Oestrogens and temperature-dependent sex determination in reptiles: all is in the gonads.

In many species of oviparous reptiles, the first steps of gonadal sex differentiation depend on the incubation temperature of the eggs. Feminization of gonads by exogenous oestrogens at a male-producing temperature and masculinization of gonads by antioestrogens and aromatase inhibitors at a female-producing temperature have irrefutably demonstrated the involvement of oestrogens in ovarian differentiation. Nevertheless, several studies performed on the entire gonad/adrenal/mesonephros complex failed to find differences between male- and female-producing temperatures in oestrogen content, aromatase activity and aromatase gene expression during the thermosensitive period for sex determination. Thus, the key role of aromatase and oestrogens in the first steps of ovarian differentiation has been questioned, and extragonadal organs or tissues, such as adrenal, mesonephros, brain or yolk, were considered as possible targets of temperature and sources of the oestrogens acting on gonadal sex differentiation. In disagreement with this view, experiments and assays carried out on the gonads alone, i.e. separated from the adrenal/mesonephros, provide evidence that the gonads themselves respond to temperature shifts by modifying their sexual differentiation and are the site of aromatase activity and oestrogen synthesis during the thermosensitive period. Oestrogens act locally on both the cortical and the medullary part of the gonad to direct ovarian differentiation. We have concluded that there is no objective reason to search for the implication of other organs in the phenomenon of temperature-dependent sex determination in reptiles. From the comparison with data obtained in other vertebrates, we propose two main directions for future research: to examine how transcription of the aromatase gene is regulated and to identify molecular and cellular targets of oestrogens in gonads during sex differentiation, in species with strict genotypic sex determination and species with temperature-dependent sex determination.

Degree of sex reversal as related to plasma steroid levels in genetic female chickens (Gallus domesticus) treated with Fadrozole.

The objectives of this work were to determine whether or not plasma levels of testosterone and estradiol reflect the various grades of sex reversal in genetic female chickens treated with Fadrozole (CGS 16949 A), a nonsteroidal aromatase inhibitor, and whether gonadal aromatase activity and plasma levels of testosterone and estradiol in treated females can or not be modified by post-hatch treatments with Fadrozole or Fadrozole + testosterone. Eggs were injected with 1 mg Fadrozole on day 4 of incubation. In females having developed sex-reversed gonads, endocrine parameters (estradiol and testosterone) at and after 13 weeks of age were indicative of the degree of sex reversal, with, for example, sex-reversed females with two testes having the highest levels of testosterone and the lowest levels of estradiol. Among these females, eight (from a total of 13) produced ejaculates with scarce and abnormal spermatozoa. Some motility was observable in the ejaculates from five of them. None of the post-hatch treatments had a significant effect on plasma levels of testosterone or estradiol (measured at 3-week intervals from week 4 to week 28 post-hatch) or on gonadal aromatase activity (measured at 12 and 28 weeks). In conclusion, these results indicate that plasma levels of testosterone and estradiol at and after 13 weeks of age are valuable indicators of the degree of sex reversal in female chickens treated with Fadrozole prior to gonadal sex differentiation. In pre-cited conditions, post-natal treatments with either Fadrozole or Fadrozole + testosterone had no apparent effect on the degree of sex reversal in these birds. Finally, the occurrence of ejaculates with motile although scarce and abnormal spermatozoa, revealed that epididymes and ducti deferens can develop and become functional in sex-reversed female chickens.

Sex reversal and aromatase in chicken.

Aromatase inhibitors administered before sexual differentiation of the gonads can induce sex reversal in female chickens. To analyze the process of sex reversal, we have followed for several months the changes induced by Fadrozole, a nonsteroidal aromatase inhibitor, in gonadal aromatase activity and in morphology and structure of the female genital system. Fadrozole was injected into eggs on day four of incubation, and its effects were examined during the embryonic development and for eight months after hatching. In control females, aromatase activity in the right and the left gonad was high in the middle third of embryonic development, and then decreased up to hatching. After hatching, aromatase activity increased in the left ovary, in particular during folliculogenesis, whereas in the right regressing gonad, it continued to decrease to reach testicular levels at one month. In treated females, masculinization of the genital system was characterized by the maintenance of the right gonad and its differentiation into a testis, and by the differentiation of the left gonad into an ovotestis or a testis; however, in all individuals, the left Müllerian duct and the posterior part of the right Müllerian duct were maintained. In testes and ovotestes, aromatase activity was lower than in gonads of control females (except in the right gonad as of one month after hatching) but remained higher than in testes of control and treated males. Moreover, in ovotestes, aromatase activity was higher in parts displaying follicles than in parts devoid of follicles. The main structural changes in the gonads during sex reversal were partial (in ovotestes) or complete (in testes) degeneration of the cortex in the left gonad, and formation of an albuginea and differentiation of testicular cords/tubes in the two gonads. Testicular cords/tubes transdifferentiated from ovarian medullary cords and lacunae whose epithelium thickened and became Sertolian. Transdifferentiation occurred all along embryonic and postnatal development; thus, new testicular cords/tubes were continuously formed while others degenerated. The sex reversed gonads were also characterized by an abundant fibrous interstitial tissue and abnormal medullary condensations of lymphoid-like cells; in the persisting testicular cords/tubes, spermatogenesis was delayed and impaired. Related to aromatase activity, persistence of too high levels of estrogens can explain the presence of oviducts, gonadal abnormalities and infertility in sex reversed females.

Expression of AMH, SF1, and SOX9 in gonads of genetic female chickens during sex reversal induced by an aromatase inhibitor.

Aromatase inhibitors administered prior to histological signs of gonadal sex differentiation can induce sex reversal of genetic female chickens. Under the effects of Fadrozole (CGS 16949A), a nonsteroidal aromatase inhibitor, the right gonad generally becomes a testis, and the left gonad a testis or an ovotestis. We have compared the expression pattern of the genes encoding AMH (the anti-Müllerian hormone), SF1 (steroidogenic factor 1), and SOX9 (a transcription factor related to SRY) in these sex-reversed gonads with that in control testes and ovaries, using in situ hybridization with riboprobes on gonadal sections. In control males, the three genes are expressed in Sertoli cells of testicular cords; however, only SOX9 is male specific, since as observed previously AMH and SF1 but not SOX9 are expressed in the control female gonads. In addition to testicular-like cords, sex-reversed gonads present many lacunae with a composite, thick and flat epithelium. We show that during embryonic and postnatal development, AMH, SF1 and SOX9 are expressed in the epithelium of testicular-like cords and in the thickened part but not in the flattened part of the epithelium of composite lacunae. AMH and SF1 but not SOX9 are expressed in follicular cells of ovotestes. Coexpression of the three genes, of which SOX9 is a specific Sertoli-cell marker, provides strong evidence for the transdifferentiation of ovarian into testicular epithelium in gonads of female chickens treated with Fadrozole.

Sex reversal and aromatase in the European pond turtle: treatment with letrozole after the thermosensitive period for sex determination.

In the European pond turtle (Emys orbicularis), gonadal sex differentiation is temperature-dependent. The temperature sensitive period (TSP) of gonadogenesis lies between stages 16 and 22 of embryonic development. Previous studies have shown that embryos incubated at 30 degrees C, a temperature yielding 100% phenotypic females, can be sex reversed by treatments with an aromatase inhibitor administered during TSP or even somewhat after TSP (as of stage 22+). The goal of the present study was to determine whether the ovary still retains male potential at later stages of embryonic development and whether the induced male characters persist after hatching. For this purpose, eggs of E. orbicularis were treated with letrozole, a nonsteroidal aromatase inhibitor, at or as of stages 23, 24 or 25, then gonadal aromatase activity in each individual and the related gonadal structure were studied at hatching (stage 26) and for one year after hatching. Two kinds of treatments were carried out: 1) repeated applications of 10 microg of letrozole in ethanolic solution onto the eggshell; and 2) a single injection of 10 microg of letrozole in olive oil. Similar results were obtained with either application or injection of the aromatase inhibitor. In treatments as of or at stage 23, individuals with gonadal aromatase activity lower than 20 fmoles/hour/gonad had ovotestes, i.e., 22% of the treated individuals. At hatching, the inner part of these ovotestes contained testicular cords and also mixed lacunae presenting various degrees of transdifferentiation of the epithelium into a Sertolian epithelium. The cortex was maintained, although some germ cells degenerated within it. These processes continued after hatching. However, at 12 months, gonads were still ovotestes displaying some follicles with a growing oocyte in the remaining parts of the cortex. In treatments as of or at stages 24 or 25, only a few individuals were masculinized. One had ovotestes; in others, the cortex was absent in some parts and when it was present oocytes were degenerating. These results show that in the European pond turtle, differentiation of ovotestes from ovaries can be induced by treatment with an aromatase inhibitor starting at late stages of embryonic development (between the end of TSP and hatching), although such differentiation is less frequent as embryonic development proceeds. Sex reversal persists for at least one year after hatching. J. Exp. Zool. 290:490-497, 2001.

Temperature-dependent sex determination and gonadal differentiation in reptiles.

In many reptile species, sexual differentiation of gonads is sensitive to temperature (temperature-dependent sex determination, TSD) during a critical period of embryonic development (thermosensitive period, TSP). Experiments carried out with different models including turtles, crocodilians and lizards have demonstrated the implication of estrogens and the key role played by aromatase (the enzyme complex that converts androgens to estrogens) in ovary differentiation during TSP and in maintenance of the ovarian structure after TSP. In some of these experiments, the occurrence of various degrees of gonadal intersexuality is related to weak differences in aromatase activity, suggesting subtle regulations of the aromatase gene at the transcription level. Temperature could intervene in these regulations. Studies presently under way deal with cloning (cDNAs) and expression (mRNAs) of genes that have been shown, or are expected, to be involved in gonadal formation and/or differentiation in mammals. Preliminary results show that homologues of the WT1, SF1, SOX9, DAX1 and AMH genes exist in TSD reptiles. However, the expression patterns of these genes during gonadal differentiation may be different between mammals and TSD reptiles and also between different reptile species. How these genes could interact with aromatase is being examined.

Temperature-dependent sex determination and gonadal differentiation in reptiles.

In many reptile species, sexual differentiation of gonads is sensitive to temperature during a critical period of embryonic development (thermosensitive period, TSP). Experiments carried out with different models among which turtles, crocodilians and lizards have demonstrated the implication of estrogens and the key role played by aromatase (the enzyme complex that converts androgens to estrogens) in ovary differentiation during TSP and in maintenance of the ovarian structure after TSP. In some of these experiments, the occurrence of various degrees of gonadal intersexuality is related to weak differences in aromatase activity, suggesting subtle regulations of the aromatase gene at the transcription level. Temperature could intervene in these regulations. Present studies deal with cloning (complementary DNAs) and expression (messenger RNAs) of genes that have been shown, or are expected, to be involved in gonadal formation and/or differentiation in mammals. Preliminary results indicate that homologues of AMH, DAX1, SF1, SOX9 and WT1 genes with the same function(s) as in mammals exist in reptiles. How these genes could interact with aromatase is being examined.

Sexual differentiation of gonads as a function of temperature in the turtle Emys orbicularis: endocrine function, intersexuality and growth.

Emys orbicularis is a freshwater turtle with temperature-dependent sex determination. Estrogens play a major role in gonadal differentiation; when they are produced at high levels during the thermosensitive period (TSP), ovaries differentiate; when their synthesis is very low, testes differentiate. Estrogens are synthesized from androgens through the activity of aromatase. We examine here two aspects of gonadal differentiation, intersexuality and growth, in E. orbicularis. For gonadal intersexuality, we studied the relationship between gonadal aromatase activity and gonadal structure at 28.5 degrees C (pivotal temperature), from the beginning of TSP to hatching, and compared results to those obtained at 30 degrees C (producing 100% females) and 25 degrees C (producing 100% males). At 28.5 degrees C, both males and females are obtained. However, histological differentiation of gonads is delayed compared to that at 25 degrees C and 30 degrees C, and an ovarian-like cortex of various thicknesses often develops at the surface of the male gonads; thus, several individuals display ovotestes at hatching. Despite important individual variations, the aromatase activity in ovaries differentiating at 28.5 degrees C increases during development as in ovaries differentiating at 30 degrees C. In most cases, however, activity is slightly lower than at 30 degrees C, and at the end of embryonic life, it becomes similar to that at 30 degrees C. In testes or ovotestes differentiating at 28.5 degrees C, aromatase activity remains low but is generally slightly higher than in testes at 25 degrees C; however, at the end of embryonic development, it becomes similar to that at 25 degrees C. Oocytes in the cortex of ovotestes begin to degenerate around hatching and continue to degenerate after hatching. Therefore, ovotestes evolve as testes. However, some oocytes may persist at the surface of testes up to the adult age. To estimate gonadal growth, the protein content was measured at different embryonic stages at 25 degrees C and at 30 degrees C. Testis growth is fast during TSP, somewhat slower after TSP, and decreases around hatching. Ovary growth is much slower than testis growth during TSP and then accelerates up to the end of embryonic development. This differential growth is well correlated with gonadal aromatase activity--much higher at 30 degrees C than at 25 degrees C--and can be explained by the fact that during TSP, testicular cords develop at 25 degrees C whereas they are inhibited at 30 degrees C; the ovarian cortex begins to form during this period but grows chiefly after TSP. Both inhibition of testicular cord development and stimulation of cortex development are under the control of endogenous estrogens. In the case of ovotestes, slight increases in estrogen synthesis, compared to that in typical testes, are sufficient to induce the transient formation of an ovarian-like cortex although they do not inhibit the development of testicular cords.

The ovary retains male potential after the thermosensitive period for sex determination in the turtle Emys orbicularis.

Emys orbicularis is a turtle with temperature-dependent sex determination. The thermosensitive period (TSP) lies between embryonic stages 16 and 22. Gonadal differentiation begins during this period involving oestrogens. Treatment with oestrogens during TSP results in the differentiation of ovaries at a male-producing temperature (25 degrees C), whereas treatment with an antioestrogen (tamoxifen) or with nonsteroidal aromatase inhibitors results in gonadal masculinization at a female-producing temperature (30 degrees C). The present study examines the effects on the ovary of inhibiting aromatase activity after TSP. Eggs of E. orbicularis incubated at 30 degrees C were given five or seven applications of 10 micrograms aromatase inhibitor Letrozole (CGS 20267) in ethanol, between stages 22+ and 24-25 when ovarian aromatase activity strongly increases. Individuals which received five applications were sacrificed at stages 24(+)-25. Those which received seven applications were sacrificed either at stage 25+ (close to hatching), or 34-36 days after hatching. Gonadal aromatase activity and related gonadal structure were studied in each individual. In the three series, the gonadal aromatase activity in individuals treated with Letrozole varied from similar or close to that in controls to much lower, and the gonadal structure varied from ovary-like to ovotestis. Ovotestes had the lowest levels of aromatase activity, under 4 fmoles/h/gonad, close to testis levels. They were found in 7 out of 26 individuals given Letrozole. Besides ovotestes, gonads presenting various degrees of masculinization, with enlarged epithelial cords and lacunae in the medulla, were found. Therefore, by inhibiting aromatase activity and thus estrogen synthesis, we were able to obtain the differentiation of testis-like cords or tubes in ovaries of E. orbicularis, after the period of temperature sensitivity. These results show that the ovary retains male potential after this period. Thus, besides their implication during the critical embryonic period for gonadal sex differentiation, oestrogens play a role in maintaining the ovarian structure after this period. A decrease in oestrogen levels could explain some other cases of ovarian masculinization known in vertebrates.

Endocrine sex reversal of gonads by the aromatase inhibitor Letrozole (CGS 20267) in Emys orbicularis, a turtle with temperature-dependent sex determination.

In embryos of Emys orbicularis, the sexual differentiation of gonads is influenced by the incubation temperature of eggs. Estrogens administered during the thermosensitive period result in the feminization of gonads at 25 degrees (male-producing temperature), whereas an antiestrogen or aromatase inhibitors masculinize the gonads at 30 degrees (female-producing temperature). The nonsteroidal aromatase inhibitor Letrozole induces gonads with different degrees of masculinization, from ovary-like to testis-like. The present study examines the endocrine function of such masculinized gonads, at the end of embryonic life. Aromatase activity (which is involved in estrogen synthesis in ovary) and the status of Müllerian ducts (the regression of which reflects the secretion of a putative anti-Müllerian hormone by the Sertoli cells) were examined. One month after treatment with Letrozole, the gonads of embryos presented various levels of aromatase activity. There was a strong correlation among aromatase activity, gonadal structure, and Müllerian duct status; high levels of aromatase (similar or close to those in control females) were found in ovary-like gonads; intermediate levels were found in gonads (masculinized ovaries or ovotestes?) exhibiting a cortex and a composite medulla containing a mixture of ovarian lacunae and testicular cord-like structures; low levels (similar or close to those in control males) were found in strongly masculinized gonads (testis-like or ovotestes). Müllerian ducts were regressing in the majority of embryos with gonads containing low levels of aromatase activity. In these individuals, gonads functioned as embryonic testes. These results confirm the implication of estrogens in gonadal differentiation. The origin of these hormones is controversial, so that the aromatase activity was compared in gonads, in the undissociated adrenal-mesonephric complex (AM), and in different parts of this complex during the thermosensitive period. At the female-producing temperature, the aromatase activity per unit of tissue increased in differentiating ovaries but it was low in AM and similar to that found in AM at male-producing temperature. In embryos whose gonads had been masculinized by early treatment with Letrozole, aromatase activity was unchanged in AM. These results suggest that the main source of estrogens involved in ovarian differentiation is the gonad itself.

Temperature-dependent gonadal differentiation in the turtle Emys orbicularis: concordance between sexual phenotype and serological H-Y antigen expression at threshold temperature.

As in many other turtles, the sexual differentiation of gonads in embryos of Emys orbicularis is temperature-sensitive, 100% phenotypic males being obtained below 27.5 degrees C and 100% phenotypic females above 29.5 degrees C. The expression of the serologically defined H-Y (SD-H-Y) antigen at both low and high temperatures has been shown to be different in gonads and in blood : in gonads, it is closely associated with ovarian structure, whereas in blood it is independent of sexual phenotype and appears to indicate sexual genotype. Both sexes differentiate at 28.5 degrees C, suggesting that at this intermediate (threshold) temperature, sexual differentiation of gonads conforms with sexual genotype. To test this hypothesis, the expression of SD-H-Y antigen has been carried out in blood cells of Emys individuals raised from eggs incubated at the threshold temperature (28.5 degrees C). All phenotypic males typed SD-H-Y negative, whereas most phenotypic females typed SD-H-Y positive. From this concordance between sexual phenotype of gonads and SD-H-Y phenotype of blood, we postulate that a ZZ male/ZW female mechanism of genotypic sex determination is revealed at the threshold temperature for gonad differentiation in Emys.

A memory effect in DNA replication.

A study of the polymerization/excision ratio in the replication of poly(dA), primed with oligo(dT), was carried out with E. coli DNA polymerase I, at various primer and enzyme concentrations. The variations in this ratio suggest that 1) the DNA polymerase is able to switch between two states of low and high exonuclease activities and 2) after dissociating from the template, the DNA polymerase drifts towards the low exonuclease state. The recovery of the high exonuclease state would require several successive incorporations.

Polymerization/excision kinetics of Escherichia coli DNA polymerase I. Stability in kinetic behaviour and variations of the rate constants with temperature and pH.

1. The incorporation and excision kinetics of Escherichia coli DNA polymerase I in the replication of (dA)n with dUTP and (dI)n with dCTP was studied at various temperatures and pH. When the incorporation/excision ratio (dy/dx) is plotted against the concentration of deoxynucleoside triphosphate [S] two kinds of curves are obtained. With (dI)n, dy/dx increases with [S], then reaches an asymptotic value. This behaviour, consistent with a kinetic amplification or kinetic proofreading mechanism, is observed at all temperatures and pH. With (dA)n, dy/dx increases with [S] but in a convex, instead of a concave manner. In this case, we approximated the curves by straight lines at the origin, in conformity with the prediction of the frayed-unfrayed model. Again a single behaviour is consistently observed at all temperatures and pH. 2. The data were analyzed in terms of ratios or products of three kinetic constants: ki for incorporation, ke for excision and, in the (dI)n system, theta for the processing of the incoming dNTP. The dNMP production in the (dI)n system is dominated by the ke theta term which increases with temperature and pH. Temperature influences excision more than incorporation, the net result being a linear decrease of ki/ke with temperature. The effect is more pronounced with (dI)n than with (dA)n and is probably related to the stability of the template-primer complex. The ki/ke term shows a bell-shaped dependency with pH in the (dI)n system. With (dA)n it remains constant between pH 7.5 and 8.5 then decreases with a transition midpoint at pH 9.0. We suggest that the pH profiles may reflect the ionization of the template in the first case, and of the substrate in the second.

H-Y antigen expression in temperature sex-reversed turtles (Emys orbicularis).

H-Y antigen has been used as a marker for the heterogametic sex and is assumed to be an organizing factor for the heterogametic gonad. In the turtle Emys orbicularis, H-Y antigen is restricted to the female cells, indicating a female heterogamety (ZZ/ZW) sex-determining mechanism. Moreover, the sexual differentiation of the gonads is temperature sensitive, and complete sex reversal can be obtained at will. In this framework the relationships between H-Y antigen, temperature, and gonadal phenotype were studied. Mouse H-Y antiserum was absorbed with blood and gonadal cells of control wild male and female adults, and with blood and gonadal cells from three lots of young turtles from eggs incubated at 25-26 degrees C (100% phenotypic males), at 30-30.5 degrees C (100% phenotypic females), or at 28.5-29 degrees C (majority of females with some males and intersexes). The residual activity of H-Y antiserum was then estimated using an immunobacterial rosette technique. In adults, both blood cells and gonadal cells were typed as H-Y negative in males and as H-Y positive in females. In each of the three lots of young, blood cells were H-Y negative in some individuals and H-Y positive in others. The proposed interpretation is that the H-Y negative individuals were genotypic males (ZZ) and the H-Y positive were genotypic females (ZW). The gonads of these animals were then pooled in different sets according to their sexual phenotype and to the presumed genotypic sex (i.e., blood H-Y phenotype). Testicular cells were typed as H-Y negative in genotypic males as well as in the presumed sex-reversed genotypic females; likewise, ovarian cells were typed as H-Y positive in genotypic females as well as in the presumed sex-reversed genotypic males. These results provide additional evidence that H-Y antigen expression is closely associated with ovarian structure in vertebrates displaying a ZZ/ZW sex-determining mechanism.

Aminoacylation of tRNA Trp from beef liver, yeast and E. coli by beef pancrease tryptophan-tRNA ligase. Stoichiometry of tRNATrp binding.

The Michaelis constants and the maximum velocities in the aminoacylation reaction of tRNATrp from beef liver, yeast and E. coli by pure beef pancreas tryptophan-tRNA ligase show that this mammalian enzyme recognizes and charges the two eucaryotic tRNAs with the same efficiency. The rate of aminoacylation of the procaryotic tRNATrp by the enzyme is three orders of magnitude lower. The pH optimum of aminoacylation is 8 for both eucaryotic tRNAs. The optimum magnesium concentration is different. The rate is maximum when magnesium concentration is stoichiometric to ATP concentration for tRNATrp from beef liver and 10 mM above ATP concentration for tRNATrp from yeast. The number of binding sites on the enzyme for the two eucaryotic tRNAs has been measured by equilibrium filtration on Sephadex G-100 and found equal to two.

Purification of tRNATrp, tRNAVal, and partial purification of tRNAIle and tRNAMfet from beef liver.

tRNATrp from beef lever has been purified by classical chromatographical methods. Total tRNA, prepared on a large scale (total aminoacid acceptance 1280 pmol/A260 unit) was submitted to chromatography on benzoylated-DEAE cellulose, then DEAE Sephadex. The major species accepting tryptophan issued from the second chromatography was aminoacylated with [14C] tryptophan and chromatographed on benzoylated-DEAE cellulose. The tRNA carrying the radioactive label was eluted in the ethanolic region. After stripping, the resulting tRNATrp has an acceptance of 1800 pmol/A260 unit. No isoacceptors could be demonstrated by chromatography of the pure species on RCP 5 in 6 M urea. The yield in pure tRNATrp was currently in the range of 25 to 30 percent of the total tryptophan acceptance of the starting curde tRNA.

Increase in the proportion of cells with the C'3 receptor in BALB/c mice bearing mammary tumors.

Transplantation of mammary tumors originally induced by dimethylbenzanthracene caused splenomegaly and an increase in the total population of spleen cells paralleling the increase in tumor size. At least part of the hypercellularity was due to in situ proliferation as evidenced by the increased number of blast forms and mitotic figures. The cells were characterized with respect to parameters generally associated with B-cells; surface immunoglobulins as detected by immunofluorescence and C'3 receptors as determined by rosette formation with sheep red blood cells (SRBC) coated with rabbit anti-SRBC and mouse complement. There was no change in the percentage of B-cells with surface immunoglobulin in tumor-bearing mice compared with the control animals. However, there was a profound change in the representation of cells bearing C'3 receptors. The percentage of the cells increased dramatically with tumor growth. Various possibilities regarding the nature and function of these cells are considered.