Comparative study of preimplantation development following distinct assisted oocyte activation protocols in a PLC-zeta knockout mouse model

Abstract Mammalian fertilization encompasses a series of Ca2+ oscillations initiated by the sperm factor phospholipase C zeta (PLCζ). Some studies have shown that altering the Ca2+ oscillatory regime at fertilization affects preimplantation blastocyst development. However, assisted oocyte activation (AOA) protocols can induce oocyte activation in a manner that diverges profoundly from the physiological Ca2+ profiling. In our study, we used the newly developed PLCζ-null sperm to investigate the independent effect of AOA on mouse preimplantation embryogenesis. Based on previous findings, we hypothesized that AOA protocols with Ca2+ oscillatory responses might improve blastocyst formation rates and differing Ca2+ profiles might alter blastocyst transcriptomes. A total of 326 MII B6D2F1-oocytes were used to describe Ca2+ profiles and to compare embryonic development and individual blastocyst transcriptomes between four control conditions: C1 (in-vivo fertilization), C2 (ICSI control sperm), C3 (parthenogenesis) and C4 (ICSI-PLCζ-KO sperm) and four AOA groups: AOA1 (human recombinant PLCζ), AOA2 (Sr2+), AOA3 (ionomycin) and AOA4 (TPEN). All groups revealed remarkable variations in their Ca2+ profiles; however, oocyte activation rates were comparable between the controls (91.1% ± 13.8%) and AOA (86.9% ± 11.1%) groups. AOA methods which enable Ca2+ oscillatory responses (AOA1: 41% and AOA2: 75%) or single Ca2+ transients (AOA3: 50%) showed no significantly different blastocyst rates compared to ICSI control group (C2: 70%). In contrast, we observed a significant decrease in compaction (53% vs. 83%) and blastocyst rates (41% vs. 70%) in the absence of an initial Ca2+ trigger (AOA4) compared with the C2 group. Transcription profiles did not identify significant differences in gene expression levels between the ICSI control group (C2) and the four AOA groups.

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P–214 Effect of artificial activation of oocytes (AOA-ICSI) on the ploidy status of the resultant blastocysts. A sibling-oocytes pilot study

Human Reproduction ◽

10.1093/humrep/deab130.213 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

E Seo. Pe. Yin

Keyword(s):

Pilot Study ◽

Fertilization Rate ◽

Control Group ◽

Oocyte Activation ◽

Fertilization Failure ◽

Factor Deficiency ◽

Sperm Factor ◽

Blastocyst Rate ◽

Artificial Activation ◽

Ploidy Status

Abstract Study question Will artificial activation of oocytes alter the ploidy status of the resultant blastocysts? A sibling-oocytes pilot study Summary answer AOA-ICSI does not increase the risk of having aneuploidy blastocysts and can improve the fertilization rate in patients with sperm factor deficiency. What is known already Despite introducing ICSI as an aid to improve chances of fertilization, fertilization failure can still occur in 2–3% of ICSI cycles. Fertilization is a complex process triggered by a cascade of events following calcium (Ca2+) oscillations. Evidence suggests that the deficiency, localization or altered structure of the sperm-derived protein PLCζ in oocyte activation may be a reason for meiotic II arrest in the oocyte. Artificial oocyte activation has been proposed to compensate for the lack of calcium oscillation and resumes meiotic progression. There are however insufficient studies to determine its effect on the chromosomal status of the resultant blastocysts. Study design, size, duration This is a prospective, randomized study conducted at our Center from August-October 2020. A total of 20 couples intended for ICSI + Preimplatation Genetic Testing for Aneuploidy (PGT-A) cycles were recruited based on fulfilling one of the following criteria: 1) previous total fertilization failure (TFF), 2) history of low fertilization rate (<30%), 3) more than 2 cycles of failed IVF cycles (no implantation) 4) poor embryo development (no blastocysts formed) and 5) severe male factor. Participants/materials, setting, methods A total of 231 MII oocytes underwent randomization in a 1:1 ratio between AOA-ICSI and control group. All oocytes are subjected to ICSI treatment. Oocytes in the AOA-ICSI group are treated in 25μl droplets 10μM ready to use bicarbonate buffered calcium ionophore (Kitazato, Japan) for 15 minutes post-ICSI. The blastocysts were biopsied and subjected to PGT-A. Primary outcome was the aneuploidy rate and secondary outcomes were fertilization rate and blastocyst rate. Main results and the role of chance There were 11 out of 40 (27.5%) aneuploid blastocysts in the AOA-ICSI group and 7 out of 23 aneuploid blastocysts (30.4%) in the control group [odds ratio (OR) = 0.87; 95% confidence interval (CI) 0.28–2.68, p = 0.8040). There was no statistically significant difference between both groups. However, fertilization rate of the AOA- ICSI group was significantly higher than the fertilization rate in the control group (68.6% vs 49.6% respectively, OR = 2.22; 95% CI, 1.31–3.81, p = 0.0034). There were 40 blastocysts formed in the AOA-ICSI group and 23 blastocysts formed in the control group. It was found that the AOA-ICSI group yielded a higher blastocyst rate (49.4%) compared to the control group (41.1%) (OR = 1.40; 95% CI, 0.71 to 2.78, p = 0.3379) but the difference was not statistically significant. Limitations, reasons for caution The possibility of TE cells biopsied may not be representative of the whole blastocyst makes it possible to have false clinical data. The dosage and time were also not evaluated in this study as exposure time was found to be a critical factor of fertilization rate in a previous study. Wider implications of the findings: This study showed that AOA-ICSI does not increase the risk of having aneuploidy blastocysts and can improve the fertilization rate in patients with sperm factor deficiency. Additional studies involving a larger number of patients with more specific indication can further justify the benefits of AOA as a therapeutic application. Trial registration number NA

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Chronic Nitric Oxide Deficiency is Associated with Altered Leutinizing Hormone and Follicle-Stimulating Hormone Release in Ovariectomized Rats1

Experimental Biology and Medicine ◽

10.1177/153537020222700915 ◽

2002 ◽

Vol 227 (9) ◽

pp. 817-822 ◽

Cited By ~ 7

Author(s):

Maria J. Barnes ◽

Karen Lapanowski ◽

Jose A. Rafols ◽

David M. Lawson ◽

Joseph C. Dunbar

Keyword(s):

Nitric Oxide ◽

Follicle Stimulating Hormone ◽

Independent Effect ◽

Control Group ◽

Gonadotropin Secretion ◽

No Donor ◽

Ovx Rats ◽

Nos Inhibitor ◽

Stimulating Hormone

Nitric oxide (NO) synthase (NOS) has been found in the gonadotrophs and folliculo-stellate cells of the anterior pituitary. Previous observations from our laboratory suggest that NO may play a role in regulating gonadotropin secretion. Because estrogen secretion by the ovary can influence gonadotropin secretion, we investigated the hypothesis that chronic in vivo NO deficiency has a direct estrogen-independent effect on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. Chronic NO deficiency was induced by adding an NOS inhibitor, N-nitro-L-arginine (L-NNA, 0.6 g/l) to the drinking water of ovariectomized (OVX) rats. The control OVX rats were untreated. After 6–8 weeks, the animals were sacrificed, and the pituitaries were removed and perfused continuously for 4 hr in the presence of pulsatile gonadotropin-releasing hormone (GnRH, 500 ng/pulse) every 30 min. S-Nitroso-l-acetyl penicillamine (SNAP, an NO donor, 0.1 mM) or l-nitro-arginine methyl ester (L-NAME, an NOS inhibitor, 0.1 mM) was added to the media and perfusate samples were collected at 10-min intervals. GnRH-stimulated LH and FSH levels were significantly lower in pituitaries from OVX/NO-deficient pituitaries compared with pituitaries from the OVX control group. The addition of SNAP significantly decreased LH and FSH secretion by pituitaries from OVX control animals, but significantly increased their secretion by pituitaries from the OVX/NO-deficient animals. L-NAME also suppressed LH and FSH secretion by pituitaries from the OVX control animals and stimulated their release by pituitaries from the NO-deficient/OVX animals. Immunohisto-chemistry of frontal sections through the hypothalamus demonstrated that OVX/NO deficiency is associated with increased GnRH in the median eminence. We conclude that NO has a chronic stimulatory effect on LH and FSH release and the subsequent altered secretory responsiveness to NO agonist or antagonist is the result of chronic NO suppression.

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Use of strontium in the activation of bovine oocytes reconstructed by somatic cell nuclear transfer

Zygote ◽

10.1017/s0967199405003333 ◽

2005 ◽

Vol 13 (4) ◽

pp. 295-302 ◽

Cited By ~ 15

Author(s):

Walt Yamazaki ◽

Christina Ramires Ferreira ◽

Simone Cristina Méo ◽

Cláudia Lima Verde Leal ◽

Flávio Vieira Meirelles ◽

...

Keyword(s):

Somatic Cell ◽

Nuclear Transfer ◽

Standard Treatment ◽

Oocyte Activation ◽

Blastocyst Development ◽

Reconstructed Embryos ◽

Developmental Capacity ◽

Bovine Oocytes

As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three different treatments for oocyte activation on the in vitro and in vivo developmental capacity of bovine reconstructed embryos: (1) strontium, which has been successfully used in mice but not yet tested in cattle; (2) ionomycin and 6-dimethylaminopurine (6-DMAP), a standard treatment used in cattle; (3) ionomycin and strontium, in place of 6-DMAP. As regards NT blastocyst development, no difference was observed when strontium (20.1%) or ionomycin/6-DMAP (14.4%) were used. However, when 6-DMAP was substituted by strontium (3), the blastocyst rate (34.8%) was superior to that in the other activation groups (p <0.05). Results of in vivo development showed the possibility of pregnancies when NT embryos activated in strontium were transferred to recipient cows (16.6%). A live female calf was obtained when ionomycin/strontium were used, but it died 30 days after birth. Our findings show that strontium can be used as an activation agent in bovine cloning procedures and that activation with a combination of strontium and ionomycin increased the in vitro developmental capacity of reconstructed embryos. This is the first report of a calf produced by adult somatic cell NT in Latin America.

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Oocyte activation and preimplantation development of bovine embryos obtained by specific inhibition of cyclin-dependent kinases

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/s0102-09352007000200002 ◽

2007 ◽

Vol 59 (2) ◽

pp. 280-287 ◽

Cited By ~ 2

Author(s):

F. Perecin ◽

S.C. Méo ◽

C.L.V. Leal ◽

J.M. Garcia

Keyword(s):

Embryonic Development ◽

Developmental Competence ◽

Oocyte Activation ◽

Blastocyst Development ◽

Parthenogenetic Activation ◽

Cyclin Dependent Kinases ◽

Activation Rate ◽

Activation Rates ◽

Bovine Oocytes

The efficiency of bohemine and roscovitine in combination with ionomycin on parthenogenetic activation and initial embryonic development of bovine oocytes was studied. Two experiments were performed: in the first, different concentrations (0, 50, 75 or 100µM) and different exposure periods (2, 4 or 6 hours) to bohemine or roscovitine were tested for activation rates of in vitro matured (IVM) bovine oocytes, which were pre-exposed to ionomycin. The best treatments, 75µM bohemine and 50µM roscovitine, both for 6h, were used in the second experiment, in which IVM bovine oocytes were exposed to ionomycin, followed or not by bohemine or roscovitine treatment, and evaluated for nuclear status, activation rate and blastocyst development were assessed. The combined treatments (ionomycin + cyclin-dependent kinases inhibitors - CDKIs) showed better results for activation rates (77.3%) and initial embryonic development (35.2%) than the single ionomycin treatment (69.4% for activation and 21.9% for development); and also lead to a more uniform activation (nearly 90% single pronucleus development). The results showed that CDKIs improve the effects of ionomycin on parthenogenetic activation and blastocyst development in bovine oocytes and could help to achieve more efficient activation protocols, increasing the developmental competence of embryos obtained by reproductive biotechniques.

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Diagnosis and Treatment of Male Infertility-Related Fertilization Failure

Journal of Clinical Medicine ◽

10.3390/jcm9123899 ◽

2020 ◽

Vol 9 (12) ◽

pp. 3899

Author(s):

Arantxa Cardona Barberán ◽

Annekatrien Boel ◽

Frauke Vanden Meerschaut ◽

Dominic Stoop ◽

Björn Heindryckx

Keyword(s):

Male Infertility ◽

Current Knowledge ◽

Oocyte Activation ◽

Fertilization Failure ◽

Sperm Abnormalities ◽

Normal Sperm ◽

Activation Mechanisms ◽

Sperm Factor ◽

Male Factors ◽

Assisted Oocyte Activation

Infertility affects approximately 15% of reproductive-aged couples worldwide, of which up to 30% of the cases are caused by male factors alone. The origin of male infertility is mostly attributed to sperm abnormalities, of which many are caused by genetic defects. The development of intracytoplasmic sperm injection (ICSI) has helped to circumvent most male infertility conditions. However, there is still a challenging group of infertile males whose sperm, although having normal sperm parameters, are unable to activate the oocyte, even after ICSI treatment. While ICSI generally allows fertilization rates of 70 to 80%, total fertilization failure (FF) still occurs in 1 to 3% of ICSI cycles. Phospholipase C zeta (PLCζ) has been demonstrated to be a critical sperm oocyte activating factor (SOAF) and the absence, reduced, or altered forms of PLCζ have been shown to cause male infertility-related FF. The purpose of this review is to (i) summarize the current knowledge on PLCζ as the critical sperm factor for successful fertilization, as well as to discuss the existence of alternative sperm-induced oocyte activation mechanisms, (ii) describe the diagnostic tests available to determine the cause of FF, and (iii) summarize the beneficial effect of assisted oocyte activation (AOA) to overcome FF.

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Supplementation of 9-cis retinoic acid in the in vitro maturation medium increase blastocyst development rate and quality

Asian Journal of Medical and Biological Research ◽

10.3329/ajmbr.v3i4.35343 ◽

2018 ◽

Vol 3 (4) ◽

pp. 516-520 ◽

Cited By ~ 1

Author(s):

Rabiul Islam ◽

Gautam Kumar Deb ◽

Md Ahsanul Kabir ◽

Md Faizul Hossain Miraz ◽

Talukder Nurun Nahar ◽

...

Keyword(s):

Retinoic Acid ◽

Embryonic Development ◽

In Vitro Maturation ◽

Control Group ◽

Blastocyst Development ◽

Media Control ◽

Maturation Medium ◽

Medium Increase

The 9-cis retinoic acid (9-cisRA) enhances early embryonic development in both in vitro and in vivo conditions. This experiment was conducted to evaluate the effect of supplementation of 9-cisRA in the in vitro maturation (IVM) medium on embryo development efficiency and embryo quality. For this purpose, immature cumulus oocyte complexes (COC) collected from slaughterhouse derived bovine ovaries were matured in three different IVM media (control group, DMSO group and DMSO+RA group). In the control group, base IVM medium were used without supplementation of 9-cisRA and DMSO. In the DMSO group, base IVM medium was supplemented with 0.5 μl DMSO per ml IVM medium without 9-cisRA. In DMSO+RA group, base medium was supplemented with 5 nm 9-cisRA dissolved in 0.5 μl DMSO. Data were analyzed using one way ANOVA method and means were compared using Duncan’s multiple range test. Results showed that, supplementation of 9-cisRA in the maturation medium has no effect on embryonic development uptocleavage stage. However, blastocyst development rates (P>0.01), total blastomere number (P> 0.01), number of apoptotic blastomere per blastocyst (P>0.05) and percent of apoptotic blastomere per blastocyst (P>0.05) were significantly influences by 9-cisRA. In conclusion, 9-cisRA may be supplemented into the maturation medium for increasing bovinein vitro blastocyst development efficiency and blastocyst quality.Asian J. Med. Biol. Res. December 2017, 3(4): 516-520

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Superovulation, fertilization and in vitro embryo development in mice after administration of an inhibin-neutralizing antiserum

Reproduction ◽

10.1530/rep.0.1220809 ◽

2001 ◽

pp. 809-816 ◽

Cited By ~ 31

Author(s):

H Wang ◽

CB Herath ◽

G Xia ◽

G Watanabe ◽

K Taya

Keyword(s):

Chorionic Gonadotrophin ◽

Developmental Competence ◽

Alpha Subunit ◽

Control Group ◽

Control Groups ◽

Blastocyst Development ◽

Embryo Production ◽

Adult Mice

The present study was conducted to investigate the effect of immunoneutralization against endogenous inhibin on oocyte and embryo production in adult and immature mice. At 12:00 h on day 2 of oestrus (day 1 of dioestrus), a single i.p. injection of inhibin antiserum (50, 100, 200 or 400 microl per animal) or equine chorionic gonadotrophin (eCG; 10 or 20 iu per animal) or control goat serum (100 microl per animal) was administered to adult female mice. After 48 h, the mice in each of the three groups were given a single i.p. injection of hCG (10 iu per animal). At 42 h after hCG injection, ova were collected from oviducts and cultured in KSOM solution. Treatments with both inhibin antiserum-hCG and eCG-hCG induced superovulation in all the animals tested. The number of oocytes in animals treated with inhibin antiserum was significantly higher (P < 0.05) compared with the control group, and the number of oocytes ovulated in animals treated with 200 or 400 ml inhibin antiserum was significantly (P < 0.05) higher than that in animals treated with 10 or 20 iu eCG. The superovulated oocytes that were fertilized normally in vivo were able to form blastocysts in vitro. The rate of blastocyst development for animals treated with 50-200 ml inhibin antiserum was significantly (P < 0.05) higher than that of the eCG-treated animals. Irrespective of the day of the oestrous cycle, 200 microl inhibin antiserum administered at 12:00 h on each of 4 days induced superovulation in all the animals tested. The rates of oocyte and embryo production by these animals were significantly (P < 0.05) higher than in the control groups. Furthermore, administration of inhibin antiserum at doses of 50, 100, 200 or 400 ml produced similar results in 26-day-old immature mice. These results indicate that passive immunoneutralization of endogenous inhibin alpha-subunit induces superovulation in immature and adult mice. The superovulated oocytes obtained by administration of inhibin antiserum have normal embryonic developmental competence. Thus, it is concluded that this inhibin antiserum method is a new practical alternative for induction of superovulation in mice instead of the more commonly used eCG-hCG protocol.

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The oocyte activation and Ca2+ oscillation-inducing abilities of mouse and human dead (sonicated) spermatozoa

Zygote ◽

10.1017/s0967199408005157 ◽

2009 ◽

Vol 17 (2) ◽

pp. 175-184 ◽

Cited By ~ 10

Author(s):

Hiroyuki Yazawa ◽

Kaoru Yanagida ◽

Shoutaro Hayashi ◽

Akira Sato

Keyword(s):

Laser Scanning ◽

Confocal Laser Scanning Microscope ◽

Confocal Laser ◽

Oocyte Activation ◽

Blastocyst Development ◽

Time Intervals ◽

Mouse Oocytes ◽

Long Time ◽

Activation Rates ◽

Selection Of

SummaryIn ICSI procedures, it is well known that the selection of viable (live) spermatozoa and certain types of immobilization prior to injection is very important for obtaining successful results, but unfortunately there are rare situations when only immotile spermatozoa are available (such as in severe asthenozoospermia or necrozoospermia). In such cases, failure of oocyte activation after ICSI often occurs and may be due to the lack of SOAF (sperm-borne oocyte activating factor) activity. In order to investigate the SOAF activities of dead spermatozoa, mouse and human spermatozoa were immobilized (killed by sonication), maintained in THF medium for varying time intervals (up to 72 h) and then injected into mature unfertilized mouse oocytes. Injected mouse oocytes were examined for their activation, development into blastocysts and Ca2+ responses by imaging and confocal laser scanning microscope. The rates of oocyte activation, blastocyst development and normal patterns of Ca2+ oscillation from the killed-sperm-injected oocytes decreased gradually in accordance with the maintenance interval between sonication and injection. For injection with mouse sonicated spermatozoa, the rate of normal Ca2+ oscillations declined first (after a 3 h maintenance interval) and then blastocyst development was gradually obstructed (after approx. 10 h). The oocyte activation-inducing ability of dead spermatozoa was maintained for a relatively long period, but began to decline after 20 h. The activation rates and Ca2+ response of the oocytes that were injected with human sonicated spermatozoa decreased earlier than those injected with mouse spermatozoa. Although the oocyte activation-inducing ability was maintained for a relatively long time after the death of the spermatozoa, embryo development into blastocysts and the rate of normal Ca2+ oscillations declined after a short maintenance interval between sonication and injection. The Ca2+ response seemed to be the most sensitive indicator for the evaluating the SOAF activity of dead (killed) spermatozoa.

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Facilitated migration of cells from a transected peripheral nerve over collagen fibers: in vivo observations

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156420 ◽

1989 ◽

Vol 47 ◽

pp. 888-889

Author(s):

Arthur J. Wasserman ◽

Azam Rizvi ◽

George Zazanis ◽

Frederick H. Silver

Keyword(s):

Sciatic Nerve ◽

Peripheral Nerve ◽

Collagen Gel ◽

Collagen Fibers ◽

Control Group ◽

Guide Tube ◽

Sprague Dawley ◽

Silicone Tube ◽

Thin Sections

In cases of peripheral nerve damage the gap between proximal and distal stumps can be closed by suturing the ends together, using a nerve graft, or by nerve tubulization. Suturing allows regeneration but does not prevent formation of painful neuromas which adhere to adjacent tissues. Autografts are not reported to be as good as tubulization and require a second surgical site with additional risks and complications. Tubulization involves implanting a nerve guide tube that will provide a stable environment for axon proliferation while simultaneously preventing formation of fibrous scar tissue. Supplementing tubes with a collagen gel or collagen plus extracellular matrix factors is reported to increase axon proliferation when compared to controls. But there is no information regarding the use of collagen fibers to guide nerve cell migration through a tube. This communication reports ultrastructural observations on rat sciatic nerve regeneration through a silicone nerve stent containing crosslinked collagen fibers.Collagen fibers were prepared as described previously. The fibers were threaded through a silicone tube to form a central plug. One cm segments of sciatic nerve were excised from Sprague Dawley rats. A control group of rats received a silicone tube implant without collagen while an experimental group received the silicone tube containing a collagen fiber plug. At 4 and 6 weeks postoperatively, the implants were removed and fixed in 2.5% glutaraldehyde buffered by 0.1 M cacodylate containing 1.5 mM CaCl2 and balanced by 0.1 M sucrose. The explants were post-fixed in 1% OSO4, block stained in 1% uranyl acetate, dehydrated and embedded in Epon. Axons were counted on montages prepared at a total magnification of 1700x. Montages were viewed through a dissecting microscope. Thin sections were sampled from the proximal, middle and distal regions of regenerating sciatic plugs.

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In-vitro- und In-vivo-Wirkung von Schilddrüsenhormon auf das Wachstum von Neuroblastomzellen

Nuklearmedizin ◽

10.1055/s-0038-1629520 ◽

1990 ◽

Vol 29 (03) ◽

pp. 120-124

Author(s):

R. P. Baum ◽

E. Rohrbach ◽

G. Hör ◽

B. Kornhuber ◽

E. Busse

Keyword(s):

Cell Differentiation ◽

Neuroblastoma Cells ◽

Control Group ◽

Initial Value ◽

Initial Rise ◽

Biphasic Effect ◽

Contrast Microscopy ◽

Morphological Sign

The effect of triiodothyronine (T3) on the differentiation of cultured neuroblastoma (NB) cells was studied after 9 days of treatment with a dose of 10-4 M/106 cells per day. Using phase contrast microscopy, 30-50% of NB cells showed formation of neurites as a morphological sign of cellular differentiation. The initial rise of the mitosis rate was followed by a plateau. Changes in cyclic nucleotide content, in the triphosphates and in the activity of the enzyme ornithine decarboxylase (ODC) were assessed in 2 human and 2 murine cell lines to serve as biochemical parameters of the cell differentiation induced by T3. Whereas the cAMP level increased significantly (3 to 7 fold compared with its initial value), the cGMP value dropped to 30 to 50% of that of the control group. ATP and GTP increased about 200%, the ODC showed a decrease of about 50%. The present studies show a biphasic effect of T3 on neuroblastoma cells: the initial rise of mitotic activity is followed by increased cell differentiation starting from day 4 of the treatment.

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