Ovarian mRNA Research Articles

Localization and expression of tissue inhibitor of metalloproteinase-4 in the immature gonadotropin-stimulated and adult rat ovary.

The tissue inhibitors of metalloproteinases (TIMPs) are important regulators of the matrix metalloproteinases (MMPs), proteolytic enzymes essential for controlling the coordinated tissue remodeling that takes place in the ovary. In the present study, we characterized the ovarian expression pattern of TIMP-4. The localization of TIMP-4 mRNA was determined by in situ hybridization in adult cycling rats. TIMP-4 mRNA on the day of estrus was expressed in a punctate pattern in stroma and in corpora lutea (CL) from previous cycles but not in newly formed CL or follicles. At metestrus, TIMP-4 mRNA was present in certain CL from the current and previous cycles and continued to exhibit a punctate pattern of expression in the stroma. By diestrus, TIMP-4 mRNA was detected in the thecal layer surrounding follicles, and a relatively high level of expression was observed in a punctate pattern within new and previous CL and in the stroma. TIMP-4 mRNA was also observed in the thecal layer at proestrus, but the punctate pattern within CL and stroma was absent. To correlate the changes in cellular localization with changes in overall TIMP-4 levels, ovarian mRNA and protein levels were examined in adult cycling rats and in gonadotropin-stimulated immature rats. In cycling rats, there was no change in mRNA or protein levels across the cycle, although there was a trend towards higher levels during estrus (P = 0.08). In gonadotropin-treated rats, there was an increase in TIMP-4 mRNA 48 h after eCG administration with a corresponding doubling of TIMP-4 protein. Although TIMP-4 mRNA and protein tended to decline after hCG treatment, this trend was not significant (P = 0.08). These findings indicate that TIMP-4 could play an important role in regulating MMPs in a localized manner in follicles and CL throughout the cycle.

Identification of growth hormone receptor in the ovary of tilapia, Oreochromis mossambicus

Growth hormone receptor (GH-R) cDNA was isolated and characterized in the tilapia, Oreochromis mossambicus. GH-R mRNA was expressed in the nucleus and cytoplasm of the young oocytes and in the follicles surrounding vitellogenic oocytes showing a correlation with ovarian IGF-I mRNA levels. However, no change was seen in liver GH-R/IGF-I mRNA levels or plasma GH/IGF-I levels during ovarian development, suggesting that GH/IGF-I axis may be involved in the ovarian development in paracrine or autocrine manner, independent of liver derived IGF-I.

Vascular endothelial growth factor receptor-2 activation induces vascular permeability in hyperstimulated rats, and this effect is prevented by receptor blockade.

The existence of a vasoactive molecule released in response to hCG is believed to be the main feature in the development of ovarian hyperstimulation syndrome (OHSS) in women, and vascular endothelial growth factor (VEGF) is the main candidate as the human chorionic gonadotropin (hCG) mediator. This study was conducted to investigate the role of VEGF in increasing vascular permeability (VP) in vivo, a characteristic of OHSS. We analyzed the source and specific isoforms of VEGF involved and developed strategies to reverse increased VP in hyperstimulated rats targeting the VEGF system. Ovarian hyperstimulation was induced with pregnant mare's serum gonadotropin, or pregnant mare's serum gonadotropin plus hCG. Time-course experiments analyzed VP and the expression of whole VEGF mRNA in the mesentery and the ovaries. VP and ovarian mRNA VEGF expression increased to peak values after 48 h. No significant change in expression was observed in the mesentery. To further prove the ovarian origin of VEGF, we showed that VP was not altered when ovariectomized rats were treated with gonadotropins. The ovary expressed VEGF(120) and VEGF(164) isoforms. Immunohistochemistry showed VEGF in granulosa and zona pellucida of preovulatory and atretic follicles and in granulosa-lutein and endothelial cells of whole corpus luteum. A specific VEGF receptor-2 inhibitor (SU5416) was administered in three different protocols: on a daily basis, every 48 h, or two injections after hCG. Increased VP was reversed when SU5416 was administered every 48 h or two injections after hCG. These results show that the ovary is the main source of VEGF(120) and VEGF(164), which act through the VEGF receptor-2 to increase VP, and provide new insights into the prevention of OHSS.

Neonatal exposure to genistein induces estrogen receptor (ER)alpha expression and multioocyte follicles in the maturing mouse ovary: evidence for ERbeta-mediated and nonestrogenic actions.

Outbred CD-1 mice were treated neonatally on Days 1-5 with the phytoestrogen, genistein (1, 10, or 100 micro g per pup per day), and ovaries were collected on Days 5, 12, and 19. Ribonuclease protection assay analysis of ovarian mRNA showed that estrogen receptor beta (ERbeta) predominated over ERalpha in controls and increased with age. Genistein treatment did not alter ERbeta expression, however, ERalpha expression was higher on Days 5 and 12. ERbeta was immunolocalized in granulosa cells, whereas ERalpha was immunolocalized in interstitial and thecal cells. Genistein treatment caused a dramatic increase in ERalpha in granulosa cells. Genistein-treated ERbeta knockout mice showed a similar induction of ERalpha, which is seen in CD-1 mice, suggesting that ERbeta does not mediate this effect. Similar ERalpha induction in granulosa cells was seen in CD-1 mice treated with lavendustin A, a tyrosine kinase inhibitor that has no known estrogenic actions, which suggests that this property of genistein may be responsible. As a functional analysis, genistein-treated mice were superovulated and the number of oocytes was counted. A statistically significant increase in the number of ovulated oocytes was observed with the lowest dose, whereas a decrease was observed with the two higher doses. This increase in ovulatory capacity with the low dose coincided with higher ERalpha expression. Histological evaluations on Day 19 revealed a dose-related increase in multioocyte follicles (MOFs) in genistein-treated mice. Tyrosine kinase inhibition was apparently not responsible for MOFs because they were not present in mice that had been treated with lavendustin; however, ERbeta must play a role, because mice lacking ERbeta showed no MOFs. These data taken together demonstrate alterations in the ovary following neonatal exposure to genistein. Given that human infants are exposed to high levels of genistein in soy-based foods, this study indicates that the effects of such exposure on the developing reproductive tract warrant further investigation.

Estrous cycle-regulated expression of CYP1B1 mRNA in the rat ovary

CYP1B1, a member of the cytochrome P450 superfamily, is expressed constitutively in the steroidogenic tissues of mammals and is inducible by peptide hormones, cAMP and aromatic hydrocarbon receptor (AHR) ligands. The mechanism of induction of this cytochrome P450 is similar to that for CYP1A1, i.e. through the aromatic hydrocarbon receptor (AHR) signaling pathway. We have recently reported that CYP1B1, but not CYP1A1, is expressed in rat granulosa cells (GC) in the absence of any external stimulus. The induction of CYP1B1 mRNA in rat GC by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in vitro was followed by an increase in AHR and estrogen receptor (ER-β) RNA levels. Estrous cycle-dependent expression of AHR, AHR-nuclear translocator (ARNT) and ER-mRNAs in the rat ovary was reported. We suggest that CYP1B1 may play a major role in the regulation of rat ovarian function/cycle but until now this has been unexplored experimentally. The present study was therefore aimed at examining the expression of CYP1A1, CYP1B1 and ER-mRNA in rat ovarian tissues throughout the estrous cycle to establish any correlation in the expressions of these mRNAs in rat ovary. Total RNA was extracted from the ovary and liver of cycling adult rats and the mRNAs were analyzed using relative RT-PCR with gene-specific primers for the target mRNA and for RPL 19 or S16 primers as an internal control. The results indicated that in the ovary, CYP1B1 mRNA increased significantly on the evening of proestrus and dramatically decreased on the morning of estrus, while ER-mRNA remained unaltered throughout the estrous cycle. CYP1A1 mRNA in the ovary and both CYP1A1 and CYP1B1 mRNAs in the liver were undetectable. That the sudden decrease of ovarian CYP1B1 mRNA on the morning of estrus was not an effect of the LH surge was verified in vitro using our short-term GC culture model. GC prepared from rats super-stimulated with equine chorionic gonadotropin (eCG) were cultured for 6 h with or without LH and TCDD. It was observed that both CYP1A1 and CYP1B1 mRNAs were induced by TCDD with no apparent effect of LH. It is suggested that the high level of CYP1B1 mRNA expression on the evening of proestrus in rat ovary might be involved in metabolism of estrogens to catecholestrogen (a known effect of CYP1B1), and that expression is unaffected in GC by LH.

Regulation of expression of ovarian mRNA encoding steroidogenic enzymes and gonadotrophin receptors by FSH and GH in hypogonadotrophic cattle

A study was conducted to determine the effects of FSH and bovine somatotrophin on the expression of mRNA encoding the gonadotrophin receptors and steroidogenic enzymes in ovarian follicles of cattle rendered hypogonadotrophic by treatment with a GnRH agonist. Hereford x Friesian heifers were allotted into two pretreatment groups: controls (n = 10) and GnRH agonist-treated (n = 20). Ovaries of control cows were removed on day 2 of the first follicular wave after synchronized oestrus. GnRH agonist-treated heifers were given either FSH or no FSH. FSH was infused at 50 microg h(-1) for 48 h. Ovaries in GnRH agonist-treated heifers were removed at the end of exogenous hormone treatment. The control, GnRH agonist and GnRH agonist plus FSH treatment groups were divided further into bovine somatotrophin or no bovine somatotrophin treatments (n = 5 per treatment). Bovine somatotrophin (25 mg day(-1) by s.c. injection) was administered for 3 days. Ovaries were scanned once a day by ultrasonography. Blood samples for hormone measurements were collected three times a day from oestrus until the time of removal of ovaries. Expression of mRNAs for the FSH and LH receptors and cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17) and cytochrome P450 aromatase (P450arom) enzymes was localized by in situ hybridization and quantified by image analysis. Ovarian follicular growth was arrested at < or = 4.5 mm in diameter in GnRH agonist-treated heifers. There was no effect of bovine somatotrophin on follicular dynamics, gonadotrophin secretion or expression of mRNA for either the gonadotrophin receptors or steroidogenic enzymes. Infusion of FSH to GnRH agonist-treated heifers increased FSH concentrations in serum to the physiological concentrations observed in controls and stimulated growth of follicles to a size similar (5.5-8.0 mm in diameter) to recruited follicles in control cows. FSH induced mRNA expression of P450scc and P450arom in granulosa cells of follicles at a smaller size (< or = 4.5 mm in diameter) than in controls and increased (P < 0.001) expression in larger (> 4.5 mm in diameter) follicles. Expression of mRNAs for P450scc and P450c17 increased (P < 0.001) with increasing follicle size and was higher (P < 0.01) in theca cells of GnRH agonist plus FSH-treated heifers than in the other groups. There were no treatment differences in expression of FSH receptor in granulosa cells or LH receptor in theca cells, but expression of both receptors increased with follicle size. There was no expression of LH receptor in the granulosa cells of cows from any treatment group. In conclusion, FSH treatment in GnRH agonist-treated heifers induced similar changes in follicular growth to those observed during the first follicular wave, but despite similar peak concentrations, prolonged exposure to high FSH induced precocious expression of mRNAs for P450scc and P450arom in granulosa cells from small follicles and markedly upregulated expression of these enzymes in granulosa cells from recruited follicles. The results of this study demonstrate the key role that FSH plays in the induction of follicular growth and differentiation.

Regulation of expression of ovarian mRNA encoding steroidogenic enzymes and gonadotrophin receptors by FSH and GH in hypogonadotrophic cattle

A study was conducted to determine the effects of FSH and bovine somatotrophin on the expression of mRNA encoding the gonadotrophin receptors and steroidogenic enzymes in ovarian follicles of cattle rendered hypogonadotrophic by treatment with a GnRH agonist. Hereford x Friesian heifers were allotted into two pretreatment groups: controls (n = 10) and GnRH agonist-treated (n = 20). Ovaries of control cows were removed on day 2 of the first follicular wave after synchronized oestrus. GnRH agonist-treated heifers were given either FSH or no FSH. FSH was infused at 50 microg h(-1) for 48 h. Ovaries in GnRH agonist-treated heifers were removed at the end of exogenous hormone treatment. The control, GnRH agonist and GnRH agonist plus FSH treatment groups were divided further into bovine somatotrophin or no bovine somatotrophin treatments (n = 5 per treatment). Bovine somatotrophin (25 mg day(-1) by s.c. injection) was administered for 3 days. Ovaries were scanned once a day by ultrasonography. Blood samples for hormone measurements were collected three times a day from oestrus until the time of removal of ovaries. Expression of mRNAs for the FSH and LH receptors and cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17) and cytochrome P450 aromatase (P450arom) enzymes was localized by in situ hybridization and quantified by image analysis. Ovarian follicular growth was arrested at < or = 4.5 mm in diameter in GnRH agonist-treated heifers. There was no effect of bovine somatotrophin on follicular dynamics, gonadotrophin secretion or expression of mRNA for either the gonadotrophin receptors or steroidogenic enzymes. Infusion of FSH to GnRH agonist-treated heifers increased FSH concentrations in serum to the physiological concentrations observed in controls and stimulated growth of follicles to a size similar (5.5-8.0 mm in diameter) to recruited follicles in control cows. FSH induced mRNA expression of P450scc and P450arom in granulosa cells of follicles at a smaller size (< or = 4.5 mm in diameter) than in controls and increased (P < 0.001) expression in larger (> 4.5 mm in diameter) follicles. Expression of mRNAs for P450scc and P450c17 increased (P < 0.001) with increasing follicle size and was higher (P < 0.01) in theca cells of GnRH agonist plus FSH-treated heifers than in the other groups. There were no treatment differences in expression of FSH receptor in granulosa cells or LH receptor in theca cells, but expression of both receptors increased with follicle size. There was no expression of LH receptor in the granulosa cells of cows from any treatment group. In conclusion, FSH treatment in GnRH agonist-treated heifers induced similar changes in follicular growth to those observed during the first follicular wave, but despite similar peak concentrations, prolonged exposure to high FSH induced precocious expression of mRNAs for P450scc and P450arom in granulosa cells from small follicles and markedly upregulated expression of these enzymes in granulosa cells from recruited follicles. The results of this study demonstrate the key role that FSH plays in the induction of follicular growth and differentiation.

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.

Aberrant expression of growth differentiation factor-9 in oocytes of women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a major cause of female infertility. Despite substantial effort, the etiology and pathogenesis of PCOS and polycystic ovaries (PCO) in women remain unknown. Recent studies in laboratory animals have documented a link between dysfunction of two oocyte growth factors, growth differentiation factor-9 (GDF-9) and bone morphogenetic factor-15 (BMP-15), and aberrant folliculogenesis. Because aberrant follicle development is a hallmark of PCOS, we wondered whether the expression patterns of these growth factors might be disrupted in PCOS and PCO oocytes. To address this issue, we compared the pattern and level of expression of GDF-9 and BMP-15 mRNA in ovaries from normal cycling (n = 12), PCOS (n = 5), and PCO (n = 7) patients. In situ hybridization studies showed that the expression of GDF-9 and BMP-15 is restricted to the oocytes in all ovaries examined. Interestingly, a decreased level of GDF-9 signal was observed in developing PCOS and PCO oocytes, compared with normal. This difference was evident throughout folliculogenesis, beginning at recruitment initiation and continuing through the small Graafian follicle stage. By contrast, there were no qualitative or quantitative changes in the expression of BMP-15 mRNA in PCOS oocytes during folliculogenesis. There were also no significant differences between normal and PCOS and PCOs in the levels of the mRNA encoding the housekeeping gene, cyclophilin. Together, these results indicate that the expression of GDF-9 mRNA is delayed and reduced in PCOS and PCO oocytes during their growth and differentiation phase. Because oocyte-derived GDF-9 is crucial for normal folliculogenesis and female fertility, we suggest that a dysregulation of oocyte GDF-9 expression may contribute to aberrant folliculogenesis in PCOS and PCO women.

Distinct expression of endothelin receptor subtypes A and B in luteinized human granulosa cells.

Endothelins (ET) are potent vasoconstrictive peptides originally isolated from vascular endothelial cells. Their biological effects are mediated through two different receptors, the endothelin-1 (ET-1)-selective endothelin receptor subtype ETA and the non-selective receptor subtype ETB. ET-1 protein has been found in human ovarian follicular fluid and ET-1 mRNA expression has been demonstrated in ovarian tissue. These findings indicate that the endothelin-system participates in the modulation of ovarian function, probably acting in an autocrine/paracrine manner. In the current study we used freshly aspirated, luteinized human granulosa cells (hGC) representing an in vitro model of the early corpus luteum. By means of RT-PCR and immunocytochemistry we investigated whether luteinized human granulosa cells express ETA and ETB receptors. Specific amplification products of ETA transcripts were detected in all samples investigated. In contrast, only after using a three-fold amount of ETB reverse transcripts we were able to demonstrate specific, but weak amplification products. In addition, immunocytochemical staining for ETA but not for ETB was found in granulosa cell preparations. The present study provides clear evidence that human granulosa cells predominantly express ETA receptor subtype mRNA and protein hinting to its possible role in follicle maturation and corpus luteum formation.

Expression and regulation of gonadotropin‐re leasing hormone (GNRH) and its receptor MRNA transcripts during the mouse ovarian development

The present study examines the expression and regulation of gonadotropin‐releasing hormone (GnRH) and its receptor (GnRH‐R) mRNA levels during mouse ovarian development. A fully processed, mature GnRH mRNA together with intron‐containing primary transcripts was expressed in the immature mouse ovary as determined by Northern blot analysis and reverse transcription‐polymerase chain reaction (RT‐PCR). The size of ovarian GnRH mRNA was similar to that of hypothalamus, but its amount was much lower than that in the hypothalamus. Quantitative RT‐PCR procedure also revealed the expression of GnRH‐R mRNA in the ovary, but the estimated amount was a thous and‐fold lower than that in the pituitary gland. We also examined the regulation of ovarian GnRH and GnRH‐R mRNA levels during the follicular development induced by pregnant mare's serum gonadotropin (PMSG) and/or human chorionic gonadotropin (hCG). Ovarian luteinizing hormone receptor (LH‐R) mRNA was abruptly increased at 48 h after the PMSG administration and r...

Quantification of rainbow trout (Oncorhynchus mykiss) estrogen receptor-alpha messenger RNA and its expression in the ovary during the reproductive cycle.

This study developed a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method to measure estrogen receptor-alpha (ERalpha) mRNA in the rainbow trout (Oncorhynchus mykiss). Using RT-PCR, and primers based on the known ERalpha DNA sequence in this species, cDNA sequences representing most of the protein coding region were obtained from ovary poly A(+) RNA. Using these DNA sequences as probes in Northern blot hybridizations confirmed that a single transcript of 4.2 kilobases in poly A(+) RNA could be detected in liver and ovary RNA. For the quantitative RT-PCR assay an internal standard RNA molecule was produced to control for inherent inter-tube differences in amplification efficiency and permit accurate quantification of ERalpha mRNAs. The quantitative RT-PCR assay proved to be highly specific for ERalpha mRNA with a detection limit of 6.9 fg, which corresponds to 273 fg ERalpha mRNA/microg total RNA. The quantitative RT-PCR assay was used to measure the levels of ERalpha mRNA in ovaries of rainbow trout at different stages of reproductive development. Ovarian ERalpha mRNA expression was found during two distinct periods of reproductive development, in pre-vitellogenic ovaries of fish with ovarian follicle diameters (OFDs) </=100 microm and in mid-vitellogenic ovaries with OFDs >1000 microm. ERalpha mRNA could not be detected in the ovaries of fish with OFDs >100 microm but </=1000 microm. The highest levels of ERalpha mRNA were found in late vitellogenic ovaries of fish with OFDs >2000 microm.

Ovarian steroidogenic responsiveness to exogenous gonadotropin stimulation in young and middle-aged female rats.

Reproductive aging in the female rat is associated with gradual declines in LH secretion and ovarian progesterone (P) production. This study examined whether the influences of aging on P levels reflect decreased ovarian responsiveness to gonadotropin stimulation, as opposed to changes in gonadotropin release. Young and middle-aged regularly cyclic female rats received sodium pentobarbital to block endogenous proestrous luteinizing hormone (LH) surges, followed by administration of various doses of human chorionic gonadotropin (hCG). Similar treatments were performed in middle-aged acyclic persistent-estrous (PE) females. Injection of hCG resulted in equivalent plasma hCG levels in each treatment group. At the lowest hCG dose tested, a significant rise in plasma P levels was observed in middle-aged cyclic rats, but not in young cyclic or middle-aged PE females. This unexpected finding may reflect accelerated follicular development in middle-aged cyclic females, as suggested by a previous study. At the intermediate dose, young and middle-aged cyclic but not PE rats displayed significantly increased P in response to hCG. At the highest dose tested, all three groups of rats displayed increased P levels after hCG stimulation. However, P concentrations were significantly lower in middle-aged PE than regularly cyclic females. Northern and slot blot hybridization analyses revealed that ovarian mRNA levels for cytochrome P450 side-chain cleavage, the rate-limiting enzyme in P synthesis, were markedly reduced in PE rats following hCG stimulation. These findings indicate that ovarian responsiveness to gonadotropin stimulation is impaired in middle-aged PE, but not regularly cyclic rats, and suggest influences of cycle status on the biochemical and molecular mechanisms regulating ovarian steroid production. Furthermore, these findings reveal that attenuated P production in middle-aged proestrous rats is due to attenuated preovulatory LH surges, rather than decreased ovarian sensitivity to LH.

A Rapidly Diverging EGF Protein Regulates Species-Specific Signal Transduction in Early Sea Urchin Development

The macromolecules mediating species-specific events during fertilization and early development and their molecular evolution are only beginning to be understood. We screened sea urchin ovary mRNA for species-specific gene products using representational differential analysis to identify unique transcripts in Strongylocentrotus franciscanus that are absent or divergent from a closely related species, S. purpuratus. One of the transcripts identified by this screening process is SfEGF-II, which contains four EGF repeats. SfEGF-II is orthologous to the previously reported genes S. purpuratus SpEGF-II and Anthocidaris crassispina AcEGF-II, encoding exogastrulation-inducing peptides (EGIP). EGF peptides derived from EGIP induce exogastrulation, a classical developmental defect, when added to embryos prior to gastrulation. The first three EGF repeats (EGF1–3) share 50 to 60% identity among the three species, but the fourth repeat (EGF4) is more divergent, displaying only 30% identity. Analysis of the sequence divergence indicates that the EGF-II genes display a relatively high nonsynonymous-to-synonymous ratio, a significant excess of radical compared to conservative amino acid substitutions, and a lack of polymorphism within SfEGF-II, indicating that these genes have been subjected to positive Darwinian selection. Recombinant EGF3 from S. franciscanus induces exogastrulation in both S. franciscanus and S. purpuratus. In contrast, recombinant EGF4 from both S. franciscanus and S. purpuratus induces exogastrula in a species-specific manner. In hybrid embryos, both species of EGF4 induce exogastrulation, suggesting that the receptor for this EGF molecule is expressed from both parental genomes during development. Both EGF3 and EGF4 induce the phosphorylation of membrane proteins of the blastula stage embryos, but EGF4 stimulates phosphorylation of proteins only in membranes prepared from homologous embryos, suggesting that it utilizes a unique pathway involving a species-specific receptor for EGF4. Thus, species-specific events of gastrulation and early development may be controlled by these rapidly diverging EGF molecules, through a novel species-specific signal transduction pathway.

Two estrogen receptors expressed in the teleost fish, Sparus aurata: cDNA cloning, characterization and tissue distribution

Estrogen is an essential hormone for many reproductive and non-reproductive functions. The function of estrogen in the reproductive cycle of seabream (Sparus aurata), a protandrous hermaphrodite teleost fish, is complex but it is understood to be involved in sex inversion, a process that occurs in some individuals during the second reproductive season. Estrogen action is mediated by two estrogen receptor (ER) subtypes designated alpha and beta. As a step to understanding the mechanisms of estrogen action during natural and induced sex reversal in seabream, we have isolated two cDNAs encoding distinct forms of ER homologous to mammalian ERalpha and ERbeta. The seabream ERalpha clone (sbERalpha1), which was truncated in the A/B domain, corresponded to a variant differing in five amino acids from another recently cloned sbERalpha. The ERbeta clone (sbERbeta) encoded a protein 559 amino acids long and showed only 40% identity to sbERalpha. Northern blot analysis of liver and ovary mRNA indicated the presence of several transcripts of the two receptor subtypes. PCR analysis showed that the two receptors differed in their expression pattern. sbERalpha had a more restricted distribution, occurring mainly in testis, liver and heart, and sbERbeta was present in most tissues, being more abundant in ovary, testis, liver, intestine and kidney. The presence in seabream of two ERs with several ER transcripts and their pattern of distribution are consistent with the widespread effects of estrogen in different tissues.

Expression of β-Amyloid Precursor Protein in the Porcine Ovary

Abstract cDNA cloning and 5′-RACE experiments were conducted on β-amyloid precursor protein (APP) using porcine ovary mRNA. The isolated cDNA clone and the clones generated by 5′RACE spanned 3,051 bp containing the complete open reading frame of APP which consisted of 770 amino acid residues. The amino acid sequence of porcine APP was 97.8, 97.1, and 97.4% homologous to those of human, mouse, and guinea pig APPs, respectively. The expression of APP in several porcine tissues was examined by Northern blot analysis. The ovaries and adrenal glands showed a strong expression of the APP mRNA, as did the granulosa cells from small and large follicles of the porcine ovary. RT-PCR analyses using two primer sets revealed that the porcine ovary expressed at least four types of APP mRNAs. Western blot analysis was conducted using the extract of granulosa cells and the fluid of ovarian follicles, and the results indicated that the follicular fluid contained soluble APP in relatively high content. These results sugges...

Expression of adrenomedullin in the human corpus luteum

Objective: Adrenomedullin (AM) is a potent hypotensive peptide found in human pheochromocytoma tissue. In the present study, the expression of AM mRNA in the human ovary was examined. Design: Ovarian mRNA was analyzed in the follicle, the corpus luteum of mid-luteal phase, and early pregnancy. Setting: Gunma University School of Medicine, Gunma, Japan. Patient(s): Premenopausal women with histologically normal ovary who were undergoing salpingoophorectomy. Intervention(s): The dominant follicle and corpora lutea were isolated and total RNA was extracted from these tissues. Main Outcome Measure(s): Northern blot analysis of AM, receptor activity–modifying protein 2 (RAMP2), and LH/hCG receptor mRNA in human samples. Result(s): An AM mRNA transcript of 1.6 kilobases (kb) was detected in corpus luteum tissue; this transcript was identical to that which has been detected in placenta and fetal membrane. The AM and LH/hCG receptor mRNA levels were low in the mature follicle but increased in the corpus luteum of the mid-luteal phase and were maintained during early pregnancy. A single transcript of 0.8 kb for RAMP2 was also seen in the follicle and corpus luteum, the level of RAMP2 mRNA was relatively high in the preovulatory follicle and RAMP2 was present in the corpus luteum. Conclusion(s): The expression of AM, its receptor, and LH/hCG receptor may be an important component in the process of development and differentiation of the corpus luteum.

Periovulatory and interleukin-1 beta-dependent up-regulation of intraovarian vascular endothelial growth factor (VEGF) in the rat: potential role for VEGF in the promotion of periovulatory angiogenesis and vascular permeability.

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen and permeability factor the role of which in ovarian angiogenesis has been the subject of increasing interest. It was the objective of this communication to explore the possibility that interleukin (IL)-1 may regulate the in vitro expression of rat ovarian VEGF mRNA, as well as to study the in vivo expression of rat ovarian VEGF transcripts during follicular maturation, ovulation, and corpora lutea formation. Taken together, our findings 1) reaffirm the rat ovary as a site of VEGF expression; 2) document an in vivo increase in VEGF transcripts before ovulation; 3) disclose a marked dependence of VEGF on IL-1 beta; 4) reveal the IL-1 beta effect to be receptor mediated and dose and time dependent and to be shared by at least two growth factors--epidermal growth factor and basic fibroblastic growth factor; and 5) demonstrate a lack of VEGF effect on ovarian progesterone biosynthesis as assessed in cultured isolated granulosa cells. It is tempting to speculate that the up-regulatory effect of IL-1 beta on VEGF transcripts may be relevant to the marked angiogenesis and increased vascular permeability displayed by the hyperemic ovarian Graafian follicle during the terminal stages of follicular development. In this context, VEGF may be joined by other IL-1-dependent angiogenesis promoters such as IL-6 or transforming growth factor beta 1. Thus, IL-1-mediated VEGF induction may constitute one of several end points through which IL-1 may coordinate and perhaps amplify the ovulatory cascade.

Characterization of ovarian carbonyl reductase gene expression during ovulation in the gonadotropin-primed immature Rat.

In this differential-display polymerase chain reaction-based study, four different primer sets generated cDNA fragments of ovarian carbonyl reductase genes that were uniquely expressed during the ovulatory process in eCG-primed immature rats. The temporal pattern of expression of this aldo-keto reductase gene was delineated by extracting ovarian RNA at 0, 2, 4, 8, 12, and 24 h after induction of ovulation via injection of the primed animals with hCG. The results showed that at least four homologous forms of this gene were transcribed during ovulation. Northern blot analyses indicated a 14-fold increase in ovarian mRNA for carbonyl reductase, with expression reaching a peak at 8 h after hCG treatment and then declining to negligible levels during the next 16 h. In situ hybridization revealed that most of the transcription was in the thecal connective tissue of the ovary and was absent from the granulosa layer of ovarian follicles. Treatment of the animals with ovulation-blocking doses of epostane (an inhibitor of progesterone synthesis) or indomethacin (an inhibitor of prostanoid synthesis) did not reduce the expression of ovarian carbonyl reductase. Nevertheless, the temporal pattern of expression of carbonyl reductase after the induction of ovulation suggests that this enzyme activity is at least indirectly associated with the ovulatory process.

Three Different Turkey Luteinizing Hormone Receptor (tLH-R) Isoforms I: Characterization of Alternatively Spliced tLH-R Isoforms and Their Regulated Expression in Diverse Tissues1

Using combinations of reverse transcription-polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends, three different, alternatively spliced, partial turkey LH receptor (tLH-R) cDNA isoforms were characterized from ovarian mRNA. The first cDNA (tLH-R(intact)) showed 98% and 72-75% similarity with chicken and mammalian LH-R sequences, respectively. The second cloned cDNA isoform (tLH-R(insert)) contained an in-frame TGA stop codon within an 86-base pair insertion that was located in the extracellular domain of the seven-transmembrane region. The tLH-R(insert) isoform could encode a truncated soluble protein isoform that lacked the transmembrane region. The third cDNA isoform truncated the transmembrane region (tLH-R(trunc)) and was derived by the deletion of the last exon by incomplete splicing. Generation of multiple transcripts by alternative splicing was elucidated by partial characterization of tLH-R genomic sequences. The differentially regulated expression of the tLH-R mRNA isoforms in nongonadal tissues and ovarian stromal tissues during various reproductive stages was quantified and analyzed by Northern blot and/or RT-PCR. Alternatively spliced tLH-R isoforms were differentially expressed in a tissue-specific manner in most of the tissues examined. The steady-state levels of tLH-R mRNA isoforms were relatively high in the hypothalamus and optic nerve and relatively low in the cortex, pituitary, and cerebellum when compared to levels in ovarian follicles. In nongonadal reproductive tissues, the steady-state levels of tLH-R mRNA isoforms were relatively high in the uterus and infundibulum and relatively low in the isthmus, oviduct, and magnum. In addition, in the nongonadal peripheral tissues, the steady-state levels of tLH-R isoforms were relatively high in the thyroid gland and relatively low in the spleen, adrenal gland, kidney, skin, bursa, and muscle. The present study suggests that the alternative splicing of LH-R transcripts occurs in a tissue-specific manner and has been evolutionarily conserved (similar results were obtained in chicken and swine). These results raise fundamental questions as to the function of LH-R isoforms in nongonadal tissues.