Progression of oocyte maturation from metaphase I to metaphase II is disturbed by previous immunological interference with cumulus cell function.

The effects of an antibody preparation reacting with preovulatory mouse cumuli oophori (anticumulus Ig) on oocyte maturation in vivo and in vitro were studied. Continuous presence of anticumulus Ig in culture medium did not impair oocyte maturation in vitro. Similarly, no effect on oocyte maturation in vivo was observed when anticumulus Ig was given to females superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) at the time of hCG treatment. However, when administered earlier, anticumulus Ig brought about serious disturbances of oocyte meiotic competence, since only immature oocytes were ovulated after anticumulus Ig injection at the time of PMSG treatment and as much as 70% of the ovulated oocytes were immature when the antibody was applied 24 hr later. Previous absorption of anticumulus Ig with isolated cumulus cells removed the inhibitory effect of this preparation on oocyte meiotic competence to the same extent as absorption with whole cumuli oophori, despite the persistence of a strong reactivity of the cumulus cell-absorbed antibody preparations with the cumulus intercellular matrix. The ability of oocytes obtained from antibody-injected animals to mature in vitro was also considerably impaired when the injection was made at the time of PMSG treatment. In all cases the maturation defect concerned the progression of meiosis from metaphase I to metaphase II, while the ability of oocytes to undergo germinal vesicle breakdown (GVBD) was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fertilizability of Superovulated Eggs by Estrous Stage-independent PMSG/hCG Treatment in Adult Wistar-Imamichi Rats

We investigated the fertilization and developmental ability of superovulated eggs obtained from adult Wistar-Imamichi (WI) rats, by using pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) treatment. Female WI rats, 11–13 weeks of age, were divided into four groups by estrous stage (metestrus [ME], diestrus [DE], proestrus [PE], or estrus [E]). PMSG (150 IU/kg) and hCG (75 IU/kg) were injected at an interval of 48 or 55 h and the female rats were mated with mature male rats. The ovulated eggs were collected 20, 24, and 27 h after hCG injection. Regardless of the estrous stage at the time of PMSG injection, the treated rats mated and ovulated similar to the untreated spontaneously ovulated rats (S group). Although the proportion of fertilized eggs in the E- and PE-treated groups was less than the S group 20 h after hCG injection, the proportion was not different among all treated and S groups 24 h after hCG injection. The proportion of fertilized eggs using in vitro fertilization and the proportion of offspring obtained from 2-cell stage embryo transfer did not differ among the treated and S groups. In comparison with PMSG/hCG-treated immature rats, mating and ovulation rate of adult rats were significantly higher. The proportion of fertilized eggs obtained from mated rats did not differ between immature and adult rats. These results demonstrate that adult WI rats are good egg donors for reproductive biotechnological studies using unfertilized or fertilized eggs.     

Mitochondrial organization in prepubertal goat oocytes during in vitro maturation and fertilization

The aim of this study was to evaluate mitochondrial distribution during in vitro maturation (at 0, 15, 20, and 27 hr of IVM) and fertilization of prepubertal goat oocytes compared to mitochondrial distribution of ovulated and in vitro fertilized oocytes from adult goats. Oocytes from prepubertal goats were recovered from a slaughterhouse and were matured in M199 with hormones and serum for 27 hr. Ovulated oocytes were collected from gonadotrophin-treated Murciana goats. Frozen-thawed spermatozoa were selected by centrifugation in Percoll gradient and were capacitated in DMH with 20% steer serum for 1 hr. Ovulated and IVM-oocytes were inseminated in DMH medium with steer serum and calcium lactate for 20 hr. Oocytes and presumptive zygotes were stained with Mitotraker Green FM and observed under a confocal laser scanning microscope. Ultrastructural morphology of oocytes and presumptive zygotes were analyzed by transmission electron microscopy (TEM). Prepubertal goat oocytes at germinal vesicle stage (GV) presented mitochondria localized in the cortical and perinuclear region. IVM-oocytes at metaphase II presented mitochondria peripheral polarized to the region opposite were the metaphase spindle is positioned and within the polar body. Ovulated oocytes presented peripheral mitochondria distribution and mitochondrial aggregation around the MII spindle. At 20 hr post-insemination, mitochondria were distributed around the two synchronous pronuclei (2PN rpar; in zygotes ovulated oocytes whereas in prepubertal 2PN-zygotes mitochondria presented a peripheral polarized distribution. Images by TEM detected that immature prepubertal goat oocytes that are less electrodense and present fewer cristae than in vitro matured prepubertal goat oocytes; these are characterized by being associated to swollen vesicles. Mol. Reprod. Dev. 73: 617-626, 2006 (c) 2006 Wiley-Liss, Inc.     

Pattern and frequency of nondisjunction in oocytes from the Djungarian hamster are determined by the stage of first meiotic spindle inhibition

In order to study the mechanisms of nondisjunction at meiosis I in oocytes gonadotropin-stimulated Djungarian hamsters were treated at two stages [4.5 and 6 h post human chorionic gonadotropin (HCG)] during the preovulatory period with 1000 mg/kg Carbendazim (MBC). The compound, known to bind fast but reversibly to mammalian tubulin, was chosen to investigate whether the stage at which spindle function is inhibited affects the pattern of nondisjunction. Ovulated oocytes were cytologically prepared and scored for hyperhaploidy, diploidy and presegregation. Application at an early spindle phase, 4.5 h post HCG, to females stimulated with a low gonadotropin dose [3 IU pregnant mares serum (PMS); 2 IU HCG] caused a high frequency of nondisjunction (40.6%) with a more or less nonspecific pattern of malsegregated bivalents. Treatment at a late stage of spindle function (6 h post HCG) resulted in a less frequent (22.5%) but highly preferential malsegregation of those A-D group bivalents thought earlier to be late segregators. On the other hand, oocytes from females primed with a high (10 IU PMS and HCG) gonadotropin dose, a treatment assumed to delay meiosis by approximately 1.5 h, responded to MBC treatment at the late stage (6 h) with a nonspecific pattern and a high frequency (71.2%) of nondisjunction. The latter result is comparable to that in which MBC was given at the early stage (4.5 h) and after a low gonadotropin dose. The high nondisjunction response additionally indicates that spindles in hypergonadotropic stimulated oocytes are more susceptible and/or that the concentration of the inhibitor is higher in such oocytes. Only few oocytes with presegregation (3.1%; 0.0%; 1.7%) and few diploid oocytes (3.3%; 1.5%; 3.2%) with complete inhibition of meiosis I were observed. We conclude, that in Djungarian hamsters (1) the segregation of bivalents at meiosis I is asynchronous with the large A-D bivalents segregating last, (2) the phase in which spindle function is inhibited determines the pattern of nondisjunction, and (3) the resumption of meiosis I — from dictyotene to metaphase II — does not follow a rigidly timed programme but depends on the conditions of follicular maturation.     

Reversal of indomethacin blockade of ovulation in gilts by prostaglandins

Prepubertal gilts given 750 IU pregnant mares′ serum gonadotropin (PMSG) followed 72 h later by 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation fail to ovulate when 10 mg/kg indomethacin (INDO) is injected 24 h after hCG administration. This study examines the effects of administration of exogenous prostaglandins F_2α and E₂ (PGF_2α and PGE₂) alone or in combination, and at various times prior to the expected time of ovulation, on the INDO blockade of ovulation in PMSG/hCG-treated gilts. Occurrence of ovulation was determined by visual observation at laparotomy 48 h after hCG. When 5 mg or 10 mg PGF_2α was injected at each of 38, 40 and 42 h after hCG injection, 63% and 79%, respectively, of preovulatory follicles ovulated. In contrast, injection of 5 mg PGE₂ or 5 mg PGE₂ plus 5 mg PGF_2α induced ovulation in 0% and 24% of preovulatory follicles, respectively. In control groups, 100% of folicles in PMSG/hCG-treated gilts ovulated whereas none did so in PMSG/hCG/INDO-treated animals. These results indicate that administration of PGF_2α can induce ovulation in the PMSG/hCG/INDO-treated prepubertal gilt and suggest that PGE₂ is ineffective and may be antagonistic to PGF_2α in overcoming the ovulation blocking effect of INDO.     

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.     

Evidence that cortical granule formation is a periovulatory event in marsupials.

Formation of cortical granules was examined in superovulated oocytes from three marsupial species, brushtail possums (Trichosurus vulpecula) tammar wallabies (Macropus eugeniii) and grey short-tailed opossums (Monodelphis domestica) and in oocytes obtained during natural cycles in Macropus eugenii. Superovulation was induced by pregnant mares' serum gonadotrophin/gonadotrophin-releasing hormone (PMSG/GnRH) protocols and natural ovulation by removal of pouch young. Oocytes were collected after ovariectomy or by laparoscopically guided follicle aspiration into Hanks balanced salt solution (HBSS) supplemented with either 2.5% fetal calf serum (FCS) or 2.5% bovine serum albumin (BSA). Ovulated oocytes were collected by removing and flushing the oviducts with HBSS and fixed immediately for electron microscopy. There were no differences in the morphology or timing of formation of cortical granules between superovulated and naturally cycling animals. Cortical granules were absent from germinal vesicle (GV) stage follicular oocytes before the luteinizing hormone (LH) surge in all species. Dark cortical granules, similar in appearance to those seen in the oocytes of eutherian mammals, were found just beneath the plasma membrane (9 per 100 microns of plasma membrane) of preovulatory oocytes at germinal vesicle, metaphase 1 or anaphase 1 stages. In addition, they contained a number of less electron-dense cortical granules (12 per 100 microns plasma membrane). The cortical cytoplasm of preovulatory oocytes was rich in Golgi complexes actively involved in vesicle formation. Large numbers of dark cortical granules (90 per 100 microns plasma membrane) were found only in ovulated oocytes. A small number of cortical granules of lighter electron density were also present in ovulated oocytes. This suggests that the marsupial oocyte is following a very different timetable for cortical granule formation and accumulation from eutherian mammals and that oocytes of marsupials may not achieve cytoplasmic maturity until after ovulation. The significance of these events for fertilization and development remains to be established.     

Vanadate,an inhibitor of tyrosine phosphatases,induced premature anaphase in oocytes and aneuploidy and polyploidy in mouse bone marrow cells

Protein tyrosine phosphatases are needed for activating maturation promoting factor, meiotic spindle assembly and spindle checkpoint inactivation. The protein phosphatase inhibitor vanadate was used to upset the kinase-phosphatase equilibrium during oocyte maturation (OM) and the metaphase anaphase transition (MAT) prior to cytogenetic analyses of mouse oocytes and bone marrow cells. ICR females received pregnant mare serum gonadotrophin (PMSG) and 48h later received human chorionic gonadotrophin (hCG). Vanadate doses of 0, 5, 15, and 25mg/kg were administered intraperitoneally immediately after hCG and ovulated oocytes and bone marrow cells were processed for cytogenetic analyses 18h after hCG. Data were analyzed by Chi-square and Fisher's exact tests. Vanadate induced different cytogenetic abnormalities in oocytes and in bone marrow cells. The frequencies of oocytes exhibiting premature anaphase (spontaneous activation) in vanadate exposed mice were significantly (P<0.01) elevated over controls; whereas, in bone marrow cells, the levels of tetraploidy, hyperploidy and premature centromere separation were significantly (P<0.01) increased by vanadate treatment. These results suggest that alteration of the kinase-phosphatase equilibrium during OM and the MAT leads to cytogenetic abnormalities that differ between oocytes and bone marrow cells.     

Interference with the preovulatory luteinizing hormone surge and blockade of ovulation in immature pregnant mare's serum gonadotropin-primed rats with the anti-androgenic drug, hydroxyflutamide

The involvement of androgens in the control of ovulation has been assessed by administration of the androgen antagonist, hydroxyflutamide, to prepubertal rats treated with pregnant mare's serum gonadotropin (PMSG) to induce first estrus and ovulation. Without human chorionic gonadotropin (hCG) injection, only 46% of rats that received six 5-mg, s.c. injections of hydroxyflutamide at 12-h intervals, beginning an hour before s.c. injection of 4 IU PMSG on Day-2 (Day 0 = the day of proestrus), had ovulated a mean of 1.3 +/- 0.4 oocytes per rat when killed on the morning of Day 1, whereas 92% of sesame oil-treated controls had ovulated a mean of 6.9 +/- 0.6 oocytes. After i.p. injection of hCG at 1600 h on Day 0, 92% of hydroxyflutamide-treated rats ovulated a mean of 8.3 +/- 1.2 oocytes compared to 100% of controls, which ovulated 7.3 +/- 0.4 oocytes per rat: these groups were not significantly different from each other, nor from control rats that received no hCG. Thus, exogenous hCG completely overcame the inhibitory effect of hydroxyflutamide on ovulation. Rats treated with PMSG and hydroxyflutamide without hCG were killed either on the morning of Day 0 to determine serum and ovarian steroid levels or on the afternoon of Day 0 to determine serum LH levels. Serum levels of estradiol-17 beta and testosterone in hydroxyflutamide-treated rats were significantly higher (178% and 75%, respectively; p less than 0.01) than levels observed in controls on the morning of Day 0. Ovarian concentrations of the steroids were also elevated in hydroxyflutamide-treated rats (p less than 0.01 for testosterone only).(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localization of tissue-type plasminogen activator in ovaries before and after induced and spontaneous ovulation in the rat

Summary The observation that tissue-type plasminogen activator (tPA) activity increased dramatically in preovulatory follicles has led to the hypothesis that plasminogen activation is causally related to follicle rupture. With immunohistochemistry, we have studied the appearance of tPA in ovaries of immature rats induced to ovulate and in adult cycling rats. Treatment of immature female rats with a single dose of pregnant mare serum gonadotropin (PMSG) induced follicular maturation. A subsequent human chorionic gonadotropin (hCG) injection resulted in follicle rupture 12–14 h later. PMSG treatment alone did not induce appearance of tPA-immunoreactive cells in any ovarian compartment. After hCG stimulation, however, theca cells, granulosa cells, and oocytes of pre- and postovulatory follicles displayed distinct tPA immunoreactivity. Fibroblastlike cells in the theca layers and tunica albuginea of the follicle apex also demonstrated localized cytoplasmic tPA reactivity. In addition to tPA synthesis in preovulatory follicles, hCG also induced tPA staining in the theca (but not granulosa) layers of non-ovulatory follicles. At 24 h after hCG treatment, there was a marked tPA staining in developing corpora lutea, ovulated ova, and oviductal epithelium. Ovaries from regularly cycling adult rats displayed a similar ovulation-related pattern of tPA immunostaining. The appearance of tPA in different cell types of the preovulatory follicle and in the fibroblast-like cells at the follicle apex, strengthens the hypothesis of a direct involvement of tPA in follicle rupture. Presence of tPA in postovulatory oocytes, cumulus cells, and surrounding oviductal epithelium may also indicate a role for tPA in the transfer of eggs in the oviduct.This work was supported by NIH Research Grants HD-14084; 12303     

Influence of ovarian hyperstimulation and ovulation induction on the cytoskeletal dynamics and developmental competence of oocytes

This study was undertaken to determine the effects of gonadotrophin on cytoskeletal dynamics and embryo development and its role in improving the retrieval of developmentally competent oocytes. Female golden hamsters were injected with human chorionic gonadotrophin (hCG; 5-, 7.5- or 15-IU) on the day 4 of estrus, pregnant mare serum gonadotrophin (PMSG; 5-, 7.5- or 15-IU) on the day 1 of estrus, or 15-IU hCG at 56 hr post-15-IU PMSG injection in any cycle except estrus. Increasing the hCG dose decreased not only retrieval rate of 2-cell embryo but development to blastocyst after subsequent in vitro culture. Whereas, although increasing the PMSG dose induced increasing the number of 2-cell embryo and blastocyst, 15-IU PMSG injection caused retardation of development to blastocyst. No 2-cell embryos were retrieved by injecting both PMSG and hCG. The injections of 15-IU hCG and 7.5- or 15-IU PMSG inhibited the proliferation of trophectodermal and inner cell mass cells, respectively. Gonadotrophin injection didn't influence microtubular spindle formation, but 5- or 15-IU hCG, 15-IU PMSG, or PMSG and hCG injections induced aberrant cortical granule (CG) and microfilament distribution. After 15-IU hCG or PMSG and hCG injections, fewer oocytes had enriched cortical actin domains, and the expression of alpha-, beta- and gamma-actin genes was greatly increased. In conclusion, a high dose of gonadotrophins alters the microfilament and CG distribution, which in turn reduces the developmental competence of oocytes. Injecting a reduced dose of PMSG to initiate ovarian hyperstimulation without triggering ovulation contributes to the efficient retrieval of developmentally competent oocytes.     

Control of meiosis by somatic cells in mice: inheritance of the meiosis I error 'diploidy' and nonexpression in sensitive NMRI/Han oocytes ovulated from chimeras

NMRI mouse and Djungarian hamster females ovulate diploid and/or hyperploid oocytes with increased frequencies after gonadotrophin stimulation, suggesting that somatic cells are involved in the failures of endocrine control resulting in aneuploidy. To study the inheritance of gonadotrophin-induced aneuploidy as well as the fate of sensitive oocytes in a resistant somatic environment and vice versa, we analysed the frequency of diploid oocytes in NMRI/Han, C57BL/6J and their F1 hybrids (C57BL/6J X NMRI/Han), (NMRI/Han X C57BL/6J) as well as in NMRI/Han in equilibrium C57BL/6J chimeric females after gonadotrophin injections. Ovulated oocytes were analysed in all females for the appearance of diploidy, characterized as premature arrest of development at metaphase I. Our data suggest that the trait of induced diploidy is genetically determined and can be transmitted either maternally or paternally. A maternal effect modulated the expression of that trait. Several mechanisms acting on the feed-back control ovary-hypothalamus/pituitary, within the ovary or even within a chimeric follicle, may be responsible that 'sensitive' oocytes ovulated from chimeras are all normal haploid. These data suggest that not only oocyte maturation but also chromosome disjunction during meiosis I is controlled by somatic cells.     

Preferential nondisjunction of specific bivalents in oocytes from Djungarian hamsters (Phodopus sungorus) following colchicine treatment

In an attempt to study the mechanisms leading to nondisjunction during meiosis I, Djungarian hamster females were treated with colchicine (3 mg/kg), which binds specifically to tubulin. The number of ovulated oocytes per female was significantly reduced following colchicine treatment (8.2 +/- 5.3, compared to 10.6 +/- 5.9 in controls receiving saline solution only). Application of colchicine rather late during oocyte maturation (ie, 5.5 h after injection of human chorionic gonadotrophin) caused a significant increase in the number of ovulated diploid (34.5%) and hyperhaploid (11.7%) oocytes, compared to the frequencies observed in the saline-treated controls (0.8% and 3.5%, respectively). Specific bivalents (viz, the large meta- and submetacentric chromosomes of groups A, B, and C) were preferentially involved in colchicine-induced nondisjunction. The same pattern of chromosomal malsegregation was previously observed in oocytes from this hamster species following hypergonadotrophic stimulation. Preferential involvement of bivalents in the process of nondisjunction, whether induced by colchicine or hypergonadotrophic stimulation, is explained by an interference with microtubular function affecting those bivalents that are the last to segregate.     

Distinctions in meiotic spindle structure and assembly during in vitro and in vivo maturation of mouse oocytes

To better understand the differences in cytoskeletal organization between in vivo (IVO) and in vitro (IVM) matured oocytes, we analyzed remodeling of the centrosome-microtubule complex in IVO and IVM mouse oocytes. Fluorescence imaging revealed dramatic differences in meiotic spindle assembly and organization between these two populations. Metaphase spindles at both meiosis I (M-I) and meiosis II (M-II) in IVO oocytes were compact, displayed focused spindle poles with distinct gamma-tubulin foci, and were composed of acetylated microtubules. In contrast, IVM oocytes exhibited barrel-shaped spindles with fewer acetylated microtubules and gamma-tubulin diffusely distributed throughout the spindle proper. With respect to meiotic progression, IVO oocytes were more synchronous in the rate and extent of anaphase to telophase of M-I and first polar body emission than were IVM counterparts. Furthermore, IVO oocytes showed a twofold increase in cytoplasmic microtubule organizing centers (MTOCs), and constitutive MTOC proteins (gamma-tubulin and pericentrin) were excluded from the first polar body. Inclusion of MTOC constitutive proteins in the polar body and diminished number of cytoplasmic MTOCs was observed in IVM oocytes. These findings were corroborated in IVO oocytes obtained from naturally ovulated and spontaneously cycling mice and highlight a fundamental distinction in the spatial and temporal regulation of microtubule dynamics between IVO and IVM oocytes     

Induction and inhibition of meiotic maturation of follicle-enclosed porcine oocytes in vitro

Isolated porcine Graafian follicles which were explanted in vitro and maintained in organ culture were used as a test-system for the meiosis-inducing action of PMSG and hCG. The addition of either PMSG or hCG alone (10 or 20 IU/ml, respectively) to the culture medium was not effective, whereas the simultaneous administration of these hormones ($\text{15}\text{15}\text{IU/ml}$) induced resumption of meiosis in 90.3% ($\text{37}\text{41}$). The same hormone concentrations were used in a second series of experiments in which the inhibition and induction of meiosis of isolated oocytes were tested by transferring them into host follicles. In host follicles containing up to 12 foreign eggs, which were cultured in control media, meiosis was prevented in 86.0% of all oocytes ($\text{104}\text{121}$). By adding PMSG (15 IU/ml) simultaneously with hCG (15 IU/ml) to the medium, meiosis was induced in 95.0% of all oocytes ($\text{133}\text{140}$).The assumption is made that the signal initiating resumption of meiosis of the isolated and transferred oocytes is mediated by the follicular fluid, since intimate contact with the membrana granulosa of the host follicle was prevented by using a roller technique.     

The effect of pregnant mare serum gonadotropin on mouse oocytes as monitored at metaphase II

Ovulated oocytes were collected from random-bred, 7-12 week old ICR mice injected with 0, 3, or 6 i.u. pregnant mare serum gonadotropin (PMSG). Analyses of 872 metaphase figures from 87 females did not show a significant increase in chromosomal imbalance with PMSG treatment. A tendency toward ovum fragmentation was noted with an increase in PMSG dose.     

Developmental capability of rat oocytes matured in vitro in defined medium

The normality of in vitro matured oocytes was compared to that of in vivo matured (ovulated) oocytes at the following stages of development: germinal-vesicle breakdown, first polar body formation, fertilization (two polar bodies and two pronuclei with a sperm tail or first cleavage), and fetal development (day 20 fetuses). At all points, the in vitro oocytes exhibited a reduced ability, with oocytes matured cumulus-free having the poorest. The exposure of oocytes to human chorionic gonadotropin (hCG) for 2 hr before collection or during incubation improved their rates of maturation and development to day 20 fetuses but not their ability to undergo fertilization. While beneficial, the exposure to gonadotropins before or during maturation was not essential, as evidenced by the production of two day 20 fetuses matured and fertilized in vitro without any gonadotropin (luteinizing hormone or hCG) treatment in vivo or in vitro. These data demonstrate that in the population of in vitro matured oocytes there exist individuals wholly competent of complete normal development, albeit in a reduced proportion in comparison to normally matured and ovulated oocytes. That the in vitro handling, treatment, and culture of the oocytes may be responsible for some of the reduced developmental ability observed is suggested by the developmental abilities of ovulated oocytes under different conditions. Ovulated oocytes fertilized in the donor had the highest rates of development (46%), followed by those fertilized after transfer into mated recipients' oviducts (20%). The lowest rate was achieved with in vitro fertilized oocytes (7%), which represented the group subject to the greatest degree of manipulation and distinction from the normal in vivo process.     

Stage of the estrous cycle at the time of pregnant mare's serum gonadotropin injection affects the quality of ovulated oocytes in the mouse

The present study aims to analyze the effect of the stage of the estrous cycle at the time of pregnant mare's serum gonadotropin (PMSG) injection on number and quality of mouse oocytes retrieved from oviducts after exogenous ovarian stimulation. Cellular and morphological traits of ovulated oocytes from hybrid (C57Bl/6JIco female X CBA/JIco male) female mice of 12, 40-42, 50-52 or 57-62 weeks of age were analyzed. Superovulation was induced by a priming injection of PMSG at different stages of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Injection of PMSG at diestrus-1 was associated with: (1) increased percentage of cumulus-free oocytes; (2) raised total percentage of oocytes without polar body; (3) increased total percentage of oocytes with intracytoplasmic mitochondrial aggregates; (4) decreased percentage of oocytes with a normal distribution of chromosomes in the metaphase II plate; and (5) raised percentage of oocytes with chromosome scattering when compared to injection at estrus, diestrus-2, and proestrus stage. On the contrary, estrus females displayed the highest percentage of oocytes with a normal distribution of chromosomes in the metaphase II plate and the lowest percentage of oocytes denuded of cumulus cells, without polar body, with intracytoplasmic mitochondrial aggregates and/or with chromosome scattering. These data suggest that administration of gonadotropins in mice should be synchronized with the innate estrous cycle of females to optimize the quality of oocytes collected from oviducts.     

Distribution of prepubertal and adult goat oocyte cortical granules during meiotic maturation and fertilisation: ultrastructural and cytochemical study

The aim of this study was evaluate cortical granule (CG) distribution during in vitro maturation (IVM) and fertilisation of prepubertal goat oocytes compared to CG distribution of ovulated and in vitro fertilised oocytes from adult goats. Oocytes from prepubertal goats were recovered from a slaughterhouse and were matured in M199 with hormones and serum for 27 hr. Ovulated oocytes were collected from gonadotrophin treated Murciana goats. Frozen-thawed spermatozoa were selected by centrifugation in percoll gradient and were capacitated in DMH with 20% steer serum for 1 hr. Ovulated and IVM-oocytes were inseminated in DMH medium with steer serum and calcium lactate for 20 hr. Oocytes and presumptive zygotes were stained with FITC-LCA (Lens culinaris agglutinin labelled with fluorescein isothiocyanate) and observed under a confocal laser scanning microscope. Ultrastructure morphology of oocytes and presumptive zygotes were analysed by transmission electron microscopy (TEM). Prepubertal goat oocytes at germinal vesicle stage show a homogeneous CG distribution in the cytoplasm. IVM-oocytes at Metaphase II (MII) and ovulated oocytes presented CGs located in the cortex with the formation of a monolayer beneath to the plasma membrane. At 20 hr postinsemination (hpi), zygotes from IVM-oocytes showed a complete CG exocytosis whereas zygotes from ovulated oocytes presented aggregates of CGs located at the cortical region. Images by TEM detected that CGs were more electrodense and compacts in oocytes from prepubertal than from adult goats.     

Rate of gonadotrophin-induced abnormalities in mouse ova is related to the site of hormone administration

Adult female mice, regardless of the stage of the oestrous cycle, were superovulated with PMSG and hCG. Ovulated oocytes were recovered 20-22 h after hCG and fertilized ova 72-74 h after hCG. Compared with the controls, the gonadotrophin treatment increased the mating rate of the females, and the incidence of abnormal ova. Regardless of the site of gonadotrophin injections, the numbers of ova were equal, but the proportion of abnormal eggs in mice injected intraperitoneally was significantly higher than in mice injected subcutaneously.