Morning Of Metestrus Research Articles

Oxidative Damage Increases and Antioxidant Gene Expression Decreases with Aging in the Mouse Ovary

Oxidative stress has been implicated in various aspects of aging, but the role of oxidative stress in ovarian aging remains unclear. Our previous studies have shown that the initiation of apoptotic cell death in ovarian follicles and granulosa cells by various stimuli is initiated by increased reactive oxygen species. Herein, we tested the hypothesis that ovarian antioxidant defenses decrease and oxidative damage increases with age in mice. Healthy, wild-type C57BL/6 female mice aged 2, 6, 9, or 12 mo from the National Institute on Aging Aged Rodent Colony were killed on the morning of metestrus. Quantitative real-time RT-PCR was used to measure ovarian mRNA levels of antioxidant genes. Immunostaining using antibodies directed against 4-hydroxynonenal (4-HNE), nitrotyrosine (NTY), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) was used to localize oxidative lipid, protein, and DNA damage, respectively, within the ovaries. TUNEL was used to localize apoptosis. Ovarian expression of glutathione peroxidase 1 (Gpx1) increased and expression of glutaredoxin 1 (Glrx1), glutathione S-transferase mu 2 (Gstm2), peroxiredoxin 3 (Prdx3), and thioredoxin 2 (Txn2) decreased in a statistically significant manner with age. Statistically significant increases in 4-HNE, NTY, and 8-OHdG immunostaining in ovarian interstitial cells and follicles were observed with increasing age. Our data suggest that the decrease in mRNA expression of mitochondrial antioxidants Prdx3 and Txn2 as well as cytosolic antioxidants Glrx1 and Gstm2 may be involved in age-related ovarian oxidative damage to lipid, protein, DNA, and other cellular components vital for maintaining ovarian function and fertility.

Dynamics of messenger RNAs encoding inhibin/activin subunits and follistatin in the ovary during the rat estrous cycle.

Quantitative changes in ovarian inhibin/activin subunit and follistatin mRNAs during the rat estrous cycle were examined by ribonuclease protection assay using digoxygenin-labeled RNA probes. Levels of ovarian inhibin alpha subunit mRNA remained low throughout estrus, metestrus, and diestrus; abruptly increased on the morning of proestrus; then rapidly decreased when the primary gonadotropin surge occurred. A similar changing pattern was observed in inhibin/activin beta(A) subunit mRNA. On the other hand, inhibin/activin beta(B) subunit mRNA showed a different changing pattern. Levels of beta(B) subunit mRNA remained constant during metestrus and diestrus, abruptly decreased on the afternoon of proestrus, then quickly recovered from the nadir by 1100 h on estrus. Throughout the rat estrous cycle, especially during the periovulatory period, alpha subunit mRNA levels were considerably higher than beta(A) and beta(B) subunit mRNA levels. In addition, changes in plasma concentrations of inhibin A and inhibin B were very similar to that in ovarian beta(A) and beta(B) subunit mRNA levels, respectively, with several-hour delays. These results suggest that levels of beta subunit mRNAs restrict secretion of dimeric inhibins. Levels of follistatin mRNA remained low from the midnight of metestrus to the midnight of diestrus, then increased until initiation of the primary gonadotropin surge. Thereafter, follistatin mRNA decreased, reached the nadir at 0200 h on estrus, then increased abruptly at 1100 h on estrus. Afterward, follistatin mRNA levels remained high until the morning of metestrus. The changing pattern of ovarian follistatin mRNA was similar to, and preceded, the changes in plasma concentrations of progesterone, suggesting that ovarian follistatin may modulate progesterone secretion during the rat estrous cycle.

Both prolactin and progesterone in proestrus are necessary for the induction of apoptosis in the regressing corpus luteum of the rat.

This study was conducted to analyze the roles of prolactin (PRL) and progesterone in the induction of luteal cell apoptosis and accumulation of macrophages in the regressing corpus luteum. We studied the number of apoptotic cells and macrophages in regressing corpora lutea in estrus 1) in cycling rats or after blocking PRL secretion with the dopaminergic agonist CB154, and 2) after blocking progesterone actions with the progesterone receptor antagonists RU-486 or ZK98299. Cells showing the morphological features characteristic of apoptosis contained fragmented DNA as indicated by in situ 3' end labeling. In cycling rats, a 100-fold increase in the number of apoptotic cells and a 4-fold increase in the number of macrophages was found from the evening (1600 h) of proestrus to the morning (1100 h) of estrus. Both increases were blocked by PRL suppression with CB154. Furthermore, blocking progesterone actions with progesterone receptor antagonists RU-486 or ZK98299 without affecting PRL secretion inhibited apoptosis but did not affect the accumulation of macrophages, whether treatment was started on the morning of metestrus (blocking diestrous and proestrous progesterone) or on proestrus (blocking only proestrous progesterone). Otherwise, exogenous progesterone was not effective in inducing apoptosis in the absence of PRL. These results indicate that both PRL and progesterone in proestrus are necessary for the induction of apoptosis in the regressing corpora lutea, whereas the accumulation of macrophages seemed to be dependent exclusively on the PRL surge.

Serum levels of GH, IGF-I, LH and ovarian steroids in cyclic and RU486-treated rats.

Rats lacking progesterone action due to RU486 treatment have been reported to show numerous endocrine and morphological similarities with respect to human polycystic ovary syndrome (PCO). Nevertheless, abnormalities on insulin or insulin-like growth factor I (IGF-I) production, a frequent finding in the polycystic disease, have not been studied in such rats yet. The aim of these experiments was to evaluate the serum concentrations of IGF-I in rats treated with 4 mg of the antiprogestagen RU486 over 4 or 8 consecutive days starting on estrus (day 1) and decapitated on the morning of day 5 and 9. Serum levels of growth hormone (GH), luteinizing hormone (LH), testosterone (T) and 17 beta estradiol (E2) were determined and correlated with those of IGF-I. Controls were rats injected with oil and killed in the morning of metestrus, diestrus, proestrus and estrus. Rats treated with RU486 had increased serum concentrations of IGF-I, LH, T and E2 with respect to control rats on estrus, while no differences were found in the serum concentrations of GH. Parallel estrous cycle related changes were observed in the serum concentrations of IGF-I, T and E2 with the highest values at proestrus. However, changes on serum concentrations of GH were not associated with those of IGF-I in control rats through the estrous cycle. The present results are consistent with the idea that beside on GH, serum concentrations of IGF-I in the rat depend also on LH or LH-dependent ovarian steroid production. Moreover, the results indicate that IGF-I production increased in RU486-treated rats, which may have a role in the ovarian abnormalities induced by RU486 treatment in the rat.

Inhibin A and inhibin B are inversely correlated to follicle-stimulating hormone, yet are discordant during the follicular phase of the rat estrous cycle, and inhibin A is expressed in a sexually dimorphic manner.

Inhibin A and inhibin B are related dimeric protein hormones and endocrine regulators of the reproductive axis. Specifically, inhibin inhibits FSH secretion from the anterior pituitary. The inhibins are synthesized by the gonads and are themselves modulated by FSH. Although the activity of these ligands has been well characterized, the circulating concentrations of dimeric inhibin A and dimeric inhibin B have not previously been reported for the rat. Our group examined the serum concentration of inhibin A and inhibin B in normally cycling female rats, male rats, and in gonadectomized animals. Both inhibin isoforms are detected in intact female rat serum. Interestingly, inhibin B, but not inhibin A, is detected in intact male rat serum. Neither inhibin isoform is detected in long-term castrate female or male rats. In normally cycling female rats, inhibin A was low on the morning of metestrus and rose steadily to a peak on proestrus. In contrast, inhibin B was elevated on the mornings of metestrus, diestrus, and proestrus. Both ligands persisted in the serum until proestrus evening. Serum inhibins then declined beginning at 2100 h (inhibin A) or 1800 h (inhibin B) on proestrus, and the concentrations reached a nadir on the morning of estrus (0600 h). The nadir coincided with the peak of the secondary FSH surge. Both inhibins rebounded later on the morning of estrus. The results of this study demonstrate that dimeric, ovarian-derived inhibin A and inhibin B circulate in the female rat. The inverse relationship of the inhibins during the secondary FSH surge is consistent with the hypothesis that they participate in the regulation of reproductive cyclicity. The differing patterns of inhibin A and inhibin B during the period of follicular development on metestrus and diestrus suggest different follicle sources or regulation of these molecules during this period. We further demonstrate that inhibin B is the dominant form of FSH regulating protein in the male rat.

Synaptic remodeling in the arcuate nucleus during the estrous cycle is induced by estrogen and precedes the preovulatory gonadotropin surge.

We have shown that the ovarian cycle is accompanied by a fall in the axosomatic synapses on randomly selected neurons of the arcuate nucleus by the morning of estrus, with a return to the preovulatory levels by the morning of metestrus, indicating a possible role in positive feedback. However, it remains to be proven that the circulating estradiol is the actual regulator of this physiological synaptic plasticity, or that estrogen-induced synaptic retraction precedes in the surge of gonadotropins at midcycle. To resolve these questions, we used an estradiol-immunoneutralization protocol and studied arcuate nucleus axosomatic synapses during the critical points of the estrous cycle. In addition to blocking positive feedback, estrogen immunoneutralization abolished synaptic retraction in the arcuate nucleus. As a positive control, the nonbinding estrogen diethylstilbestrol maintained the gonadotropin surge and synaptic retraction in the antiestradiol-treated animals. Furthermore, in the diluent-treated cycling control females, the synaptic retraction was found to precede the preovulatory LH surge. We demonstrated that the midcycle synaptic retraction of arcuate nucleus synapses is induced by the preovulatory estradiol surge, and that these morphological events precede the preovulatory gonadotropin surge. Taken together, these observations strongly suggest that the hypothalamic mechanism underlying the physiological disinhibition of gonadotropins at midcycle (positive feedback) requires estrogen-induced synaptic retraction in the arcuate nucleus.

RU486 on an estrogen background blocks the rise in serum follicle-stimulating hormone induced by antiserum to inhibin or ovariectomy.

We used passive immunization with an antiserum to the alpha-subunit of inhibin (anti-I) or acute ovariectomy to investigate the relationship between serum inhibin levels and FSH secretion in the presence of the progesterone/glucocorticoid antagonist RU486. We demonstrated previously that 1) anti-I administered at 1700 h causes serum FSH to rise on the morning of estrus, even in the presence of a GnRH antagonist, when the two treatments are delivered on proestrus; and that 2) RU486 given on proestrus (1230 h), a time when serum estradiol levels are high, not only blocks the natural secondary FSH surge, but also suppresses the anti-I-induced rise in serum FSH on the morning of estrus. We have now extended our studies of the relationship between inhibin and RU486 to investigate treatment with RU486 and anti-I on a different day of the cycle, estrus, when serum estradiol levels are low. When both RU486 and anti-I were given on estrus (1230 and 1700 h, respectively), RU486 failed to block the anti-I-induced rise in serum FSH on the next morning of metestrus, in contrast to the blockade seen with RU486 treatment on the day of proestrus. However, pretreatment with estradiol benzoate (50 microgram) on the evening of proestrus, before the RU486 and anti-I treatment on estrus, caused RU486 to suppress the effects of anti-I on serum FSH, as it does when given on proestrus. We then repeated the study, using ovariectomy on proestrus or estrus (1700 h) to raise serum FSH, and assessed the effects of RU486 treatment at proestrus and estrus and estradiol benzoate treatment on proestrus. Our results indicate that treatment with RU486 can block the postovariectomy rise in serum FSH only in the presence of high circulating estradiol levels. We conclude that the inhibitory action of RU486 on FSH secretion after a fall in serum inhibin depends on a precedent estradiol background, probably due to induction of progesterone receptors by estradiol.

Gonadotropin-releasing hormone receptor messenger ribonucleic acid expression in the ovary during the rat estrous cycle

Recent evidence indicates that the GnRH receptor (GnRH-R) gene is expressed in a number of tissues besides the anterior pituitary gland, suggesting that GnRH may serve other functions in addition to its role as a hypothalamic releasing factor. In particular, high levels of GnRH-R transcripts have been detected in rat and human ovarian granulosa cells. To better understand the role of the GnRH-R in the ovary under physiological conditions and to determine which follicles are potentially responsive to the actions of GnRH, we used in situ hybridization histochemistry and quantitative reverse transcriptase-polymerase chain reaction for measurement of ovarian GnRH-R messenger RNA (mRNA) expression during the rat ovulatory cycle. Reverse transcriptase-polymerase chain reaction analyses revealed that total ovarian GnRH-R mRNA levels were elevated significantly at 1800 h on proestrus and again at 0900 and 1800 h on estrus compared to metestrus 0900 h levels. In situ hybridization analysis of GnRH-R gene expression at different stages of follicular maturation revealed significant variation in GnRH-R mRNA levels with respect to the degree of follicular development as well as the estrous cycle stage. GnRH-R gene expression was greatest in the granulosa cells of Graafian and atretic follicles, with lower levels of expression present in preantral and small antral follicles and corpora lutea. GnRH-R mRNA levels in atretic follicles showed substantial variation across the 4-day rat estrous cycle, with mRNA levels increasing 3-fold on the day of proestrus coincident with the preovulatory gonadotropin surges. A second peak of expression in atretic follicles was observed on the morning of estrus. Levels of GnRH-R gene expression in corpora lutea also varied significantly during the estrous cycle, with gene expression increasing 3-fold between the morning of metestrus and the afternoon of proestrus. These results demonstrate that the level and localization of ovarian GnRH-R mRNAs change significantly during the rat ovulatory cycle. The finding that atretic follicles exhibit the greatest degree of GnRH-R gene expression is consistent with a role for GnRH in the induction of follicular atresia.

Gonadotropin-releasing hormone receptor messenger ribonucleic acid expression in the ovary during the rat estrous cycle.

Recent evidence indicates that the GnRH receptor (GnRH-R) gene is expressed in a number of tissues besides the anterior pituitary gland, suggesting that GnRH may serve other functions in addition to its role as a hypothalamic releasing factor. In particular, high levels of GnRH-R transcripts have been detected in rat and human ovarian granulosa cells. To better understand the role of the GnRH-R in the ovary under physiological conditions and to determine which follicles are potentially responsive to the actions of GnRH, we used in situ hybridization histochemistry and quantitative reverse transcriptase-polymerase chain reaction for measurement of ovarian GnRH-R messenger RNA (mRNA) expression during the rat ovulatory cycle. Reverse transcriptase-polymerase chain reaction analyses revealed that total ovarian GnRH-R mRNA levels were elevated significantly at 1800 h on proestrus and again at 0900 and 1800 h on estrus compared to metestrus 0900 h levels. In situ hybridization analysis of GnRH-R gene expression at different stages of follicular maturation revealed significant variation in GnRH-R mRNA levels with respect to the degree of follicular development as well as the estrous cycle stage. GnRH-R gene expression was greatest in the granulosa cells of Graafian and atretic follicles, with lower levels of expression present in preantral and small antral follicles and corpora lutea. GnRH-R mRNA levels in atretic follicles showed substantial variation across the 4-day rat estrous cycle, with mRNA levels increasing 3-fold on the day of proestrus coincident with the preovulatory gonadotropin surges. A second peak of expression in atretic follicles was observed on the morning of estrus. Levels of GnRH-R gene expression in corpora lutea also varied significantly during the estrous cycle, with gene expression increasing 3-fold between the morning of metestrus and the afternoon of proestrus. These results demonstrate that the level and localization of ovarian GnRH-R mRNAs change significantly during the rat ovulatory cycle. The finding that atretic follicles exhibit the greatest degree of GnRH-R gene expression is consistent with a role for GnRH in the induction of follicular atresia.

Dynamic regulation of gonadotropin-releasing hormone receptor mRNA levels in the anterior pituitary gland during the rat estrous cycle.

The number of gonadotropin-releasing hormone (GnRH) receptors on pituitary gonadotropes varies substantially during the rat estrous cycle and may modulate pituitary responsiveness to GnRH. The present studies were undertaken to determine to what extent these changes in GnRH receptor number reflect a change in GnRH receptor mRNA expression in the anterior pituitary gland. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), pituitary GnRH receptor mRNA levels were measured at various timepoints throughout the rat estrous cycle. There was a three-fold increase in GnRH receptor mRNA levels on the afternoon of proestrus (PRO) when compared to levels observed on the morning of metestrus (MET). This rise preceded the onset of the LH surge by 6h (1200h). GnRH receptor mRNA levels remained elevated through 2100h PRO, after which they dropped dramatically, and by 2400h PRO were not significantly different from levels observed at 0900h MET. A two-fold increase in GnRH receptor mRNA expression was also observed during the early stages of the estrous cycle (0900h to 1800h MET), and this increase was sustained until 1800h on diestrus, at which time mRNA levels decreased to levels observed at 0900h MET. These results demonstrate that pituitary GnRH receptor mRNAs are dynamically regulated during the rat estrous cycle, with receptor mRNA expression being greatest on the afternoon of PRO, the time of the estrous cycle at which gonadotropes are most sensitive to GnRH stimulation.

The role of estradiol and progesterone in phased synaptic remodelling of the rat arcuate nucleus

During the estrous cycle there is a phasic synaptic remodelling in the hypothalamic arcuate nucleus, consisting in a loss and regain of axo-somatic synapses during the 48 h period between the morning of proestrus and the morning of metestrus. Synaptic changes are accompanied by cyclic modifications in the number of intramembrane particles in the plasma membrane of arcuate neuronal somas. To test the effect of the ovarian steroids on arcuate axo-somatic synapses we treated castrated females either with oil vehicle, 17β-estradiol, progesterone, or a combination of estradiol and progesterone, and observed them for 48 h. The number of axo-somatic synaptic profiles showed a 33% fall by 24 h after estradiol treatment and returned to control levels by 48 h. The effect of estradiol on axo-somatic synapses was accompanied by a marked and reversible modification of the number of intramembrane particles in the plasma membrane of arcuate neuronal somas. Progesterone alone did not affect the number of axo-somatic synaptic profiles nor the number of intramembrane particles, but when administered together with estradiol, blocked the effects of estradiol on neuronal membrane and synapses.

Estradiol-17 beta reduces number of ovulations in adult rats: direct action on the ovary?

The specific role of estrogen and other steroids in folliculogenesis is unclear since both inhibitory and stimulatory effects have been described. We reported that atresia of the preovulatory follicle was induced when estradiol-17 beta (E2) or progesterone was administered peripherally in rhesus monkeys, presumably due to a direct effect at the ovarian level. The present study was designed to determine if a similar direct action of E2 and other steroids occurs in rats. Minicapsules of Silastic containing E2, progesterone or dihydrotestosterone in amounts of 12.5% to 100% mixed with cholesterol, were placed unilaterally under the ovarian bursa on the morning of metestrus in rats having 4-day cycles. At autopsy on the morning of estrus, the number of oocytes ovulated from treated and untreated contralateral ovaries was determined. Ovaries treated with E2 averaged 3.1 +/- 0.4 oocytes while untreated ovaries in the same animals averaged 6.4 +/- 0.4 oocytes (P less than 0.001 by paired t test, n = 20). Results were similar for all amounts of E2 used and serum levels of E2 were not elevated at autopsy by this local treatment. Cholesterol alone did not alter the number of oocytes. Results of similar experiments with progesterone and dihydrotesterone were less conclusive than for E2. In additional trials, ovaries were treated with E2 as above, and preovulatory follicles were explanted on the morning of proestrus to determine their steroidogenic capability in vitro. Follicles from treated ovaries released somewhat less E2 and progesterone into luteinizing hormone (LH)-free medium than follicles from untreated ovaries, but not when LH was added to the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum LH, FSH and Prolactin Levels During the Estrous Cycle in the Hamster

SummarySerum LH, FSH and prolactin was measured by radioimmunoassays in 21 mature female golden hamsters at 9 am on each morning of the 4-day estrous cycle and also at 6 pm on the day of proestrus. All 3 hormones showed peak values at 6 pm on proestrous day, and by the following morning (estrus) LH and prolactin had returned to basal levels. Serum FSH remained as high on the morning of estrus as on the afternoon of proestrus, and although it fell on the morning of metestrus it was still higher than on the mornings of diestrus and proestrus. The persistence of high FSH release on the mornings of estrus and metestrus may be related to the luteotropic function ascribed to FSH in the hamster. The peak rise in all 3 hormones on the afternoon of proestrus in the hamster is similar to that observed for these 3 hormones in the serum of cycling female rats.

Changes in Ovarian Intravascular Compartment Prior to Superovulation in X-Irradiated Rats

Ovarian retention of radioiodinated serum albumin (RISA) was used as an index of the intravascular compartment of that organ during the estrous cycle in normal rats and in animals exposed to a regimen of x-irradiation known to induce superovulation (400 R whole-body at metestrus). In unirradiated animals, the intravascular space was highest on the morning of metestrus, decreased significantly during diestrus, then increased with the approach of the next estrus. In irradiated animals the large metestrus intravascular compartment persisted throughout the cycle. Choice of anesthesia significantly affected actual ovarian RISA retention, but not the pattern of change through the cycle. Possible role of this radiation response in x-ray-induced superovulation in the rat is discussed.