Cytoplasmic maturation of the marsupial oocyte during the periovulatory period

1996 ◽  
Vol 8 (4) ◽  
pp. 509 ◽  
Author(s):  
KE Mate
Keyword(s):  
Zona Pellucida ◽  
Polar Body ◽  
Monodelphis Domestica ◽  
Cortical Granules ◽  
Nuclear Maturation ◽  
Bettongia Penicillata ◽  
Maturation In Vitro ◽  
First Polar Body

During the period immediately before ovulation, the oocytes of most eutherian and marsupial mammals complete the first meiotic maturation division and extrude the first polar body. In marsupials, this phase of nuclear maturation is accompanied by an increase in size of the egg and maturation of cytoplasmic components. Oocytes from at least four marsupial species, Trichosurus vulpecula, Macropus eugenii, Bettongia penicillata and Monodelphis domestica, continue to grow after formation of the follicular antrum and, although the rate of growth slows in larger follicles, it continues into the period immediately before ovulation. The basis of this growth is unknown but may include accumulation of fluid and/or yolk-like material. Maturational changes within the cytoplasm of the oocyte also occur during the periovulatory period, including the accumulation of cortical granules. Differences in the structure of the zona pellucida are also evident between follicular and ovulated eggs; these differences are suggestive of compression of the zona pellucida, but may involve the addition of extra material. These findings suggest that the marsupial oocyte may not achieve complete cytoplasmic maturity until after ovulation; however, their relevance to fertilization and embryonic development require further investigation. Like those of eutherian mammals, marsupial oocytes undergo spontaneous nuclear maturation once removed from the follicular environment, suggesting a basically similar control system. It is not known whether the preovulatory cytoplasmic changes seen in marsupial oocytes matured in vivo also occur during maturation in vitro.

scholarly journals THE COMPARATIVE BEHAVIOR OF MAMMALIAN EGGS IN VIVO AND IN VITRO

1935 ◽  
Vol 62 (5) ◽  
pp. 665-675 ◽  
Author(s):  
Gregory Pincus ◽  
E. V. Enzmann
Keyword(s):  
Polar Body ◽  
Pituitary Hormones ◽  
Follicle Cells ◽  
Nuclear Maturation ◽  
Sequence Of Events ◽  
First Polar Body ◽  
Chronological Sequence ◽  
Mammalian Eggs

1. A definite chronological sequence of events occurs in the eggs and follicles of rabbits after mating or after the injection of ovulation-inducing substances. The follicle secretes secondary liquor folliculi, and there occurs a separation of the corona radiata from strands connecting it to the follicle cells. The ovum goes through nuclear maturation with as climax the production of the first polar body by the 8th hour after copulation. 2. Thyroxin injections cause indirectly the same effects as mating or pituitary injections but no ovulation occurs. The thyroxin effect occurs later than the pituitary effect and is due to an initiation of atresia in the follicles. 3. Explantation of ova results in typical maturation phenomena which are apparently unaffected by the presence of pituitary hormones or of thyroxin in the culture medium. 4. It is concluded that maturation of the ovum can be obtained simply by isolating it from the normal follicular environment. 5. Normal fertilization can be secured with eggs removed from the follicles.

251 IS THERE A GAP BETWEEN NUCLEAR AND CYTOPLASMIC MATURATION IN IN VITRO-MATURED EQUINE OOCYTES?

2015 ◽  
Vol 27 (1) ◽  
pp. 215
Author(s):  
E. Claes ◽  
K. Smits ◽  
C. De Schauwer ◽  
B. Leemans ◽  
E. Wydooghe ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Polar Body ◽  
Cortical Granules ◽  
In Vitro Production ◽  
General Belief ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Cytoplasmic Maturation ◽  
Specific Stimulation

It is a general belief that as soon as the oocyte is recovered from the follicular environment, the nuclear maturation starts spontaneously in vitro, while specific stimulation for the cytoplasmic maturation is lacking (Gilchrist and Thompson 2007 Theriogenology 67, 6–15; Albuz et al. 2010 Hum. Reprod. 25, 2999–3011). As both nuclear and cytoplasmic maturation are required to prepare the oocyte for successful fertilization and embryonic development, a defective cytoplasmic maturation might be an important cause of low blastocyst rates in vitro (Albuz et al. 2010 Hum. Reprod. 25, 2999–3011). Nuclear and cytoplasmic maturation can be evaluated using fluorescent dyes. Assessment of nuclear maturation is typically based on the visualisation of chromatin, whereas cytoplasmic maturation is evaluated by the localization of cytoplasmic organelles [i.e. the cortical granules (CG)]. Equine oocytes were recovered from ovaries of slaughtered mares. After in vitro maturation (IVM; Smits et al. 2010 Vlaams Diergen. Tijds. 79, 134–138), oocytes were fixed and permeabilized. Subsequently, CG were labelled by incubation in 10 µg mL–1 FITC-labelled lens culinaris agglutinin during 15 min at RT. Chromatin was counterstained to verify the nuclear status with 20 µg mL–1 Hoechst 33342 during 15 min at RT. Stained oocytes with no or dispersed chromatin were classified as degenerated. Based on the absence or presence of the first polar body (PB), non-degenerated oocytes were either classified as nuclear immature (MI, no PB present) or nuclear mature (MII, PB present). The non-degenerated oocytes were further subdivided in 3 categories based on the migration of the CG: 1) cytoplasmic immature oocytes with (clusters of) CG randomly distributed throughout the ooplasm, 2) oocytes in transition stage with progressing CG migration to the oocyte cortex, and 3) cytoplasmic mature oocytes with a clearly visible CG monolayer just underneath the oolemma. The mean and standard deviation of nuclear and cytoplasmic parameters were calculated using Excel (Excel 2007, Microsoft Corp., Redmond, WA, USA). In 3 replicates, 86.6 ± 2.75% of all oocytes (131/151) demonstrated a corresponding nuclear and cytoplasmic maturation pattern (MI corresponding to CG1 and 2; MII corresponding to CG3). Only 12.0 ± 2.82% of the oocytes (16/133) revealed a cytoplasmic maturation pattern (CG 1 or 2) that lagged behind the nuclear maturation (MII). On the other hand, 22.2 ± 9.8% of the oocytes (4/18) were already cytoplasmic (CG3) but not yet nuclear matured (MI). Since most of the equine in vitro matured oocytes exhibited, surprisingly, a corresponding nuclear and cytoplasmic maturation pattern, it can be concluded that the gap between the nuclear and cytoplasmic maturation in vitro is less important than is generally believed. Consequently, the IVM step is not the main obstacle to increase the efficiency of the in vitro production process in horses.

Triphenyltin chloride induces spindle microtubule depolymerisation and inhibits meiotic maturation in mouse oocytes

2014 ◽  
Vol 26 (8) ◽  
pp. 1084 ◽  
Author(s):  
Yu-Ting Shen ◽  
Yue-Qiang Song ◽  
Xiao-Qin He ◽  
Fei Zhang ◽  
Xin Huang ◽  
...  
Keyword(s):  
Polar Body ◽  
Meiotic Maturation ◽  
Dependent Manner ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes ◽  
First Polar Body ◽  
Triphenyltin Chloride ◽  
Dose Dependent

Meiosis produces haploid gametes for sexual reproduction. Triphenyltin chloride (TPTCL) is a highly bioaccumulated and toxic environmental oestrogen; however, its effect on oocyte meiosis remains unknown. We examined the effect of TPTCL on mouse oocyte meiotic maturation in vitro and in vivo. In vitro, TPTCL inhibited germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) in a dose-dependent manner. The spindle microtubules completely disassembled and the chromosomes condensed after oocytes were exposed to 5 or 10 μg mL–1 TPTCL. γ-Tubulin protein was abnormally localised near chromosomes rather than on the spindle poles. In vivo, mice received TPTCL by oral gavage for 10 days. The general condition of the mice deteriorated and the ovary coefficient was reduced (P < 0.05). The number of secondary and mature ovarian follicles was significantly reduced by 10 mg kg–1 TPTCL (P < 0.05). GVBD decreased in a non-significant, dose-dependent manner (P > 0.05). PBE was inhibited with 10 mg kg–1 TPTCL (P < 0.05). The spindles of in vitro and in vivo metaphase II oocytes were disassembled with 10 mg kg–1 TPTCL. These results suggest that TPTCL seriously affects meiotic maturation by disturbing cell-cycle progression, disturbing the microtubule cytoskeleton and inhibiting follicle development in mouse oocytes.

44 PRODUCTION OF LIVE PIGLETS AFTER CRYOPRESERVATION OF IMMATURE PORCINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 136
Author(s):  
T. Somfai ◽  
K. Kikuchi ◽  
K. Yoshioka ◽  
F. Tanihara ◽  
H. Kaneko ◽  
...  
Keyword(s):  
Polar Body ◽  
Petri Dish ◽  
Developmental Competence ◽  
Basic Medium ◽  
High Survival ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Vitrification Solution

Development to term of vitrified porcine follicular oocytes is reported in the present study. Immature cumulus-oocyte complexes (COC) were collected from slaughtered prepubertal gilts and were vitrified according to our method published recently (Somfai et al. 2013 J. Reprod. Dev., in press). Briefly, after pretreatment with 7.5 μg mL–1 of cytochalasin B (CB) for 30 min in modified NCSU-37 (a basic medium, BM) at 38.5°C, groups of 88 to 121 COC were equilibrated in a mixture of 2% ethylene glycol (EG), 2% propylene glycol (PG), and 7.5 μg mL–1 CB for 13 to 15 min. Then, COC were washed in vitrification solution (17.5% EG, 17.5% PG, 5% polyvinyl pyrrolidone, and 0.3 M trehalose in BM) and then dropped with 2 μL of vitrification solution onto the surface of aluminum foil floating on liquid nitrogen (LN2). Microdroplets (each containing 10–25 COC) were transferred into cryotubes. After storage in LN2 for 2 to 4 weeks, the oocytes were warmed by dropping the microdroplets directly into 2.5 mL of warming solution (0.4 M trehalose in BM) kept in a 35-mm Petri dish on a 42°C hotplate for less than 1 min. Then, the warming dish was placed on a 38°C hotplate and COC were consecutively transferred for 1-min periods into BM containing 0.2, 0.1, or 0.05 M trehalose at 38°C. The COC were matured in vitro for 44 h using porcine oocyte medium (POM) supplemented with 10% follicular fluid (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Then, oocytes were denuded, and their live/dead status and nuclear maturation were determined by their morphology and the presence of the first polar body, respectively. To assess their developmental competence, vitrified and non-vitrified (control) oocytes were in vitro fertilized (IVF; Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) and then in vitro cultured in porcine zygote medium-5 (PZM-5; Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Blastocyst rates were recorded on Days 5, 6, and 7 of culture (Day 0 = the day of IVF). The experiment was replicated 4 times. Data were analysed with 1-way ANOVA and the Tukey test. The results revealed that 86.4% (364/424) of oocytes survived after vitrification, which was significantly lower (P < 0.05) than that of controls [100% (326/326)]. Live oocytes in vitrified and control groups did not differ statistically in terms of nuclear maturation (63.9 v. 65.3%). Blastocyst rates of surviving vitrified oocytes were significantly lower compared with controls on Days 5 (2.4 v. 12.7%), 6 (4.8 v. 17.6%), and 7 (5.6 v. 18.4%). To test their ability to develop to term, 16 and 27 blastocysts on Day 5 developing from vitrified COC were transferred into 2 recipients. Both recipients became pregnant and farrowed a total of 10 live piglets (4 and 6 piglets, respectively). These data demonstrate that large groups of immature porcine oocytes could be cryopreserved by this method showing high survival and maturation rates. Furthermore, despite a low rate of blastocyst development, transfer of Day-5 blastocysts generated from vitrified oocytes resulted in piglet production for the first time in the world. Partially supported by JSPS and HAS under the Japan-Hungary Research Cooperative Program.

74 IN VIVO SURVIVAL OF DOMESTIC CAT OOCYTES AFTER VITRIFICATION, INTRACYTOPLASMIC SPERM INJECTION, AND TRANSFER TO RECIPIENTS

2008 ◽  
Vol 20 (1) ◽  
pp. 118 ◽  
Author(s):  
M. C. Gómez ◽  
N. Kagawa ◽  
C. E. Pope ◽  
M. Kuwayama ◽  
S. P. Leibo ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Oocyte Retrieval ◽  
Cumulus Cells ◽  
Domestic Cat ◽  
Minimal Amount ◽  
Vitro Fertilization ◽  
First Polar Body

The ability to cryopreserve female gametes efficiently holds immense economic and genetic implications. The purpose of the present project was to determine if domestic cat oocytes could be cryopreserved successfully by use of the Cryotop method. We evaluated (a) cleavage frequency after in vitro fertilization (IVF) v. intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification, and (b) fetal development after transfer of resultant embryos into recipients. In vivo-matured cumulus–oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment, denuded of cumulus cells, and examined for the presence of the first polar body (PB). In vitro-matured COCs were obtained from ovaries donated by local clinics and placed into maturation medium for 24 h before cumulus cells were removed and PB status was determined. Oocytes were cryopreserved by the Cryotop method (Kuwayama et al. 2005 Reprod. Biomed. Online 11, 608–614) in a vitrification solution consisting of 15% DMSO, 15% ethylene glycol, and 18% sucrose. For IVF, oocytes were co-incubated with 1 � 106 motile spermatozoa mL–1 in droplets of modified Tyrode's medium in 5% CO2/air at 38�C (Pope et al. 2006 Theriogenology 66, 59–71). For ICSI, an immobilized spermatozoon was loaded into the injection pipette, which was then pushed through the zona pellucida into the ooplasm. After a minimal amount of ooplasm was aspirated into the pipette, the spermatozoon was carefully expelled, along with the aspirated ooplasm. After ICSI, or at 5 or 18 h post-insemination, in vivo- and in vitro-matured oocytes, respectively, were rinsed and placed in IVC-1 medium (Pope et al. 2006). As assessed by normal morphological appearance after liquefaction, the survival rate of both in vivo- and in vitro-matured oocytes was >90% (93–97%). For in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 73% (16/22) and 53% (30/57), respectively, as compared to 68% (19/28) after ICSI of vitrified oocytes (P > 0.05). For in vivo-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 55% (18/33) and 35% (6/17), respectively, compared to 50% (10/20) after ICSI of vitrified oocytes (P > 0.05). At 18–20 h after ICSI, 18 presumptive zygotes and four 2-cell embryos derived from vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo-matured and 12 in vitro-matured vitrified oocytes were transferred by laparoscopy into the oviducts of two recipients at 24–26 h after oocyte retrieval. The two recipients were 9-month-old IVF/ET-derived females produced with X-sperm sorted by flow cytometry. At ultrasonography on Day 22, both recipients were pregnant, with three live fetuses observed in one recipient and one live fetus seen in the second recipient. On Day 63 and Day 66 of gestation, four live kittens were born, without assistance, to the two recipients. The one male and three female kittens weighed an average of 131 g. In summary, in vivo viability of zygotes/embryos produced by ICSI of cat oocytes vitrified by the Cryotop method was demonstrated by the birth of live kittens following transfer to recipients.

228 microRNA REGULATION OF GENES IN BOVINE OOCYTES AND EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 272
Author(s):  
J. P. Barfield ◽  
G. J. Bouma ◽  
G. E. Seidel Jr
Keyword(s):  
Mirna Expression ◽  
Polar Body ◽  
Prostaglandin F2α ◽  
Defined Medium ◽  
Tight Junction Formation ◽  
First Polar Body ◽  
Lysis Buffer ◽  
Bovine Oocytes

Little is known about expression of microRNA (miRNA) in bovine oocytes and pre-implantation embryos. These molecules likely have an important role in regulating development. For example, differences in quality of oocytes matured in vivo v. in vitro might be due, in part, to altered miRNA expression. In Experiment 1, in vivo-matured COC were collected by transvaginal aspiration of 7 superstimulated cows 21 to 23 h after GnRH injection, given 48 h after prostaglandin F2α and the last of 6 FSH injections given b.i.d. Oocytes aspirated from abattoir ovaries were matured in vitro for 23 h in a chemically defined medium. After vortexing, maturation of both groups of oocytes was confirmed by visualization of the first polar body, and oocytes were snap frozen in mirVana lysis buffer (Applied Biosciences, Foster City, CA, USA). In Experiment 2, in vitro-matured oocytes were generated as described. Subsets were fertilized in vitro or activated parthenogenetically by incubation in 5-μM ionomycin for 5 min followed by 10 μg mL-1 cycloheximide plus 5 μg mL-1 cytochalasin B for 5 h. After 18 h and 12 h, respectively, fertilized and activated oocytes were centrifuged at 10 000 × g for 10 min to enable visualization of pronuclei. Zygotes with 2 polar bodies and 2 pronuclei and parthenotes with 2 pronuclei were snap frozen in mirVana lysis buffer. Total RNA was extracted from 30 pooled oocytes for each replicate using the mirVana MiRNA Isolation Kit (Ambion, Inc., Austin, TX, USA). Reverse transcription of RNA was performed using the QuantiMir RT kit (System Biosciences, Mountain View, CA, USA), and miRNA expression was evaluated by real-time PCR using the Mouse miRNome Profiler plate, which contains primers for 384 miRNA (System Biosciences). Three plates were analyzed for each group (30 oocytes per plate). Changes in relative expression levels were analyzed with a t-test of values normalized to miR-181a, which was consistently expressed in all samples. In Experiment 1, compared with in vitro-matured oocytes, in vivo-matured oocytes had 11-fold higher (P = 0.02) expression of miR-375, which targets numerous genes involved in electron transport chain and oxidative phosphorylation pathways according to the bioinformatic database mirGator. MiR-291a-5p, miR-494, miR-539, and miR-547 were expressed in in vivo-matured oocytes only; the converse was found for miR-575-5p. Results from Experiment 2 are in the table. Major pathways associated with potential targets of the detected miRNA include TGF-beta signaling, Wnt signaling, tight junction formation, DNA replication reactome, steroid biosynthesis, mRNA processing binding reactome, and glutamate metabolism. Several of these candidate miRNA might be important for regulation of bovine oocyte maturation and embryo development. Table 1.Experiment 2: Fold change expression of miRNA

032. Cdh1: A CELL CYCLE PROTEIN INVOLVED IN FEMALE MEIOSIS AND PREVENTION OF ANEUPLOIDY

2010 ◽  
Vol 22 (9) ◽  
pp. 10
Author(s):  
K. T. Jones
Keyword(s):  
Meiotic Division ◽  
Polar Body ◽  
Single Copy ◽  
Cyclin B1 ◽  
Cell Cycle Protein ◽  
Prophase I ◽  
First Polar Body ◽  
A Cell

Mammalian oocytes are arrested at the dictyate stage of prophase I in the ovary. In growing follicles, oocytes can become responsive to Luteinising Hormone and will undergo meiotic resumption just before ovulation. During the first meiotic division, homologous chromosomes are segregated, a process that is very error prone in human oocytes. By ovulation the oocyte has extruded its first polar body and has re-arrested at metaphase of the first meiotic division. Recent work from our lab has established that the protein Cdh1 is involved uniquely in both in the process of prophase I arrest and the correct segregation of homologs in meiosis I. Thus in cultured oocytes, in vitro antisense knockdown of Cdh1 induces both meiotic resumption and high rates of aneuploidy as a result of non-disjunction during first meiosis. Cdh1 causes prophase I arrest by inducing cyclin B1 degradation and maintaining low levels of the kinase CDK1, whose activity induces meiotic resumption. Cdh1 is an activator of the Anaphase-Promoting Complex (APC), a ubiquitin ligase that earmarks proteins such as cyclin B1 for proteolysis. Cdh1 prevents aneuploidy by causing the degradation of Cdc20, a protein that is responsible for activating the APC once all homologs are correctly aligned at metaphase. Thus loss of Cdh1 seems to prematurely activate APC(Cdc20) activity. It is interesting that a single protein can affect two important meiotic transitions in oocytes. However to explore its functions more fully, and confirm that an in vitro knockdown is faithfully replicated by in vivo loss, a targeted knockout of Cdh1 is needed. Therefore we have generated an oocyte specific Cdh1 knockout by ZP3 promoter driven Cre- recombinase activity in oocytes carrying loxP insertions in the single copy Cdh1 gene. This talk will therefore focus on the effects of an in vivo Cdh1 knockout.

scholarly journals Ultrastructure of in vitro oocyte maturation in buffalo (Bubalus bubalis)

Zygote ◽  
2010 ◽  
Vol 18 (4) ◽  
pp. 309-314 ◽  
Author(s):  
Rafael Gianella Mondadori ◽  
Tiago Rollemberg Santin ◽  
Andrei Antonioni Guedes Fidelis ◽  
Khesller Patrícia Olázia Name ◽  
Juliana Souza da Silva ◽  
...  
Keyword(s):  
Polar Body ◽  
Bubalus Bubalis ◽  
Ultrastructural Changes ◽  
Oocyte Nucleus ◽  
Cortical Granules ◽  
Immature Oocytes ◽  
Transmission Electron ◽  
High Lipid ◽  
First Polar Body

SummaryThe objective of the present study was to describe ultrastructural changes in the nucleus and cytoplasmic organelles during in vitro maturation (IVM) of buffalo cumulus–oocyte complexes (COCs). The structures were collected by ovum pick-up (OPU). Some COCs, removed from maturation medium at 0, 6, 12, 18 and 24 h, were processed for transmission electron microscopy. The average number of COCs collected by OPU/animal/session was 6.4, and 44% of them were viable. Immature oocytes had a peripherally located nucleus, Golgi complex and mitochondrial clusters, as well as a large number of coalescent lipid vacuoles. After 6 h of IVM, the oocyte nucleus morphology changed from round to a flatter shape, and the granulosa cells (GC) lost most of their contact with zona pellucida (ZP). At 12 h the first polar body was extruded and the aspect of lipid droplet changed to dark, probably denoting lipid oxidation. Cortical granules were clearly visible at 18 h of maturation, always located along the oocyte periphery. At 24 h of IVM the number of cortical granules increased. Ultrastructure studies revealed that: (1) immature oocytes have a high lipid content; (2) the perivitelline space (PS) increases during IVM; (3) Golgi complexes and mitochondrial clusters migrate to oocyte periphery during IVM; (4) 6 h of IVM are enough to lose contact between GC and ZP; (5) the oocyte lipid droplets’ appearance changes between 6 and 12 h of IVM.

scholarly journals Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽  
2002 ◽  
pp. 455-465 ◽  
Author(s):  
YH Choi ◽  
CC Love ◽  
LB Love ◽  
DD Varner ◽  
S Brinsko ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

scholarly journals Variations in modifications of sugar residues in hamster zona pellucida after in vivo fertilization and in vitro egg activation

Reproduction ◽  
2002 ◽  
pp. 671-682 ◽  
Author(s):  
M El-Mestrah ◽  
FW Kan
Keyword(s):  
Quantitative Analysis ◽  
Zona Pellucida ◽  
Hydrolytic Enzymes ◽  
Outer Region ◽  
Egg Activation ◽  
Cortical Granules ◽  
Neuraminidase Treatment ◽  
Thin Sections

Lectins were used as probes in conjunction with quantitative analysis to investigate the distribution of different carbohydrate residues in hamster zona pellucida and their possible modification patterns after in vivo fertilization and in vitro egg activation. Several lectins including HPA, WGA, RCA-I, PNA, DSA, BSAIB(4), DBA, AAA and MAA were used to label the zona pellucida of both unfertilized and fertilized eggs. With the exception of PNA and BSAIB(4), the same lectins were also used to label the zona pellucida of oocytes activated in vitro. A multicomparison quantitative analysis of the density of labelling in the inner and outer regions of the zona pellucida before and after fertilization in vivo, as well as after in vitro egg activation, was performed. Of all the lectins studied, preferential localization of labelling by RCA-I and DSA to the inner zona pellucida of unfertilized eggs was observed. After in vivo fertilization, there was an increase in labelling in the inner region of the zona pellucida when thin sections of fertilized oocytes were incubated with HPA, BSAIB(4) and AAA. Although increased labelling by RCA-I was observed, a significant decrease in labelling intensity was obtained with WGA and the sequence Neu-WGA in both the inner and outer zona pellucida of fertilized oocytes. A significant increase in the density of labelling with WGA was also observed after digestion with neuraminidase. In parallel, when hamster oocytes activated in vitro were compared with those fertilized in vivo, a difference in lectin-gold labelling was observed in both the inner and outer region of the zona pellucida. Labelling with HPA, WGA, DSA and MAA increased significantly in both the inner and outer regions of the zona pellucida, whereas labelling by DBA significantly decreased in the inner portion of the zona pellucida. After neuraminidase treatment, a significant increase in labelling density was observed when thin sections of in vitro-activated oocytes were incubated with WGA. These results demonstrate: (i) the post-fertilization modifications of major saccharidic determinants that may play a role in the sperm-egg interaction process of fertilization in vivo; and (ii) that the modified properties of zonae pellucidae of fertilized and in vitro-activated eggs resulting from the action of hydrolytic enzymes, as well as glycoproteins released through exocytosis of cortical granules, are not identical.

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