Metaphase I arrest and spontaneous parthenogenetic activation of strain LTXBO oocytes: chimeric reaggregated ovaries establish primary lesion in oocytes

Oocytes of strain LT mice, and related strains such as LTXBO, exhibit a high incidence of arrest in the progression of meiosis at metaphase I (MI) and in spontaneous parthenogenetic activation. Activation of these oocytes within the ovary leads to the formation of ovarian teratomas. In this study, the role of the oocyte's companion granulosa cells, the cumulus cells, was investigated using fully grown oocytes matured in vitro after isolation from LTXBO mice. Results showed that the role of cumulus cells in MI arrest is dichotomous. Cumulus cells temporarily helped to sustain MI arrest, but they also promoted a delayed progression to metaphase II. Cumulus cells also promoted parthenogenetic activation that occurred in association with the delayed progression to metaphase II. Next, the question of whether the lesion(s) promoting MI arrest and spontaneous activation is due to defects in the somatic cells or is intrinsic to the oocyte was addressed using chimeric reaggregated ovaries. An improved method for completely exchanging the germ cell and the somatic cell compartments of ovaries from newborn mice is described. These chimeric reaggregated ovaries, grafted beneath the renal capsule of SCID mice, allowed the complete development of LTXBO oocytes to occur in association with somatic cells from control (B6SJLF(1)) ovaries and development of control oocytes in association with LTXBO somatic cells. Oocyte growth and follicular development appeared generally normal in reaggregated ovaries. High incidences of MI arrest and spontaneous activation of LTXBO oocytes occurred regardless of the genotype of the somatic cells. Moreover, there was a low incidence of MI arrest and spontaneous activation of control oocytes, even though they underwent complete development and maturation associated with LTXBO somatic cells. It is concluded that the phenotypes of MI arrest and parthenogenetic activation in LTXBO oocytes are defects caused by lesions intrinsic to the oocyte. Nevertheless, the oocyte's companion somatic cells play crucial roles in the expression of these lesions.     

Calcium-regulated plasma membrane rigidification during corpus luteum regression in the rat

Plasma membrane fractions were prepared from ovaries of superovulated rats and examined for structural changes during luteolysis. Using fluorescence polarization, we observed a rapid rigidification in vitro of samples obtained from ovaries undergoing spontaneous or prostaglandin F2 alpha-induced regression. The rigidification, manifested by a 72% polarization increase over 50 min, is calcium and calmodulin dependent, temperature sensitive and protein mediated. This increase in polarization did not appear in fractions from nonregressing ovaries; however, addition of phospholipase A2 caused virtually identical changes in polarization results as in samples prepared from regressing ovaries. These results suggest that calcium-calmodulin-dependent phospholipase A2 plays a role in membrane deterioration during luteolysis.     

Assessment of the activity of tumor necrosis factor in normal state and in ovarian tumors

The cytotoxic activity of tumor necrosis factor of healthy individuals and patients with cystadenoma and adenocarcinoma of ovaries was investigated. Tumor necrosis factor (TNF) spontaneous activities of healthy donors and patients with cystadenoma of ovaries was the same. The level of TNF spontaneous activity of patients with adenocarcinoma was increased. LPS-induced TNF cytotoxicity in patients with cystadenoma was decreased. Mononuclear cells of patients with adenocarcinoma did not respond to LPS stimulation by increased TNF production.     

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.     

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.     

Evidence for a role of intrauterine infections in the pathogenesis of cystic ovaries in postpartum dairy cows

Patterns of uterine microbial recovery and serum concentrations of prostaglandin F(2alpha) metabolite (PGFM), cortisol, luteinizing hormone (LH) and progesterone (P4) were determined in normal postpartum dairy cows (n = 12) and in postpartum cows that developed cystic ovaries (n = 12). Palpation per rectum, ultrasound examinations of the reproductive tract and endometrial swab harvest were carried out once every 4 d during the first month postpartum. Daily blood samples were collected during the first 30 d postpartum and every other day until Day 60 postpartum for hormone assay. Bacterial growth densities of cultures of uterine swabs were high in cows that developed cystic ovaries. Cysts were detected between Day 8 and 16 postpartum and persisted for a period of 18.6 +/- 9.9 d (range 8 to 41 d), followed by spontaneous regression and ovulations. Serum PGFM and cortisol were elevated for several days prior to detection of cystic ovaries but not prior to first postpartum ovulations. Serum LH concentrations in cystic cows were at basal concentrations prior to discovery of cysts. The results suggest that postpartum intrauterine infections may provoke increased secretion of prostaglandin F(2alpha) and cortisol associated with the formation of cystic ovaries in dairy cows.     

Effects of delayed excision of oviducts/ovaries on mouse oocytes and embryos

To achieve the best and reproducible results of experiments, effects of delayed excision of oviducts/ovaries on mouse ovarian/ovulated oocytes and embryos have been studied. Oviducts/ovaries were excised at different times after death of mice and effects of the postmortem interval on ovarian/ovulated oocytes and embryos were analyzed. When oviduct excision was delayed 10 min, many ovulated oocytes lysed or underwent in vitro spontaneous activation, and this postmortem effect aggravated with the extension of postmortem interval and oocyte aging. Oocytes from different mouse strains responded differently to delayed oviduct removal. Delayed oviduct excision did not cause lysis of zygotes or embryos but compromised their developmental potential. When ovaries were excised at 30 min after death, percentages of atretic follicles increased while blastocyst cell number declined significantly after oocyte maturation in vitro. Preservation of oviducts in vitro, in intact or opened abdomen at different temperatures and histological analysis of oviducts from different treatments suggested that toxic substance(s) were secreted from the dying oviducts which induced oocyte lysis and spontaneous activation and both this effect itself and the sensitivity of oocytes to this effect was temperature dependent. It is concluded that a short delay of oviduct/ovary removal had marked detrimental effects on oocytes and embryos. This must be taken into account in experiments using oocytes or embryos from slaughtered animals. The data may also be important for estimation of the time of death in forensic medicine and for rescue of oocytes from deceased valuable or endangered mammals.     

Antiestrogen therapies affect tissue homeostasis of the gerbil (Meriones unguiculatus) female prostate and ovaries

The present work aims to evaluate the response of the adult gerbil female prostate (paraurethral glands) and ovaries to short-term exposure to antiestrogenic agents, consisting of daily oral doses of letrozole (1 mg kg(-1) day(-1)) or intradermal doses of tamoxifen (1 mg/kg) every other day for 21 days. The serum levels of testosterone and estradiol were monitored, and the prostates and ovaries collected for structural, ultrastructural, and immunocytochemical analyses. The letrozole treatment resulted in increases of serum testosterone levels and secretory activity as well as in glandular hyperplasia and dysplastic growth, simulating the effects caused by the exogenous androgens. The effects caused by tamoxifen indicate that this endocrine agent acted as an estrogenic agonist on the prostate, causing glandular hypertrophy, secretory activity decrease, and the development of prostatic lesions. Therefore, it is possible to conclude that the letrozole and tamoxifen therapies result in a series of complex effects that endanger the physiology of hormone-dependent organs, including the female prostate and ovaries. The hormonal imbalance caused by administration of these drugs resulted in considerable changes in prostatic morphology, in a manner very similar to what occurs during the development of prostatic lesions in aged postmenopausal women. Thus, these therapies must be chosen carefully since long-term treatments can result in female prostate dysplasic lesions.     

Effects of reciprocal ovary transplantation on reproductive performance of lethal yellow mice (Ay/a; C57BL/6J)

This study was conducted to determine whether reproductive failures in ageing, obese lethal yellow (Ay/a) females are due primarily to defects within Ay/a ovaries or to systemic defects which may operate outside the ovaries. Reciprocal ovary transplantation between control (a/a) and lethal yellow (Ay/a) females provided an experimental system to test the reproductive potential of not only Ay/a ovaries in control (a/a) females but also control (a/a) ovaries in mutant (Ay/a) females. Results on reproductive performance of all four combinations of grafts between Ay/a and a/a mice proved that Ay-induced reproductive failures are not due to intrinsic ovarian lesions but rather to defects operating extrinsically to the ovary. The hypothalamo-pituitary axis is a likely site for this reproductive lesion.     

Total monoamine oxidase activity in the hypothalamus, ovary and uterus of rats with an extreme number of ovarian corpora lutea

The activity of total monoamine oxidase (MAO) in the rat ovary and uterus fluctuates significantly under various physiological conditions. We analyzed total MAO activity in the hypothalamus, uterus and ovary in adult rats, having an extreme number of corpora lutea (hyperluteinized ovaries) resulting from the mechanical lesions in the posterior hypothalamic region of neonatal rats. Total MAO activity in the hypothalamus (30.21 +/- 1.53 pmol/mg tissue/min) and uterus (3.16 +/- 0.61 pmol/mg tissue/min) of rats with hyperluteinized ovaries did not show a significant difference as compared to that of intact controls (31.09 +/- 1.72 and 2.90 +/- 0.40 pmol/mg tissue/min, respectively). In contrast, in the ovaries of hyperluteinized rats, total MAO activity (21.16 +/- 1.70 pmol/mg tissue/min) was significantly higher (p<0.01) when compared to that of intact controls (13.61 +/- 1.30 pmol/mg tissue/min). The increased MAO activity in the hyperluteinized ovaries may be attributed to the increased number of transformed and accumulated corpora lutes as a consequence of diminished luteolysis.     

Ovarian localization by embryonal teratocarcinoma cells derived from female germ gells

Embryonal carcinoma cells derived from several different spontaneous ovarian teratocarcinomas of strain LT mice form tumors that are located exclusively, in many cases, in the ovaries of female mice. Embryonal cells previously unselected for site specificity localize in the ovaries regardless of route of entry of the cells, and produce very few tumors in males following intraperitoneal injections. The ovary tumors have been verified as originating from the injected cells by chromosomal and drug resistance markers, as well as by general in vitro growth characteristics. Cell-cell adhesion studies suggest specificity at the level of tumor cell-ovary organ cell interaction.     

Growth and regression of ovarian follicles during the follicular phase of the oestrous cycle in heifers undergoing spontaneous and PGF-2 alpha-induced luteolysis

Ultrasonography was used to monitor the growth, ovulation and regression of individual ovarian follicles greater than or equal to 5 mm during the late luteal and follicular phases of the oestrous cycle in heifers treated with injections of PGF-2 alpha to induce luteolysis and in heifers undergoing spontaneous luteolysis. Six heifers were given a single injection of PGF-2 alpha between Day 12 and 15 of the oestrous cycle and their ovaries were examined daily by transrectal ultrasonography until ovulation occurred. Another group of 5 heifers was examined daily by ultrasound from Day 14 or 15 of the cycle through spontaneous luteolysis and ovulation. Blood samples were taken twice daily from this group and analysed for progesterone to determine when luteolysis occurred. All heifers were checked for oestrous behaviour twice daily. Mean diameters of ovulatory follicles on each of the 3 days before oestrus were not different between PGF-2 alpha-treated and untreated heifers. In both groups there was large variation among heifers in the sizes and growth rates of the ovulatory follicles. At 3 days before oestrus the diameters of ovulatory follicles were between 7.5 and 11 mm in PGF-2 alpha-treated heifers and between 6 and 11.5 mm in untreated heifers. Non-ovulatory follicles decreased in size during the 3 days before oestrus and the number of non-ovulatory follicles within the size ranges of ovulatory follicles decreased. The ovulatory follicle was not consistently the largest follicle on the ovaries until the day of oestrus but was always one of the 2 largest follicles during the 3 days before oestrus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laparoscopic evaluation of the reproductive organs and abdominal cavity content of the lowland gorilla

Laparoscopy was used to examine the abdominal cavity and evaluate reproductive competence in six adult female gorillas which had not demonstrated copulatory activity within 12 months of examination. Blood samples were obtained on the day of laparoscopy and analyzed for serum estradiol-17β and progesterone. The uterus and oviducts of each gorilla appeared morphologically normal and free of lesions. The ovaries of three animals contained anatomical evidence of activity as indicated by the presence of luteal scars and a prominent corpus hemorrhagicum, corpus luteum, or developing follicles. No visible luteal or follicular tissue was observed on the ovaries of three gorillas. In the eldest female (age, 22 years) the ovaries were flaccid in texture and demonstrated irregularities in ovarian surface integrity, similar to that observed in women during reproductive senescence. Serum concentrations of estradiol-17β and progesterone on the day of laparoscopy corresponded with observed ovarian activity. All six females had extensive adhesions in the abdominal cavity exclusive of the reproductive organs. Numerous gut, omentum, and liver to peritoneum adhesions were observed. The results indicated that laparoscopy was a safe and effective method for evaluating anatomical competence in the gorilla. Uterine and ovarian integrity appeared morphologically normal, but only three of the six gorillas showed evidence of ongoing ovarian activity.     

Egg laying inAplysia

1. Central pathways for bag cell activation were identified by examining the frequency of spontaneous egg laying episodes in animals with central connective lesions. Bilateral lesions of the cerebropleural (but not the cerebropedal) connectives abolished spontaneous egg laying. In contrast, bilateral lesions of all cerebral ganglion peripheral nerves did not abolish spontaneous egg laying, suggesting that sensory input to the cerebral ganglion is not necessary for activating the bag cells. 2. Backfilling either pleuroabdominal connective labelled cell bodies in the cerebral ganglia (via the ipsilateral cerebropleural connective) that could project to the bag cells. Focal extracellular stimulation of these stained clusters activated the bag cells in isolated brains. 3. Central pathways for initiating egg laying behaviors were identified by selectively eliciting bag cell discharges in animals with central connective lesions. Bilateral lesions of the cerebropedal (but not the cerebropleural) connectives completely abolished elicited egg laying behaviors. 4. Pathways for motor output during rhythmic head and neck movements were identified by eliciting bag cell discharges in animals with peripheral nerve lesions. Bilateral lesions of the four tegumentary nerves in combination with the anterior pedal nerve completely abolished elicited egg laying behaviors, indicating that these nerves are necessary for normal motor output. A normal pattern of egg laying behaviors occurred when the four tegumentary and the anterior pedal nerves were left intact and all other pedal ganglion nerves were lesioned bilaterally, indicating that these nerves are also sufficient for normal motor output.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of the development of uterine adenomyosis by danazol treatment in mice.

Inhibitory effects of danazol, an isoxazol derivative of synthetic steroid 17 alpha-ethinyl-testosterone, on the development of uterine adenomyosis, a pathological disorder of endometrial tissue defined as the presence of endometrial glands and stroma in the myometrium, were investigated in mice of SHN strain. Mice treated with 0.5 microgram danazol for 5 weeks during 4-9 weeks of age and killed at 21 weeks of age showed significantly lower incidence of the spontaneous development of adenomyosis than the age-matched intact control mice. The inhibitory effects of danazol were also evident in mice bearing pituitary isografts which were effective in inducing an early and a high incidence of adenomyosis. Furthermore, the treatment with danazol resulted in the decrease of serum levels of luteinizing hormone (LH) and prolactin (PRL) associated with hypofunction of ovaries and persistent diestrus. These results support the usefulness of danazol for the clinical treatment of gynecological disorders except for hypofunction of ovaries.     

Possible MIS Production by Follicle Cells in Spontaneous Oocyte Maturation of the Ascidian, Halocynthia roretzi

Outer and inner follicle cell-enclosed oocytes (oocyte complexes) of Halocynthia roretzi underwent germinal vesicle breakdown (GVBD) within 2 hr when transferred from ovaries to normal seawater of pH 8 (NSW). Extrusion of test cells (TC) into the perivitelline space and elevation of the chorion also occurred. This phenomenon was designated as spontaneous oocyte maturation.
Seawater of low pH, protease inhibitors such as leupeptin or soybean trypsin inhibitor (SBTI), and calcium deficiency inhibited the spontaneous maturation only when introduced to the NSW during the first 10 minutes of incubation. GVBD-blocked complexes underwent GVBD after addition of trypsin regardless of pH or the absence of calcium ions. The oocytes from which follicle cells were removed with glycosidase did not undergo GVBD in NSW, but addition of trypsin triggered GVBD in these defolliculated oocytes (TC oocytes). Furthermore, incubation media in which spontaneous maturation had occurred, induced GVBD in the TC oocytes. This GVBD-inducing activity was heat-labile and was inhibited by leupeptin.
These results indicate that in the first step of the spontaneous oocyte maturation, outer and/or inner follicle cells give a signal to the oocyte itself or TC oocyte. This signal is likely to be trypsin-like.     

Phenomenon of pseudomutagenesis: experiments with modifiers of cell metabolism in human lymphocytes

The term "pseudomutagens" is suggested for factors that increase the spontaneous mutation level not by induction of DNA molecule changes but as a result of inhibition of the repair of spontaneous pre-mutation lesions. The expected regularities of pseudomutagen effects, which are suitable as criteria for their identification, are considered. The experimental results on the effects of six modifiers of cell metabolism in human lymphocytes are analyzed. Four of the modifiers (5-fluorodeoxyuridine, sodium cyanide, sodium fluoride and monoiodoacetic acid) behaved as pseudomutagens according to all the criteria applied. 2,4-Dinitrophenol proved to be a true mutagen. Adenosine diphosphate revealed unusual properties: it inhibited the repair of spontaneous lesions but decreased the frequency of structural mutations induced by radiation. The significance of the phenomenon of the pseudomutagenesis for the problem of environmental mutagens/carcinogens is discussed.     

Heterotopic autotransplantation of ovarian cortex in cynomolgus monkeys

In recent years, removal of ova or ovaries before chemotherapy or radiation therapy has been investigated in young female cancer patients to avoid the adverse effects of treatment. Orthotopic autotransplantation of ovarian cortex has advantages such as easy collection of ova and the possibility of spontaneous pregnancy. Although children have been born after successful orthotopic autotransplantation into the residual ovaries, some patients cannot undergo this procedure such as those who need bilateral ovariectomy or pelvic radiation therapy, therefore it is still necessary to investigate suitable heterotopic autotransplantation sites. The present study was performed in primates (cynomolgus monkeys) with the objective of determining the optimum site for heterotopic autotransplantation of ovarian cortex to enhance the clinical application of this method. The retroperitoneal iliac fossa and omentum were selected as sites for heterotopic autotransplantation. Two cynomolgus monkeys were subjected to laparotomy under anesthesia. After resection of the bilateral adnexae, the ovaries were cut into 0.5 cm cubes that were transplanted. Blood levels of follicle-stimulating hormone, luteinizing hormone, estradiol, and progesterone were monitored, and monkeys with a regular estrus cycle underwent superovulation and egg collection. In both monkeys studied, recovery of a regular estrus cycle was confirmed after heterotopic autotransplantation of ovarian tissue. MII phase ova were successfully collected from tissues transplanted into the retroperitoneal iliac fossa or omentum. Development to the early blastocyst stage was confirmed after microfertilization. We established an appropriate method of heterotopic autotransplantation using ovarian cortex into the retroperitoneal iliac fossa or omentum in primates.     

Incipient spontaneous granulosa cell tumour in the gerbil, Meriones unguiculatus

In 167 cases of granulosa cell tumour (GCT) in the gerbil, Meriones unguiculatus, 42.5% (71 cases) were in the ovaries, with no evidence of macroscopic lesion. In this paper, we describe the common findings in the incipient--microscopic--type of this spontaneous ovarian tumour occurring in gerbils younger than two years of age.     

Parthenogenetic development of Mos-deficient mouse oocytes

Ovarian teratomas develop in Mos^−/− mutant mice produced by homologous recombination. These teratomas are probably derived from oocytes that undergo spontaneous parthenogenetic activation within the ovaries. However, it is not clear how the activated eggs develop into teratomas since embryonic development beyond the four-cell stage was not observed either in vitro or in vivo. In this study, Mos^−/− parthenotes derived from in vitro-matured oocytes were cultured using a recently developed medium, KSOM/AA, which promotes a high frequency of preimplantation development by normal embryos. In total, 5% of the Mos^−/− oocytes developed to the blastocyst stage. Preimplantation-like and early postimplantation-like embryos were observed in the ovaries of 60–63-day-old Mos^−/− mice. These observations support the hypothesis that Mos^−/− teratomas are derived from parthenogenetically activated oocytes that undergo early embryonic development up to early postimplantation-like stages within the ovaries. Aberrant meiotic divisions commonly observed in Mos^−/− oocytes in vitro may adversely affect preimplantation development and reduce the frequency of blastocyst formation even under the best culture conditions. Mol. Reprod. Dev. 48:391–396, 1997. © 1997 Wiley-Liss, Inc.