Changes in interleukin-1beta mRNA expression in the rat ovary during the estrous cycle in response to lipopolysaccharide.

The changes in interleukin-1 (IL-1) beta mRNA expression and the number of macrophages were studied in the ovary during the estrous cycle in rats and after intraperitoneal injection of lipopolysaccharide (LPS, 2 mg/body) 2 hours before autopsy. IL-1beta mRNA expression was very low in the ovary, and there was no statistically significant change during the estrous cycle. Hybridization signals of IL-1beta mRNA were localized intensely in the thecal layer, moderately in the corpora lutea, and slightly in granulosa cells of the ovary during the cycle. The number of macrophages seen mainly in the hilum and interstitium significantly increased on proestrus compared with other estrous days. LPS significantly increased IL-1beta mRNA expression on each day with the highest response to LPS at 1500 h on proestrus, and caused an increase in the number of macrophages in the ovary within 2 hours. These results indicate that IL-1beta mRNA expressions are low during the estrous cycle in rats, and proestrus is the day of maximal IL-1beta synthesis in response to LPS. The increase in IL-1beta synthesis caused by LPS might be due to at least the influx of macrophages into the ovary.     

Follicular steroidogenesis and gonadotropin binding to ovine follicles during the estrous cycle

The interrelationships among [125I]hCG binding in thecal and granulosa cells, antral fluid steroid concentrations, follicular size, and ovarian steroid secretion were examined at three different stages of the estrous cycle. Group 1 ewes were ovariectomized during the luteal phase of the estrous cycle, and the other groups were ovariectomized before (group 2), or after (group 3) the peak of the preovulatory LH surge. Times of luteolysis and the LH surge were assessed by measurement of peripheral concentrations of progesterone and LH. Three patterns of [125I]hCG binding to follicles were noted: 1) binding to both thecal and granulosa cells (activated follicle), 2) binding to the thecal cell layer only, and 3) no observed binding. In general, there was one active follicle per ewe, or one per ovary, and the number of active follicles was not different from the number of corpora lutea in each of the three groups. The active follicles were significantly larger than the other two classes of follicles. Antral fluid estradiol concentrations were significantly greater in the active follicles and were higher in group 2 ewes than in the other two groups. In group 2, antral fluid testosterone concentrations were significantly higher in follicles with LH receptors in the thecal cell layer only. Ovarian secretion of testosterone and estradiol increased during the early follicular phase (group 2), with the major secretion coming from the ovary containing the active follicle. Ovarian progesterone secretion was high in ovaries containing active corpora lutea which prevented the assessment of ovarian follicular secretion of progesterone. The follicle with LH receptors in thecal and granulosa cells was responsible for the increased estradiol secretion observed during the preovulatory period and is presumed to be the ovulatory follicle.     

The lh-hcg receptor of human ovary at various stages of the menstrual cycle.

Receptors specific for hCG were found in human corpora lutea and follicles. hCG and LH were found to bind at a similar receptor site. The dissociation constant for hCG ranged from 10-minus 10 to 10-minus 11 mol/1 in human corpora lutea. The number of binding sites for 125-I-hCG ranged from 10-minus 14 to 10-minus 15 moles/mg protein in human corpora lutea. The binding of 125-I-hCG to ovary was found to vary at different stages of the menstrual cycle. The binding of 125-I-hCG to human ovaries increased on days 13-15 of the cycle, then declined slightly, and increased again on days 22-23. Following day 23, there was a slow decline until day 27 when binding activity could no longer be measured. No binding could be measured by the corpus luteum after the onset of menstruation or in corpora albicans.     

Cyclic changes in follistatin messenger ribonucleic acid and its protein in the rat ovary during the estrous cycle.

The purpose of this research was to characterize the localization of follistatin mRNA and protein in the adult rat ovary during the 4-day estrous cycle. Analysis of ovarian sections using in situ hybridization and immunohistochemistry demonstrated the presence of follistatin messenger RNA (mRNA) and its protein in granulosa and luteal cells; no follistatin (message or protein) was detected in any of the other ovarian cell types. An important observation was that the intensity of follistatin signals changed during granulosa differentiation and the estrous cycle. During folliculogenesis, the first detectable hybridization signal appeared in the granulosa cells of secondary follicles, but the signal was weak. However, when a preantral follicle reached the early tertiary stage (beginning antrum formation), the message signal was very strong, being expressed in all granulosa cells of all such follicles (300-400 microns in diameter). In atretic follicles, follistatin mRNA was localized to granulosa cells, but only during the early stages. The above hybridization pattern of follistatin mRNA in prenatral and atretic follicles appeared constant throughout the estrous cycle. Interestingly, immunohistochemistry studies showed that the follistatin protein was detected only in certain follicles, being restricted to those which were healthy. On the morning of estrus, the follistatin protein was localized to a subpopulation of early tertiary follicles, presumably the dominant follicles selected to ovulate in the next cycle. As the dominant preovulatory follicles matured through diestrus and proestrus, the follistatin mRNA and protein signals appeared more intense in the granulosa cells. After ovulation, the hybridization and immunohistochemical signals continued to be strong in the newly formed corpora lutea on estrus morning. After luteolysis on diestrus-I, neither the follistatin message nor the protein was detectable in the corpora lutea. In conclusion, these results suggest that the follistatin message is present in all the granulosa cells of every developing follicle throughout the estrous cycle, but the follistatin protein appears to be present in only the selected dominant follicles. Accordingly, the possibility that follistatin might be an important regulatory molecule for selection/atresia should be considered.     

Demonstration of immunoreactive beta-endorphin- and gamma 3-melanocyte-stimulating hormone-related peptides in the ovaries of neonatal, cyclic, and pregnant mice

Antisera against proopiomelanocortin (POMC)-derived peptides have previously been employed to demonstrate immunostainable materials in the male reproductive tract and in the corpus luteum of rat ovary. The present study was designed to determine how the distribution of such stainable materials varies in mouse ovary as a function of the reproductive status of the animal. Peptide-like activities were localized with the unlabeled antibody peroxidase-antiperoxidase (PAP) technique in ovaries removed from mice during fetal and neonatal development, during different stages of estrous cycle, and during pregnancy, with antisera against beta-endorphin, gamma 3MSH, and an extended N-terminal portion of POMC (16 K). beta-endorphin-like activity was also quantified in ovarian extracts by RIA. Immunostainable beta-endorphin, gamma 3MSH, and 16 K fragment-like activities were present in ovaries of pregnant and normally cycling (but not immature) mice. Intense staining was found predominantly in the corpora lutea. Less intense staining was observed in the interstitium and in the following parts of large follicles: parietal granulosa, corona radiata, and cumulus oophorus. When neonatal mice were injected with hCG, immunostainable beta-endorphin-like material in the ovarian interstitial area increased. Treatment with PMSG increased staining in both secondary follicles and the interstitium. Immunoassayable beta-endorphin-like activity was twice as high (per g wet wt) at pregnancy as during the cycle. We conclude that peptides similar or identical to POMC and/or its components are present in ovarian cells and that the concentration of such material appears to be regulated by gonadotropins.     

Progesterone secretion as affected by 17 beta-estradiol after hysterectomy in the pig

Serum levels of progesterone in hysterectomized gilts were determined to evaluate the effects of exogenous 17 beta-estradiol (17 beta-E2) on the secretory activity of aging corpora lutea. Gilts were hysterectomized on day 6 of the estrous cycle and given im injections of 17 beta-E2 to mimic blood levels of endogenous estrogen during normal pregnancy. Serum progesterone was determined every third or sixth day, and estrone and 17 beta-E2 were measured at 12-day intervals from days 42-192. Progesterone decreased (P less than 0.01) after day 104 in hysterectomized gilts given sesame oil. Exogenous estrogen consistently decreased (P less than 0.02) progesterone secretion during an extended period (days 45-108) in aging corpora lutea of hysterectomized gilts. Abrupt decreases in estrone, 17 beta-E2, and progesterone levels occurred after termination of estrogen injections on day 114. The decrease in the secretion of these steroids in hysterectomized gilts was similar to that observed in previous studies at parturition and during the early postpartum period. Estrogen treatment beyond day 114 inhibits follicular growth and suppresses ovulation, but behavioral estrus was induced during the terminal stages of luteal activity. In the absence of the uterus, aging corpora lutea respond to exogenous estrogen by decreased progesterone secretion, which is similar to that observed during normal pregnancy in the pig.     

Expression of ghrelin in the cyclic and pregnant rat ovary

Ghrelin, a 28-amino acid acylated peptide, has been recently identified as the endogenous ligand for the GH secretagogue receptor. Previous studies demonstrated that ghrelin, acting centrally, strongly stimulates GH release and food intake. In this study we provide novel evidence for the expression of ghrelin in the cyclic and pregnant rat ovary. Persistent expression of ghrelin gene was demonstrated in rat ovary throughout the estrous cycle, although its relative mRNA levels varied depending on the stage of the cycle, with the lowest levels in proestrus and peak expression values on diestrous d 1, i.e. during the luteal phase of the cycle. Ghrelin immunoreactivity was predominantly located in the luteal compartment of the ovary; with intense immunostaining being detected in steroidogenic cells from corpus luteum of the current cycle as well as in all generations of regressing corpora lutea. Indeed, predominant expression of ghrelin in the corpus luteum was confirmed using a pseudopregnant rat model, where maximum ghrelin mRNA levels were detected in dissected luteal tissue. To note, the cyclicity in the profile of ovarian expression of ghrelin appeared to be tissue specific, as it was not detected in the stomach, nor was it observed in terms of circulating ghrelin levels. In addition, cyclic expression of ovarian ghrelin mRNA was disrupted by blockade of the preovulatory gonadotropin surge and ovulation by means of administration of a potent GnRH antagonist. Finally, ghrelin mRNA expression was persistently detected in rat ovary throughout pregnancy, with higher levels in early pregnancy and lower expression during the later part of gestation. In conclusion, our data provide novel evidence for the expression of ghrelin in the cyclic and pregnant rat ovary. Dynamic changes in the profile of ghrelin expression were detected during the estrous cycle and throughout pregnancy, thus suggesting a precise regulation of ovarian expression of ghrelin. Overall, our present findings may represent an additional link between body weight homeostasis and female reproductive function.     

Localization of liver-type lipase in rat ovaries and its activity during the estrous cycle and lactation

The conditions for an in vitro assay of liver-type lipase, i.e. an enzyme resembling the lipase releasable from the liver by heparin (liver lipase), in rat ovaries were established. The liver-type lipase activity in the ovaries was almost completely (greater than 95%) located in the corpora lutea and its activity ranged from 0.44 to 0.77 mU per corpus luteum of (pseudo)pregnant rats. Preovulatory ovarian follicles contained very low lipase activity. During the estrous cycle the pattern of lipase activity was similar to that of serum progesterone levels (maximal at diestrus 1 and minimal at diestrus 2). In the individual rats liver-type lipase activity in the ovaries was strongly correlated with serum progesterone and 20 alpha-hydroxyprogesterone. The activity of liver-type lipase also varied during lactation. It was relatively low at an early stage (2-3 days) but increased during later stages of lactation. The serum progesterone level was relatively low in rats lactating for 2-3 or 22-24 days. During the intervening time, its concentrations was elevated. Since serum 20 alpha-hydroxyprogesterone levels varied inversely to progesterone, the total amount of progestagens in blood during lactation remained constant. The cholesterol content of the corpora lutea of the lactating rats was initially high and decreased during the lactation.     

Leptin and leptin receptor expression in the rat ovary

Leptin is an important satiety hormone and reproductive regulator and is found, along with its receptors, throughout the ovary. To date, the changes in ovarian expression of both of these proteins throughout the estrous cycle has not been studied, and the examination of protein expression has not distinguished between different forms of the receptor. In this study leptin mRNA expression in the immature gonadotropin-primed rat ovary increased 3-fold after human chorionic gonadotropin administration, followed by a dramatic increase in mRNA for both the short form (Ob-Ra) and the long form (Ob-Rb) of the leptin receptor (approximately 8- and 7-fold, respectively). A corresponding increase in mRNA expression of the receptor was not observed in isolated preovulatory follicles. Using immunohistochemistry, we observed protein expression of the long form of the leptin receptor (Ob-Rb) in the ovary, with high intensities observed in oocytes and endothelial cells as well as thecal cells and corpora lutea. These results suggest that ovarian expression of leptin and its receptor is regulated across the cycle by gonadotropins, with peak expression at ovulation, indicating a possible involvement in oocyte maturation, angiogenesis, follicle rupture, or subsequent corpus luteum formation.     

Dynamic regulation of mouse ovarian stanniocalcin expression during gestation and lactation

Stanniocalcin is a glycoprotein hormone that appears to play a paracine/autocrine role in several mammalian tissues. Recently studies have shown that stanniocalcin is highly expressed in the ovaries of mice and humans and we have investigated its expression in the mouse ovary during several physiological states to identify potential functional relationships. During postnatal development the pattern of stanniocalcin (STC) gene expression begins to become thecal-restricted as early as day 5 and achieves the adult pattern of expression by two weeks of age. During postnatal development the primary sites of STC protein localization are the theca and oocytes and after maturation it is also strongly concentrated in the corpora lutea. Over the estrous cycle the pattern of both STC gene expression and protein localization do not show dramatic changes though STC immunoreactivity (STCir) staining appears to be greatest during metestrus I. In the superovulation model, however, we observed a significant increase in STC messenger RNA (mRNA) levels after treatment with hCG implying regulation by LH. During gestation the expression of ovarian STC increases 15-fold and is localized to the theca-interstitial cells with lower expression also being found in the corpora lutea. STC also becomes detectable in the serum for the first time suggesting an endocrine role for STC during gestation. Interestingly, the presence of a nursing litter appears to up-regulate STC gene expression in lactating mice suggesting a role for ovarian STC in lactation. Also striking is the intense STCir staining found in oocytes as they are devoid of STC mRNA, thus implying a role for STC in oocyte maturation. Stanniocalcin, to our knowledge, is unique because no other secreted proteins produced by the ovarian thecal-interstitial compartment are significantly induced during mouse pregnancy. In summary, our data provide evidence for the active regulation of STC expression in the ovary during gestation and lactation and therefore implies that STC is a new regulator of the gestational and nursing state.     

Differential expression of heparin-binding epidermal growth factor-like growth factor in the rat ovary

We investigated the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and its receptors in the rat ovary to define the role of HB-EGF in the ovarian function. The expression pattern of HB-EGF mRNA and protein were studied by semi-quantitative RT-PCR and immuno-histochemistry using an antibody that was specifically stained for the precursor form of HB-EGF in naturally cycling rats and immature pseudo-pregnant rat models. The immuno-histochemical study showed that in naturally cycling rats, HB-EGF was expressed in most granulosa cells of early follicles and all the developing follicles but not in preovulatory follicles. This was supported by the semi-quantitative RT-PCR results in that the lowest level of HB-EGF mRNA during the estrous cycle was found in the evening of proestrous when the HB-EGF negative preovulatory follicles were most prominent. The results suggest that HB-EGF might be a mitogen for granulosa cells and down regulation of its expression may be necessary for the final maturation of follicles. In corpora lutea, luteal cells of older generation stained stronger than those of younger generation. Moreover, luteal cells of late luteal phase stained stronger than those of the mid and early luteal phases in the immature pseudo-pregnant rat models, indicating that the precursor form may be associated with death of luteal cells. Finally, of the two cognate receptors for HB-EGF, erbB1 was expressed in the rat ovary, but erbB4 was specifically not expressed in this organ. The spatial and temporal pattern of HB-EGF expression suggest that HB-EGF may an important local regulator of ovarian function and structure.     

Changes in 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase messenger ribonucleic acid, activity and protein levels during the estrous cycle in the bovine ovary

A 1169 base pair fragment of bovine 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) cDNA was used to quantitate 3 beta-HSD messenger RNA (mRNA) levels in the bovine ovary during the estrous cycle. The content of 3 beta-HSD protein was measured by immunoblot analysis using an antiserum developed in rabbits against human 3 beta-HSD, whereas 3 beta-HSD activity was measured using [3H]pregnenolone, [3H] dehydroepiandrosterone, and [3H]androst-5-ene-3 beta,17 beta-diol as substrates. There was a parallel increase in 3 beta-HSD mRNA, protein content, and enzymatic activity levels from days 1-3 after estrus to maximal values at 50-100% above control on days 8-11 after estrus. Thereafter, all values decreased progressively until days 16-17 before a dramatic fall to 5% or less than maximal values on days 18-20 after estrus. Almost superimposable results of enzymatic activity were obtained with the three substrates, thus suggesting a unique 3 beta-HSD or parallel changes in the activity of multiple 3 beta-HSDs. The above-described changes observed during the luteal phase are almost exclusively due to variations in corpora lutea. In fact, 3 beta-HSD activity in ovarian follicles was approximately 10,000 lower than that measured in corpora lutea. The close correlation observed over a wide range of 3 beta-HSD mRNA, protein content, and activity levels suggests that changes of ovarian 3 beta-HSD activity are controlled at the level of 3 beta-HSD gene expression and/or 3 beta-HSD mRNA stability.     

The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy.

The hormonal factors associated with converting a corpus luteum of estrous cycle into a corpus luteum of pseudopregnancy were studied by measuring LH and FSH prolactin, estradiol and progesterone levels in decapitated rats during the 4-day estrous cycle and a comparable time of pseudopregnancy (lights on 0600-0800 hr.). During the estrous cycle, prolactin, LH and FSH remained low and unchanging except on the afternoon of proestrus, when typical proestrous surges were observed. In contrast, estradiol levels began to increase on D-1, from baseline values of 7 pg/ml to approximately 15-20 pg/ml. These levels were maintained until the afternoon of D-2 when estradiol further increased to reach peak levels of 40-50 pg/ml by 0900 hr on proestrus. Estradiol then declined in relation to the increase in LH secreation and had returned to baseline by estrus. Progesterone secretion by the corpora lutea of the cycle also increased on the afternoon of D-1 and reached a maximum value of 25-30 ng/ml early on the morning of D-2. At this time, a precipitious fall in progesterone occurred, returning to baseline values of 5-1- ng/ml by 0700 on D-2 signifying the regression of the corpora lutea of the cycle. Progesterone remained low thereafter until the afternoon of proestrus when levels increased in response to the proestrus when levels increased in response to the proestrous surge of LH. Following cervical stimulation at 1900 hr on proestrus, no differences were noted, with respect to the estrous cycle, in LH, FSH or estradiol secreation through the afternoon of D-2. Surprisingly, progesterone levels did not differ in the cycle and pseudopregnancy until the early morning of D-29 instead of progesterone levels falling to baseline as they had during the cycle, the corpora lutea of pseudopregnancy were rescused, progesterone increasing dramatically to reach levels of 45-50 ng/ml by 1700 hr on that same day. The only difference in hormone secretion that was noted which could account for this marked divergence in progesterone secretion was the pattern of prolactin secretion following cervical stimulation. In contrast to the low levels seen during the estrous cycle, biphasio surges of prolactin secretion occured each day, one being nocturnal (0100-0900 hr) and the other diurnal (1500-2100 hr). The rescue of the corpus luteum occured in association with the nocturnal surge on D-2. These results suggest that nocturnal surge on D-2, PROLACTIN IS THE MAJOR Luteotropic stimulus which transforms and estrous cycle into pseudopregnancy by prolonging progesterone secretion from the corpus luteum. Moreover, if LH is important for progesterone secretion, no changes were observed in the pattern of LH secretion which can account for the rescue of the corpus luteum.     

Intraovarian excess of nerve growth factor increases androgen secretion and disrupts estrous cyclicity in the rat

A single injection of estradiol valerate induces a form of cystic ovary resembling some aspects of the human polycystic ovarian syndrome. Preceding the development of follicular cysts, there is an increase in intraovarian synthesis of nerve growth factor (NGF) and the low affinity NGF receptor (p75 NGFR). Selective blockade of NGF actions and p75 NGFR synthesis in the ovary restored estrous cyclicity and ovulatory capacity in estradiol valerate-treated rats, suggesting that an increase in NGF-dependent, p75 NGFR-mediated actions within the ovary contributes to the development of cystic ovarian disease. We have tested this hypothesis by grafting NGF-producing neural progenitor cells into the ovary of juvenile rats that have been induced to ovulate precociously by a single injection of PMSG. The NGF-producing cells, detected by their content of immunoreactive p75 NGFR material, were found scattered throughout the ovary with some of them infiltrating the granulosa cell compartment of large, precystic follicles. Ovarian NGF content was 2-fold higher than in the ovary of rats receiving control cells. Estrous cyclicity was disrupted, with the animals showing prolonged periods of persistent estrus, and an almost continuous background of vaginal cornified cells at other phases of the estrous cycle. Morphometric analysis revealed that the presence of NGF-producing cells neither reduced the total number of corpora lutea per ovary nor significantly increased the formation of follicular cysts. However, the ovaries receiving these cells showed an increased incidence of precystic, type III follicles, accompanied by a reduced number of healthy antral follicles, and an increased size of both healthy and atretic follicles. These changes in follicular dynamics were accompanied by a selective increase in serum androstenedione levels. The results show that an abnormally elevated production of NGF within the ovary suffices to initiate several of the structural and functional alterations associated with the development of follicular cysts in the rat ovary.     

Expression of the GATA-4 and GATA-6 transcription factors in the fetal rat gonad and in the ovary during postnatal development and pregnancy

Immunohistochemical studies were undertaken to determine the distribution of GATA-4 and GATA-6 in rat fetal gonad and the postnatal ovary during development and pregnancy. In the undifferentiated gonad, GATA-4 was expressed in the somatic cells of both sexes. After differentiation of the ovary and testis, GATA-4 expression continued in both ovarian and testicular somatic cells; whereas, GATA-6 was expressed in both somatic and germ cells. In the ovary of postnatal rats, granulosa and thecal cells of healthy follicles expressed both GATA factors. In the adult rat, GATA-4 expression was lower in corpora lutea as compared to follicles; whereas, GATA-6 was strongly expressed in both structures. GATA-4 expression was greater in functional corpora lutea than regressing corpora lutea. GATA-6 was expressed in both functional and regressing corpora lutea. In all postnatal ovaries, the expression of P450scc localized with tissue expressing GATA-4 and/or GATA-6, but GATA expression also occurred in P450scc negative cells.     

Gap junctional proteins, connexin 26, 32, and 43 in sheep ovaries throughout the estrous cycle

Ovarian follicles from days 13, 14, 15, and 16 and corpora lutea (CL) from days 2, 4, 8, 12, and 15 of the estrous cycle were evaluated for the presence of connexins by immunohistochemistry. In addition, CL from days 5, 10, and 15 of the estrous cycle were used for immunofluorescent detection of Cx43 followed by image analysis, and for Western immunoblot. In all tissues, staining for all connexins appeared punctate, indicating the presence of assembled gap junctions. Cx26 was present in the ovarian surface epithelium, stroma, and blood vessels within the stroma and hilus, and in the CL. In healthy antral follicles, Cx26 was present only in the theca layer, whereas Cx43 was present in granulosa and theca layers. In the majority of atretic follicles, connexins were not detected, but in 13% of the atretic follicles, Cx43 was present in the theca layer. Cx32 was detected in the blood vessels of ovarian stroma and in the CL, and Cx43 was detected in the CL. Localization and/or expression of connexins depended on stage of luteal development. Western analysis demonstrated that expression of Cx32 in luteal tissues was similar across the estrous cycle. The area of positive staining for Cx43 and expression of Cx43 in luteal tissues decreased (p < 0.05) as the estrous cycle progressed. The pattern of expression of connexins indicates that gap junctional proteins may be important in the regulation of folliculogenesis and follicular atresia, as well as growth, differentiation, and regression of the CL.     

Pseudopregnancy in Nectophrynoides occidentalis Angel

Pseudopregnancy in N. occidentalis is the condition obtained by isolating females before ovulation from all males. Ovulation occurs spontaneously in these females and the unfertilized uterine eggs are retained in the maternal uterus for a length of time comparable to that of pregnancy. The ovulated ova leave a lasting trace in the form of corpora lutea. If by their structure, size, and activity the pseudopregnant corpora lutea are comparable to those of pregnancy, the developmental processes of the pseudopregnant ovary are much more rapid than those of the pregnant ovary. Regression of the corpora lutea occurs very early in the cycle and correlatively, oocyte growth is precocious. In consequence, the ovarian cycle lasts only 9 months in pseudopregnant females instead of 12 months as in normal females.