Effects of clomiphene citrate on ovarian function in hypophysectomized rats

On Days 28-30 of age, hypophysectomized rats were treated with oestradiol-17 beta (0.1 mg/day) and/or clomiphene citrate (0.1 mg/day). Subsequent treatment with PMSG (10 i.u., on Day 31) and hCG (10 i.u., on Day 33) was identical for all animals. Rats were killed on Day 34. Treatment with oestradiol-17 beta alone resulted in ovulations of 45.1 +/- 5.5 oocytes/rat (mean +/- s.e.m.). There were no ovulations among animals treated with clomiphene citrate alone but treatment with oestradiol-17 beta and clomiphene citrate resulted in a significant (P less than 0.05) reduction (23.1 +/- 7.6 oocytes/rat) in ovulatory response. Similarly, ovarian weights and serum progesterone concentrations were highest in the oestradiol-17 beta-treated rats, intermediate in those given oestradiol plus clomiphene citrate and the lowest in rats receiving clomiphene citrate alone. We suggest that clomiphene citrate exerts direct ovarian antiovulatory and oestrogen-antagonist actions.     

Effects of follicle-stimulating hormone and ovarian steroids during vitro meiotic maturation on fertilization of rat oocytes

The present study examined the effects of gonadotropins and ovarian steroids during in vitro meiotic maturation of rat oocytes on their ability to undergo in vitro fertilization. Fully grown oocytes were isolated from antral follicles of immature rats and cultured as oocyte-cumulus cell complexes (OCC) under conditions in which completion of meiotic maturation occurs spontaneously. They were then exposed to spermatozoa under conditions in which oocytes matured in vivo exhibit high fertilization rates. Compared with oocytes from pregnant mare's serum gonadotropin (PMSG) or follicle-stimulating hormone (FSH)-treated rats, a simiiar proportion of the oocytes (>80%) from untreated rats underwent germinal vesicle breakdown, but such oocytes had a lower rate of fertilization (70% vs. 20%). The presence of FSH during in vitro maturation restored the fertilization rate for oocytes from untreated rats, while a cytochrome P450 inhibitor, aminoglutethimide phosphate abolished this beneficial effect of FSH. The addition of progesterone during the in vitro maturation period duplicated the beneficial effect of FSH on fertilization rate of oocytes from untreated rats; oestradiol-17β was less effective in this regard, and 5α-dihydrotestosterone was ineffective. These findings indicate that FSH and progesterone, although having no apparent effect on nuclear maturation of the oocyte, play an important role during oocyte maturation in enabling normal fertilization to occur.     

Ultrastructural features of preovulatory oocyte maturation in superovulated cattle

Cows and heifers were induced to superovulate by treatment with PMSG or FSH. The ultrastructural features of the oocytes were related to the time of the LH peak and the progesterone/oestradiol-17 beta ratios in the follicular fluid. At 0-2 h after the LH peak the perivitelline space developed; at 9-12 h there was disconnection of the junctions between cumulus cell projections and oolemma, and the concomitant breakdown of the oocyte nucleus; at approximately 15 h there were spatial rearrangements in the ooplasm of (a) mitochondrial clusters from a peripheral to an even distribution and (b) vesicles from an even distribution to a more central location; at approximately 19 h there was abstriction of the first polar body with dislocation of mitochondrial clusters and vesicles towards the site of polar body formation; at 21-22 h there was migration of cortical granules to solitary positions along the oolemma and decrease in the sizes of Golgi complexes and, on some occasions, the smooth endoplasmic reticulum. These ultrastructural changes were accompanied by an increase in progesterone/oestradiol ratios in the follicular fluids. It is concluded that preovulatory oocyte maturation in gonadotrophin-stimulated cattle comprises nuclear as well as cytoplasmic changes accompanied by steroidogenic changes in the follicle, each of which are closely related to the time of the LH peak. However, some variation existed between animals, between follicular and oocyte maturation and even within oocytes between nuclear and cytoplasmic maturation.     

Developmental capacity of mouse oocytes that undergo maturation in vitro: Effect of the hormonal state of the oocyte donor

In a previous study, it was shown that cumulus cell-enclosed germinal vesicle (GV)-stage oocytes, isolated from pregnant mares' serum gonadotropin (PMSG)-primed immature (22–24 day old) mice and that underwent spontaneous maturation in vitro, exhibited frequencies of embryonic development similar to oocytes stimulated to mature and ovulate in vivo by administration of gonadotropins [Schroeder AC, Eppig JJ, (1984) Dev Biol 102:493–497]. In the present study, the effect of the hormonal state of the oocyte donor on the capacity of in vitro matured oocytes to be fertilized and undergo pre- and post-implantation development was explored further. Oocytes were isolated at the GV-stage from the following groups of mice: 1) unprimed immature mice; 2) adult cycling mice; 3) unprimed Snell dwarf (dw) mice that have undetectable levels of growth hormone (GH), prolactin, and thyroid-stimulating hormone (TSH); and 4) primed and unprimed hypogonadal (hpg) mice that have undetectable levels of circulating gonadotropins. Oocytes maturing in vitro after isolation from normal unprimed immature or adult mice at all stages of the estrous cycle acquired full developmental capacity. GV-stage oocytes isolated from dwarf mice showed embryonic development equivalent to normal ( + /?) littermate controls. Therefore, GH, TSH, or prolactin are not required during oogencsis in vivo to promote the acquisition of competence to complete embryogenesis after maturation in vitro. Oocytes from hypogonadal mice had a much reduced capacity for preimplantation development when compared with normal littermates. Administration of PMSG to the hypogonadal mice significantly increased the developmental capacity of oocytes that underwent maturation in vitro. Gonadotropins, therefore, have a beneficial effect on the oocytc's capacity for embryonic development.     

Changes in the plasma concentrations of free and conjugated oestrogens in heifers after treatment to induce superovulation and the relationship with number of ovulations

Oestradiol-17 beta and conjugated oestrone, oestradiol-17 beta and oestradiol-17 alpha were measured in peripheral plasma of heifers treated with PMSG/PGF-2 alpha to induce superovulation. Changes in the concentrations of each hormone were synchronous, the highest level being near oestrus. For a given number of ovulations the hormone with the highest concentration was total oestradiol-17 alpha, then came total oestrone, total oestradiol-17 beta and oestradiol-17 beta. For each oestrogen, the maximum preovulatory concentration measured was significantly correlated with the number of ovulations; the regression line for total oestradiol-17 alpha was twice as steep as that for oestradiol-17 beta. It is concluded that in animals treated to induce superovulation assay of total oestradiol-17 alpha gives a better induction of the number of follicles induced to ovulate than does the more conventional assay of oestradiol-17 beta.     

Effects of passive immunization against oestradiol on mouse ovum transport

The ability of anti-oestradiol immunoglobulin to withhold oestradiol-17 beta from its target tissue was examined. The total oestradiol-17 beta binding capacity present in in-vitro incubations or injected into mice intravenously was related to the amount of [3H]oestradiol present in the media or intravenously injected into the animals respectively. When the ratio of binding capacity to [3H]oestradiol was above 74:1, [3H]oestradiol was successfully withheld from uterine tissue in vitro and in vivo. Injecting anti-oestradiol immunoglobulin into mice before administration of a tube-locking dose of oestradiol-17 beta ensured normal passage of ova through the oviduct. Daily administration of anti-oestradiol immunoglobulin to PMSG-hCG stimulated mice (starting 72 h before hCG injection) induced retention of ova for at least 2 days beyond the time when all ova had left the oviducts of control animals. The binding capacity: oestradiol-17 beta concentration ratios of sera from these animals were greater than 250:1 throughout the experimental period. Non-specific immunoglobulin had no such effects, indicating the specificity of the anti-oestradiol immunoglobulin response.     

Effects of gonadotropins on oocyte maturation and progesterone production by porcine ovarian follicles cultured in vitro

Effects of gonadotropins on the maturation of isolated oocytes and production of progesterone by porcine ovarian follicles from gonadotropin treated gilts have been studied in vitro. The addition of gonadotropins (2 I. U./ml, PMSG, HGC or 2 mg/ml FSH) to the culture medium resulted in increasing the number (84 - 90 %) of isolated oocytes which reached metaphase II. Expansion of the whole cumulus mass was observed only in media containing PMSG, whereas FSH or HCG alone did not cause these marked changes in the cumulus cells. Denudation of the eggs prior to culture gave no significant differences in the maturation rates between oocytes cultured in media with or without gonadotropins. In vitro maturation of follicle-enclosed oocytes took place only in HCG treated animals. Removing the ovary at 15 or 60 minutes after intravenous HCG administration induced oocyte maturation only in 22% and 17% respectively. A sharp increase in the number of oocytes which resume meiosis during follicle culture was observed 4 hours after HCG injection (84 %) and all of the oocytes of the gilts ovariectomized at 8 hours after HCG injection matured during the culture period. The progesterone production of isolated follicles from control gilts (only PMSG injected) increased slowly during a 96-hour culture period (from 48 to 240 ng progesterone/follicle), whereas the secretion of progesterone was drastically increased after a 15 minute interval between HCG injection and ovariectomy (from 42 to 950 ng progesterone/follicle). Follicles removed 24 hours after HCG injection showed a further increase in steroid production (2000 ng progesterone/follicle) and consistently secreted large amounts of progesterone during the culture period.     

Changes in DNA, cyclic nucleotides and steroids during induced follicular atresia in the hamster

Atresia was induced in antral follicles of hypophysectomized PMSG (30 i.u.)-treated hamsters by an i.p. injection of PMSG antiserum. Mature antral follicles were isolated and incubated for 1 h. When expressed per follicle, cAMP increased 108% above control levels at 2 h, while cGMP rose 117% at 4 h; both nucleotides then declined (12-72 h). DNA per follicle fell below control values by 24 h and reached its lowest point at 48 h. Serum concentrations of oestradiol-17 beta and testosterone were 50% lower by 1 h after injections of PMSG antiserum. Oestradiol and testosterone in serum, incubation medium and tissue dropped rapidly to reach basal values by 12 h. Serum progesterone showed a sharp increase at 4 h then dropped below control levels by 12 h and remained constant thereafter. In tissue and medium, progesterone rose to peak values at 8 h, then dropped to control levels with no significant changes for the remaining 64 h. The precipitous decreases in both oestradiol and testosterone in the serum, medium and tissue indicate inhibition of steroid synthesis at some point after progesterone formation. These results demonstrate the usefulness of this model for following atretic changes in a synchronized population of Graafian follicles.     

An attempt at intracytoplasmic sperm injection of frozen-thawed minke whale (Balaenoptera bonaerensis) oocytes.

Little is known about the characteristics of fertilisation events in minke whales. Cryopreserved minke whale oocytes and spermatozoa do not fertilise in a standard IVF. This study was conducted to investigate the pronucleus formation ability of cryopreserved minke whale oocytes and their subsequent development following intracytoplasmic sperm injection (ICSI). In experiment 1, frozen-thawed minke whale immature oocytes were cultured for in vitro maturation (IVM) in a maturation medium (TCM199) supplemented with either porcine follicle stimulating hormone (pFSH)/estradiol-17beta (E2) or pregnant mare's serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG). After 120 h of IVM, oocyte survival was examined before ICSI, and showed no significant difference in morphological normality (24-36%) between the two IVM media. Two-cell embryos (two oocytes from 21 sperm-injected oocytes) were obtained when the maturation medium was supplemented with pFSH/E2 or PMSG/hCG. In experiment 2, cryopreserved maturing oocytes were investigated for the effects of repeat-culture (2 h or 24 h) on survival before ICSI. Pronuclear formation and development were examined for the effects of sperm pretreatment with dithiothreitol (DTT) and oocyte activation with ethanol at ICSI. A frequency of 49-69% of frozen-thawed maturing oocytes was used for ICSI. Although oocyte activation did not produce a significant difference in survival, pronucleus formation and embryonic development, 2- and 4-cell cleaved oocytes were observed after injection of sperm pretreated with DTT.     

Superovulation and in vitro oocyte maturation in three species of mice (Mus musculus, Mus spretus and Mus spicilegus)

Mouse oocytes can be obtained via superovulation or using in vitro maturation although several factors, including genetic background, may affect response. Our previous studies have identified various mouse species as models to understand the role of sexual selection on the evolution of sperm traits and function. In order to do comparative studies of sperm-oocyte interaction, we sought reliable methods for oocyte superovulation and in vitro maturation in mature females of three mouse species (genus Mus). When 5IU pregnant mare's serum gonadotrophin (PMSG) and 5IU human chorionic gonadotrophin (hCG) were injected 48h apart, and oocytes collected 14h post-hCG, good responses were obtained in Mus musculus (18+/-1.3oocytes/female; mean+/-S.E.M.) and Mus spretus (12+/-0.8), but no ovulation was seen in Mus spicilegus. Changes in PMSG or hCG doses, or longer post-hCG intervals, did not improve results. Use of PMSG/luteinizing hormone (LH) resulted in good responses in M. musculus (19+/-1.2) and M. spretus (12+/-1.1) but not in M. spicilegus (5+/-0.9) with ovulation not increasing with higher LH doses. Follicular puncture 48h after PMSG followed by in vitro maturation led to a high oocyte yield in the three species (M. musculus, 23+/-0.9; M. spretus, 17+/-1.1; M. spicilegus, 10+/-0.9) with a consistently high maturation rates. In vitro fertilization of both superovulated and in vitro matured oocytes resulted in a high proportion of fertilization (range: 83-87%) in the three species. Thus, in vitro maturation led to high yields in all three species. These results will allow future studies on gamete interaction in these closely related species and the role of sexual selection in gamete compatibility.     

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.     

In vitro and in vivo maturation of oocytes from gonadotrophin-treated brushtail possums

The time course of nuclear maturation of oocytes was examined in brushtail possums, Trichosurus vulpecula. Oocytes were recovered from ovarian follicles > 2 mm in diameter after pregnant mares' serum gonadotrophin/porcine luteinizing hormone (PMSG/LH) treatment (in vivo matured) or 72 hr after PMSG treatment (in vitro matured). Oocytes recovered from small (< 2 mm) and large (> 2 mm) follicles were also assessed for their ability to mature in vitro. Staining with the DNA-specific dye Hoechst 33342 was used to assess the stage of nuclear development by fluorescence microscopy. The process of nuclear maturation progressed rapidly in vivo, as oocytes collected at 20-27 hr post-LH all had a GV, but by 28-29.5 hr post-LH approximately a third of eggs were MII. By 30-hr post-LH, more than 70% of oocytes had reached MII stage and all ovulated eggs were MII. In vitro, all oocytes were at germinal vesicle stage at the start of culture. After 24 hr of culture, 67% of oocytes had progressed to metaphase I/anaphase I of meiosis. After 36 hr, 25% of oocytes had completed maturation to metaphase II, increasing to 52% after 48 hr. Maturation of oocytes after 48 hr in culture was unaffected by the presence or absence of granulosa cells, PMSG or LH/porcine follicle stimulating hormone (FSH). More oocytes from large follicles (55%) completed maturation by 48 hr than from small follicles (15%). The potential of oocytes to mature after 48 hr in culture was dependent on the follicle harvested having reaching a critical diameter of 1.5 mm.     

In-vitro zona-lytic activity in uterine fluid from ovariectomized mice treated with oestradiol-17 beta and progesterone

Mouse embryos collected before implantation were incubated in vitro for 24 h with fluid rinsed from the uteri of ovariectomized female mice injected with progesterone, oestradiol-17 beta + progesterone, oestradiol-17 beta + progesterone, or oestradiol-17 beta alone. Although none of the zonae was completely dissolved, those incubated in fluid from animals treated with oestradiol + progesterone were subsequently more soluble in sodium thiocyanate (NaSCN) than those incubated similarly in control buffer, indicating a sublytic change during the incubation with uterine washings. Zonae incubated in fluid from animals injected with either hormone alone did not undergo such a change.     

Fertilization in vitro of rat oocytes undergoing maturation in response to a GnRH analogue

Oocytes were exposed to GnRHa to induce their maturation both in vivo, by administration of the hormone to hypophysectomized rats, and in vitro, in cultures of intact ovarian follicles. Mature oocytes obtained under both these conditions were then exposed in vitro to a sperm suspension for fertilization. Fertilization of control groups of oocytes, isolated from intact or hypophysectomized PMSG-primed hCG-induced ovulators, was 88.3 +/- 3.3% (n = 331) and 90.0 +/- 2.8% (n = 427), respectively, as compared to 82.8 +/- 3.2% (n = 413) for oocytes isolated from hypophysectomized PMSG-primed GnRHa-induced ovulators. Fertilization rate in oocytes treated by GnRHa in vitro was 78.5 +/- 3.1% (n = 247) as compared to 79.3 +/- 4.1% (n = 261) in LH-treated oocytes. These results demonstrate that fertilizability of oocytes undergoing maturation in response to GnRHa is similar to that of oocytes induced to mature by LH. No differences could be detected in the proportions of abnormal oocytes (polyspermic, fragmented and dead) and the zygotes obtained after fertilization of GnRHa- or LH-treated oocytes showed similar ability to cleave.     

Studies on oocyte maturation of the medaka, Oryzias latipes. V. On the structure of steroids that induce maturation in vitro

The response of oocytes within isolated follicles (800-950 micron in diameter) to various steroids was examined with the teleost fish, Oryzias latipes. Continuous exposure of oocytes, which were removed from ovarian investments 17 hours before predicted germinal vesicle breakdown (GVBD), to C19- or C21-steroids brought about maturation in vitro but never triggered ovulation. The steroids effective in inducing maturation have in common a C=0 (or alpha-OH) group at 3C and a beta-OH group at 17C in the C19-steroids, and a C=O (or beta-OH) group at 3C and a C=O (or alpha-OH) group at 20C in the C21-steroids, in addition to an delta4- or delta5-unsaturated for 5alpha-saturated configuration. The orientation of the hydrogen at 5C seems to be critical in determining the ability of a particular steroid to stimulate oocyte maturation. Maturation of oocytes in the ovaries of hypophysectomized females was induced by administering progesterone, but the mature oocytes did not subsequently undergo ovulation. Thus the steroid hormone is capable of inducing oocyte maturation but apparently does not participate directly in the ovulation of Oryzias latipes oocytes.     

Timing of sequential changes in cumulus cells and first polar body extrusion during in vitro maturation of buffalo oocytes

Studies were conducted to investigate the degree of the cumulus cell expansion and expulsion of the first polar body in relation to time of incubation in three different culture media during in vitro maturation of buffalo oocytes and to suggest a suitable practical method for assessment of in vitro maturation rate of buffalo oocytes. Buffalo oocytes were aspirated from ovaries collected from a local slaughterhouse. Only oocytes with more than two layers of cumulus cells and homogenous ooplasm were cultured into 50 microl droplets of three different culture systems: (1) TCM-199 + steer serum (10%): (2) TCM-199 + steer serum (10%) + PMSG (40 IU/ml); and (3) TCM-199 + steer serum (10%) + PMSG (40 IU/ml) + estradiol 17beta (1 microg/ml) in a 35 mm Petri dish. The droplets were covered with warm (39 degrees C) mineral oil and incubated in a CO2 incubator (39 degrees C, 5% CO2 in air, 90-95% relative humidity) for 16-18, 20, 22, and 24 h. The maturation rate was assessed by evaluation of degree of cumulus cells expansion and identifying first polar body extrusion into the perivitelline space under stereo zoom microscope. Matured oocytes were inseminated in vitro with 9-10 million sperm/ml of Brackett and Oliphant (BO) medium. Cleaved embryos were cultured in TCM-199 supplemented with steer serum (10%) for 8 days. Cumulus expansion and extrusion of first polar body commenced at 16 and 17 h, respectively, of buffalo oocyte culture. These events mainly exhibited during 22-24 h of culture. Oocytes with Degrees 1 and 2 cumulus cells expansion and extruded first polar body in degree 0 oocytes may be considered as matured and can be used in IVF studies.     

Suppression of spontaneous maturation of isolated cumulus-free mouse oocytes by a calmodulin antagonist

Oocytes isolated from antral follicles undergo spontaneous maturation when cultured in vitro. W7, a calmodulin antagonist, at concentration of more than 50 microM blocked the occurrence of spontaneous germinal vesicle breakdown (GVBD) in isolated cumulus-free mouse oocytes. The inhibition of maturation was observed in more than 90% of oocytes when W7 was added within 15 min after the initiation of incubation of the oocytes. The block was partially reversible. Hypoxanthine, estradiol-17 beta, testosterone and progesterone did not influence the inhibition induced with W7. The present results suggest that calmodulin is involved in the early stage of mouse oocyte maturation.     

Ovarian responses and oocyte recovery in prepubertal swamp buffalo (Bubalus bubalis) calves after FSH or PMSG treatment

Stimulation of follicular growth was examined using two different gonadotropin treatments in 10 prepubertal swamp buffalo calves (8 to 12 mo old). Each calf received an ear implant consisting of 3 mg norgestromet and 5 mg estradiol valerate during hormonal treatment. Five calves were additionally administered FSH (24 mg, im) and, 2 mo later, PMSG (3,000 IU). The remaining 5 calves were first treated with PMSG followed by FSH. Ovarian responses to treatments were examined by laparotomy, 72 h after ear implant removal, and by the number of follicles (diameter > or = 0.8 cm) and corpora hemorrhagica present. Ovaries had more significant response to FSH than PMSG treatment (13.9+/-8.6 vs 5.9+/-3.3 follicles; P<0.01). Although the recovery rate tended to be lower for FSH treated (64%) than PMSG-treated (82%) animals, more oocytes/animal were harvested in the PMSG treatment (8.3+/-5.0 vs 4.6+/-3.2, respectively). The immature oocytes (n = 38) were cultured for 24 to 25 h in maturation medium (TCM-199 NaHCO3+10% fetal calf serum [FCS] in 5%CO2 in air at 39 degrees C). Oocyte maturation was assessed after fixation and staining with aceto orcein. The in vitro maturation rate was 52.6% (20/38). This study shows the possibility of harvesting oocytes from prepubertal swamp buffalo calves and maturing the oocyte in vitro.     

In vitro maturation of oocytes from a marsupial,the tammar wallaby (Macropus eugenii)

This study examined the competence of oocytes from the tammar wallaby, Macropus eugeniio mature in vitro. Oocytes were collected from follicles >1 mm diameter 24 h after pregnant mare serum gonadotrophin (PMSG) treatment and incubated in Eagle's minimum essential medium supplemented with 10% fetal calf serum, at 35°C in 5% CO₂ in air for 24, 36, or 48 h. Oocytes were incubated either granulosa cell-intact or granulosa cell-free or in the presence of 10 IU ml^?1 PMSG or 10 μg ml^?1 porcine luteinizing hormone (LH) + 10 μg ml^?1 porcine follicle stimulating hormone (FSH). The ability of oocytes recovered from small (<1.5-mm-diameter) and large (≥1.5-mm-diameter) follicles to mature in vitro was also examined. The nuclear status of oocytes was assessed using the DNA-specific dye Hoechst 33342. Initially, all oocytes examined contained a germinal vesicle. After 24 h of culture, 60% of oocytes had progressed to metaphase I or anaphase I. After 36 h, approximately 20% of oocytes possessed metaphase II chromosomes, and 20% of oocytes were at metaphase I or anaphase I. At the completion of the 48 h culture period, 40% of oocytes had completed maturation to the metaphase II stage. In vitro oocyte maturation after 48 h was not affected by the presence of granulosa cells, PMSG, or LH and FSH. More oocytes from large follicles (55%) completed maturation by 48 h than from small follicles (20%). Approximately 50% of oocytes remained at the GV stage at all times under all conditions. Marsupial oocytes thus undergo spontaneous nuclear maturation once removed from the follicular environment, suggesting a basically similar control system to that in placental mammals. © 1993 Wiley-Liss, Inc.     

Oestrogen and progesterone concentrations in plasma and oviductal tissue of ewes exhibiting a natural or induced oestrus

Synchronisation of oestrus in Karagouniki ewes by administration of the standard dose of progesterone results in lower fertility than observed when these ewes ovulate naturally. This suggests that the optimum dose of progesterone may be breed dependent. The exogenous progesterone may perturb the concentrations of oestradiol-17beta and progesterone in blood plasma and the oviductal wall. This possibility was investigated using Karagouniki ewes allocated at random to three treatments (n=4 per treatment). Ewes were allowed to exhibit natural oestrus (N) or oestrus was synchronised by administration of 250 mg (LP) or 375 mg (HP) progesterone (subcutaneous implants) followed by PMSG at 8 mg/kg live weight i.m. 14 days later. Oestrus was observed using teaser rams. Blood samples were collected for plasma oestradiol-17beta and progesterone assay from the onset to the end of oestrus at 2 h intervals. The uterus of each ewe was recovered at the end of oestrus and samples of the oviductal wall were taken from both oviducts and prepared, separately, for progesterone and oestradiol-17beta assay. Statistical analysis was performed using univariate analysis of variance. Plasma oestradiol-17beta concentrations from the onset to the end of oestrus were highest for N ewes and lowest for HP ewes with the values for LP ewes occupying an intermediate position. The differences were significant (P<0.05) between HP and the other two treatments from 4 to 12 h after the onset of oestrus and then between all treatments until the end of oestrus. Plasma progesterone levels were similar and fairly constant from the onset to the end of oestrus for N and LP. The plasma progesterone levels for HP were significantly (P<0.05) higher than for the other two treatments throughout oestrus. In oviductal wall samples, the oestradiol-17beta concentration was significantly (P<0.05) higher for N ewes than for synchronised ewes and the levels were similar for LP and HP ewes. The concentration of oestradiol-17beta differed (P<0.05) between right and left oviducts for N ewes but not for ewes of either of the synchronised oestrus treatments. Progesterone concentrations in oviductal wall samples were highest (P<0.05) for HP ewes and the values for N and LP ewes were similar. The concentration of progesterone did not differ between right and left oviductal wall samples within treatments. It was concluded that the higher dose of exogenous progesterone perturbed the levels of oestradiol-17beta and progesterone in blood plasma and the oviductal wall, and this could explain the lower levels of fertility (relative to naturally occurring oestrus) observed when this protocol is used for Karagouniki ewes in practice.