Sexual dimorphic miRNA-mediated response of bovine elongated embryos to the maternal microenvironment.

A skewed male-to-female ratio in cattle is believed to be due to the biased embryo losses during pregnancy. The changes in biochemical secretion such as miRNAs by the embryo due to altered maternal environment could cause a sex biased selective implantation resulting in a skewed male to female ratio at birth. Nevertheless, it is still not clear whether the male and female embryos could modify their miRNA expression patterns differently in response to altered physiological developmental conditions. Therefore, this study was focused on identifying sex specific miRNA expression patterns induced in the embryo during the elongation period in response to the maternal environment. For this, in vitro produced day female and male embryos were transferred to Holsteins Frisian cows and heifers. The elongated female and male embryos were then recovered at day 13 of the gestation period. Total RNA including the miRNAs was isolated from each group of elongated embryo samples were subjected to the next generation miRNA sequencing. Sequence alignment, identification and quantification of miRNAs were done using the miRDeep2 software package and differential miRNA expression analyses were performed using the edgeR bioconductor package. The recovery rate of viable elongating embryos at day 13 of the gestation period was 26.6%. In cows, 2.8 more viable elongating male embryos were recovered than female embryos, while in heifers the sex ratio of the recovered elongating embryos was close to one (1.05). The miRNA analysis showed that 254 miRNAs were detected in both male and female elongated embryos developed either in cows or heifers, of which 14 miRNAs including bta-miR-10b, bta-miR-148a, bta-miR-26a, and bta-miR-30d were highly expressed. Moreover, the expression level of 32 miRNAs including bta-let-7c, bta-let-7b, bta-let-7g, bta-let-7d and bta-let-7e was significantly different between the male and female embryos developed in cows, but the expression level of only 4 miRNAs (bta-miR-10, bta-mR-100, bta-miR-155 and bta-miR-6119-5p) was different between the male and female embryos that were developed in heifers. Furthermore, 19 miRNAs including those involved in cellular energy homeostasis pathways were differentially expressed between the male embryos developed in cows and heifers, but no significantly differentially expressed miRNAs were detected between the female embryos of cows and heifers. Thus, this study revealed that the sex ratio skewed towards males in embryos developed in cows was accompanied by increased embryonic sexual dimorphic miRNA expression divergence in embryos developed in cows compared to those developed in heifers. Moreover, male embryos are more sensitive to respond to the maternal reproductive microenvironment by modulating their miRNA expression.

Endometrial DNA methylation signatures during the time of breeding in relation to the pregnancy outcome in postpartum dairy cows fed a control diet or supplemented with rumen-protected methionine.

Post calving metabolic stress reduces the fertility of high producing dairy cows possibly by altering the expression of genes in the maternal environment via epigenetic modifications. Therefore, this study was conducted to identify endometrial DNA methylation marks that can be associated with pregnancy outcomes in postpartum cows at the time of breeding. For this, twelve days post-calving, cows were either offered a control diet or supplemented daily with rumen-protected methionine. Cows showing heat 50-64 days postpartum were artificially inseminated. Endometrial cytobrush samples were collected 4-8 h after artificial insemination and classified based on the pregnancy out comes as those derived from cows that resulted in pregnancy or resulted in no pregnancy. The DNAs isolated from endometrial samples were then subject to reduced representative bisulfite sequencing for DNA methylation analysis. Results showed that in the control diet group, 1,958 differentially methylated CpG sites (DMCGs) were identified between cows that resulted in pregnancy and those that resulted in no pregnancy of which 890 DMCGs were located on chr 27: 6217254-6225600 bp. A total of 537 DMCGs were overlapped with 313 annotated genes that were involved in various pathways including signal transduction, signalling by GPCR, aldosterone synthesis and secretion. Likewise, in methionine supplemented group, 3,430 CpG sites were differentially methylated between the two cow groups of which 18.7% were located on Chr27: 6217254-6225600 bp. A total of 1,781 DMCGS were overlapped with 890 genes which involved in developmental and signalling related pathways including WNT-signalling, focal adhesion and ECM receptor interaction. Interestingly, 149 genes involved in signal transduction, axon guidance and non-integrin membrane-ECM interactions were differentially methylated between the two cow groups irrespective of their feeding regime, while 453 genes involved in axon guidance, notch signalling and collagen formation were differentially methylated between cows that received rumen protected methionine and control diet irrespective of their fertility status. Overall, this study indicated that postpartum cows that could potentially become pregnant could be distinguishable based on their endometrial DNA methylation patterns at the time of breeding.

The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events.

At the embryonic level, CRISPR technologies have been used to edit genomes reliably and efficiently in various mammalian models, with Ribonucleoprotein (RNP) electroporation potentially representing a superior delivery method into mammalian zygotes. However, detailed insights of the interactions between varying technical settings as well as the time point of electroporation in a bovine zygote's cell cycle on developmental metrics and the frequency and type of editing events are largely unknown. The present study uncovers that increasing pulse lengths result in higher Full Edit rates, with Mosaicism in Full-Edit embryos being significantly affected by adjusting RNP-electroporation relative to zygote cell cycle. A considerable proportion of Full Edit embryos demonstrated loss-of-heterozygosity after RNP-electroporation prior to S-phase. Some of these loss-of-heterozygosity events are a consequence of chromosomal disruptions along large sections of the target chromosomes making it necessary to check for their presence prior use of this technique in animal breeding. One out of 2 of these loss-of-heterozygosity events, however, was not associated with loss of an entire chromosome or chromosomal sections. Whether analysed loss-of-heterozygosity in these cases, however, was a false negative result due to loss of PCR primer sequences after INDEL formation at the target side or indeed due to interhomolog recombination needs to be clarified in follow up studies since the latter would for sure offer attractive options for future breeding schedules.

Evaluation of a small volume oil-free in vitro production system for bovine embryos.

BACKGROUND: Embryos are usually produced in culture systems with an oil overlay, which conveys protection against the evaporation of water and microbial contamination. The oil can also release toxic substances and absorb essential components, such as hormones, which adversely affect the quality of the oocytes and the development of embryos in vitro. OBJECTIVE: The aim of this study was to validate an oil-free bovine in vitro production (IVP) system. METHOD: Cumulus-oocyte complexes collected from abattoir-derived ovaries were matured, fertilized and cultured employing a standard system. The quantity of medium in both groups (with and without an oil overlay) and throughout all stages of IVP was maintained at a volume of 100 µl. The oil group was covered with paraffin oil. The maturation stage of oocytes was assessed using fluorescence staining after 24 hr and developmental stages of embryos were evaluated on day 8. The expanded day 8 blastocysts were assessed by live-dead staining. RESULTS: Oocytes matured in the absence of an oil overlay had significantly higher maturation rates when compared against matured oocytes in medium with an oil overlay. Steroid concentration is higher in medium after maturation without oil cover. The developmental rate was significantly higher after culture without oil overlay. The total cell number and the live-dead ratio was not significantly different. The osmolality did not differ between both groups during maturation and slightly decreased during culture without oil. CONCLUSION: Based on the current study, bovine oil-free IVP systems can be suggested as an alternative to oil-covered medium.

Profile of bile acid subspecies is similar in blood and follicular fluid of cattle.

The composition of follicular fluid (FF) has an impact on the developmental capacity of the oocyte and the resulting embryo. FF is composed of blood plasma constituents which cross the blood follicular barrier and the secretory components of granulosa and theca cells. Moreover, it has been shown recently that follicular cells have the ability to synthesize bile acids (BAs). BAs are present in several fluids of mammals especially in bile, blood and urine. FF is an essential impacting factor on the oocyte quality and therefore resulting embryos. To achieve a better understanding of this subject, the presence and concentration of BAs were measured in fluid collected from bovine follicles, categorized according to their size, throughout two entire oestrus cycles and compared to those in blood and urine. The body fluids were collected during the same examination procedure and in total samples from four heifers were obtained. A broad spectrum of 11 BA derivatives was measured applying liquid chromatography-tandem mass spectrometry (LC-MS/MS). The simultaneous and direct quantification of BAs in different body fluids of cattle are reported. Within the follicular fluid, blood and urine, cholic acid and glycocholic acid are the dominant BA subspecies irrespective of the oestrus cycle stage. Moreover, BA concentrations in blood compared to those in the FF were similar. For the first time these results clearly highlight the presence of different BA subspecies in FF, blood and urine during the oestrus cycle in cattle.

Temporal expression pattern of steroid-metabolizing enzymes in bovine COC during in vitro maturation employing different gonadotropin concentrations.

Steroid hormones are regulators in the fine-tuned process of follicular development. During final maturation in vivo a switch from oestradiol (E2) to progesterone (P4) dominance within the follicle is well-described. This change is accompanied by the resumption of meiosis and results in the maturation of the oocyte. It also suggests the important role of these hormones. However, present in vitro maturation (IVM) systems do not completely mimic the in vivo situation, resulting in oocytes of reduced quality. Aim of the study was to determine the temporal pattern of steroid hormone concentrations in the IVM medium of bovine cumulus-oocyte-complexes (COC) at defined time points. The influence of different gonadotropin supplementations during IVM on oocyte maturation, as well as the molecular quality of the oocytes and their corresponding cumulus cells was investigated. COCs were obtained from abattoir-derived ovaries and matured in medium added with different compounds of gonadotropins (eCG/hCG; FSH/LH, each at 0.05 IU or 0.01 IU; only FSH; without gonadotropins) employing a standard protocol without oil overlay. In experiment 1, medium, oocytes and cumulus cells were collected at different time points (0 h [control], 4 h, 8 h, 12 h, 16 h, 20 h, 24 h) after IVM in just eCG/hCG-supplemented medium. In experiment 2, medium, oocytes and cumulus cells were collected at 0 h (control) and after 24 h of IVM with all above-named supplements. The E2 concentration remained similar during IVM whereas P4 concentration increased during experiment 1. No significant changes could be determined after the addition of different gonadotropins (experiment 2). These results suggest that during IVM the temporal pattern of E2 and P4 did not correspond with the pattern during final maturation in vivo. RT-qPCR was used to assess the relative abundance of developmentally important genes in oocytes (BMP15; GDF9; ZAR1; PGR; PGRMC1/2; G6PD; StAR; ESR1/2; SULT1E1; STS; SOAT) and cumulus cells (ESR1/2; FSHR; LHCGR; CYP19A1; HSD3B1; PGR; PGRMC1/2; SULT1E1; STS; SOAT) at all collection points in both experiments. Most transcripts follow a time-regulated mRNA expression pattern during the entire in vitro maturation period. In addition, the expression of the analyzed transcripts was not influenced by the different gonadotropin supplementations during the IVM period. In all, this underlines that present conditions of IVM do not reflect the in vivo situation and require further optimisation.

Occurrence of sulfonated steroids and ovarian expression of steroid sulfatase and SULT1E1 in cyclic cows.

Historically sulfonated steroids were primarily considered as inactive metabolites destined for elimination. However, more recently they have been increasingly recognized as precursors for the production of bioactive steroids in target tissues and as functional molecules without preceding hydrolysis. In order to comprehensively characterize their occurrence in cyclic cows and their formation and hydrolysis in bovine ovarian steroidogenesis, ovaries from cyclic cows were screened for the expression of oestrogen sulfotransferase (SULTE1) and steroid sulfatase (STS) by Western blot and immunohistochemistry. Moreover, a broad spectrum of 13 sulfonated steroids was measured applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) in blood samples collected from three cycling heifers during defined stages of the ovarian cycle and in fluid obtained from ovarian follicles of different size. SULT1E1 was undetectable in ovarian tissues. For STS only a weak immunostaining was found predominantly in granulosa cells of larger follicles. However, no specific band occurred in Western blot. In blood, concentrations of all sulfonated steroids investigated were below the limit of quantification (LOQ). In follicular fluid, only cholesterol sulfate was measured in considerable concentrations (328.3 ±â€¯63.8 ng/ml). However, the role of cholesterol sulfate in bovine follicular steroidogenesis remains unclear as concentrations were obviously unrelated to follicular size. The remaining sulfonated steroids investigated were undetectable or only slightly exceeded LOQ in a minor proportion of samples. The results are clearly contrary to a role of sulfonated steroids as important precursors, intermediates or products of bovine ovarian steroidogenesis.

The role of sulfated steroid hormones in reproductive processes.

Sulfated steroid hormones, such as dehydroepiandrosterone sulfate or estrone-3-sulfate, have long been regarded as inactive metabolites as they cannot activate classical steroid receptors. Some of them are present in the blood circulation at quite high concentrations, but generally sulfated steroids exhibit low membrane permeation due to their hydrophilic properties. However, sulfated steroid hormones can actively be imported into specific target cells via uptake carriers, such as the sodium-dependent organic anion transporter SOAT, and, after hydrolysis by the steroid sulfatase (so-called sulfatase pathway), contribute to the overall regulation of steroid responsive organs. To investigate the biological significance of sulfated steroid hormones for reproductive processes in humans and animals, the research group "Sulfated Steroids in Reproduction" was established by the German Research Foundation DFG (FOR1369). Projects of this group deal with transport of sulfated steroids, sulfation of free steroids, desulfation by the steroid sulfatase, effects of sulfated steroids on steroid biosynthesis and membrane receptors as well as MS-based profiling of sulfated steroids in biological samples. This review and concept paper presents key findings from all these projects and provides a broad overview over the current research on sulfated steroid hormones in the field of reproduction.