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- Atikuzzaman, Mohammad, 1977-
(författare)
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Seminal Influence on the Oviduct : Mating and/or semen components induce gene expression changes in the pre-ovulatory functional sperm reservoir in poultry and pigs
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Internal fertilization occurs in birds and eutherian mammals. Foetal development, however, is either extra- respectively intra-corpore (egg vs uterus). In these animal classes, the female genital tract stores ejaculated spermatozoa into a restricted oviductal segment; the functional pre-ovulatory sperm reservoir, where they survive until ovulation/s occur. Paradoxically, this immunologically foreign sperm suspension in seminal fluid/plasma, often microbiologically contaminated, ought to be promptly eliminated by the female local immune defence which, instead, tolerates its presence. The female immune tolerance is presumably signalled via a biochemical interplay of spermatozoa, as well as the peptides and proteins of the extracellular seminal fluid, with female epithelial and immune cells. Such interplay can result in gene expression shifts in the sperm reservoir in relation to variations in fertility. To further aid our understanding of the underlying mechanisms, this thesis studied the proteome of the seminal fluid (using 2D SDS-PAGE and mass spectrometry) including cytokine content (using Luminex and/or ELISA) of healthy, sexually mature and fertile boars and cocks. As well, gene expression changes (using cDNA microarray) in the oviductal sperm reservoirs of sexually-mature females, mated or artificially infused with homologous sperm-free seminal fluid/plasma were studied. Pigs were of commercial, fertility-selected modern breeds (Landrace), while chicken belonged to the ancestor Red Junglefowl (RJF, low egg laying-capacity), a selected egg-layer White Leghorn (WL) and of their Advanced Intercross Line (AIL). Ejaculates were manually collected as single sample in cocks or as the sperm-rich fraction [SRF] and the post- SRF fraction in boars to harvest seminal fluid/plasma for proteome/cytokine and infusion-studies. Oviducts were retrieved for gene-expression analyses via microarray immediately post-mortem (chicken) or at surgery (pig), 24 h after mating or genital infusion. In pigs, the protein-rich seminal plasma showed the highest amounts of cytokines [interferon-γ, interferon gamma-induced protein 10 (IP-10/CXCL10), macrophage derived chemokine (MDC/CCL22), growth-regulated oncogene (GRO/CXCL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemo-attractant protein-1 (MCP-1/ CCL2), interleukin (IL)-6, IL-8/CXCL8, IL-10, IL-15, IL-17 and transforming growth factor (TGF)-β1-3) in the larger, protein-rich and sperm-poor post-SRF, indicating its main immune signalling influence. Chicken showed also a plethora of seminal fluid proteins with serum albumin and ovotransferrin being conserved through selection/evolution. However, they showed fewer cytokines than pigs, as the anti-inflammatory/immune-modulatory TGF-β2 or the pro-inflammatory CXCL10. The RJF contained fewer immune system process proteins and lacked TGF-β2 compared to WL and AIL, suggesting selection for increased fertility could be associated with higher expression of immune-regulating peptides/proteins. The oviductal sperm reservoir reacted in vivo to semen exposure. In chicken, mating significantly changed the expression of immune-modulatory and pH-regulatory genes in AIL. Moreover, modern fertile pigs (Landrace) and chicken (WL), albeit being taxonomically distant, shared gene functions for preservation of viable sperm in the oviduct. Mating or SP/SF-infusion were able to change the expression of comparable genes involved in pH-regulation (SLC16A2, SLC4A9, SLC13A1, SLC35F1, ATP8B3, ATP13A3) or immune-modulation (IFIT5, IFI16, MMP27, ADAMTS3, MMP3, MMP12). The results of the thesis demonstrate that both mating and components of the sperm-free seminal fluid/plasma elicit gene expression changes in the pre-ovulatory female sperm reservoir of chickens and pigs, some conserved over domestication and fertility-selection.
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| 2. |
- Liffner, Susanne, 1974-
(författare)
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Infertility in Men in Relation to Their Birth Characteristics
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: About 10-15 % of couples worldwide suffer from infertility, the inability to achieve a pregnancy after more than 12 months of unprotected intercourse. Causes can be related to female factors, male factors, or both. In about 20 % of the couples, the cause remains unexplained. Male factor infertility is based on semen sample evaluation and is defined as poor semen quantity or quality, preferably in two or more samples. When assisted reproductive technology (ART) is considered for a couple with male factor infertility, intra-cytoplasmic sperm injection (ICSI) where the sperm is injected into the oocyte, is often needed to achieve embryo development.Men born small for gestational age (SGA), with low birth weight (LBW, £2,5000g) and/or prematurely (before the 37th gestational week) do not become fathers as often as men born with appropriate size for gestational age (AGA) at term. The aims of the present thesis were to investigate if this lower likelihood to become a father for men born SGA, preterm, or with low birth weight could be related to an increased risk of male factor infertility, if ICSI and sperm donation more often were needed and if the chance of conceiving by ART was lower than in men born AGA. An additional aim was to investigate if sperm DNA damage was more common in men born SGA compared with men born with normal size for gestational age.Material and Methods: Paper I and II were based on retrospective case control studies on men becoming fathers by ART in study I, and infertile men, patients at the Centre of Reproductive Medicine, University Hospital, Linköping, Sweden (RMC), in study II. Paper III was based on a cohort study on all men born in Sweden between 1973 and 1993. All three studies used data from national registers held by the National Board of Health and Welfare and Statistics Sweden. Study III also used information from the Swedish National Quality Register of Assisted Reproduction.Paper IV was based on a case series study of sperm DNA fragmentation in infertile patients and sperm donors at RMC, where two different methods of measuring sperm DNA fragmentation were compared, Halosperm® (easier to perform) and Sperm Chromatin Structure Assay (reference method). Paper V was based on a cohort study of men undergoing ART at RMC. The proportion of sperm with DNA fragmentation was compared between men born SGA and men born AGA.Results and conclusions: Men becoming fathers after ART were more often born with low birth weight than men conceiving naturally. Men becoming fathers after ICSI, presuming a poor semen sample, were more often born SGA than men conceiving by conventional IVF, or conceiving without treatment. ICSI had to be performed in 63% of men born SGA but only in 45% of men born AGA. Men with male factor infertility were more often born SGA or with LBW than men with female factor infertility, or when the infertility was unexplained. The results after ART were not affected by birth weight or size at birth.The likelihood of becoming a father was lower for men born SGA or with LBW compared with men born with normal birth weight. These men were also more often single (never married or with a registered partner) and had a higher risk of being born with undescended testicles, which increases the risk of infertility. Men born SGA more often needed ICSI or sperm donation.The two different methods to analyse sperm DNA fragmentation had high correlation and agreement, and Halosperm® could be considered a reliable and more easily handled method in the clinical setting. The median proportion of sperm with DNA fragmentation were higher for men born SGA (16.6%) compared with men born AGA (6.4%). The result was not statistically significant as the absolute number of men born SGA was much smaller than expected in this sample of 550 men. Larger studies are needed to confirm the hypothesis that DNA fragmentation contributes to a lower likelihood to become a father in men born SGA.
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| 3. |
- Martinez, Cristina A., et al.
(författare)
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Prenatal stress, anxiety and depression alter transcripts, proteins and pathways associated with immune responses at the maternal-fetal interface
- 2022
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Ingår i: Biology of Reproduction. - : Oxford University Press. - 0006-3363 .- 1529-7268. ; 106:3, s. 449-462
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Tidskriftsartikel (refereegranskat)abstract
- During pregnancy, the immune system is modified to allow developmental tolerance of the semi-allogeneic fetus and placenta to term. Pregnant women suffering from stress, anxiety, and depression show dysfunctions of their immune system that may be responsible for fetal and/or newborn disorders, provided that placental gene regulation is compromised. The present study explored the effects of maternal chronic self-perceived stress, anxiety, and depression during pregnancy on the expression of immune-related genes and pathways in term placenta. Pregnancies were clinically monitored with the Beck Anxiety Inventory (BAI) and Edinburgh Postnatal Depression Scale (EPDS). A cutoff threshold for BAI/EPDS of 10 divided patients into two groups: Index group (>10, n = 11) and a Control group (<10, n = 11), whose placentae were sampled at delivery. The placental samples were subjected to RNA-Sequencing, demonstrating that stress, anxiety, and depression during pregnancy induced a major downregulation of placental transcripts related to immune processes such as T-cell regulation, interleukin and cytokine signaling, or innate immune responses. Expression differences of main immune-related genes, such as CD46, CD15, CD8 alpha & beta ILR7 alpha, and CCR4 among others, were found in the Index group (P < 0.05). Moreover, the key immune-like pathway involved in humoral and cellular immunity named "Primary immunodeficiency" was significantly downregulated in the Index group compared with Controls. Our results show that mechanisms ruling immune system functions are compromised at the maternal-fetal interface following self-perceived depressive symptoms and anxiety during pregnancy. These findings may help unveil mechanisms ruling the impact of maternal psychiatric symptoms and lead to new prevention/intervention strategies in complicated pregnancies. Summary Sentence Mechanisms ruling immune system functions are compromised at the maternal-fetal interface following self-perceived depressive symptoms and anxiety during pregnancy.
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| 4. |
- Vicente Carrillo, Alejandro, 1989-
(författare)
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Sperm Membrane Channels, Receptors and Kinematics : Using boar spermatozoa for drug toxicity screening
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Internal fertilization usually implies that a spermatozoon, with intact attributes for zygote formation, passes all hurdles during its transport through the female genitalia and reaches the oocyte. During this journey, millions to billions of other spermatozoa perish. Spermatozoa are highly differentiated motile cells without synthetic capabilities. They generate energy via glycolysis and oxidative phosphorylation to sustain motility and to maintain the stability and functionality of their plasma membrane. In vivo, they spend their short lifespan bathing in female genital tract fluids of different origins, or are in vitro exposed to defined media during diverse sperm handling i.e. extension, cryopreservation, in vitro fertilization, etc. Being excitable cells, spermatozoa respond in vivo to various stimuli during pre-fertilization (capacitation, hyperactivation, oocyte location) and fertilization (acrosome reaction, interaction with the oocyte) events, mediated via diverse membrane ion-conducting channels and ligand-gated receptors. The present Thesis has mapped the presence and reactivity (sperm intactness and kinematics) of selected receptors, water and ion channels in ejaculated boar spermatozoa. The final aim was to find a relevant alternative cell type for in vitro bioassays that could ease the early scrutiny of candidate drugs as well as decreasing our needs for experimental animals according to the 3R principles. Spermatozoa are often extended, cooled and thawed to warrant their availability as fertile gametes for breeding or in vitro testing. Such manipulations stress the cells via osmotic variations and hence spermatozoa need to maintain membrane intactness by controlling the exchange of water and the common cryoprotectant glycerol, via aquaporins (AQPs). Both AQPs-7 and -9 were studied for membrane domain changes in cauda- and ejaculated spermatozoa (un-processed, extended, chilled or frozen-thawed). While AQP-9 maintained location through source and handling, thawing of ejaculated spermatozoa clearly relocated the labelling of AQP-7, thus appearing as a relevant marker for non-empirical studies of sperm cryopreservation. Alongside water, spermatozoa interact with calcium (Ca2+) via the main Ca2+ sperm channel CatSper. Increments in intracellular Ca2+ initiate motility hyperactivation and the acrosome reaction. The four subunits of the CatSper channel were present in boar spermatozoa, mediating changes in sperm motility under in vitro capacitation-inducing conditions (increased extracellular Ca2+ availability and bicarbonate) or challenge by the CatSper antagonists mibefradil and NNC 55-0396. Uterine and oviduct fluids are richest in endogenous opioids as β-endorphins during mating and ovulation. Both μ- and δ- opioid receptors were present in boar spermatozoa modulating sperm motility, as in vitro challenge with known agonists (μ: morphine; δ: DPDPE and κ: U 50488) and antagonists (μ: naloxone; δ: naltrindole and κ: nor-binaltrorphimine) showed that the μ-opioid receptor maintained or increased motility while the δ-opioid receptor mediated decreased motility over time. Finally, boar spermatozoa depicted dose-response effects on sperm kinematics and mitochondrial potential following in vitro challenge with 130 pharmacological drugs and toxic compounds as well as with eight known mito-toxic compounds. In conclusion, boar spermatozoa expressing functional water (AQPs-7 and -9) and ion (CatSper 1-4) channels as well as μ- and δ-opioid receptors are able to adapt to stressful environmental variations, capacitation and pharmacological compounds and drug components. Ejaculated sperm suspensions are easily and painlessly obtained from breeding boars, and are suitable biosensors for in vitro drug-induced testing, complying with the 3R principles of reduction and replacement of experimental animals, during early toxicology screening.
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