Bovine Sperm Sexing Alters Sperm Morphokinetics and Subsequent Early Embryonic Development.
ABSTRACT: In artificial insemination the use of sex-sorted bovine sperm results in reduced conception, the causes of which are only partly understood. Therefore, we set out to investigate the effects of sexing on bovine sperm function and early embryonic development. Computer-assisted semen analysis (CASA) of sperm of the same bulls (n?=?5), before and after sexing, demonstrated significantly reduced fast (A) and slow (B) progressively motile sperm (p?
Project description:BACKGROUND:Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. RESULTS:The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). CONCLUSIONS:A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples.
Project description:Darwin's finches are an iconic example of adaptive radiation and evolution under natural selection. Comparative genetic studies using embryos of Darwin's finches have shed light on the possible evolutionary processes underlying the speciation of this clade. Molecular identification of the sex of embryonic samples is important for such studies, where this information often cannot be inferred otherwise. We tested a fast and simple chicken embryo protocol to extract DNA from Darwin's finch embryos. In addition, we applied minor modifications to two of the previously reported PCR primer sets for CHD1, a gene used for sexing adult passerine birds. The sex of all 29 tested embryos of six species of Darwin's finches was determined successfully by PCR, using both primer sets. Next to embryos, hatchlings and fledglings are also impossible to distinguish visually. This extends to juveniles of sexually dimorphic species which are yet to moult in adult-like plumage and beak colouration. Furthermore, four species of Darwin's finches are monomorphic, males and females looking alike. Therefore, sex assessment in the field can be a source of error, especially with respect to juveniles and mature monomorphic birds outside of the mating season. We caught 567 juveniles and adults belonging to six species of Darwin's finches and only 44% had unambiguous sex-specific morphology. We sexed 363 birds by PCR: individuals sexed based on marginal sex specific morphological traits; and birds which were impossible to classify in the field. PCR revealed that for birds with marginal sex specific traits, sexing in the field produced a 13% error rate. This demonstrates that PCR based sexing can improve field studies on Darwin's finches, especially when individuals with unclear sex-related morphology are involved. The protocols used here provide an easy and reliable way to sex Darwin's finches throughout ontogeny, from embryos to adults.
Project description:To date sperm-oviduct interactions have largely been investigated under in vitro conditions. Therefore we set out to characterize the behaviour of bovine spermatozoa within the sperm reservoir under near in vivo conditions and in real-time using a novel live cell imaging technology and a newly established fluorescent sperm binding assay. Sperm structure and tubal reactions after sperm binding were analysed using scanning and transmission electron microscopy and histochemistry. As a model to specify the impact of stress on sperm-oviduct interactions, frozen-thawed conventional and sex-sorted spermatozoa from the same bulls (n?=?7) were co-incubated with oviducts obtained from cows immediately after slaughter. Our studies revealed that within the oviductal sperm reservoir agile (bound at a tangential angle of about 30°, actively beating undulating tail), lagging (bound at a lower angle, reduced tail movement), immotile (absence of tail movement) and hyperactivated (whip-like movement of tail) spermatozoa occur, the prevalence of which changes in a time-dependent pattern. After formation of the sperm reservoir, tubal ciliary beat frequency is significantly increased (p?=?0.022) and the epithelial cells show increased activity of endoplasmic reticula. After sex sorting, spermatozoa occasionally display abnormal movement patterns characterized by a 360° rotating head and tail. Sperm binding in the oviduct is significantly reduced (p?=?0.008) following sexing. Sex-sorted spermatozoa reveal deformations in the head, sharp bends in the tail and a significantly increased prevalence of damaged mitochondria (p?<?0.001). Our results imply that the oviductal cells specifically react to the binding of spermatozoa, maintaining sperm survival within the tubal reservoir. The sex-sorting process, which is associated with mechanical, chemical and time stress, impacts sperm binding to the oviduct and mitochondrial integrity affecting sperm motility and function.
Project description:Parental cannabis use has been associated with adverse neurodevelopmental outcomes in offspring, but how such phenotypes are transmitted is largely unknown. Using reduced representation bisulphite sequencing (RRBS), we recently demonstrated that cannabis use is associated with widespread DNA methylation changes in human and rat sperm. Discs-Large Associated Protein 2 (DLGAP2), involved in synapse organization, neuronal signaling, and strongly implicated in autism, exhibited significant hypomethylation (p < 0.05) at 17 CpG sites in human sperm. We successfully validated the differential methylation present in DLGAP2 for nine CpG sites located in intron seven (p < 0.05) using quantitative bisulphite pyrosequencing. Intron 7 DNA methylation and DLGAP2 expression in human conceptal brain tissue were inversely correlated (p < 0.01). Adult male rats exposed to delta-9-tetrahydrocannabinol (THC) showed differential DNA methylation at Dlgap2 in sperm (p < 0.03), as did the nucleus accumbens of rats whose fathers were exposed to THC prior to conception (p < 0.05). Altogether, these results warrant further investigation into the effects of preconception cannabis use in males and the potential effects on subsequent generations.
Project description:PURPOSE: Partial molar pregnancies are rare conceptions characterized by having 69 rather than 46 chromosomes, the additional chromosome complement usually occurring as a result of fertilization of the ovum by two sperm. Although assisted conception with intracytoplasmic sperm injection (ICSI) should prevent the development of a partial molar pregnancy, occasional cases have been described after assisted conception using ICSI. The objective of this study was to investigate the cause of partial molar pregnancy in a couple who had undertaken assisted conception with ICSI. METHODS: Fluorescent microsatellite genotyping of DNA from the couple and tissue from their partial molar pregnancy was performed in order to confirm diagnosis and investigate the origin of the additional chromosome set. RESULTS: Genotyping confirmed that the partial molar tissue was triploid with an additional chromosome complement from the father. Genotyping of additional loci proximal to the centromere demonstrated that the two paternal sets of chromosomes originated in a single sperm with a double complement of paternal DNA resulting from non-reduction at the second meiotic division. CONCLUSIONS: This study confirms that partial molar pregnancy may occur after assisted conception with ICSI and that this occurs as a result of fertilization with a diploid sperm.
Project description:PURPOSE:Recent studies have shown that improved clinical outcomes can be achieved by transferring blastocysts rather than cleavage-stage embryos. However, blastocyst transfer is not performed in all patients. The aim of this study was to compare clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles using testicular sperm (TE) with those of ICSI cycles using ejaculated sperm (EJ). METHODS:ICSI was performed using EJ in 141 cycles and TE in 37 cycles. Embryos were cultured for 5 days. The quality of embryos was assessed on days 3 and 5 before embryo transfer. RESULTS:Fertilization rate was 77.3% in the EJ group and 69.6% in the TE group (p?<?0.05). The good-quality embryos on day 3 and 5 were not different between the EJ and TE groups. Embryos did not develop to blastocyst stage in 7 cycles of the EJ group (5.0%) and 2 cycles of the TE group (5.4%). There were no significant differences in blastocyst formation and blastocyst quality (46.1% vs. 47.5% and 5.7% vs 5.8%, respectively) on day 5 between both groups. Embryos were transferred in all cycles. Implantation (22.8 vs. 24.7%), clinical pregnancy (44.7 vs. 43.2%), miscarriage (21.7 vs. 33.3%), and delivery (76.5 vs. 66.7%) did not differ between EJ group and TE group. Clinical outcomes of ICSI were not different between the EJ and TE groups. CONCLUSIONS:In conclusion, the potential of testicular sperm supporting embryonic development to blastocysts is comparable to that of ejaculated sperm. Therefore, this study suggests that blastocyst transfer can be a very useful assisted reproductive technique in the ICSI cycles that require the use of testicular sperm, and the clinical outcomes of the cycles are comparable to those of ICSI cycles using ejaculated sperm.
Project description:Genomic instability is common in human embryos, but the underlying causes are largely unknown. Here, we examined the consequences of sperm DNA damage on the embryonic genome by single-cell whole-genome sequencing of individual blastomeres from bovine embryos produced with sperm damaged by ?-radiation. Sperm DNA damage primarily leads to fragmentation of the paternal chromosomes followed by random distribution of the chromosomal fragments over the two sister cells in the first cell division. An unexpected secondary effect of sperm DNA damage is the induction of direct unequal cleavages, which include the poorly understood heterogoneic cell divisions. As a result, chaotic mosaicism is common in embryos derived from fertilizations with damaged sperm. The mosaic aneuploidies, uniparental disomies, and de novo structural variation induced by sperm DNA damage may compromise fertility and lead to rare congenital disorders when embryos escape developmental arrest.
Project description:Artificial insemination with cryopreserved spermatozoa is a major assisted reproductive technology in many species. In horses, as in humans, insemination with cryopreserved sperm is associated with lower pregnancy rates than those for fresh sperm, however, direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored. The aim of this study was to investigate differences in gene expression between embryos resulting from fertilization with fresh or cryopreserved sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares inseminated post-ovulation on successive cycles with either fresh sperm or frozen-thawed sperm from the same stallion, providing matched embryo pairs at each day. RNA was isolated from two matched pairs (4 embryos) for each day, and cDNA libraries were built and sequenced. Significant differences in transcripts per kilobase million (TPM) were determined using (i) genes for which the expression difference between treatments was higher than 99% of that in the random case (P < 0.01), and (ii) genes for which the fold change was ? 2, to avoid expression bias in selection of the candidate genes. Molecular pathways were explored using the DAVID webserver, followed by network analyses using STRING, with a threshold of 0.700 for positive interactions. The transcriptional profile of embryos obtained with frozen-thawed sperm differed significantly from that for embryos derived from fresh sperm on all days, showing significant down-regulation of genes involved in biological pathways related to oxidative phosphorylation, DNA binding, DNA replication, and immune response. Many genes with reduced expression were orthologs of genes known to be embryonic lethal in mice. This study, for the first time, provides evidence of altered transcription in embryos resulting from fertilization with cryopreserved spermatozoa in any species. As sperm cryopreservation is commonly used in many species, including human, the effect of this intervention on expression of developmentally important genes in resulting embryos warrants attention.
Project description:Parental age at time of offspring conception is increasing in developed countries. Advanced male age is associated with decreased reproductive success and increased risk of adverse neurodevelopmental outcomes in offspring. Mechanisms for these male age effects remain unclear, but changes in sperm DNA methylation over time is one potential explanation. We assessed genome-wide methylation of sperm DNA from 47 semen samples collected from male participants of couples seeking infertility treatment. We report that higher male age was associated with lower likelihood of fertilization and live birth, and poor embryo development (p?<?0.05). Furthermore, our multivariable linear models showed male age was associated with alterations in sperm methylation at 1698 CpGs and 1146 regions (q?<?0.05), which were associated with?>?750 genes enriched in embryonic development, behavior and neurodevelopment among others. High dimensional mediation analyses identified four genes (DEFB126, TPI1P3, PLCH2 and DLGAP2) with age-related sperm differential methylation that accounted for 64% (95% CI 0.42-0.86%; p?<?0.05) of the effect of male age on lower fertilization rate. Our findings from this modest IVF population provide evidence for sperm methylation as a mechanism of age-induced poor reproductive outcomes and identifies possible candidate genes for mediating these effects.
Project description:Growing evidence suggests that paternal obesity may decrease male fertility potential. During infertility treatment with intra-cytoplasmic sperm injection (ICSI), a morphologically normal motile spermatozoon is injected into a mature egg, when possible. However, sperm motility and morphology per se do not reflect the sperm molecular composition. In this study, we aimed to assess the quality of motile spermatozoa in the context of obesity by analysing their conventional and molecular characteristics as well as their ability to promote early embryonic development. A prospective study was conducted on 128 infertile men divided into three groups: 40 lean, 42 overweight, and 46 obese men. Conventional sperm parameters (concentration, motility and morphology) and sperm molecular status (chromatin composition and integrity, 5-methycytosine (5-mC) and 5-hydroxycytosine (5-hmC) contents and oxidative stress level) were analysed on raw semen and/or on motile spermatozoa selected by density gradient or swim-up techniques. Morphokinetic analysis of the embryos derived from ICSI was performed using the Embryoviewer software. Our results showed that the motile sperm-enriched fraction from obese men exhibited higher levels of retained histones (p<0.001), elevated percentage of altered chromatin integrity (p<0.001), and decreased contents of 5-hmC (p<0.001), and 5-mC (p<0.05) levels as compared to that from lean men. Importantly, there were no statistically significant correlations between these molecular parameters and the percentages of morphologically normal motile spermatozoa. Regarding embryo morphokinetics, the CC1 (p<0.05) and CC3 (p<0.05) embryonic cell cycles were significantly delayed in the cleavage embryos of the obese group as compared to the embryos of the lean group. Our data is of particular interest because, besides demonstrating the negative impacts of obesity on motile spermatozoa molecular composition, it also highlights the possible risk of disturbing early embryonic cell cycles kinetics in the context of paternal obesity.