final maturation
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2022 ◽  
Author(s):  
Julia Puffal ◽  
Ian L. Sparks ◽  
James R. Brenner ◽  
Xuni Li ◽  
John D. Leszyk ◽  
...  

The intracellular membrane domain (IMD) is a metabolically active and laterally discrete membrane domain initially discovered in Mycobacterium smegmatis. The IMD correlates both temporally and spatially with the polar cell envelope elongation in M. smegmatis. Whether or not a similar membrane domain exists in pathogenic species remains unknown. Here we show that the IMD is a conserved membrane structure found in Mycobacterium tuberculosis. We used two independent approaches, density gradient fractionation of membrane domains and visualization of IMD-associated proteins through fluorescence microscopy, to determine the characteristics of the plasma membrane compartmentalization in M. tuberculosis. Proteomic analysis revealed that the IMD is enriched in metabolic enzymes that are involved in the synthesis of conserved cell envelope components such as peptidoglycan, arabinogalactan, and phosphatidylinositol mannosides. Using a fluorescent protein fusion of IMD-associated proteins, we demonstrated that this domain is concentrated in the polar region of the rod-shaped cells, where active cell envelope biosynthesis is taking place. Proteomic analysis further revealed the enrichment of enzymes involved in synthesis of phthiocerol dimycocerosates and phenolic glycolipids in the IMD. We validated the IMD association of two enzymes, α1,3-fucosyltransferase and fucosyl 4-O-methyltransferase, which are involved in the final maturation steps of phenolic glycolipid biosynthesis. Taken together, these data indicate that functional compartmentalization of membrane is an evolutionarily conserved feature found in both M. tuberculosis and M. smegmatis, and M. tuberculosis utilizes this membrane location for the synthesis of its surface-exposed lipid virulence factors.


2021 ◽  
pp. 167397
Author(s):  
Adrian Drazic ◽  
Evy Timmerman ◽  
Ulrike Kajan ◽  
Michaël Marie ◽  
Sylvia Varland ◽  
...  

2021 ◽  
Author(s):  
◽  
Zaramasina Clark

<p>The number of cycles of assisted reproductive technologies (ART) performed increased by ~9.5 % globally between 2008 and 2010. In spite of this, the success rate in terms of delivery was only ~19.0 % (Dyer et al., 2016). This discrepancy between the demand for, and success of, these technologies necessitates the development of tools to improve ART efficiency. To facilitate this, a better understanding of how the microenvironment changes within the developing follicle to culminate in a mature, developmentally-competent oocyte is required. This study employed an in vivo and in vitro ovine model to investigate the relationship between the surrounding microenvironment and oocyte maturation, and in particular, the attainment of oocyte developmental competency and high-quality embryos.  The first objective of this PhD study was to comprehensively investigate the changing microenvironment of in vivo matured, presumptive preovulatory (PPOV) follicles from wild-type (++) and high ovulation rate (OR; I+B+) ewes. The high OR ewes were heterozygous carriers of mutations in BMP15 (I+) and BMPRIB (B+). Functional differences in follicular somatic (granulosa and cumulus) cells between these genotypes, including differential gonadotropin responsiveness of granulosa cells, composition of follicular fluid and gene expression profiles in cumulus cells were evident. These differences emerged as part of a compensatory mechanism by which oocytes from smaller follicles, containing fewer granulosa cells, achieved developmental competency in I+B+ ewes.  The second objective of this PhD study was to develop new approaches for improving current in vitro maturation (IVM) strategies. The first approach utilised in this study focused on developing biomarkers that could be used to improve prediction of developmental competency in oocytes and in vitro produced embryos. This involved interrogating the hypothesis that a combination of molecular and morphokinetic biomarkers would better predict the developmental competency of oocytes and embryos compared to using these biomarkers alone. The second approach utilised in this PhD study tested the effects of modulating IVM conditions to better mimic the follicular microenvironment of a high, compared to a low, OR species on oocyte developmental competency and embryo quality. This involved supplementing IVM media with different ratios of two oocyte-secreted growth factors, i.e. GDF9:BMP15, that were representative of low or high OR species. These approaches demonstrated significant potential and warrant further investigation.  The most significant finding of this study was that despite variances in the surrounding microenvironment during in vivo and in vitro oocyte maturation that culminated in differential gene expression patterns in cumulus cells, and divergent gonadotropin-responsiveness of granulosa cells, the gene expression signatures of developmentally-competent oocytes and the morphokinetics of high-quality embryos were unaltered. This confirms the value of developing such biomarkers for oocyte development competency and embryo quality that remain unaltered despite a changing surrounding environment. Interestingly, simulating the ratio of GDF9:BMP15 that oocytes from high OR species are exposed to during maturation improved developmental competency in oocytes as demonstrated by increased blastocyst rates. Furthermore, this study has demonstrated that combinations of molecular (cumulus cell gene expression) and morphokinetic biomarkers improved the ability to predict developmental competency in oocytes and embryos. Overall, this study revealed novel information regarding the follicular microenvironment during final maturation and identified several novel approaches to improving the efficiency of ART.</p>


2021 ◽  
Author(s):  
◽  
Zaramasina Clark

<p>The number of cycles of assisted reproductive technologies (ART) performed increased by ~9.5 % globally between 2008 and 2010. In spite of this, the success rate in terms of delivery was only ~19.0 % (Dyer et al., 2016). This discrepancy between the demand for, and success of, these technologies necessitates the development of tools to improve ART efficiency. To facilitate this, a better understanding of how the microenvironment changes within the developing follicle to culminate in a mature, developmentally-competent oocyte is required. This study employed an in vivo and in vitro ovine model to investigate the relationship between the surrounding microenvironment and oocyte maturation, and in particular, the attainment of oocyte developmental competency and high-quality embryos.  The first objective of this PhD study was to comprehensively investigate the changing microenvironment of in vivo matured, presumptive preovulatory (PPOV) follicles from wild-type (++) and high ovulation rate (OR; I+B+) ewes. The high OR ewes were heterozygous carriers of mutations in BMP15 (I+) and BMPRIB (B+). Functional differences in follicular somatic (granulosa and cumulus) cells between these genotypes, including differential gonadotropin responsiveness of granulosa cells, composition of follicular fluid and gene expression profiles in cumulus cells were evident. These differences emerged as part of a compensatory mechanism by which oocytes from smaller follicles, containing fewer granulosa cells, achieved developmental competency in I+B+ ewes.  The second objective of this PhD study was to develop new approaches for improving current in vitro maturation (IVM) strategies. The first approach utilised in this study focused on developing biomarkers that could be used to improve prediction of developmental competency in oocytes and in vitro produced embryos. This involved interrogating the hypothesis that a combination of molecular and morphokinetic biomarkers would better predict the developmental competency of oocytes and embryos compared to using these biomarkers alone. The second approach utilised in this PhD study tested the effects of modulating IVM conditions to better mimic the follicular microenvironment of a high, compared to a low, OR species on oocyte developmental competency and embryo quality. This involved supplementing IVM media with different ratios of two oocyte-secreted growth factors, i.e. GDF9:BMP15, that were representative of low or high OR species. These approaches demonstrated significant potential and warrant further investigation.  The most significant finding of this study was that despite variances in the surrounding microenvironment during in vivo and in vitro oocyte maturation that culminated in differential gene expression patterns in cumulus cells, and divergent gonadotropin-responsiveness of granulosa cells, the gene expression signatures of developmentally-competent oocytes and the morphokinetics of high-quality embryos were unaltered. This confirms the value of developing such biomarkers for oocyte development competency and embryo quality that remain unaltered despite a changing surrounding environment. Interestingly, simulating the ratio of GDF9:BMP15 that oocytes from high OR species are exposed to during maturation improved developmental competency in oocytes as demonstrated by increased blastocyst rates. Furthermore, this study has demonstrated that combinations of molecular (cumulus cell gene expression) and morphokinetic biomarkers improved the ability to predict developmental competency in oocytes and embryos. Overall, this study revealed novel information regarding the follicular microenvironment during final maturation and identified several novel approaches to improving the efficiency of ART.</p>


Author(s):  
Nabaa Afeef A. AL-Nawab ◽  
Israa Hasan Ali ◽  
Lobna Kadim Jassim AL-Khafaji

Infertility can be detected when the couples have not completed pregnancy after a year or more of normal coitus. So, in order to treat infertility, there are many supported reproductive techniques are in practice. The success rate of these techniques depends upon the way by which preparation of the paternal semen sample. Over the past 30 years, the manual has been standard as providing global standards and has been used extensively by research and clinical laboratories throughout the world. The spermatozoa of all placental (eutherian) mammals, including humans, are in a protective, no labile formal at ejaculation and are incapable of fertilization even if they are placed in direct contact with an oocyte. Accordingly, they must undergo a subsequent period of final maturation. during which they acquire the capacity to interact with the oocyte cumulus complex and achieve fertilization. In this article we tried to explain the most important analysis and techniques necessary for semen preparation to produce healthy and normal offspring.


2021 ◽  
Vol 22 (3) ◽  
pp. 466
Author(s):  
STYLIANOS SOMARAKIS ◽  
MARIA GIANNOULAKI ◽  
KONSTANTINOS MARKAKIS ◽  
KOSTAS TSIARAS ◽  
EUDOXIA SCHISMENOU ◽  
...  

The Golani’s round herring Etrumeus golanii is an Erythraean small pelagic fish (lessepsian migrant) that entered into the Mediterranean Sea through the Suez Canal. It has expanded its distribution from the east to the western Mediterranean with well-established local populations. We investigated basic aspects of its reproductive biology off the island of Crete (eastern Mediterranean) using ovarian histology and analysis of oocyte size-frequency distributions. The species exhibited a protracted breeding period (winter to early summer), with all ovaries examined during the main spawning season having markers of recent (postovulatory follicles, POFs) or imminent spawning (advanced oocyte batch in germinal vesicle migration or hydration). The advanced batch (AB) increased rapidly in size and was fully separated from the remainder, less developed oocytes in 95% of females with “old” POFs (POFs with signs of degeneration) and all females in final maturation. The growth of the subsequent batch (SB) was arrested at sizes <630 µm until full maturation of the AB. Mean diameter of hydrated oocytes ranged from 1181 to 1325 µm and relative batch fecundity was low ranging from 56 to 157 eggs g-1. The simulation of a coupled hydrodynamic/biogeochemical model (POM/ERSEM) provided evidence that E. golanii takes advantage of the seasonal cycle of planktonic production to reproduce and exhibits monthly changes in batch fecundity that appear to be closely related with the seasonal cycle of mesozooplankton concentration.     


GYNECOLOGY ◽  
2021 ◽  
Vol 23 (3) ◽  
pp. 230-235
Author(s):  
Zaira K. Kumykova ◽  
Elena V. Uvarova ◽  
Zalina K. Batyrova

The article presents an overview of the physiological role of anti-Mullerian hormone (AMH) during persons lifetime, the fluctuations of its values from birth to the reproductive period in women, the significance of its determination in the diagnosis and prognosis of treatment outcomes for many gynecological diseases. AMH is produced mainly by preantral and early antral follicles and decreases during the final maturation and luteinization. AMH plays a potential role in preserving the ovarian reserve by performing dual actions. AMH inhibits the initial recruitment of follicles, preventing the influence of stimulating growth factors for recruitment (KIT-ligand, the main fibroblast growth factor). From the moment of puberty, AMH reduces the sensitivity of primary follicles to the follicle-stimulating hormone, reducing the likelihood of their cyclic recruitment. The systematic review data presented the characteristics of the ovarian reserve in a healthy female population aged 0 to 19 years. At birth, very low AMH values were noted with an increase in its level in the first 3 months of life, followed by a monthly increase of 31%. A significant increase in AMH secretion was revealed by the beginning of puberty, then most authors noted a constant level of serum AMH in the adolescent period. The level of AMH in the blood serum decreases with an increase in chronological age by 68% annually and varies during the menstrual cycle. AMH is an important diagnostic tool in pediatric practice when differentiating various causes of puberty disorders, virilization and menstrual cycle disorders in girls.


2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
J Garratt ◽  
B Raikundalia ◽  
M Rimington ◽  
K Ahuja ◽  
N Macklon ◽  
...  

Abstract Study question Which clinical parameters predict a high oocyte maturation rate in patients undergoing IVF treatment? Summary answer Time between oocyte collection and insemination demonstrated significant association with oocyte maturation and represents a parameter that could be optimised in IVF cycles. What is known already Oocyte maturation is an important factor determining IVF outcomes and can be a rate-limiting step for patients undergoing treatment. A number of clinical and laboratory variables may affect this process, including the choice of trigger prior to oocyte collection, and certain laboratory procedures. Identification of which of these are predictors of maturation in individual centres enables local protocols to be optimised. Study design, size, duration This is a retrospective study of 714 oocyte collections from 661 women between January 2020 to November 2020 treated in a large, single centre in the UK. Subsequent fertilisation on fresh oocytes consisted of 371 IVF and 343 ICSI cycles. Participants/materials, setting, methods Patient and treatment data was collected by clinical staff at time of treatment. Either GnRH agonist, hCG or double trigger were administered 36 hours before collection. Prior to ICSI, oocyte maturation was assessed by visualisation of polar body (PB) extrusion. After IVF, the number of 2PNs plus unfertilised oocytes with PB extrusion were assessed. Univariate analyses consisted of Mann-Whitney test, t-test, Fisher’s Exact test or ANOVA. Potential predictors were investigated by logistic regression. Main results and the role of chance The end point was maturation rate, defined as high (greater or equal to 70%) or low (less than 70%). Factors predictive of a high rate included insemination more than 4 hours after collection. Oocytes inseminated over 4 hours post-collection displayed significantly higher maturation rates than oocytes inseminated less than 2 hours after collection (69% and 61% respectively; P = 0.01). Oocytes inseminated between 2–4 hours also had higher maturation than those inseminated less than 2 hours post-collection, but this did not reach significance (67% and 61%, respectively; P = 0.06). Further, oocytes fertilised by ICSI had significantly higher maturation than conventional IVF (77% and 67%, respectively, P &lt; 0.001). No significant difference in oocyte maturation between triggers was observed. Similarly, neither age, AMH, a diagnosis of PCOS or number of oocytes collected predicted oocyte maturation in univariate analysis. Logistic regression analysis showed only time between oocyte collection and insemination (aOR 2.12; 95% CI 1.03–4.38; P = 0.04) to be a significant independent predictor. Limitations, reasons for caution Varying means of data collection across clinics and between clinical staff inevitably leads to provision of incomplete data and should be taken into consideration alongside interpretation. Prescription bias of specific triggers to certain patient demographics should be noted. Wider implications of the findings: Collectively, these results suggest that greater time between oocyte collection and insemination could be recommended to IVF clinics that wish to optimise their oocyte maturation. Triggering final maturation with GnRH agonist versus hCG or dual trigger did not have a significant effect on oocyte maturation when adjusted for confounders. Trial registration number Not applicable


2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A C Herta ◽  
L Vo. Mengden ◽  
N Akin ◽  
K Billooye ◽  
J Va. Leersum ◽  
...  

Abstract Study question Are there significant differences in carbohydrate metabolism trends between in vivo and in vitro grown mouse antral follicles during oocyte final maturation? Summary answer Glucose metabolism characterization during GV to MII transition revealed altered metabolic patterns mainly in cumulus cells of in vitro grown and matured mouse antral follicles. What is known already For some cancer patients fertility restoration is dependent on using efficient in vitro follicle culture systems. As human donor ovarian tissue available for research is limited, establishing such culture systems relies on data generated from animal models. The culture system previously developed in our laboratory supports in vitro growth of mouse preantral follicles with good oocyte maturation rates but lower developmental competence compared to in vivo grown oocytes. Tracking and comparing the metabolic changes after meiotic maturation in in vitro and in vivo follicles could serve as a screening tool for improving culture conditions and identifying metabolic quality markers. Study design, size, duration Mouse secondary follicle culture was performed. In vitro grown oocytes, their corresponding cumulus (CC) and granulosa cells (GC) were collected from antral follicles, at germinal vesicle stage (GV) on day 9, and at metaphase 2 (MII) on day 10, after hCG/EGF stimulation. In vivo age-matched controls were obtained after intraperitoneal injections with eCG for GV, or with eCG and hCG for MII. In vivo GC after ovulation were not included. Participants/materials, setting, methods Glucose metabolism trends were compared during final maturation between in vitro grown antral follicles and their in vivo controls. Follicles that failed to resume meiosis in vitro were also included. Enzymatic spectrophotometric assays were used to measure glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, and the antioxidant capacity in individual cell types. Pools of 5 oocytes and corresponding somatic cells were collected, from 3 independent experiments. Unpaired t-test was performed with significance when p &lt; 0.05. Main results and the role of chance Important differences were detected between in vivo and in vitro conditions. GV to MII transition in in vivo follicles leads to a metabolic boost in CC as indicated by: i. significant increase in glycolysis, PPP and TCA cycle activity; ii. higher total antioxidant capacity (TAC) (p &lt; 0.05) and small molecule antioxidant capacity (SMAC) (p &lt; 0.01). After ovulation, the only significant change in oocytes was an increase in nicotinamide adenine dinucleotide phosphate (NADP+) level (p &lt; 0.01), possibly due to increased reduced-NADP recycling. Meiotic maturation triggered no significant differences in any of the metabolic pathways for in vitro grown oocytes. Contrary to their in vivo controls, in vitro CC showed significant upregulations limited to aconitase, lactate dehydrogenase (LDH) and glutathione-s-transferase (GST) activity (p &lt; 0.05). In vitro GC showed increased G6PDH activity (p &lt; 0.05), suggesting PPP upregulation. Significant differences were detected between in vivo GV follicles and the in vitro failed-to-mature ones. Oocytes from impaired follicles have higher NADP+ levels (p &lt; 0.0001) than their in vivo immature counterparts. CC showed higher phosphofructokinase (PFK), LDH, catalase activity and increased NADP + (p &lt; 0.01), TAC and SMAC (p &lt; 0.05) compared to in vivo GV CCs. GCs from failed-to-mature follicles have significantly higher LDH and superoxide dismutase (SOD) activity than in vivo GV GC (p &lt; 0.05). Limitations, reasons for caution The altered metabolic patterns described here in in vitro follicles during oocyte GV to MII transition are probably the cumulative effects of both growth and maturation in vitro. Wider implications of the findings: We explored extensively and directly, for the first time, several enzymes and metabolites involved in follicle glucose and redox metabolism in different cell types separately. Understanding of the follicle metabolic requirements is essential for the optimization of follicle culture systems and could lead to development of oocyte quality markers. Trial registration number Not applicable


2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Eli Sellem ◽  
Sylvain Marthey ◽  
Andrea Rau ◽  
Luc Jouneau ◽  
Aurelie Bonnet ◽  
...  

Abstract Background During epididymal transit, spermatozoa go through several functional maturation steps, resulting from interactions with epididymal secretomes specific to each region. In particular, the sperm membrane is under constant remodeling, with sequential attachment and shedding of various molecules provided by the epididymal lumen fluid and epididymosomes, which also deliver sncRNA cargo to sperm. As a result, the payload of sperm sncRNAs changes during the transit from the epididymis caput to the cauda. This work was designed to study the dynamics of cattle sperm sncRNAs from spermatogenesis to final maturation. Results Comprehensive catalogues of sperm sncRNAs were obtained from testicular parenchyma, epididymal caput, corpus and cauda, as well as ejaculated semen from three Holstein bulls. The primary cattle sncRNA sperm content is markedly remodeled as sperm mature along the epididymis. Expression of piRNAs, which are abundant in testis parenchyma, decreases dramatically at epididymis. Conversely, sperm progressively acquires miRNAs, rsRNAs, and tsRNAs along epididymis, with regional specificities. For instance, miRNAs and tsRNAs are enriched in epididymis cauda and ejaculated sperm, while rsRNA expression peaks at epididymis corpus. In addition, epididymis corpus contains mainly 20 nt long piRNAs, instead of 30 nt in all other locations. Beyond the bulk differences in abundance of sncRNAs classes, K-means clustering was performed to study their spatiotemporal expression profile, highlighting differences in specific sncRNAs and providing insights into their putative biological role at each maturation stage. For instance, Gene Ontology analyses using miRNA targets highlighted enriched processes such as cell cycle regulation, response to stress and ubiquitination processes in testicular parenchyma, protein metabolism in epididymal sperm, and embryonic morphogenesis in ejaculated sperm. Conclusions Our findings confirm that the sperm sncRNAome does not simply reflect a legacy of spermatogenesis. Instead, sperm sncRNA expression shows a remarkable level of plasticity resulting probably from the combination of multiple factors such as loss of the cytoplasmic droplet, interaction with epididymosomes, and more surprisingly, the putative in situ production and/or modification of sncRNAs by sperm. Given the suggested role of sncRNA in epigenetic trans-generational inheritance, our detailed spatiotemporal analysis may pave the way for a study of sperm sncRNAs role in embryo development.


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