Fertilisation in the Rabbit

1932 ◽  
Vol 9 (4) ◽  
pp. 403-408
Author(s):  
G. PINCUS ◽  
E. V. ENZMANN
Keyword(s):  
Zona Pellucida ◽  
Critical Period ◽  
Polar Body ◽  
Follicle Cells ◽  
Fallopian Tubes ◽  
Sperm Penetration ◽  
Pass Through ◽  
Second Polar Body

The series of events occurring in the Fallopian tubes of rabbit does mated to fertile bucks may be summarised as follows: The ova liberated from the ovaries and surrounded by the follicle cells become massed together. Sperm penetrate the massed follicle cells, which fall away as the sperm pass through them. At from 1½-3 hours after ovulation the spermatozoa reach the egg. A number of spermatozoa pass through the zona pellucida, but only one, apparently, enters the egg. At the time of sperm penetration the egg shrinks slightly but definitely. The second polar body is given off 45 min. or longer after sperm penetration. The pronuclei are formed after the formation of the second polar body and, at the earliest, 3 hours after ovulation. The critical period for sperm penetration appears to occur at 2-3 hours after ovulation (cf. Pincus,1930).

The effect of osmolarity on mouse parthenogenesis

Development ◽  
1974 ◽  
Vol 31 (2) ◽  
pp. 513-526
Author(s):  
M. H. Kaufman ◽  
M. A. H. Surani
Keyword(s):  
Protein Synthesis ◽  
Culture Medium ◽  
Culture Media ◽  
Polar Body ◽  
Amino Acid Mixture ◽  
Culture Conditions ◽  
Follicle Cells ◽  
Acid Mixture ◽  
Second Polar Body

Eggs from (C57B1 × A2G)F1 mice were activated by treatment with hyaluronidase, which removed the follicle cells, and cultured in vitro. Observations were made 6–8 h after hyaluronidase treatment to determine the frequency of activation and the types of parthenogenones induced. Cumulus-free eggs resulting from hyaluronidase treatment were incubated for 2¼ h in culture media of various osmolarities. The frequency of activation was found to be dependent on the postovulatory age of oocytes, while the types of parthenogenones induced were dependent on the osmolarity of the in vitro culture medium and their postovulatory age. Culture in low osmolar medium suppressed the extrusion of the second polar body (2PB). This decreased the incidence of haploid eggs with a single pronucleus and 2PB and immediately cleaved eggs from 97·5% to 42·3% of the activated population. Where 2PB extrusion had been suppressed, 97·4% of parthenogenones contained two haploid pronuclei. Very few were observed with a single and presumably diploid pronucleus. Serial observations from 11 to 18 h after hyaluronidase treatment were made on populations of activated eggs as they entered the first cleavage mitosis after 2¼ h incubation in medium either of normal (0·287 osmol) or low (0·168 osmol) osmolarity. A delay in the time of entry into the first cleavage mitosis similar to the duration of incubation in low osmolar medium was observed. Further, eggs were incubated in control and low osmolar culture media containing uniformly labelled [U-14C]amino acid mixture to examine the extent of protein synthesis in recently activated eggs subjected to these culture conditions. An hypothesis is presented to explain the effect of incubation in low osmolar culture medium in delaying the first cleavage mitosis.

133 VITRIFICATION CAUSES DAMAGE OF THE ANTIOXIDANT DEFENSE SYSTEM IN IVM PORCINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 184 ◽  
Author(s):  
T. Somfai ◽  
M. Ozawa ◽  
J. Noguchi ◽  
H. Kaneko ◽  
K. Ohnuma ◽  
...  
Keyword(s):  
Polar Body ◽  
Antioxidant Defense System ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Cleavage Rate ◽  
Sperm Penetration ◽  
Second Polar Body ◽  
Polar Body Extrusion

The present study investigated the ability of in vitro-matured (IVM) porcine oocytes to be fertilized in vitro after vitrification. Oocytes matured in vitro for 46 h according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) were cryopreserved by solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) or subjected to the steps of SSV without cooling (toxicity control, TC). Oocyte viability was assessed 2 h after treatment by morphology and fluorescein diacetate staining. Live oocytes were in vitro-fertilized (IVF) and cultured (IVC) for 6 days according to Kikuchi et al. (2002). Fertilization and pronuclear development of oocytes were assessed 10 h after IVF by aceto-orcein staining. Cleavage and blastocyst rates were recorded during IVC. Glutathione (GSH) and hydrogen peroxide levels in oocytes were analyzed by DTNB-glutathione disulfide reductase recycling assay and 20,70-dichlorofluorescein fluorescence assay, respectively. Data were analyzed by ANOVA and paired t-test. The rate of live oocytes after SSV was lower compared to the control and the TC groups (54.4%, 100%, and 100%, respectively; P < 0.05). Sperm penetration rates of SSV oocytes were lower than those of the control group (51.9% and 67.8%, respectively; P < 0.05). Significantly fewer penetrated oocytes in the SSV group formed male pronuclei than those in the control and the TC groups (66.7%, 96.5%, and 98.5%, respectively; P < 0.05). There were no differences in second polar body extrusion and monospermy rates between the treatment groups. The cleavage rate of SSV oocytes was significantly lower than that of the control and the TC groups (13.3%, 46.6%, and 47.7%, respectively; P < 0.05). Blastocyst rates of control and TC oocytes were similar (20.7% and 23.6%, respectively), whereas only a single embryo developed to the blastocyst stage in the SSV group. GSH content of SSV oocytes was significantly lower than that of the control oocytes (7.3 pM and 10.5 pM, respectively), whereas the peroxide level was higher in SSV oocytes than in the control oocytes (59.0 and 50.5 FIU, respectively; P < 0.05). Our results reveal a cryopreservation-related drop of intracellular GSH level in oocytes, which may cause their decreased ability to form a male pronucleus and their increased sensitivity to oxidative stress. These factors might contribute to the low developmental competence of vitrified oocytes. This work was supported by a grant-in-aid for the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Foreign Researchers (P05648) and the Bilateral Scientific and Technological Collaboration Grant between Hungary and Japan (TET, no. JAP-11/02).

Timing of the First Cleavage Division of Haploid Mouse Eggs, and the Duration of its Component Stages

1973 ◽  
Vol 13 (2) ◽  
pp. 553-566 ◽  
Author(s):  
M. H. KAUFMAN
Keyword(s):  
Polar Body ◽  
Time Lapse ◽  
Follicle Cells ◽  
Cleavage Division ◽  
Cell Stage ◽  
Cell Embryo ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Mouse Eggs

Mouse eggs were activated by treatment with hyaluronidase which removed the follicle cells, followed by culture in vitro, and examined at the first cleavage mitosis. Second polar body extrusion usually occurred and haploid parthenogenesis was initiated. Air-dried chromosome preparations were made between 11 and 15.5 h after activation. Out of the 308 eggs examined 74 had already progressed to the 2-cell stage; the remaining 234 at the 1-cell stage were examined in detail. All chromosome preparations of the first cleavage mitosis were classified into groups corresponding with the stages of prometaphase, metaphase (early or ‘pre-chromatid’, ‘chromatid’ and ‘late chromatid’) and anaphase. An indirect estimate was made of the duration of the first cleavage mitosis and of its component stages from the incidence of stages observed at different time intervals after activation. Similar eggs were also observed at 37 °C by time-lapse cine-photography and the interval between the disappearance of the pronucleus to the beginning of telophase of the first cleavage division was determined. The results of timing studies on the haploid eggs were compared with results obtained from similar observations on the first cleavage division of fertilized eggs which would of course normally be diploid. Artificially activated eggs with 2 pronuclei, resulting from second polar body suppression, were also examined, and serial chromosome preparations during mitosis showed that the 2 pronuclear chromosome groups unite on the first cleavage spindle and divide to give a hetero-zygous diploid 2-cell embryo.

Effects of extremely low osmolarity on fertilized mouse eggs

Development ◽  
1977 ◽  
Vol 37 (1) ◽  
pp. 65-77
Author(s):  
Jolanta Opas
Keyword(s):  
Water Treatment ◽  
Zona Pellucida ◽  
Polar Body ◽  
Preimplantation Development ◽  
Distilled Water ◽  
Second Polar Body ◽  
Mouse Eggs

When fertilized one-cell eggs are subjected to distilled water treatment for 2–6 min, cytoplasm bulges through the sperm-slit in the zona pellucida and forms a cytoplasmic fragment (CF). CFs were observed in 86·5 % of eggs; in 20·9 % of cases CFs contained a pronucleus (or pronuclei). In 53·4 % of eggs permanent incorporation of the second polar body (2 P.B.) into the egg cytoplasm occurred. These phenomena occurring in different combinations produced 6·2 % of haploid eggs, 10·3 % of diploid eggs with a pronucleus replaced by 2 P.B. nucleus, and 43·1 % of triploid eggs. 4·4 % t of eggs were enucleated. The remaining group comprised diploid eggs which were either not affected by the treatment (6·4 %) or lost a certain amount of cytoplasm by formation of an anucleate CF (29·6%). The frequencies of the types of reaction were related to the post-fertilization stage of eggs. All eggs except the enucleated ones were able to develop to the stage of morula or blastocyst. Triploids developed until the 12th day of pregnancy and diploids that had lost up to 15 % of the cytoplasm developed to term. There was a twofold reduction in the percentage of preimplantation development when treated eggs originated from induced rather than spontaneous ovulation.

scholarly journals Roles of heterotrimeric and monomeric G proteins in sperm-induced activation of mouse eggs

Development ◽  
1994 ◽  
Vol 120 (11) ◽  
pp. 3313-3323 ◽  
Author(s):  
G.D. Moore ◽  
T. Ayabe ◽  
P.E. Visconti ◽  
R.M. Schultz ◽  
G.S. Kopf
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Zona Pellucida ◽  
Polar Body ◽  
Active Form ◽  
Dominant Negative ◽  
Egg Activation ◽  
Cell Stage ◽  
Second Polar Body ◽  
Mouse Eggs

Results of several lines of experimentation suggest that sperm-induced egg activation has several features in common with G protein-coupled receptor signal transduction mechanisms. We report that microinjection of GDP beta S into metaphase II-arrested mouse eggs blocks sperm-induced egg activation. Since GDP beta S inactivates both heterotrimeric and monomeric classes of G proteins, the involvement of members of each of these families in sperm-induced egg activation was evaluated. Neither pertussis toxin treatment of eggs nor microinjection of eggs with inhibitory antibodies toward G alpha q blocked sperm-induced egg activation. Nevertheless, microinjection of phosducin, a protein that binds tightly to free G protein beta gamma subunits, specifically inhibited second polar body emission, the fertilization evoked decrease of H1 kinase activity and pronucleus formation. Microinjection of phosducin, however, did not inhibit the fertilization-induced modifications of the zona pellucida and microinjection of beta gamma t did not result in egg activation in the absence of sperm. Inactivation of the monomeric Rho family of G proteins with C3 transferase from Clostridium botulinum inhibited emission of the second polar body and cleavage to the 2-cell stage, but did not affect the modifications of the zona pellucida or pronucleus formation. Microinjection of Rasval12, which is a constitutively active form of Ras, did not result in egg activation in the absence of sperm. Moreover, microinjection of either an anti-Ras neutralizing antibody (Y13-259) or a dominant negative form of Ras (RasT) did not affect events of sperm-induced egg activation. In contrast, microinjection of RasT inhibited embryo cleavage to the 2-cell stage. These results suggest that both heterotrimeric and monomeric G proteins are involved in various aspects of sperm-induced egg activation.

scholarly journals Acrosin is essential for sperm penetration through the zona pellucida in hamsters

2020 ◽  
Vol 117 (5) ◽  
pp. 2513-2518 ◽  
Author(s):  
Michiko Hirose ◽  
Arata Honda ◽  
Helena Fulka ◽  
Miwa Tamura-Nakano ◽  
Shogo Matoba ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Female Genital Tract ◽  
Vitro Fertilization ◽  
Sperm Penetration ◽  
New Gene ◽  
Pass Through ◽  
Mammalian Spermatozoa ◽  
Female Genital

During natural fertilization, mammalian spermatozoa must pass through the zona pellucida before reaching the plasma membrane of the oocyte. It is assumed that this step involves partial lysis of the zona by sperm acrosomal enzymes, but there has been no unequivocal evidence to support this view. Here we present evidence that acrosin, an acrosomal serine protease, plays an essential role in sperm penetration of the zona. We generated acrosin-knockout (KO) hamsters, using an in vivo transfection CRISPR/Cas9 system. Homozygous mutant males were completely sterile. Acrosin-KO spermatozoa ascended the female genital tract and reached ovulated oocytes in the oviduct ampulla, but never fertilized them. In vitro fertilization (IVF) experiments revealed that mutant spermatozoa attached to the zona, but failed to penetrate it. When the zona pellucida was removed before IVF, all oocytes were fertilized. This indicates that in hamsters, acrosin plays an indispensable role in allowing fertilizing spermatozoa to penetrate the zona. This study also suggests that the KO hamster system would be a useful model for identifying new gene functions or analyzing human and animal disorders because of its technical facility and reproducibility.

Later stages of sperm penetration and second polar body and blastodisc formation in the egg ofFundulus heteroclitus

1985 ◽  
Vol 234 (3) ◽  
pp. 423-439 ◽  
Author(s):  
Anna Ruth Brummett ◽  
James N. Dumont ◽  
Carole S. Richter
Keyword(s):  
Polar Body ◽  
Sperm Penetration ◽  
Second Polar Body

scholarly journals P57 A new technique for polar body biopsy with laser tweezers and femtosecond laser zona pellucida drilling

2010 ◽  
Vol 20 ◽  
pp. S41-S42
Author(s):  
S.A. Sergeev ◽  
M.M. Rakityanskiy ◽  
Y.V. Khramova ◽  
M.L. Semenova ◽  
A.A. Smirnova ◽  
...  
Keyword(s):  
Femtosecond Laser ◽  
Zona Pellucida ◽  
Polar Body ◽  
New Technique ◽  
Laser Tweezers ◽  
Polar Body Biopsy ◽  
A New Technique

Preservation of sperm within the mouse cauda epididymidis in salt or sugars at room temperature

Zygote ◽  
2010 ◽  
Vol 18 (3) ◽  
pp. 245-256 ◽  
Author(s):  
Tetsuo Ono ◽  
Eiji Mizutani ◽  
Chong Li ◽  
Teruhiko Wakayama
Keyword(s):  
Room Temperature ◽  
Polar Body ◽  
Methylation Status ◽  
Cost Effective ◽  
Oocyte Activation ◽  
Male Gametes ◽  
Effective Manner ◽  
Cell Embryo ◽  
Cauda Epididymidis ◽  
Second Polar Body

SummaryThe development of preservation techniques for male gametes at room temperature might allow us to store them in a simple and cost-effective manner. In this study, we studied the use of pure salt or sugar to preserve the whole cauda epididymidis, because it is known that food can be preserved in this way at room temperature for long periods. Mouse epididymides were placed directly in powdered salt (NaCl) or sugars (glucose or raffinose) for 1 day to 1 year at room temperature. Spermatozoa were recovered from the preserved organs after being rehydrated with medium and then isolated sperm heads were microinjected into fresh oocytes. Importantly, the oocyte activation capacity of spermatozoa was maintained after epididymal storage in NaCl for 1 year, whereas most untreated spermatozoa failed to activate oocytes within 1 month of storage. Pronuclear morphology, the rate of extrusion of a second polar body and the methylation status of histone H3 lysine 9 (H3K9me3) in those zygotes were similar to those of zygotes fertilized with fresh spermatozoa. However, the developmental ability of the zygotes decreased within 1 day of sperm storage. This effect led to nuclear fragmentation at the 2-cell embryo stage, irrespective of the storage method used. Thus, although the preserved sperm failed to allow embryo development, their oocyte activation factors were maintained by salt storage of the epididymis for up to 1 year at room temperature.

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