Rodent Ovary Research Articles

Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary.

Recent data support a role for apoptosis, under tight regulatory control by bcl-2, oxidative stress response, tumor suppressor, and CASP gene family members, in mediating granulosa cell demise during follicular atresia in the rodent and avian ovary. Herein we evaluated the occurrence of apoptosis in the human and baboon ovary relative to follicular health status, and analyzed expression of several cell death genes in these tissues. In situlocalization of DNA strand breaks in fixed human and baboon ovarian tissue sections indicated that apoptosis was essentially restricted to granulosa cells of atretic antral follicles. Biochemical analysis of DNA oligonucleosomes in individual follicles isolated from baboon ovaries during the ovulatory phase revealed the presence of apoptotic DNA fragments in subordinate but not dominant follicles, thus substantiating the in situ labeling studies. Messenger RNA transcripts encoded by the bax death susceptibility gene, the bcl-xlong survival gene, the bcl-xshort pro-apoptosis gene, the p53 tumor suppressor gene, and two members of the CASP gene family (CASP-2/Ich-1, CASP-3/CPP32), were detected by Northern blot analysis of total RNA prepared either from human ovaries or from Percoll-purified granulosa-lutein cells obtained from patients undergoing assisted reproductive technologies. Lastly, immunohistochemical localization of the BAX death-susceptibility protein in the human ovary revealed abundant expression in granulosa cells of early atretic follicles, whereas BAX protein was extremely low or non-detectable in healthy or grossly-atretic follicles. We conclude that apoptosis occurs during, and is probably responsible for, folicular atresia in the human and baboon ovary. Moreover, apoptosis in the human ovary is likely controlled by altered expression of the same cohort of cell death regulatory factors recently implicated as primary determinants of apoptosis induction or suppression in the rodent ovary.

Gonadotropin suppression of apoptosis in follicle somatic cells.

In each oestrous cycle only a limited number of follicles are selected for ovulation whereas the remaining majority undergo atresia. The earliest and most prominent feature of atresia is the death of granulosa cells. Recent biochemical evidence has demonstrated that granulosa cell death during follicular atresia in swine (Tillyet al., 1992), bovine (Jollyet al., 1994) and rodent (Tillyet al., 1991) ovaries occurs by apoptosis, a process whereby cells die in a controlled manner. A biochemical event considered to be characteristic of apoptotic cell death is the intranucleosomal cleavage of genomic DNA into fragments 180–200 bp in size, which separate into a distinctive ladder-like pattern on agarose gel electrophoresis. Detection of this pattern of oligonucleosomes in DNA provides a marker of apoptotic cell death.

Expression of superoxide dismutase, catalase and glutathione peroxidase in the bovine corpus luteum: evidence supporting a role for oxidative stress in luteolysis

Apoptosis, a type of physiological or active cell death, has been implicated as a mechanism underlying regression of the corpus luteum (CL) in the rat, bovine, rabbit and ovine ovary. Previousin vitro studies of cultured luteal cells have also provided evidence which suggests that reactive oxygen species play an important role in luteolysis in the rodent ovary. To further evaluate the potential role of oxidative stress in luteal cell demise, changes in the expression of several enzymes known to protect cells from oxidative stress were investigated using bovine CL collected from ovaries of non-pregnant (day 21 of the estrous cycle; regressed CL) and pregnant (day 21 of pregnancy; functional CL) animals. Biochemical analysis of genomic DNA extracted from these two pools of CL demonstrated the presence of extensive levels of internucleosomal DNA cleavage characteristic of cell death via apoptosis in regressed, but not in functional, CL. Northern blot analysis of total RNA indicated that functional CL expressed significantly higher levels of mRNA encoding secreted superoxide dismutase (SEC-SOD, 1.9 kb) and manganese-containing or mitochondrial SOD (Mn-SOD, multiple transcripts) as compared to regressed CL. Similarly, levels of mRNA encoding catalase (2.1 kb), an enzyme responsible for detoxification of peroxides to water, were significantly higher in functional versus regressed CL. From these data, we conclude that a decline in expression of specific oxidative response genes occurs during luteolysis, and that maintained expression of these genes in the CL during pregnancy may prevent oxidative damage and delay regression.

Rat granulosa cells express the gonadotrophin-releasing hormone gene: evidence from in-situ hybridization histochemistry.

There is still debate as to whether natural sequence gonadotrophin-releasing hormone (GnRH) is produced in the mammalian gonads and concerning its potential role as a paracrine modulator of gonadal function. To address this question, we have used in-situ hybridization histochemistry with an oligonucleotide probe complementary to the GnRH decapeptide coding sequence, to determine the cellular site(s) of expression of the GnRH gene in rodent ovaries. GnRH mRNA was detected in granulosa and thecal cells from ovarian follicles at all stages of development (primary-->Graafian), with no significant change in grain density during follicular development. The granulosa cell compartment always contained more mRNA than the thecal cell compartment. Corpora lutea expressed the GnRH gene to the same extent as thecal cells. These results indicate that preproGnRH mRNA is detectable under physiological conditions in the mammalian ovary, though whether this produces authentic GnRH decapeptide or an alternative protein product is not known. The physiological significance of these findings remains to be determined.

Histamine, mast cells and ovarian function.

Mast cells, endothelial cells, basophils and platelets are potential sources of histamine in the ovary. Little is known about the role of the latter three cell types in ovarian function. Several studies have revealed changes in the number and degranulation (release of histamine) of mast cells in the ovary during the cycle. Mast cells degranulate on pro-oestrus in the rodent ovary, and mast cells numbers increase in the theca externa of the dominant follicle in the bovine ovary. In rodents, mast cells are limited to the ovarian hilum and are not observed in follicles, corpora lutea and interstitium; this contrasts with larger species such as man, cows and monkeys where mast cells are observed throughout the ovary. Evidence is accumulating that mast cell degranulation in the ovary may be regulated by neuronal input. Neurones have been shown to have close morphological relationships with mast cells in the ovary. Histamine participates in regulating capillary permeability and blood flow in the ovary. These actions are induced by injections of LH, yet the mechanism by which LH induces mast cell degranulation is unknown. Histamine stimulates ovarian contractility, ovulation and follicular progesterone secretion in vitro. Whether these actions of histamine occur in vivo are currently unknown. This review gives a chronological description of the discoveries of the effects of histamine on ovarian function and makes suggestions for future research in this area.

Gonadal Steroids Regulate Proenkephalin Gene Expression in a Tissue-Specific Manner within the Female Reproductive System

Proenkephalin gene expression undergoes marked changes within the female reproductive system of rodents during the estrous cycle and in pregnancy. In order to define the factors responsible for this regulation, the effects of 17-beta-estradiol (E2) and progesterone (P4) have been examined in the ovary and uterus. In the ovary of the rat and hamster, E2 and P4 were without effect on proenkephalin RNA levels when injected individually. However, P4 increased ovarian transcript abundance 2- to 3-fold after pretreatment of animals with E2. In the uterus of either species, E2 had little effect but P4 alone stimulated both proenkephalin RNA abundance and total content severalfold. Glucocorticoids and androgen reproduced this stimulatory effect on proenkephalin transcript levels. The interaction between E2 and P4 on proenkephalin gene expression in the uterus varied with species. In the rat, E2 inhibited stimulation by P4, while in the hamster uterus the two hormones had a synergistic effect, producing a 15-fold elevation of proenkephalin RNA abundance and a 50-fold increase in total uterine content. These distinct steroid responses appear to account for tissue- and species-related differences in the variation of proenkephalin gene expression during the estrous cycle in the rodent ovary and uterus. The stimulatory effect of P4 was shown to involve direct steroid action on the uterus and to be inhibited both by the steroid antagonist RU-486 and the transcriptional inhibitor actinomycin D. These data are consistent with receptor-mediated activation of proenkephalin gene transcription in uterine cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization of aromatase in ovaries of some rodents, cow and human.

In order to clarify the in vivo localization of aromatase in the ovarian tissues of rat, mouse, guinea pig, golden hamster, cow and human, immunocytochemical investigations using the anti-human placental aromatase II cytochrome P-450 antibody were carried out by the PAP or indirect fluorescent antibody method.Immunoreactive aromatase was detected on the theca interna, as well as the interstitial gland cells of both immature and mature rodent ovaries and the corpus luteum cell of mature rodent ovaries. The immunoreactivity was also demonstrated on the theca interna cell and corpus luteum cell of cow and human ovaries. Only in large preovulatory follicles of the mature rodent ovary did the granulosa cell react with the antibody of this enzyme, while granulosa cells in all the other follicles were consistently negative in this immunoreaction.These results might imply that the theca interna cell and interstitial gland cell presumably have a fundamental ability of the aromatization from androgens to estrogens in the mature and immature ovaries.

SMOKING AND INDUSTRIAL POLLUTION, AND THEIR EFFECTS ON MENOPAUSE AND OVARIAN CANCER

The rodent ovary contains an enzyme system(s) capable of metabolising polycyclic aromatic hydrocarbons to reactive electrophilic intermediates known to cause cytotoxicity, mutation, and cancer. If the human ovary contains similar enzyme systems, metabolic activation of environmental chemicals could explain the earlier menopause in cigarette smokers and the higher incidence of ovarian cancer in industrialised areas.

The effect of a spleen dialysate on the enzymatic pattern of the rodent ovary.

The stimulating effect of a spleen dialysate on the testicles and the accessory genital organs motivated the authors to test this dialysate on the ovaries. Also on the ovaries a different influence was observed.In comparison to the control animals, the enzyme pattern: lactate dehydrogenase, steroid-3-β-ol-dehydrogenase, alkaline phosphatase, succinate dehydrogenase, and, glucose-6-phosphate-dehydrogenase is significantly changed. The effect is manifested by an increase of the tertiary follicles, increased follicular atresia and disquamating granulosa. On the basis of these findings we assume that the spleen dialysate is able to increase the gonadotropin effect on the ovaries.

Comparative studies on free amino acids in female reproductive tissues

The free amino acid pools of human and animal female reproductive tissues were quantitatively determined. Human ovary had higher levels of taurine, nitrogenous phospholipid components, and aspartic acid than uterus. Endometrium tended to be lower in glutamine and aspartic and glutamic acids than uterus. Cervix was high in ammonia. Comparative analyses of ovary and uterus from the rabbit, dog, cat, rat, mouse, hamster, guinea pig, and monkey were also carried out and some species differences were noted. Animal tissues resembled the human samples except that taurine was higher in uterus than ovary. Despite the variety of animals investigated, with differing diets and breeding habits, the free amino acid pools did not deviate greatly from a common pattern. Large and repeated doses of testosterone and estradiol had little effect on the free amino acids of rat ovary. Hypophysectomy reduced free amino acids in rat ovary and uterus to the same extent despite an increase in serum amino acids. Rodent ovaries were found to contain appreciable amounts of an amino acid with the same column elution time as gamma aminobutyric acid.

20- ALPHA-HYDROXYPREGN-4-EN-3-ONE, AN INTERFERING FLUOROGEN IN ASSAY OF CORTICOSTERONE.

SummaryA comparison of 20α-hydroxypregn-4-en-3-one and corticosterone reveals that their fluorescence properties are similar in concentrated sulphuric acid. Since rodent ovaries secrete 20α-hydroxypregn-4-en-3-one, this steroid may interfere with the assay of corticosterone by fluorimetric techniques; however, interference may be reduced by the use of a petroleum ether wash. It is suggested that a fluorescence method may be used as a sensitive assay for 20α-hydroxypregn-4-en-3-one.

Formation of 20α-Hydroxy-Δ4-pregnen-3-one and Δ4-Androsten-3;17-dione in Rodent Ovaries

Formation of 20α-Hydroxy-Δ⁴-pregnen-3-one and Δ⁴-Androsten-3;17-dione in Rodent Ovaries

Growth and Sexual Development of Prenatally Irradiated Cattle

Pregnant Hereford and Hereford-Angus cows received 400 r (air dose) of gamma radiation from Co/sup 60/ at a rate of 24.9 r/hr. The average stage of gestation at irradiation was 202 days (range 122 to 235 days). Fetal irradiation caused a small decrease in growth rate, and 90% of the prenatally irradiated heifers were fertile when bred to normal bulls. Testis weights of the prenatally irradiated bulls were approximates 50% less than those of the control bulls when castrated at 187 days of age. In addition, bulls prenatally irradiated in the 5th or 8th morth of fetal life produced 42% and 52% as many sperm as control bulls. Metabolic activity of the sperm from irradiated bulls did not differ from that of the control bulls. Six % of the dams died from acute radiation sickness (average 23 days postirradiation), whereas no mortality was observed in 34 bulls irradiated with 400 r. The failure to observe decreased fertility in mature cows within 3 or 5 years after 400 r, or 3 years after an accumulated dose of 800 r (400 r and 400 r 2 years later) is in contrast to decreased fertility in rodents following lower doses. Thus suggests thatmore » the ovaries of females of the larger species are less sensitive to irradiation than are the ovaries of rodents. (H.H.D.)« less

Microscopic histochemical demonstration of steroid-3 beta-ol dehydrogenase in tissue sections.

A procedure for the microscopic histochemical demonstration of steroid-3β-ol dehydrogenase has been described. In agreement with reported quantitative biochemical data, positive reactions have been obtained in the adrenal cortex, corpus luteum, placenta, and interstitial cells of the testis. In addition, a strongly positive reaction is obtained in the interstitial cells of the rodent ovary.

Effect of antigonadotrophic serum on recent intrasplenic ovarian implants of castrate mice.

IF TECHNICAL error is excluded, tumors appear with consistent regularity in the laboratory rodent ovary when it is transplanted to the spleen of a castrate animal. This frequently confirmed response was first recorded by Biskind and Biskind (1), who suggested that tumors arise in ovaries transplanted to the spleen as a result of excessive and continuous gonadotrophic stimulation. In their opinion, enhanced gonadotrophic secretion follows hepatic inactivation of gonadal steroids which must pass through the liver en route to the general circulation from the ovarian implant in the spleen. The ensuing loss of the well-known inhibiting effect of ovarian hormones on the gonadotrophic secretions of the pituitary gland leads to exposure of the intrasplenic grafts to heightened gonadotrophic stimulation which eventually terminates in tumor formation. Support for the postulation of Biskind and Biskind has been derived from widely different experimental approaches. The literature has been summarized (3, 6, 7, 8).

Inhibition of Estrous Cycle in the Rodent with Post-partum Urine and Commercial Prolactin

In attempting to ascertain the effect of urine from lactating women upon mammary glands of rats and mice, it was observed that there was an inhibition of estrus in these animals. Animals with normally recurring estrus were used, and these had daily vaginal smears for at least 3 consecutive cycles as a means of control. Injections of post-partum urine were then started. It was found that at least oney usually 2, and occasionally 3 cycles were suppressed, after which the animal resumed its normal estrous rhythm in spite of continuation or increase in the amount of injected urine. Controls, injected with the urine of normal human males and females had no alteration of the rhythm. The rodent's ovaries during this period of induced diestrus contained active corpora lutea which were similar to the ovaries of lactating animals. If injections were discontinued during this period of induced diestrus, a vaginal smear indicative of estrus was obtained within 48 hours. These findings suggested that a substance, possi...

Changes in the Hypophyses of Castrate and Cryptorchid Guinea Pigs.

Engle has shown that the anterior hypophysis of the castrate rat is more potent in gonad-stimulating hormone than that of the normal animal. Evans and Simpson confirmed this and demonstrated that glands from cryptorchid rats have more than normal potency. Severinghaus showed that the same holds for the castrate guinea pig. In the present experiments anterior lobes from normal and castrate (4 to 12 months) adult male and female, and experimental cryptorchid (6 to 14 months) guinea pigs were implanted intramuscularly into immature female rats and mice. In all, 235 animals were used as donors and 83 as hosts. The relative impotency of the guinea pig pituitary in gonadotropic hormone made it necessary to implant considerable amounts in the rat. The amount of guinea pig anterior lobe (fresh gland weight) required to induce a significant increase in the immature rodent ovary was found to be approximately 4 times that of rat pituitary. In the case of the five types of guinea pig pituitaries mentioned above from 3 to 5 times as much, by weight, was required in the rat as in the mouse. Similar results have been seen following the administration of sheep pituitary extracts. It was shown that the ovarian response following implantation of equal amounts of normal male and female pituitaries is approximately 50% greater in the case of the male. Pituitaries from castrate and cryptorchid males and spayed females were of equal potency, about 70% greater than normal male glands, in each instance. Pituitaries from females subjected to extreme partial castration were only slightly less potent than those from totally spayed animals. Glands from gonadectomized males and females receiving daily injections of estrin (25 to 100 R.U.) and from castrate and cryptorchid males bearing functional ovarian grafts were somewhat less potent than normal pituitaries.

On the Protein Nature of Prolactin and of Follicle-Stimulating Hormones

SummaryProlactin is a protein-like substance which at pH 8.0 is almost completely destroyed by trypsin in 2 hours at 37°C. In confirmation of previous work though using the bird testis instead of the rodent ovary to test potency, it is found that the follicle-stimulating hormone obtained from the anterior pituitaries of cattle is very rapidly destroyed by trypsin under the above-named conditions.The methods used in the separation and purification of prolactin suggest that this hormone is a protein, but since it has not yet been obtained in a pure state the possibility of its adsorption on inert protein is not thus excluded. It is therefore desirable to learn whether prolactin is affected by tryptic digestion.That prolactin is rapidly destroyed by trypsin is shown by the data of Table I. Almost complete destruction in 2 hours by a purified trypsin is obtained. The prolactin preparation used in this test was relatively pure and highly potent. Both the control and digest were, at the beginning of the test, a...

Hyperplasia of Mammary Apparatus of Adult Virginal Females Induced by Anterior Hypophyseal Hormones.

The mammary tree of adult virginal rats invariably undergoes some hyperplasia after a month of daily treatment with alkaline extracts of the anterior hypophysis, solutions very rich in growth hormone and almost completely devoid of the hormone which stimulates the ovaries of immature rodents to precocious development. By more chronic treatment with such an extract this can be brought to true milk secretion. Mammary hyperplasia takes place with more speed after treatment with the anterior hypophyseal ovary-stimulating hormone (bovine or rat hypophysis implant, or extracts of placenta and of the urine of pregnant women). The hyperplasia is, in fact, remarkable after only 10 days dosage with the latter methods.