Cardiological effects of catecholamine-secreting tumours

Ovaries (N = 250) from slaughtered buffaloes were collected to study follicular population and compare methods of oocyte retrieval. The number and size of surface follicles were recorded and grouped into different categories. Different sized follicles in relation to oocyte diameter were studied histologically. Yield of oocytes per ovary were less (P < 0.05) from ovaries bearing a corpus luteum (CL). Techniques used for oocyte recovery included slicing, follicle puncture and aspiration. The oocyte recovery rate was greatest (P < 0.05) using slicing. The average number of visible surface follicles was 5.20 +/- 0.97 with mean numbers of 2.5, 1.2, 0.82 and 0.62 per ovary for follicles sized 4, 8, 12 and 12mm respectively. Histological studies revealed large numbers of primordial follicles in prepubertal and atretic follicles in senile buffaloes. They also established a biphasic relationship of growth between oocyte diameter and follicular size.     

Empty follicle syndrome as cause of unsuccessful in vitro fertilization

The authors report a case, where ultrasound guided transvaginal follicle aspiration was carried out for oocyte retrieval during the source of in vitro fertilization. Despite the favourable situation described by ultrasound and prognosed by serum hormone levels aspiration of oocytes proved unsuccessful even after lavage of follicules. The authors give an overview of the literature of "empty" follicle syndrome, its possible aetiology and methods of treatment.     

Transfer of immature oocytes to a preovulatory follicle: an alternative to in vitro maturation in the mare?

In the mare, success rates for the in vitro maturation of oocytes are low. Accordingly, we attempted to determine if immature oocytes could be matured in vivo by injecting them into a preovulatory follicle. Groups of 3-9 oocytes collected from donor mares were transferred under ultrasound control into the preovulatory follicle of a recipient mare that was treated with crude equine pituitary gonadotrophin (CEG) to induce ovulation. Just before ovulation (34 h post treatment) the preovulatory follicle of the recipient mare was punctured to collect both the transferred and the indigenous oocytes to analyse the stages of nuclear maturation. The transfer technique did not impair significantly the final maturation of the recipient preovulatory follicle. The indigenous oocytes within the recipient follicles were recognisable by their larger expanded cumulus of yellow colouration due to high hyaluronic acid content; 7/12 of these oocytes were mature (metaphase II). Around half (42/86; 49%) of the oocytes transferred to preovulatory follicles were recovered subsequently. Most of them showed cumulus expansion (41/42, 6 of which were rich in hyaluronic acid), 13 (32%) were mature, 15 (36%) were immature and 13 (32%) were degenerate. When the indigenous oocyte of the recipient mare was mature, 38% of the transferred oocytes were mature, this rate being no different from the in vitro maturation rate of 46%. This study showed that in vivo maturation of immature oocytes by transfer into a preovulatory follicle in a recipient mare is possible. The maturation rate is not different from the in vitro maturation rate. The technique allows the generation of mature oocytes that have an expanded cumulus rich in hyaluronic acid, similar to the situation in preovulatory oocytes. This result has not been obtained in vitro previously.     

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.     

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.     

The correlation between follicular measurements, oocyte morphology, and fertilization rates in an in vitro fertilization program

OBJECTIVE: To explore the relationship between follicle size and the morphology of the oocyte-cumulus-corona complex with fertilization rates in stimulated cycles of IVF. DESIGN: Retrospective comparison of measurements and observations of 2,429 oocytes from 215 patients undergoing 324 stimulated IVF cycles. SETTING: A large hospital-based IVF program. MAIN OUTCOME MEASURES: Individual follicles were measured by ultrasound before transvaginal aspiration and the size was recorded. The oocyte-cumulus-corona complex from each follicle was examined and classified. The oocytes were checked for evidence of fertilization 17 to 22 hours after insemination. RESULTS: The fertilization rate of all oocytes regardless of morphological type revealed a positive linear correlation with increasing follicle diameter. The fertilization rates of type I oocytes was marginally higher than type II oocytes, controlling for follicle diameter; however, this difference did not achieve statistical significance. Oocytes from follicles with a mean diameter > or = 16 mm had significantly higher fertilization rates than did oocytes from follicles with a mean diameter < or = 14 mm. CONCLUSIONS: Follicle size is a better predictor of fertilization than is morphological characterization of the oocyte-cumulus-corona complex in IVF.     

Prospective randomized trial of the effect of two flushing media on oocyte collection and fertilization rates after in vitro fertilization

OBJECTIVE: To assess the value of heparinized saline as a flushing medium for oocyte recovery. DESIGN: Prospective randomized study. SETTING: Academic tertiary referral center for fertility treatment. PATIENT(S): Thirty-five patients, with both ovaries intact having IVF-ET. INTERVENTION(S): Patients were randomized either to have the follicles of the left or right ovary flushed with heparinized normal saline at the time of oocyte recovery for IVF-ET. The contralateral ovary was flushed with heparinized culture medium. Oocytes obtained from each side were cultured separately and assessed for fertilization 18-21 hours after insemination. MAIN OUTCOME MEASURE(S): Collection and fertilization rates. RESULT(S): A total of 481 follicles were aspirated yielding 366 oocytes. Of these, 240 fertilized. From the side flushed with saline 185 oocytes were collected from 237 follicles, which was not significantly different from 181 oocytes collected from 244 follicles on the side flushed with culture medium (odds ratio = 1.23; 95% confidence interval = 0.79-1.92). Similarly, there was no significant difference observed in fertilization rates between oocytes obtained after saline (median 71.4%) and culture medium flush (median 75.0%) (odds ratio = 1.08; 95% confidence interval = 0.68-1.72). CONCLUSION(S): Heparinized normal saline is an equally good but cheaper and more convenient medium than standard heparinized culture medium and could replace it for flushing follicles during oocyte recovery for IVF-ET procedures.     

Oocyte maturation and intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI), treatment of severe male infertility allows an accurate evaluation of oocyte maturity at recovery after corona-cell removal. In cycles comprising a GnRH analog desensitization and a stimulation by hMG or FSH, 12% of oocytes aspirated from follicles (> 14 mm), 34 hours post-hCG are still immature, in prophase or metaphase 1. They are able to achieve meiosis in vitro in 66% of the cases and will be fertilized (2 PN) by ICSI in 51% of the cases as the in vivo mature oocytes of the same cohort. Nevertheless, the quality of cytoplasmic maturation and consequently of embryonic viability remains to be assessed as there still are few pregnancies arising from in vitro matured oocytes. ICSI also represents the only way to obtain normal fertilization in some exceptional but observed anomalies of oocyte maturation, particularly when there is a lack of zona reaction leading to repetitive polyspermy in conventional IVF.     

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.     

Effects of follicular aspiration and flushing, and the genotype of the fetus on circulating progesterone levels during pregnancy in the mare

When aspirating ovarian follicles in pregnant mares to obtain oocytes for in vitro fertilisation (IVF), the effect of the manipulation on circulating concentrations of progesterone may be an important consideration in terms of the maintenance of pregnancy. The object of this study was to compare the effects of 3 different forms of transvaginal ultrasound-guided follicle aspiration (Treatment 1, no aspiration, n = 4; Treatment 2, aspirate only follicles > or =20 mm in diameter, n = 7; Treatment 3, aspirate all visible follicles, n = 7) on peripheral plasma progesterone concentrations between Days 21 and 150 of gestation in 9 mares carrying intraspecies horse and 9 mares carrying interspecies mule conceptuses. The 3 follicle aspiration treatments were applied at the peak of each follicular wave as determined by follicular mapping by means of transrectal ultrasonography on alternate days. The plasma progesterone profile in mares undergoing Treatment 1 was in close agreement with those reported previously in pregnant mares. A decline in plasma progesterone levels occurred after Day 53 of gestation in Treatments 2 and 3 mares, indicating that the follicular aspiration procedures did interfere with the formation of secondary corpora lutea. However, the levels in individual mares never dropped low enough to endanger the pregnancy. Mares carrying mule pregnancies exhibited higher mean plasma progesterone concentrations between Days 39 and 45 of gestation than mares carrying horse pregnancies, equivalent levels between Days 46 and 66 despite the lower circulating concentrations of chorionic gonadotrophin (mule CG) in their blood during this period and lower progesterone levels between Days 67 and 150 of gestation. The results indicate that the primary corpus luteum in the pregnant mare may be more sensitive to mule CG than horse CG. Furthermore, the earlier disappearance of CG from the circulation in mares carrying mule fetuses is reflected by an earlier decline in plasma progesterone concentrations in this type of equine pregnancy.     

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.     

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.     

The chicken homologue of zona pellucida protein-3 is synthesized by granulosa cells

Oocyte development within avian ovarian follicles is an intricate process involving yolk deposition and the formation of extraoocytic matrices. Of these, the perivitelline membrane (pvm) not only plays a role in sperm binding but also provides mechanical support for the large oocyte's journey through the oviduct after ovulation. To date we have focused on the mechanisms for uptake of yolk precursors into oocytes of the chicken; now we extend our studies to a detailed analysis of the pvm. In the course of characterization of its major components, we obtained partial protein sequences; comparison with the GenBank database revealed that one of the pvm proteins is the homologue of mammalian zona pellucida glycoprotein 3 (ZP3), a key component in sperm binding. Following a nomenclature based on gene structure, the protein is referred to as chicken ZPC (chZPC). The chicken protein (444 residues) and murine ZP3 (424 residues) are highly conserved, with 41% of the amino acids identical. As shown by Northern blot analysis, the avian ZPC gene is expressed exclusively in the granulosa cells surrounding the oocyte, in contrast to murine ZP3, which is synthesized by the oocyte. Upon reaching a size larger than 1.5 mm in diameter, follicles accumulate chZPC in highly polarized fashion, i.e., in the space intercalated between the oocyte and the granulosa cells, as revealed by immunohistochemistry of follicle sections. ChZPC synthesis and secretion by granulosa cells was demonstrated directly by metabolic labeling and immunoprecipitation from the culture medium of granulosa cell sheets isolated ex vivo from follicles. Immunoblot analysis and glycosidase treatment of chZPC from preovulatory and freshly ovulated oocytes, as well as laid eggs, revealed that the primary product undergoes a two-step decrease in size from follicle to laid egg that is unlikely to be due to modification of the carbohydrate moiety.     

Effect of luteinizing hormone on the pattern of steroid production by preovulatory follicles of pregnant mare's serum gonadotropin-injected immature rats

Preovulatory follicles were explanted on the day before ovulation from immature rats given a single injection of Pregnant Mare's Serum gonadotropin (PMS) 2 days earlier. The follicles were incubated for 4 h in modified Krebs bicarbonate buffer containing glucose and albumin in absence or presence of ovine luteinizing hormone (NIH-LH-S18; 0.1-10 mug/ml). The accumulation of progresterone, androstenedione and 17beta-estradiol in the medium was determined by radioimmunoassay. As in indicator of LH exposure the meiotic stage of the follicle-enclosed oocyte was determined at recovery by interference contrast microscopy. The first group of follicles were explanted in the morning, before the endogenous gonadotrophin surge. In hormone-free medium the oocytes remained in the dictyate stage, whereas addition of LH induced oocyte maturation. These follicles, when incubated in hormone-free medium, secreted predominantly androstenedione and estradiol and only low amounts of progesterone. In the presence of LH the secretion of all steroids was enhanced. The second group of follicles were explanted in the evening, 2-4 h after the endogenous gonadotrophin surge. After incubation in hormone-free medium the follicle-enclosed oocytes had matured. The steroid secretion by the follicles was different from that of the first group. In hormone-free medium they secreted predominantly progesterone and low amounts of androstenedione and estradiol. Addition of LH to the medium caused further enhancement of progesterone secretion, but had no effect on androstenedione and estradiol secretion. The third group of follicles were explanted in the evening from rats in which the preovulatory gonadotrophin surge had been prevented by Nembutal treatment. Oocyte maturation and steroid secretion did not differ from that found for the first group of follicles explanted in the morning. The results are compatible with the hypothesis that LH, after a transitory stimulation, inhibits androgen and estrogen secretion and stimulates progesterone secretion by the preovulatory ovarian follicle.     

Drosophila oocyte localization is mediated by differential cadherin-based adhesion

In a Drosophila follicle the oocyte always occupies a posterior position among a group of sixteen germline cells. Although the importance of this cell arrangement for the subsequent formation of the anterior-posterior axis of the embryo is well documented, the molecular mechanism responsible for the posterior localization of the oocyte was unknown. Here we show that the homophilic adhesion molecule DE-cadherin mediates oocyte positioning. During follicle biogenesis, DE-cadherin is expressed in germline (including oocyte) and surrounding follicle cells, with the highest concentration of DE-cadherin being found at the interface between oocyte and posterior follicle cells. Mosaic analysis shows that DE-cadherin is required in both germline and follicle cells for correct oocyte localization, indicating that germline-soma interactions may be involved in this process. By analysing the behaviour of the oocyte in follicles with a chimaeric follicular epithelium, we find that the position of the oocyte is determined by the position of DE-cadherin-expressing follicle cells, to which the oocyte attaches itself selectively. Among the DE-cadherin positive follicle cells, the oocyte preferentially contacts those cells that express higher levels of DE-cadherin. On the basis of these data, we propose that in wild-type follicles the oocyte competes successfully with its sister germline cells for contact to the posterior follicle cells, a sorting process driven by different concentrations of DE-cadherin. This is, to our knowledge, the first in vivo example of a cell-sorting process that depends on differential adhesion mediated by a cadherin.     

Leucine transport in Xenopus laevis oocytes: functional and morphological analysis of different defolliculation procedures

L-leucine uptake in stage V Xenopus laevis oocytes was affected by the specific methods used to remove the follicle cells. In the presence of 100 mM NaCl, L-leucine uptake was reduced by 67.5% +/- 5.7 when defolliculation was performed enzymatically by collagenase treatment, whereas the reduction was 30.5% +/- 6.4 after mechanical defolliculation. The Na(+)-dependent uptake of 0.1 mM L-leucine was 18.6 +/- 4.6 pmol oocyte-1 40 min-1 in folliculated oocytes and 5.6 +/- 1.9 in collagenase defolliculated oocytes (means +/- SE). L-leucine uptake was not affected by the removal of the follicular layer if defolliculation occurred after the transport period; radiolabeled L-leucine is therefore not taken up into a compartment that is removed by the defolliculation process. The different L-leucine uptake rates observed in folliculated and defolliculated oocytes were not due to non-specific L-leucine binding to membranes. L-leucine kinetics showed that the L-leucine Vmax and Km values were lower in oocytes deprived of the follicular layer than in control oocytes enveloped in intact follicular layers. The Vmax and Km values of Na(+)-dependent L-leucine transport, calculated from data obtained the day after defolliculation by collagenase treatment, were: 16 +/- 1.5 pmol oocyte-1 40 min-1 and 57 +/- 21 mumol (mean +/- SD). The Na(+)-activation curve of 0.1 mM L-leucine was hyperbolic in folliculated oocytes and sigmoidal in defolliculated oocytes. The morphological analysis performed in parallel with the transport experiments showed that after defolliculation, the fibers forming the vitelline membrane tended to be arranged in a more regular orthogonal array, and the number of oocyte microvilli was reduced after collagenase treatment. Mechanical defolliculation did not appreciably affect the oocyte microvilli, however this procedure did not completely remove all follicle cells. The damage to collagenase treated oocytes was reversible, and the functional and structural features of most oocytes improved upon subsequent in vitro incubation. The recovery process seemed to involve protein synthesis in view of the increased value of L-leucine Vmax, and microscopic observation showing recovery of the microvillar apparatus.     

Folliculogenesis in the prenatal period of human development

Quantitative analysis of the follicular formation has been performed in ovaries of 97 human embryos and fetuses (6--40-week-old). The first oocytes at the stage of diplotene appear in 11.5--12-week-old fetuses. Primordial follicles are formed around the oocyte as early as the stage of diplotene. Follicles are intensively formed after the 14th--15th week. Single primordial follicles begin to transform into the growing primary ones in 17-week-old fetuses and the process becomes active after the 19th--20th week of the development. As a rule, the oocyte reaches the stage of dictyotene only after the follicle has transformed into the primary one. By the time of birth most of oocytes are enclosed by the primary, and in less number--by the primordial follicles. The number of secondary and tertiary cavitary follicles is small. The formation rate of follicles with binuclear oocytes and that of polyoocytic follicles is stated. The data on quantitative analysis of oocyte degeneration at the stages of diplotene and dictyotene are presented.     

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.     

A new source of human oocytes: preliminary report on the identification and maturation of human preantral follicles from follicular aspirates

This study aimed to investigate the development of human preantral follicles and oocyte maturation in vitro. Preantral follicles were obtained from follicular aspirates during egg retrieval carried out during an in-vitro fertilization (IVF) programme. They were first incubated in Ham's F10 medium with 15% fetal cord serum (FCS). After 28 days, the medium was supplemented with different doses of human menopausal gonadotrophin (HMG), human follicular fluid (hFF) and epidermal growth factor (EGF) by orthogonal design. Promotion of final maturation was completed in the presence of HMG and hFF. Development from preantral to antral follicles was found within 6-12 days of culture. With time, the proportion of follicles with diameters of >300 microm increased at 21-28 days of culture (P < 0.005). The maximum number of oocytes extruded, and first polar body formation, occurred in the presence of 0.15 IU/ml HMG 40% (v/v) hFF and 6 ng/ml EGF. We conclude that follicular aspirates obtained during egg retrieval in an IVF programme contain many preantral follicles which could develop into antral follicles with extrusion of oocytes in culture, and that the oocytes can mature in vitro. Hence, a new source of human oocytes is available.