Maintenance and characterization of human myometrial smooth muscle cells in monolayer culture

Summary Human myometrial cells were dispersed from uterine tissue by limited enzymatic digestion of myometrium that was obtained at the time of hysterectomy. The dispersed myometrial cells that are obtained in this manner can be maintained in monolayer culture in the presence of medium that contains fetal bovine serum. In primary culture, as well as after passage, the characteristics of these cells are morphologically and biochemically similar to those of smooth muscle cells and myometrial tissue. This investigation was supported, in part, by U.S. Public Health Service Grants 5-P50-HD11149 and 5-P01-AG00306.     

Effect of progesterone and oestradiol on expression of connexin43 in cultured human myometrium cells

In human myometrium, the formation of gap junctions at various stages of labour and in correlation with the concentration of progesterone and oestradiol in maternal blood was described previously by electron microscopy and laser confocal microscopy of immunohistochemically stained myometrial sections. The present investigation focused on the effect of continuous exposure of isolated myometrial tissue to progesterone and oestradiol on the number of gap junction plaques in human myometrium cells in vitro. The presence of gap junctions was evaluated by immunocytochemistry with antibodies against gap junction protein, connexin43 (Cx43). Human myometrial cells were isolated from biopsies obtained from term pregnant women who had an elective caesarean operation in the 37th or 40th week of pregnancy. The dispersed myometrial cells that were obtained by limited enzymatic digestion of the myometrial samples were maintained in monolayer culture for 1, 3 and 6 days in the presence of medium that contained foetal bovine serum and the steroids at different concentration. In primary culture, as well as after several passages, the characteristics of these cells were morphologically and biochemically similar to those of smooth muscle cells and myometrial tissue. The obtained results showed that the cells in culture responded synchronously to the increased concentrations of oestradiol/progesterone mixtures. The number of gap junctions increased significantly on days 1, 3 and 6 in culture and showed positive correlation (p < 0.05) with the cell number when the concentration of oestradiol was raised to 1 microgram/mL in the progesterone ratio (1.0 microgram/0.5 microgram/mL). No significant correlation, however, in connexin43 gap junction number versus cell number was observed between the six experimental groups treated with progesterone only.     

Biochemical markers of contraction in human myometrial smooth muscle cells in culture

Summary Phosphorylation of a light chain subunit of myosin by Ca²⁺ and calmodulin-dependent myosin light chain kinase is believed to be essential for smooth muscle contraction. The biochemical properties of the myosin phosphorylation system in human myometrial smooth muscle cells in monolayer culture were compared with those of human myometrial tissue and nonmuscle cells in culture. Native myosin was isolated from other cellular proteins of crude homogenates by polyacrylamide gel electrophoresis (in the presence of pyrophosphate) and quantified by densitometry. The myosin content of myometrial smooth muscle cells in culture and that of myometrial tissue were similar and four- to five-fold greater than that of human endometrial stromal cells or skin fibroblasts in culture. The specific activities of myosin light chain kinase in homogenates of myometrial smooth muscle cells that were maintained in culture and in myometrial tissue were similar (2.05±0.18 and 1.60±0.37 nmol phosphate incorporated per min per mg protein, respectively). On the other hand, enzyme activity in skin fibroblasts was only 5% of that in myometrial smooth muscle cells. Myosin light chain kinase activity in myometrial smooth muscle cells was dependent upon Ca²⁺ and was inhibited reversibly by the calmodulin antagonist, calmidazolium. The intracellular Ca²⁺ concentration measured by quin2 fluorescence was 0.12 μM in resting cells and increased in a concentration-dependent manner with KC1 to a maximal value of 0.47 μM. These results indicate that biochemical processes important for smooth muscle contraction are retained in human myometrial smooth muscle cells in culture. This research was supported by grants HL26043, HD11149, and GM07062 from the National Institutes of Health, Bethesda, MD.     

Evolution of cAMP phosphodiesterase activity in cultured myometrial cells: Effects of steroids and of successive subcultures

Cyclic AMP phosphodiesterase (PDE) activity was characterized in culture of ewe myometrial cells and its sensitivity to steroid hormones was tested. Cultured myometrial cells were maintained from the first to the 20th subculture in the presence of 2% of serum in a medium supplemented with 1 μM of insulin. It was found that myometrial cells possess a PDE activity with atypical kinetics. The nonlinear responses in Lineweaver-Burke plots suggest the presence of high- and low-affinity PDE activities. In cell culture, apparent Km values were similar to those obtained from the original myometrium. Vmax values increased with successive subcultures, revealing an increase in the capacity of the cells to degrade cAMP; in parallel, the growth rate decreased. The PDE specific activity in cultured myometrial cells was inhibited by estra-diol or progesterone. When added together, no synergistic effect was obtained. The rate of inhibition for both steroids was constant during successive passages for both low- and high-affinity conditions. Results obtained in myometrial cell long-term culture were compatible with reports in other species in vivo. Considering the role of cAMP in the regulation of uterine functions, subcultured myometrial cells provided us a useful experimental system with which to study the cAMP metabolism process.     

Effect of a diabetic state on myometrial ultrastructure and isolated uterine contractions in the rat

Alterations in rat myometrial ultrastructure and in vivo uterine contractile responses to oxytocin were examined in estradiol-treated (40 micrograms/kg) euglycemic and streptozotocin-induced (85 mg/kg) diabetic rats. Myometrial morphology was examined 18, 24, and 36 hr after estradiol administration. At the time points examined, nuclei of myometrial cells from euglycemic and diabetic rats were pleomorphic and contained large areas of heterochromatin dispersed throughout the nuclei. Mitochondria were round to oval in shape and contained a dense matrix with cristae that extended across the mitochondria. Myofilaments were found in both euglycemic and diabetic cells but the relative number of myofilaments in diabetic cells appeared to be less than the number found in myometrial cells removed from euglycemic animals. The number of free cytoplasmic ribosomes in diabetic cells also appeared to be less than those found in euglycemic cells. In order to determine if apparent differences in the number of myofilament found in diabetic myometrial cells could be correlated with changes in uterine contractile responses to hormones, oxytocin dose-response curves (10(-8) to 10(-5) M) were examined in isolated uteri removed from saline-injected and estradiol-injected (24-hr pretreatments) euglycemic and diabetic rats. The maximal contractile responses (milligrams tension developed per milligrams tissue) in saline-injected euglycemic and diabetic rats were 49 +/- 5 and 36 +/- 4, respectively, while maximal contractile responses in estradiol-injected euglycemic and diabetic rats were 68 +/- 7 and 45 +/- 5, respectively. Maximal contractile responses induced by oxytocin in estradiol-treated diabetic uteri were significantly smaller than the contractile responses measured in euglycemic estradiol-treated uteri. This study demonstrates that estradiol-induced changes in both myometrial cell morphology and in vitro uterine contractile responses to oxytocin are altered in diabetic rats.     

The effect of progesterone on oxytocin-stimulated intracellular mobilization of Ca2+ and prostaglandin E2 and F2alpha secretion from porcine myometrial cells

Our past studies have shown that porcine myometrium produce prostaglandins (PG) during luteolysis and early pregnancy and that oxytocin (OT) and its receptor (OTr) support myometrial secretion of prostaglandins E2 and F2alpha (PGE2 and PGF2alpha) during luteolysis. This study investigates the role of intracellular Ca2+ [Ca2+]i as a mediator of OT effects on PG secretion from isolated myometrial cells in the presence or absence of progesterone (P4). Basal [Ca2+]i was similar in myometrial cells from cyclic and pregnant pigs (days 14-16). OT (10(-7)M) increased [Ca2+]i in myometrial cells of cyclic and pregnant pigs, although this effect was delayed in myometrium from pregnant females. After pre-incubation of the myocytes with P4 (10(-5)M) the influence of OT on [Ca2+]i)was delayed during luteolysis and inhibited during pregnancy. Myometrial cells in culture produce more PGE2 than PGF2alpha regardless of reproductive state of the female. OT (10(-7)M) increased PGE2 secretion after 6 and 12 h incubation for the tissue harvested during luteolysis and after 12 h incubation when myometrium from gravid females was used. In the presence of P4 (10(-5)M), the stimulatory effect of OT on PG secretion was diminished. In conclusion: (1) porcine myometrial cells in culture secrete PG preferentially during early pregnancy and produce more PGE2 than PGF2alpha, (2) OT controls myometrial PGF2alpha secretion during luteolysis, (3) release of [Ca2+]i is associated with the influence of OT on PG secretion, and (4) the effects of OT on PG secretion and Ca2+ accumulation are delayed by P4 during luteolysis and completely inhibited by P4 during pregnancy.     

Transposition of elements of the 412, copia and 297 dispersed repeated gene families in Drosophila.

The stability of elements of three different dispersed repeated gene families in the genome of Drosophila tissue culture cells has been examined. Different amounts of sequences homologous to elements of 412, copia and 297 dispersed repeated gene families are found in the genomes of D. melanogaster embryonic and tissue culture cells. In general the amount of these sequences is increased in the cell lines. The additional sequences homologous to 412, copia and 297 occur as intact elements and are dispersed to new sites in the cell culture genome. It appears that these elements can insert at many alternative sites. We also describe a DNA sequence arrangement found in the D. melanogaster embryo genome which appears to result from a transposition of an element of the copia dispersed repeated gene family into a new chromosomal site. The mechanism of insertion of this copia element is precise to within 90 bp and may involve a region of weak sequence homology between the site of insertion and the direct terminal repeats of the copia element.     

Glucocorticosteroid regulation of prostaglandin biosynthesis in human myometrial smooth muscle cells in monolayer culture

In the present investigation, we evaluated the production of prostaglandins by human myometrial smooth muscle cells maintained in monolayer culture in the absence or presence of glucocorticosteroids. In the presence of cortisol (10(-7) M) or dexamethasone (10(-8) M), the rate of production of prostacyclin (PGI2) by these cells was decreased significantly. The glucocorticosteroid-mediated inhibition of prostaglandin production was attenuated when cortisol-21-mesylate (10(-6) M), a glucocorticosteroid antagonist, was present in the culture medium. The rate of conversion of radiolabeled arachidonic acid to radiolabeled prostaglandins as determined by use of sonicates of myometrial cells and optimal assay conditions, however, was not affected significantly by treatment with cortisol or dexamethasone in concentrations sufficient to inhibit prostaglandin formation by more than 80%. These findings are suggestive that glucocorticosteroids act in human myometrial smooth muscle cells in culture to inhibit prostaglandin formation by way of a receptor-mediated process that does not involve inhibition of enzyme activities that are involved in the biosynthesis of prostaglandins, i.e. the conversion of arachidonic acid to prostaglandin.     

Regulation of prostaglandin production by nitric oxide in rat smooth muscle myometrial cells.

Smooth muscle myometrial cells isolated by an enzymatic method from estrogenized rats were used after 7-10 days of culture. They were incubated for 24 h with two distinct competitive nitric oxide (NO) inhibitors: NG-monomethyl-L-arginine (L-NMMA: 300 microM) and L-nitro-arginine methylester (L-NAME: 600 microM, 5 mM and 10 mM). Afterwards, the supernatants were separated in order to measure nitrite production and prostaglandin PGE synthesis. In the present report, we demonstrate that myometrial cells from estrogenized rats are able to produce NO, since all the inhibitors significantly decrease the production of nitrites in the culture media. Furthermore, we report that both inhibitors inhibited PGE synthesis by myometrial cells. We also used a donor of NO in the incubation medium for 24 h, sodium nitroprusside (NP), obtaining an strong (P< 0.001) increase in both nitrite and PGE production. We conclude that myometrial cells can produce NO and that one possible role of the NO synthetized by this cells may be the modulation of PGE production.     

Interaction between nitric oxide and prostaglandin E pathways in rat smooth muscle myometrial cells

Previously, we demonstrated the presence of a nitric oxide (NO) prostaglandin (PG) pathway in myometrial cells obtained from uterine rat tissue. This pathway was modulated by estrogen and one possible function could be to modulate uterine relaxation. In the present study, we investigated the role of progesterone in the regulation of NO synthesis and the uterotonic PGE production by myometrial cells from uterine rat tissue. We worked with two groups of rats: (i) ovariectomizcd (OV) rats, without influence of sex hormones and (ii) OV rats injected with progesterone (4 mg) s.c. Myometrial uterine cells were obtained by a selective enzymatic digestion. In the incubation medium of these cells, nitrite concentration (as a measure of NO production) and PGE production were evaluated. To ensure a specific response, a competitive NOs inhibitor, N(G)-monomethyl-L-arginine; L-NMMA (300 microM) was used. We found that at 48 h of the incubation period, cells obtained from progesterone-primed uterine tissue presented an increase in the nitrite concentration concomitant with a decrease in the PGE production. When L-NMMA was added to the cells, nitrite production and PGE synthesis returned to control values. The fact that this effect had not been observed in the group of cells obtained from OV rats suggests that progesterone was responsible for it. These data provide strong evidence that in spite of the fact that estrogen and progesterone modulate the NO-PG pathway in the uterine rat tissue, the two hormones have opposite effects.     

Calponin. Developmental isoforms and a low molecular weight variant

Two-dimensional gel analysis of basic proteins in developing human smooth muscle identifies calponin as a prominent marker of the differentiated phenotype. Adult tissue (human and mouse) typically expresses up to four calponin isoforms, three of which appear sequentially during fetal development: adult myometrial cells express the same three isoforms in primary culture in vitro and these are down-regulated, in reverse order, during the subsequent modulation of phenotype. Monospecific, polyclonal antibodies against calponin identify a lower molecular weight variant of calponin (L-calponin) that is strongly and specifically expressed in adult smooth muscles of the human urogenital tract. L-calponin is down-regulated in benign smooth muscle derived tumors (leiomyoma) and is not expressed in primary cultures of normal uterine tissue.     

Uterine peristalsis-induced stresses within the uterine wall may sprout adenomyosis

Adenomyosis is a disease in which ectopic endometrial glands and stromal cells appear in the uterine myometrium. This pathology is common among women of reproductive age, and in addition to chronic pelvic pain and heavy periods it may also cause infertility. The ‘tissue injury and repair’ mechanism in response to increased intrauterine pressures was proposed as the etiology for migration of fragments of basal endometrium into the myometrial wall. In order to investigate this mechanism, a conceptual two-dimensional model of the uterine wall subjected to intrauterine pressures was implemented using ADINA commercial software. The stress field within the uterine wall was examined for a variety of intrauterine sinusoidal pressure waves with varying frequencies. The results revealed that: (1) as the wavelength of the subjected pressure wave decreased, high concentration of stresses developed near the inner uterine cavity; (2) as the pressure wave frequency increased, high gradients of the stresses were obtained; (3) at menstrual phase, the highest stresses obtained at the endometrial–myometrial interface. Therefore, increased uterine activity results in high stresses which may lead to tissue lesions and detachment of endometrial cells.     

Induction of platelet-derived growth factor receptor expression in smooth muscle cells and fibroblasts upon tissue culturing

The expression of platelet-derived growth factor (PDGF) receptors in porcine uterus and human skin in situ, was compared with that of cultured primary cells isolated from the same tissues. PDGF receptor expression was examined by monoclonal antibodies specific for the B type PDGF receptor and by RNA/RNA in situ hybridization with a probe constructed from a cDNA clone encoding the B type PDGF receptor. In porcine uterus tissue both mRNA and the protein product for the PDGF receptor were detected in the endometrium; the myometrium, in contrast, contained much lower amounts. Moreover, freshly isolated myometrial cells were devoid of PDGF receptors. However, after 1 d in culture receptors appeared, and after 2 wk of culturing essentially all of the myometrial cells stained positively with the anti-PDGF receptor antibodies and contained PDGF receptor mRNA. Similarly, B type PDGF receptors were not detected in normal human skin, but fibroblast-like cells from explant cultures of human skin possessed PDGF receptors. When determined by immunoblotting, porcine uterus myometrial membranes contained approximately 20% of the PDGF receptor antigen compared with the amount found in endometrial membranes. In addition, PDGF stimulated the phosphorylation of a 175-kD component, most likely representing autophosphorylation of the B type PDGF receptor in endometrial membranes, whereas only a marginal phosphorylation was seen in myometrial membranes. Taken together, these results demonstrate that PDGF receptor expression varies in normal tissues and that fibroblasts and smooth muscle cells do not uniformly express the receptor in situ. Furthermore, fibroblasts and smooth muscle cells that are released from tissues are induced to express PDGF receptors in response to cell culturing. The data suggest that, in addition to the availability of the ligand, PDGF-mediated cell growth in vivo is dependent on factors regulating expression of the receptor.     

Origins of the differences in function of rat adrenal zona glomerulosa cells incubated as intact tissue and as collagenase-prepared cell suspensions

While in vitro incubation of dispersed cell preparations of adrenal cell types has been widely used as an experimental model, few studies have addressed the possibility that the enzymic and mechanical treatments involved may affect tissue functions. Using rat adrenal whole capsule tissue, consisting of glomerulosa cells still attached to the connective tissue capsule together with some fasciculata cells, and dispersed glomerulosa cell preparations formed by a variety of enzymic and incubation treatments, striking differences have been demonstrated between the functions of the various preparations in vitro. Under ACTH stimulation, whole capsules produced (ng per pair ± s.e.) 405 ± 35 ng aldosterone, 650 ± 60 ng 18-hydroxycorticosterone (18-OH-B) and 850 ± 90 ng corticosterone. In cells dispersed by collagenase incubation followed by repeated pipetting and filtration, aldosterone and 18-OH-B yields under ACTH stimulation fell to values less than 10% of those produced by whole tissue, whereas corticosterone values were unchanged. Omitting the filtration step gave a less well marked decline in aldosterone and 18-OH-B to 50% of intact tissue values. When the tissue was not dispersed after collagenase incubation, aldosterone and 18-OH-B outputs were similar in the two preparations. The decline in aldosterone and 18-OH-B is not attributable to loss in cell–cell contact alone, since short term culture of collagenase dispersed cells on contracting collagen discs did not restore the capacity to produce these steroids, and a decline in their output also occurred in similar culture of intact capsule tissue. In acute incubations, hyaluronidase had similar effects to collagenase, whereas trypsin, papain and a bacterial protease evoked aldosterone release during the preincubation period, but did not affect subsequent yields of aldosterone and 18-OH-B in incubations of dispersed (but not filtered tissue) in the presence of ACTH. Chymo-trypsin had no effect on preincubation but eliminated subsequent response to ACTH in all incubation conditions. Together with previously published data on the effects of trypsin, the results support the view that in intact rat adrenal glomerulosa tissue, aldosterone and 18-OH-B are sequestered into intracellular stores in the form of novel steroid-protein complexes. These are hydrolysed by trypsin and other preoteases with consequent release of steroid, but are virtually eliminated by conventional methods of cell suspension preparations, using collagenase preincubation with subsequent mechanical dispersal and filtration.     

Immunoreactive dynorphin-A in bovine anterior pituitary and its in vitro release

The anterior lobe (AL) of the bovine pituitary contained and released, during in vitro culture, a form of immunoreactive dynorphin-A (ir-DYN-A) larger than that occurring in neural tissue. Bovine AL tissue from intact females contained less ir-DYN-A than did AL tissue from castrated males. Enzymatically dispersed AL cells contained and released ir-DYN-A in vitro. Preincubation of dispersed AL cells for 18 hr, rather than 1.5 hr, increased the content and release of ir-DYN-A as well as LH. Addition of gonadotropin-releasing hormone (GnRH) to tissue slices or dispersed cells stimulated release of LH, but in contrast to published observations from rat AL, GnRH had no effect on release of ir-DYN-A. Addition of estradiol-17 beta, with or without progesterone, increased release of ir-DYN-A but not LH during 2-hr cultures. In summary, bovine AL contains and releases in vitro a large molecular weight form of ir-DYN-A. Although this ir-DYN-A was not coreleased with LH, a reproductive role was suggested by in vivo and in vitro effects of gonadal hormones on ir-DYN-A in the bovine anterior pituitary.     

Control of aldosterone secretion in zona glomerulosa cell suspensions and in the perfused adrenal gland of the rat

The secretion of aldosterone and its responses to stimulation have been studied in rat adrenal zona glomerulosa tissue incubated as intact capsules or as collagenase-dispersed cell suspensions, and in intact perfused rat adrenal glands. Several differences are apparent in the functions of the various preparations. Aldosterone secretion rates are similar in incubated intact capsules and in the perfused gland. Relative to corticosterone, lower yields of aldosterone are obtained in dispersed glomerulosa cell in vitro. This may be related to the loss in the dispersed cells of a pool of tissue steroid (aldosterone or a precursor) which is revealed only in intact tissue incubations by trypsin stimulation of aldosterone secretion. Trypsin-released aldosterone is increased by prior dietary sodium restriction. In addition, differences occur in the responses of dispersed cells and perfused glands to stimulation. Perfused glands from animals on a normal diet are less sensitive to stimulation by ACTH or alpha-MSH, but more sensitive than dispersed cells to angiotensin II amide. In the perfused gland, sensitivity of response (lowest effective concentration) to all three stimulants is increased by prior dietary sodium restriction, in contrast to dispersed cells in which increased sensitivity has been reported only to alpha-MSH. The perfused gland is particularly sensitive to angiotensin II amide, and a bolus administration of 1 amol gives significant stimulation in glands from animals on low sodium intake. Electrical (field) stimulation or dopamine administration at 10(-6) mol/l (which is ineffective in dispersed cells) both depress aldosterone secretion by the perfused gland. The data suggest that the sequestered pool of steroid is utilized in the perfused gland for aldosterone secretion. They furthermore suggest that in the intact gland there are mechanisms, which involve neural components, for intraglandular regulation of aldosterone secretion, which are lost in dispersed cells in vitro. Such mechanisms may be involved in sensitivity increases in sodium depletion.     

Cultured myometrial cells establish communicating gap junctions

Myometrial cells were isolated and cultured from term rat uterus. The myometrial origin of the cultures was verified by antibody staining of cellular desmin and alpha-smooth muscle actin. The presence of functional gap junctions was indicated by transfer of radiolabeled nucleotide and microinjected Lucifer yellow dye. The cultured cells expressed mRNA recognized by a connexin43 gap junction cDNA probe. To our knowledge, this is the first report that isolated myometrial cells form gap junctions in culture.     

Gap junctions in myometrial cell cultures: evidence for modulation by cyclic adenosine 3':5'-monophosphate.

Primary cultures of myometrial cells from juvenile rats, continuous cultures maintained by serial passage, and a pSV3neotransfected myometrial cell line were established and utilized for the study of development and modulation of gap junctional intercellular communication (GJIC) in vitro. The smooth muscle origin and homogeneity of the cultures were verified by immunofluorescence staining of alpha-smooth muscle actin and cellular desmin. Although gap junctions were not detected in thin sections of juvenile and adult myometrial tissues by transmission electron microscopy, they were detected in cultured myometrial cells derived from juvenile and adult animals. The presence of GJIC in cultured cells was confirmed using a fluorescence recovery after photo-bleaching assay. Administration of exogenous estradiol-17 beta (10(-7) M) resulted in an increase in GJIC in primary and passage 9 myometrial cultures, whereas pSV3neo-transfected myometrial cells were not significantly different from untreated controls. The lack of estrogen responsiveness in pSV3neo-transfected cultures correlated with lower levels of estrogen receptors than in primary cultures. Addition of 1 mM 8-bromo-cAMP resulted in rapid (within 2 min) increases in dye transfer in both control and estradiol-17 beta-primed primary cultures. Uncoupling of cells by treatment with 1 mM 1-octanol, followed by addition of 1 mM 8-bromo-cAMP, resulted in increased GJIC in control and estradiol-17 beta-primed cultures, although up-regulation of GJIC in estradiol-17 beta-primed cultures was much greater than in control cultures. Comparative experiments carried out on a spontaneously immortalized rat granulosa cell line (SIGC), which expresses the same connexin43 species as myometrial cells, exhibited similar responses to exogenous 8-bromo-cAMP following uncoupling of gap junctions with octanol. While the results of these investigations may not be extrapolated to myometrium in vivo, they suggest that myometrial cell culture may offer additional opportunities to explore the temporal expression and modulation of GJIC in myometrium.     

Characterization of molt-inhibiting hormone (MIH) action on crustacean Y-organ segments and dispersed cells in culture and a bioassay for MIH activity

Ecdysteroid secretion in vitro by gland quarters and dispersed cells of ecdysial glands (Y-organs) of the crab, Cancer antennarius Stimpson, was characterized. Optimum culture conditions are reported for maximum, sustained (72 hr) secretion and maintenance of cell viability in activated Y-organs obtained from de-eyestalked donors. Addition in vitro of eyestalk ganglia extracts containing the putative molt-inhibiting hormone (MIH) inhibited ecdysteroid production dose-dependently in the range of 0.1-4.0 and 0.01-4.0 eyestalk equivalents of MIH for gland quarters and dispersed cells, respectively. Inhibition by MIH was reversible, tissue specific as to source of MIH activity, and did not affect cell viability relative to controls. The results of replicate incubations of gland quarters with MIH were analyzed with formal statistics of parallel-line assay. The inhibitory action on ecdysteroid secretion is shown to be reproducibly linear and parallel in the dosage range, 0.1-4.0 eyestalk equivalents, amenable to calculation of relative potency among successive extracts, and of sufficiently high precision to serve as an MIH bioassay. Also, the results of these studies support the hypothesis that control of Y-organs by the eyestalks is physiologically direct.