FSH withdrawal improves developmental competence of oocytes in the bovine model

Combinations of genetic, environmental, and management factors are suspected to explain the loss in fertility observed for over 20 years in dairy cows. In some cases, IVF is used. When compared with in vivo embryo production, IVF resulted in low success rates until the FSH coasting process (FSH starvation after superstimulation) was introduced in 2002. Increased competence associated with FSH withdrawal of aspirated oocyte for in vitro maturation and IVF has not been optimized nor explained yet. The goal here was to determine and characterize the optimal oocyte competence acquisition window during the coasting period by determining blastocyst rates and follicular cohort development. Commercial milking cycling cows (n=6) were stimulated with 3 days of FSH (6×40 mg NIH Folltropin-V given at 12 h intervals) followed by a coasting period of 20, 44, 68, or 92 h. Each animal was exposed to the four conditions and served as its own control. At the scheduled time, transvaginal aspirations of immature oocytes were performed followed by IVF of half the oocytes. The outcomes were as follows: i) FSH coasting was optimal at a defined period: between 44 and 68 h of coasting; ii) The best estimated coasting duration was ∼54±7 h; iii) Under these conditions, the best statistical blastocyst rate estimation was ∼70%; iv) Between 44 and 68 h of coasting, follicle size group proportions were similar; v) Follicle diameter was not linearly associated with competence. In conclusion, coasting duration is critical to harvest the oocytes at the right moment of follicular differentiation.

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Maturation of human oocytes in vitro and their developmental competence

Reproduction ◽

10.1530/rep.0.1210051 ◽

2001 ◽

pp. 51-75 ◽

Cited By ~ 221

Author(s):

A Trounson ◽

C Anderiesz ◽

G Jones

Keyword(s):

Embryo Development ◽

Oocyte Maturation ◽

Growth Phase ◽

Developmental Competence ◽

Minimum Size ◽

Success Rates ◽

Human Oocytes ◽

Maturation In Vitro

Complete maturation of oocytes is essential for the developmental competence of embryos. Any interventions in the growth phase of the oocyte and the follicle in the ovary will affect oocyte maturation, fertilization and subsequent embryo development. Oocyte size is associated with maturation and embryo development in most species examined and this may indicate that a certain size is necessary to initiate the molecular cascade of normal nuclear and cytoplasmic maturation. The minimum size of follicle required for developmental competence in humans is 5-7 mm in diameter. Maturation in vitro can be accomplished in humans, but is associated with a loss of developmental competence unless the oocyte is near completion of its preovulatory growth phase. This loss of developmental competence is associated with the absence of specific proteins in oocytes cultured to metaphase II in vitro. The composition of culture medium used successfully for maturation of human oocytes is surprisingly similar to that originally developed for maturation of oocytes in follicle culture in vitro. The presence of follicle support cells in culture is necessary for the gonadotrophin-mediated response required to mature oocytes in vitro. Gonadotrophin concentration and the sequence of FSH and FSH-LH exposure may be important for human oocytes, particularly those not exposed to the gonadotrophin surge in vivo. More research is needed to describe the molecular and cellular events, the presence of checkpoints and the role of gene expression, translation and protein uptake on completing oocyte maturation in vitro and in vivo. In the meantime, there are very clear applications for maturing oocytes in human reproductive medicine and the success rates achieved in some of these special applications are clinically valuable.

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Effects of in vitro maturation on gene expression in rhesus monkey oocytes

Physiological Genomics ◽

10.1152/physiolgenomics.90281.2008 ◽

2008 ◽

Vol 35 (2) ◽

pp. 145-158 ◽

Cited By ~ 37

Author(s):

Young S. Lee ◽

Keith E. Latham ◽

Catherine A. VandeVoort

Keyword(s):

Oocyte Maturation ◽

Nonhuman Primates ◽

Cdna Array ◽

Cell Types ◽

Cell Interactions ◽

Developmental Competence ◽

Great Promise ◽

Success Rates

In vitro oocyte maturation (IVM) holds great promise as a tool for enhancing clinical treatment of infertility, enhancing availability of nonhuman primates for development of disease models, and facilitating endangered species preservation. However, IVM outcomes have remained significantly below the success rates obtained with in vivo matured (VVM) oocytes from humans and nonhuman primates. A cDNA array-based analysis is presented, comparing the transcriptomes of VVM oocytes with IVM oocytes. We observe a small set of just 59 mRNAs that are differentially expressed between the two cell types. These mRNAs are related to cellular homeostasis, cell-cell interactions including growth factor and hormone stimulation and cell adhesion, and other functions such as mRNA stability and translation. Additionally, we observe in IVM oocytes overexpression of PLAGL1 and MEST, two maternally imprinted genes, indicating a possible interruption or loss of correct epigenetic programming. These results indicate that, under certain IVM conditions, oocytes that are molecularly highly similar to VVM oocytes can be obtained; however, the interruption of normal oocyte-somatic cell interactions during the final hours of oocyte maturation may preclude the establishment of full developmental competence.

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Effect of follicle size on mRNA expression in cumulus cells and oocytes of Bos indicus: an approach to identify marker genes for developmental competence

Reproduction Fertility and Development ◽

10.1071/rd08201 ◽

2009 ◽

Vol 21 (5) ◽

pp. 655 ◽

Cited By ~ 60

Author(s):

Ester Siqueira Caixeta ◽

Paula Ripamonte ◽

Maurício Machaim Franco ◽

José Buratini Junior ◽

Margot Alves Nunes Dode

Keyword(s):

Gene Expression ◽

Bos Indicus ◽

Germinal Vesicle ◽

Cumulus Cells ◽

Developmental Competence ◽

Marker Genes ◽

Oocyte Competence ◽

Follicular Size ◽

Follicle Size

To identify the genes related to oocyte competence, we quantified transcripts for candidate genes in oocytes (H1Foo, H2A, H3A, GHR, GDF9, BMP15, OOSP1) and cumulus cells (FSHR, EGFR, GHR, PTX3, IGFII) using the follicle size model to select oocytes of better developmental quality. Follicles were dissected and distributed into four groups according to diameter as follows: 1.0–3.0, 3.1–6.0, 6.1–8.0 and ≥8.1 mm. Cumulus–oocyte complexes (COCs) were released, classified morphologically, matured, fertilised and cultured in vitro or denuded for measurement of diameter and determination of gene expression. Denuded germinal vesicle oocytes and their cumulus cells were used for gene expression analysis by reverse transcription–polymerase chain reaction. The blastocyst rate was highest for oocytes recovered from follicles >6 mm in diameter. In the oocyte, expression of the H2A transcript only increased gradually according to follicle size, being greater (P < 0.05) in oocytes from follicles ≥8.1 mm in diameter than in oocytes from follicles <6.0 mm in diameter. In cumulus cells, expression of FSHR, EGFR and GHR mRNA increased with follicular size. In conclusion, we confirmed the importance of H2A for developmental competence and identified important genes in cumulus cells that may be associated with oocyte competence.

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Failure to launch: aberrant cumulus gene expression during oocyte in vitro maturation

Reproduction ◽

10.1530/rep-16-0426 ◽

2017 ◽

Vol 153 (3) ◽

pp. R109-R120 ◽

Cited By ~ 16

Author(s):

Hannah M Brown ◽

Kylie R Dunning ◽

Melanie Sutton-McDowall ◽

Robert B Gilchrist ◽

Jeremy G Thompson ◽

...

Keyword(s):

Gene Expression ◽

Cell Metabolism ◽

Cumulus Cell ◽

Cumulus Cells ◽

Developmental Competence ◽

Oocyte Competence ◽

Oocyte Developmental Competence ◽

Growth Factor Signalling

In vitro maturation (IVM) offers significant benefits for human infertility treatment and animal breeding, but this potential is yet to be fully realised due to reduced oocyte developmental competence in comparison with in vivo matured oocytes. Cumulus cells occupy an essential position in determining oocyte developmental competence. Here we have examined the areas of deficient gene expression, as determined within microarrays primarily from cumulus cells of mouse COCs, but also other species, between in vivo matured and in vitro matured oocytes. By retrospectively analysing the literature, directed by focussing on downregulated genes, we provide an insight as to why the in vitro cumulus cells fail to support full oocyte potential and dissect molecular pathways that have important roles in oocyte competence. We conclude that the roles of epidermal growth factor signalling, the expanded extracellular matrix, cumulus cell metabolism and the immune system are critical deficiencies in cumulus cells of IVM COCs.

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hCG Priming Before Ovary Collection Increasing The Oocyte Quality In The Domestic Cat

Acta Veterinaria Indonesiana ◽

10.29244/avi...123-126 ◽

2021 ◽

pp. 123-126

Author(s):

Karisma Mardatillah ◽

Rini Widyastuti ◽

Diah Nugrahani Pristihadi ◽

Wahyudin ◽

Sigit Prastowo ◽

...

Keyword(s):

Oocyte Quality ◽

Domestic Cat ◽

Developmental Competence ◽

Control Group ◽

Oocyte Competence ◽

Slicing Method ◽

Determining Factor

Oocyte competence is a determining factor that influences the embryo development. Embryos produced in vitro have a reduced developmental competence than embryos produced in vivo. Therefore, human Chorionic Gonadotropin (hCG) injection was carried out to improve the quality of the oocytes. The objective of this study was to evaluate the effect of ovarian stimulation with hCG before ovary collection on oocyte quality in the domestic cat. Oocyte donors were either 1) treated with a single dose of 200 IU hCG four days before ovary collection (hCG group), or, 2) no treatment before ovary collection (control group). The oocytes were collected by the slicing method. Immature cumulus oophorus complexes (COCs) from both groups were pooled and matured in vitro for 24-26 hours. Then mature oocytes were fertilized with epididymal sperm and cultured in vitro for seven days. The results study showed that the number of the dominant follicle (DF) and the number of COCs in the hCG group was higher than the control group in right and left ovaries (p<0.05). The morulae and blastocyst rates from cleavage embryos were 88% and 75%, respectively. These results demonstrate that hCG priming of oocytes donors before ovary collection improve oocyte quality.

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Microarray analysis of mRNA from cumulus cells following in vivo or in vitro maturation of mouse cumulus–oocyte complexes

Reproduction Fertility and Development ◽

10.1071/rd11305 ◽

2013 ◽

Vol 25 (2) ◽

pp. 426 ◽

Cited By ~ 23

Author(s):

Karen L. Kind ◽

Kelly M. Banwell ◽

Kathryn M. Gebhardt ◽

Anne Macpherson ◽

Ashley Gauld ◽

...

Keyword(s):

Microarray Analysis ◽

Cell Function ◽

Cumulus Cell ◽

Cumulus Cells ◽

Chorionic Gonadotrophin ◽

Developmental Competence ◽

Rt Pcr ◽

Oocyte Competence

The IVM of mammalian cumulus–oocyte complexes (COCs) yields reduced oocyte developmental competence compared with oocytes matured in vivo. Altered cumulus cell function during IVM is implicated as one cause for this difference. We have conducted a microarray analysis of cumulus cell mRNA following IVM or in vivo maturation (IVV). Mouse COCs were sourced from ovaries of 21-day-old CBAB6F1 mice 46 h after equine chorionic gonadotrophin (5 IU, i.p.) or from oviducts following treatment with 5 IU eCG (61 h) and 5 IU human chorionic gonadotrophin (13 h). IVM was performed in α-Minimal Essential Medium with 50 mIU FSH for 17 h. Three independent RNA samples were assessed using the Affymetrix Gene Chip Mouse Genome 430 2.0 array (Affymetrix, Santa Clara, CA, USA). In total, 1593 genes were differentially expressed, with 811 genes upregulated and 782 genes downregulated in IVM compared with IVV cumulus cells; selected genes were validated by real-time reverse transcription–polymerase chain reaction (RT-PCR). Surprisingly, haemoglobin α (Hba-a1) was highly expressed in IVV relative to IVM cumulus cells, which was verified by both RT-PCR and western blot analysis. Because haemoglobin regulates O2 and/or nitric oxide availability, we postulate that it may contribute to regulation of these gases during the ovulatory period in vivo. These data will provide a useful resource to determine differences in cumulus cell function that are possibly linked to oocyte competence.

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In vitro maturation alters gene expression in bovine oocytes

Zygote ◽

10.1017/s0967199415000672 ◽

2016 ◽

Vol 24 (4) ◽

pp. 624-633 ◽

Cited By ~ 12

Author(s):

Paulo R. Adona ◽

Cláudia L.V. Leal ◽

Fernando H. Biase ◽

Tiago H. De Bem ◽

Lígia G. Mesquita ◽

...

Keyword(s):

Gene Expression ◽

Protein Interactions ◽

Regulatory Networks ◽

Developmental Competence ◽

Protein Protein Interactions ◽

Developmental Potential ◽

Oocyte Competence ◽

Bovine Oocytes

SummaryGene expression profiling of in vivo- and in vitro-matured bovine oocytes can identify transcripts related to the developmental potential of oocytes. Nonetheless, the effects of in vitro culturing oocytes are yet to be fully understood. We tested the effects of in vitro maturation on the transcript profile of oocytes collected from Bos taurus indicus cows. We quantified the expression of 1488 genes in in vivo- and in vitro-matured oocytes. Of these, 51 genes were up-regulated, whereas 56 were down-regulated (≥2-fold) in in vivo-matured oocytes in comparison with in vitro-matured oocytes. Quantitative real-time polymerase chain reaction (PCR) of nine genes confirmed the microarray results of differential expression between in vivo- and in vitro-matured oocytes (EZR, EPN1, PSEN2, FST, IGFBP3, RBBP4, STAT3, FDPS and IRS1). We interrogated the results for enrichment of Gene Ontology categories and overlap with protein–protein interactions. The results revealed that the genes altered by in vitro maturation are mostly related to the regulation of oocyte metabolism. Additionally, analysis of protein–protein interactions uncovered two regulatory networks affected by the in vitro culture system. We propose that the differentially expressed genes are candidates for biomarkers of oocyte competence. In vitro oocyte maturation can affect the abundance of specific transcripts and are likely to deplete the developmental competence.

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Evaluation of 99mTc-Label led Vitamin K4 as an Agent for Testicular Imaging

Nuklearmedizin ◽

10.1055/s-0038-1629552 ◽

1991 ◽

Vol 30 (01) ◽

pp. 35-39 ◽

Cited By ~ 1

Author(s):

H. S. Durak ◽

M. Kitapgi ◽

B. E. Caner ◽

R. Senekowitsch ◽

M. T. Ercan

Keyword(s):

Soft Tissue ◽

Room Temperature ◽

Normal Subjects ◽

Left Testis ◽

Wide Range ◽

Activity Ratios ◽

The Right ◽

Radioactivity Levels

Vitamin K4 was labelled with 99mTc with an efficiency higher than 97%. The compound was stable up to 24 h at room temperature, and its biodistribution in NMRI mice indicated its in vivo stability. Blood radioactivity levels were high over a wide range. 10% of the injected activity remained in blood after 24 h. Excretion was mostly via kidneys. Only the liver and kidneys concentrated appreciable amounts of radioactivity. Testis/soft tissue ratios were 1.4 and 1.57 at 6 and 24 h, respectively. Testis/blood ratios were lower than 1. In vitro studies with mouse blood indicated that 33.9 ±9.6% of the radioactivity was associated with RBCs; it was washed out almost completely with saline. Protein binding was 28.7 ±6.3% as determined by TCA precipitation. Blood clearance of 99mTc-l<4 in normal subjects showed a slow decrease of radioactivity, reaching a plateau after 16 h at 20% of the injected activity. In scintigraphic images in men the testes could be well visualized. The right/left testis ratio was 1.08 ±0.13. Testis/soft tissue and testis/blood activity ratios were highest at 3 h. These ratios were higher than those obtained with pertechnetate at 20 min post injection.99mTc-l<4 appears to be a promising radiopharmaceutical for the scintigraphic visualization of testes.

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Enhanced Increases in Cytosolic Ca2+ in ADP-Stimulated Platelets from Patients with Delta-Storage Pool Deficiency – A Possible Indicator of Interactions between Granule-Bound ADP and the Membrane ADP Receptor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655971 ◽

1997 ◽

Vol 77 (02) ◽

pp. 376-382 ◽

Cited By ~ 15

Author(s):

Bruce Lages ◽

Harvey J Weiss

Keyword(s):

Platelet Rich Plasma ◽

Limited Range ◽

Dense Granule ◽

Receptor Desensitization ◽

Fura 2 ◽

Storage Pool ◽

Low Levels ◽

The Right

SummaryThe possible involvement of secreted platelet substances in agonist- induced [Ca2+]i increases was investigated by comparing these increases in aspirin-treated, fura-2-loaded normal platelets and platelets from patients with storage pool deficiencies (SPD). In the presence and absence of extracellular calcium, the [Ca2+]i response induced by 10 µM ADP, but not those induced by 0.1 unit/ml thrombin, 3.3 µM U46619, or 20 µM serotonin, was significantly greater in SPD platelets than in normal platelets, and was increased to the greatest extent in SPD patients with Hermansky-Pudlak syndrome (HPS), in whom the dense granule deficiencies are the most severe. Pre-incubation of SPD-HPS and normal platelets with 0.005-5 µM ADP produced a dose-dependent inhibition of the [Ca2+]i response induced by 10 µ M ADP, but did not alter the [Ca2+]i increases induced by thrombin or U46619. Within a limited range of ADP concentrations, the dose-inhibition curve of the [Ca2+]i response to 10 µM ADP was significantly shifted to the right in SPD-HPS platelets, indicating that pre-incubation with greater amounts of ADP were required to achieve the same extent of inhibition as in normal platelets. These results are consistent with a hypothesis that the smaller ADP-induced [Ca2+]i increases seen in normal platelets may result from prior interactions of dense granule ADP, released via leakage or low levels of activation, with membrane ADP receptors, causing receptor desensitization. Addition of apyrase to platelet-rich plasma prior to fura-2 loading increased the ADP-induced [Ca2+]i response in both normal and SPD-HPS platelets, suggesting that some release of ADP derived from both dense granule and non-granular sources occurs during in vitro fura-2 loading and platelet washing procedures. However, this [Ca2+]i response was also greater in SPD-HPS platelets when blood was collected with minimal manipulation directly into anticoagulant containing apyrase, raising the possibility that release of dense granule ADP resulting in receptor desensitization may also occur in vivo. Thus, in addition to enhancing platelet activation, dense granule ADP could also act to limit the ADP-mediated reactivity of platelets exposed in vivo to low levels of stimulation.

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Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

Microorganisms ◽

10.3390/microorganisms9051107 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1107

Author(s):

Wonho Choi ◽

Yoshihiro Yamaguchi ◽

Ji-Young Park ◽

Sang-Hyun Park ◽

Hyeok-Won Lee ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Physiological Function ◽

Functional Characterization ◽

Site Directed Mutagenesis ◽

In Vitro Assays ◽

Cell Growth Inhibition ◽

The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

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