150 Effect of bovine seminal plasma and sperm on endometrial gene expression

2019 ◽  
Vol 31 (1) ◽  
pp. 200
Author(s):  
B. Fernandez-Fuertes ◽  
J. M. Sanchez ◽  
S. Bages ◽  
P. Lonergan
Keyword(s):  
Gene Expression ◽  
Seminal Plasma ◽  
Reproductive Tract ◽  
Epididymal Sperm ◽  
Rna Integrity ◽  
Rna Quality ◽  
Rna Integrity Number ◽  
Maternal Immune System ◽  
Negative Effect

In cattle, most pregnancy losses are sustained before implantation. Many factors are involved in implantation failure, but in mice, pigs and humans there is increased evidence of a role for the maternal immune system and its regulation by seminal plasma (SP). However, there is little evidence for a role of SP in bovine fertility, where dilution or removal of SP before AI is routine. Therefore, the aim of this work was to determine the effect of bull SP or sperm on endometrial gene expression. To this end, 6 heifers were oestrous synchronised and slaughtered 12h after the onset of oestrus. Five endometrial explants from the horn ipsilateral to the preovulatory follicle were obtained from each animal. Explants were incubated with (1) RPMI medium (control); (2) epididymal sperm (106 epididymal sperm mL−1); (3) complete ejaculate (106 ejaculated sperm mL−1+25% SP); (4) ejaculated sperm alone (106 ejaculated sperm mL−1); and (5) SP alone (25% SP). Epididymal sperm were collected and pooled from the cauda epididymis of 3 bulls slaughtered in a commercial abattoir. In addition, complete ejaculates were obtained from 3 other bulls using an artificial vagina. After pooling the samples, a small volume was washed through a density gradient to obtain the ejaculated sperm, and the rest of the ejaculate was filtered to obtain sperm-free SP. The RPMI media was used to dilute sperm and SP to the working concentrations. After 6h of incubation, explants were snap frozen. The RNA quality was assessed with an Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA) before RT-qPCR analysis. Interestingly, SP had a dramatic effect on endometrium RNA integrity, as evidenced by a lower RNA integrity number in explants exposed to a complete ejaculate (2.4±0.14) or SP (2.4±0.06), in comparison with the control, epididymal sperm, or ejaculated sperm treatments (6.7±0.43, 6.9±0.32, 6.7±0.30, respectively; P<0.05). Due to the low RNA quality, those treatments were excluded from further analysis. However, this finding is currently being explored, along with the possibility of this effect being inherent to species that ejaculate intravaginally. We then compared the ability of ejaculated sperm (which have been exposed to SP) and epididymal sperm (which have never had contact with SP) to regulate the endometrial expression of IL1A, IL1B, IL8, IL6, PTGES2, TNFA, and LIF. Although IL6, IL1A and LIF increased in all animals when exposed to either ejaculated or epididymal sperm, there was no effect of treatment. In conclusion, these data did not support the notion that exposure of sperm to SP is important for the immune regulation of the bovine uterus. In addition, the negative effect of SP on the endometrium, together with the fact that bulls are intravaginal ejaculators, suggests that any putative immunoregulatory role of this fluid in the cattle uterus is indirect. Further analysis of the effect of SP on the vagina and cervix will help elucidate whether this response is present in this species and whether it can propagate to more distal regions of the reproductive tract. This work was supported by Science Foundation Ireland (13/IA/1983, 16/IA/4474).

scholarly journals A robust method for RNA extraction and purification from a single adult mouse tendon

2018 ◽  
Author(s):  
M Grinstein ◽  
HL Dingwall ◽  
RR Shah ◽  
TD Capellini ◽  
JL Galloway
Keyword(s):  
Gene Expression ◽  
Adult Mouse ◽  
Rna Extraction ◽  
Cost Effective ◽  
Biological Variation ◽  
Mouse Genetics ◽  
Rna Integrity ◽  
Rna Quality ◽  
Rna Analysis ◽  
Rna Integrity Number

AbstractBackgroundMechanistic understanding of tendon molecular and cellular biology is crucial towards furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts.MethodsSingle Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between 3-5 months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed.ResultsAfter testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis of Scx gene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyze Sox9 and Col1a2 gene expression changes in injured compared with uninjured control tendons.DiscussionOur work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RNA-seq and RT-qPCR. We show this can reduce biological variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues as well as precious human samples.

scholarly journals The spatial RNA integrity number assay for in situ evaluation of transcriptome quality

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Linda Kvastad ◽  
Konstantin Carlberg ◽  
Ludvig Larsson ◽  
Eva Gracia Villacampa ◽  
Alexander Stuckey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Spatial Variation ◽  
Tissue Quality ◽  
Rna Integrity ◽  
Quality Metric ◽  
Rna Quality ◽  
Rna Integrity Number ◽  
Cellular Resolution

AbstractThe RNA integrity number (RIN) is a frequently used quality metric to assess the completeness of rRNA, as a proxy for the corresponding mRNA in a tissue. Current methods operate at bulk resolution and provide a single average estimate for the whole sample. Spatial transcriptomics technologies have emerged and shown their value by placing gene expression into a tissue context, resulting in transcriptional information from all tissue regions. Thus, the ability to estimate RNA quality in situ has become of utmost importance to overcome the limitation with a bulk rRNA measurement. Here we show a new tool, the spatial RNA integrity number (sRIN) assay, to assess the rRNA completeness in a tissue wide manner at cellular resolution. We demonstrate the use of sRIN to identify spatial variation in tissue quality prior to more comprehensive spatial transcriptomics workflows.

scholarly journals A robust method for RNA extraction and purification from a single adult mouse tendon

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4664 ◽  
Author(s):  
Mor Grinstein ◽  
Heather L. Dingwall ◽  
Rishita R. Shah ◽  
Terence D. Capellini ◽  
Jenna L. Galloway
Keyword(s):  
Gene Expression ◽  
Adult Mouse ◽  
Rna Extraction ◽  
Cost Effective ◽  
Mouse Genetics ◽  
Rna Integrity ◽  
Rna Quality ◽  
Rna Analysis ◽  
Rna Integrity Number ◽  
Extraction And Purification

BackgroundMechanistic understanding of tendon molecular and cellular biology is crucial toward furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts.MethodsSingle Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between three and five months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed.ResultsAfter testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis ofScxgene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyzeSox9andCol1a2gene expression changes in injured compared with uninjured control tendons.DiscussionOur work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RT-qPCR as well as RNA-seq. We show this can reduce variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues, as well as precious human samples.

scholarly journals Semen Modulates the Expression of NGF, ABHD2, VCAN, and CTEN in the Reproductive Tract of Female Rabbits

Genes ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 758 ◽  
Author(s):  
Jaume Gardela ◽  
Amaia Jauregi-Miguel ◽  
Cristina A. Martinez ◽  
Heriberto Rodriguez-Martinez ◽  
Manel Lopez-Bejar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Seminal Plasma ◽  
Gene Expression Analysis ◽  
Reproductive Tract ◽  
Plasma Deposition ◽  
Mating Experiment ◽  
Maternal Environment ◽  
Expression Of Genes ◽  
Treatment Experiment ◽  
Ngf Gene

Semen changes the gene expression in endometrial and oviductal tissues modulating important processes for reproduction. We tested the hypothesis that mating and/or sperm-free seminal plasma deposition in the reproductive tract affect the expression of genes associated with sperm-lining epithelium interactions, ovulation, and pre-implantation effects (nerve growth factor, NGF; α/β hydrolase domain-containing protein 2, ABHD2; C-terminal tensin-like protein, CTEN or TNS4; and versican, VCAN) in the period 10–72 h post-mating. In Experiment 1, does (n = 9) were treated with gonadotropin-releasing hormone (GnRH) (control), GnRH-stimulated, and vaginally infused with sperm-free seminal plasma (SP-AI), or GnRH-stimulated and naturally mated (NM). In Experiment 2, does (n = 15) were GnRH-stimulated and naturally mated. Samples were retrieved from the internal reproductive tracts (cervix-to-infundibulum) 20 h post-treatment (Experiment 1) or sequentially collected at 10, 24, 36, 68, or 72 h post-mating (Experiment 2, 3 does/period). All samples were processed for gene expression analysis by quantitative PCR. Data showed an upregulation of endometrial CTEN and NGF by NM, but not by SP-AI. The findings suggest that the NGF gene affects the reproductive tract of the doe during ovulation and beyond, influencing the maternal environment during early embryonic development.

scholarly journals Lysophosphatic acid modulates prostaglandin secretion in the bovine uterus

Reproduction ◽  
2009 ◽  
Vol 137 (1) ◽  
pp. 95-105 ◽  
Author(s):  
Izabela Woclawek-Potocka ◽  
Junichi Komiyama ◽  
Jean Sebastian Saulnier-Blache ◽  
Edyta Brzezicka ◽  
Mamadou Moussa Bah ◽  
...  
Keyword(s):  
Gene Expression ◽  
Estrous Cycle ◽  
Dehydrogenase Activity ◽  
Lysophosphatidic Acid ◽  
Early Pregnancy ◽  
Reproductive Tract ◽  
Hydroxysteroid Dehydrogenase ◽  
Lpa Receptor

Lysophosphatidic acid (LPA) modulates prostaglandin (PG) synthesis via LPA receptor 3 (LPAR3) in the murine endometrium. The lack of functional LPAR3 in mice may lead to embryo mortality. In the present study, we examined the role of LPA in the bovine uterus. We confirmed that LPA is locally produced and released from the bovine endometrium. Moreover, there are enzymes involved in LPA synthesis (phospholipase(PL)D2and PLA2G1B) in the bovine endometrium during estrous cycle and early pregnancy. Expression of the receptor for LPA (LPAR1) was positively correlated with the expression ofPGE2synthase(PGES) and negatively correlated with the expression ofPGF2αsynthase(aldose reductase with 20 α-hydroxysteroid dehydrogenase activity –PGFS) during early pregnancy.In vivoLPA induced P4 and PGE2secretion was inhibited by LPAR1 antagonist (Ki16425). The overall results indicate that LPA is locally produced and released from the bovine endometrium. Moreover,LPAR1gene expression in the endometrium during the estrous cycle and early pregnancy indicates that LPA may play autocrine and/or paracrine roles in the bovine uterus.LPAR1gene expression is positively correlated with the expression of the enzyme responsible for luteotropic PGE2production (PGES) in endometrium. In cow, LPA stimulates P4 and PGE2secretion. Thus, LPA in the bovine reproductive tract may indirectly (via endometrium) or directly support corpus luteum action via the increase of P4 synthesis and the increase of PGE2/PGF2αratio. It suggests that LPA may serve as an important factor in the maintenance of early pregnancy in cow.

scholarly journals 46 Endometrial transcriptome profiles associated with bovine preimplantation pregnancy outcome and parity

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 26-27
Author(s):  
Hannah Sweett ◽  
Murilo R Carvalho ◽  
Jose Felipe W Spricigo ◽  
Elvis Ticiani ◽  
Eduardo S Ribeiro ◽  
...  
Keyword(s):  
Pregnancy Outcome ◽  
Reproductive Tract ◽  
Reference Genome ◽  
Enrichment Analysis ◽  
Pathway Enrichment Analysis ◽  
Interferon Tau ◽  
Illumina Hiseq ◽  
Rna Integrity ◽  
Rna Integrity Number ◽  
Regulator Genes

Abstract Pregnancy failure in dairy cattle appears to be attributable to losses during embryogenesis, mainly within the first month. Models have estimated that pregnancies lost after day 30 of fertilization cost producers US $550/cow. The reasons for these losses are not well understood, and the transcriptome profile of the reproductive tract during the establishment of pregnancy may point to mechanisms. Thirty-eight cows in lactations 1 to 3 were artificially inseminated, and endometrial biopsies were collected on day 15 after artificial insemination to investigate the effect of parity on the preimplantation pregnancy outcome. Pregnancy status was determined by the presence of interferon-tau in the uterine flushing, revealing 19 pregnant (P) and 19 non-pregnant (NP) cows. Nineteen biopsy samples [9 cows in lactation 1 (6 P and 3 NP), 4 in lactation 2 (2 P and 2 NP), and 6 in lactation 3 (3P and 3NP)] with an average RNA integrity number of 7 were selected for RNA sequencing with an Illumina HiSeq analyzer. Sequence reads were assembled to the ARS_USD1.2.99 bovine reference genome using the CLC genomics workbench software. On average, the samples generated ~56 million reads and 94.99% were mapped to the reference genome. Differential gene expression analysis between P and NP cows identified 187, 60, and 136 differentially expressed genes (DEG) in lactation 1, 2 and 3, respectively (P < 0.01, FDR < 0.05, FC > ±2). Metabolic pathway enrichment analysis identified several DEG upregulated in lactations 2 and 3 associated with IL-17 signaling pathway, although expression of IL-17 itself was not different. This pathway is part of the protective response against extracellular bacteria and likely participates in pregnancy maintenance through local regulation of immune function. Further functional genomic analyses will be performed to determine additional metabolic pathways, key regulator genes, and functional SNPs associated with the establishment of pregnancy.

Lack of correlation between placental gene expression and RNA integrity number (RIN) or time to collection

Placenta ◽  
2014 ◽  
Vol 35 (9) ◽  
pp. A46-A47 ◽  
Author(s):  
Christopher J. Stodgell ◽  
Richard K. Miller ◽  
Linda Salamone ◽  
Jeffrey Murray ◽  
Jia Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Integrity ◽  
Rna Integrity Number

scholarly journals Detection of osteopontin on Holstein bull spermatozoa, in cauda epididymal fluid and testis homogenates, and its potential role in bovine fertilization

Reproduction ◽  
2007 ◽  
Vol 133 (5) ◽  
pp. 909-917 ◽  
Author(s):  
David W Erikson ◽  
Amy L Way ◽  
David A Chapman ◽  
Gary J Killian
Keyword(s):  
Reproductive Tract ◽  
Bovine Milk ◽  
High Fertility ◽  
Epididymal Sperm ◽  
Holstein Bull ◽  
Sds Page ◽  
Sperm Membrane ◽  
Epididymal Fluid

Osteopontin (OPN) is a secreted extracellular matrix phosphoprotein identified in various tissues and fluids including those of the male and female reproductive tracts. OPN was previously identified as a 55 kDa high fertility marker in Holstein bull seminal plasma, produced by the ampulla and the vesicular gland. The objectives of this study were to characterize OPN on ejaculated and cauda epididymal sperm using immunofluorescence and western blot analysis, and to assess the role of sperm OPN in fertilization. Solubilized sperm membrane proteins from ejaculated and cauda epididymal sperm were separated by 1D SDS-PAGE, transferred to nitrocellulose, and probed with an antibody to bovine milk OPN. A 35 kDa protein was detected by this antibody in both ejaculated and cauda epididymal sperm membranes. Analyses also recognized OPN at 55 and 25 kDa in cauda epididymal fluid and testicular parenchyma homogenates respectively. Immunofluorescent analysis of ejaculated and cauda epididymal sperm showed OPN localization in a well-defined band in the postacrosomal region of the sperm head and also on the midpiece. Results ofin vitrofertilization experiments showed that sperm treated with an antibody to OPN fertilized fewer oocytes than sperm treated with control medium while increasing incidence of polyspermy, suggesting a role of sperm-associated OPN in fertilization and a block to polyspermy. These studies demonstrate that OPN exists at multiple molecular weight forms in the bull reproductive tract and its presence on ejaculated sperm may signal its importance in fertilization by interacting with integrins or other proteins on the oocyte plasma membrane.

Characterization and biological role of cysteine-rich venom protein belonging to CRISPs from turkey seminal plasma

2021 ◽  
Author(s):  
Mariola Słowińska ◽  
Laura Pardyak ◽  
Ewa Liszewska ◽  
Sylwia Judycka ◽  
Joanna Bukowska ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Seminal Plasma ◽  
Reproductive Tract ◽  
Protein Localization ◽  
Mass Spectrometry Analysis ◽  
Etiologic Factor ◽  
Secretory Proteins ◽  
Ductus Deferens ◽  
Venom Protein

Abstract Turkey semen contains cysteine-rich secretory proteins (CRISPs) that belong to the dominant seminal plasma proteins. We aimed to isolate and characterize CRISP from turkey seminal plasma and evaluate its possible involvement in yellow semen syndrome (YSS). YSS, which is well characterized, causes reduced fertility and hatchability. The protein was purified using hydrophobic interaction, gel filtration, and reverse phase chromatography. It then was subjected to identification by mass spectrometry, analysis of physicochemical properties and specific antibody production. The biological function of the isolated protein was tested and included its effects on sperm motility and migration and sperm-egg interactions. Sperm motility was measured with the CASA system using Hobson Sperm Tracker. The reproductive tract of turkey toms was analyzed for gene expression; immunohistochemistry was used for protein localization in the male reproductive tract, spermatozoa, and inner perivitelline layer. The isolated protein was identified as cysteine-rich venom protein-like isoform X2 (CRVP X2; XP_010706464.1) and contained feature motifs of CRISP family proteins. Turkey CRVP X2 was present in both spermatozoa and seminal plasma. The extensive secretion of CRVP X2 by the epithelial cells of the epididymis and ductus deferens suggests its involvement in post-testicular sperm maturation. The internally localized CRVP X2 in the proximal part of the sperm tail might be responsible for stimulation of sperm motility. CRVP X2 on the sperm head might be involved in several events prior to fusion and may also participate in gamete fusion itself. Although the mechanisms by which CRPV X2 mediates fertilization are still unknown, the involvement of complementary sites cannot be excluded. The disturbance of CRVP X2 expression can serve as an etiologic factor of YSS in the turkey. This study expands the understanding of the detailed mechanism of fertilization in birds by clarifying the specific role of CRVP X2.

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