Annexin V labelling and terminal transferase-mediated DNA end labelling (TUNEL) assay in human arrested embryos

Confocal laser scanning microscopy was used to observe human arrested and fragmented preimplantation embryos obtained by in-vitro fertilization. Observation of the cellular actin cortex and chromatin showed a high frequency of embryos with blastomeres exhibiting two or more nuclei, while others had nuclei displaying chromatin condensation and fragmentation patterns. Many of the abnormal chromatin images could be due to the process of programmed cell death (apoptosis). The possible link between abnormalities of the blastomeres and apoptosis was investigated using two detection methods for cells undergoing apoptosis. Detection of phosphatidylserine exposure was performed using annexin V; the chromosomal breakdown preceding the nuclear collapse of apoptotic nuclei was tested using the terminal transferase-mediated DNA end labelling (TUNEL) assay. Annexin V staining was observed in all arrested and/or fragmented human embryos, but not in cryopreserved embryos which continued to develop normally after thawing. The TUNEL assay was positive in 30% (15/50) of arrested embryos, all of which had cytoplasmic fragments. In contrast, embryos showing regular size blastomeres without fragments were TUNEL negative.     

Timing of in vivo maturation of equine preovulatory oocytes and competence for in vitro maturation of immature oocytes collected simultaneously

The objects of this study were to monitor the development of the cumulus complex and nuclear maturation in oocytes recovered from preovulatory follicles following treatment to induce ovulation and to investigate the in vitro maturation competence of oocytes recovered from smaller nonpreovulatory follicles of varying size. All follicles > or =5 mm in pony mares were individually punctured at 0, 6, 12, 24 and 35 h after an injection of LH to induce ovulation. The recovery rates of oocytes were 64% from 55 preovulatory follicles, 22% from 32 subordinate follicles and 52% from 227 small follicles. Cumulus expansion of the preovulatory oocytes occurred at 12 h post LH treatment while the metaphase I and II components of nuclear maturation were not completed until 24 and 35 h post LH respectively. For nonpreovulatory follicles, the frequency of atresia and oocyte competence for in vitro nuclear maturation both increased with increasing follicular size.     

Significance of diet therapy in pediatrics

Changes in the ovaries of chicken embryos (17th day of incubation) and in 2-day-old chickens at different terms (2, 24, 72 h) were studied by light and electron microscopic methods after x-ray irradiation in the doses of 606 and 1200 rad. In oocytes of 17 embryos x-ray irradiation acts as an additional factor, enhancing and increasing the degree of chromatin condensation and producing earlier appearance of pachynema stage. In the ovarian oocytes, at diplonema stage, in 2-day-old chickens after irradiation, condensation of chromosomal substance is observed; in this case, irradiation produces a reverse action to that of normal despiralization. Ultrastructural changes in cellular organelles, in nuclear membrane, in particular, depend on the degree of oocyte differentiation and the irradiation dose applied. Within the terms of investigation, x-ray irradiation does not produce any considerable ultrastructural changes in the oocytes, but its disturbes the contacts between them and follicular cells, which is supposed to be the cause of oocyte destruction.     

Influence of light intensity on methanotrophic bacterial activity in Petit Saut Reservoir, French Guiana

We have determined that the tolerance of in vitro matured/in vitro fertilized (IVM/IVF) bovine embryos to cryopreservation at the pre-morula stage can be improved by removal of cytoplasmic lipid droplets by centrifugation. Nucleus transfer was also performed using cryopreserved, delipated (lipid droplets removed) 8- to 16-cell-stage blastomeres of IVM/IVF embryos as donor nuclei. In vitro developmental ability of the delipated embryos to the blastocyst stage (20 of 126) was found to be equal to that of undelipated embryos (35 of 176); and of 53 delipated embryos cryopreserved at the 8- to 16-cell stage, 12 developed into blastocysts in vitro after thawing. On the other hand, only 2 of 43 undelipated embryos and 5 of 59 sham-operated embryos survived (p < 0.05). When blastomeres isolated from cryopreserved, delipated 8- to 16-cell-stage embryos were used for nucleus transfer, 57 of 80 successfully fused with enucleated oocytes, which was significantly lower than the fusion rate obtained with blastomeres of unfrozen, undelipated embryos (93 of 104, p < 0.01). However, the developmental rate to the blastocyst stage for nucleus transfer embryos reconstituted with frozen, delipated blastomeres (9 of 57) was not different from that of the nucleus transfer embryos with unfrozen, undelipated embryos (23 of 93). These results confirm that removal of cytoplasmic lipid droplets from bovine IVM/IVF zygotes allows for successful cryopreservation at the 8- to 16-cell stage and that blastomeres from these embryos can be used as donors of karyoplasts for nucleus transfer.     

Human menopausal gonadotropin during in vitro maturation of human oocytes retrieved from small follicles enhances in vitro fertilization and cleavage rates

OBJECTIVES: To compare the IVF rates of oocytes retrieved from small follicles (< 2 mL in volume) with those of oocytes retrieved from large follicles and to test the effect of adding gonadotropins to the IVF medium on the fertilization rates of oocytes from small follicles. DESIGN: Oocytes were retrieved with endovaginal ultrasound (US) guidance from patients undergoing infertility treatment in our IVF program. Oocytes were grouped according to the volume of the originating follicle and subjected to our routine procedure for IVF. HMG was added to the IVF medium for some of the oocytes from small follicles. SETTING: Toronto Fertility and Sterility Institute is affiliated with the University of Western Ontario and University of Toronto and is equipped for RIA, endovaginal US monitoring and oocyte retrieval, and for processing and culturing gametes and embryos. PATIENTS: Infertile patients admitted to our IVF program. INTERVENTIONS: Patients underwent ovarian stimulation with hMG before oocyte retrieval. No other interventions were introduced to the processing and culturing the gametes and embryos except the addition of hMG to the medium of some of the small follicle-originated oocytes with the informed consent from the patients. MAIN OUTCOME MEASURES: Rates of fertilization, cleavage of the fertilized embryos before replacement, and meiotic status of some of the oocytes from small follicles. RESULTS: Most of the oocytes from small follicles did not complete the first meiotic division; they had low rates of fertilization and cleavage compared with oocytes from large follicles, and these rates were improved by the addition of hMG to the IVF medium. CONCLUSIONS: Oocytes from small follicles are probably less mature and require a more physiological environment to achieve normal rates of fertilization and cleavage.     

The relationship between follicular fluid aspirate volume and oocyte maturity in in-vitro fertilization cycles

As a consequence of multiple follicular growth during ovarian stimulation for in-vitro fertilization (IVF), follicles of varying sizes often yield oocytes that vary in maturity and morphology of the oocyte-cumulus-corona complex. The objective of this prospective study was to explore the relationship between follicular fluid aspirate volume and the oocyte's developmental potential in an IVF treatment cycle. In total 9933 follicles were studied from 400 patients who underwent 535 consecutive IVF treatment cycles at St James's University Hospital, Leeds, UK, between February 1995 and February 1996. The volume of each individual follicle aspirated was recorded and related to the probability of obtaining an oocyte, its fertilizing capacity, the cleavage rate and the quality of embryos derived. We found no statistically significant difference in oocyte recovery rates between follicles with an aspirate volume < or = 1 ml and follicles with a volume > 1 ml. Although oocytes obtained from follicles with an aspirate volume > or = 1 ml showed a significantly lower fertilization rate, they went on to cleave at the same rate as oocytes obtained from larger follicles and resulted in embryos of comparable quality. Furthermore, there was no statistically significant difference in the implantation, clinical pregnancy or live birth rates per cycle between embryos derived from follicles with an aspirate volume < or = 1 ml and those derived from follicles with an aspirate volume > 1 ml. We conclude that follicular size and the oocyte's developmental potential in the stimulated ovary are not closely related and can be independent. This is in contrast to the Graafian follicle and the pre-ovulatory oocyte in the natural cycle.     

Structural and endocrine aspects of equine oocyte maturation in vivo

The objectives were to describe the ultrastructure of equine oocytes aspirated from small and preovulatory follicles, and to relate the ultrastructural features to follicle size and follicular fluid steroid concentrations. Mares were examined every second day by transrectal ultrasonography, and follicles measuring > 30 mm were aspirated (in vivo) using a 20-cm-long 12-gauge needle through the flank. Following slaughter, both large and small follicles were aspirated (in vitro) from six mares. The oocytes were isolated under a stereomicroscope and processed for transmission electron microscopy, and the follicular fluid was assayed for progesterone (P4) amd estradiol-17 beta (E2). A total of 29 oocytes (32% recovery rate) were aspirated in vivo, and 15 oocytes were recovered in vitro. According to the stage of nuclear maturation, the oocytes could be divided into the following six categories: 1) the central oocyte nucleus (CON) stage, 2) the peripheral spherical oocyte nucleus (PON-I) stage, 3) the peripheral flattened oocyte nucleus (PON-II) stage, 4) the oocyte nucleus breakdown (ONBD) stage, 5) the metaphase I (M-I) stage, and 6) the metaphase II (M-II) stage. The maturation of the preovulatory follicle was reflected by alterations in the follicular fluid concentrations of steroid hormones. E2 was high in all preovulatory follicles, whereas P4 concentration exhibited a 10-fold increase during follicle maturation, particularly associated with the progression from M-I- to M-II-stage oocytes. The nuclear oocyte maturation included flattening of the spherical oocyte nucleus, followed by increasing undulation of the nuclear envelope, formation of the metaphase plate of the first meiotic division, and, finally, the extrusion of the first polar body and the subsequent formation of the metaphase plate of the second meiotic division. The cytoplasmic oocyte maturation changes comprised breakdown of the intermediate junctions between the cumulus cell projections and the oolemma, enlargement of the perivitelline space, the formation and arrangement of a large number of cortical granules immediately beneath the oolemma, the rearrangement of mitochondria from a predominantly peripheral distribution to a more central or semilunar domain, and the rearrangement of membrane-bound vesicles and lipid droplets from an even distribution to an often semilunar domain, giving the ooplasm a polarized appearance. It is concluded that the final equine oocyte maturation includes a series of well-defined nuclear and cytoplasmic changes that are paralleled by an increase in P4 concentration in the follicular fluid, whereas E2 concentration remains constantly high.     

Dynamic nuclear polarization at very low magnetic fields

Non-mosaic Klinefelter patients are generally azoospermic due to primary testicular failure. Nevertheless, in some cases, testicular spermatozoa may be recovered and utilized to fertilize oocytes via intracytoplasmic sperm injection (ICSI). As the risk for an increased number of gonosomes in these spermatozoa is unclear, preimplantation genetic diagnosis (PGD) may be attempted in the resulting embryos. In the present study, we report our experience with the combined approach of sperm retrieval by testicular fine needle aspiration (FNA), ICSI and PGD in seven consecutive non-mosaic Klinefelter individuals. In four patients, between one and five spermatozoa were retrieved in five out of nine consecutive attempts. In a fifth patient, only 10 round spermatids could be isolated. Mature spermatozoa were injected into a total of 16 metaphase-II oocytes, of which 11 (69%) remained intact. Two distinct pronuclei (2PN) were observed in four oocytes (36%) while a single pronucleus (1PN) was documented in two oocytes. Five cleavage stage embryos developed from the oocytes of two couples. Upon the request of one couple, their three embryos (two derived from 1PN oocytes) were transferred without PGD but pregnancy was not achieved. PGD by fluorescence in-situ hybridization (FISH) was performed in the two embryos of the other couple which were derived from normal fertilization. PGD results of one embryo were 18,18,X,X,Y, the embryo was not transferred and FISH analysis of the remaining blastomeres identified variable chromosome numbers in the nuclei. The second embryo was diagnosed as normal and was transferred, resulting in a successful pregnancy and birth. In conclusion, the results of this report indicate that a pregnancy and birth may be attained in azoospermic non-mosaic Klinefelter individuals by testicular FNA combined with ICSI. Due to the unknown risk of gonosomes aneuploidy in embryos from Klinefelter patients, PGD or prenatal diagnosis should be recommended.     

Therapeutic drug monitoring: antiarrhythmic drugs

Two experiments were carried out to test the hypothesis that follicles recovered from Meishan animals may provide a more favourable environment for oocyte maturation in vitro than follicles recovered from Large-White hybrid animals. In Experiment 1, all follicles > or = 4 mm were recovered from six Meishan and seven Large-White hybrid gilts in the late follicular phase and healthy oocyte cumulus complexes recovered. Cumulus oocyte complexes were randomly divided into two groups, and each group cultured for 27 or 34 h (62 and 64; 56 and 56 for Meishan and Large-White hybrid, respectively) in defined medium in the presence of either of the two largest follicle shells per animal. Subsequent examination of oocyte nuclear maturation showed that although maturation did not differ significantly between the breeds after 27 h, more (P < 0.01) Meishan oocytes co-cultured with Meishan follicles developed to metaphase II stage than Large-White hybrid oocytes co-cultured with Large-White hybrid follicles after 34 h. The next eight largest follicles per animal were cultured for 34 h to produce conditioned media. In Experiment 2, oocytes recovered from the slaughterhouse were matured for 46 h in the presence of conditioned media from Meishan (612 oocytes) or Large-White hybrid (731 oocytes) follicles, or in fresh medium in the presence of a follicle shell from slaughterhouse ovaries. Oocytes were then inseminated and 12 h later examined for penetration and male pronuclear formation. A higher (P < 0.05) percentage of oocytes cultured in Meishan follicle conditioned medium underwent sperm penetration and male pronuclear formation than oocytes cultured in conditioned media from Large-White hybrid animals. Concentration of oestradiol and progesterone in the conditioned media did not differ between the breeds (P > 0.1). In conclusion, these results suggest that (1) Meishan oocytes have advanced maturational capacity when cultured with Meishan preovulatory follicle shells and (2) differences in follicle maturation in the Meishan compared to the Large-White hybrid pig may result in an improved ability of the follicles, via conditioned media, to support oocyte maturation and fertilization in vitro.     

Embryo cloning in cattle: the use of in vitro matured oocytes

In vitro matured (IVM) bovine oocytes were examined to determine their potential viability in embryo cloning. Activation competence, as monitored by pronuclear formation, increased with oocyte age. Oocytes readily formed a pronucleus when challenged with an electrical pulse 30 h after the onset of maturation. Developmental competence of IVM oocytes tended to increase with oocyte age (P = 0.079). Selection of IVM oocytes on the basis of the presence of a polar body 24 h after the onset of maturation and the size of the follicle from which the oocyte was derived improved development of nuclear transfer embryos (polar body positive 25% versus polar body negative 10%, P < 0.05; large follicle oocytes 31% versus small follicle oocytes 14%, P < 0.05). When selected, IVM oocytes were compared with in vivo matured oocytes recovered from superovulated cows and heifers; no difference was detected for the frequency of embryos produced, pregnancies confirmed between days 50 and 60 of gestation, or the number of calves born. We conclude that selected IVM oocytes are equivalent to in vivo matured oocytes when used for bovine embryo cloning.     

Effect of follicular cells, IGF-I and tyrosine kinase blockers on oocyte maturation

The aim of this study was to test the following hypotheses: (i) that oocyte maturation is controlled by surrounding follicular cells; (ii) that a meiosis-regulating factor of follicular origin is not species-specific; (iii) that one of the follicular regulators of oocyte maturation is IGF-I; and, (iv) that Cumulus oophorus and tyrosine kinase-dependent intracellular mechanisms do not mediate IGF-I action on oocytes. It was found that co-culture of cumulus-enclosed bovine oocytes with isolated bovine ovarian follicles or with isolated porcine ovarian follicles significantly increased the proportion of matured oocytes (at metaphase II of meiosis) after culture. Porcine oocytes without cumulus investments had lower maturation rates than cumulus-enclosed oocytes. Co-culture with isolated porcine ovarian follicles resulted in stimulation of maturation of both cumulus-free and cumulus-enclosed porcine oocytes. These observations suggest that follicular cells (whole follicles or Cumulus oophorus) support bovine and porcine oocyte maturation, and that follicular maturation-promoting factor is not species-specific. The release of significant amounts of IGF-I by cultured bovine and porcine isolated follicles and granulosa cells was demonstrated. Addition of IGF-I to culture medium at 10 or 100 (but not 1000) ng/ml stimulated meiotic maturation of both cumulus-enclosed and cumulus-free porcine oocytes. Neither of the tyrosine kinase blockers, genistein or lavendustin (100 ng/ml medium), changed the stimulating effect of IGF-I on porcine oocytes. The present data suggest that at least one of the follicular stimulators of oocyte nuclear maturation is IGF-I, and that its effect is probably not mediated by cumulus investment or by tyrosine kinase-dependent intracellular mechanisms.     

Development of parthenogenetic and cloned ovine embryos: effect of activation protocols

Preliminary experiments carried out on ovine oocytes were designed to establish correlations between activation protocols and subsequent rates of embryonic development. The best activation protocols were thereafter used in studies on ovine parthenogenesis and cloning. The first study established that chemical activators induce pronuclear development at a slightly higher rate than physical activation (ionomycin, 96%; ethanol, 95%; electro activation, 80%). Inhibition of second polar body extrusion and one single pronucleus were observed in the majority of the oocytes (approximately 90%) treated for 3 h with 6-dimethylaminopurine (6-DMAP) following either ionomycin or ethanol activation. While over 80% of these oocytes cleaved after transfer to the oviducts of recipients, progression to the blastocyst stage was higher after ionomycin as compared with ethanol activation (58% vs. 19%). The ionomycin plus 6-DMAP activation protocol was used to produce parthenogenetic blastocysts whose subsequent development was monitored both by ultrasonography and by direct fetal examination. Over 70% of parthenogenotes were viable on Day 21 of pregnancy but dead by Day 25. The effects of 6-DMAP on nuclear remodeling and fetal development of cloned embryos was then investigated. Control cloned embryos underwent nuclear envelope breakdown (NEBD), premature chromatin condensation (PCC), and inhibition of DNA synthesis. By contrast, reconstructed embryos treated with 6-DMAP exhibited intact nuclear membranes, interphase chromatin, and no interference on DNA synthesis. Moreover, cloned embryos developed to blastocyst stage in higher percentage after 6-DMAP treatment (83% vs. 25%). We conclude that ionomycin followed by 6-DMAP incubation yields high percentages of diploid parthenogenetic embryos that develop to Day 25 before dying. Cloned embryos activated by the ionomycin-6-DMAP protocol develop readily to term.     

Equine oocyte-cumulus morphology as affected by follicular size

From the ovaries of 256 slaughtered mares a total of 1713 follicles were isolated from which 1641 (95.8%) oocytes were recovered (6.4/mare). A total of 564 follicles and oocytes were evaluated for the degree of vascularisation of the follicle wall, the appearance of the follicular fluid and the location and morphology of the cumulus-oocyte-complex. Follicles with a diameter of >10 mm displayed more numerous, well branched and more pronounced blood vessels than the smaller ones (4-10 mm diameter) and most of them contained clear, yellowish fluid with few granulosa cells. The percentage of oocytes with compact cumuli increased significantly with an increasing diameter of the follicle, being 233%, 43.9%, 55.6% and 64.2% (P<0.01) for the follicles with diameters of 4-10, 11-15, 16-20 and 21-35 mm, respectively. The percentage of oocytes attached to the follicle wall also increased with increasing follicle size, being 48.0%, 59.6%, 81.5% and 90.1% (P<0.01), respectively. On the contrary, the percentage of oocytes floating in the follicular fluid decreased with increasing follicle diameter, from 52.0% in the smallest follicles to 9.9% in the biggest ones. A significantly greater percentage of oocytes found on the follicular wall than in the follicular fluid had a compact cumulus (56.6 versus 21.3%; P<0.01). For in vitro culture were accepted 30.4%, 54.3%, 60.7% and 77.8% (P<0.01) of oocytes from the follicles with diameters of 4-10, 11-15, 16-20 and 21-35 mm, respectively. After culture for 28-40 h in TCM 199 medium, 90 of a total of 165 (54.5%) oocytes reached the metaphase II stage of maturation.     

Detection of apoptosis in human and rat ovarian follicles

OBJECTIVE: The purpose of this study was to determine whether cells acquired from individual human preovulatory follicles undergo apoptosis (physiologic cell death) and, if so, to correlate the degree of apoptosis with characteristics of the follicles or the oocytes derived from the follicles. METHODS: We devised a sensitive nonradioactive method for detecting apoptotic DNA fragmentation in small numbers of cells derived from rat atretic follicles and follicular aspirates of patients undergoing assisted reproductive technologies. RESULTS: Using this method, apoptotic DNA was detected in rat atretic follicles, with optimal detection at 10-100 ng. Furthermore, apoptotic DNA was detected in some, but not all individual human follicular aspirates from several patients, and was found in follicles that produced oocytes that fertilized and developed into embryos. CONCLUSION: Apoptosis occurs in cells from human ovarian preovulatory follicles and may be a normal physiologic process of the follicle during luteinization.     

Isolation and characterization of maturation inhibiting compound in bovine follicular fluid

The protein fraction which is responsible for the inhibition of maturation of bovine oocytes in vitro was isolated from cow follicular fluid by means of column chromatography on a Sephadex G-200 and a Sepharose 4B, both in 0.1 M ammonium acetate, pH 6.7. The molecular weight of the maturation inhibiting protein fraction is approximately 60 kDa. At a concentration of 2.0 mg/mL in cultivation medium, 100% of the oocytes were arrested at the germinal vesicle stage. At a concentration of 0.25 mg/mL, the protein fraction still had some meiosis inhibiting effects, but 56% of the oocytes were capable of maturing to the metaphase of the second meiosis (MII). Without compact cumulus the inhibiting fraction had no meiosis retarding effect on the oocytes. Cow follicular fluid also exhibited this inhibitory effect on oocyte maturation in vitro. However, the follicular fluid from follicles of 2.5-5.0 mm diameter showed higher meiosis inhibiting effects than the follicular fluid from follicles of 5-10 mm diameter.     

Impact of patient consciousness on the intensity of the do-not-resuscitate therapeutic plan

The pluri- or totipotency of gonial cells, isolated from rabbit fetuses at 18-20 days of pregnancy, has been investigated by transferring their nuclei into enucleated oocytes and following the development of the resulting reconstituted embryos both in vitro (in a total of 726 embryos) and in vivo (in 135 embryos). The gonial cells exhibited pseudopodial activity like that of primordial germ cells and ultrastructural studies confirmed that neither male nor female cells had entered meiosis. When the gonial cells were used immediately after isolation, about 37% of the reconstituted embryos of both sexes cleaved, with no significant difference according to sex. However, after a further 4-day culture of the cleaved embryos, the blastocyst formation rate was four times higher in those made with male (16%) than with female (4%) gonial cells. No implantation sites were detected following transfer of reconstituted embryos into recipient females. These results show that the nuclei of male and female rabbit diploid germ cells differ in their capability to be "reprogrammed" and bring about development to the blastocyst stage following nuclear transfer. The origin of this difference, which is evidenced long before the onset of meiosis is discussed.     

Effect of naftidrofuryl (LS129) on axonal growth

Wistar rats, eight days old, were subjected to permanent bilateral forebrain ischemia, followed by hypoxia for 15 minutes. A cerebral infarct, mainly involving the cerebral neocortex, hippocampus, amygdala, striatum and subcortical white matter was produced. Neurons and glia showing punctate chromatin condensation and karyorrhectic cells were observed 12 hours after hypoxia-ischemia. Their number increased during the first two days and recruitment of cells with degenerating nuclei occurred until day five. In situ labeling of nuclear DNA fragmentation stained many normal-appearing nuclei, as well as punctate chromatin condensations and nuclear fragments in karyorrhectic cells. Delayed neuronal death in the CA1 area of the hippocampus was observed after 20 minutes of transient forebrain ischemia in the adult gerbil. In situ labeling of nuclear DNA fragmentation demonstrated stained punctate chromatin condensation in a few degenerating cells at 48 hours post-ischemia. Substantial labeling of CA1 neurons occurred in the fourth day. Agarose gel electrophoresis of extracted brain DNA from ischemic infant rats and adult gerbils showed a ladder-type pattern which is typical of nuclear DNA fragmentation into oligonucleosomal fragments (internucleosomal cleavage). These findings suggest that endonuclease(s) activation may play a role in cell death induced by different forms of hypoxia-ischemia.     

Expression of a soluble form of LFA-1 and demonstration of its binding activity with ICAM-1

The mouse homologues of the breast cancer susceptibility genes, Brca1 and Brca2, are expressed in a cell cycle-dependent fashion in vitro and appear to be regulated by similar or overlapping pathways. Therefore, we compared the non isotopic in situ hybridization expression patterns of Brca1 and Brca2 mRNA in vivo in mitotic and meiotic cells during mouse embryogenesis, mammary gland development, and in adult tissues including testes, ovaries, and hormonally altered ovaries. Brca1 and Brca2 are expressed concordantly in proliferating cells of embryos, and the mammary gland undergoing morphogenesis and in most adult tissues. The expression pattern of Brca1 and Brca2 correlates with the localization of proliferating cell nuclear antigen, an indicator of proliferative activity. In the ovary, Brca1 and Brca2 exhibited a comparable hormone-independent pattern of expression in oocytes, granulosa cells and thecal cells of developing follicles. In the testes, Brca1 and Brca2 were expressed in mitotic spermatogonia and early meiotic prophase spermatocytes. Northern analyses of prepubertal mouse testes revealed that the time course of Brca2 expression was delayed in spermatogonia relative to Brca1. Thus, while Brca1 and Brca2 share concordant cell-specific patterns of expression in most proliferating tissues, these observations suggest that they may have distinct roles during meiosis.     

Relationships among oocyte-cumulus morphology, follicular atresia, initial chromatin configuration, and oocyte meiotic competence in the horse

Horse oocytes with expanded (EX) cumuli appear to have greater meiotic competence than do horse oocytes with compact (CP) cumuli but are thought to come from atretic follicles. We evaluated the relationships among cumulus expansion, follicle viability, initial chromatin configuration, and meiotic competence of horse oocytes. Follicle walls were sectioned for histological examination, and the follicles were scraped to obtain the oocytes. Half of the oocytes were evaluated immediately and half were matured for 24 h in vitro. Cumulus expansion was significantly associated with follicle atresia. Initially, significantly more EX than CP oocytes had chromatin condensed into one mass within the germinal vesicle (CC configuration; 61% vs. 32%). After culture, significantly more EX than CP oocytes had matured (74% vs. 30%). The proportion of oocytes with the CC configuration was lowest in viable follicles and increased in follicles with slight to moderate atresia. The maturation rate of oocytes from viable follicles was significantly lower than for oocytes from follicles with slight or moderate atresia. The CC chromatin configuration appears to be associated with meiotic competence in horse oocytes. The association of follicle atresia with increased meiotic competence suggests that acquisition of meiotic competence is related to a loss of suppressive activity by the degenerating follicle.