Atretic Ovarian Follicles Research Articles

A 13-week subchronic toxicity study of dietary administered saponin-rich and isoflavones-containing soybean extract in F344 rats

A subchronic toxicity study of soybean extract was performed in F344 rats with dietary administration at concentrations of 0%, 1.25%, 2.5% and 5% for 13 weeks. No mortality or abnormal clinical signs in any group were observed. Body weight gains were decreased with a tendency for reduction of feed intake in the 1.25% and above female and 5% male groups. In males, absolute and relative liver weights were increased in the 1.25% and above groups. In females relative kidney weights were increased in the 1.25% and above groups. Other significant changes such as decreased RBC and hematocrit and increased urea nitrogen were detected in the 2.5% and/or 5% groups. On histopathological observation, atrophy of the ventral prostate was observed in all animals in the 5% male group. Mucification and atrophy of the vaginal epithelium and increased atretic follicles in ovaries were noted in 2.5% and 5% female rats. Based on the above findings the lowest-observed-adverse-effect level for male and female rats was estimated to be 1.25% (707.2 and 751.8 mg/kg b.w./day, respectively).

Ultrastructural Features of Atretic Follicles in the Sexually Immature Ostrich (Struthio Camelus)

The aim of this study was to describe the ultrastructural features of atresia in follicles of the immature ostrich (12-14 months old); a ratite that displays seasonal, precocious ovarian activity. The early stage of atresia in primordial, pre-vitellogenic and vitellogenic follicles was characterized by the accumulation of lipid droplets in the granulosa cells. Granulosa cells with condensed cytoplasm and nuclei were a prominent feature during the intermediate phase of atresia. The degenerating follicles were then infiltrated by stroma during the terminal stages of atresia. The results of this study provide further information on the morphology of atretic ovarian follicles in the immature ostrich.

Role of Nitric Oxide on the Generation of Atretic Follicles in the Rat Ovaries

The aim of this study was to investigate the role of Nitric Oxide (NO) in the atresia of ovarian follicles in an animal model. Twenty adult, female rats (90 days old with body weights of210 +/- 10 g in the beginning of the experiments) were divided into 4 groups of 5 each. They were treated twice daily from the subcutaneous route for 21 successive days with either of the following chemicals: nitroglycerine, L-arginine, L-NAME, or saline. On day 22, all animals were sacrificed. Ovaries were dissected out free of connected tissue and were fixed in formaline 10%. Later, paraffine blocks were prepared and serial sections were made by means of H and E routine staining method. Intact and atretic follicles were counted separately. In addition, damages were analyzed qualitatively from the points of view of appearance and morphologic changes. In the evaluation of ovarian follicular structures, different types of healthy as well as atretic follicles were observed. In most of atretic follicles, the oocytes were abnormally elongated and increnation of their outlines were obvious. There were numerous macrophages around and inside of the atretic follicles. Our investigation regarding the distribution of atretic follicles in the ovaries of test groups revealed that atretic follicles in the L-NAME treated group were increased in comparison to the control group. Conversely, however, in the arginine-treated group, the atretic follicles were reduced compared to the control animals. Treatment with nitroglycerine of the rats decreased the number of atretic follicles significantly (p < 0.05) in comparison to the control group. In conclusion, enhanced NO, either from endogenous or exogenous origins, prevents atresia phenomenon, while inhibition of NO exerts an opposite effect.

A 90-day subchronic toxicity study of nivalenol, a trichothecene mycotoxin, in F344 rats

A subchronic toxicity study of nivalenol (NIV), a trichothecene mycotoxin, was conducted in male and female F344 rats fed diet containing 0, 6.25, 25 or 100 ppm concentration for 90 days. Decrease of body weight and loose stools were observed at 100 ppm in both sexes from the start of the experiment, and body weight reduction was also observed at 25 ppm in males from week 6. At necropsy, many organs demonstrated reduced absolute weights at 100 ppm in both sexes, mostly due to the reduction in the body growth, with reduction of relative thymus weight also being evident in females. Hematologically, decrease of the white blood cell count was found at 100 ppm in males and from 6.25 ppm in females. In addition, decreased platelet counts in both sexes, red blood cell counts in males, and the hemoglobin concentration in females were detected at 100 ppm. Histopathologically, treatment-related changes were predominantly observed in the hematopoietic and immune organs and the anterior pituitary in both sexes and female reproductive organs at 100 ppm, such as thymic atrophy, hypocellularity in the bone marrow, diffuse hypertrophy of basophilic cells with increase of castration cells in the anterior pituitary, and increase of ovarian atretic follicles. Based on the hematological data, the no-observed-adverse-effect level of NIV was determined to be less than 6.25 ppm (0.4 mg/kg body weight/day for both males and females).

Fine structural and cytochemical analysis of the processes of cell death of oocytes in atretic follicles in new born and prepubertal rats

The process of cell death of oocytes was studied in atretic ovarian follicles of rats aged from 1 to 28 days using light and electron microscope and cytochemical methods. These methods were TUNEL procedure for DNA breaks, active caspase-3 and lysosome-associated membrane protein 1 (LAMP-1) immunolocalizations. The structural features of the process of oocyte death are mainly characterized by the presence of abundant clear vacuoles and autophagosomes, as well as by the absence of large clumps of compact chromatin associated to the nuclear envelope and apoptotic bodies. These features are common to oocytes in all types of follicles studied. Cytochemical features consisting in positive reactions to TUNEL method, active caspase-3 and LAMP-1 immunolocalizations, are common to the cell death of oocytes in all types of follicles. Particular features of the process of cell death of oocytes are found in different types of follicles. Two morphological patterns of cell death occur in pre-follicular oocytes of the new born and in primordial follicles in 1 to 5 days old rats. One is distinguished by clear nucleoli and moderate compaction of chromatin in clumps frequently resembling meiotic bivalents. The second pattern is characterized by nucleolar condensation and by the absence of compact chromatin. The process of cell death of oocytes in antral follicles is characterized by ribonucleoprotein ribbon-like cytoplasmic structures, pseudo-segmentation, and loss of contact with granulosa cells.

Impaired Regulation of Gonadotropins Leads to the Atrophy of the Female Reproductive System in klotho-Deficient Mice

klotho-Deficient mice exhibit a syndrome resembling human premature ageing, with multiple pathological phenotypes in tissues including reproductive organs. It was proposed that Klotho might possess the hormonal effects on many organs. In this study, the female reproductive system of klotho mice was examined to reveal the mechanism that brought the female sterility by histological and molecular approaches. We observed cessation of ovarian follicular maturation at the preantral stage and the presence of numerous atretic ovarian follicles and atrophic uteri. In situ hybridization analysis revealed that LH receptor and aromatase P450 were not expressed in the ovaries. These results suggest the impairment of gonadal development during the antral transition process. We next addressed the responsible organs for the failure of antral transition. Transplantation of klotho ovaries to wild-type mice resulted in the ability to bear offspring. Administration of FSH or GnRH induced advanced maturation of ovaries and uteri in klotho mice. These results indicate that the female reproductive organs in klotho mice are potentially functional and that klotho gene deficiency leads to the atrophy of reproductive organs via impairment of the hypothalamic-pituitary axis. Absence of the estrus cycle and constant low trends of both FSH and LH levels were found in female klotho mice. Immunohistochemical analysis revealed that the production of both FSH and LH were decreased in pituitary gland. Taken together, our findings suggest the involvement of klotho in the regulatory control of pituitary hormones.

Duration of the reproductive period decreases the frequency of preovulatory luteinizing hormone surges in heavy weight-sire line turkey hens

The frequency of preovulatory luteinizing hormone (LH) surges is an important determinant of ovulation and oviposition rates in turkeys. Egg production rate is relatively poor in heavy weight-sire line type turkey hens and declines with advancing duration of the reproductive period. The purpose of this study was to measure frequency and characteristics of preovulatory LH surges in turkey hens of a heavy weight-sire line type early, at peak of egg production (Early), and late, after egg production rate had declined (Late), in a reproductive period. The Early hens were photostimulated with a continuous photoperiod [24 h light (L):0 h dark (D)] at 40 weeks of age and sampled during peak egg production at about 47 weeks of age. The Late hens were photostimulated at 40 weeks of age with a long day photoperiod (14L:10D). After a 27-week egg production period, the Late hens were switched to the 24L:0D photoperiod and sampled at 74 weeks of age. Continuous lighting was used during blood sampling to allow the rhythm of preovulatory LH surges to free run. All hens were cannulated 3–5 days before starting sampling and hourly blood samples were collected for 200 h. All hens were necropsied and ovarian and oviductal morphologies were measured after serial bleeding. The Late hens had a longer interval between intra-clutch preovulatory LH surges than the Early hens, and a higher incidence of atretic ovarian follicles. The Early hens had higher baseline and surge amplitude LH concentrations but lower progesterone (P 4) surge amplitude concentrations than the Late hens. The duration of preovulatory LH surges, incidence of “blind” preovulatory LH surges, baseline P 4 concentrations, and overall estradiol-17β (E 2) concentrations were not different between Early and Late hens. In conclusion, a longer interval between preovulatory LH surges, lower LH baseline and surge amplitude concentrations, a higher incidence of atretic follicles, and higher P 4 surge amplitude concentration were associated with the decline in egg production late in the reproductive period in a heavy weight-sire line of turkey hens.

The effect of twenty-eight-hour ahemeral day lengths on carcass and reproductive characteristics of broiler breeder hens late in lay

The effect of increasing the day length from 24 to 28 h on the reproductive and carcass characteristics of old broiler breeder hens was examined. A total of 128 birds aged 47 wk of age from three different strains; Classic, Experimental (EXP), and Feather Sexable Yield (FSY) were used. Selection was based on target BW and egg production rates; birds were housed in light-tight environment chamber facilities. Birds were feed restricted, and feed was allocated according to actual weekly BW relative to target BW. At 55 wk of age 125 broiler breeders were killed and dissected, and carcass and reproductive traits were examined. Overall, ahemeral day length negatively affected carcass and reproductive parameters. No significant differences were found for BW, yolk, albumen and shell weights, normal and atretic ovarian follicles, and breast, fat pad, and liver weights. Significantly lower production rates, higher amounts of defective eggs, lower oviduct and ovary weights, lower F1 follicle weight, poorer eggshell quality, and higher egg weights were found. Subsequently, broiler breeder hens increased egg weight at the expense of egg production when subjected to an ahemeral day length. It is not recommended that day length be increased this late in production because it results in reduced egg productivity.

Increased cellular apoptosis after chronic aqueous exposure to nonylphenol and quercetin in adult medaka ( Oryzias latipes)

Increasing evidence suggests that sublethal effects of natural or xenobiotic chemicals in the environment may be mediated via the stimulation of apoptosis. To investigate whether apoptosis can be induced in fish by weakly estrogenic and androgenic chemicals, adult male Japanese medaka ( Oryzias latipes) were exposed to 100 ppb of the estrogenic alkylphenol, 4-nonylphenol, and adult female medaka were exposed to 100 ppb of the aromatase-inhibiting bioflavonoid, quercetin, for 6 weeks. Exposure to nonylphenol and quercetin had no significant effect on the length, weight or condition factors compared to solvent (acetone) controls in male or female medaka. Apoptosis was evaluated in blinded histological sections of whole medaka using terminal dideoxynucleotidyl transferase dUTP nick end labeling (TUNEL) that labels nuclei of cells containing apoptotic (fragmented) DNA. There was a six-fold greater extent of apoptosis in spermatocytes, Sertoli cells and Leydig-homologue cells, but not in spermatids of testes from nonylphenol-exposed male medaka compared to testes of solvent controls. No significant differences in the extent of apoptosis were detected in intestine, liver or kidney from the same male fish. Quercetin-treated female medaka had a significantly increased number of atretic ovarian follicles, but no significant differences in the extent of apoptosis in intestine, liver or kidney. These results suggest that nonylphenol caused testicular degeneration via increased testicular cell apoptosis, while quercetin may be ovotoxic via increased follicular atresia.

Tubedown-1, a novel acetyltransferase associated with blood vessel development.

We have used an embryonic endothelial cell line (IEM cells) as an experimental system for identifying and characterizing new molecules which are regulated during blood vessel development. A novel gene isolated from IEM cells, tubedown-1 (tbdn-1), is expressed at high levels in unstimulated IEM cells and is downregulated during formation of capillary tube structures by the IEM cells induced by basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) in vitro. Tbdn-1 is also downregulated in M1 myeloid leukemia cells after differentiation in response to LIF in vitro. Tbdn-1 is homologous to the yeast NAT-1 N-terminal acetyltransferases and encodes a novel protein of approximately 69 kDa associated with an acetyltransferase activity. Levels and distribution of tbdn-1 expression are regulated in both endothelial and hematopoietic cells during development in tissues such as the yolk sac blood islands, heart, and liver blood vessels. In the adult, tbdn-1 expression is low or undetected in most organs examined with the exception of the atrial endocardium, the endothelial and myeloid compartments of bone marrow, and the remodeling vascular bed of atretic ovarian follicles. The distribution and regulation of expression of tbdn-1 suggest that this novel acetyltransferase may be involved in regulating vascular and hematopoietic development and physiologic angiogenesis.

Syndecan-4 expression is associated with follicular atresia in mouse ovary.

Ovarian granulosa cells synthesize heparan sulfate proteoglycans (HSPGs), that have anticoagulant properties. Moreover, HSPGs greatly increase in the granulosa cells during follicular atresia. However, the species of ovarian HSPGs have not yet been identified. Syndecan-4 (ryudocan, amphiglycan) is a membrane-spanning HSPG and a member of the syndecan family. Herein, we demonstrate that syndecan-4 is expressed in the granulosa cells of type 4-5b follicles and, most intensely, in those of the atretic follicles in the mouse ovary, as revealed by in situ hybridization. There is no relationship between syndecan-4 expression and age or sexual cycle stage. Compared with syndecan-4 expression, syndecan-1 and -3 are expressed more abundantly in postovulatory follicles and the corpora lutea, but less in the type 4-5b follicles and much less in the atretic follicles. Immunohistochemistry also demonstrates syndecan-4 expression in atretic follicles with apoptosis. The present study has revealed the distinct modes of expression of the syndecan family members, and the association of syndecan-4 expression and apoptosis in ovarian atretic follicles.

Hormonal control of insulin-like growth factor-binding protein-5 production in the involuting mammary gland of the rat.

We have demonstrated a 50-fold increase in the concentration of insulin-like growth factor-binding protein-5 (IGFBP-5) in milk after 2 days of mammary involution induced by removal of the suckling young. IGFBP-5 was identified by its immunoreactivity with an antiserum to IGFBP-5 and was shown by in situ hybridization to be synthesized by the secretory epithelial cells undergoing apoptosis. Smaller increases in IGFBP-2 and -4 messenger RNAs (mRNAs) were also evident, but neither protein could be detected on Western ligand blots of milk. Preliminary evidence failed to detect mRNAs for IGFBP-1, -3, or -6. The large increase in IGFBP-5 concentrations in milk from involuting mammary glands was inhibited by 90% if the dams received concurrent PRL injections for 2 days, but was unaffected by GH, progesterone, corticosterone, or an antiserum to insulin-like growth factor I (IGF-I). In lactating rats allowed to continue nursing their young, 17beta-estradiol failed to affect IGFBP-5 concentrations, whereas in animals that had half the teats sealed to prevent milk removal, IGFBP-5 concentrations increased 5- to 10-fold in the sealed gland compared with those in the contralateral gland where milk removal continued. The changes in IGFBP-5 concentrations in milk were accompanied by similar changes in steady state mRNA levels of IGFBP-5 in mammary tissue. We have previously shown that PRL inhibits apoptosis and involution of the mammary gland, whereas teat sealing has the opposite effect. We, therefore, propose that IGFBP-5 serves to inhibit IGF-I-mediated cell survival, but that it is normally suppressed by PRL and milk removal. Although IGFBP-5, when bound to extracellular matrix, augments the action of IGF, we believe that in the involuting mammary gland IGFBP-5 inhibits IGF action by interacting with casein micelles, which contain calcium phosphate nanoclusters, thereby preventing IGF interaction with IGF receptors. This is analogous to the interaction of IGFBP-5 with hydroxyapatite, which serves to sequester IGFs in bone. IGFBP-5 may, in fact, play a central role in inducing apoptosis, as it is also up-regulated in involuting prostate and thyroid glands as well as in atretic ovarian follicles.

Expression of Fas ligand in murine ovary.

Corresponding to the expression of Fas in the ovarian oocytes as previously reported (Guo et al., Biochem Biophys Res Commun 1994; 203:1438-1446; Mori et al., JSIR 1995; 9:49-50), the expression of Fas ligand (FasL) in the ovarian follicle was found to be restricted in the area of granulosa cells by the indirect immunofluorescence (IIF) test. Reverse transcriptase/polymerase chain reaction (RT/PCR) technique coupled with Southern blot hybridization analysis showed that the highest level of FasL mRNA was demonstrated in murine ovaries and granulosa cells 1 day after the administration of pregnant mare's serum gonadotropin (PMSG), while the level of FasL mRNA became very weak on the day 5, respectively. The observed gradual decrease in FasL mRNA could not be attributed to a generalized degradation of cellular RNA during atresia, as evidenced by the presence of constitutive expression of elongation factor 1 alpha (EF-1 alpha) mRNA in murine ovaries and granulosa cells treated with PMSG. Furthermore, in situ hybridization analysis with a FasL-specific probe confirmed that FasL was specifically localized in the granulosa cells of most follicles and its expression was regulated by PMSG administration. FasL localized in granulosa cells might possibly play an important role in the formation of the ovarian atretic follicles, most likely depending on PMSG administration.

Genomic organization and polymorphism of human angiotensin II type 2 receptor: no evidence for its gene mutation in two families of human premature ovarian failure syndrome

Angiotensin II type 2 (AT 2) receptor is highly expressed in the fetal tissues and decreases rapidly after birth. AT 2 receptor is re-expressed in the adult atretic ovarian follicles. Recently, it has been reported that AT 2 receptor mediates apoptosis. Primarily, we have cloned human AT 2 receptor cDNA and mapped it to the X-chromosome. To further analyze the organization and function of the AT 2 receptor gene, in this study we cloned the human AT 2 receptor genomic DNA. Human AT 2 receptor gene is composed of three exons and two introns. Primer extension analysis revealed a putative transcription initiation site at 24 bp downstream from TATA box. Furthermore, we identified a polymorphism (C–A) in 3′ untranslated region of exon 3, which may be a useful genetic marker for genetic analysis of human X-linked inherited disease. In this study, we postulated that the patients with premature ovarian failure, which has been reported to be linked with X-chromosome abnormality, have AT 2 receptor mutation that may contribute to the early onset of atresia. We examined the entire coding sequence of this receptor in two different families of sisters with premature ovarian failure (POF) but found no changes in nucleotide sequences.

Elimination of apoptotic granulosa cells by intact granulosa cells and macrophages in atretic mature follicles of the guinea pig ovary.

Phagocytic cells disposing of dying granulosa cells in the atretic ovarian follicles were morphologically studied in guinea pig ovaries at various stages of estrous cycle. Epon embedded semithin sections stained with toluidine blue were observed with a light microscope, and ultrathin sections were examined under a transmission electron microscope. Frozen sections were processed for acid phosphatase histochemistry and MR-1 (a monoclonal antibody against guinea pig macrophages) immunohistochemistry. In animals during the estrus period (days 1 and 2) as well as the second half of the estrous cycle (days 11 and 16), there were numerous mature follicles in which massive groups of granulosa cells were undergoing apoptosis. Two kinds of phagocytic cells were identified in these follicles of the initial stage of atresia: one was intact granulosa cells ingesting neighboring dead granulosa cells, and the other was large round cells identified as macrophages due to their strong acid phosphatase activity and MR-1 immunoreactivity. Mature follicles of the advanced stage of atresia were frequently recognized during the metestrus period (days 4 and 5). Small stellate cells were regarded as surviving granulosa cells, while large round cells showing intense reactions for acid phosphatase and MR-1 were identified as macrophages. This study demonstrates that both intact granulosa cells and macrophages participate in the elimination of apoptotic granulosa cells in atretic mature follicles of the guinea pig ovary, and remain even in the advanced stages of atresia.

Functional aspects of angiotensin type 2 receptor.

Angiotensin II (AngII) exerts various actions in its diverse target tissues controlling vascular tone, hormone secretion, tissue growth, and neuronal activities. Extensive pharmacological evidence indicates that most of the known effects of AngII in adult tissues are mediated by the seven-transmembrane G-protein coupled Angll type 1 (AT1) receptor. Recently, a second receptor subtype known as type 2 (AT2) receptor has been described and cloned by us and others (1,2). However, little is known about the regulation and physiological function(s) of this novel receptor. The intracellular signal transduction mechanism after the activation of AT2 receptor is not also well defined. The AT2 receptor is abundantly and widely expressed in fetal tissues and immature brain, but present only in at low levels in adult tissues such as adrenal gland, specific brain regions, uterine myometrium, and atretic ovarian follicles (3–5). The highly abundant expression of this receptor during embryonic and neonatal growth and quick disappearance after birth has led us to the suggestion that this receptor may be involved in growth, development and/or differentiation.

Subtypes of active cell death in the granulosa of ovarian atretic follicles in the quail (Coturnix coturnix japonica)

Follicular atresia in the ovaries of Japanese quail was studied by cytochemistry and electron microscopy. Three different types of cell death coexisted in the granulosa. A large number of cells showed signs of apoptosis. The DNA fragmentation in these cells was demonstrated in a previous study using in situ end-labeling. A second and non-negligible type of cell death consisted of extensive autophagocytosis of the cytoplasm occurring simultaneously with late nuclear alterations. Finally, a few detached cells displayed cytoplasmic disintegration and small irregular clumps of chromatin condensation indicative of primary cell necrosis. Apoptotic versus autophagic cell death revealed a different pattern of acid phosphatase activity (lysosomal versus cytoplasmic). We propose that these observations may be linked to the existence of distinct subpopulations in the granulosa as has been shown by others. This study confirms the biochemical data on granulosa cell death, but demonstrates that apoptosis is not the exclusive mode of active cell death in follicular atresia.

Expression cloning of type 2 angiotension II receptor reveals a unique class of seven-transmembrane receptors.

Angiotensin II acts on at least two distinct receptor subtypes (AT1 and AT2). Most known effects of angiotensin II in adult tissues are attributable to the AT1 receptor. The function of AT2 receptor is undefined, but its abundant expressions in fetal tissues, immature brain, skin wound, and atretic ovarian follicles suggest a role in growth and development. Previous studies suggested that AT2 receptor may not be G protein-coupled. Here, from a rat fetus expression library, we cloned a cDNA encoding a unique 363-amino acid protein with pharmacological specificity, tissue distribution, and developmental pattern of the AT2 receptor. It is 34% identical in sequence to the AT1 receptor, sharing a seven-transmembrane domain topology. A review of prior data on other receptors suggests that this receptor may belong to a unique class of seven-transmembrane receptors (including somatostatin SSTR1, dopamine D3, and frizzled protein Fz) for which G protein coupling has not been demonstrated. All members of this class exhibit fetal and developmental and/or neuronal-specific expression. A conserved motif in the third intracellular loop, distinguishing this class from "classical" G protein-coupled receptors, may mediate novel intracellular effects.

Identification of Fas Antigen Associated with Apoptotic Cell Death in Murine Ovary

The majority of ovarian follicles including oocytes undergo atresia through a mechanism involving apoptotic cell death. The mechanisms underlying atresia remain to be clarified. In the present study, we detected the expression of the Fas antigen (Fas), which is a cell-surface protein to modulate apoptosis, in murine ovarian oocytes and hyperovulated eggs as well as in several control tissues. Substantial decline in Fas mRNA was found in atretic follicles which were injected with pregnant mare′s serum gonadotropin (PMSG) on day 3. The observed decreases in mRNA of Fas could not be attributed to a generalized degradation of cellular RNA during atresia, as evidenced by the presence of intact 18S and 28S ribosomal RNA as well as constitutive expression of EF-1α mRNA in atretic follicles. The data obtained indicate that apoptotic cell death of oocytes seemed to be associated with internucleosomal DNA fragmentation regulated by Fas molecule expressed in atretic ovarian follicles.

Expression cloning of type 2 angiotensin II receptor reveals a unique class of seven-transmembrane receptors.

Angiotensin II acts on at least two distinct receptor subtypes (AT1 and AT2). Most known effects of angiotensin II in adult tissues are attributable to the AT1 receptor. The function of AT2 receptor is undefined, but its abundant expressions in fetal tissues, immature brain, skin wound, and atretic ovarian follicles suggest a role in growth and development. Previous studies suggested that AT2 receptor may not be G protein-coupled. Here, from a rat fetus expression library, we cloned a cDNA encoding a unique 363-amino acid protein with pharmacological specificity, tissue distribution, and developmental pattern of the AT2 receptor. It is 34% identical in sequence to the AT1 receptor, sharing a seven-transmembrane domain topology. A review of prior data on other receptors suggests that this receptor may belong to a unique class of seven-transmembrane receptors (including somatostatin SSTR1, dopamine D3, and frizzled protein Fz) for which G protein coupling has not been demonstrated. All members of this class exhibit fetal and developmental and/or neuronal-specific expression. A conserved motif in the third intracellular loop, distinguishing this class from "classical" G protein-coupled receptors, may mediate novel intracellular effects.