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Cholesteryl ester depletion from the ovaries of superovulated female rats fed a normal or essential fatty acid deficient diet.

The cholesteryl ester content of the ovaries was determined in rats diets containing corn oil or hydrogenated coconut oil (essential fatty acid (EFA) deficient) and subjected to superovulation by injection of luteinizing hormone and follicle-stimulating hormone. Superovulation increased ovarian weight; the effect was greater in animals fed corn oil. Superovulation significantly decreased total ovarian cholesteryl ester concentration in animals fed corn oil, with disproportionately large decreases occurring in the esters of 20:1, 20:2, 22:5w6, and 22:6w3. Significant decreases were observed in these esters when the data were expressed on a unit mass of tissue basis or in relation to total ovarian mass. In superovulated, EFA-deficient rats, esters of 18:1, 20:1, 22:5w6, and 22:6w3 were significantly lower per unit mass of tissue but this was due, in all cases except that of 22:6w3, to the increased mass of ovarian tissue; there was no decrease in total esters per ovary weight during superovulation of deficient rats. The pattern and degree of selective changes in ovarian cholesteryl esters during superovulation were different from those previously reported for adrenal esters of stressed rats.

Animals

Plasma oestrogen and progesterone in relation to superovulation and egg recovery in the cow.

Pregnant mares serum gonadotrophin (PMSG) was used in combination with prostaglandin F2alpha or its analogues to induce superovulation in 25 heifers. Total unconjugated oestrogen and progesterone were determined in peripheral plasma of these superovulated animals, and the levels compared with those found during the normal oestrous cycle. A very high level of oestrogen was found between day 3 and 6 after superovulation, and it seems likely that large unovulated follicles were responsible for the excess steroid. Similarly, progesterone levels were raised in the superovulated animal presumably due to production by the numerous corpora lutea. In two heifers, in which overstimulation of follicular development had occurred, there were no ovulations in one of the animals while in the other animal there were 16 ovulations but early regression of corpora lutea was indicated. It is concluded that the poor recovery of eggs on day 6 after superovulation could have been due to deleterious effects of high levels of oestrogen on either the motility of the genital tract causing the premature transport of eggs, or the properties of the zona pellucida.

Animals

Development of mouse oocytes superovulated at different ages.

The development of oocytes superovulated at 25, 50, or 90 days in four mouse strains (C57BL/6N, DBA/2N, ICR, and B6D2F1) was examined using the techniques of in vitro fertilization, culture, and transfer of two-cell embryos to pseudopregnant recipients. The highest number of ova were obtained from superovulated 25-day-old mice in all strains. Approximately 80% of oocytes developed to the two-cell stage after in vitro fertilization. Of these living oocytes, 60% developed to weanling stage through the recipient. These results suggested that donor age among 25, 50, or 90-day-old mice has no influence on the viability of superovulated oocytes. Consequently, we conclude that superovulated 25-day-old mice offer an economical and efficient source of viable oocytes for the production of transgenic mice.

Age Factors

Embryonic mortality during the pre- and post-implantation periods of pregnancy in mature mice after superovulation.

Sexually mature mice were stimulated to superovulate by giving exogenous gonadotrophins at known stages of the oestrous cycle. Untreated animals which ovulated spontaneously served as controls. The number of oocytes ovulated by each female was estimated from counts of the number of CL of pregnancy, and the incidence of embryonic mortality during the pre- and post-implantation stages of pregnancy was assessed from the number of zygotes recovered from the reproductive tract at 2-0 and 4-0 days post coitum and of conceptuses examined at 7-5 and 11-5 days post coitum. The mean number of oocytes ovulated by treated animals was 39-54, compared with 12-80 in controls: in mice which had superovulated, 44% of the ova were lost before implantation compared with about 10% in the controls. Further losses occurred about the time of implantation and at mid-pregnancy and thus the number of embryos classified as normal rarely exceeded the maximum found in controls. Death at mid-pregnancy seemed to be preceded by developmental retardation. The possibility that genetic and environmental factors play a role in embryonic loss after superovulation is discussed.

Animals

[Changes in antiproteolytic activity in the blood and cervical mucus after superovulation stimulation].

The function of proteins, peptides, proteases and inhibitors of proteases in modulations of regulation mechanisms of gonadotrophins during the development of ovarian folicles has not been fully explained up to now. We can see difference reactions of ewes to superovulation stimulations in oestrous and anoestrous periods as shown by the variation of the antiproteolytic activity of blood plasma and cervical mucus. Trypsin is used as a model for serine protease, and trypsin inhibitory activity (TIA) was measured from the reduced rate of trypsin hydrolysis of the chromogenic substrate N-alpha-tosyl-L-arginine-4-nitroanilide (TAPA, Bartík et al., 1974). Full hydrolytic activity was determined as a change in absorbency at 405 nm = 1.0 after ten-minute incubation at 25 degrees C and pH = 8.1, and inhibition was expressed in percentage of full activity. Statistical analyses were performed by Student's t-test. Twenty-three ewes in anoestrus and twenty-eight ewes in oestrus were included in this experiment. They were of the Slovak Merino breed, two to three years old, with the mean live weight of thirty to forty kg. The ewes were treated with Ageline vaginal sponges (20 mg chlorsuperlutin/sponge) to provide for synchronization of ovarian activity, in the interval of eleven to twelve days. After removal of sponges, the ewes were stimulated for superovulation as shown in Tab. I. Heparinized blood plasma samples were stored at -25 degrees C. Low molecular TIA activities were determined in HClO4--treated blood plasma. Samples of cervical mucus were taken on cotton-wool tampons which were evaluated in 0.2 M Tris-HCl buffer, pH = 8.1. Figs. 1-6 shows TIA changes in blood plasma and cervical mucus on particular days. Fig. 7 shows the mean values of controls (I-initial) after synchronization (A) and after stimulation (S). TIA changes were different in anoestrous and oestrous periods. Differences in TIA changes in blood plasma and cervical mucus in oestrous period suggested certain local regulation mechanisms of synthesis and/or secretion of this activity in the cervix. Some values of TIA were maximum at the time of expected heat or ovulation and may be some of the factor(s) which influenced fertility of females after superovulation stimulations.

Animals

Luteinizing hormone, progesterone and the morphological development of normal and superovulated corpora lutea in sheep.

The development of granulosa-lutein cells was studied in 27 normal and 32 superovulated ewes between days 0-4(day 0 began with the preovulatory LH peak in normal animals and the HCG injection in superovulated ewes). The pattern of differentiation was similar in both groups. Following initial hormonal stimulation (0-12 hours after LH or HCG), granulosa cells were approximately 100 mu2 and contained small, pleomorphic nuclei with large amounts of clumped chromatin. Elongate cells lining the basement membrane possessed large, heterogeneous dense bodies, and a well-developed Golgi apparatus. Mitotic figures were observed up to 6 hours prior to ovulation. Sixteen to 20 hours following the LH surge or HCG injection, hypertrophy of granulosa cells was evident. Nuclei contained definitive nucleoli. Blood vessels in the theca interna were abundant and highly dilated. Ovulation occurred approximately 24 hours after the LH peak or HCG injection. Visible signs of luteinization were evident 6-12 hours after ovulation. A slight increase in serum progesterone levels was detected. The second post-ovulatory day was characterized by continuing hypertrophy of granulosa cells and extensive proliferation of smooth endoplasmic reticulum and mitochondria. Nuclei of granulosa cells were larger and possessed extremely large nucleoli. Numerous mitotic figures were apparent within the corpus luteum. Serum progesterone concentrations began increasing at 60-72 hours after hormone stimulation. By the end of the third post-ovulatory day, the corpus luteum consisted of large, pleomorphic, parenchymal cells, interspersed between capillaries and connective tissue elements. Only an occasional mitotic figure was apparent within the corpus luteum at 100 hours. Light microscopic autoradiography of 5, 10, and 15 day corpora lutea taken from ewes pulsed with 3H thymidine at specific times before and after ovulation revealed that granulosa cells did not undergo secondary mitoses following ovulation. In contrast, thecal, mesenchymal and endothelial cells did mitose on day 3.

Animals

Free and esterified cholesterol concentration and cholesteryl ester composition in the ovaries of maturing and superovulated immature rats.

The cholesterol and cholesteryl ester concentration and cholesteryl ester composition were determined in the ovaries of immature rats, sexually mature rats and superovulated immature rats. The immature rat ovary accumulated cholesteryl esters, and long-chain polyunsaturated fatty acids were preferentially incorporated into these esters. The cholesteryl esters decreased in concentration and changed in composition with the onset of the first estrous cycle. Superovulation of immature rats, by injection of 50 I.U. pregnant mare serum gonadotropin, caused a decrease in the cholesteryl ester concentration of the ovary within 24 h and specific depletion of some esters, particularly those of 20 : 1 and 22 : 6 acids. Human choriogonadotropin, administered 54 h later, induced synchronous luteinization of the ovaries and was followed by increases in the concentration of free and esterified cholesterol and preferential accumulation of the esters of 20 : 4, 22 : 4, 4, 22 : 5 and 22 : 6 acids. Acute stimulation of luteinized ovaries by a second injection of the rats with 25 I.U. of human choriogonadotropin resulted in preferential hydrolysis of the esters of 18 : 1, 20 : 4, 22 : 4 and 22 : 5 acids.

Animals

Techniques of surgical and non-surgical ova collection of superovulated cows.

A comparison is made of the results of ova collection from 65 superovulated cows of varying ages using the conventional surgical technique and a non-surgical (transcervical) technique. Two types of apparatus, rigid and flexible, were developed for non-surgical collections. The problems associated with these techniques are discussed and some remedial measures suggested. A mean of 9.8 ova per donor cow were recovered by the surgical method compared to 2.9 and 3.0 ova recovered by the transcervical method using flexible and rigid apparatus respectively. Eight young cows, which had not been previosly subjected to superovulation and embryo collection, yielded an average of 5.6 ova per donor by the non-surgical technique.

Animals

Interactions of gonadotropins with corpus luteum membranes. I. Properties and distributions of some marker enzyme activities after subcellular fractionation of the superovulated rat ovary.

The properties of a number of enzyme activities of the superovulated rat ovary have been studied to establish optimal assay conditions and specific assay procedures for each activity. The activities were chosen on the basis of their extensive use in other tissues of the rat as marker enzymes for the major cell organelles. Homogenates of superovulated rat ovaries were subjected to fractionation by differential rate centrifugation, and sedimentation profiles were constructed for each marker enzyme activity. The various subcellular fractions were also monitored by electron microscopy. The enrichment of fractions with particular organelles by electron microscopy, and enrichment of the appropriate organelle marker enzyme activities correlated well. Sedimentation profiles of a number of plasma membrane marker enzymes demonstrated a marked discrepancy between hCG-binding activity, and 5'-nucleotidase-, alkaline phosphatase-, and Mg2+-dependent ATP-ase on the one hand, and basal, hCG-stimulated, and fluoride-stimulated adenylate cyclase activities on the other hand. Fractions enriched in hCG-binding and adenylate cyclase activities were subjected to further fractionation on discontinuous sucrose density gradients. The distributions of the various plasma membrane markers again indicated a partial dissociations between hCG-binding and adenylate cyclase activities of luteinized rat ovaries, suggesting the existence of two distinct major plasma membrane populations, with different buoyant densities, marker enzyme profiles and adenylate cyclase and hormone-binding levels.

Adenylyl Cyclases

Interactions of gonadotropins with corpus luteum membranes. II. The identification of two distinct surface membrane fractions from superovulated rat ovaries.

Fractions enriched in hCG-binding activity were prepared by differential rate centrifugation of superovulated rat ovarian homogenates and were applied to continuous sucrose density gradients (20-55%). After centrifugation at 63,000 x gav for 3.5 h, fractions of each gradient were collected and assayed for a range of marker enzyme activities characteristic of surface membranes and subcellular organelles. Mitochondria, lysosomes, and rough and smooth endoplasmic reticulum membranes accumulated in the gradient between 38-41% sucrose (1.165-1.180 g/cm3). Nuclei passed through the gradient. However, the various surface membrane markers concentrated in two distinct regions of the gradient. Alkaline phosphatase, phosphodiesterase, (Na+ + K+)ATPase I, and hCG-binding activity concentrated at 29-32% sucrose (1.120-1.135 g/cm3), whereas 5'-nucleotidase, Mg2+-dependent ATPase, and adenylate cyclase activities (and minor peaks of hCG-binding and phosphodiesterase activities) were enriched at 36-38% sucrose (1.16-1.17 g/cm3). A second ATPase, [(Na+ + K+)ATPase II], was also observed in this region of the gradient, which could be distinguished from (Na+ + K+)ATPase I of the light membrane fraction by its sensitivity to the Ca2+-chelating agent, ethylene glycol bis-(aminoethyl)tetraacetic acid (EGTA). The kinetics of binding of radioiodinated hCG to the gonadotropin receptors of the light and heavy membrane fractions were very similar. It is suggested that fractionation of superovulated rat ovaries yields two distinct populations of surface membrane material which have distinct densities and marker enzyme profiles. Furthermore, in contrast to the heavy membrane fraction, light membranes seem to possess considerable amounts of hCG receptor activity but very little adenylate cyclase.

Animals

[Use of different superovulation regimes and laparoscopic studies of ovarian function in African dwarf goats].

17 African dwarf goats were treated with 4 different superovulation regimes. The results of the treatments were assessed spectroscopically and checked laparoscopically. The quantitative basis of the investigation is too small to allow statistically well-founded statements, but is indicative of distinctive trends. A proportion of 73% CL and CLA in the overall ovarian changes after treatment with 750 IE PMSG (2 days before removal of the sponge) and 125 micrograms PGF2 alpha (at the time of the sponge removal) proved an acceptable method of treating African dwarf goats as regards the requirement of labour and material as well as the superovulation effect.

Administration, Intravaginal

[Catecholamine blood levels in sheep during superovulation in estrus].

The effects of a serum gonadotropin (SG) superovulation hormonal preparation were investigated on catecholamine levels (norepinephrine, dopamine and epinephrine) in the blood plasma of ewes with synchronized oestrus in the oestrus period. In this trial the blood plasma of eleven ewes of the Slovak Merino breed was analyzed to detect catecholamines in the oestrus period. Superovulation was induced by an i.m. administration of 1500 IU SG as soon as oestrus synchronization with Agelin vaginal tampons finished (20 mg chlorsuperlutin) which lasted for 10 days. Catecholamines were detected in the blood plasma before synchronization, on the day of Agelin vaginal tampons application, and in 48 and 72 hours after the hormone administration--on the days of the expected ovulation. Catecholamine concentrations in the blood plasma were determined by a radioenzymatic assay using a Catechola test (Praha). The results indicate that synchronization and hormonal stimulation influence plasma catecholamine levels. The norepinephrine (NE) concentration in the blood plasma of the control samples has the value of 8.31 +/- 0.732 pmol/ml. An insignificant increase in the NE levels (13.12 +/- 0.120 pmol/ml; Fig. 1) was recorded on day 1 of the trial, after the start of synchronization. On the day of the expected ovulation the NE concentrations rose to 17.12 +/- 1.289 pmol/ml (P less than 0.001) and they remained significantly increased (P less than 0.01) at the level of 12.89 +/- 1.020 pmol/ml on the following day of the experiment. The dopamine level (DA) in the plasma of a control sample is 6.42 +/- 0.350 pmol/ml (Fig. 2).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Oestradiol-17beta, progesterone, FSH and LH in prepubertal calves induced to superovulate.

Fluorogestone acetate (vaginal sponge for 4 days) and PMSG (i.m. injection at the time of sponge insertion) treatment was administered to seven 3-month-old calves to induce superovulation. Samples of peripheral plasma were taken every 4 h during treatment (4 days) and then every 2 h for 7 days. FSH, LH, oestradiol and progesterone were measured by radioimmunoassays. In all calves oestradiol concentrations increased 24 h after PMSG injection and reached the highest levels (41-502 pg/ml) during the preovulatory surge of both gonadotropins. The surge of LH and FSH occurred from 12 to 22 h after cessation of treatment. The maximum levels of LH and FSH were 11-72 ng/ml and 23-40 ng/ml respectively and occurred within 4 h of each other. Between 40 and 68 h after the LH peak the concentrations of progesterone began to increase from basal values, reaching 24.0-101.7 ng/ml when the animals were killed. A quantitative relationship was found between plasma oestradiol concentration and the numbers of ovulating follicles. Progesterone levels seemed to be related to the numbers of corpora lutea and also to the numbers of unovulated follicles. Gonadotrophin output was not quantitatively related to ovarian activity or to steroid secretion.

Animals

Estrone 3-glucuronide chemiluminescence immunoassay (LIA) and 17beta estradiol radioimmunoassay (RIA) in the monitoring of superovulation for in vitro fertilization (IVF): correlation with follicular parameters and oocyte maturity.

Many works in the literature of the last years had reported that urinary approach to superovulation study is a suitable method to evaluate ovarian response to pharmacological stimulation. Before applying urinary determination of hormonal levels with a chemiluminescence immuno assay (LIA) method in early morning urine (EMU) samples, we had studied the correlation of RIA-LIA procedures with reference to follicular volumes at hCG day and to recovered oocyte maturity; in fact follicular growth and oocyte morphological features are the main parameters to evaluate a successful induced cycle. In our department the IVF cycles are daily monitored with RIA seric E2 and LIA E1-3G determination, besides ultrasound examination of follicular growth. We have studied E2 and E1-3G levels on the hCG administration day and their correlation with follicular areas and volumes; moreover, we have evaluated hormonal values on oocyte pick-up day with reference to recovered oocyte number and maturity. We have assumed as good timing for oocyte pick-up when more than 50% of recovered oocytes were of good quality (maturity score 4). We have observed that the highest pre ovulatory E1-3G value is consistent with the best timing for oocyte pick-up; it's possible to obtain a conversion coefficient follicular volumes and urinary E1-3G excretion. We have not found significant differences between plasmatic and urinary estrogenic parameters. It is important to remember the advantages connected by a not isotopic and not invasive method. The absence of discomfort for the patients may be a decisive factor to choose the monitoring method and LIA procedure may represent a valid alternative to RIA.

Adult

Estradiol dynamics during superovulation are associated with oocyte quantity and maturation stage in cynomolgus macaques.

OBJECTIVE: To examine whether dynamic changes in circulating estradiol during ovarian stimulation are associated with the number and maturation status of oocytes recovered in cynomolgus macaques. DESIGN: Observational study based on retrospective analysis of ovarian stimulation cycles. SUBJECTS: Sixty-six ovarian stimulation cycles from adult female cynomolgus macaques (Macaca fascicularis) housed at the Primate Resources Center. EXPOSURE: Animals underwent a standardized gonadotropin-based ovarian stimulation protocol. Circulating estradiol concentrations were measured at multiple time points during the late follicular phase before oocyte recovery, and estradiol dynamics were defined as the difference between the maximum and minimum values observed across these measurements. MAIN OUTCOME MEASURES: Total number of oocytes recovered per stimulation cycle, number of mature oocytes at the metaphase two stage, and proportional distribution of oocyte maturation stages. RESULTS: Dynamic changes in estradiol concentrations were positively associated with the total number of oocytes recovered per cycle (correlation coefficient 0.45). A similar but weaker association was observed with the number of mature oocytes recovered (correlation coefficient 0.36). In contrast, estradiol dynamics were not associated with the proportional distribution of oocyte maturation stages. Donor age and body weight were not significantly associated with oocyte recovery outcomes. CONCLUSION: Dynamic changes in circulating estradiol during ovarian stimulation primarily reflect the quantitative dimension of ovarian response in cynomolgus macaques, with limited relevance for the maturation stage composition of recovered oocytes.

Animals