scholarly journals Successful blastocyst production by intracytoplasmic injection of sperm after in vitro maturation of follicular oocytes obtained from immature female squirrel monkeys (Saimiri boliviensis)

2021 ◽  
Author(s):  
Shinichi WATANABE ◽  
Megumi MIURA ◽  
Hiromi MORITA ◽  
Moeka NISHI ◽  
Shin-ichi YOKOTA ◽  
...  
Keyword(s):  
Squirrel Monkeys ◽  
Immature Female ◽  
Intracytoplasmic Injection ◽  
Saimiri Boliviensis

IN-VITRO STUDIES OF THE EFFECTS OF OESTROGEN PRETREATMENT ON THE SENSITIVITY OF THE IMMATURE FEMALE RAT PITUITARY GLAND TO STIMULATION WITH GONADOTROPHIN RELEASING HORMONE

1977 ◽  
Vol 74 (1) ◽  
pp. 11-21 ◽  
Author(s):  
M. WILKINSON ◽  
D. DE ZIEGLER ◽  
DANIELLE CASSARD ◽  
K. B. RUF
Keyword(s):  
Pituitary Gland ◽  
Brain Lesion ◽  
Culture Technique ◽  
Female Rats ◽  
Female Rat ◽  
Immature Female ◽  
Releasing Hormone ◽  
Gonadotrophin Releasing Hormone ◽  
Unilateral Brain Lesion

The effects of oestrogen priming on the sensitivity of the anterior pituitary gland to stimulation with gonadotrophin releasing hormone (GnRH) was investigated in immature female rats using a new organ culture technique. Hemipituitary glands obtained from animals primed with a single dose of oestradiol benzoate (OB; 20 μg/100 g body weight) released significantly more LH when pulsed with GnRH (4 nmol/l) than did control hemipituitary glands. This potentiating effect was detectable as early as 5 days after birth. After a second stimulation, LH secretion remained high. These results were compared with those obtained from animals treated to induce increased levels of endogenous oestrogen on day 26 of life. Thus, hemipituitary glands were obtained from animals given two injections of OB, an injection of pregnant mare serum gonadotrophin (PMSG) or a unilateral brain lesion placed in the basal hypothalamus. Pituitary tissue was stimulated as before with a pulse of GnRH. Two injections of OB enhanced the sensitivity to stimulation. Conversely, both PMSG and lesion treatment severely reduced the sensitivity to GnRH, although PMSG-treated and lesioned animals have been used as models for the study of ovulation.

scholarly journals The Intriguing Effects of Substituents in the N-Phenethyl Moiety of Norhydromorphone: A Bifunctional Opioid from a Set of “Tail Wags Dog” Experiments

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2640 ◽  
Author(s):  
Meining Wang ◽  
Thomas C. Irvin ◽  
Christine A. Herdman ◽  
Ramsey D. Hanna ◽  
Sergio A. Hassan ◽  
...  
Keyword(s):  
Partial Agonist ◽  
Squirrel Monkeys ◽  
The Body ◽  
In Vitro Assays ◽  
Potency Ratio ◽  
Gtpγs Binding ◽  
Potent Agonist ◽  
Optically Pure ◽  
Stimulation Of

(−)-N-Phenethyl analogs of optically pure N-norhydromorphone were synthesized and pharmacologically evaluated in several in vitro assays (opioid receptor binding, stimulation of [35S]GTPγS binding, forskolin-induced cAMP accumulation assay, and MOR-mediated β-arrestin recruitment assays). “Body” and “tail” interactions with opioid receptors (a subset of Portoghese’s message-address theory) were used for molecular modeling and simulations, where the “address” can be considered the “body” of the hydromorphone molecule and the “message” delivered by the substituent (tail) on the aromatic ring of the N-phenethyl moiety. One compound, N-p-chloro-phenethynorhydromorphone ((7aR,12bS)-3-(4-chlorophenethyl)-9-hydroxy-2,3,4,4a,5,6-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7(7aH)-one, 2i), was found to have nanomolar binding affinity at MOR and DOR. It was a potent partial agonist at MOR and a full potent agonist at DOR with a δ/μ potency ratio of 1.2 in the ([35S]GTPγS) assay. Bifunctional opioids that interact with MOR and DOR, the latter as agonists or antagonists, have been reported to have fewer side-effects than MOR agonists. The p-chlorophenethyl compound 2i was evaluated for its effect on respiration in both mice and squirrel monkeys. Compound 2i did not depress respiration (using normal air) in mice or squirrel monkeys. However, under conditions of hypercapnia (using air mixed with 5% CO2), respiration was depressed in squirrel monkeys.

Development and implementation of intracytoplasmic sperm injection (ICSI)

1995 ◽  
Vol 7 (2) ◽  
pp. 211 ◽  
Author(s):  
GD Palermo ◽  
J Cohen ◽  
M Alikani ◽  
A Adler ◽  
Z Rosenwaks
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Human Sperm ◽  
Fertilization Rate ◽  
Semen Parameters ◽  
Ongoing Pregnancy Rate ◽  
Vitro Fertilization ◽  
Human Oocytes ◽  
Intracytoplasmic Injection ◽  
Previously Treated

The purpose of this paper is to elucidate the experimental steps that led to the development of intracytoplasmic sperm injection (ICSI) and its application in the human. ICSI has become the most successful micromanipulation procedure for treating male infertility. A total of 355 in vitro fertilization (IVF) cycles utilizing ICSI are described; 180 couples were previously treated in 509 IVF cycles but achieved no fertilization and 175 couples could not be treated by IVF because of extremely poor semen parameters. Of the 3063 metaphase II (M II) oocytes retrieved, 2970 were injected with a survival rate of 93.6%, yielding 1917 bipronuclear zygotes (64.5%). In 148 patients, a foetal heart was evidenced by ultrasound; 11 of these patients miscarried between 7 and 13 weeks of gestation. The ongoing pregnancy rate was 38.6% (137/355) per retrieval and 40.5% (137/338) per embryo replacement. At the time of writing, there were 22 deliveries and one therapeutic abortion for a trisomy 21 chromosomal abnormality. In addition, 66 singleton, 37 twin, 10 triplet and 1 quadruplet pregnancies were ongoing. The concentration of motile spermatozoa in the ejaculate only slightly influenced the fertilization rate (P < 0.001) and the pregnancy outcome (P < 0.01). A preliminary injection procedure utilizing intracytoplasmic injection of isolated sperm heads was performed in 35 M II human oocytes with resultant fertilization and cleavage rates of 74% and 73% respectively. Skills in ICSI were acquired by injecting hamster and unfertilized human oocytes with human sperm. ICSI can be used to successfully treat couples who have failed IVF or who have too few spermatozoa for conventional in vitro insemination.(ABSTRACT TRUNCATED AT 250 WORDS)

47 ACTIVATION WITH IONOMYCIN FOLLOWED BY DEHYDROLEUCODINE AND CYTOCHALASIN B OF CLONED BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 181
Author(s):  
N. Canel ◽  
R. Bevacqua ◽  
D. Salamone
Keyword(s):  
Cytochalasin B ◽  
Combined Treatment ◽  
Cumulus Cells ◽  
Normal Embryo ◽  
Intracytoplasmic Injection ◽  
Glass Slides ◽  
Cloned Bovine ◽  
Pronuclear Formation ◽  
Vitro Development

A combined treatment of dehydroleucodine (DhL) and cytochalasin B (CB) was previously demonstrated to induce pronuclear formation of bovine oocytes (Canel and Salamone 2008 Reprod. Fertil. Dev. 21, 214-215). The aim of this study was to evaluate the potential of DhL combined with CB to induce diploid activation of parthenogenetic embryos and to employ this treatment to assist cloning by intracytoplasmic injection of whole cumulus cells. To do that, COCs were collected from cow ovaries obtained from a slaughterhouse and in vitro-matured in TCM-199, at 39°C under 6% CO2 in air for 24 h. After removal of cumulus cells, metaphase II (MII) oocytes were treated with 5 μM ionomycin (Io) for 4 min and randomly assigned to the following activation groups: a) DhL/CB (incubation with 1 μM DhL and 5 μg mL-1 CB, for 3 h); b) DhL/long CB (treatment DhL/CB for 3 h, followed by exposure to 5 μg mL-1 CB alone, for 3 additional hours); and c) DMAP (incubation with 2 mM 6-DMAP for 3 h). In experiment 1, activated oocytes underwent IVC for 48 h and cleaved embryos were treated with 1 μg mL-1 colchicine for 6 h, fixed on glass slides, and stained with 5% vol/vol Giemsa solution to assess chromosomal complements. In experiment 2, MII oocytes were mechanically enucleated and injected with whole cumulus cells obtained from IVM COCs. After 2 h, reconstructed eggs were treated with 5 μM Io for 4 min and randomly exposed to the activation treatments a, b, or c. Parthenogenetic control groups were also included. All embryos were cultured in SOF medium and rates of cleavage, morulae, and blastocysts were evaluated on Days 2, 5, and 8 (Table 1). Results showed that DhL/long CB diploidy rates were significantly higher than those of DhL/CB and DMAP (63.8, 40. and 31.6%, respectively; Fisher’s test, P < 0.05). Both DhL treatments induced polyploidy rates lower than DMAP (5.2, 10.6, and 31.6%, respectively; P < 0.05). Finally, Io followed by DhL/CB or DhL/long CB was able to induce cloned blastocyst rates not statistically different from Io plus DMAP (P > 0.05), but presumably with a higher degree of normal embryo ploidy. Table 1.In vitro development of bovine cloned embryos activated with DhL and CB

240 QUALITY AND VIABILITY OF IVF BOVINE EMBRYOS AFTER INTRACYTOPLASMIC INJECTION OF DNA–LIPOSOME COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 232
Author(s):  
L. N. Moro ◽  
G. Vichera ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Dna Fragmentation ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Transgenic Animals ◽  
Egfp Expression ◽  
Bovine Embryos ◽  
Exogenous Dna ◽  
Intracytoplasmic Injection

Transgenic animals have important applications in agriculture and human medicine; nevertheless the available techniques still remain inefficient and technically difficult. We have recently developed a novel method to transfect bovine embryos that consists of intracytoplasmic injection of exogenous DNA–liposome complexes (eDNA-LC) in IVF zygotes. This study was designed to evaluate the quality and viability of IVF bovine embryos, after intracytoplasmic injection of pCX-EGFP–liposome complexes (EGFP-LC) or pBCKIP2.8-liposome complexes (plasmid that codifies the human insulin gene, HI-LC). First, we evaluated embryo development and enhanced green fluorescent protein (EGFP) expression of IVF embryos injected with both plasmids separately. This treatment was analysed by Fisher's Exact test (P ≤ 0.05). Cleavage rates for EGFP-LC, HI-LC and IVF embryos injected with liposomes alone (IVF-L) and IVF control (IVF-C) were 62% (63/102), 67% (67/100), 66% (67/101) and 79% (98/124); blastocysts rates were 17% (17/102), 21% (21/100), 21% (21/101) and 23% (28/124), respectively. No statistical differences were seen among groups. The percentage of EGFP-positive embryos (EGFP+) after EGFP-LC injection was 42.9% after 3 days of culture and 41.8% at the blastocyst stage. In the second experiment, the blastocysts obtained, EGFP+ or EGFP-negative (EGFP–), were analysed by TUNEL assay at Day 6 (Bd6), 7 (Bd7) and 8 (Bd8) of in vitro culture, in order to evaluate the effect of the transgene and culture length, on DNA fragmentation. This treatment was analysed by the difference of proportions test (P ≤ 0.05) using statistical INFOSTAT software. All EGFP+ blastocysts showed TUNEL positive cells (T+). The percentage of T+ in Bd6, Bd7 and Bd8 were 91, 73.7 and 99.5%, respectively (P ≤ 0.05). EGFP– blastocysts showed lower fragmented nuclei (0, 44.6 and 85%, respectively; P ≤ 0.05). Groups IVF-L and IVF-C were also evaluated. In both groups, there was no evidence of DNA fragmentation in Bd6 and Bd7, but T+ were detected in Bd8 (66.4 and 85.8%, respectively; P ≤ 0.05). In the third experiment, bovine blastocysts obtained from the HI-LC group were individually transferred to recipient cows after 6 (n = 11), 7 (n = 5) and 8 (n = 5) days of culture post-IVF and HI-LC injection. The pregnancies obtained were from Bd6 [18.2% (2/11)] and Bd7 [40% (2/5)], although none of the recipients receiving Bd8 were diagnosed pregnant. Two pregnancies developed to term, one derived from Bd6 and the other from Bd7. Analysis by PCR determined that none of the born cows were transgenic. In summary, IVF bovine embryos could be easily transfected after the injection of eDNA-LC and the technique did not affect offspring viability. The results indicate that extended time in in vitro culture increases the percentage of fragmented nuclei in blastocysts. Moreover, this parameter increases in blastocysts with transgene expression compared with those without expression. Finally, more transfers are required in order to obtain the real efficiency of this new technique and to overcome the drawbacks generated by in vitro culture length and transgene expression.

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