Ultrasound-guided follicle aspiration: the collection of bovine cumulus-oocyte complexes from ovaries of slaughtered or live cows

To increase the collection efficiency of bovine cumulus-oocyte-complexes (COCs) by transvaginal aspiration, the effects of aspiration pressure and needle diameter on bovine follicular oocyte collection were assessed. Oocytes were aspirated from ovaries of slaughtered cows using 2 different diameter needles (18- or 21-gauge) with 4 different aspiration pressures (40, 80, 120 or 160 mmHg) and of live cows using 18-gauge needles with 40 or 80 mmHg, or using 21-gauge needles with 80 or 120 mmHg. The recovered oocytes were divided into 4 categories according to the surrounding cumulus cells and quality of oocytes: 1) 4 or more layers, 2) between 1 and 3 layers, 3) completely or partially denuded and 4) all others, including expanded cumulus cells and degenerated oocytes. The highest oocyte recovery rates from Categories 1 and 2 were obtained using 18-gauge needles with 40 mmHg pressure and 21-gauge needles with 120 mmHg pressure, respectively, from the ovaries of slaughtered cows. When oocytes were collected from live cows, the highest recovery rates for Categories 1 and 2 were obtained using an 18-gauge needle and 40 mmHg pressure, and 21-gauge needle and 80 mmHg, respectively. In addition, the proportion of oocytes in each category were compared between ovaries from slaughtered and live cows. The proportion of Category 1 oocytes collected from live cows was lower than from slaughtered cows when 18-gauge needles at 80 mmHg (P<0.05). The results show that the combination of aspiration pressure and needle diameter is crucial for COC collection, and they suggest that optimal aspiration conditions for ovaries of slaughtered cows are not necessarily applicable to live cows.     

Studies on the morphology and histology of the ovary of red palm weevil female irradiated with gamma rays

The present study involves red palm weevil adults Rhynchophorus ferrugineus (Oliver) (Coleoptera: Curculionidae) irradiated with 5, 10 or 15 Gy of gamma radiation. The biological effects of gamma irradiation on the F₁ adult females, descendant of irradiated parental male pupae, were studied. The percentage egg hatch decreased significantly, as the dose increased, compared with the untreated control.The effect of gamma irradiation on the morphology of the ovaries showed a remarkable effect on size, shape and measurement of the paired ovaries.Additionally, histological studies showed some damages by irradiation of the oocytes maturation, which increased with increasing dose. These symptoms were elongation of the terminal filament, rupture, separation, or shrinkage of external sheath and follicular epithelium, degenerated or absent of nurse cells, and ruptured oocytes at 15 Gy.Vacuolation appeared in different degrees inside the oocytes and the nurse cells were absent in some areas. The damage in the oocytes was more severe as the dose was increased. The follicular epithelium was thin, oocytes clumped together throughout the ovariole causing some oocytes become abnormal or rectangular in shape.     

The histological and histochemical studies on the ovary in relation to vitellogenesis in the dragonfly, Orthetrum chrysis Selys (Libellulidae: Odonata).

The ovaries consist of large number of panoistic ovarioles in the last instar nymph and the adult dragonfly Orthetrum chrysis (Selys). In the nymph the vitellaria are compactly filled with the primary oocytes and the vitellogenesis takes place only in the adult stage. During vitellogenesis oocytes change widely in their shape, size and cytological organisation and their developmental stages can be divided into pre-vitellogenic, early-vitellogenic, vitellogenic, late-vitellogenic and maturation age. PAS-positive material appears first around the germinal vesicle in the early-vitellogenic stage and lateron it migrates towards the periphery. Glycogen appears in the late-vitellogenic stage. DNA is abundantly present in the nuclei of the oocytes during the pre-vitellogenic and completely absent in early-vitellogenic, vitellogenic, late-vitellogenic and maturation stages. It is observed in the nuclei of follicular epithelial cells of all the stages. RNA is abundantly present in cytoplasm of the pre-vitellogenic oocytes but lateron is gradually decreases. During the early-vitellogenic and vitellogenic stages high concentration of RNA in the follicular epithelial cells has been observed. The protein bodies appear first in the interfollicular spaces and towards the periphery of the oocytes just near the enveloping follicular epithelial cells, during the early-vitellogenic stage suggesting the formation of yolk proteins from the haemolymph. In Orthetrum chrysis the sudanophilic bodies appear first in the follicular cells and then lie in the peripheral region of the oocytes suggesting the incorporation of yolk lipid either from the follicular epithelium or from the haemolymph through the follicular epithelium. The phospholipids are synthesised in pre-vitellogenic to the late-vitellogenic stages. In the late-vitellogenic stages the phospholipid granules are present abundantly in the follicular epithelium while in the maturation stage they disappear suggesting their utilisation in the formation of membranes like vitelline and chorion. The neutral fats are present in the form of large number of droplets in the oocytes during the maturation stage.     

Induction of meiotic maturation of follicle-enclosed oocytes of rabbits by a transient increase followed by an abrupt decrease in cyclic AMP concentration.

The involvement of cyclic adenosine monophosphate (cAMP) in mammalian oocyte maturation was assessed using cultures of rabbit cumulus-oocyte complexes and perfused rabbit ovaries. Rabbit cumulus-oocyte complexes were cultured in Brackett's medium with or without forskolin at 10(-4), 10(-5) or 10(-6) mol l-1 for 3-6 h. At 3 or 4 h spontaneous meiotic maturation was significantly (P < 0.05) inhibited by forskolin at 10(-4) mol l-1. With prolonged incubation, spontaneous maturation progressed despite exposure to forskolin. In the second experiment ovaries were perfused for 12 h with forskolin (10(-4), 10(-5) or 10(-6) mol l-1) or medium alone. Neither ovulation nor degeneration of follicular oocytes occurred in any perfused ovary. The percentage of follicular oocytes achieving germinal vesicle breakdown was significantly (P < 0.001) increased in response to forskolin in a dose-related manner. In an additional experiment, ovaries were perfused with forskolin at 10(-4) mol l-1. A significant increase in the cAMP content in the follicle was observed within 30 min, but the ability to produce cAMP in response to forskolin decreased as the duration of perfusion was increased. Intraoocyte cAMP increased significantly within 30 min and reached its maximum 2 h after exposure to forskolin. Thereafter, cAMP levels in the oocytes decreased abruptly. This drop in intraoocyte cAMP concentration was followed by the resumption of meiosis. The alterations of intraoocyte cAMP contents following exposure to hCG in vivo paralleled those observed in the ovaries perfused with forskolin. These data suggest that a transient, but not continuous, increase in cAMP concentration after the gonadotrophin surge may be required to initiate oocyte maturation.     

Localization of an activin/activin receptor system in the porcine ovary.

The aim of this study was to locate a possible activin/activin receptor system within porcine ovaries containing functional corpora lutea. In situ hybridization was used to assess the gene expression of beta(A)- and beta(B)-activin subunits, and immunohistochemical studies were done to detect activin-A protein and activin receptor type II. mRNA expression of the beta(A)- and beta(B)-activin subunits was found in the granulosa from the unilaminar follicle stage onward, in the developing thecal layer of multilaminar and small antral follicles, in the theca interna of mid-sized antral follicles, in corpora lutea, and in the ovarian surface epithelium. Immunoreactive activin A protein could be detected at the same ovarian sites, but in thecal tissue of small antral follicles only. This protein was also demonstrated at the peripheral zone of oocytes from multilaminar and antral follicles. A positive immunoreaction for activin receptor was found in granulosa cells from multilaminar and older follicles and in oocytes from the earliest stages of follicular development onward. In late multilaminar follicles and in antral follicles, the oolemma was stained. Except for small antral follicles, a positive activin receptor immunoreaction was absent in the follicular theca. Activin receptor immunoreaction was furthermore present in corpora lutea and in the ovarian surface epithelium. It is concluded that, within porcine ovaries containing functional corpora lutea, an activin/activin receptor system is present in all intact follicles, the corpora lutea and the surface epithelium. Within follicles, granulosa and theca cells are the main sites of activin synthesis, while oocytes and granulosa cells are the main activin binding sites.     

Selection of follicles, preculture oocyte evaluation, and duration of culture for in vitro maturation of equine oocytes

Equine oocytes (n = 537) were collected from slaughterhouse ovaries (n = 118 mares) by scraping the internal follicular wall. Preculture record was made of the appearance of oocyte investments (no cumulus, corona radiata only, compact cumulus, expanded cumulus), appearance of cytoplasm (homogeneous, condensed heterogeneous/fragmented), and nuclear maturation stages (germinal vesicle, germinal-vesicle breakdown, metaphase I, metaphase II, degenerated). There was no difference between follicles > 30 mm and follicles < or = 30 mm in the preculture frequency distribution among the 5 nuclear stages; 96% were at either the germinal vesicle or germinal-vesicle breakdown stages. Oocytes from follicles 5 to 30 mm were cultured in modified TCM-199 for 18, 24, 36 and 48 h. Postculture nuclear maturation classifications were immature (germinal vesicle, germinal-vesicle breakdown, and metaphase I), mature (metaphase II or secondary oocyte), and degenerated. The frequency distribution of oocytes among the 3 postculture maturation classifications changed (P < 0.05) at 18 h (15% mature oocytes), changed (P < 0.05) further at 24 h (55% mature oocytes), with no additional change for 36 or 48 h. The only preculture cytoplasm group that affected the postculture results was the heterogeneous/fragmentation group which had a high proportion of postculture degenerated oocytes (67%); however, only 4% of oocytes were in this group. Luteal status of the mare had an effect (P < 0.05) on the frequencies of the maturation classifications, but not enough to be useful in selecting oocytes. Consistency of the follicle and the type of oocyte investment did not alter significantly the maturation frequencies. The frequency of degenerated oocytes after culture was high under the following conditions: 1) diameter of the follicle from which the oocyte was selected was 5 to 10 mm (44% degenerated oocytes), 2) the largest follicle per pair of ovaries was < or = 10 mm (63%), and 3) the mare was pregnant (66%). These results were probably related to the reported high frequency of atretic follicles in the 5- to 10-mm population. In summary, oocytes from individual follicles < or = 10 mm or from follicles in which the largest follicle per mare was < or = 10 mm were the poorest candidates for in vitro maturation.     

In vitro maturation of follicular oocytes of the Giant Panda (Ailuropoda melanoleuca): a case report

The Giant Panda is an endangered species that would benefit from biotechnological assistance in reproduction. However, because there are only a few of these animals left in the world, scientists hesitate to use them for research procedures. We were fortunate to obtain ovaries from a Giant Panda that died of hepatic cirrhosis during the nonbreeding season. Oocytes were harvested within 4 h of death by dissecting the ovarian cortex in physiological saline and collecting the cumulus-oocyte complexes from the fluid, and then were classified into large (> 125 microns) and small (100 to 124 microns) follicular oocytes and placed in TCM199 supplemented with FSH (10 micrograms/mL) and LH (20 micrograms/mL). After culture for 22 h at 37 degrees C in air with 5% CO2, response was evaluated by growth of oocytes and presence of the first polar body. Of the 26 large follicular oocytes that were harvested, 12 were considered suitable for IVM, and 14 were degenerated, had a broken zona pellucida or had lost some cytoplasm. Of the 12 cultured oocytes, all grew to a mean diameter of 141.1(SD = +/- 6.7, n = 12), and 4 released the first polar body. None of the small follicular oocytes showed growth or other signs of maturation. We conclude from our preliminary results that it is possible to obtain functional Giant Panda oocytes from ovaries obtained post mortem during the nonbreeding season.     

Comparative studies of ovarian histopathology and the mating behaviour of Periplaneta americana, Linn. (Orthoptera) treated with sublethal concentrations of BHC and DDT.

Cockroaches are familiar pests of grain stores. They are prolific breeders and can also transmit various diseases. Adult females were therefore treated with sublethal BHC and DDT concentrations to control their fecundity and viability. Ovulation was arrested as a result of damage to and resorption of the terminal oocytes. Histopathological changes were clearly expressed in the follicular epithelium of pathological oocytes and also in immature oocytes. Vitellogenesis was arrested and protein yolk precursors were broken down and destroyed by the pycnotic follicular epithelium. Fibrosis and thickening of the tunica propria gradually diminished. Prolonged treatment resulted in atrophy of the ovaries, together with a large number of immature and pathological oocytes. Since the effect of DDT is cumulative, the histopathological changes it caused were severer than those induced by BHC. Treated adults kept in pairs showed prolonged duration of mating, followed by a 2-3 days' delay in ootheca formation; no first instar nymphs emerged from these oothecae, however, owing to the failure of fertilized eggs to develop any further in the oothecal lumen.     

Penetration by bull spermatozoa into the zona pellucida of dead bovine oocytes recovered from frozen-thawed ovaries

The present study was carried out to determine if the zona pellucida of dead bovine oocytes obtained from ovaries stored at -196 degrees C could be used to assess penetrability of capacitated bull spermatozoa. Follicular oocytes were recovered from bovine ovaries which were frozen slowly in a box containing dry ice, plunged into liquid nitrogen, and thawed at 37 degrees C. The dead oocytes were inseminated with various concentrations of spermatozoa preincubated for 0 to 4 h. Sperm penetration rates of the dead oocytes were significantly altered by sperm concentration and preincubation time. Dead and living oocytes matured in vitro (control) gave similar patterns of penetrability based on sperm preincubation time. When sperm concentration was increased, the rate of multiple sperm penetration into the dead oocytes also increased significantly, but the rate of penetration into living oocytes did not alter significantly. All dead oocytes from ovaries stored at -196 degrees C for 1 d to 3 mo were penetrated at similar rates by spermatozoa preincubated for 1-h. Thus, we conclude that dead follicular oocytes recovered from frozen ovaries are useful for the assessment of sperm capacitation and/or the acrosome reaction in cattle.     

Relationship between growth hormone concentrations in bovine oocytes and follicular fluid and oocyte developmental competence

In the last few years, several works suggest that Growth Hormone (GH) is involved in follicular development and oocyte maturation. These actions may reflect endocrine roles of pituitary GH and also account for local autocrine or paracrine activities of GH produced in reproductive tissue. This study was aimed to verify whether the developmental competence of bovine female gametes might be related to ovarian GH. We evaluated the localisation and distribution of GH in the cumulus oocytes complexes (COCs) and the concentration of GH in the oocytes and in the follicular fluids (FF) from ovaries classified on the basis of the follicles number. Oocytes retrieved from ovaries with more than 10 follicles of 2 to 5 mm in diameter (High ovaries, Hi) show higher rate of maturation and blastocyst formation than those retrieved from ovaries with less than 10 follicles (Low ovaries, Lo). At the same time we measured Estrogen (E2) and Progesterone (P4) concentrations in FF, to relate oocytes quality, GH concentration and follicle health. GH localization in COCs and oocytes was performed by indirect immunofluorescence and its concentration within the ooplasm was evaluated by microspectrophotometer analysis. GH, E2 and P4 concentrations in FF were measured by an Enzyme Linked ImmunoSorbent assay (ELISA). We observed a positive, diffuse signal at cytoplasmic level in most of the cumulus cells, with no differences between COCs collected from Hi and Lo ovaries. On the contrary, GH level was significantly higher in the oocytes collected from Lo ovaries than in those recovered from Hi ovaries. Finally we found that also GH level in the FF was inversely related to the oocytes developmental capability. We suggest that the increase of GH in the oocytes and in the FF derived from Lo ovaries might be interpreted as attempt of the follicular environment to improve ovarian activity and in turn oocytes developmental competence in a autocrine-paracrine manner. Moreover, E2, and P4 levels in FF suggest that, in our model, atresia processes are also involved in oocyte developmental capability and that the highest level of GH may represent a local reaction to these phenomena.     

Morphology of the ovaries of Sphaerodema (Diplonychus) rusticum (Heteroptera,Belostomatidae)

The female reproductive system of Sphaerodema rusticum consists of a pair of ovaries, two lateral oviducts, a median common oviduct, and a median spermatheca. Accessory glands are absent. Each ovary has five free ovarioles branching from the oviduct. Each ovariole consists of a terminal filament, germarium, vitellarium, brown mass, and an exceptionally long pedicel. The terminal filament consists of a central core, interstitial cells, and an outer sheath. In the germarium, which consists of trophic and prefollicular regions, the trophic region or nurse cell chamber is divided into four histologically differentiated zones, distinguished as zones I–IV. Nutritive cords, originating from the posterior end of the trophic core in zone IV extend centrally and join the developing oocytes in the prefollicular chamber and the vitellarium. The compact prefollicular tissue at the base of the trophic core gives rise to prefollicular cells which, after encircling the young oocytes, become modified into follicular epithelial cells, the interfollicular plug, and epithelial plug. The young oocytes descend into the vitellarium and gradually develop into mature oocytes. A compound corpus luteum is observed simultaneously in all the ovarioles of both ovaries after ovulation. Below the epithelial plug there is an accumulation of material, the “brown mass,” which develops cyclically in correlation with the ovulation cycle. Each pedicel stores five mature chorionated eggs ready for oviposition. The epithelium of the anterior region of the pedicel secretes a PAS-positive material. General morphology and histology of the subdivisions of the ovarioles are described.     

Yolk sphere formation is initiated in oocytes before development of patency in follicles of the moth,Plodia interpunctella

We describe a provitellogenic stage, a previously unrecognized stage of follicle development in moths, and show that oocytes begin yolk sphere formation prior to the development of patency by the follicular epithelium. The vitellogenic activities of follicles from pharate adult femalePlodia interpunctella (Hübner) were determined by visualizing the subunits of vitellin (YP1 and YP3) and the follicular epithelium yolk protein (YP2 and YP4) using monospecific antisera to each subunit to immunolabel whole-mounted ovaries or ultrathin sections. At 92 h after pupation, yolk spheres that contained only YP2 began to proliferate in the oocytes. The inter-follicular epithelial cell spaces were closed at 92 h making vitellogenin inaccessible to the oocyte, and consequently, the vitellin subunits were not observed in the yolk spheres. YP2 uptake most likely occurred across the brush border from the follicular epithelial cells to the oocyte at this time. At 105 h, the inter-follicular epithelial cell spaces appeared closed yet trace amounts of labeling for vitellin were observed in the spaces and also in the yolk spheres along with YP2. Equivalent labeling for all four YPs in yolk spheres was finally observed at 112 h after pupation when the follicular epithelium had become patent. These data indicate that the provitellogenic stage is an extended transition period between the previtellogenic and vitellogenic stages that lasts for approximately 13 h, and it is marked at the beginning by YP2 yolk sphere formation in the oocyte and at the end by patency in the follicular epithelium.     

The co-culture of cumulus-enclosed bovine oocytes and hemi-sections of follicles: Effects on meiotic resumption

To prolong the culture of oocytes, it is essential to know how the follicle maintains meiotic arrest. This study was undertaken to evaluate the short-term effects (24 h) of the co-culture of follicular hemi-sections, including theca and granulosa cells, with cumulus-enclosed primary oocytes on meiotic resumption. Bovine oocytes were collected from 1 to 5-mm follicles from ovaries kept at 35 degrees C. Follicular hemi-sections were prepared by careful dissection of another group of follicles of the same size but from ovaries transported on ice. Following 24 h of co-culture, the oocytes were either fixed for determination of nuclear maturation or matured for an additional 24 h to evaluate reversibility of inhibition. The inhibitory action of the hemi-sections on meiotic resumption of oocytes was directly related to the amount of tissue and did not require direct physical contact between the cumulus and the follicular wall. The inhibition was reversible after 24 h of co-culture. Therefore, follicular tissue can be used to maintain meiotic arrest for at least 24 h, thus allowing for the study of changes in developmental competence during late folliculogenesis.     

成体雌性小熊猫卵巢组织学观察

2000 年至2009 年,12 只固定于10% 福尔马林中非生殖系统疾病死亡的小熊猫卵巢组织,按常规组织学技术制作组织切片,HE 染色,光学显微镜观察。结果:(1)不同发情时期卵巢均有原始卵泡、初级卵泡和次级卵泡分布。发情期的卵巢未观察到典型的成熟卵泡和卵母细胞; (2)原始卵泡数量较少,初级卵泡数量较多,多数初级卵泡和大多数的次级卵泡都处在闭锁状态;(3)卵泡腔出现之前,卵母细胞的直径和卵泡直径同时增长;卵泡腔出现之后,卵母细胞直径增长较慢,卵泡直径增长较快; (4)不同发情时期的小熊猫卵巢均存在大量的间质腺细胞;(5)妊娠小熊猫和发情间期无妊娠小熊猫的卵巢均有发育正常的黄体;(6)卵泡细胞发育呈低柱状至柱状时出现透明带。结论:(1) 卵泡闭锁主要发生在初级卵泡阶段,仅少数卵泡能发育至次级卵泡;(2)卵母细胞和卵泡生长呈双相生长的趋势; (3) 不同发情时期的小熊猫卵巢间质腺都发达; (4)发情排卵后,非妊娠黄体与妊娠黄体维持的时间相似,证实了小熊猫存在假孕现象。     

Developmental regulation of baboon fetal ovarian maturation by estrogen

Ovarian function in adult human and nonhuman primates is dependent on events that take place during fetal development, including the envelopment of oocytes by granulosa (i.e., folliculogenesis). However, our understanding of fetal ovarian folliculogenesis is incomplete. During baboon pregnancy, placental production and secretion of estradiol into the fetus increases with advancing gestation, and the fetal ovary expresses estrogen receptors alpha and beta in mesenchymal-epithelial cells (i.e., pregranulosa) as early as midgestation. Therefore, the current study determined whether estrogen regulates fetal ovarian follicular development. Pregnant baboons were untreated or treated with the aromatase inhibitor CGS 20267, or with CGS 20267 plus estradiol benzoate administered s.c. to the mother on Days 100-164 (term = Day 184). On Day 165, baboon fetuses were delivered by cesarean section and the number of total follicles and interfollicular nests consisting of oocytes and mesenchymal-epithelial cells in areas (0.33 mm(2)) of the outer and inner cortices of each fetal ovary were quantified using image analysis. Maternal and umbilical serum estradiol levels were decreased by >95% with CGS 20267. Treatment with CGS 20267 and estrogen restored maternal estradiol to normal and fetal estradiol to 30% of normal. Although fetal ovarian weight was unaltered, the mean number of follicles +/- SEM/0.33 mm(2) in the inner (59.0 +/- 1.7) and outer (95.3 +/- 2.4) cortical regions of fetal ovaries in untreated animals was 35%-50% lower (P < 0.01) in estrogen-depleted baboons (25.9 +/- 1.4, inner cortex; 62.5 +/- 2.7, outer cortex) and was restored to normal by treatment with CGS 20267 and estrogen. In contrast, the number of interfollicular nests was 2-fold greater (P < 0.01) in fetal ovaries of estrogen-suppressed animals, a change that was prevented by treatment with estrogen. In summary, fetal ovarian follicular development was significantly altered in baboons in which estrogen was depleted during the second half of gestation and restored to normal by estradiol. We propose that estrogen plays an integral role in regulating, and perhaps programming, primate fetal ovarian development.     

Developmental potential of mouse follicular epithelial cells and cumulus cells after nuclear transfer.

The developmental potential after nuclear transfer of mouse follicular epithelial cells cultured in vitro was examined. Follicular epithelial cells surrounding growing oocytes (type 5, diameter of oocytes, 62.6 +/- 5.9 microm; n = 14) were obtained from ovaries of adult mice. Before nuclear transfer, cells were cultured for several passages and subjected to serum starvation for several days. When the nuclear-transfer oocytes were at the 2-cell stage, serial nuclear transfer was performed. Additionally, cumulus cells surrounding ovulated oocytes were used as nuclear donors, with or without thermal stimulation (from -25 degrees C to 60 degrees C for 10 min) before nuclear transfer. Nuclear-transfer oocytes with follicular epithelial cells developed into blastocysts (34%) after serial nuclear transfer, and 4 living fetuses on Day 10.5 (25%, 16 transferred) and 1 dead fetus on Day 19.5 of pregnancy (3%, 30 transferred) were obtained after transfer to recipients. Although blastocysts (20%) were obtained after serial nuclear transfer of cumulus cells, only one implantation site without a fetus was observed on Day 10.5 of pregnancy. Thermal stimulation of cumulus cells before nuclear transfer did not enhance the ability to develop into fetuses or blastocysts.     

Effects of estradiol-17beta and progesterone supplementation on in vitro nuclear maturation of canine oocytes

Unlike in other domestic animals, in vitro maturation (IVM) of canine oocytes has had limited success. The present study investigated the effect of the estrous cycle and estradiol-17beta (E2) or progesterone (P4) supplementation on in vitro nuclear maturation of canine oocytes recovered from domestic dog ovaries in various reproductive states (follicular, luteal or anestrous stages). Oocytes were cultured in serum-free tissue culture medium (TCM)-199 supplemented with various concentrations of E2 (Exp. 1: 0, 0.1, 1.0 or 2.0 microg/ml) or P4 (Exp. 2; 0, 0.5, 1.0 or 2.0 microg/ml) for 72 h to determine the effective concentration of hormones. In Exp. 3, in order to investigate the synergistic effect of E2 and P4 supplementation, three groups of oocytes were cultured with 2 microg/ml E2 plus various concentrations of P4 (0, 0.5, 1.0 or 2.0 microg/ml). As results, the rate of maturation to metaphase II (MII) stage was significantly higher (P < 0.05) in oocytes from the follicular stage supplemented with 2 microg/ml E2 (14.7%) compared to the other groups (1.5-8.2%). Significantly higher (P < 0.05) maturation rate to MII stage was observed in oocytes from the follicular stage supplemented with 1.0 (10.0%) or 2.0 microg/ml (10.8%) P4 compared to the other groups (0-4.8%). Furthermore, more (P < 0.05) oocytes from the follicular stage supplemented with 2.0 microg/ml of E2 and P4 (16.6%) were matured to MII stage compared to oocytes from the follicular stage supplemented with 2.0 microg/ml E2 alone (10.4%) or the other groups of oocytes (0-7.8%). Interestingly, compared to 2.0 microg/ml E2 alone (10.4%), supplementation of 2 microg/ml E2 + 0.5 microg/ml P4 (3.4%) decreased the maturation of oocytes from the follicular stage to MII stage. In conclusion, the present study demonstrated that supplementation of the culture medium with E2 or P4 alone significantly increased maturation of canine oocyte to MII and that P4 supplementation with E2 further promote or decrease oocyte maturation compared to E2 alone depending on P4 concentration.     

Effects of dibutyryl cyclic AMP on oocyte maturation and ovulation in the perfused rabbit ovary

The involvement of cyclic AMP (cAMP) in mammalian oocyte maturation was assessed using cultures of rabbit cumulus-oocyte complexes and in-vitro perfused rabbit ovaries. Rabbit cumulus-oocyte complexes were cultured in Brackett's medium with or without dibutyryl cyclic AMP [Bu)2cAMP) at 10(-3), 10(-4) or 10(-5) M for 4-12 h. At 4 h spontaneous meiotic maturation was significantly inhibited by (Bu)2cAMP (P less than 0.025). With prolonged incubation, spontaneous maturation progressed despite exposure to (Bu)2cAMP. When ovaries were continuously perfused in vitro for 12 h with (Bu)2cAMP (10(-3) M) or medium alone, (Bu)2cAMP stimulated ovarian progesterone production, but did not affect ovulation or maturation of follicular oocytes. When ovaries were perfused in vitro with or without (Bu)2cAMP (10(-3), 10(-4) or 10(-5) M) for the first 2 h and then transferred to medium without (Bu)2cAMP for an additional 10 h, ovulation did not occur, but transient exposure to (Bu)2cAMP stimulated a dose-related increase in maturation of follicular oocytes. Degeneration of follicle-enclosed oocytes and cumulus-oocyte complexes was not affected by exposure to (Bu)2cAMP. These results suggest that transient, but not continuous, elevation of cAMP after the gonadotrophin surge may be required for the initiation of oocyte maturation.     

In vitro maturation,fertilization and development of domestic cat oocytes recovered from ovaries collected at three stages of the reproductive cycle

This study was conducted to examine the effect of the donor cat's reproductive cycle stage on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro development of oocytes recovered from ovaries that were collected and stored at 35 degrees C for a short period (1-6 h). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular, or luteal stages. Nuclear status of 161 cumulus-oocyte complexes (COCs) were examined immediately after recovery; 91.3% of the oocytes were found to be at the immature germinal vesicle (GV) stage, and 3.7% of the oocytes were at metaphase II (MII) stage. The percentage of the oocytes at the GV stage was significantly lower in the follicular stage than in the inactive stage (P < 0.01). Of the oocytes from the follicular stage, 9.1% were at MII stage. After culture for 24 h, however, the proportions of oocytes that reached metaphase I and MII were not different among the reproductive cycle stages of the ovaries collected (P > 0.05). After co-incubation with sperm, 63.1% of oocytes were fertilized, but there were no significant differences among the reproductive cycle stages of the ovaries with respect to the proportions of normal and polyspermic fertilization. However, the number of oocytes reaching cleavage stage and development to the morula and blastocyst stages from follicular stage ovaries were significantly lower (P < 0.05) than those obtained from inactive and luteal stage ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries, stored at 35 degrees C, has no apparent effects on the frequencies of maturation and fertilization of oocytes, but influences developmental competence of the oocytes following IVM or IVF.