In vitro fertilization and embryo transfer in the rhesus monkey

Twenty-three rhesus monkeys were subjected to 9 days of ovarian hyperstimulation with sequential exposure to human follicle-stimulating hormone (hFSH) and then human luteinizing hormone (hLH) + hFSH. Six animals (26%) did not exhibit sustained, elevated levels of circulating estradiol, primarily due to the occurrence of a premature surge of endogenous LH (n = 4). Seventeen animals (74%) responded with supraphysiologic levels of circulating estradiol (peak value: means = 4480 pg/ml) and received human chorionic gonadotropin (hCG) on Day 10. Oocytes were collected 26 h later by aspiration of large antral follicles. Oocyte quantity (means = 18/animal) and quality (63% mature) were evaluated by in vitro fertilization (IVF), embryonic development, and embryo transfer to foster mothers. Modified conditions for the successful fertilization of oocytes used a Tyrode's augmented (TALP) medium supplemented with 0.3% bovine serum albumin (BSA). Oocytes were inseminated at the metaphase II stage with ejaculated, washed sperm (50 100 x 10(3)/ml) preexposed at ambient temperature to caffeine and dibutyryl cyclic adenosine 3'5'-monophosphate. Successful fertilization ranged from 26% to 75%. In one experiment, 5 of 11 embryos produced by IVF developed in vitro to hatched blastocysts. Embryo freezing employed a propanediol-based protocol and was applied to early cleavage-stage embryos with 100% (5 of 5) post-thaw survival. Two frozen-thawed embryos were transferred transtubally on 3 occasions into rhesus monkeys during the early luteal phase of spontaneous menstrual cycles. One pregnancy resulted, which proceeded normally to the unassisted delivery of a male offspring 170 days after the LH surge. We conclude that this sequential regimen of human gonadotropins provides a cohort of oocytes from rhesus monkeys that will complete meiotic maturation and fertilize in vitro, with embryonic development proceeding in vitro and in vivo. The production of putative antibodies to human gonadotropins, assessed by the presence of Protein A-precipitated hCG binding components in sera, limits the repeated use of monkeys in the hyperstimulation protocol. Nevertheless, this model system should facilitate further studies on oocyte maturation, fertilization, and early embryogenesis in primates.     

Correlation of zona-binding with oocyte maturation and sperm motility in rhesus monkeys by hemizona assay

The hemizona assay (HZA) in Rhesus monkeys was employed to study the correlation of zona-binding ability with sperm motility or with naturally developing oocytes at various maturational stages. Oocytes from unstimulated ovaries were retrieved within 2 hr from monkeys sacrificed for vaccine production (in reproductive season, but with their menstrual cycles not determined). Oocytes were divided into four groups based on their morphological maturation: 1) Oocytes surrounded by more than one cumulus layer (MC); 2) Oocytes retaining intact germinal vesicle nuclei (GV); 3) Oocytes with germinal vesicle breakdown showing distinct perivitelline space (PVS); and 4) Oocytes extruding the first polar body (PBI). The mean numbers of sperm bound to hemizona for PB1, PVS, GV, and MC groups were 132.9 ± 12.0, 71.5 ± 10.1, 36.1 ± 4.0, and 20.1 ± 2.9 (Mean ± SE), respectively. The four groups showed significant differences from each other in sperm/egg binding ability (P < 0.01). The number of bound sperm significantly increased with oocyte maturation. The present study also showed that zona-binding ability was also affected by sperm motility. For sperm with 67.7% motility and sperm with 31.2% motility, the average numbers of bound sperm were 43.5 ± 2.2 and 25.3 ± 2.9 (Mean ± SE), respectively. There was significantly higher binding ability for sperm with higher motility (P < 0.01). The results suggest that: 1) The rhesus monkey model can serve as a very sensitive model for studying sperm/egg interaction by HZA; 2) Sperm motility positively correlated with sperm/egg binding; and 3) Sperm/egg binding ability increases with oocyte maturation. The binding ability is highest when oocytes matured to the PB1 stage, which is also the best opportunity for fertilization. This is strong evidence for the “zona maturation” hypothesis. © 1994 Wiley-Liss, Inc.     

Dissociation of human follicle-stimulating hormone. Comparison with luteinizing hormone.

Rat testis tissue receptor assays were utilized to study the kinetics of dissociation of human follicle-stimulating hormone (hFSH) and luteinizing hormone (hLH) under varying conditions of urea concentration and pH. In these competitive protein binding assays, 125I-hFSH and 125I-hLH were the radioligands and hormone dissociation was followed by a decrease in the ability of the dissociating hormone to inhibit uptake of the radioligand by tissue receptors. Rate data for dissociation of the gonadotropins were analyzed for quality of fit to first or second order integrated rate equations by nonlinear regression analysis. Treatment of hFSH with 4 M urea at pH 8 and 25 degrees for 22 hours did not result in significant dissociation, whereas in 8 M urea, over 90% dissociation was observed. The rate of dissociation of hFSH in 8 M urea was increased approximately 4-fold by raising the temperature from 25 to 37 degrees. Similar results were obtained when dissociation of hFSH was followed through use of an accepted whole animal bioassay for FSH, thus confirming the reliability of the tissue receptor assay for such dissociation studies. Kinetic studies showed that hFSH was undissociated by incubation in 6 M urea of pH 8 after 4 hours at 25 degrees. In contrast, hLH was 90% dissociated under similar conditions. This differential rate of inactivation of hLH allowed preparation of hFSH having significant reduced levels of contaminating LH activity, as determined by tissue receptor assays and by whole animal bioassays. Marked differences were noted in the rate of dissociation of hFSH and hLH under acid conditions. hFSH completely dissociated after approximately 2 min of incubation of pH 2 (25 degrees), and over 90% dissociated after 15 min of incubation at pH 3. In contrast, hLH was dissociated 60% after 20 min of incubation at pH 2 (25 degrees) and 40% dissociated after 60 min at pH 3. Neither hormone was significantly dissociated at pH 4.4 after 60 min, but hFSH showed a slightly greater rate of dissociation than did LH in the period between 1 and 23 hours of incubation at that pH. hFSH and hLH were relatively resistant to dissociation after incubation at pH 12 for 1 hour, bu;t dissociated significantly after incubation for 22 hours at that pH. The time course for dissociation of hFSH or hLH under the various conditions described above did not conform clearly to either first or second order kinetics, indicating that the over-all dissociation process represents a mixed order reaction. It appears that urea or acid-induced denaturation of one or both subunits of hLH and hFSH may occur prior to their dissociation. The very rapid rate of dissociation at acid pH values, particularly of hFSH, indicate that ionic interactions contribute importantly to the subunit association phenomenon.     

Comparison of the steroidogenic response of luteinized granulosa cells from rhesus monkeys to luteinizing hormone and chorionic gonadotropin.

The dynamics of the steroidogenic response of nonprimate gonadal cells to gonadotropins suggests that the biologic action of pituitary LH differs from that of placental CG. To compare the response to LH and CG in primate species, luteinized granulosa cells (LGCs) obtained from rhesus monkeys following follicle stimulation were cultured in vitro. The pattern and levels of progesterone (P) produced during culture was influenced by the concentration (0-10%) and type (fetal bovine or macaque) of serum in the medium and whether LGCs were plated on plastic or extracellular matrix from bovine corneal endothelial cells. After 2-3 days of culture, LGCs were exposed acutely (15-30 min) or chronically (6 h) to 1 or 100 ng/ml human LH (hLH, NIH 1-2) or hCG (CR123), 50 micrograms/ml ovine LH (oLH, NIH-oLH-25), or incubated in the absence of gonadotropins (controls). After the first 15-30 min, the media were changed at 30-min intervals. Both acute and chronic exposure to hLH, hCG, and oLH increased (p less than 0.05) P concentrations above control levels within 15-30 min. There were no differences in the patterns or levels of P elicited by hLH or hCG over time for each treatment condition. Chronic exposure to 1 and 100 ng/ml hLH or hCG and 50 micrograms/ml oLH sustained P levels above that of controls for the 6-h interval. Acute exposure to 1 ng/ml hLH or hCG failed to maintain elevated P levels throughout the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization

Serum, gonadotrophins, growth factors, and steroid hormones stimulate the in vitro maturation (IVM) of competent oocytes, acting, directly or indirectly, upon the adenylate cyclase pathway to produce the intracellular messenger, cAMP. The intracellular levels of cAMP in cattle cumulus‐oocyte complexes (COC) were manipulated by adding to the collection and maturation media invasive adenylate cyclase (iAC), a toxin produced by the bacterium, Bordetella pertussis. High concentrations of iAC (1 or 5 μg/ml) in the maturation medium inhibited the resumption of meiosis, while low concentrations (0.1 or 0.01 μg/ml) resulted in high rates of maturation to the MII stage (92.6 ± 2.5 and 98.5 ± 1.4% respectively). The same low concentrations of iAC in the maturation medium resulted in rates of development to the blastocyst stage 8 days post insemination (30.1 ± 4.2 and 45.1 ± 3.9%, respectively), which were either not different, or significantly better, than those obtained after IVM in medium supplemented only with serum and gonadotrophins (36.1 ± 2.9%). Finally, the addition of 0.1 μg/ml iAC and 0.5 mM 3‐isobutyl 1‐methylxanthine (IBMX) in the collection medium significantly improved the blastocyst rate when IVM was performed in control medium or medium supplemented with 0.01 μg/ml iAC (31.9 ± 5.5 vs. 12.1 ± 1.6 and 45.5 ± 2.9 vs. 19.1 ± 2.3% respectively). It is concluded that the maintenance of an optimal intracellular concentration of cAMP before and during IVM ensures a high developmental competence of bovine oocytes matured in medium without serum and hormones. Mol. Reprod. Dev. 54:86–91,1999. © 1999 Wiley‐Liss, Inc.     

Anti-human gonadotropin antibodies generated during in vitro fertilization (IVF)-related cycles: Effect on fertility of rhesus macaques

Administration of human gonadotropins such as hFSH, hLH, and hCG to rhesus macaques can result in formation of anti-human gonadotropin antibodies. To determine whether the presence of these antibodies interferes with subsequent fertility, sixteen female rhesus macaques (Macaca mulatta) with known antibody levels were bred with male rhesus macaques. The presence of antibodies did not interfere with conception or maintenance of pregnancy. Furthermore, antibody titers did not increase during gestation or following the resolution of pregnancy.     

Comparison of methods to stimulate ovarian follicular growth in cynomolgus and African green monkeys for collection of mature oocytes

The objective was to compare various gonadotropin-based methods to stimulate ovarian follicular growth in female cynomolgus (n=16) and African green monkeys (n=8) for collection of mature oocytes. On the 1st day of menstruation, the monkeys were treated with 3.75 mg leuprorelin acetate (a GnRH agonist). Starting 2-3 weeks later, ovarian follicular growth was stimulated as follows: (a) 25 IU/kg of human FSH (hFSH) in a glycerol solution given once daily for 9 d; (b) 200 IU of eCG given six times during a 9-d interval; (c) 75 IU/kg hFSH in a glycerol solution given three times (72 h intervals) during a 6-d interval. In addition, the monkeys were given 1200 or 4000 IU of hCG 36 h (Methods A and B) or 60 h (Method C) after the last gonadotropin treatment, and oocyte collection was attempted 36-38 h after hCG. Although there were no significant differences among methods in the number of oocytes collected, in cynomolgus monkeys, hFSH (Methods A and C) was better than eCG (Method B; 12 and 10 versus 7 mature oocytes, respectively), whereas in African green monkeys, eCG (Method B) was more effective than hFSH (Method A; 12 versus 7 mature oocytes). Furthermore, in cynomolgus monkeys, Method C was nearly as effective as Method A; using a glycerol solution as a solvent decreased the frequency of hFSH administration from nine to three times. In conclusion, in cynomolgus and African green monkeys, ovarian response depended on the species and on the individual, and in cynomolgus monkeys, hFSH in a glycerol solvent was effective.     

Contraceptive potential of an antiprogestin ZK 98.734: reversal of ZK 98.734--induced blockade of folliculogenesis with FSH and LH and their differential effects in bonnet monkeys.

Studies were undertaken in adult bonnet monkeys to investigate whether treatment with an antiprogestin ZK 98.734 at weekly intervals, starting from day one of menstrual cycle, could arrest ovulation and also to determine if ZK 98.734 induced blockade of ovulation could be reversed with gonadotropins. Adult animals have ovulatory menstrual cycles of normal duration were treated at weekly intervals with ZK 98.734 (25 mg/dose, sc, oil base) for 10 consecutive weeks and its effects on serum levels of estradiol, bioactive LH and progesterone, and endometrial histology were investigated. Following treatment with the antiprogestin they were treated with hMG or hFSH alone. Ovulation was blocked during treatment period in all the animals (n = 14). Typical follicular phase rise in estradiol levels was inhibited, mid cycle surge in the levels of bioactive LH was abolished and serum progesterone levels remained below 1 ng/ml throughout the treatment period. However, prolonged treatment had no significant effect on the basal levels of estradiol which were around 50 pg/ml. ZK 98.734 also had no significant effect on cortisol levels. In animals (n = 4) followed for recovery after the last dose, the treatment cycle length was increased to 117.8 + 6.8 days. In three animals the treatment cycles were anovulatory, whereas in one delayed ovulation with luteal insufficiency was observed. The endometrium had become atrophic. Treatment with hMG (Pergonal: 35 I.U. hLH and 35 I.U. hFSH) or hFSH (Metrodin, 35 I.U.) for 7 consecutive days initiated folliculogenesis and the animals ovulated either spontaneously or after a single im injection of hCG (100 I.U.) on day 8 in ZK 98.734 treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin binding factor(s). Extraction of high affinity gonadotropin binding sites from rat testis and partial characterization of their interaction with human follitropin, lutropin, and choriogonadotropin.

Factor(s) that bind gonadotropins have been extracted from rat testis by 30% ethanol (v/v) in water and their interaction with human lutropin (hLH) and human follitropin (hFSH) have been investigated by a new assay using dextran-coated charcoal. These studies reveal that: 1. Maximal binding of gonadotropin with soluble factors was observed over a broad range of pH from 6.0 to 8.0 with a relative decline in binding at extremes of pH. The binding was independent of the ionic strength of the buffer and reached equilibrium within 5 min at 4 degrees, 27 degrees, and 37 degrees. 2. The soluble factors have marked thermostability, a point of distinction from detergent-solubilized receptors. 3. The equilibrium dissociation constant (Kd) of 125I-hFSH binding to the soluble factor was 6.0 +/- 0.58 X 10(-10) M, consistent with the values obtained from the membrane binding studies. Similarly, the Kd value for 125I-hLH to the soluble factor(s) was 3.33 +/- 0.3 X 10(-9) M, comparable to the values obtained from the membrane binding studies. Hill plots demonstrated a lack of a cooperative relationship with an apparent Hill coefficient of 1.071 for hLH and 0.909 for hFSH. Furthermore, two classes of binding sites for 125I-human choriogonadotropin (hCG) were clearly discernible by both Lineweaver-Burk and Hill plots with an equilibrium dissociation constant of 2.4 +/- 0.5 X 10(-11) M and 1.35 +/- 1.2 X 10(-9) M. The apparent Hill coefficient of interaction of 125I-hCG with the soluble factors was found to be 0.923 for high affinity and 1.09 for low affinity binding sites. 4. The binding of 125I-hLH and 125I-hFSH with respect to concentrations of soluble factor(s) was found to be a saturable process, yielding an expected 4.4-fold higher Kd for hLH (294 +/- 13.8 mug/ml) compared to hFSH (66.6 +/- 4 mug/ml). These findings are comparable with the equilibrium dissociation constants, thus confirming a 5-fold higher affinity of hFSH as compared to hLH for the soluble factors, i.e. the ratio of 3.0 X 10(-9) M to 6.0 X 10(-10) M versus the ratio of 294 mug/ml to 66.6 mug/ml. 5. The hormone specificity of the interaction has been studied by using radiolabeled hFSH, hLH, hCG, prolactin, growth hormone, and bovine serum albumin. The binding of FSH at low factor concentrations was found to be 5- to 10-fold greater than prolactin, growth hormone, and albumin. 6. The soluble factors are found in higher concentration in testis compared to liver, kidney, and blood. 7. The effect of ethanol upon solubilization of the factor(s) has been investigated. The factor(s) can be extracted with buffer or water alone. However, 10 to 25% of ethanol (v/v) facilitates the process of solubilization. The treatment with 70% ethanol (v/v) or more did not extract any factor activity from testes. The factor(s) were insoluble in petroleum ether, chloroform, absolute ethanol, methanol, or lipid solvent. 8. Finally the effect of soluble factors on classical membrane binding was investigated...     

Parthenogenetic development of bovine oocytes matured in vitro for 24 hr and activated by ethanol and cycloheximide

This research was undertaken to improve development of parthenogenetic embryos following various combined treatments of ethanol and cycloheximide. In Experiment 1 in vitro matured oocytes (IVM, 24 hr) were treated with 7% ethanol for 5 min followed by incubation in 10 μg/ml cycloheximide in Medium 199 for 0 (control), 5, 10, and 20 hr. Development to 2–8 cells following culture for 3 days was similar among treated groups (32–41%; P > 0.05), which was higher than that of controls (6%; P < 0.05). Experiment 2 compared pre-ethanol exposures for 0, 1, 2.5, and 5 min, followed by 5 hr cycloheximide treatment on activation development. One- to 5-min groups resulted in 42–44% cleavage contrasted to 1–12% for controls (P < 0.05). Experiment 3 examined the effect on oocyte development of ethanol and different concentrations of cycloheximide (0, 1, 5, and 10 μg/ml). Cleavage to 2–8 cells was similar among the 5 and 10 μg/ml cycloheximide groups (36% and 42%, P > 0.05) but lower (P < 0.05) for the 1 μg/ml group (24%) and the controls (2–13%). When 5 μg/ml cycloheximide was used (Experiment 4), pre-exposure to ethanol (1, 2.5, and 5 min) resulted in more oocytes cleaved (38–41%) than in the cycloheximide alone group (0%) or the control (0%, P < 0.05). Experiment 5 tested blastocyst development of the activated oocytes with or without cytochalasin B treatment. Oocytes developed to blastocyts were 0%, 14%, 3%, and 3% (P < 0.05), respectively, for control, treatment with ethanol and cycloheximide in the presence, or absence of cytochalasin B, or electrical pulse plus cycloheximide. In conclusion, the combined ethanol and cycloheximide treatment supported high rates of parthenogenetic development using 24 hr IVM bovine oocytes. Blastocyst rate was significantly higher when cytochalasin B was added to the combined activation regimen. © 1994 Wiley-Liss, Inc.     

Colcemid‐treatment of heifer oocytes enhances nuclear transfer embryonic development,establishment of pregnancy and development to term

Four experiments were designed to examine the effects of colcemid, a microtubule assembly inhibitor, on the development of bovine nuclear transfer (NT) embryos in vitro and in vivo. Recipient oocytes matured at different times were exposed to colcemid. Approximately 80–93% of the exposed oocytes, with or without the first polar body (PB1), developed obvious membrane projections. In Experiment 1, oocytes matured for either 14–15 or 16–17 hr, treated with colcemid and used as recipient cytoplasm for NT resulted in over 40% blastocyst development. In Experiment 2, oocytes matured for 16–17 hr were treated with either 0.2 or 0.4 µg/ml colcemid for 2–3 or 5–6 hr, respectively. The percentages of blastocyst development (39–42%) were not statistically different among the different colcemid treatment groups, but were both higher (P < 0.05) than the control group (30%). Colcemid concentrations and length of colcemid treatment of oocytes did not affect their ability to support NT embryo development to the blastocyst and hatched blastocyst stages. Results from Experiment 3 indicate that semi‐defined medium increases morula and blastocyst development of NT embryos derived fromcolcemid‐treated oocytes under 5% CO₂ in air atmosphere. In addition, cell numbers of blastocysts in colcemid‐treated groups were numerically higher than the control groups. After embryo transfer, higher (P < 0.05) pregnant rates were obtained from the colcemid‐treated group than the nontreated group. Five of 40 recipients (12.5%) which received embryos from colcemid‐treated oocytes delivered healthy calves, significantly higher than those recipients (3.3%) that received embryos derived from nontreated oocytes. Mol. Reprod. Dev. 76: 620–628, 2009. © 2009 Wiley‐Liss, Inc.     

Time course of meiotic progression after transferring primary spermatocyte into ooplasm at different stages

This study attempted to investigate the time course of meiotic progression after transferring primary spermatocyte (PS) into ooplasm at different maturing stages. In present experiments, PSs were introduced into maturing ooplasts or oocytes by electrofusion. Higher fusion rate was obtained by phytohemagglutinin (PHA) agglutination than by perivitelline space (PVS) insertion. When the ooplasms prepared at 0, 2, 5, and 8.5 hr of in vitro maturation (IVM) were used as recipients and PSs were used as donors, the reconstructed cells extruded the first polar body (PB1) approximately 8.5, 7, 5.5, and 3 hr after electrofusion, respectively. Especially, when ooplasm cultured for 8.5 hr in vitro after GV removal was fused with PS, the PB1 was emitted 7-11 hr after electrofusion. Additionally, the PB1 extrusions of GV and pro-MI oocytes fertilized with PSs were 2.5 hr earlier than control oocytes. The results suggest that (1) PSs undergo the first meiosis in different time courses when introduced into ooplasm at different maturing stages; (2) GV material plays an important role in determining the timing of PB1 extrusion; and (3) first meiotic division of GV and pro-MI oocytes can be accelerated by introducing PS.     

γ-glutamyl transpeptidase of spermatozoa may decrease oocyte glutathione content at fertilization in pigs

The presence of γ-glutamyl transpeptidase (GGT) in boar spermatozoa and the potential role of the GGT at sperm penetration were examined using in vitro matured porcine oocytes. In the first experiment, GGT of boar spermatozoa was examined using a histochemical stain. GGT was detected in the midpiece and the acrosome regions of boar spermatozoa. In the second experiment, porcine oocytes matured in vitro were injected with approximately 40 pl of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT or 1 mM guanosine-5′-0-(3′-thiotriphosphate) (GTP-γ-S; G-protein activator). When GGT was injected into oocytes, the incidence of oocytes activated (23.7 ± 1.4%) was not different (P > 0.05) from HEPES-injected controls (24.9 ± 1.3%) at 6 h after injection. Injected GTP-γ-S, however, activated 76.0 ± 5.3% of oocytes at 6 h after injection, but extrusion of the second polar body was very low (2.8 ± 4.8%). Total content of glutathione (GSH) and glutathione disulfide (GSSG) did not differ (P > 0.05) between GTP-γ-S injected oocytes (4.2 ± 0.7 pmol/oocyte) and noninjected oocytes (4.0 ± 0.1 pmol/oocyte) at 6 h after injection. However, the total content of GSH and GSSG was lower (P < 0.01) in GGT-injected oocytes (2.1 ± 0.2 pmol/oocyte) than HEPES-injected oocytes (3.4 ± 0.2 pmol/oocyte) at 6 h after injection. In the third experiment, in vitro matured porcine oocytes were injected with about 40 pl of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT and then inseminated. At 12 h after insemination, the incidence of male pronuclear formation was significantly lower in oocytes injected with GGT as compared with injected control oocytes. These results demonstrated that (1) GGT was present on the surface of spermatozoa, (2) total oocyte content of GSH and GSSG was decreased by microinjection of GGT but not by that of GTP-γ-S, and (3) male pronuclear formation was inhibited in GGT-injected oocytes. These results suggest that sperm GGT may be a limiting factor for male pronuclear formation in polyspermic oocytes. © 1996 Wiley-Liss, Inc.     

Different fates of oocytes with DNA double-strand breaks in vitro and in vivo

In female mice, despite the presence of slight DNA double-strand breaks (DSBs), fully grown oocytes are able to undergo meiosis resumption as indicated by germinal vesicle breakdown (GVBD); however, severe DNA DSBs do reduce and delay entry into M phase through activation of the DNA damage checkpoint. But little is known about the effect of severe DNA DSBs on the spindle assembly checkpoint (SAC) during oocyte maturation. We showed that nearly no first polar body (PB1) was extruded at 12 h of in vitro maturation (IVM) in severe DNA DSBs oocytes, and the limited number of oocytes with PB1 were actually at telophase. However, about 60% of the severe DNA DSBs oocytes which underwent GVBD at 2 h of IVM released a PB1 at 18 h of IVM and these oocytes did reach the second metaphase (MII) stage. Chromosome spread at MI and MII stages showed that chromosomes fragmented after GVBD in severe DNA DSBs oocytes. The delayed PB1 extrusion was due to the disrupted attachment of microtubules to kinetochores and activation of the SAC. At the same time, misaligned chromosome fragments became obvious at the first metaphase (MI) in severe DNA DSBs oocytes. These data implied that the inactivation of SAC during the metaphase-anaphase transition of first meiosis was independent of chromosome integrity. Next, we induced DNA DSBs in vivo, and found that the number of superovulated oocytes per mouse was significantly reduced; moreover, this treatment increased the percentage of apoptotic oocytes. These results suggest that DNA DSBs oocytes undergo apoptosis in vivo.     

Bovine parthenogenesis is characterized by abnormal chromosomal complements: Implications for maternal and paternal co-dependence during early bovine development

The present study was conducted to examine the karyotypes of parthenogenetic bovine embryos arising from the application of standard oocyte activation and diploidization methods. Bovine cumulus–oocyte complexes were collected and matured in vitro for 24 hr prior to oocyte activation with either 5 μM ionomycin or 7% ethanol for 5 min. Groups of activated oocytes were further treated with 5 μg/ml cytochalasin D or 1.9 mM 6-dimethylaminopurine (DMAP) for 6 hr. Cleavage varied significantly (P < .05) among the treatment groups with 68.0% of the ethanol- and DMAP-treated oocytes dividing. Blastocyst development did not vary with 18.4 ± 2.5% of all treated oocytes progressing to this stage. Blastocyst development did not occur in groups subjected to oocyte activation alone. Blastocysts displayed haploid (2.3%), diploid (11.4%), tetraploid (40.9%), octaploid (4.5%), and mixoploid chromosomal complements (40.9%). Two-cell stage parthenogenotes resulting from ethanol or ionomycin treatment alone displayed haploid (66.7%), diploid (16.7%), tetraploid (4.2%), and mixoploid (12.5%) complements. Our results demonstrate that diploid bovine parthenogenotes arising from these procedures are a minority, with the majority of parthenogenotes displaying polyploid and mixoploid chromosomal complements. The events contributing to these abnormal chromosomal complements occur as early as completion of the first cell cycle, possibly linking these events with the absence of a paternally supplied centrosome. Dev. Genet. 21:160–166, 1997. © 1997 Wiley-Liss, Inc.     

Comparative analysis of calf and cow oocytes during in vitro maturation

To determine possible causes of reported differences between developmental competence of oocytes isolated from prepubertal (10- to 14-week-old calves) and adult cows, three parameters were analysed, comparatively, during in vitro maturation (IVM): (1) oocyte diameter, (2) oocyte energy metabolism, and (3) protein synthesis of oocytes and cumulus cells. Cumulus-oocyte complexes were isolated from follicles of 3–5 mm in diameter in both age groups. Mean oocyte diameter was smaller (P < 0.02) in calves than in cows (118.04 ± 1.15 versus 122.83 ± 0.74 μm). During the first 3 hr of IVM, calf oocytes metabolised glutamine and pyruvate at lower rates than adult oocytes, but after 24 hr of culture, both molecules were metabolised at the same rate as for adult oocytes. A significant decrease in protein synthesis, as measured by [³⁵S]methionine and [³⁵S]cysteine incorporation was recorded after 9 hr of IVM in calf oocytes, while in adult oocytes a significant decrease in protein synthesis was detected only after 24 hr. After the first 3 hr of maturation, proteins of 130, 26, and 24 kDa were more abundant in adult than in calf oocytes, while a protein of 55 kDa was more visible in calf than in adult oocytes. At the same time, among proteins newly synthesised by cumulus cells, molecules of 405, 146, 101, and 77 kDa were more abundant in adults than in calves. In conclusion, calf oocytes and cumulus cells showed several differences when compared with their adult counterparts, which are consistent with their reported lower developmental competence. Mol. Reprod. Dev. 49:168–175, 1998. © 1998 Wiley-Liss, Inc.     

Gonads of males and females of the mangrove tree crab Aratus pisonii (Grapsidae: Brachyura: Decapoda): a histological and histochemical view

Nicolau, C.F., Nascimento, A.A., Machado‐Santos, C., Sales, A. and Oshiro, L.M.Y. 2011. Gonads of males and females of the mangrove tree crab Aratus pisonii (Grapsidae: Brachyura: Decapoda): a histological and histochemical view. —Acta Zoologica (Stockholm) 00 :1–9. This study describes the microscopic anatomy of the male and female gonads and the spermatogenesis and oogenesis of the mangrove tree crab Aratus pisonii. Males and females were captured in a mangrove marsh in Guaratiba (23°04′S, 44°10′W), Rio de Janeiro State, Brazil. The testes are composed of spermatogonia I (7.82 ± 0.84 μm), spermatogonia II (6.12 ± 0.72 μm), spermatocytes I (5.62 ± 0.71 μm), spermatocytes II (5.00 ± 0.42 μm), spermatids (4.01 ± 0.33 μm), and spermatozoa (2.58 ± 0.18 μm). The spermatozoids are sent to the vas deferens, which is divided into three regions: anterior vas deferens, middle vas deferens and posterior vas deferens. There are no indications of development as the production of male gametes was continuous throughout the study period. In the females, there are four ovary development stages: previtellogenesis, early‐stage vitellogenesis, mature vitellogenesis, and postspawning. Five types of cells were found in the gonads: oogonia (5.23 ± 1.31 μm), oocytes in early development (19.84 ± 5.16 μm), previtellogenic oocytes (49.49 ± 6.87 μm), vitellogenic oocytes (87.51 ± 10.23 μm), and mature oocytes (174.78 ± 29.46 μm). The findings of this study indicate that A. pisonii females lay eggs on multiple occasions throughout the study period.     

Procyanidin B1 promotes in vitro maturation of pig oocytes by reducing oxidative stress

Oxidative stress negatively affects the in vitro maturation (IVM) of oocytes. Procyanidin B1 (PB1) is a natural polyphenolic compound that has antioxidant properties. In this study, we investigated the effect of PB1 supplementation during IVM of porcine oocytes. Treatment with 100 μM PB1 significantly increased the MII oocytes rate (p <0.05), the parthenogenetic (PA) blastocyst rate (p <0.01) and the total cell number in the PA blastocyst (p < 0.01) which were cultured in regular in vitro culture (IVC) medium. The PA blastocyst rate of regular MII oocytes activated and cultured in IVC medium supplemented with 100 and 150 μM PB1 significantly increased compared with control (p < 0.01 and p < 0.05). We also evaluated the reactive oxygen species (ROS) levels, mitochondrial membrane potential (Δψm) levels, glutathione (GSH) levels, and apoptotic levels in MII oocytes and cumulus cells following 100 μM PB1 treatment. The results showed that the PB1 supplementation decreased ROS production and apoptotic levels. In addition, PB1 was found to increase Δψm levels and GSH levels. In conclusion, PB1 inhibited apoptosis of oocytes and cumulus cells by reducing oxidative stress. Moreover, PB1 improved the quality of oocytes and promoted PA embryo development. Taken together, our results suggest that PB1 is a promising antioxidant additive for IVM of oocytes.     

Effects of gonadotropins and steroids on the subsequent fertilizability of extrafollicular bovine oocytes cultured in vitro

Effects of gonadotropins (2 i.u./ml follicle stimulating hormone, FSH and 10 μg/ml luteinizing hormone, LH) and steroids (1 μg/ml oestradiol, E and progesterone, P) on the fertilizability of extrafollicular bovine oocytes cultured in vitro and transferred in either the rabbit oviduct (Experiment I) or glass test tubes (Experiment II) were investigated. Bovine oocytes collected from follicles of 2–5 mm in diameter were cultured in vitro for 27 h in a medium containing Ham's F-12, 20% (v/v) bovine fetal serum and antibiotics. The combination of the hormones added to the medium was as follows; (1) none (control), (2) E, (3) LH, (4) LH + E, (5) FSH + LH + E, and (6) FSH + LH + E + P. All oocytes were recovered 24 h after insemination and examined for the presence of the pronuclei and a sperm tail with the midpiece in the oocyte cytoplasm, and the extrusion of the second polar body.In Experiment I, 630 of 704 transferred oocytes (85.7%) were recovered from the rabbit oviduct. The maturation rates of these oocytes (overall 61.1%) were not significantly affected by gonadotropins and steroids. Of the 741 of 920 oocytes recovered from test tubes in Experiment II, the maturation rates of them (overall 64.2%) were significantly increased (P < 0.05) by addition of LH (72.9%) and FSH + LH + E + P (74.1%) as compared with controls (55.4%). Fertilization rates were increased (P < 0.05) by the addition of FSH + LH + E compared with the controls in both Experiments I (31.1% and 14.0%) and II (36.2% and 20.8%). Furthermore, the proportion of fertilized eggs in hormone treated groups was the highest in each experiment. The present study indicates that the addition of FSH, LH and E to a medium has improved the fertilizability of extrafollicular bovine oocytes cultured in vitro.     

Differential effects of protein synthesis inhibitors on porcine oocyte activation

This study was designed to evaluate the effects of cycloheximide and puromycin on activation and protein synthesis of porcine oocytes. When matured oocytes were electrostimulated, then cultured in the presence of cycloheximide (5 μ/ml) for 6 or 24 hr, 92% of oocytes were activated as indicated by pronuclear formation, vs. 2.8% for untreated oocytes, 5.3% for oocytes not electrostimulated but cultured with cycloheximide, and 60.0% for those only electrostimulated. When cultured with L-[³⁵S]methionine in the presence of cycloheximide, puromycin (100 μg/ml), or no protein synthesis inhibitor for 24 hr, oocytes had mean radiolabeled incorporation rates of 36.5, 2.21, and 32.0 fmol/4 hr/oocyte, respectively. Thus, cycloheximide had little effect on protein synthesis after 24 hr of culture. A 1D-SDS PAGE showed that oocytes cultured with puromycin or cycloheximide are not activated, while electrostimulated oocytes are activated, as characterized by the conversion of a 25-kDa polypeptide to a 22-kDa polypeptide. The radiolabeling experiment was repeated, except that oocytes were cultured for 4 or 24 hr. At 4 hr, mean incorporation rates were lower in the cycloheximide group (2.34 fmol/4 hr/oocyte), but similar in the puromycin (15.7 fmol/4 hr/oocyte) and control groups (18.9 fmol/4 hr/oocyte). At 24 hr, the puromycin group (5.73 fmol/4 hr/oocyte) had a lower rate of incorporation, while the cycloheximide (22.6 fmol/4 hr/oocyte) and control (26.0 fmol/4 hr/oocyte) groups were similar. Cycloheximide was more effective earlier during culture, while puromycin was more effective later. When combined with ES, puromycin did have a higher rate (P = 0.10) of activation (87.8%) than with electrostimulation alone (73.0%). A final experiment evaluated the development to blastocyst after transfer to a ligated oviduct. Cycloheximide treatment in conjunction with an electric pulse did not increase the rate of compact morula or blastocyst formation. In conclusion, puromycin and cycloheximide have differential effects on protein synthesis, and although cycloheximide alone will not induce activation in porcine oocytes, it is very effective in generating activated oocytes in combination with electrostimulation. © 1995 Wiley-Liss, Inc.