Impaired development of CD4+ CD8+ thymoyctes by csk-'knock-in' into fyn locus.

p59fyn is one of the Src-family kinases thought to play an important role in signaling through T cell receptor. However, Fyn deficiency has caused no overt defects in vivo on T cell development, nor has it caused any changes in the phosphorylation status of molecules such as ZAP-70 which have been proposed as p59fyn substrates. This could be explained as being due to compensation of Fyn deficiency by other Src-family kinases. Here, we have 'knocked-in' the csk gene, a negative regulator of Src-family kinases, into fyn locus to challenge the problem of redundant functions among Src-family kinases. The csk-'knock-in' mice displayed atrophy of the thymic cortex and impaired development of CD4+ CD8+ thymocytes. This was concomitant with decrease in tyrosine phosphorylation of ZAP-70 and p120cbl.

Effects of inhibin on rat gonadal differentiation and development in vitro.

Previously we examined that inhibin-alpha subunit, transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF) were expressed in sex-, cell- and stage-specific manners in perinatal rat gonads. To clarify effects of these growth factors on the rat gonadal differentiation and development, indifferent gonadal primordia with mesonephric tubules on gestational day 13 were cultured in vitro for 4 days in serum-free CMRL-1066 medium with inhibin, TGF-beta 1, EGF, anti-sera against these growth factors, testosterone or estradiol-17 beta, and then morphologically examined with reference to seminiferous tubule formation, germ cell division, Wolffian and Müllerian duct development. In male gonads, anti-inhibin-alpha serum suppressed the seminiferous tubule formation but inhibin, TGF-beta 1, EGF or steroid hormones did not affect on the tubule formation, germ cell proliferation nor gonoduct development. Seminiferous tubules in male gonads cultured in the medium containing anti-inhibin-alpha serum were incomplete and irregular in shape. On the other hand, in female gonads, inhibin suppressed the germ cell division and anti-inhibin-alpha serum led to the necrosis of germ cells, but other factors affected to neither sex cord formation nor germ cell division. Testosterone and estradiol-17 beta stimulated female Wolffian and Müllerian duct development, respectively. These results indicate that inhibin induces the seminiferous tubule formation and suppresses the female germ cell division in developing rat gonads in vitro.

p59fyn-p125FAK cooperation in development of CD4+CD8+ thymocytes.

p59fyn is an Src family nonreceptor tyrosine kinase that has been suggested to play an important role in T-cell development and function. p125FAK is a unique nonreceptor tyrosine kinase and has been known to respond to integrin-extracellular matrix interactions. To examine their roles in thymocytes, heterozygous fak mutation was introduced into homozygous Fyn deficiency. The double mutation, but neither Fyn deficiency nor FAK heterozygosity alone, displayed impaired development of CD4+CD8+ thymocytes with atrophy of the thymic cortex, suggesting a unique cooperation between p59fyn and p125FAK in CD4+CD8+ T-cell development.

Immunohistochemical localization of insulin-like growth factor-II in the perinatal rat gonad.

To clarify the participation of insulin-like growth factor type-II (IGF-II) in rat gonadal differentiation, immunohistochemical localization of IGF-II was chronologically studied in Sprague-Dawley rat gonads from gestational day (GD) 13 to postnatal day (PD) 21 by using avidin-biotin complex technique. In male gonads, most cells were negative to IGF-II immunostaining during the perinatal period. Only Leydig/interstitial cells expressed positive reactivity from GD 21 to PD 11: the intensity of staining and the number of positive cells were gradually increased until PD 11. In female gonads, almost all cells showed negative immunoreactivity. Mesonephric tubules in both sexes exhibited slight or moderate reactivity on GD 13. These results indicate that IGF-II is likely to participate in the regression of fetal-type Leydig cells and/or the proliferation of adult-type Leydig cells around birth, and in the development of mesonephric tubules in the initial stage of gonadal differentiation.

Integrin stimulation decreases tyrosine phosphorylation and activity of focal adhesion kinase in thymocytes.

FAK, focal adhesion kinase, is expressed in a variety of cell types and has been suggested to transduce signals brought about by integrin-extracellular matrix (ECM) interactions. Integrin stimulation increases tyrosine phosphorylation and activity of FAK in all the cells examined to date. In contrast, in thymocytes stimulation of VLA-4 (alpha 4 beta 1) and LFA-1 (alpha L beta 2) resulted in a marked decrease in tyrosine phosphorylation and activity of FAK.

Immunohistochemical detection of the expression of the alpha subunit of inhibin, TGF-beta, basic-FGF and IGF-II in fetal ovarian grafts grown with fetal testes beneath the kidney capsule of adult castrated male rats.

In order to characterize the participation of growth factors in gonadal differentiation and development, we examined patterns of expression of the alpha subunit of inhibin, transforming growth factor-beta (TGF-beta), basic fibroblast growth factor (basic FGF) and insulin-like growth factor-II (IGF-II) immunohistochemically in experimentally induced ovotestes. Ovotestes were derived from ovaries of fetal rats on gestational day (GD) 13 that had been co-crafted with fetal testes (GD 17) beneath the kidney capsule of adult castrated males and examined on the 7th, 14th and 21st days after transplantation (TD). Reactivity with antibodies against the alpha subunit of inhibin and basic FGF was observed in the Sertoli cells in both ovotestes and testes on TD 14 and on TD7, 14 and 21, respectively. Expression of IGF-II was also recognized in the Leydig/interstitial cells in both types of graft on TD 14 and 21. Therefore, the gonadal somatic cells in the testicular region of the ovotestes had immunohistochemical properties similar to those in the cografted testes. However, the somatic cells in the ovarian region of the ovotestes had immunohistochemical profiles different from those in solitary grafted ovaries. The germ cells in the ovotestes showed some differences in patterns of expression when compared with those in cografted testes and solitary grafted ovaries: expression of basic FGF was recognized in the germ cells in ovotestes on TD 21 but not in co-grafted testes; expression of IGF-II was recognized in the germ cells in ovotestes on TD 21 but not in solitary grafted ovaries. These results indicate that the immunohistochemical properties that reflect expression of growth factors in female gonadal somatic cells were changed to properties that resemble those of male gonads by the co-grafted fetal testes.

Developmental pattern of androgen receptor immunoreactivity in the mouse submandibular gland.

We studied the immunohistochemical localization of androgen receptor in the mouse submandibular gland, and developmental profiles of its expression using polyclonal human androgen receptor antibody. In the submandibular glands of both sexes, specific immunoreactivity appeared only in cell nuclei of the acini, the intercalated ducts, the granular convoluted tubules (GCT) and the excretory striated ducts. The percentage of immunoreactive cells in each region gradually declined with age during the first 90 days of postnatal development studied. The sexual difference in the percentage of immunoreactive cells was observed in the acini on days 20 and 30 and in the GCT on day 30. Incidence of immunoreactive cells in the female was significantly smaller than that in the male. On day 60, the percentage of immunoreactive cells of these two regions turned to increase slightly in the female but continued to decline in the male, and then it became higher in the female than in the male. In addition, one-week castration did not cause any changes in the intracellular distribution of androgen receptor and the percentage of immunoreactive cells in each region of the adult gland. These results suggest that androgen receptor is localized primarily in cell nuclei in all four regions of the mouse submandibular gland in situ, and that its expression in acini and GCT is superior in the male around days 20 to 30, when sex difference of the gland becomes evident.

Extrusion of rotating microtubules on the dynein-track from a microtubule-dynein gamma-complex.

Applying a new in vitro motility assay system for microtubules and 22S dynein, we recently reported on an ATP-induced extrusion of microtubules from microtubule-dynein alpha- and beta-complexes [Mimori and Miki-Noumura, 1994: Cell Motil. Cytoskeleton 27:180-191]. In the present study, we prepared a gamma-complex by copolymerizing porcine brain tubulin and Tetrahymena ciliary 22S dynein, and examined the ATP-induced microtubule movement from the gamma-complex. The extrusion process appeared quite similar to that of the beta-complex. The sliding velocity was 18.39 +/- 2.20 microns/sec, which was a value comparable to that of trypsin-digested flagellar axonemes [Yano and Miki-Noumura, 1980: J. Cell Sci. 44:169-186]. Higher velocity may be due to a densely arranged dynein-track with the same polarity, which was detached from the gamma-complex and absorbed in rows on a glass surface of the slide. Sometimes a free-floating microtubule in the perfusion chamber was observed riding and sliding on the dynein-track remaining on the slide after extrusion. Unexpectedly, we found that when the front part of the microtubule was fixed to a glass surface, a continuous sliding microtubule at the rear part on the dynein-track often transformed into a left-handed helix, and subsequently a twisted helix with several turns. The helix formation may be due to some rigidity in the microtubule and a right-handed torque component in the sliding force of 22S dynein. The addition of ATP may release some distortion accumulated in the complex structure during copolymerization of tubulin and 22S dynein, inducing reverse rotation of the microtubule.

Regulatory role of nucleotides in axonemal function.

Axonemal sliding involves both sliding velocity and the extent of sliding, that is how many doublets slide. It is clear that axonemes cannot beat if all doublets were to slide simultaneously, thus sliding extent is important. Using the turbidimetric assay of sliding disintegration of Tetrahymena axonemes, we examined the sliding extent and th effect of APD, ATP, and ATP analogs on the sliding extent. Of course, ATP is necessary to produce sliding disintegration, but ATP alone did not produce extensive sliding disintegration. The additions of higher ATP concentration even in the presence of ADP inhibited sliding disintegration. We also observed sliding disintegration using ribose-modified ATP analogs, anthraniloylATP, and methylanthraniloylATP. The extent of sliding disintegration was proportional to the analog concentration. Thus in contrast to ATP, higher analog concentration was not inhibitory. These results indicate that high ATP concentration acts to inhibit the extent of sliding disintegration and that ADP relieves this inhibition. We propose a model in which the affinity of of multiple cooperative active sites are regulated by the binding of ATP or ADP to a regulatory site. This model provides a mechanism by which nucleotides regulate the extent of sliding necessary for effective axonemal bending.

Cell- and stage-specific expression of basic FGF in the developing rat gonads.

In order to clarify the participation of basic fibroblast growth factor (bFGF) in rat gonadal differentiation and development, immunohistochemical localizations of bFGF were chronologically studied in Sprague-Dawley rat gonads from gestational day (GD) 13 to postnatal day (PD) 21 by using avidin-biotin complex technique. Immunohistochemical reactivity to bFGF antibody was positive in the germ cells. Slight or moderate immunostaining was seen in male germ cells from GD 18 to PD 5, and in female germ cells from PD 5 to 21. Leydig/interstitial cells in male gonads were slightly or moderately stained from GD 16 to PD 21. On the other hand, other types of gonadal cells were not stained during the perinatal period. These results indicate that bFGF shows discrete cell- and stage-specific patterns of expression in gonads during the perinatal period and is likely to participate in control of gonadal development in rats.

ATP-induced sliding of microtubules on tracks of 22S dynein molecules aligned with the same polarity.

Chlamydomonas and Tetrahymena axonemal dyneins have previously been found to bind to porcine brain microtubules to produce a microtubule-dynein complex. At appropriate microtubule:dynein concentration, microtubules in the complex became covered to saturation by dynein arms of the same polarity and at a spacing of 24 nm [Haimo et al., 1979; Haimo and Fenton, 1988; Haimo, 1989; Porter and Johnson, 1983a]. In the present study, two different types of microtubule-dynein complexes (alpha- and beta-complexes) were prepared from Tetrahymena ciliary 22S dynein and porcine brain tubulin. The characteristics of the adenosine triphosphate (ATP)-induced extrusion of microtubules from these complexes were analyzed, as a simple and direct in vitro assay for the ATP-induced extrusion of singlet microtubules. The alpha-complex prepared by adding dynein to microtubules showed an interrupted sliding movement, which would stop and start several times following the addition of ATP. In the beta-complex, prepared by adding dynein bound to DEAE-tubulin to pre-assembled microtubules, microtubules became covered with dynein molecules whose orientation and binding were uniform with respect to microtubule polarity. The microtubules in the beta-complex extruded at 12 microns/second following the addition of ATP. Dark-field and electron microscopy indicated that the extruded microtubules had undergone sliding on a dynein-track that had become detached from the complexes and had been absorbed onto the surface of the glass slide. At higher light intensity under a dark-field microscope, the dynein-track was seen to be composed of rows of dynein molecules arranged densely. The orientation of dynein molecules in rows appeared to be uniform, considering the images of bound dynein in the beta-complex under electron microscope. The higher sliding velocity of the microtubules on these dynein-tracks compared to that seen on slides coated at random with dynein [Vale and Toyoshima, 1988, 1989], may be due to more efficient force generation by this dense arrangement of dynein molecules with the same polarity on the tracks.

Stabilization of microtubules by dynein-binding in vitro. Stability of microtubule-dynein complex.

We have studied the effects of dynein binding on the stability of microtubules in vitro, using Tetrahymena ciliary dynein and microtubules (three-cycled purified microtubules: 3 X-Mts and phosphocellulose-column purified microtubules: PC-Mts). To determine the relative stability of the microtubules, we first prepared the microtubules bound with dynein (Mts--dynein complex) and subjected the Mts-dynein complex to treatments that depolymerize the microtubules, such as dilution to below critical concentration of tubulin, calcium ions and lower temperature. Dark-field microscopy revealed that the microtubules in the Mts--dynein complex appeared intact under conditions which otherwise result in microtubule depolymerization. However, when dynein was dissociated from the Mts--dynein complex with addition of ATP, no microtubule was found in the specimens under the same conditions. That is, the microtubules in the Mts--dynein complex did not depolymerize upon dilution with the buffer solution to below critical concentration of tubulin. However, addition of ATP to the diluted specimen caused dynein to become separated from the Mts, resulting in complete depolymerization of the microtubules. Stability of the microtubules was also studied by the turbidity changes and was confirmed by the patterns of stained gel bands in electrophoresis. With the addition of calcium ion, the Mts--dynein complex decomposed into separate molecules dynein and tubulin. At the lower temperature of 0 degrees C, the 3 X-Mts--dynein complex was decomposed into dynein and tubulin, while the microtubules in the PC-Mts--dynein complex did not depolymerize. Although we have not yet studied the effects of cytoplasmic dynein binding on the microtubules, the results suggest that the stabilizing effect of dynein binding to the microtubules is one of the important functions of dynein in vivo.

Age-related changes in the mitogenic activity of heparin-binding growth factors in rat sera.

Sera from rats of either sex and different ages were examined for their ability to stimulate DNA synthesis in BALB/c 3T3 cells. The activity levels of sera from male and female rats were almost the same, with age-related changes in activity also being quite similar. Activity was considerably higher in infant rats (1-month-old), but then, at a young age (6-7 months), decreased drastically for male rats, but not significantly for female rats. It increased again in middle-aged rats (12-13 months old) and was maintained at the same level toward old age (24-26 months old) for both sexes. In order to determine what kinds of growth factors were responsible for these changes, we carried out heparin affinity chromatography on the sera of male rats. Four peaks were obtained for all sera, with individual peaks exhibiting specific age-related changes in activity. Among them a peak which was eluted at 1.1 M NaCl had very high activity. It showed a similar age-related change to that of the whole sera, except for a significant increase at old age, and the factor(s) included in the peak was found to be derived from platelets. These results suggested that the factor(s) in the peak was responsible for maintaining serum mitogenic activity at an old age. The experiments undertaken to characterize this factor suggested that it is a novel one.

Type I collagen promotes modulation of cultured rabbit arterial smooth muscle cells from a contractile to a synthetic phenotype.

The phenotypic transition of smooth muscle cells (SMC) from a contractile to a synthetic state appears to be an early event in the pathogenesis of atherosclerosis. We examined the effects of extracellular matrix components on the phenotypic modulation of rabbit arterial SMC in primary culture by flow cytometry. The results demonstrate that freshly isolated SMC attached, spread, and started to proliferate on type I collagen as well as on fibronectin. Moreover, type I collagen was as efficient as fibronectin in promoting the transition of the cells into the synthetic phenotype without exogenous mitogens. However, unlike on fibronectin, the synthetic peptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) and the peptide KDGEA (Lys-Asp-Gly-Glu-Ala), which contains the recognition sequence for alpha 2 beta 1 integrin in type I collagen, interfered little with the attachment, spreading, and phenotypic modulation of the cells on type I collagen. On the other hand, the phenotypic modulation of the cells was counteracted by the anti-beta 1 integrin antibody. These findings indicate that type I collagen promotes the phenotypic transition of the rabbit arterial SMC by interacting with a cell surface receptor (beta 1 integrin family) for a cell-binding sequence without RGD and DGEA. In contrast, elastin, a major constituent of the media, suppressed the cell attachment and spreading and maintained the cells in the contractile phenotype as laminin. These results suggest diverse roles of type I collagen and elastin as well as of fibronectin and laminin in the control of the differentiated properties of arterial SMC.

Inhibition of gliding movement by calcium in doublet microtubules on Tetrahymena ciliary dyneins in vitro.

We examined the effects of Ca ions on the gliding movement of Tetrahymena ciliary doublet microtubules induced by 14S or 22S dyneins in an in vitro motility assay system. The doublet microtubule appeared as circular-arc in solution, about 5 to 6 microns in length [1]. The doublet microtubules glided distal-end first on a 14S or 22S dynein-coated glass surface either clockwise or counterclockwise following the addition of ATP. The diameter of the circular path changed according to Ca concentration in the solution. Gliding velocity was from 1 to 5 microns/s. The addition of 0.1% Nonidet P-40 was necessary to induce the gliding movement on 22S dynein. This movement on 22S dynein was strongly inhibited above 0.5 mM ATP in the presence of 10(-9) M Ca, and at 0.05 to 1 mM ATP in the presence of 10(-3) M Ca. Many studies have indicated that Ca ions regulate ciliary movement [2-8] in which dyneins and doublet microtubule in the axoneme may play an essential role. The inhibition of the gliding movement of doublet microtubule on dyneins at appropriate concentrations of Ca and ATP as observed in this study may be the key for understanding Ca regulation of ciliary motility.

Effects of sex steroids on the development of sexual dimorphism in mouse innominate bone.

The sexual dimorphism of the innominate bone was examined in 14 strains of mice. In female mice of all strains, the pubis was significantly longer and thinner than that in the strain-matched males. In 13 of 14 strains, the ischium in the male was longer and thicker than in the female. In the testicular-feminized male (Tfm) mouse, the ischium was longer and thinner than that in the wild-type male, resembling that of the wild-type female. The pubis of the Tfm mouse was longer than in the wild-type males. The pubis width in the Tfm mouse was between those of the wild-type male and female. Gonadectomy at ages of 5, 10, 20, 30, and 60 days in both sexes showed that the ischium develops as the female type when sex hormones are absent. In contrast, postnatal testicular androgen induces the male-type ischium. Gonadectomy at 60 days had a slight effect on the pubis, indicating that sexual dimorphism of the pubis was determined before 60 days of age. Estrogen receptors (ER) were immunohistochemically demonstrated in bone cells of 0- to 60-day-old mice. ER was found exclusively in the periosteum of the pubis at the day of birth; however, it appeared in bone cells of all parts of pelvis at 10-60 days. These results indicate that sexual dimorphism of the pubis is consistent for the 14 mouse strains examined, and that the shape of the pubis is determined by sex steroids before 60 days of age. Since ER exist in the bone cells, morphogenesis of the pelvis may be regulated by these sex steroids.

Disassembly of F-actin filaments in human endothelial cells cultured on type V collagen.

We examined the inhibitory activity of type V collagen on cell attachment and cell growth and the role of stress fibers and beta 1 integrin in cultured human endothelial cells. Human endothelial cells cultured on type V collagen attached temporarily to the substrate and formed stress fibers. However, the cells failed to proliferate and gradually detached from the substrate. After 24 h, the cells on type V collagen lacked discernible stress fibers (F-actin filaments) and exhibited dots in small aggregates of F-actin. In addition, the cells expressed little or no proteins as focal adhesions, including vinculin and beta 1 integrin. In contrast, the cells on fibronectin and type I collagen formed complete F-actin filaments, exhibited sufficient vinculin and beta 1 integrin, and grew logarithmically from 2 days. On the other hand, human smooth muscle cells formed complete F-actin filaments, revealed typical focal adhesions, and started to proliferate rapidly after 24 h on type V collagen as well as on fibronectin and type I collagen. Thus, the disassembly of F-actin filaments was observed as a specific phenomenon in human endothelial cells cultured on type V collagen. Moreover, the F-actin filaments disappeared from endothelial cells treated with cytochalasin D after 24 h and the cells detached from fibronectin and type I collagen with time, a result consistent with the observations on type V collagen. Accordingly, the disassembly of F-actin filaments in focal adhesions may result in the detachment of endothelial cells from type V collagen.

ATPase sites in two-headed fragment of Tetrahymena 22S ciliary dynein.

Three-headed Tetrahymena 22S ciliary dynein was found to consist of three heavy chains (HCs) and decompose into two-headed and single-headed fragments upon chymotrypsin digestion. The three HCs (A alpha, A beta, and A gamma) were immunologically different, and presumed to be located on each of the head regions. The two-headed fragment contained A beta and A gamma HCs, while the A alpha HC originated in the single-headed fragment. Both fragments were associated with ATPase activity (Toyoshima, Y. (1987a) J. Cell Biol. 105, 887-895 and Toyoshima, Y. (1987b) J. Cell Biol. 105, 897-901). Using the two-headed dynein fragment, we attempted to determine the site of ATP hydrolysis in the fragment. After digestion of the fragment with 100 micrograms/ml thermolysin for 45 min, we noted eight thermolysin-digested polypeptides (TH 1, 2, 3, 4, 5 alpha, 5 beta, 6 alpha, and 6 beta). By precisely analyzing the degradation process and the products using peptide mapping, immunoblotting and high pressure liquid chromatography, it appeared that the two-headed fragment is dissociated as two separate fragments, each of which contained A beta or A gamma HC. Thermolysin digests, TH 1, 2, 5 alpha and 6 beta were found to be derived from A beta HC, while TH 3, 4, 5 beta and 6 alpha originated in the A gamma HC. Based on the measurements of ATPase activity of these polypeptides, we concluded that the ATPase site is located in the A beta and A gamma HCs, which may have their origins in each head of the two-headed fragment of Tetrahymena 22S ciliary dynein.

Identification of the phenotypic modulation of rabbit arterial smooth muscle cells in primary culture by flow cytometry.

In atherosclerotic lesions, smooth muscle cells (SMC) change from a contractile to a synthetic phenotype. The in vivo and in vitro phenotypic transformations of SMC have been confirmed by transmission electron microscopy (TEM), but the relationship between this change and the cell cycle is still unknown. We demonstrated the structural modulation of rabbit arterial SMC in primary culture by TEM and immunocytochemistry and simultaneously studied changes in two-dimensional histograms of the relative DNA and RNA contents by flow cytometry. During the first day of primary culture, the cells exhibited the contractile phenotype and were composed of a population in the G0 phase characterized by low contents of DNA and RNA. On the second day of culture, some of the cells (18.2%) had started but not completed the transition into the synthetic phenotype and a cell population in the G1A phase with an RNA content above the G0 level appeared in almost the same proportion. This cell population could be categorized as an "intermediate" type. Moreover, after 3 days when about three-quarters of the cells had undergone structural transition, the same proportion of cells had entered into the cycling phase, while some cells still remained in the G0 and G1A phases. Thus, cell cycle analysis by flow cytometry corresponded well with the observations obtained by TEM and immunocytochemistry. These results show that flow cytometry can rapidly and relatively conveniently monitor the process of phenotypic modulation in SMC and is a useful method for the analysis of such transitions.

Sexually dimorphic duct system of the submandibular gland in mouse with testicular feminization mutation (Tfm/Y).

The X-linked testicular feminization mutation (Tfm/Y) in the mouse is characterized by androgen insensitivity of the target cells. The aim of this study was to examine sexually dimorphic development of the submandibular gland of Tfm/Y mutant mice in comparison with those of wild-type male, wild-type female and heterozygous Tfm female mice. In either 30- or 90-day-old wild-type male mice, the granular convoluted tubules (GCT) of the glands were more developed, and the relative occupied areas (ROA) of GCT were superior to those of the age-matched wild-type and heterozygous Tfm females. In androgen-insensitive Tfm/Y mice, the glandular structures rather resembled the female glands, showing lower values of the ROA of the GCT. Sex differences in the mitotic rate were observed at 30 days of age, being significantly higher in the wild-type male GCT than in the female GCT. Thereafter, the mitotic rate of the wild-type male GCT declined to the female levels by 90 days of age. The mitotic rate of GCT in Tfm/Y mutants was as low as those of the females during observation periods. An other three regions, the acini, the intercalated ducts and the excretory striated ducts, were not significantly different in either the ROA or the mitotic rate among wild-type males and females, and Tfm/Y. On the other hand, either the ROA or the mitotic activity of GCT of the glands in Tfm/Y mutants was completely unaffected by 5 alpha-dihydrotestosterone (DHT).(ABSTRACT TRUNCATED AT 250 WORDS)