scholarly journals Isolation of living sperm cells and in vitro fusion of Torenia fournieri gametes

2006 ◽  
Vol 12 (4) ◽  
Author(s):  
P. Vági ◽  
K. Imre ◽  
Z. Kristóf
Keyword(s):  
Male Gametophyte ◽  
Embryo Sac ◽  
Pollen Tubes ◽  
Sperm Cells ◽  
Torenia Fournieri ◽  
Isolation Medium ◽  
Vitro Fertilization ◽  
Crucial Part

In contrast to most angiosperms, Torenia contains a naked embryo sac and therefore has been considered since many years as an exciting model plant to study the double fertilization process of flowering seed plants. It is thus not surprising that the isolation of protoplasts from the female gametophyte has been reported already 20 years ago by Mol, the isolation of megaspores and megagametophytes has been published by the authors of this manuscript in 1996 and in 1999. The isolation of the male gametophyte and of sperm cells was published by the authors in 2004. The isolation of viable Torenia sperm cells is a crucial part of the elaboration of an in vitro fertilization system. Torenia sperm cells were isolated from in vivo — in vitro cultured pollen tubes. In this system pollen tubes first grow inside a cut style then follow their elongation in a solid isolation medium. The medium contained agarose in order to detain pollen tube contents. Released sperm cells and enzymatically isolated egg cells were collected and handled using glass micropipettes and transmitted to an electrofusion apparatus or polyethylene glycol containing media for fusion probes.

scholarly journals Isolation of Sperm and Egg Cells from Pepper

2018 ◽  
Vol 143 (4) ◽  
pp. 310-315 ◽  
Author(s):  
Wei Deng ◽  
Yunling Xie ◽  
Yilan Qiu
Keyword(s):  
Generative Cell ◽  
Embryo Sac ◽  
Pollen Tubes ◽  
Enzymatic Digestion ◽  
Sperm Cells ◽  
Free Solution ◽  
In Vitro Method ◽  
Viable Embryo

Pepper (Capsicum annuum) pollen is bicellular and contains a vegetative cell and a generative cell, which divides in pollen tubes to form two sperm cells. Sperm cells of pepper were isolated using an in vivo–in vitro method. Hand-pollinated styles were first grown in vivo for several hours, then cut from their base and cultured in vitro until pollen tubes grew from the cut end. When the pollen tubes were transferred to a breaking solution, sperm cells were released from broken tubes. Viable embryo sac cells of pepper were isolated using enzymatic digestion and mechanical dissection. Isolated ovules were digested using cellulase and pectinase for 40 minutes and then transferred to an enzyme-free solution for mechanical dissection. Three cells of the egg apparatus and a central cell were released from a cut at the chalazal end of each ovule by pressing on the micropylar area of the ovule with a microneedle. Optimal isolation conditions included 11% mannitol, 0.04% CaCl2, 1% bovine serum albumin (BSA), 1% cellulase, 1% pectinase, and 0.3% pectolyase. Using this protocol, populations of pepper egg cells, synergids, and central cells were isolated.

scholarly journals Seminal Plasma: Relevant for Fertility?

2021 ◽  
Vol 22 (9) ◽  
pp. 4368
Author(s):  
Heriberto Rodriguez-Martinez ◽  
Emilio A. Martinez ◽  
Juan J. Calvete ◽  
Fernando J. Peña Vega ◽  
Jordi Roca
Keyword(s):  
Seminal Plasma ◽  
Current Knowledge ◽  
Inorganic Ions ◽  
Vitro Fertilization ◽  
Dna And Rna ◽  
Model Animal ◽  
Sexual Glands ◽  
Species Specific

Seminal plasma (SP), the non-cellular component of semen, is a heterogeneous composite fluid built by secretions of the testis, the epididymis and the accessory sexual glands. Its composition, despite species-specific anatomical peculiarities, consistently contains inorganic ions, specific hormones, proteins and peptides, including cytokines and enzymes, cholesterol, DNA and RNA—the latter often protected within epididymis- or prostate-derived extracellular vesicles. It is beyond question that the SP participates in diverse aspects of sperm function pre-fertilization events. The SP also interacts with the various compartments of the tubular genital tract, triggering changes in gene function that prepares for an eventual successful pregnancy; thus, it ultimately modulates fertility. Despite these concepts, it is imperative to remember that SP-free spermatozoa (epididymal or washed ejaculated) are still fertile, so this review shall focus on the differences between the in vivo roles of the SP following semen deposition in the female and those regarding additions of SP on spermatozoa handled for artificial reproduction, including cryopreservation, from artificial insemination to in vitro fertilization. This review attempts, including our own results on model animal species, to critically summarize the current knowledge of the reproductive roles played by SP components, particularly in our own species, which is increasingly affected by infertility. The ultimate goal is to reconcile the delicate balance between the SP molecular concentration and their concerted effects after temporal exposure in vivo. We aim to appraise the functions of the SP components, their relevance as diagnostic biomarkers and their value as eventual additives to refine reproductive strategies, including biotechnologies, in livestock models and humans.

scholarly journals Protein Analysis of Pollen Tubes after the Treatments of Membrane Trafficking Inhibitors Gains Insights on Molecular Mechanism Underlying Pollen Tube Polar Growth

2021 ◽  
Vol 40 (2) ◽  
pp. 205-222
Author(s):  
Monica Scali ◽  
Alessandra Moscatelli ◽  
Luca Bini ◽  
Elisabetta Onelli ◽  
Rita Vignani ◽  
...  
Keyword(s):  
Pollen Tube ◽  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Tip Growth ◽  
Male Gametophyte ◽  
Pollen Tubes ◽  
Structural Features ◽  
Two Dimensional Gel Electrophoresis ◽  
Depth Analysis

AbstractPollen tube elongation is characterized by a highly-polarized tip growth process dependent on an efficient vesicular transport system and largely mobilized by actin cytoskeleton. Pollen tubes are an ideal model system to study exocytosis, endocytosis, membrane recycling, and signaling network coordinating cellular processes, structural organization and vesicular trafficking activities required for tip growth. Proteomic analysis was applied to identifyNicotiana tabacumDifferentially Abundant Proteins (DAPs) after in vitro pollen tube treatment with membrane trafficking inhibitors Brefeldin A, Ikarugamycin and Wortmannin. Among roughly 360 proteins separated in two-dimensional gel electrophoresis, a total of 40 spots visibly changing between treated and control samples were identified by MALDI-TOF MS and LC–ESI–MS/MS analysis. The identified proteins were classified according to biological processes, and most proteins were related to pollen tube energy metabolism, including ammino acid synthesis and lipid metabolism, structural features of pollen tube growth as well modification and actin cytoskeleton organization, stress response, and protein degradation. In-depth analysis of proteins corresponding to energy-related pathways revealed the male gametophyte to be a reliable model of energy reservoir and dynamics.

scholarly journals Graphene and Reproduction: A Love-Hate Relationship

Nanomaterials ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 547
Author(s):  
Marina Ramal-Sanchez ◽  
Antonella Fontana ◽  
Luca Valbonetti ◽  
Alessandra Ordinelli ◽  
Nicola Bernabò ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Reproductive Function ◽  
Scientometric Analysis ◽  
Potential Toxicity ◽  
Good Tool ◽  
Vitro Fertilization ◽  
Number Of Publications ◽  
The Individual

Since its discovery, graphene and its multiple derivatives have been extensively used in many fields and with different applications, even in biomedicine. Numerous efforts have been made to elucidate the potential toxicity derived from their use, giving rise to an adequate number of publications with varied results. On this basis, the study of the reproductive function constitutes a good tool to evaluate not only the toxic effects derived from the use of these materials directly on the individual, but also the potential toxicity passed on to the offspring. By providing a detailed scientometric analysis, the present review provides an updated overview gathering all the research studies focused on the use of graphene and graphene-based materials in the reproductive field, highlighting the consequences and effects reported to date from experiments performed in vivo and in vitro and in different animal species (from Archea to mammals). Special attention is given to the oxidized form of graphene, graphene oxide, which has been recently investigated for its ability to increase the in vitro fertilization outcomes. Thus, the potential use of graphene oxide against infertility is hypothesized here, probably by engineering the spermatozoa and thus manipulating them in a safer and more efficient way.

scholarly journals Novel Insights on the Role of Nitric Oxide in the Ovary: A Review of the Literature

2021 ◽  
Vol 18 (3) ◽  
pp. 980
Author(s):  
Maria Cristina Budani ◽  
Gian Mario Tiboni
Keyword(s):  
Nitric Oxide ◽  
In Vivo Studies ◽  
Published Data ◽  
Vitro Fertilization ◽  
Peripheral Neurons ◽  
Relevant Role ◽  
Oocyte Meiotic Maturation

Nitric oxide (NO) is formed during the oxidation of L-arginine to L-citrulline by the action of multiple isoenzymes of NO synthase (NOS): neuronal NOS (nNOS), endotelial NOS (eNOS), and inducible NOS (iNOS). NO plays a relevant role in the vascular endothelium, in central and peripheral neurons, and in immunity and inflammatory systems. In addition, several authors showed a consistent contribution of NO to different aspects of the reproductive physiology. The aim of the present review is to analyse the published data on the role of NO within the ovary. It has been demonstrated that the multiple isoenzymes of NOS are expressed and localized in the ovary of different species. More to the point, a consistent role was ascribed to NO in the processes of steroidogenesis, folliculogenesis, and oocyte meiotic maturation in in vitro and in vivo studies using animal models. Unfortunately, there are few nitric oxide data for humans; there are preliminary data on the implication of nitric oxide for oocyte/embryo quality and in-vitro fertilization/embryo transfer (IVF/ET) parameters. NO plays a remarkable role in the ovary, but more investigation is needed, in particular in the context of human ovarian physiology.

scholarly journals Calcium: A Critical Factor in Pollen Germination and Tube Elongation

2019 ◽  
Vol 20 (2) ◽  
pp. 420 ◽  
Author(s):  
Ren Zheng ◽  
Shun Su ◽  
Hui Xiao ◽  
Hui Tian
Keyword(s):  
Calcium Ion ◽  
Pollen Germination ◽  
Higher Plants ◽  
Male Gametophyte ◽  
Embryo Sac ◽  
Critical Factor ◽  
Sperm Cells ◽  
Critical Element ◽  
Major Function ◽  
Spatial And Temporal Characteristics

Pollen is the male gametophyte of higher plants. Its major function is to deliver sperm cells to the ovule to ensure successful fertilization. During this process, many interactions occur among pollen tubes and pistil cells and tissues, and calcium ion (Ca2+) dynamics mediate these interactions among cells to ensure that pollen reaches the embryo sac. Although the precise functions of Ca2+ dynamics in the cells are unknown, we can speculate about its roles on the basis of its spatial and temporal characteristics during these interactions. The results of many studies indicate that calcium is a critical element that is strongly related to pollen germination and pollen tube growth.

scholarly journals FOS, a Critical Downstream Mediator of PGR and EGF Signaling Necessary for Ovulatory Prostaglandins in the Human Ovary

2018 ◽  
Vol 103 (11) ◽  
pp. 4241-4252 ◽  
Author(s):  
Yohan Choi ◽  
Katherine L Rosewell ◽  
Mats Brännström ◽  
James W Akin ◽  
Thomas E Curry ◽  
...  
Keyword(s):  
Target Genes ◽  
Activator Protein 1 ◽  
Egf Receptors ◽  
Human Ovary ◽  
Promoter Regions ◽  
Vitro Fertilization ◽  
Egf Signaling ◽  
Periovulatory Follicles

Abstract Context Fos null mice failed to ovulate and form a corpus luteum (CL) even when given exogenous gonadotropins, suggesting that ovarian Fos expression is critical for successful ovulation and CL formation. However, little is known about FOS in the human ovary. Objectives To determine the expression, regulation, and function of FOS in human periovulatory follicles. Design/Participants Timed periovulatory follicles were obtained from normally cycling women. Granulosa/lutein cells were collected from in vitro fertilization patients. Main Outcome Measures The in vivo expression after human chorionic gonadotropin (hCG) administration and in vitro regulation of FOS, JUN, JUNB, and JUND was evaluated at the mRNA and protein level. Binding of progesterone receptor (PGR) and FOS to their target genes was assessed by chromatin immunoprecipitation analyses. Prostaglandin E2 (PGE2) and progesterone were measured. Results The expression of FOS, JUNB, and JUND drastically increased in ovulatory follicles after hCG administration. In human granulosa/lutein cell cultures, hCG increased the expression of FOS and JUN proteins. Inhibitors of PGR and epidermal growth factor (EGF) receptors reduced hCG-induced increases in the expression and phosphorylation of FOS. PGR bound to the FOS gene. A selective FOS inhibitor blocked hCG-induced increases in PGE2 and the expression of prostaglandin (PG) synthases and transporters (PTGES, SLCO2A1, and ABCC1). FOS bound to the promoter regions of these genes. Conclusions The increase of FOS/activator protein 1 in human periovulatory follicles after hCG administration is mediated by collaborative actions of PGR and EGF signaling and critical for the upregulated expression of key ovulatory genes required for the rise in ovulatory PG in human granulosa cells.

scholarly journals Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG

2021 ◽  
Author(s):  
Shuhong Ma ◽  
Wenjian Jiang ◽  
Xujie Liu ◽  
Wen-Jing Lu ◽  
Tao Qi ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Mouse Model ◽  
Pathogenic Mutation ◽  
Adeno Associated Virus ◽  
Vitro Fertilization ◽  
Base Editing ◽  
Dna And Rna ◽  
Hereditary Disorders

Rationale: Genetic editing has shown great potential for the treatment of human hereditary disorders via the elimination of mutations in embryos. However, the efficiency and safety of germline gene editing are not well understood. Objective: We aimed to examine the preclinical efficacy/safety of embryonic base editing in a mouse model of hypertrophic cardiomyopathy (HCM) using a novel adenine base editor (ABE) platform. Methods and Results: Here, we described the use of an ABEmax-NG to directly correct the pathogenic R404Q/+ mutation (Myh6 c.1211C>T) in embryos for a mouse model of HCM, increasing the number of wild-type embryos for in vitro fertilization. Delivery of the ABEmax-NG mRNA to embryos from R404Q/+ HCM mice resulted in 62.5-70.8% correction of the Myh6 c.1211C>T, reducing the level of mutant RNA and eliminating HCM in the post-natal mice as well as their offspring. In addition, the same sgRNA was also used to target an intronic locus (TGG PAM) with an overall editing rate of 86.7%, thus confirming that ABEmax-NG can efficiently edit target loci with different PAMs (NG) and genomic distribution in vivo. Compared with CRISPR/ssODN-mediated correction, ABEmax-NG displayed a much higher correction rate without introducing indels. DNA and RNA off-target analysis did not detect off-target editing in treated embryos and founder mice. In utero injection of adeno-associated virus 9 (AAV9) encoding the ABEmax-NG also resulted in around 25.3% correction of the pathogenic mutation and reduced of mutant RNA, thereby indicating ABEmax-NG has the potential to correct the HCM mutation in vivo. Conclusions: We developed an ABEmax-NG system, which efficiently corrected a pathogenic Myh6 HCM mutation in mouse embryos without off target lesions, thus safely eliminating HCM in derived mice and their progeny.

scholarly journals In Vitro Fertilization of In Vitro and In Vivo Matured Oocytes from Prepubertal Meishan Gilts

1990 ◽  
Vol 61 (11) ◽  
pp. 1011-1016
Author(s):  
Takashi MIYANO ◽  
Kiyoshi YOSHIKAWA ◽  
Seishiro KATO ◽  
Hiroshi HARAYAMA ◽  
Iwao NANJO ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Vitro Fertilization
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