, adult male mice presented no anatomical anomalies and had no evidence of retained Müllerian duct structures, suggesting that AMH levels, although markedly reduced, were sufficient to masculinize the male embryo. In contrast to males, GATA binding to the Amh promoter was dispensable for Amh expression in the adult ovary. These results provide conclusive evidence that in males, GATA4 is a positive modulator of Amh expression that works in concert with other key transcription factors to ensure that the Amh gene is sufficiently expressed in a correct spatiotemporal manner during fetal and prepubertal testis development.

Whole Genome Amplification of Day 3 or Day 5 Human Embryos Biopsies Provides a Suitable DNA Template for PCR-Based Techniques for Genotyping, a Complement of Preimplantation Genetic Testing

Our objective was to determine if whole genome amplification (WGA) provides suitable DNA for qPCR-based genotyping for human embryos. Single blastomeres (Day 3) or trophoblastic cells (Day 5) were isolated from 342 embryos for WGA. Comparative Genomic Hybridization determined embryo sex as well as Trisomy 18 or Trisomy 21. To determine the embryo’s sex, qPCR melting curve analysis for SRY and DYS14 was used. Logistic regression indicated a 4.4%, 57.1%, or 98.8% probability of a male embryo when neither gene, SRY only, or both genes were detected, respectively (accuracy = 94.1%, kappa = 0.882, andp<0.001). Fluorescent Capillary Electrophoresis for the amelogenin genes (AMEL) was also used to determine sex. AMELY peak’s height was higher and this peak’s presence was highly predictive of male embryos (AUC = 0.93, accuracy = 81.7%, kappa = 0.974, andp<0.001). Trisomy 18 and Trisomy 21 were determined using the threshold cycle difference for RPL17 and TTC3, respectively, which were significantly lower in the corresponding embryos. The Ct difference for TTC3 specifically determined Trisomy 21 (AUC = 0.89) and RPL17 for Trisomy 18 (AUC = 0.94). Here, WGA provides adequate DNA for PCR-based techniques for preimplantation genotyping.

142 MORPHOLOGICAL EVALUATION OF CANINE EMBRYOS OBTAINED IN VIVO

The aim of this study was to evaluate re-expansion and ultrastructure of in vivo-produced canine embryos immediately after collection (Co; n = 6) and 24 h after in vitro culture (Co24; n = 6). For embryo collection, two bitches in pro-oestrous were monitored every 48 h by vaginal cytology up to the detection of 80 to 90% of superficial cells. Then, they were inseminated 3 times on alternate days with fresh semen from 1 fertile male. Embryo collections were performed after ovariohysterectomies and uterine flushing 12 days after the first artificial insemination. Each uterine horn was flushed 3 times with 60 mL of DPBS at 36 to 37°C and collecting embyos in Petri dishes. Embryos culture were performed at 38.5°C under an atmosphere with 5% CO2 and maximum humidity, using SOFaa media added with 20% of fetal bovine serum (FBS). Embryo reexpansion was evaluated after 24 h of culture and the embryos were classified as re-expanded or not re-expanded, according the appearance of the blastocoele by stereomicroscopy (Leica MZ 12.5, Leica Microsystems, Wetzlar, Germany). Twelve embryos were collected, 5 from bitch A and 7 from bitch B; 100% of embryos (6/6) showed re-expansion after 24 h of culture. Blastocoele reexpansion was used as an embryo viability marker. The group Co showed perivitelline space and apical surface of blastomeres covered with microvilli, elongated mitochondria, rough endoplasmic reticulum (RER), tight junctions, and a large amount of lipid droplets that was similar to the results previously described in others mammals species. The Co24 group showed the same characteristcs of the group Co at the time of collection, however with a reduction of lipid droplets and the presence of myelinic structures. In conclusion, the lipid droplet reduction and presence of myelinic structures after 24 h of in vitro culture may indicate lipid consumption associated with embryo expansion.

90 TRANSCRIPTION OF USP9Y AND ZFY IN DEVELOPING AND ARRESTED BOVINE EMBRYOS IN VITRO

Sexual dimorphisms such as differences in growth rate and metabolism have been observed in the early embryo, suggesting that sex chromosome-linked gene expression may play an active role in early embryo development. Furthermore, in vitro sex ratios are often skewed toward males, indicating that Y-linked genes may benefit development. While little attention has been paid to the Y chromosome, expression of some Y-linked genes such as SRY and ZFY has been identified in the early embryo, and only a few studies have systematically examined early stages. Identification of transcripts of Y-linked genes in the early embryo may provide insights into male development and provide markers of embryonic genome activation in male embryos. The objectives of this study were i) to examine the timing of transcription of 2 Y chromosome-linked genes involved with sperm production and male development, ubiquitin-specific peptidase 9 (USP9Y) and zinc finger protein (ZFY), in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst stage and ii) to determine if USP9Y and ZFY transcripts are present in in vitro-produced embryos arrested at the 2- to 8-cell stages. To examine the chronology of transcription of these genes, pools of 30 embryos for each developmental stage, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst, were produced by bovine standard in vitro embryo production (Ashkar et al. 2010 Hum. Reprod. 252, 334–344) using semen from a single bull. Pools of 30 were used to balance sex ratios and to account for naturally arresting embryos. Embryos for each developmental stage were harvested and snap frozen. Total RNA was extracted from each pool, reverse transcribed to cDNA and by using PCR, and transcripts of USP9Y and ZFY were detected as positive or negative. In addition pools of 30 embryos arrested at the 2- to 8-cell stage harvested 7 days after IVF were processed and analysed in the same way to determine if transcripts from the Y chromosomes are present in developmentally arrested embryos. Transcripts of USP9Y and ZFY were detected in the pooled embryos from the 8-cell stage through to the blastocyst stage, but none were detected in the 2-cell or 4-cell pools. Transcripts of ZFY were detected in the arrested 2- to 8-cell embryo pool, but transcripts of USP9Y were not detected. Given that these Y genes begin expression at the 8-cell stage, coincident with embryonic genome activation, it was concluded that these genes may be important for early male embryo development. Furthermore, the results suggest that arrested embryos that have stopped cleaving before the major activation of the embryonic genome are still capable of transcribing at least some of these genes. The absence of USP9Y transcripts in the arrested embryos suggests that it may be important for early male embryo development. Funding was provided by NSERC, the CRC program, and the OVC scholarship program.

146 BOVINE EMBRYO AGGREGATION TO MULTIPLY ELITE GENOTYPES

Each in vitro produced bovine embryo has only a limited chance of developing into a live calf. As genetically superior embryos can now be identified (Humblot et al. 2010 Vet. Med. Int.), maximizing these odds is desirable. We aim to multiply the elite genetics of selected male embryos by aggregating them with female embryos and generating germ line chimaeras. We postulate that this approach will result in functionally male cattle that produce sperm derived from the embryo of high genetic value. In this study, we evaluated the number and developmental stage of male cleavage-stage blastomeres required for efficient sex conversion and chimaerism. On Day 4, at the 12- to 16-cell stage, each presumptive male embryo donor (generated from Y-chromosome-sorted sperm) was dissociated into 1/4 (3–4 cells), 1/2 (6–8 cells) or 3/4 (9–12 cells) portions. One-quarter of each donor embryo was kept for future genotyping. Each donor portion was aggregated with a whole Day 4 presumptive female embryo generated from X-chromosome-sorted sperm from a different sire. On Day 7, blastocysts were assessed for blastomere incorporation and suitable quality embryos transferred individually to recipients. Development of individual aggregates to suitable blastocysts was not significantly different between 1/4-, 1/2-, and 3/4-aggregates or control whole male embryos (11/49 = 22%, 20/49 = 41% 15/50 = 30%, and 11/37 = 30%, respectively). However, aggregating three 1/4 embryos resulted in a 2.3-fold increase in the number of blastocysts produced per donor embryo compared to controls (11/16 = 69% v. 11/37 = 30%). Following embryo transfer of different aggregate groups (1/4, 1/2, and 3/4) and controls, there was neither a significant difference for pregnancy establishment on Day 35 (5/11 = 45%, 9/15 = 60%, 8/13 = 62%, and 5/10 = 50%, respectively), nor on Day 150 of gestation (2/11 = 18%, 3/15 = 20%, 4/13 = 31%, and 4/10 = 40%, respectively). Most of the aggregate fetuses recovered around Day 150 were phenotypically male (1/4 = 2 of 2, 1/2 = 1 of 3, and 3/4 = 4 of 4 fetuses; 7/9 = 78% overall). Expression of the female-specific mRNA for XIST (X-inactive specific transcript) was detected in liver and/or heart from aggregate male fetuses (3 from 3/4 aggregates and 1 from a 1/4 aggregate; 4/7 = 57% overall), indicating somatic cell sex chimaerism. Using sex-specific sequences of the amelogenin gene (AMELX and AMELY), no Y-chromosomal contribution was detected in liver or heart of the two phenotypically female aggregate fetuses. A recent asynchronous aggregation experiment, whereby three 1/4 male Day 5 embryos were each aggregated with a single Day 4 female embryo, resulted in a 4.6-fold increase in the number of blastocysts produced per donor embryo compared to controls (39/17 = 229% v. 22/44 = 50%). In summary, embryo aggregation more than doubled the number of blastocysts that potentially contained the desired genetics of a donor embryo. The majority of the resulting fetuses were male and developed to Day 150 of gestation at rates comparable to controls. Future studies will determine the minimum number of male blastomeres required to ensure sex conversion and colonisation of the germ line to reliably generate bull calves. Supported by MSI C10X1002.