Effect of follicle diameter on oocyte apoptosis, embryo development and chromosomal ploidy in prepubertal goats

The aim of this study was to assess the following parameters in prepubertal goat oocytes of different follicle diameter (≥3 mm, <3 mm, control): oocyte diameter, early (Annexin-V) and late (TUNEL) apoptosis, embryo development and chromosomal ploidy of these blastocysts using Fluorescence In Situ Hybridization (FISH). Before in vitro maturation, oocytes were measured and stained with Annexin-V or TUNEL. The rest of the oocytes were matured, fertilized, and cultured in vitro for 8 days. Oocytes from follicles of ≥3 mm showed greater mean oocyte diameter (128.27 ± 7.20 μm vs. 125.35 ± 7.59 μm), higher percentages of TUNEL positive (42.86 vs. 24.23%), higher cleavage (47.85 ± 3.98 vs. 23.07 ± 2.44 %) and blastocyst rates (19.77 ± 3.04 vs. 4.11 ± 1.10 %) than oocytes from follicles of <3 mm.. Blastocyst mean cell numbers did not show differences between follicular groups (123.83 ± 49.62 vs. 104.29 ± 36.09 for follicles of ≥3 mm and <3 mm, respectively). A total of 54 blastocysts with 7084 nuclei were hybridized with specific probes to chromosomes X and Y. Ninety-eight percent (98%) of the embryos presented at least one cell carrying an abnormal number of chromosomes, but 78% of them presented less than 25% of chromosomal abnormal cells. No differences in the percentage of blastocysts with abnormal ploidy were found in embryos produced from oocytes of different follicle diameter.     

Effect of blood plasma collected after adrenocorticotropic hormone administration during the preovulatory period in the sow on oocyte in vitro maturation

Reproduction may be affected by stressful events changing the female endocrine or metabolic profile. An altered environment during oocyte development could influence the delicate process of oocyte maturation. Here, the effect of simulated stress by media supplementation with blood plasma from sows after adrenocorticotropic hormone (ACTH) administration during the preovulatory period was assessed. Oocytes were matured for 46 hours in the presence of plasma from ACTH-treated sows, or plasma from NaCl-treated control sows, or medium without plasma (BSA group). The plasma used had been collected at 36 and 12 hours (±2 hours) before ovulation (for the first 24 hours + last 22 hours of maturation, respectively). Subsequent fertilization and embryo development were evaluated. Actin cytoskeleton and mitochondrial patterns were studied by confocal microscopy both in the oocytes and the resulting blastocysts. Nuclear maturation did not differ between treatments. Subtle differences were observed in the actin microfilaments in oocytes; however, mitochondrial patterns were associated with the treatment (P < 0.001). These differences in mitochondrial patterns were not reflected by in vitro outcomes, which were similar in all groups. In conclusion, an altered hormonal environment provided by a brief exposure to plasma from ACTH-treated sows during in vitro oocyte maturation could induce alterations in actin cytoskeleton and mitochondrial patterns in oocytes. However, these changes might not hamper the subsequent in vitro embryo development.     

Fertilization and development of quail oocytes after intracytoplasmic sperm injection

The present study was conducted to establish the intracytoplasmic sperm injection (ICSI) method for in vitro fertilization and development in quail. The efficiency of fertilization of oocytes was compared 1) between spontaneous and premature ovulation and 2) among testicular round spermatids, elongated spermatids, and immature and mature spermatozoa. The oocytes were injected with a single spermatozoon or spermatid and cultured for 24 h. Cell division was histologically observed with hematoxylin-eosin (HE) and a nucleus-specific fluorescent dye (DAPI). Five of 30 (16.6%) and 4 of 30 (13.3%) oocytes injected with mature sperm were fertilized in the spontaneous and induced ovulation group, respectively. Those embryos showed development at stages II-VII. Half the number (three of six) of the oocytes injected with testicular spermatozoa were fertilized and developed to stages IV-VII, and two of five oocytes injected with elongated spermatids were fertilized and developed to stage VI. All ooocytes injected with round spermatids were unfertilized. The results demonstrate that intracytoplasmic injection of a single sperm into quail oocyte can activate the oocyte and lead to fertilization. Oocytes prematurely ovulated are capable of fertilizing with mature sperm as are those spontaneously ovulated. In addition, the results suggest that the testicular round spermatids may not possess sufficient oocyte-activating potency but that the elongated spermatids and immature spermatozoa are competent to participate in fertilization and early embryonic development in quail.     

Supplementation with vascular endothelial growth factor during in vitro maturation of porcine cumulus oocyte complexes and subsequent developmental competence after in vitro fertilization

The aim of the present study was to investigate whether the effects of vascular endothelial growth factor (VEGF) on porcine cumulus oocyte complexes (COCs) and subsequent blastocyst formation following in vitro fertilization are attributable to improved fertilization and cytoplasmic maturation. Porcine COCs were cultured for 42 h in TCM199 medium with 5 ng/mL human recombinant VEGF, and the resultant metaphase II oocytes were fertilized in vitro. COCs without VEGF supplementation served controls. Supplementation with VEGF during in vitro maturation (IVM) significantly (P < 0.05) improved the blastocyst formation rate and total cell number (46.7 ± 3.1% and 82.8 ± 6.7, respectively) compared with controls (32.5 ± 3.4% and 64.1 ± 5.6, respectively). On day 2, the percentage of four-cell stage embryos was significantly higher in the VEGF-matured group (49.1 ± 2.7%) than in the control (33.1 ± 5.8%), and the percentage of two-cell stage embryos was significantly higher in the control group (10.4 ± 1.4%) than in the VEGF-matured group (6.6 ± 0.9%). At 10 h after the onset of in vitro fertilization (IVF), oocytes with two pronuclei were considered as monospermically or normally fertilized, and oocytes with more than two pronuclei were considered as polyspermically fertilized. Monospermy was significantly higher in VEGF-matured oocytes (47.2 ± 4.3%) than in the control (20.0 ± 2.4%), and polyspermy and sperm penetration per oocyte were significantly higher in the control group (54.4 ± 3.8% and 2.3 ± 0.1, respectively) than in the VEGF-matured oocytes (43.9 ± 3.6% and 1.8 ± 0.1, respectively). Supplementation with VEGF during IVM significantly (P < 0.05) improved male pronuclear formation as compared with the control (91.1 ± 1.9 vs 74.4 ± 3.8%). Type III cortical granule distribution in oocytes was more common in VEGF-matured oocytes (78.0%) than in the control (52.1%). These results suggest that VEGF supplementation during IVM enhanced the developmental potential of porcine IVF embryos through higher male pronuclear formation and higher monospermic fertilization rates as a consequence of improved cytoplasmic maturation.     

Improvement in in?vitro fertilization outcome following in?vivo synchronization of oocyte maturation in mice

Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2–4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients.     

Ovarian activity reversibility after the use of deslorelin acetate as a short-term contraceptive in domestic queens

The objective was to evaluate ovarian activity reversibility in domestic queens after short-term contraceptive treatment with deslorelin acetate. Ten mature queens were used. In all queens, the estrous cycle was evaluated every 72 h by vaginal cytology (VC) and behavior assessments. When queens had VC characteristic of interestrus or diestrus, one deslorelin acetate implant (4.7 mg) was placed in the subcutaneous tissue of the interscapular region (day of insertion = Day 0). Thereafter, VC was performed every 48 h and on Day 90, implants were removed. At Day 100, estrus and ovulation were induced with 100 IU eCG (im), followed by 100 IU hCG (im), 84 h later (Day 103.5). Queens were ovariohysterectomized on Day 106. Corpora lutea (CL) were counted, oviducts were flushed, and oocytes were identified, isolated and stained to assess viability. In all queens, blood samples for plasma progesterone concentrations were collected once a week, from Days −21 to 106. After deslorelin acetate application, four queens had VC and behavior typical of estrus, and one ovulated. Furthermore, ovulation occurred in three queens that did not have VC or behavior consistent with estrus. After the initial ovarian stimulation, all females had anestrous VC during the deslorelin treatment period. Implants were readily removed. Following implant removal, all females responded to treatments to induce estrus and ovulation. There were (mean ± SEM) 13.1 ± 5.5 CL and 8.1 ± 5.5 oocytes per queen; the oocyte recovery rate was 56.8 ± 25.4% and all recovered oocytes were viable. We concluded that deslorelin acetate can be used as a reversible short-term contraceptive in domestic cats, because estrus and ovulation were successfully induced following implant removal.     

Maturation-associated changes in the rat zona pellucida

Rat follicular oocytes, arrested at prophase I, cannot be fertilized in vitro. This capacity is acquired following resumption of meiosis and a series of changes involving both the oocyte and the cumulus cells surrounding it. Oocytes exposed to sperm at different hours before ovulation show a gradual increase in the permeability of their zona pellucida (ZP). Our study examined whether the ZP, in response to the physiological stimulus for maturation and concomitant with the other oocyte--cumulus components, undergoes maturational changes. Two ZP characteristics were assessed, sensitivity to proteolysis and sperm binding. ZP surrounding oocytes and eggs were collected from five sources: 1) germinal vesicle (GV)-intact oocytes, 2) preovulatory eggs, 3) ovulated eggs isolated from oviducts of immature females, 4) fertilized eggs, 5) ovulated eggs isolated from oviducts of mature females. All ZP surrounding oocytes/eggs from groups 1-5 were dissolved by trypsin. When solubility by pronase and alpha-chymotrypsin was examined, a large variation between groups was found. All ZP from group 2 were dissolved by 0.001% pronase, compared to 0% solubility in group 4. Only 10% of the ZP surrounding GV-intact oocytes (group 1) were dissolved by this enzyme, compared to 82% in group 3. Solubility in 0.01% alpha-chymotrypsin showed a similar pattern. Capacitated sperm were incubated with eggs from groups 1 and 3. The number of sperm binding to ZP in group 3 was repeatedly higher than that in group 1. In both tests it was found that the ZP surrounding the mature eggs differ in their characteristics from ZP of GV-intact oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dimethyl sulfoxide inhibits spontaneous oocyte fragmentation and delays inactivation of maturation promoting factor (MPF) during the prolonged culture of ovulated murine oocytes in vitro

In this study, the effects of dimethyl sulfoxide (DMSO) on the spontaneous aging of ovulated murine oocyte were evaluated in vitro. When ovulated oocytes were cultured continuously in vitro without fertilization stimulation, they underwent several phenotypic changes, including non-activation, activation, fragmentation, and lysis. To investigate the effects of DMSO on these changes, I cultured ovulated oocytes with various concentrations of DMSO and evaluated the phenotypic changes for up to 3 days. After 3 days of culture, the frequency of oocyte fragmentation was significantly lower in oocytes treated with 2 and 4% DMSO (7 and 5%, respectively) than in control oocytes (80%). All control oocytes were activated or fragmented after 3 days of culture in vitro. However, more than 80% of the oocytes cultured with 4% DMSO for 3 days contained spindles and condensed chromosomes, although they displayed abnormal spindle structures. Next Cdk1 activity in DMSO-treated oocytes was examined. The results showed that DMSO treatment prevented the reduction in Cdk1 activity during prolonged culture. Moreover, DMSO inhibited the degradation of cyclin B. These results suggest that DMSO inhibits spontaneous oocyte fragmentation and maintains Cdk1 activity in ovulated murine oocytes during prolonged culture in vitro, possibly by inhibiting cyclin B degradation.     

Influence of caffeine pretreatment on biphasic in vitro maturation of dog oocytes

Phosphodiesterase (PDE) inhibitors have been utilized for in vitro maturation (IVM) of oocytes to manipulate the meiotic resumption and progression. Premature chromatin condensation and DNA replication of the oocytes, immediately after the decrease in the cAMP level, are the difficulties in canine IVM. Caffeine, a nonselective competitive PDE inhibitor, due to its structural similarity to adenosine molecule maintains the cAMP level by occupying PDE enzymes such as PDE-3A inside the oocyte and PDE-4 and PDE-5 in the cumulus cells. In this study, the effects of 12-hour caffeine pretreatment in a biphasic IVM protocol were assessed on maturation rates of canine oocytes. Sixty hours of culture after a 12-hour of 10 mM caffeine pretreatment resulted in 16.9% ± 2.4 of the oocytes reaching metaphase II stage (MII) and 25.9% ± 5.2 degeneration rate compared with the control group with 2.2% ± 2.2 MII and 37.6% ± 4.3 degeneration rates (P < 0.05). Caffeine pretreatment induced higher mitogen-activated protein kinases (MAPK1 and MAPK3) phosphorylation and maturation-promoting factor activity at 12 hours and activated MAPK1 and maturation-promoting factor at 48 hours after culture in cumulus-oocyte complexes (COCs) compared with the control group (P < 0.05). Fresh canine COCs were also analyzed before IVM using brilliant cresyl blue (BCB) staining. Oocytes showed difference in meiotic resumption (MI-MII) (BCB+ = 16.11% ± 5.5, BCB− = 9.86% ± 5.0; P < 0.05) after 60 hours of culture following 12-hour caffeine pretreatment. The BCB+ canine oocytes had higher MII rate than the BCB− group under caffeine pretreatment (10.2% ± 2.9 vs. 1.1% ± 1.1, respectively; P < 0.05). Results indicated that 12-hour caffeine pretreatment of canine COCs improves the MII maturation rates at 72 hours and BCB+ oocytes have higher competency in vitro for nuclear maturation.     

Ovum pick up, in vitro embryo production, and pregnancy rates from a large-scale commercial program using Nelore cattle (Bos indicus) donors

The objective was to clarify in vitro production of bovine embryos in Brazil. Data from 656 ovum pick-up/in vitro production (OPU/IVP) procedures, performed on 317 Nelore (Bos indicus) donors, without hormone stimulation or control of ovarian follicular waves, were analysed. Donors were subjected to OPU from one to nine times (no specific schedule), with < 15 d between consecutive procedures. There were 20,848 oocytes, of which 15,747 (75.53%) were considered viable, 5,446 embryos were obtained, 5,398 embryos were immediately transferred, resulting in 1,974 pregnancies (36.57%) at Day 30 and 1,788 (33.12%) pregnancies at Day 60. The average number of total and viable oocytes produced per OPU session was (mean ± SEM) 30.84 ± 0.88 and 23.35 ± 0.7 (average of 8.1 ± 0.3 embryos and 3.0 ± 0.1 pregnancies per OPU-IVP procedure). Since oocyte production varied widely among donor, they were designated as very high, high, intermediate, and low, with 58.94 ± 2.04, 32.61 ± 0.50, 22.13 ± 0.50, and 10.26 ± 0.57 oocytes, respectively, produced by 78, 80, 79, and 80 donors. The number of viable oocytes recovered ranged from 0 to 128; since donors with numerous viable oocytes produced many viable embryos and pregnancies, oocyte production was useful for donor selection. However, there was no significant effect of the number of OPU sessions per donor on mean numbers of oocytes produced. In conclusion, we confirmed field reports of high oocyte production by some Nelore donors and demonstrated individual variation in oocyte yield, which was associated with embryo production and pregnancy rates.     

Pattern of sea bass oocyte development after ovarian stimulation by LHRHa

The cyclic pattern of oocyte development in the sea bass, Dicentrarchus labrax L., was studied after induction of spawning by two injections, 24 h apart, of a luteinizing hormone releasing-hormone analog (LHRHa) administered at the end of vitellogenesis. The first difference in the developmental stage of the ovary and in the size-frequency distribution of oocytes between the LHRHa treated group and the control group, was detected 32 h after the first injection, the LHRHa group showing a higher proportion of the 900 μm diameter oocyte class (maturing oocytes) ( P <0.01). At 48 h LHRHa-treated females showed an increase in the 1000 and 1100 μm classes (maturing oocyte and ovulated eggs) ( P <0.01) and at 72 h these females exhibited a bimodal pattern, reaching the highest proportions in the 1100 (27.4%) and the 600 (14.7%) μm classes (ovulated eggs and advanced vitellogenic oocytes, respectively). Bimodal distributions were present in 80% of the LHRHa-treated females. Once oocyte final maturation was triggered by LHRHa the time needed for ovulation was about 48 h and the interval between consecutive ovulations and spawnings seemed to be 48–72 h.     

A combination of FSH and dibutyryl cyclic AMP promote growth and acquisition of meiotic competence of oocytes from early porcine antral follicles

Growing porcine oocytes from early antral follicles (1.2-1.5 mm in diameter) do not mature to metaphase II (MII, 4%) under culture conditions which supported maturation (MII, 95%) of fully grown oocytes from large (4-6 mm) antral follicles. We hypothesized that FSH and dbcAMP supported growth and acquisition of meiotic competence. Growing oocytes (113.0 ± 0.4 μm, mean ± SEM) were cultured for 5 d in medium supplemented with 1 mM dbcAMP, 0.01 IU/mL FSH or both; in these media, oocytes reached, 120.5 ± 0.4, 123.5 ± 0.4 and 125.7 ± 0.2 μm, respectively, after 5 d, and then were matured in vitro for 48 h. Oocytes remained enclosed by cumulus cells when cultured with FSH (82%) or both FSH and dbcAMP (80%), but not with dbcAMP alone (0%). Furthermore, oocytes cultured with FSH maintained trans-zonal projections of cumulus cells. Oocytes remained at the GV stage at higher rates when cultured with dbcAMP and FSH (99%), or dbcAMP (97%), than with FSH (64%), or without either (75%). Following in vitro maturation, oocytes reached MII after in vitro growth with dbcAMP (19%), FSH (11%), or both (68%). When oocytes were cultured with both FSH and dbcAMP, activation of Cdc2 and MAP kinases in growing oocytes was similar to fully grown oocytes. In conclusion, growing porcine oocytes grew and acquired meiotic competence in medium supplemented with dbcAMP and FSH; the former maintained oocytes in meiotic arrest, whereas the latter maintained trans-zonal projections of cumulus cells to oocytes during in vitro growth culture.     

Effects of Hoechst 33342 staining and ultraviolet irradiation on the developmental competence of in vitro-matured porcine oocytes

Hoechst 33342 (H342) in combination with ultraviolet (UV) irradiation is frequently used to assist the enucleation of porcine oocytes in somatic cell nuclear transfer programs. This work evaluated the effects of H342 (5 μg/mL for 12 min) staining and/or exposure to UV irradiation on fertilisability and developmental capacity of porcine oocytes matured in vitro. In Experiment 1, a total of 1388 mature oocytes were distributed in the following groups: Group 1: oocytes without treatment (Control), Group 2: oocytes stained with H342, Group 3: oocytes stained with H342 and UV irradiated for 30 sec, and Group 4: oocytes UV irradiated for 30 sec. Oocytes from each group were exposed to thawed spermatozoa and cultured for 18 h to assess fertilization parameters or for 7 d to evaluate embryo development. Sperm penetration (P < 0.001) and monospermy (P < 0.04) were lower in oocytes exposed to H342/UV (80.7 ± 4.5% and 30.7 ± 5.4%, respectively) than in oocytes from the control group (94.9 ± 4.3 and 50.0 ± 4.9, respectively). The oocytes exposed to H342/UV showed lower (P < 0.001) cleavage (49.8 ± 2.9%) and blastocyst (7.7 ± 2.9%) rates than oocytes from the other groups (range: 73.8 ± 2.9% to 77.7 ± 2.9% and 22.3 ± 2.9% to 30.9 ± 3.0%, respectively). Experiment 2 was designed to evaluate the effect of shorter UV irradiation (5 sec). A total of 1835 mature oocytes were separated into the same groups as those of Experiment 1. The fertilization parameters and the cleavage rates were not influenced by the different treatments. However, the oocytes exposed to H342 and UV irradiation for 5 sec showed a lower (P < 0.02) rate of blastocyst formation (15.2 ± 4.5%) than the oocytes from other groups (range: 26.1 ± 4.5% to 30.7 ± 4.5%). In conclusion, our results demonstrate that the combination of H342 staining with UV irradiation has a clear deleterious effect on the developmental ability of oocytes, with the effects being more intense with increased exposure to UV irradiation.     

Role of hydration in ovulation of common frog oocytes in vitro

Stimulation of ovulation of the common frog Rana temporaria oocytes with homologous pituitary extract caused an increase in their volume. Factors that are known to inhibit hydration in teleostean oocytes (potassium-free Ringer solution and inhibitor of Na⁺,K⁺-ATPase—ouabain), as well as aquaporin inhibitors (mercuric chloride and methylmethanethiosulphonate) inhibited also homologous pituitary extract-induced volume increase in follicle-enclosed oocytes and led to reduced percentage of ovulated oocytes. Volume of denuded oocytes remained unchanged in the course of maturation when exposed to progesterone or other treatments. The data obtained suggest that stimulation of oocyte ovulation in the common frog caused an increase in their hydration that is necessary for their ovulation but this did not occur in denuded cells.     

Parthenogenetic development of domestic cat oocytes treated with ionomycin, cycloheximide, roscovitine and strontium

The objective was to evaluate the parthenogenetic activation of domestic cat oocytes. Cumulus-oocyte complexes matured for 36 h were subjected to three protocols of parthenogenetic activation: Group 1 - ionomycin + cycloheximide; Group 2 - ionomycin + roscovitine; and Group 3 - ionomycin + strontium. As a control, a fourth group of oocytes were cultured in the absence of any activation agent. In all groups, embryos were cultured in SOFaa for 72 h after activation and evaluated for activation rate, cleavage, and embryonic development using Hoechst33342. There were no significant differences among the three treated groups for rates of activated oocytes (70.1 ± 4.3, 75.5 ± 4.7, and 61.9 ± 7.2%, for Treatments 1, 2, and 3 respectively; mean ± SEM), or cleavage (48.1 ± 5.9, 47.4 ± 3.8, and 33.3 ± 6.8%). However, activation and cleavage rates were higher (P < 0.05) than those in the control group (35.5 ± 6.4 and 11.8 ± 4.0%). There were no significant differences among treatment groups for proportion of embryos with 2-10 cells, 10-16 cells, and morulas. In the Control group, the embryo production rate was lower (P < 0.05), although the activation rate was high. The authors concluded that all three treatments effectively induced parthenogenetic activation of domestic cat oocytes. However, to optimize the use of strontium and roscovitine, a dose response and the effect of the presence of Ca⁺⁺ in the medium requires further study.     

Effects of hyperstimulation with gonadotrophins and age of females on oocytes and their metaphase II status in rats

The present study was undertaken to evaluate the effects of hyperstimulation and aging on the number and proportion of oocytes in the metaphase II stage in female Wistar rats. It explored the validity of the hypothesis that a combination of hyperstimulation with pregnant mare serum gonadotrophins (PMSG) and age could compromise, to a greater extent, the oocyte quality as indicated by the proportion of ovulated oocytes in the metaphase II stage. Female Wistar rats were stimulated with varying doses of PMSG and human chorionic gonadotrophins (hCG) and the number and proportion of ovulated oocytes in the metaphase II stage were examined and compared between different groups of young adult (8-10 weeks old) and aging (30-32 weeks old) female rats. While spontaneous ovulation occurred in all young adult rats, only 50% of the aging rats did. The ovulation rate in aging rats was increased from 50 to 93% when non-PMSG-stimulated rats were given a dose of 10 IU of hCG at proestrus. The lower number of ovulated oocytes noted, even in those hyperstimulated with high doses of PMSG/hCG, also indicated a reduction in fertility in aging rats. Under the influence of high doses of PMSG, all aging rats ovulated, but as with the young adult rats, a higher dose of hCG was needed to achieve the maximum number of ovulated oocytes from the PMSG-induced expanded pool of preovulatory follicles. However, the average number of ovulated oocytes in aging rats was, nevertheless, still significantly lower than in young adult rats even when approximation of weight was considered. No consistent significant difference in proportion of normal oocytes was noted within groups and between young adult and aging rats. A lower proportion of ovulated oocytes was arrested at the metaphase II stages when rats, whether they were young adult or aging, were hyperstimulated with 40 IU of PMSG. However, this proportion was restored to normal (about 100%) when a higher dose of hCG, which is a signal responsible for initiating oocyte maturation, was used. Results of the present study showed that there appears to be an age-related reduction of sensitivity of the preovulatory follicles to the ovulation induction signal of hCG and thus higher doses of hCG were needed to ovulate the PMSG-induced expanded pool of dominant follicles. In older rats, apart from the obvious depletion of the pool of follicles, the evidence from the present study suggests that some of these older rats do have follicles, but that these were unable to develop to preovulatory follicles, probably because of the absence of sufficiently high levels of gonadotrophins essential for the initiation of folliculogenesis. PMSG-hyperstimulation can affect nuclear maturation; the proportion of ovulated oocytes not arrested at the metaphase II stage was higher. However, the proportion of ovulated oocytes at the metaphase II was restored to normal by increasing the dose of hCG use. Hence, meiotic aberration in rats is not age-dependent but rather dependent on the amplitude of the luteinizing hormone (LH)/hCG surge present. The results from this study nullified the hypothesis that hyperstimulation in combination with aging would lead to a higher proportion of abnormality in ovulated oocytes with respect to their being at inappropriate meiotic stages.     

Azadirachta indica A. Juss (neem) induced morphological changes on oocytes of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) tick females

Many studies have been conducted with plants whose extracts have the potential to be used for pest control. One of these plants is Azadirachta indica (neem), whose main active ingredient is azadirachtin, a compound shown to have acaricide and insecticide activity. Rhipicephalus sanguineus (brown dog tick) is currently considered to be an “urban pest,” because of its high levels of infestation and its ability to attack humans. In the present study partially and fully engorged R. sanguineus females were exposed to aqueous extracts of neem at concentrations of 10% and 20%, and to a control treatment. The results showed that differently from what was observed in the control, the pedicel cells of females exposed to neem at both concentrations lost their original shape. In the latter cases, the cytoplasm of the cells became fully vacuolated, especially near the germinal vesicle (oocyte nuclei) and in the oocyte pole, which is in contact with the cells of the pedicel. Oocytes in early stages of development (I and II) of ticks treated with both concentrations had irregular germinal vesicle, including the presence of two nucleoli as well as fragments of these. Oocytes in stages IV and V of exposed individuals showed full granular cytoplasm with bigger yolk granules when compared to the early stages. Chorion of mature oocytes was also altered, showing folds and deformations along their entire extension. The observed changes in cells of the reproductive system of R. sanguineus, especially in the oocytes, indicated the potential of neem as a new alternative method to control these ectoparasites.     

Changes in the reciprocal position of the first polar body and oocyte chromosome set in golden hamsters

The golden hamster is an attractive model organism for studying reproductive physiology, oncology, genetics and virology. In an effort to establish experimental protocols necessary for cloning golden hamsters, we examined changes in the reciprocal position of the FPB (first polar body) and chromosome set of MII (the second meiotic metaphase) oocytes of golden hamsters. Oocytes were collected under three different conditions: (i) oocyte direct recovery from the oviduct of hormonally treated donor; (ii) oocyte recovery from the oviduct of hormonally treated donor followed by 5 h/10 h in vitro culture; and (iii) oocyte recovery from ovaries of hormonally treated donors and in vitro maturation. Then oocyte recovery was performed from the oviduct of hormonally treated donors, followed by 5 h in vitro culture with colchicine and/or CB (cytochalasin B). Denuded oocytes were stained with Hoechst 33342 and propidium iodide and evaluated under a microscope. Our results demonstrate that the change in FPB position in relation to the MII oocyte chromosome set increases with age of in vivo-matured oocytes. Cumulus cells can protect the FPB of in vitro-cultured oocytes from degeneration but do not significantly affect its repositioning, and in vitro-matured oocytes age slower. The colchicine has a stronger effect on cytoplasmic protrusions of golden hamster oocytes when compared with CB. These results define conditions for changes in FPB position relative to the MII oocyte chromosome set. Early ovulated oocytes, in vitro-matured oocytes and oocytes treated with colchicine should improve the effectiveness of the cloning procedure in golden hamsters as an animal model for human diseases.     

Ovulated oocytes collected from PMSG/hCG-treated and cycling Djungarian or Siberian hamsters (Phodopus sungorus) are structurally similar with no evidence of polar body formation,indicating arrest in meiosis I

This study was undertaken (1) to devise a method of inducing multiple follicular development and subsequent ovulation in the Djungarian or Siberian hamster (Phodopus sungorus) and (2) to assess the quality of ovulated oocytes collected from PMSG/hCG treated animals in comparison to naturally ovulating animals. Hamsters (4–5 weeks; n = 70) received 5 IU PMSG followed 50–52 hr later by 10 IU hCG. Ovulated oocytes were collected 14–20 hr after hCG injection. Ovulated oocytes were flushed from oviducts of cycling animals (7–12 weeks; n = 30) exhibiting two consecutive estrous cycles. Oocytes were fixed and subjected to triple fluorescence immunostaining using anti-tubulin antibodies, fluorescein phalloidin, and Hoechst 33258. The mean number of ovulated oocytes collected from cycling animals was 4.8 ± 0.4 (range 1–7). Ovulation occurred in 73% of the PMSG/hCG-stimulated animals. The mean number of oocytes ovulated from stimulated animals was 9.2 ± 0.8 (range 0–22). The ovaries of animals that did not ovulate or that ovulated few oocytes did respond to PMSG, as indicated by the presence of multiple follicular development and pre-ovulatory stigmata. There was no evidence of a polar body in ovulated oocytes collected from PMSG/hCG-treated or cycling animals, indicating that oocytes were arrested in meiosis I. In the majority (80%) of ovulated oocytes from PMSG/hCG-treated and cycling animals, cortically placed chromosomes were aligned on a metaphase plate equidistant from a bipolar spindle. Sparse f-actin staining was observed in the region of the ooplasm surrounding the chromosomes. As the interval between hCG injection and the time of collection increased, chromosomes lost their proper alignment and migrated away from the cortex of the oocyte concomitant with a disruption of spindle integrity. This collapse of proper chromosome alignment and disruption of spindle architecture also characterized aging oocytes collected from cycling animals. These data show that in the Djungarian or Siberian hamster (Phodopus sungorus), (1) there is individual animal variation in responsiveness to hCG following PMSG treatment, (2) there are no cytological differences in the quality of oocytes collected from hormonally treated animals when compared to cycling animals, and (3) oocytes are ovulated in meiosis I. © 1995 Wiley-Liss, Inc.     

Developmental competence and mRNA expression of preimplantation in vitro-produced embryos from prepubertal and postpubertal cattle and their relationship with apoptosis after intraovarian administration of IGF-1

Recombinant human Insulin-like growth factor-I (hIGF-1) was administered to one ovary of prepubertal and postpubertal cattle to determine its effects on (1) oocyte developmental competence, (2) the expression pattern of six developmentally important genes (GLUT3, GLUT8, AKT1, BCL-XL, BAD, and BAX), and (3) its relationship with apoptosis (female Holstein-Friesian). Oocytes were retrieved from 7- to 10-mo-old prepubertal dairy calves (preP), 11- to 18-mo-old postpubertal heifers (postP), and cows via ultrasound-guided follicular aspiration. Immature oocytes were matured in vitro then fertilized and cultured up to the blastocyst stage. Apoptosis was determined by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) in 8-d blastocysts. Similar low blastocyst yields were observed in the IGF-1-treated preP group (11.2 ± 2.4%), the control preP group (10.4 ± 3.0%), and in the IGF-1 postP group (10.9 ± 2.3%). These were lower (P ≤ 0.01) compared with the control postP group (21.2 ± 3.8%) and with cows (23 ± 3.7%). The expression profile of the six genes was partly affected by age and IGF-1 treatment. Apoptosis was correlated with the age of the oocyte donors and was increased in blastocysts derived from prepubertal heifers. Results show that apoptosis is a critical feature of the acquisition of developmental competence of oocytes from prepubertal cattle and that IGF-1 did not beneficially affect oocyte developmental competence.