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The Journal of Reproduction and... May 2024Intracytoplasmic sperm injection (ICSI) is clinically used to treat obstructive/nonobstructive azoospermia. This study compared the efficacy of ICSI with cauda...
Intracytoplasmic sperm injection (ICSI) is clinically used to treat obstructive/nonobstructive azoospermia. This study compared the efficacy of ICSI with cauda epididymal and testicular sperm in Wistar (WI) and Brown-Norway (BN) rats. The transfer of ICSI oocytes with cryopreserved epididymal and testicular WI sperm resulted in offspring production of 26.2% and 3.7%-4.7%, respectively (P < 0.05). Treatments for artificial oocyte activation (AOA) and acrosome removal improved pronuclear formation in BN-ICSI oocytes; however, only AOA treatment was effective in producing offspring (3.7%-6.5%). In the case of ICSI with testicular sperm (TESE-ICSI), one offspring (0.6%) was derived from the BN-TESE-ICSI oocytes. The application of AOA or a hypo-osmotic sperm suspension did not improve the production of TESE-ICSI offspring. Thus, outbred WI rat offspring can be produced by using ICSI and less efficiently by using TESE-ICSI. Challenges in producing offspring by using ICSI/TESE-ICSI in inbred BN strains require further investigation.
PubMed: 38735740
DOI: 10.1262/jrd.2024-030 -
Scientific Reports May 2024Within the medical field of human assisted reproductive technology, a method for interpretable, non-invasive, and objective oocyte evaluation is lacking. To address this...
Within the medical field of human assisted reproductive technology, a method for interpretable, non-invasive, and objective oocyte evaluation is lacking. To address this clinical gap, a workflow utilizing machine learning techniques has been developed involving automatic multi-class segmentation of two-dimensional images, morphometric analysis, and prediction of developmental outcomes of mature denuded oocytes based on feature extraction and clinical variables. Two separate models have been developed for this purpose-a model to perform multiclass segmentation, and a classifier model to classify oocytes as likely or unlikely to develop into a blastocyst (Day 5-7 embryo). The segmentation model is highly accurate at segmenting the oocyte, ensuring high-quality segmented images (masks) are utilized as inputs for the classifier model (mask model). The mask model displayed an area under the curve (AUC) of 0.63, a sensitivity of 0.51, and a specificity of 0.66 on the test set. The AUC underwent a reduction to 0.57 when features extracted from the ooplasm were removed, suggesting the ooplasm holds the information most pertinent to oocyte developmental competence. The mask model was further compared to a deep learning model, which also utilized the segmented images as inputs. The performance of both models combined in an ensemble model was evaluated, showing an improvement (AUC 0.67) compared to either model alone. The results of this study indicate that direct assessments of the oocyte are warranted, providing the first objective insights into key features for developmental competence, a step above the current standard of care-solely utilizing oocyte age as a proxy for quality.
Topics: Humans; Machine Learning; Blastocyst; Oocytes; Female; Embryonic Development; Adult; Fertilization in Vitro; Image Processing, Computer-Assisted
PubMed: 38719918
DOI: 10.1038/s41598-024-60901-1 -
Scientific Reports Apr 2024Oocytes of both vertebrates and invertebrates often contain an intricate organelle assemblage, termed the Balbiani body (Bb). It has previously been suggested that this...
Oocytes of both vertebrates and invertebrates often contain an intricate organelle assemblage, termed the Balbiani body (Bb). It has previously been suggested that this assemblage is involved in the delivery of organelles and macromolecules to the germ plasm, formation of oocyte reserve materials, and transfer of mitochondria to the next generation. To gain further insight into the function of the Bb, we performed a series of analyses and experiments, including computer-aided 3-dimensional reconstructions, detection of DNA (mtDNA) synthesis as well as immunolocalization studies. We showed that in orthopteran Meconema meridionale, the Bb comprises a network of mitochondria and perinuclear nuage aggregations. As oogenesis progresses, the network expands filling almost entire ooplasm, then partitions into several smaller entities, termed micro-networks, and ultimately into individual mitochondria. As in somatic cells, this process involves microfilaments and elements of endoplasmic reticulum. We showed also that at least some of the individual mitochondria are surrounded by phagophores and eliminated via mitophagy. These findings support the idea that the Bb is implicated in the multiplication and selective elimination of (defective) mitochondria and therefore may participate in the transfer of undamaged (healthy) mitochondria to the next generation.
Topics: Animals; Oocytes; Oogenesis; Mitochondria; Insecta; Endoplasmic Reticulum; Orthoptera
PubMed: 38594333
DOI: 10.1038/s41598-024-58997-6 -
BioRxiv : the Preprint Server For... Jan 2024Genetic screens for recessive alleles induce mutations, make the mutated chromosomes homozygous, and then assay those homozygotes for the phenotype of interest. When...
Genetic screens for recessive alleles induce mutations, make the mutated chromosomes homozygous, and then assay those homozygotes for the phenotype of interest. When screening for genes required for female meiosis, the phenotype of interest has typically been nondisjunction from chromosome segregation errors. As this requires that mutant females be viable and fertile, any mutants that are lethal or sterile when homozygous cannot be recovered by this approach. To overcome these limitations, our lab has screened the VALIUM22 collection produced by the Harvard TRiP Project, which contains RNAi constructs targeting genes known to be expressed in the germline in a vector optimized for germline expression. By driving RNAi with GAL4 under control of a germline-specific promoter ( or , we can test genes that would be lethal if knocked down in all cells, and by examining unfertilized metaphase-arrested mature oocytes, we can identify defects associated with genes whose knockdown results in sterility or causes other errors besides nondisjunction. We screened this collection to identify genes that disrupt either of two phenotypes when knocked down: the ability of meiotic chromosomes to congress to a single mass at the end of prometaphase, and the sequestration of Mps1-GFP to ooplasmic filaments in response to hypoxia. After screening >1450 lines of the collection, we obtained multiple hits for both phenotypes, identified novel meiotic phenotypes for genes that had been previously characterized in other processes, and identified the first phenotypes to be associated with several previously uncharacterized genes.
PubMed: 38293152
DOI: 10.1101/2024.01.12.575435 -
Frontiers in Endocrinology 2023The average age of childbearing has increased over the years contributing to infertility, miscarriages, and chromosomal abnormalities largely invoked by an age-related...
BACKGROUND
The average age of childbearing has increased over the years contributing to infertility, miscarriages, and chromosomal abnormalities largely invoked by an age-related decline in oocyte quality. In this study, we investigate the role of nitric oxide (NO) insufficiency and protein nitration in oocyte chronological aging.
METHODS
Mouse oocytes were retrieved from young breeders (YB, 8-14 weeks [w]), retired breeders (RB, 48-52w) and old animals (OA, 80-84w) at 13.5 and 17 hours after ovulation trigger. They were assessed for zona pellucida dissolution time (ZPDT); ooplasmic microtubule dynamics (OMD); cortical granule (CG) status and spindle morphology (SM), as markers of oocyte quality. Sibling oocytes from RB were exposed to NO supplementation and assessed for aging phenomena (AP). All oocyte cumulus complexes were subjected to fluorescence nitrotyrosine (NT) immunocytochemistry and confocal microscopy to assess morphology and protein nitration.
RESULTS
At 13.5 h from hCG trigger, oocytes from RB compared to YB had significantly increased ZPDT (37.8 ± 11.9 vs 22.1 ± 4.1 seconds [s]), OMD (46.9 vs 0%), CG loss (39.4 vs 0%), and decreased normal SM (30.3 vs 81.3%), indicating premature AP that worsened among oocytes from RB at 17 hours post-hCG trigger. When exposed to SNAP, RB AP significantly decreased (ZPDT: 35.1 ± 5.5 vs 46.3 ± 8.9s, OMD: 13.3 vs 75.0% and CG loss: 50.0 vs 93.3%) and SM improved (80.0 vs 14.3%). The incidence of NT positivity was significantly higher in cumulus cells (13.5 h, 46.7 ± 4.5 vs 3.4 ± 0.7%; 17 h, 82.2 ± 2.9 vs 23.3 ± 3.6%) and oocytes (13.5 h, 57.1 vs 0%; 17 h, 100.0 vs 55.5%) from RB compared to YB. Oocytes retrieved decreased with advancing age (29.8 ± 4.1 per animal in the YB group compared to 10.2 ± 2.1 in RB and 4.0 ± 1.6 in OA). Oocytes from OA displayed increased ZPDT, major CG loss, increased OMD and spindle abnormalities, as well as pronuclear formation, confirming spontaneous meiosis to interphase transition.
CONCLUSIONS
Oocytes undergo zona pellucida hardening, altered spindle and ooplasmic microtubules, and premature cortical granule release, indicative of spontaneous meiosis-interphase transition, as a function of chronological aging. These changes are also associated with NO insufficiency and protein nitration and may be alleviated through supplementation with an NO-donor.
Topics: Female; Mice; Animals; Oocytes; Aging; Zona Pellucida; Nitric Oxide Donors; Nitric Oxide
PubMed: 38149097
DOI: 10.3389/fendo.2023.1251102 -
Biological Research Nov 2023The decline in the quantity and quality of mitochondria are closely associated with infertility, particularly in advanced maternal age. Transferring autologous...
BACKGROUND
The decline in the quantity and quality of mitochondria are closely associated with infertility, particularly in advanced maternal age. Transferring autologous mitochondria into the oocytes of infertile females represents an innovative and viable strategy for treating infertility, with no concerns regarding ethical considerations. As the donor cells of mitochondria, stem cells have biological advantages but research and evidence in this area are quite scarce.
METHODS
To screen out suitable human autologous ooplasmic mitochondrial donor cells, we performed comprehensive assessment of mitochondrial physiology, function and metabolic capacity on a varity of autologous adipose, marrow, and urine-derived mesenchymal stromal cells (ADSC, BMSC and USC) and ovarian germline granulosa cells (GC). Further, to explore the biosafety, effect and mechanism of stem cell-derived mitochondria transfer on human early embryo development, randomized in-vitro basic studies were performed in both of the young and aged oocytes from infertile females.
RESULTS
Compared with other types of mesenchymal stromal cells, USC demonstrated a non-fused spherical mitochondrial morphology and low oxidative stress status which resembled the oocyte stage. Moreover, USC mitochondrial content, activity and function were all higher than other cell types and less affected by age, and it also exhibited a biphasic metabolic pattern similar to the pre-implantation stage of embryonic development. After the biosafety identification of the USC mitochondrial genome, early embryos after USC mitochondrial transfer showed improvements in mitochondrial content, activity, and cytoplasmic Ca levels. Further, aging embryos also showed improvements in embryonic morphological indicators, euploidy rates, and oxidative stress status.
CONCLUSION
Autologous non-invasively derived USC mitochondria transfer may be an effective strategy to improve embryonic development and metabolism, especially in infertile females with advanced age or repeated pregnancy failure. It provides evidence and possibility for the autologous treatment of infertile females without invasive and ethical concerns.
Topics: Female; Humans; Pregnancy; Aging; Infertility, Female; Mitochondria; Oocytes; Stem Cells
PubMed: 37978575
DOI: 10.1186/s40659-023-00470-1 -
Brazilian Journal of Biology = Revista... 2023Specimens of Cnemidocarpa amphora were collected monthly from the Arabian Gulf from September 2017 to August 2018. Parts of their gonads were prepared for histological...
Gonadal proliferation and reproductive cycle of the exotic sea squirt Cnemidocarpa amphora () (Pleurogona, Styelidae) sampled for the first time from the northern coast of Arabian Gulf in Saudi Arabia.
Specimens of Cnemidocarpa amphora were collected monthly from the Arabian Gulf from September 2017 to August 2018. Parts of their gonads were prepared for histological testing. The gonads' diameters varied by month. Each gonad contained many ovarian follicles with different morphologies and was surrounded by several testicular follicles. The ovarian and testicular follicles were separate, although the latter were always present near the former. Repeated measures ANOVA tests were conducted to investigate monthly changes in oocyte stages. In squirts measuring 12-13 cm in length, the gonads measured 30-50 mm from July to August; 20-25 mm from September to October; 15-20 mm from November to February; and 25-40 mm from March to June. Oogonia budded from the germinal epithelium with diameters of 20-30 µm. Previtellogenic oocytes measuring 70-120 µm were characterized by the deposition of small granules of protein around the nucleus, a continuous layer of follicular cuboidal epithelium, and scattered vacuoles in the ooplasm. The measurement of gonads and oocyte diameters were performed by image analysis (Image scope 2.3, Image Line, Inc.) and stage micrometer. The vitellogenic oocytes measured 130-220 µm and the follicular epithelium consisted of flattened and cuboidal layers. Beneath the vitelline membrane, scattered test cells appeared in the ooplasm and different granules of protein and MPS were deposited in the ooplasm. In the later phase, lipid droplets began to appear in the ooplasm. Yolk bodies formed after the impregnation of various granules together and the oocyte was ready to be shed. Before spawning, a yolk membrane appeared above the ooplasm. Post-vitellogenic oocytes, in which the homogeneity of ooplasm was restored, underwent gradual lysis and entered the atretic phase. Different stages of sperm development were present year-round in different follicles of the same squirt; hence, the testes were always mature.
Topics: Animals; Female; Male; Urochordata; Saudi Arabia; Semen; Oocytes; Ovary; Cell Proliferation
PubMed: 37970899
DOI: 10.1590/1519-6984.273666 -
Animals : An Open Access Journal From... Oct 2023This study sought to examine the ovarian cellular and stromal components of the zebrafish () throughout the spawning season using light and electron microscopic tools....
This study sought to examine the ovarian cellular and stromal components of the zebrafish () throughout the spawning season using light and electron microscopic tools. The ovaries of zebrafish showed oocytes in all stages of follicular development and degeneration (atresia). Six stages of oogenesis were demonstrated: oogonia, early oocytes, late oocytes, vacuolated follicles, the yolk globule stage (vitellogenesis), and mature follicles. The SOX9 protein was expressed in the ooplasm of the primary and previtellogenic oocytes and the theca cell layer of the mature follicles. Myostatin was expressed in the granulosa and theca cells. Many stem cells in the ovarian stroma expressed myostatin and SOX9. During the spawning season, the EM results indicated that the zona radiata increased in thickness and was crossed perpendicularly by pore canals that contained processes from both oocytes and zona granulosa. The granulosa cells contained many mitochondria, rER, sER, and vesicles. Meanwhile, the thecal layer consisted of fibroblast-like cells. Atretic follicles could be demonstrated that involved both oocytes and their follicular walls. Several types of cells were distinguished in the ovarian stroma, including mast cells, telocytes, lymphocytes, fibroblasts, endocrine cells, macrophages, adipocytes, dendritic cells, and steroidogenic (stromal) cells. The ovary of the zebrafish serves as a model to investigate follicular development.
PubMed: 37958117
DOI: 10.3390/ani13213362 -
Science Advances Oct 2023In worms, epigenetic information transmits transgenerationally. Still, it is unknown whether the effects transfer to the next generation inside or outside of the...
In worms, epigenetic information transmits transgenerationally. Still, it is unknown whether the effects transfer to the next generation inside or outside of the nucleus. Here, we use the tractability of gene-specific double-stranded RNA-induced silencing to demonstrate that RNA interference can be inherited independently of any nuclear factors via mothers that are genetically engineered to transmit only their ooplasm but not the oocytes' nuclei to the next generation. We characterize the mechanisms and, using RNA sequencing, chimeric worms, and sequence polymorphism between different isolates, identify endogenous small RNAs which, similarly to exogenous siRNAs, are inherited in a nucleus-independent manner. From a historical perspective, these results might be regarded as partial vindication of discredited cytoplasmic inheritance theories from the 19th century, such as Darwin's "pangenesis" theory.
Topics: Animals; Caenorhabditis elegans; RNA, Small Interfering; RNA Interference; Caenorhabditis elegans Proteins; Gene Silencing; RNA, Double-Stranded
PubMed: 37878696
DOI: 10.1126/sciadv.adj8618 -
BMC Pregnancy and Childbirth Sep 2023Previous studies looked into the connections between pregnancy and the Zona Pellucida (ZP) thickness and Zona Pellucida Thickness Variation (ZPTV), as well as the...
BACKGROUND
Previous studies looked into the connections between pregnancy and the Zona Pellucida (ZP) thickness and Zona Pellucida Thickness Variation (ZPTV), as well as the embryo's radius, circumference, perimeter and global symmetry. However, no research has linked embryo implantation and pregnancy to the percentage of ZP thinning, the reduction in ooplasm volume, and the increase in perivitelline space (PVS) volume. Our objective is to correlate the percentage of ZP thinning, the percentage of ooplasm volume shrinkage and the percentage of PVS increase to the implantation. These data will be used for embryo selection as well as it can be put into a software that will assist embryo selection.
MATERIALS AND METHODS
Retrospective study included 281 patients, all of them had 2 embryos transferred, 149 patients got pregnant with two gestation sacs and 132 patients did not get pregnant. All of the transferred embryos had the ZP thickness measured several times from time of ICSI till Embryo Transfer (ET), the ooplasm volume was calculated from time of ICSI till two Pronuclei (2PN) fading and the PVS was calculated from the ICSI time till the 2PN fading.
RESULTS
The first characteristic is the change in the average ZP thickness that decreased by 32.7% + 5.3% at 70 h for the implanted embryos (Group 1) versus 23.6% + 4.8% for non-implanted embryos (Group 2) p = 0.000. The second characteristic is the average reduction in the volume of the ooplasm which is 20.5% + 4.3% in Group 1 versus 15.1% + 5.2% in Group 2, p = 0.000. The third characteristic is the increase in the volume of the PVS which was 38.1% + 7.6% in Group 1 versus 31.6% + 9.7% in Group 2 p = 0.000.
CONCLUSION
The implanted embryos showed higher percent of ZP thinning, higher percent of ooplasm reduction and higher percent of PVS increase.
Topics: Pregnancy; Female; Humans; Retrospective Studies; Sperm Injections, Intracytoplasmic; Embryo Implantation; Embryo Transfer; Zona Pellucida
PubMed: 37770819
DOI: 10.1186/s12884-023-06025-2