Extracellular vesicles (EVs) are of growing interest due to their potential diagnostic, disease surveillance, and therapeutic applications. While several studies have evaluated EV isolation methods ...in various biofluids, there are few if any data on these techniques when applied to stool. The latter is an ideal biospecimen for studying EVs and colorectal cancer (CRC) because the release of tumour markers by luminal exfoliation into stool occurs earlier than vascular invasion. Since EV release is a conserved mechanism, bacteria in stool contribute to the overall EV population. In this study, we assessed five EV separation methods (ultracentrifugation UC, precipitation EQ-O, EQ-TC, size exclusion chromatography SEC, and ultrafiltration UF) for total recovery, reproducibility, purity, RNA composition, and protein expression in stool supernatant. CD63, TSG101, and ompA proteins were present in EV fractions from all methods except UC. Human (18s) and bacterial (16s) rRNA was detected in stool EV preparations. Enzymatic treatment prior to extraction is necessary to avoid non-vesicular RNA contamination. Ultrafiltration had the highest recovery, RNA, and protein yield. After assessing purity further, SEC was the isolation method of choice. These findings serve as the groundwork for future studies that use high throughput omics technologies to investigate the potential of stool-derived EVs as a source for novel biomarkers for early CRC detection.
Embryo survival and pregnancy success is increased among animals that exhibit estrus prior to fixed time-artificial insemination, but there are no differences in conceptus survival to d16. The ...objective of this study was to determine effects of preovulatory estradiol on uterine transcriptomes, select trophectoderm (TE) transcripts, and uterine luminal fluid proteins. Beef cows/heifers were synchronized, artificially inseminated (d0), and grouped into either high (highE2) or low (lowE2) preovulatory estradiol. Uteri were flushed (d16); conceptuses and endometrial biopsies (n = 29) were collected. RNA sequencing was performed on endometrium. Real-time polymerase chain reaction (RT-PCR) was performed on TE (n = 21) RNA to measure relative abundance of IFNT, PTGS2, TM4SF1, C3, FGFR2, and GAPDH. Uterine fluid was analyzed using 2D Liquid Chromatography with tandem mass spectrometry-based Isobaric tags for relative and absolute quantitation (iTRAQ) method. RT-PCR data were analyzed using the MIXED procedure in SAS. There were no differences in messenger RNA (mRNA) abundances in TE, but there were 432 differentially expressed genes (253 downregulated, 179 upregulated) in highE2/conceptus versus lowE2/conceptus groups. There were also 48 differentially expressed proteins (19 upregulated, 29 downregulated); 6 of these were differentially expressed (FDR < 0.10) at the mRNA level. Similar pathways for mRNA and proteins included: calcium signaling, protein kinase A signaling, and corticotropin-releasing hormone signaling. These differences in uterine function may be preparing the conceptus for improved likelihood of survival after d16 among highE2 animals. Summary sentence Preovulatory estradiol did not impact conceptus survival to d16; however, it did influence uterine gene/protein expressions related to adhesion, endometrial remodeling, metabolism, and immune regulation, which may explain improved pregnancy success.
The uterine environment must provide sufficient endocrine conditions and nutrients for pregnancy maintenance and conceptus survival. The objective of this study was to determine the effects of ...preovulatory estradiol and conceptus presence on uterine transcripts and uterine luminal fluid (ULF) proteins. Beef cows/heifers were synchronized and artificially inseminated (d 0). Uteri were flushed (d 16); conceptuses and endometrial biopsies were collected. Total cellular RNA was extracted from endometrium for RNA sequencing and RT-PCR validation. There were two independent ULF pools made for each of the following groups: highE2/conceptus, highE2/noconceptus, lowE2/conceptus, and lowE2/noconceptus that were analyzed using the 2D LC-MS/MS based iTRAQ method. There were 64 differentially expressed genes (DEGs) and 77 differentially expressed proteins (DEPs) in common among the highE2/conceptus vs highE2/noconceptus and lowE2/conceptus vs lowE2/noconceptus groups. In summary, the interaction between preovulatory estradiol and the conceptus induces the expression of genes, proteins, and pathways necessary for pregnancy.
•There were 64 DEGs in common among the highE2/conceptus vs highE2/noconceptus groups and lowE2/conceptus vs lowE2/noconceptus groups.•There were 77 DEPs in common among highE2/conceptus vs highE2/noconceptus and lowE2/conceptus vs lowE2/noconceptus.•The interaction between preovulatory estradiol and conceptus presence influences uterine gene and protein expression.
Increased antral follicles are associated with greater fertility and a uterine environment that is more supportive of early embryonic development in beef heifers. Glucose is a primary energy source ...for embryos, and glucose concentrations are elevated in uterine luminal fluid (ULF) of pregnant heifers. We hypothesized that ULF glucose concentrations and endometrial transcript abundance for glucose transporters on d16 after insemination would be greater in heifers with increased numbers of antral follicles. Heifers classified with either increased or diminished antral follicle counts were artificially inseminated following the CO-Synch protocol (d0). On d16 after insemination, reproductive tracts of heifers were collected at an abattoir to retrieve conceptuses to determine pregnancy. Uterine luminal fluid was collected, endometrium was biopsied, total RNA was extracted and glucose transporter transcript abundances were determined. Data were analyzed using the MIXED procedure of SAS with antral follicle group, pregnancy status, and the interaction as fixed effects. Glucose concentrations in ULF were greater in heifers with increased antral follicle numbers. Glucose ULF concentrations increased in pregnant heifers. Facilitated glucose transporter member 1 (SLC2A1) transcript abundance was increased in the endometrium of pregnant heifers but was not different due to antral follicle number or the interaction. Differences in uterine concentrations of glucose associated with antral follicle number could be due to another mechanism, since glucose transporters were not different between antral follicle numbers. Therefore, heifers with increased number of antral follicles have increased energy availability in the uterus to support trophoblast proliferation and function.
•Increased antral follicles associated with greater uterine glucose concentrations.•Pregnancy status influences uterine glucose abundance at day 16 of gestation.•Antral follicle number does not impact abundance of glucose transporters.
Nutritional changes immediately after insemination cause increased embryonic mortality, but the mechanisms controlling this are not well known. Our objective was to evaluate the impact of nutritional ...change on estrus expression, steroid concentrations, peripheral and uterine luminal fluid metabolites, and embryo quality in beef heifers. Heifers (n = 139) were assigned to one of two pre-artificial insemination (AI) dietary treatments: LOW (≤ 90% NEm) or HIGH (≥ 139% NEm). Heifers were on treatment for 33–36 days before AI (d0) when half of the heifers in each treatment were randomly reassigned to generate four treatments; HIGH-HIGH, HIGH-LOW, LOW-HIGH, and LOW-LOW. Heifers remained on treatments until embryo collection (d 6–8). Negative energy balance was achieved among LOW heifers as demonstrated by body weight loss and increased NEFA concentrations (P < 0.05). Pre-AI treatment influenced expression of estrus (P = 0.05; HIGH 80.4 ± 4.0% vs. LOW 69.4 ± 4.2%). Estradiol concentrations and interval to estrus were not affected by treatment (P > 0.55); however, progesterone concentrations were reduced among LOW compared to HIGH (3.57 ± 0.27, 4.64 ± 0.26 ng/mL, respectively; P = 0.004), and heifers maintained on the HIGH pre-AI diet had consistently greater concentrations of progesterone from d 0 to d 8 (P = 0.014). Pre-AI treatment influenced embryo stage (P = 0.05; HIGH 3.61 ± 0.32 vs. LOW 2.72 ± 0.30). Post-AI treatment affected embryo grade (P = 0.02; HIGH 1.78 ± 0.23 vs. LOW 2.64 ± 0.27). In summary, pre-AI nutrient restriction caused decreased expression of estrus, reduced progesterone concentrations after AI, and negatively impacted embryo development, while post-AI restriction hindered embryo quality.
•Size of the pre-ovulatory follicle and expression of estrus is reduced following nutrient restriction.•Progesterone production after insemination is compromised in heifers experiencing negative energy balance before breeding.•Nutrient restriction prior to insemination delayed embryo development, while restriction after AI reduced embryo quality.
Abstract
Impairment of bovine reproductive function and decreased pregnancy rates have been reported in modified-live vaccinated females. The objective of this study was to determine if virus could ...be found in various areas of the bovine reproductive tract following vaccination. Previously vaccinated beef females (n = 50) were administered a single dose of one of three treatments on d0 of the estrous cycle; BoviShield (n = 20), Titanium 5 (n = 20), or ViraShield (n = 10) based on estrus expression. Blood samples were collected on d0, 15, and 30 to evaluate Infectious Bovine-Rhinotracheitis (IBR) and Bovine Viral Diarrhea Virus (BVDV) titers and progesterone concentrations. On d15/16, half the animals from each treatment were non-surgically flushed with sterile saline (Uterine Fluid), and uterine biopsies (Uterine Tissue) were collected. On d17, follicular fluid and granulosa cells were collected by follicle aspirations. Remaining animals had uteri flushed and uterine tissue collected on d29, and follicles aspirated on d31. All samples were evaluated for presence of virus. Data were analyzed using the MIXED procedure of SAS. Sub-luteal progesterone concentrations were observed in 15% of the BoviShield and Titanium treatments on d15 and d30, versus 0% in the ViraShield treatment. Virus isolation and RT-PCR results were negative in all samples for all animals. Titers for BVDV-1 did not differ between treatments (P = 0.99) or treatment by time (P = 0.48). Titers for IBR tended to differ by treatment (P = 0.09) and treatment by time (P = 0.06), while BVDV-2 titers were affected by treatment (P < 0.01) and treatment x time (P < 0.01). Sub-luteal progesterone animals had a rapid rise in IBR and BVDV-2 titers from d0 to d15 and then tended (P < 0.07) to decrease to d30, compared to animals that had normal cycles where titers increased from d0 to d15 and remained elevated on d30. Collectively, these results indicate an alternative mechanism of impacting reproductive efficiency, possibly through an immune response.
Abstract
Transcript abundance of two forms of GnRH and its receptor have been characterized in bovine ovaries. The objective was to investigate the relationship of GnRH-1, GnRH-2, GnRH-R, ...intrafollicular estradiol (E2) and progesterone (P4) during follicular development. Ovaries were collected from beef cows at specific stages of follicular development pre-selection (PRE, n = 9), post-selection (POST, n = 9), and post-selection 24h after luteal regression (PG, n = 9). The largest follicle per stage was aspirated to obtain granulosa cells (GC) and follicular fluid (FF). Total cellular RNA was extracted from GC and RT-PCR was performed for GnRH-1, GnRH-2, GnRH-R and GAPDH. Radioimmunoassays were performed to determine FF concentrations of E2 and P4. Data were analyzed using the MIXED and REG procedures in SAS. There was no difference (P ≥ 0.23) in mRNA abundance of GnRH-2, GnRH-R or FF concentrations of P4 across follicular stages. There was an effect of stage on FF E2 (PRE: 17,925±20,273, POST: 28,458±21,503, and PG: 252,616±21,503 pg/mL; P < 0.01). Stage affected GnRH-1 mRNA abundance (PRE: 2.28±0.55, POST: 0.92±0.55, and PG: 0.11±0.55; P = 0.03). There was no relationship of GnRH-1 and GnRH-2 mRNA abundance or effect of FF P4 on GnRH-R mRNA abundance (P ≥ 0.23). There was no effect of FF P4 on GnRH-1 mRNA abundance (P = 0.68). There was no effect of FF E2 on GnRH-2 mRNA abundance (P = 0.66). As FF E2 increased GnRH-R mRNA abundance tended to increase (P = 0.10;r2=0.13). As FF P4 concentrations increased GnRH-2 mRNA abundance tended to decrease (P = 0.09;r2=0.12). As FF E2 increased GnRH-1 mRNA abundance decreased (P = 0.02;r2=0.20). In conclusion, there were differences in peptide and receptor mRNA abundance of GnRH in relation to FF E2 and P4 at specific stages of follicular development. This is supportive of a regulatory relationship between ovarian GnRH and steroidogenesis. This work is supported by AFRI Grant No.2018-67016-27578 from USDA. USDA is an equal opportunity provider and employer.
Abstract
Increased numbers of antral follicles are associated with greater fertility and a uterine environment that is more supportive of early embryonic development in beef heifers. Glucose is a ...primary energy source for embryos and glucose concentrations are elevated in uterine luminal fluid (ULF) of pregnant heifers. We hypothesized that ULF glucose concentrations and endometrial transcript abundance for glucose transporters at d16 after insemination would be greater in heifers with increased numbers of antral follicles. Heifers classified with either increased (32 ± 1.1) or diminished (14.7 ± 1.1) antral follicle counts were selected and artificially inseminated following the Select Synch protocol (d0). At d16 after insemination, heifers were sent to the abattoir and reproductive tracts were collected to retrieve conceptuses to determine pregnancy. Uterine luminal fluid was collected, the endometrium was biopsied, total RNA was extracted and glucose transporter transcript abundance was determined. Data were analyzed using the MIXED procedure of SAS with antral follicle group, pregnancy status, and the interaction as fixed effects. Glucose concentrations in ULF were greater (P < 0.05) in heifers with increased antral follicle numbers compared to heifers with diminished numbers (122.65 ± 11.91 vs 84.12 ± 12.42 mg/dL). Glucose ULF concentrations were increased (P < 0.05) in pregnant vs. non-pregnant heifers (124.84 ± 12.81 vs 81.93 ± 11.50 mg/dL). Endometrial glucose transporter 1 (GLUT1) transcript abundance was increased in pregnant heifers (P < 0.01) but was not different due to antral follicle number or the interaction. Therefore, differences in glucose concentrations associated with antral follicle number may be due to differences in GLUT1 transcription before d16 or due to differences in protein abundance or functionality. Taken together, heifers with increased number of antral follicles may have increased energy availability in the uterus for trophoblast proliferation and function. USDA is an equal opportunity provider and employer.
Abstract
The relationship of ovarian reserve parameters to fertility in mammalian females continues to be debated. We demonstrated improved uterine function, creating an environment that is more ...supportive of early embryonic development in beef heifers with increased numbers of follicles. In the present study we hypothesized that timing of luteolysis differs between heifers with increased compared to heifers with diminished numbers of follicles. Angus heifers (n = 20/ group) were classified as low (14.7 ± 1.1) or high (32 ± 1.1) antral follicle number based on ovarian ultrasonography, and artificially inseminated (d0) following a Select Synch protocol. At d16 after insemination, heifers were slaughtered, and reproductive tracts collected. Tracts were flushed with 20 mL of physiological saline to determine pregnancy status by presence of a conceptus and endometrial samples from the horn ipsilateral to the CL were frozen for determination of transcript abundance. Real-time RT-PCR was performed to determine relative transcript abundance of oxytocin receptor (OXTR) and interferon-stimulated gene-15 (ISG15) with glyceraldehyde-3-phospate dehydrogenase as the endogenous reference gene. Transcript abundance was analyzed using the MIXED procedure of SAS with follicle group, pregnancy status and the interaction as fixed effects. Transcript abundance of ISG15 was greater in pregnant endometrium than non-pregnant endometrium (2.75 ± 0.25 vs 0.16 ± 0.22-fold; P < 0.0001) with no influence of follicle group or the interaction. The interaction of follicle group and pregnancy status influenced OXTR transcript abundance (P = 0.06). Luteolytic mechanisms were activated in non-pregnant heifers with diminished numbers of follicles (1.46 ± 0.25-fold) and were not activated in pregnant heifers (0.06 ± 0.22-fold) or heifers with increased numbers of follicles that were not pregnant (0.32 ± 0.30-fold). These data indicate a wider window of recognition of pregnancy in beef heifers with increased numbers of ovarian follicles. USDA is an equal opportunity provider and employer.
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