Biomedical subjects

A M Dharmarajan

Publications and source records attributed to A M Dharmarajan.

At least 19 recordsLinked to original sources

PubMed2005

The expression of apoptosis related genes in the first trimester human placenta using a short term in vitro model.

Using in vitro model for studying the induction and inhibition of spontaneous apoptosis in human first trimester placental villi, mediated by the free radical scavenger SOD, we have examined the expression of bcl-xL, bax, Caspase-3 and PARP (Poly ADP-ribosyl). An increase in apoptosis was associated with activation of PARP and an increase and activation of Caspase-3. There was no significant change in bcl-x or bax. Therefore bcl-x and bax do not appear to have a significant role in apoptosis in the first trimester in vitro. Cleavage of Caspase-3 rather than transcriptional regulation appears to be the main determinant of Caspase-3 activity in first trimester placental villi.

Apoptosis↗

PubMed2004

Inhibition of nitric oxide synthesis potentiates apoptosis in the rabbit corpus luteum.

To determine if nitric oxide (NO) plays a role in corpus luteum (CL) physiology by affecting progesterone secretion or luteal apoptosis, an in-vitro pseudopregnant rabbit ovarian perfusion system was used to measure the effects of an inhibitor of NO synthesis, NG-nitro-L-arginine methyl ester (L-NAME), on progesterone secretion and corpus luteal apoptosis as measured by internucleosomal DNA breakdown. Pseudopregnant rabbit ovaries perfused in vitro with L-NAME did not demonstrate any significant differences compared with control ovaries in progesterone secretion. However, apoptosis, as measured by internucleosomal breakdown, was significantly increased in L-NAME-perfused CL compared with controls. While NO does not appear to directly affect progesterone secretion, there does appear to be a role for NO in CL maintenance, or a role for inhibition of NO production in CL regression.

Animals↗

PubMed2004

Role of apoptosis in functional luteolysis in the pregnant rabbit corpus luteum: evidence of a role for placental-derived factors in promoting luteal cell survival.

Corpora lutea (CL) were isolated from one rabbit ovary on days 4, 8, 16 (peak luteal function), 28 (functional regression) and 30 of pregnancy and processed for biochemical analysis of DNA integrity. Analysis of DNA integrity revealed the presence of oligonucleosomal fragments in day 28 and day 30 CL but not in day 16 CL. The extent of low molecular weight (<15 kb) DNA labeling was 6.6 +/- 0.84 fold higher in day 30 as compared to day 16 CL (mean +/- SEM; n = 4, P < 0.01). In a second series of experiments, healthy CL collected from day 16 pregnant rabbits were incubated for 2 h in the absence or presence of 250 microg/ml of placental extract (PE) obtained from day 16 and/or day 30 placentas. Analysis of DNA integrity revealed that extensive apoptosis occurred in CL incubated in medium alone and in medium containing day 30 PE. In contrast, day 16 PE significantly suppressed apoptosis vs control (70 +/- 4%). In a third series of experiments, expression of mRNA for bcl-x and bax was measured by Northern analysis of CL treated without and with day 16 PE using cRNA probes for bcl-x and bax developed in our laboratory by RT-PCR. Treatment with PE significantly reduced bax mRNA levels but did not change bcl-x mRNA levels. These studies provide evidence that functional luteolysis in the pregnant rabbit CL is correlated with the occurrence of apoptosis. The data suggest that a factor(s) derived from the placenta may be responsible for the prolongation of CL life span during pregnancy by its ability to alter the bax:bcl -x rheostat and suppress apoptosis.

Animals↗

PubMed2003

The role of sFRP4, a secreted frizzled-related protein, in ovulation.

Ovulation is a complex, multi-factorial event that involves the degeneration of a specific area of the follicular and ovarian surface via apoptosis. Many apoptosis related genes have been identified in the ovary. Secreted Frizzled Related Protein 4 (sFRP4) is a protein that appears to antagonize a molecular pathway for cell survival. sFRP4 gene expression is known to be upregulated with apoptosis in the ovarian corpus luteum. In this study, ovulation was hormonally induced in immature Wistar rats and their ovaries collected for analysis of apoptosis and sFRP4. TUNEL staining identified a greater amount of dying cells in the thecal layer of treated rat ovaries compared to controls. The results of 3'-end labelling revealed a significant increase (p < 0.01) in apoptosis at 12 hours following treatment compared to other time points and control. In situ hybridization exhibited a visible increase in amounts of sFRP4 mRNA expression in the thecal layers of follicles from treated rats compared to controls. Quantitative RT-PCR revealed no significant difference in sFRP4 expression levels between treated and control tissues although a clear trend towards an increase was observed in the treated group. This study demonstrates an association between sFRP4 and apoptosis in rat ovulation.

Animals↗

PubMed2003

Role of DDC-4/sFRP-4, a secreted frizzled-related protein, at the onset of apoptosis in mammary involution.

Using differential display, we isolated DDC-4, a secreted frizzled-related protein (sFRP), which is induced in the physiological apoptosis of hormonally regulated, reproductive tissues such as mammary gland, prostate, corpus luteum and uterus. The role of this gene in apoptosis was studied in animals overexpressing ectopic DDC-4/sFRP-4. Transgenic mice bearing the DDC-4/sFRP-4 cDNA under the control of the MMTV-LTR promoter showed lactational insufficiency and many apoptotic cells in the alveoli between day 19 of pregnancy and day 4 of lactation as demonstrated by TUNEL reaction and the presence of activated caspase-3. We performed a PKB/Akt kinase assay and studied several of its substrates using phosphorylation-specific antibodies to show reduced phosphorylation in PKB/Akt itself, as well as in glycogen synthetase kinase-3beta (GSK-3beta), BAD, and Forkhead. Taken together, our results show a role for DDC-4/sFRP-4 in abrogating an epithelial cell survival pathway at the onset of mammary gland involution.

Animals↗

PubMed2001

Patterns of apoptosis in the corpora lutea of the rat during the oestrous cycle, pregnancy and in vitro culture.

Apoptosis is associated with regression of corpora lutea (CL) in a number of species. The present work compared apoptotic rates, assessed by low molecular weight DNA labelling, in rat CL of the oestrous cycle, pregnancy, the immediate post-partum period, and in vitro culture. Apoptosis was significantly related to cycle phase with peak activity at oestrus. This pattern was similar in CL formed from the most recent ovulation and from the two previous generations. Apoptosis was evident during pregnancy, albeit at low rates. It declined on the day of parturition and increased on the following day. Apoptosis was greatly increased in cultured CL to reach 20-fold higher levels than those achieved during the oestrous cycle or pregnancy. Collectively, these results suggest that apoptosis has lesser significance in rat CL functional regression than in other species with more precipitate structural changes. However, rat CL clearly retain the potential for high rates of apoptosis and so present a useful model for examining molecular mechanisms of apoptotic cell death.

Animals↗

PubMed2001

Inhibitors of caspase homologues suppress an apoptotic phenotype in cultured rabbit corpora lutea.

Apoptosis is a morphologically defined type of cell death initiated by various stimuli that results in the activation of caspases (cysteine-containing aspartate-specific proteases). In the present study, it was determined that caspases are present during, and play a role in, corpus luteum (CL) apoptosis in vitro. Pseudopregnancy was induced in rabbits with 100 IU human chorionic gonadotrophin. On Day 11 of pseudopregnancy, CL were isolated and cultured for 0, 2, 4, 6, and 8 h in the absence of trophic support to induce spontaneous apoptosis. Total RNA was extracted and analysed for caspase-I expression by Northern blot analysis. The results demonstrated caspase-I expression from 4 h. In the second part of the study, CL were incubated without trophic support for 4 h with increasing concentrations of three general caspase inhibitors, sodium aurothiomalate (SAM), iodoacetic acid (IAA) and N-tosyl-L-phenylalanylchloromethylketone (TPCK), and two specific caspase inhibitors, N-acetyl (Ac)-Tyr-Val-Ala-Asp (YVAD)-chloromethylketone (CMK) (Ac-YVAD-CMK) and Ac-Asp-Glu-Val-Asp (DEVD)-aldehyde (CHO) (Ac-DEVD-CHO). At completion, DNA was isolated and integrity assessed. Treatment of CL with SAM, IAA or Ac-DEVD-CHO effectively suppressed apoptotic DNA fragmentation. The final component of the study was to examine caspase-3 protein expression. Western blot analysis revealed a significant increase in caspase-3 expression over the experimental time-course. The results of the present study clearly demonstrate a time-dependent link between the caspases, specifically caspase-3 and spontaneous apoptosis in the rabbit CL.

Amino Acid Chloromethyl Ketones↗

PubMed2000

Cloning and characterization of an apoptosis-associated gene in the human placenta.

Placenta is a transient feto-maternal association that develops during mammalian pregnancies. Human placental tissue during the first trimester of pregnancy is an actively dividing and differentiating tissue, while near term, it represents a fully differentiated unit performing many life-sustaining functions for the fetus. Previous studies have demonstrated that the percentage of placental cells that undergo apoptosis is greater at full term as compared to the first trimester of pregnancy. In this study, we undertook a study aimed at gaining an insight into the kind of genes expressed in the two developmentally distinct stages of gestation ie, the first trimester and term using Differential Display RT-PCR. Cloning and sequencing of one of the differentially expressed cDNAs from term placental tissue revealed that it is a novel gene, referred to as T-18 in the text. In this study, we also examined the regulation of this gene during apoptosis in the human placenta. A model for analysis of placental apoptosis was established by incubating placental villi in serum-free culture medium. It was observed that apoptosis occurred rapidly following incubation of placental villi without tropic support, and the proposed free-radical scavenger, superoxide dismutase (SOD) suppressed apoptosis in the placenta. Interestingly, the levels of T-18 mRNA increased significantly during spontaneous induction of apoptosis and decreased when apoptosis was blocked by SOD. These data clearly suggest that there is a strong correlation between the expression of T-18 and placental apoptosis and that T-18, may play a significant role in this process. Furthermore, the establishment of a defined in vitro explant culture model should facilitate elucidation of factors, which regulate apoptosis in human placenta.

Apoptosis↗

PubMed2000

Apoptosis in rat placenta is zone-dependent and stimulated by glucocorticoids.

Apoptosis, or physiological cell death, is elevated in the placenta of human pregnancies complicated by fetal growth retardation, suggesting that placental apoptosis may be a key factor in the overall control of feto-placental growth. The present study used DNA internucleosomal fragmentation analysis to characterize apoptosis in the two morphologically and functionally distinct regions of the rat placenta, the basal and labyrinth zones, during the last week of pregnancy (Days 16, 22, and 23). In addition, because glucocorticoids are potent inhibitors of feto-placental growth and can stimulate apoptosis in other tissues, we examined whether dexamethasone treatment in vivo induces placental apoptosis. DNA fragmentation was clearly evident in both placental zones at each stage of pregnancy, with higher levels evident in the basal zone compared with the labyrinth zone on Days 22 and 23. TUNEL analysis, which identifies dying cells in situ, demonstrated positive staining of cells in the basal zone, particularly giant trophoblast cells. Dexamethasone treatment increased DNA fragmentation in the basal zone but not the labyrinth zone. Similarly, maternal treatment with carbenoxolone, which can enhance local concentrations of endogenous glucocorticoid by inhibition of 11 beta-hydroxysteroid dehydrogenase, also increased DNA fragmentation in the basal zone but not in the labyrinth zone. These effects of dexamethasone and carbenoxolone on placental apoptosis were associated with reduced placental and fetal weights. In conclusion, this study shows that apoptosis occurs in both zones of the rat placenta, particularly in the basal zone near term, and is elevated after increased glucocorticoid exposure in vivo. These data support the hypothesis that placental apoptosis is an important player in the regulation of feto-placental growth, and establish the rat as a useful model to study the endocrine control of placental apoptosis.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1999

Mitochondrial peripheral-type benzodiazepine receptor expression. Correlation with gonadotropin-releasing hormone (GnRH) agonist-induced apoptosis in the corpus luteum.

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) decreases the expression of the mitochondrial peripheral-type benzodiazepine receptor (PBR) and increases the rate of DNA degradation in a time-dependent manner in the corpora lutea of pregnant rats. In the present study, we show in situ the GnRH-Ag-induced DNA fragmentation and correlate the increase of the rate of DNA degradation with the decrease in mitochondrial PBR ligand binding (r = 0.89). The GnRH-Ag-induced decrease in the 18-kDa PBR protein also correlated with the reduction in the Bcl-X(L), but not Bcl-2 (cell survival), gene product levels and the increase in the Bax (cell death) gene product expression in the luteal mitochondrial preparations. Considering the function of PBR in cholesterol uptake and intramitochondrial movement, we propose that decreased PBR expression may lead to reduced levels of mitochondrial membrane cholesterol, which, together with the ability of Bcl-X(L) and Bax to form ion channels, produces breaks in the outer membranes allowing the exit of cytochrome c, thus triggering apoptosis. Alternatively, PBR may exert an as yet unidentified anti-apoptotic function.

Animals↗

PubMed1999

Physiological apoptosis in hormone-dependent tissues: involvement of caspases.

Physiological apoptosis in mammals is a type of programmed cell death, an important element in the developmental repertoire ensuring tissue homeostasis and proper disposal of cells that are no longer needed, such as milk-producing epithelial cells in the mammary gland after lactation, luteal cells in the post partum Corpus luteum or secretory cells in the prostate after castration. Although incompletely described, apoptosis in hormone-dependent tissues is apparently initiated and executed using common biochemical strategies. These include survival pathways governed by local and systemic factors and hormones, diverse regulatory pathways and caspase-dependent execution pathways. Using an antibody that recognizes processed effector caspases or a fluorogenic caspase substrate, we present for the first time evidence that caspases are activated in the mammary gland, in the prostate and in the ovary at the time when apoptosis occurs. Most likely phagocytosis of apoptotic cells by neighboring cells may represent an important step, since only a modest involvement of professional phagocytes is apparent. Here, we will summarize and discuss recent data and will attempt to draw a generalized picture of how physiological apoptosis may occur in these organs.

Animals↗

PubMed1999

Fas and Fas ligand messenger ribonucleic acid and protein expression in the rat corpus luteum during apoptosis-mediated luteolysis.

Apoptosis has been found to occur during regression of the corpus luteum (CL) in many species. The Fas (APO-1/CD95) receptor, a transmembrane protein that induces apoptosis in the cell when bound to Fas ligand (FasL), may be involved. This study established and quantitated the presence and regulation of Fas receptor and FasL in the rat CL during pregnancy and postpartum. Using immunohistochemistry, FasL was localized in CL during pregnancy and postpartum. Fas was localized at Day 1 of pregnancy and at the time of luteolysis. Both Fas and FasL mRNA were found to be expressed throughout pregnancy and postpartum using reverse transcription-polymerase chain reaction (RT-PCR). Relative quantitative RT-PCR established that expression of FasL mRNA increased significantly at Day 22 of pregnancy and decreased by Day 3 postpartum. Spontaneous apoptosis of rat CL placed in an in vitro culture model with serum-free medium was examined by analysis of extracted DNA using 3' end-labeling. Treatment with an anti-rat Fas monoclonal antibody demonstrated a reduction in the occurrence of spontaneous apoptosis. These data support a role for Fas receptor and FasL in rat CL apoptosis during luteolysis.

Animals↗

PubMed1999

Antioxidants mimic the ability of chorionic gonadotropin to suppress apoptosis in the rabbit corpus luteum in vitro: a novel role for superoxide dismutase in regulating bax expression.

We have recently reported that members of the bcl-2 gene family are expressed and estradiol regulated in rabbit luteal cells during corpus luteum (CL) regression, and that estradiol and hCG are effective inhibitors of apoptosis in the rabbit CL in vivo and in vitro. As Bcl-2 and related proteins are known to regulate levels of reactive oxygen species or their intermediates in cells as one possible mechanism to control apoptosis, the present studies were designed to examine if oxidative stress plays a role in luteal cell apoptosis during CL regression in the rabbit. In the first set of experiments, healthy CL obtained from day 11 pseudopregnant rabbits were incubated in serum-free medium for 2 h in the absence or presence of superoxide dismutase (SOD; 1.5-150 U/ml), ascorbic acid (1-100 mM), N-acetyl-L-cysteine (25 and 50 mM), or catalase (10-1000 U/ml). Cells within CL incubated in medium alone exhibited extensive apoptosis (examined by analysis of extracted DNA using 3'-end labeling), and this onset of apoptosis was blocked in a dose-dependent fashion by treatment with SOD, ascorbic acid, N-acetyl-L-cysteine, or catalase. In the second set of experiments, expression of bax and bcl-x in CL after in vitro treatment without and with 100 U/ml SOD was examined. Although SOD treatment did not alter the levels of bcl-x messenger RNA (mRNA) over the 2-h incubation period, this antioxidant enzyme significantly reduced the levels of bax mRNA in incubated CL. In the final set of experiments, we observed that expression of mitochondrial- or manganese-containing SOD was significantly increased by treatment of isolated CL with 1 microg/ml hCG in vitro, whereas bax mRNA levels were significantly reduced under the same culture conditions. Collectively, these data indicate that the gonadotropin-mediated inhibition of apoptosis in rabbit luteal cells involves enhanced expression of the oxidative stress response gene, manganese-containing SOD, whose protein product may then function to protect luteal cells directly from the damaging effect of reactive oxygen species and/or indirectly by acutely down-regulating expression of Bax, a prooxidant member of the Bcl-2 protein family.

Animals↗

PubMed1999

Evidence for alternative pathways of granulosa cell death in healthy and slightly atretic bovine antral follicles.

Granulosa cell death is an early feature of atresia; however, there are many apparent contradictions in the literature concerning the mode of granulosa cell death. We have therefore examined this process in bovine healthy and atretic antral follicles, using a variety of established techniques. Light and electron microscopic observations indicated the presence of pyknotic or shrunken nuclei in both the membrana granulosa and the antrum. In the membrana granulosa, these nuclei were frequently crescent shaped and uniformly electron dense and were approximately the same size as healthy nuclei, all of which are typical of early apoptosis. However, these nuclei were within the membranes of a healthy granulosa cell, suggesting that phagocytosis by a neighboring granulosa cell is an unusually early event in the apoptotic pathway of granulosa cells. In the membrana granulosa, pyknotic nuclei stained intensely with hematoxylin but weakly with the DNA-intercalating stain propidium iodide. A percentage of these pyknotic nuclei stained by TUNEL (terminal deoxy-UTP nick end-labeling). However, in the antrum, the pyknotic nuclei and larger globules of DNA stained intensely with both hematoxylin and propidium iodide, but were not TUNEL positive. The comet assay of cell death produced a streak tail of randomly nicked DNA, rather than the plume of low mol wt apoptotic DNA. Globules collected from fresh follicular fluid stained intensely with propidium iodide and were shown by PAGE to contain DNA, the majority of which was high mol wt. In conclusion, granulosa cells within the membrana granulosa die by apoptosis, with phagocytosis by a neighboring cell preceding any potential budding of the nucleus or cell itself. Granulosa cells near the antrum are sloughed off into the antrum, and their death has features more consistent with that of other cell types that undergo death as a result of terminal differentiation.

Animals↗

PubMed1998

Induction of apoptosis by a gonadotropin-releasing hormone agonist during early pregnancy in the rat.

We have demonstrated that continuous administration of a GnRH-agonist (GnRH-Ag) in the rat during early pregnancy suppressed plasma progesterone levels within 8 h after the commencement of treatment. The purpose of the present study is to evaluate the hypothesis that in vivo GnRH-Ag treatment induces apoptotic cell death during early pregnancy. Rats were treated individually on day 8 of pregnancy with 5 microg/day of GnRH-Ag via an osmotic minipump. Sham-controlled rats received no treatment. Rats were killed at 4, 8 or 24 h after the treatment. At autopsy, blood samples were obtained and ovaries were removed. The corpora lutea (CL) from one ovary were removed for DNA laddering and RNA studies. As early as 8 h after the commencement of treatment, GnRH-Ag suppressed serum progesterone levels as expected, and increased the degree of DNA degradation in the CL that was time-dependent. At 24 h after the treatment, GnRH-Ag increased the Bax, a cell death gene, expression in the CL. Collectively, these data suggest that GnRH-Ag treatment induces apoptosis during early pregnancy in the rat.

Journal Article↗

PubMed1998

Apoptosis-associated gene expression in the corpus luteum of the rat.

The involution of the corpus luteum (CL) at parturition is an example of physiological apoptosis, a complex process involving massive vascular regression while luteal cells undergo apoptosis. In the present study, changes in gene expression associated with physiological apoptosis were examined. Three genes isolated in our laboratory because of their association with apoptotic processes in the ovary, mammary gland, and prostate served as the focus of our investigation: Y81, Gas-1, and the gene IAP encoding integrin-associated protein. Y81 is a novel gene for which three transcripts are apparent. A Y81 cDNA clone representing the longest transcript has been isolated; it shows an open reading frame exhibiting a region of very high homology with members of the frizzled family, the prototypes of which are cell autonomous polarity genes encoding seven-pass transmembrane receptor proteins, for example the receptor for Wingless. Gas-1 is known as a growth-arrest gene that inhibits DNA synthesis when microinjected into cells. Integrin-associated protein is a beta 3-integrin-binding protein for which, recently, a thrombo-spondin-binding activity has been recognized. These three genes, all sharply up-regulated in the course of physiological involution processes in the ovarian CL, in mammary gland, and in prostate, seem promising candidates-by virtue of their specific expression in distinct tissues undergoing programmed cell death-as mediators of stimuli leading to apoptosis and subsequent phagocytosis. In this study, sulfated glycoprotein-2, previously observed in many instances of physiological apoptosis, was further employed as an indicator for incipient apoptosis, and stromelysin was followed as a marker for the tissue remodeling activity that is intimately associated with apoptosis during involution.

Amino Acid Sequence↗

PubMed1998

Estradiol-mediated suppression of apoptosis in the rabbit corpus luteum is associated with a shift in expression of bcl-2 family members favoring cellular survival.

In the rabbit, estradiol is the primary luteotropic hormone. Estradiol withdrawal results in a rapid decline in serum progesterone and eventually in corpus luteum (CL) regression. The objective of this study was to determine whether estradiol modulates luteal cell apoptosis. In the first experiment, rabbits were randomly assigned to one of five experimental groups. An empty capsule (control) or estradiol-filled Silastic capsule was inserted s.c. on Day 0 of pseudopregnancy (day of hCG administration). On Day 11 of pseudopregnancy, some of the group I (control) and group II (estradiol capsule) rabbits were subjected to laparotomy, and one ovary from each rabbit was perfused in vitro to determine progesterone secretion rates. The CL from the contralateral ovary were dissected, snap-frozen, and stored at -70 degrees C until analyzed for internucleosomal DNA cleavage (apoptosis). Estradiol-containing capsules were removed from some of the remaining rabbits on Days 8, 9, and 10 to initiate estradiol deprivation. Rabbits were then subjected to laparotomy 24, 48, or 72 h after capsule removal (groups III, IV, and V, respectively), and ovaries or CL were processed as described above. Deprivation of estradiol for 24 (group III), 48 (group IV), or 72 (group V) h in vivo reduced in vitro progesterone secretion rates by more than 90% as compared to that in ovaries collected from estradiol capsule-intact animals. After in vivo endogenous estradiol suppression, withdrawal of exogenous estradiol resulted in luteal cell apoptosis, which increased in a time-dependent manner. Northern blot analysis revealed an increase in bax mRNA levels and a decrease in bcl-x mRNA levels coincident with luteal cell apoptosis induced by estradiol withdrawal. These data demonstrate that changes in progesterone production caused by estradiol exposure and deprivation are in part related to luteal cell apoptosis, and alterations in the expression of bcl-2 gene family members may be one of the mechanisms by which estradiol exerts its luteotropic effect in the rabbit CL.

Animals↗

PubMed1997

DDC-4, an apoptosis-associated gene, is a secreted frizzled relative.

The differential display method has been used in our laboratory as a coincidence analysis to isolate genes expressed in common in each of three different rat tissues undergoing physiological apoptosis: mammary gland, ovarian corpus luteum and ventral prostate. The most interesting of these isolates, DDC-4, shows a clear association with apoptosis, its expression being confined to these three organs, and only during their involution. Using DDC-4 as probe, we screened a rat ovarian cDNA library to obtain full-length isolates. One isolate, Y81 clone 40, gives rise to a protein of approximately 40 kDa with coupled in vitro transcription/translation. Sequencing of this clone indicates an open reading frame of 1044 nucleotides encoding a protein of 39.7 kDa with a putative signal sequence. This clone exhibits a high homology with the cysteine-rich domain, i.e. the ligand-binding domain, of the fizzled gene family originally defined as tissue polarity genes in Drosophila. The homology of Y81 clone 40 is most extensive with the newly described secreted frizzled relatives, the frzb subfamily.

Amino Acid Sequence↗