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- Godakumara, K, et al.
(författare)
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Trophoblast derived extracellular vesicles specifically alter the transcriptome of endometrial cells and may constitute a critical component of embryo-maternal communication
- 2021
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Ingår i: Reproductive biology and endocrinology : RB&E. - : Springer Science and Business Media LLC. - 1477-7827. ; 19:1, s. 115-
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundThe period of time when the embryo and the endometrium undergo significant morphological alterations to facilitate a successful implantation—known as “window of implantation”—is a critical moment in human reproduction. Embryo and the endometrium communicate extensively during this period, and lipid bilayer bound nanoscale extracellular vesicles (EVs) are purported to be integral to this communication.MethodsTo investigate the nature of the EV-mediated embryo-maternal communication, we have supplemented trophoblast analogue spheroid (JAr) derived EVs to an endometrial analogue (RL 95–2) cell layer and characterized the transcriptomic alterations using RNA sequencing. EVs derived from non-trophoblast cells (HEK293) were used as a negative control. The cargo of the EVs were also investigated through mRNA and miRNA sequencing.ResultsTrophoblast spheroid derived EVs induced drastic transcriptomic alterations in the endometrial cells while the non-trophoblast cell derived EVs failed to induce such changes demonstrating functional specificity in terms of EV origin. Through gene set enrichment analysis (GSEA), we found that the response in endometrial cells was focused on extracellular matrix remodelling and G protein-coupled receptors’ signalling, both of which are of known functional relevance to endometrial receptivity. Approximately 9% of genes downregulated in endometrial cells were high-confidence predicted targets of miRNAs detected exclusively in trophoblast analogue-derived EVs, suggesting that only a small proportion of reduced expression in endometrial cells can be attributed directly to gene silencing by miRNAs carried as cargo in the EVs.ConclusionOur study reveals that trophoblast derived EVs have the ability to modify the endometrial gene expression, potentially with functional importance for embryo-maternal communication during implantation, although the exact underlying signalling mechanisms remain to be elucidated.
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- Hallap, T, et al.
(författare)
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Changes in semen quality in Estonian Holstein AI bulls at 3, 5 and 7 years of age
- 2006
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Ingår i: Reproduction in domestic animals. - : Wiley-Blackwell. - 0936-6768 .- 1439-0531. ; 41:3, s. 214-218
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Tidskriftsartikel (refereegranskat)abstract
- The predictability of semen quality of mature sires from measurements at an early age is not well established. The aim of the present study was to determine age-dependent changes in the quality of bull semen from six Estonian Holstein (EHF) bulls, processed when the sires were 3, 5 and 7 years old. Fertility data such as 60-day non-return to oestrus rates (60d-NRRs) were available for 3-year-old bulls. From each batch, semen straws were analysed immediately after thawing [i.e. post-thaw (PT)] (controls) and after a swim-up (SU) procedure. The analyses comprised subjective and computerized measurements of sperm motility using computer-assisted sperm analysis (CASA) as well as estimations of sperm concentration, morphology and membrane integrity. There was a significant (p less than 0.05) increase in the percentage of sperm motility (SU), membrane integrity (PT, SU) and normal tail and acrosome morphology (SU) with an increase in the age of the sires. The percentage of total motile spermatozoa PT measured by CASA correlated between 3- and 7-, and between 5- and 7-year-old bulls (p less than 0.05). In addition, the proportion of head abnormalities tended to correlate between all three age groups both PT and after SU (p less than 0.1). The sperm parameters correlating with fertility were average path velocity (VAP) (p less than 0.001), total motility as measured by CASA (p less than 0.01) linearly motile spermatozoa (p less than 0.05) and CASA-assessed numbers of motile spermatozoa (p less than 0.05), all after SU selection. The results showed that overall semen quality examined at 3 years of age is related to the semen parameters later in bulls life. Moreover, CASA-assessed motility after SU seems to be a reliable marker for semen quality assessment as it shows correlation not only between the ages, but also to field fertility.
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- Hallap, T, et al.
(författare)
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Mitochondrial activity of frozen-thawed spermatozoa assessed by Mitotracker Deep Red 633
- 2005
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Ingår i: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 63:8, s. 2311-2322
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Tidskriftsartikel (refereegranskat)abstract
- The present study was conducted to find a more objective method of evaluating sperm quality than the current subjective motility evaluations by testing the applicability of a novel fluorescent probe, Mitotracker Deep Red 633 (M-22426), for measuring the mitochondrial activity of spermatozoa both after freezing/thawing (PT) and after swim-up selection (SU), using flow cytometry (FC). The results from FC were compared to those of conventional microscopic motility evaluations and of computer-assisted sperm analysis (CASA) as well as to the fertility obtained after AI in the field. Semen from six Estonian Holstein bulls, processed when the sires were aged 3, 5, and 7 years, was included in the experiment. Sperm motility (measured either subjectively or by means of CASA) was always significantly (p less than 0.01-0.001) higher in the spermatozoa recovered by SU, for any of the age groups considered, or even when combining the age groups. Motility (measured subjectively) after SU was significantly (p less than 0.05) higher when bulls reached 7 years of age, compared to earlier collection ages, but no differences were registered between ages for CASA-assessed motility, either after SU or immediately PT. The numbers of spermatozoa. with high red fluorescence also increased after SU: p less than 0.05 (for semen from bulls aged 3 years), p less than 0.001 (5 years), p less than 0.001 (7 years), and p less than 0.001 when all age groups were combined. The proportion of spermatozoa with high mitochondrial activity as determined by Mitotracker Deep Red 633 correlated with sperm motility (p less than 0.01) both PT and after SU, but not with the fertility results. In conclusion, MitoTracker Deep Red 633 seems to be a reliable marker for frozen-thawed bovine semen viability both PT and after SU. (c) 2004 Elsevier Inc. All rights reserved.
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- Hallap, T, et al.
(författare)
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Usefulness of a triple fluorochrome combination Merocyanine 540/Yo-Pro 1/Hoechst 33342 in assessing membrane stability of viable frozen-thawed spermatozoa from Estonian Holstein Al bulls
- 2006
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Ingår i: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 65:6, s. 1122-1136
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Tidskriftsartikel (refereegranskat)abstract
- In a situation where technology allows for the simultaneous measurement of numerous parameters of a single sperm cell, it becomes crucial to choose those parameters which may be useful in estimating in vivo fertility. Sperm membrane destabilization is believed to occur during chilling of semen, although its effect on the post-thaw (PT) fertility of the spermatozoa has not yet been fully assessed. For this reason, we tested a new combination of fluorophores, Merocyanine 540 (M540)/Yo-Pro 1/Hoechst 33342 (H33342), to detect sperm plasma membrane destabilization in bull spermatozoa conventionally processed for artificial insemination (AI). The samples were tested by flow cytometry (FC), both immediately PT and following an in vitro swimup (SU) technique, and results were thereafter compared with conventional sperm quality Measurements (of concentration, motility, morphology, and membrane integrity), including in vivo fertility. Semen samples from six Estonian Holstein (EHF) AI bulls, frozen when the sires were aged 3, 5, and 7 years, allowed us to test the effect of bull age on quality of semen. Plasma membrane stability correlated to motility, normal head morphology (p less than 0.05), and membrane integrity (p less than 0.01). Following the SU selection, motility, membrane integrity (p less than 0.001). and membrane instability increased (p less than 0.01), as did stability (p less than 0.05). Bull age did not influence the degree of sperm membrane destabilization, except for the 3-year sample versus 7-year sample, in which the proportion of spermatozoa with destabilized plasma lemma increased PT (p less than 0.05) without affecting membrane integrity. Only parameters measured after SU, such as proportion of total motile and linearly motile spermatozoa, assessed with computer-assisted sperm analysis (CASA) (p less than 0.01), average path velocity (VAP) (p less than 0.001), and percentage of spermatozoa with unstable plasma lemma (p less than 0.05), had a significant relationship with non-return rate (NRR). The results indicate that it triple combination of the fluorophores M540/-Pro 1/H33342 is suitable for monitoring the status of membrane stability in frozen-thawed (FT) bull spermatozoa. As well, a SU preselection method seems helpful in distinguishing relationships between sperm quality and fertility among bulls in it homogenous sire population.(c) 2005 Elsevier Inc. All rights reserved.
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