Surface alterations of the bovine oocyte and its investments during and after maturation and fertilization in vitro

Surface characteristics of the bovine oocyte and its investments before, during, and after maturation, and fertilization in vitro were evaluated by scanning electron microscopy (SEM). Oocyte diameters were also measured during SEM analysis of the oocyte. The cumulus cells manifested a compact structure with minimal intercellular spaces among them in the immature oocytes. These became fully expanded with increased intercellular spaces after maturation in vitro, but contracted again after fertilization. The zona pellucida (ZP) showed a fibrous, open mesh-like structure in the maturing and matured oocytes. The size and number of meshes on the ZP decreased dramatically after fertilization. The vitelline surface of immature oocytes was characterized by distribution of tongue-shaped protrusions (TSPs) varying in density. After 10 and 22 hr of maturation incubation, oocyte surface microvilli (MV) increased to become the predominant surface structure, and TSPs decreased substantially. The vitelline surface of fertilized oocytes (at 6 and 20 hr) was similar to that of the matured oocytes, but unfertilized oocytes had less dense MV than did fertilized oocytes (at 20 hr). The diameter of the oocytes decreased from 99 to 80 μm during maturation and increased to 106 μm after insemination (P < 0.05). Membrane maturation was characterized by surface changes from a TSP-predominant pattern to a MV-predominant pattern. Thus, the bovine oocyte maturation process was found to involve the expansion of cumulus cells and the maturation of the ZP, which changes dramatically upon fertilization. Also, volumetric changes occurred in ooplasm processed for SEM following oocyte maturation and insemination. © 1994 Wiley-Liss, Inc.     

Cryopreservation of equine oocytes by 2-step freezing

Immature equine oocytes were frozen-thawed with ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GL) in PBS and cultured to assess the rate of in vitro maturation (Experiment 1). Compact-cumulus oocyte complexes were collected from slaughterhouse ovaries and equilibrated for 10 min in the freezing medium containing 10% (V/V) cryoprotectant and 0.1 M sucrose. The 0.25-ml straws, loaded with 10 to 30 oocytes, were seeded at -6 degrees C and cooled to -35 degrees C at 0.3 degrees C/min before being plunged into liquid nitrogen. The straws were thawed rapidly in a 37 degrees C waterbath for 20 sec. The proportions of frozen-thawed oocytes reaching Metaphase II (MII) stage after in vitro maturation of 32 h were 15.8% (EG), 5.8% (PD) and 0% (GL), while 63.3% of the nonfrozen control oocytes matured in vitro. The fertilizing ability of immature and mature oocytes after freezing in EG was tested by the insemination of zona-free oocytes with stallion spermatozoa (Experiment 2). Spermatozoa were preincubated for 3 h with 5 mM caffeine, treated with 0.1 mu M ionophore A23187, and inseminated for 20 h at the concentration of 1 to 2 x 10(7)/ml with 6 to 10 oocytes in 50 mu l of Brackett and Oliphant (BO) medium. Immature oocytes (Group 1) were matured in vitro after thawing and then their zona pellucida removed using 0.5% protease. The zona of mature oocytes were removed immediately after thawing (Group 2) or maturation (nonfrozen controls). The oocytes, which had mechanically damaged plasma membrane or lost by artifact, were not examined for insemination. Significantly more control oocytes exhibited a polar body at the time of insemination (53.5%) than either frozen-thawed immature or mature oocytes (25.8 and 27.3%, respectively). Similar proportion of frozen-thawed and control oocytes were penetrated by spermatozoa (71.8 to 79.1%) and exhibited 2 or more pronuclei (73.6 to 80.8%). The mean numbers of spermatozoa per penetrated oocyte were 1.9, 3.0 and 2.5, respectively, for Groups 1 and 2 and for the control oocytes. These results indicate that immature equine oocytes mature to the MII stage in vitro following freezing and thawing in EG or PD but not in GL. Stallion spermatozoa can penetrate zona-free immature and mature oocytes following freezing/thawing in EG and form morphologically normal pronuclei.     

Effect of vitrification on the zona pellucida hardening and follistatin and cathepsin B genes expression and developmental competence of in vitro matured bovine oocytes

The purpose of our study was to assess the effect of vitrification with or without the presence of calcium in the vitrification solution on the: 1) diameter of oocytes and thickness of the zona pellucida, 2) zona pellucida hardening, 3) expression of mRNA follistatin (FST) and cathepsin B (CTSB) in oocytes and 4) developmental competence of embryos derived from in vitro matured and vitrified oocytes.The results of our study demonstrate, that vitrification did not alter thickness of the zona pellucida and diameter of the oocytes, however it triggered hardening of the zona pellucida. The presence of calcium in the vitrification solutions intensified hardening of zona in immature and mature oocytes (P < 0.04, P < 0.001, respectively) and provoked increased mRNA FST expression in oocytes matured in vitro compared to immature oocytes (P < 0.01) and those vitrified without calcium (P < 0.004). CTSB mRNA expression was increased in immature oocytes and oocytes vitrified with calcium compare to mature oocytes (P < 0.02). The developmental potential of vitrified oocytes was impaired compared to non-vitrified oocytes, being more evident in oocytes vitrified with calcium.In summary, vitrification did not change the oocyte diameter and thickness of the zona pellucida and expression of FST and CTSB mRNA. It diminished developmental potential of the vitrified oocytes. The presence of calcium in the vitrification solutions increased hardening of zona pellucida as well as affected the level of FST and CTSB mRNA in oocytes and developmental potential of these oocytes.     

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)     

Influence of follicle size on the penetrability of immature pig oocytes for homologous in vitro penetration assay

In order to improve the performance of homologous in vitro penetration (hIVP) assays using immature oocytes to assess the penetrating ability of boar sperm, the present study was designed to evaluate the influence of oocyte and follicle size on the penetrability of immature pig oocytes obtained from slaughterhouse ovaries. Nonatretic antral follicles were isolated, measured with a computerized image analysis system and grouped according to their diameter: Group 1 (0.40-0.99 mm), Group 2 (1.00-2.19 mm), Group 3 (2.20-2.79 mm), and Group 4 (2.80-6.50 mm). After sperm coincubation and before penetrability evaluation, the immature oocytes were classified into four size categories according to their diameter excluding zona pellucida: <105, 105-109, 110-114, and > or =115 microm. As regards follicle size, the highest viability and penetrability were obtained with oocytes from follicles >2.20 mm (P>0.05). Regarding oocyte size, significant differences (P<0.05) were observed for all parameters evaluated between oocytes with a diameter above or below 110 microm. However, our results revealed that such differences were due to follicle size rather than oocyte diameter, since oocytes with the same diameter but from different follicle size groups showed different penetration rates. With increasing follicle size, the percentage of penetrated oocytes increased (P<0.05). Finally, our results showed that the greater penetrability of immature oocytes from larger follicles is not due to variations in the thickness of the zona pellucida. There were no significant differences in zona pellucida thickness between oocytes from the four follicular size groups. In summary, these results indicate that follicle size directly affects the penetrability of immature pig oocytes used in hIVP.     

Maturity and fertility of rhesus monkey oocytes collected at different intervals after an ovulatory stimulus (human chorionic gonadotropin) in in vitro fertilization cycles

In rhesus monkeys undergoing ovarian stimulation for in vitro fertilization (IVF), a midcycle injection of human chorionic gonadotropin (hCG) substitutes for the LH surge and induces preovulatory oocyte maturation. The time interval between injection and oocyte collection, ideally, allows for the completion of oocyte maturation without ovulation, which would reduce the number of oocytes available for harvest. To evaluate the influence of this time interval on oocyte parameters following hCG administration, we conducted a series of gonadotropin treatment protocols in 51 animals in which the interval from hCG administration to follicular aspiration was systematically varied from 27 to 36 hr. Follicle number and size, evaluated prior to hCG administration by sonography, did not vary significantly or consistently with preovulatory maturation time. Oocytes were harvested by laparotomy or laparoscopy, and scored for maturity before insemination. The percentage of mature, metaphase II (MII) oocytes at recovery increased significantly with increasing preovulatory time and was inversely proportional to that of metaphase I (MI) oocytes. However, oocyte yield tended toward a progressive decrease with increasing preovulatory maturation times from a high of 27 oocytes at 27 hr to a low of 17 oocytes/animal at the 36 hr time interval. Fertilization levels declined significantly from a high of 50% at 27 hr to a low of 30% at 36 hr. Thus, although higher percentages of mature oocytes were recovered at the longer time intervals, optimal oocyte/embryo harvests were realized after the shorter time intervals (27 and 32 hr) and are most compatible with the goal of achieving high yields of fertile oocytes and embryos following gonadotropin stimulation in rhesus monkeys. © 1996 Wiley-Liss, Inc.     

Role of cumulus cells for rat oocyte maturation and metabolism

Oocytes collected from immature PMSG-treated rats on the morning of proestrus were allowed to mature in culture either surrounded by their cumulus cells or after denudation. It was found that the time course of oocyte nuclear maturation was similar whether the cumulus cells were present or not. The oxygen consumption of noncultured oocytes was 0.12 nl/hr/oocyte and increased by 40% after four to eight hours in culture with intact cumulus. Respiration of oocytes cultured without cumulus remained constant throughout the culture, except for a transient decrease after four hours. It is concluted that the cumulus cells do not affect the spontaneous nuclear maturation in vitro, but that the metabolism in oocytes cultured with intact cumulus is different from that of cultured denuded oocytes. Furthermore, it appears that the rise in oocyte oxygen consumption is not a prerequisite for nucler maturation.     

Maturation of pig and rat oocytes transplanted into surrogate pig follicles in vitro

Pig oocytes obtained from slaughterhouse material and rat oocytes obtained from PMSG-treated immature females were incubated as isolated oocytes or injected into explanted pig follicles (5–8 mm). Free oocytes of both species, with or without their cumulus investment or gonadotropins during culture, matured at high rates after 30 hr or 9–10 hr of culture, respectively. Gonadotropic stimulation was necessary for maturation of both the native and injected cumulus-intact pig oocytes in follicle culture. Cumulus-free pig oocytes injected into follicle failed to mature in response to gonadotropic stimulation, suggesting an inability to perceive or respond to stimulation. Injected rat oocytes, however, matured irrespective of cumulus investment or gonadotropic stimulation. Their maturation was delayed and reduced at 9 hr. These results in the rat suggest that the pig follicular environment is incapable of regulating rat oocyte maturation but rather presents a permissive or supportive environment for their maturation. The explanted surrogate follicles from the pig or other species may provide a useful model for the study of oocyte-follicle interactions in oocyte maturation within or between species.     

Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.     

A potential role for triglyceride as an energy source during bovine oocyte maturation and early embryo development

The potential role of endogenous triglyceride in bovine oocyte maturation and preimplantation development has been investigated. Bovine immature oocytes were recovered from abattoir-derived ovaries, matured and fertilised in vitro and the zygotes grown to the blastocyst stage in SOFaaBSA. Methyl palmoxirate (MP) blocks the oxidation of fatty acids by inhibiting mitochondrial carnitine palmitoyltransferase A. The development of zygotes exposed to MP during oocyte maturation, and of zygotes exposed to MP during embryo culture has been assessed in terms of oxygen consumption by oocytes and embryos during a 4-6 hr incubation period in the presence of MP and as blastocyst formation and cell number. Immature oocytes exposed to MP during maturation had reduced capacity to form blastocysts after fertilisation; the same effect was apparent, but to a lesser extent, in zygotes exposed to MP during embryo development. Oxygen consumption values of oocytes and blastocysts in the absence of exogenous substrates were similar to those in control medium containing nutrients. MP-inhibited oxygen consumption of immature oocytes, mature oocytes, cleavage stages embryos and blastocysts by 64, 45, 12 and 13%, respectively. The data are consistent with a role for triglyceride as a key energy source during bovine oocyte maturation and potentially, during preimplantation embryo development.     

FORMATION OF THE FERTILIZATION MEMBRANE BY INSEMINATION OF IMMATURE STARFISH OOCYTES PRETREATED WITH CALCIUM-FREE SEAWATER

When immature oocytes of the starfish, Asterina pectinifera , were treated with calcium-free seawater for 1 hr and then inseminated in normal seawater, they formed several blisters, indicative of polyspermy, and raised fertilization membranes. These oocytes continued to have intact germinal vesicles. Electron microscopic study revealed that the egg surface remained virtually unchanged after the treatment with calcium-free seawater. Upon insemination, however, the cortical granules broke down and the fertilization membrane was formed. These immature oocytes with ferilization membranes underwent maturation (germinal vesicle breakdown) after treatment with 1-methyladenine.
The treatment with calcium-free seawater seems to bring about some physiological change on the surface of immature oocyte, which bestows some attributes of maturation but is insufficient to mature the oocytes completely.     

Differential Emergence of Cortical Granule Breakdown and Electrophysiological Responses during Meiotic Maturation of Toad Oocytes

Oocytes of the toad, Bufo bufo japonicus , at various stages of progesterone-induced maturation were stimulated by pricking or treatment with Ca-ionophore A23187. Upon pricking, oocytes 14 hr after hormone treatment (PHT) underwent sequential activation responses, such as development of an activation potential, cortical granule breakdown (CGBD), and formation of a perivitelline space (PVS), like those of mature oocytes (18 hr PHT). When oocytes were pricked at 14 hr PHT, it took about 10 min for the wave of CGBD to spread over all the oocyte surface, in contrast to the case with mature oocytes in which it took about 150 sec. The rate of spread of CGBD was significantly less in the vegetal hemisphere than in the animal hemisphere in both mature and immature oocytes. Treatment with A23187 (1 μM) for 5 min induced an activation potential, and PVS formation by the oocytes from 10 hr PHT, which was 3–4 hr earlier than the time when these responses could be induced by pricking. Oocytes at 8–9 hr PHT also showed CGBD in response to A23187, but without formation of an activation potential. Several patches of local PVS caused by the non-propagating CGBD were observed in oocytes treated with the ionophore 5–7 hr PHT. When a high concentration (10 μM) of A23187 was employed, CGBD without PVS formation was induced even in oocytes at 0 hr PHT. These results indicate that the responsiveness to a Ca²⁺ surge that is a prerequisite for both CGBD and genesis of an activation potential is acquired for the repective responses at different stages of oocyte maturation.     

Morphologic comparison of ovulated and in vitro–matured porcine oocytes,with particular reference to polyspermy after in vitro fertilization

This study was conducted to evaluate morphologic differences in pig oocytes matured in vivo and in vitro, with particular reference to the potential relationship between oocyte morphology and the occurrence of polyspermy after in vitro fertilization (IVF). In vivo–matured oocytes were surgically recovered from the oviducts of gilts with ovulated follicles on day 2 of estrus, and in vitro–matured oocytes were obtained by culturing follicular oocytes in a oocyte maturation system that has resulted previously in production of live offspring following IVF. Comparisons were made of the cytoplasm density, the diameter of oocytes with or without zona pellucida (ZP), the thickness of the ZP, the size of the perivitelline space (PVS), ZP dissolution time, and cortical granule (CG) distribution before IVF, and CG exocytosis and polyspermic penetration after IVF. Oviductal oocytes have clear areas in the cytoplasm cortex, while in vitro–matured oocytes have very dense cortex. The diameter of ovulated oocytes with ZPs was significantly (P < 0.001) greater than that of in vitro–matured oocytes. However, no difference was observed in the diameter of the oocyte proper. Significantly (P < 0.001) thicker ZPs and wider PVSs were observed in the ovulated oocytes. The ZPs of ovulated oocytes were not dissolved by exposure to 0.1% pronase within 2 hr, but the ZPs of in vitro–matured oocytes were dissolved within 131.7 ± 7.6 sec. The ZPs of ovulated oocytes, but not of in vitro–matured oocytes, were strongly labeled by a lectin from archis hypogaea that is specific for β-D-Gal(1–3)-D-GalNAc. Polyspermy rate was significantly (P < 0.01) higher for in vitro–matured oocytes (65%) than for ovulated oocytes (28%). CGs of oviductal oocytes appeared more aggregated than those of in vitro–matured oocytes. Most of CGs were released from both groups of oocytes 6 hr after IVF regardless of whether they were polyspermic or monospermic oocytes. These results indicate that in vitro–matured and in vivo–matured pig oocytes possess equal ability to release CGs on sperm penetration. Unknown changes in the extracellular matrix and/or cytoplasm of the oocytes while in the oviduct may play an important role(s) in the establishment of a functional block to polyspermy in pig oocytes. Mol. Reprod. Dev. 49:308–316, 1998. © 1998 Wiley-Liss, Inc.     

Assessment of Mouse Germinal Vesicle Stage Oocyte Quality by Evaluating the Cumulus Layer,Zona Pellucida,and Perivitelline Space

To improve the outcome of assisted reproductive technology (ART) for patients with ovulation problems, it is necessary to retrieve and select germinal vesicle (GV) stage oocytes with high developmental potential. Oocytes with high developmental potential are characterized by their ability to undergo proper maturation, fertilization, and embryo development. In this study, we analyzed morphological traits of GV stage mouse oocytes, including cumulus cell layer thickness, zona pellucida thickness, and perivitelline space width. Then, we assessed the corresponding developmental potential of each of these oocytes and found that it varies across the range measured for each morphological trait. Furthermore, by manipulating these morphological traits in vitro, we were able to determine the influence of morphological variation on oocyte developmental potential. Manually altering the thickness of the cumulus layer showed strong effects on the fertilization and embryo development potentials of oocytes, whereas manipulation of zona pellucida thickness effected the oocyte maturation potential. Our results provide a systematic detailed method for selecting GV stage oocytes based on a morphological assessment approach that would benefit for several downstream ART applications.     

Comparison of the penetrability of the egg vestments in follicular oocytes, unfertilized and fertilized ova of the rabbit

Mixed populations of rabbit ovulated eggs and follicular oocytes, one labelled with a fluorescent marker, were transferred to the same tubal ampulla of an inseminated recipient female and were then recovered 3 hr later. There was no significant difference in the number spermatozoa penetrating to the perivitelline space or within the substance of the zona pellucida of follicular oocytes (immature or atretic) and mature ovulated ova. In contrast to mature ovulated ova, however, none of the spermatozoa reaching the perivitelline space of vesicular (dictyate) oocytes had attached to or penetrated the vitelline surface to enter the ooplasm.The same approach involving transfer of nonpenetrated eggs together with eggs penetrated previously in a donor female, demonstrated that prior entry of spermatozoa does not reduce the penetrability or receptivity of the rabbit zona pellucida to subsequent spermatozoa.These experiments indicate: (a) that the penetrability of the granulosa cell investment and/or zona pellucida of the rabbit follicular oocyte does not change from the time of antrum formation until the point at which follicular atresia ensures; (b) that between the time of initial LH stimulation and ovulation important changes mediating the onset of the fertizability of the dictyate oocyte of the rabbit probably occur at the vitelline surface; and (c) that in neither a qualitative nor quantitative sense has the demonstrably greater resistance of the rabbit zona pellucida to proteolysis following fertilization any physiological significance for sperm penetration.     

Establishment of a basic method for manipulating preantral follicles: effects of retrieval method on in vitro growth of preantral follicles and intrafollicular oocytes

The aim of this study was to establish a basic manipulation protocol of preantral follicles for deriving developmentally competent oocytes. Primary, early and late secondary follicles retrieved from the ovaries of 14-day-old F1 (C57BL/6 x DBA2) female mice mechanically or enzymatically were cultured singly and in vitro growth of the follicles and maturation of intrafollicular oocytes were subsequently monitored. A mechanical method retrieved more (p < 0.0001) follicles (339 +/- 48 vs. 202 +/- 28) than an enzymatic method. However, the enzymatic method collected more singly isolated follicles that could be provided for subsequent culture (102 +/- 26 vs. 202 +/- 28). When an enzymatic method was employed, early and late secondary follicles required 9 and 6 days for reaching the maximal incidence of the pseudoantral stage. However, primary follicles were not possible to develop into the pseudoantral stage. The optimal duration of oocyte maturation from the onset of follicle culture was 7 days and 5-7 days for early and late secondary follicles, respectively. A general decrease in oocyte diameter (65.2-65.53 microm vs. 75 microm) and zona thickness (5.41-5.74 microm vs. 7.76 microm) was detected in in vitro-derived compared with in vivo-derived matured oocytes. Pronuclear formation was detected in 86-94% of mature oocytes after parthenogenetic activation and no significant difference was detected among groups. These results showed that preantral follicles retrieved by an enzymatic method underwent step-by-step growth in vitro, which could yield mature oocytes.     

A histochemical study of the changes occurring in the protein-carbohydrate composition of the cumulus-oocyte complex and zona pellucida in immature mice in response to gonadotrophin stimulation

Summary A histochemical account is presented of the changes that occur in the protein—carbohydrate composition of the cumulus—oocyte complex in immature mice after gonadotrophin treatment. The distribution and nature of the glycosaminoglycans (GAG) present was established by enzymic digestion of tissue sections with testicular orStreptomyces hyaluronidase prior to staining with periodic—acid Schiff (PAS) or Alcian Blue. Treatment with exogenous gonadotrophins [pregnant mare's serum and human chorionic gonadotrophin (hCG)] induced gross changes in the appearance of the zona pellucida (and in the histochemical staining of the cumulus—oocyte complex). A reduction was observed in the amount of PAS-positive material present within the zona pellucida of oocytes located in large Graafian follicles examined 40 h after stimulation with pregnant mare's serum. After the injection of hCG, the zona pellucida was further depleted of PAS-positive naterial. Most of the PAS-positive material became confined to the plasma membrane of the oocyte, while the oocyte itself also became increasingly PAS-positive. All the GAGs disappeared from zona pellucida within 4 h of hCG stimulation. The changes observed in the protein—carbohydrate composition of the zona pellucida in preovulatory oocytes immediately prior to ovulation may be a prerequisite for successful sperm-egg interactions.     

The relationship between cumulus cell-oocyte coupling,oocyte meiotic maturation,and cumulus expansion

The timing of the reduction of cumulus cell-oocyte coupling was correlated with oocyte meiotic maturation and the expansion (mucification) of the cumulus oophorus using immature mice treated with gonadotropins. Three hours after the injection of an ovulatory dose of human chorionic gonadotropin (hCG), more than 90% of the oocytes isolated from large Graafian follicles had undergone germinal vesicle breakdown, indicating that oocyte meiotic maturation had been initiated. However, no cumulus expansion or reduction of intercellular coupling was detected at this time. By 6 hr after hCG injection, the index of oocyte-cumulus cell coupling was still not less than that found in oocyte-cumulus cell complexes isolated from control mice not receiving hCG. Cumulus expansion at 6 hr post-hCG was limited to the outer cumulus cells while those adjacent to the oocyte were still tightly packed. Cumulus expansion appeared complete by 9 hr after hCG injection and the cumulus cell-oocyte coupling index was greatly reduced. These results show that oocyte meiotic maturation in the mouse is not initiated by a reduction in cumulus cell-oocyte coupling or by cumulus expansion. However, the results suggest that the reduction of intercellular coupling in vivo may be a result of cumulus expansion.     

Involvement of the metabolic hormones leptin, ghrelin, obestatin, IGF-I and of MAP kinase in control of porcine oocyte maturation

The general aim of our in vitro experiments was to study the role of the metabolic hormones leptin, ghrelin, obestatin and IGF-I and mitogen-activated protein kinase (MAPK)-dependent intracellular mechanisms in the control of nuclear maturation of porcine oocytes. For this purpose, porcine oocytes were isolated from the ovary and cultured in the presence of leptin, ghrelin, obestatin, IGF-I, MAPK blocker PD98059 and the combinations of hormones with PD98059. Proportions of matured oocytes (at metaphase II of meiosis, determined by DAPI staining) and of oocytes containing MAPK/ERK1-2 (determined by immunocytochemistry) were measured before and after culture. It was observed that the majority of oocytes isolated from the ovary before culture were immature and did not contain visible MAPK, but some oocytes were mature, and the majority of these oocytes contained MAPK. Incubation of oocytes resulted in a significant increase in the proportion of matured oocytes and in the percentage of oocytes containing MAPK in both the matured and not matured groups. Addition of IGF-I to the culture medium increased the proportion of matured oocytes, addition of leptin decreased it, and ghrelin and obestatin did not oocyte maturation. Addition of hormones did not affect the expression of MAPK in either immature or mature oocytes. PD98059, when given alone, suppressed the maturation and accumulation of MAPK in both mature and immature oocytes. When given together with hormones, PD98059 was able to reduce the stimulatory effect of IGF-I, to invert the inhibitory action of leptin to stimulatory and to induce the stimulatory action of ghrelin and obestatin on meiosis. IGF-I, ghrelin and obestatin, but not leptin, when given together with PD98059, increased the accumulation of MAPK in both immature and mature oocytes. Association of nuclear maturation and expression of MAPK in oocytes before, but not after culture, as well as the prevention of oocyte maturation by MAPK blocker suggests the involvement of MAPK-dependent intracellular mechanisms in the promotion of reinitiation, but not completion of meiosis. The effect of hormonal additions on meiosis of oocytes suggests that IGF-I is a stimulator, leptin can be an inhibitor, while ghrelin and obestatin probably do not control oocyte maturation. The ability of PD98059 to modify the effect of hormones on oocyte maturation and on MAPK expression suggests possible interference of hormones and MAPK-dependent intracellular mechanisms in oocytes. However, no influence of hormones on MAPK and lack of association between action of hormones and PD98059 on MAPK and meiosis suggest that MAPK is probably not a mediator of effect of IGF-I, leptin, ghrelin and obestatin on porcine oocyte nuclear maturation.     

Zona pellucida characteristics and sperm-binding patterns of in vivo and in vitro produced porcine oocytes inseminated with differently prepared spermatozoa

IVF of porcine oocytes has been carried out in many laboratories. However, polyspermic fertilization is still a major issue to be solved. It is well known that besides the nucleus, oocyte organelles and the cytoplasm have to undergo a final maturation process before they become fully competent for fertilization. Until now, it is still uncertain whether the zona pellucida (ZP) must also undergo a maturation process and what impact the maturation status may have on sperm recognition and monospermic fertilization. Our data show that the ZP undergoes biochemical changes in the final maturation phase of the oocyte prior to fertilization. During zona maturation, the induction of the acrosome reaction in spermatozoa bound to the zona pellucida shows a different time pattern. Additionally, it was shown by 2D gel electrophoresis that after maturation, ZPA moved 0.8 pI units and ZPB/ZPC 1.3 pI units in the direction of the anode, indicating increased acidity. These preliminary studies indicate that the maturation processes of the oocyte involves biochemical and functional alterations in the zona pellucida. In addition, the morphology of the porcine ZP was investigated before and after maturation at the GVI and metaphase II stage as well as 1h after onset of IVF. No significant consistent structural changes were seen between immature oocytes and those matured in vitro for 48 h. However, at 24 h, the zona structures were more similar to those in in vivo matured oocytes. This phenomenon needs to be elucidated. So far, the only way to avoid polyspermic penetration is to reduce the number of spermatozoa per oocyte used for IVF. The amount depends on the treatment of the sperm and has to be set for each individual boar.