Transferrin expression by placental trophoblastic cells.

Transferrin (TF), a 76-80 kDa glycoprotein, is responsible for the transport of iron to cells within both the fetal and maternal systems, but it does not cross the multiple cell layer barrier of the placenta. Recent findings that both rat and human placental cells produce TF indicated that placental TF may function in some manner to transport or regulate iron passage across this barrier. However, placental production of TF was brought into question because the cell preparations used to identify TF were obtained using dispersed tissue and may have contained non-placental contaminating elements. In this study, cultures of phenotypically distinct cell types containing only placental cells were used to firmly establish whether or not TF is expressed, and if so to begin to identify the cell(s) associated with its synthesis. Utilizing RT-PCR, in situ hybridization, and Western blot analysis, we identified TF mRNA and protein in three trophoblast cell types, HRP-1 (rat), Rcho-1 (rat), and BeWo (human) cells. Additionally, TF mRNA and protein were found in Giant cells, the differentiated form of Rcho-1 cells. When taken together, these results demonstrate clearly that TF is expressed by both differentiated and non-differentiated placental cells, and when viewed in light of previous findings, strengthen the possibility that placental TF may be central to the passage of iron from the mother to the fetus during development.

Relaxin-like factor (RLF) serum concentrations and gubernaculum RLF receptor display in relation to pre- and neonatal development of rats.

Deletion of the relaxin-like factor (RLF) gene in mice causes retention of testicles and infertility. The development of a synthetic RLF has made it possible to investigate the events that connect the genomic event and the basic biological responses that cause gonadal positioning. Anti-RLF antibodies were raised against synthetic RLF, allowing determination of RLF concentrations during the critical period, testing for RLF receptors on the gubernaculum and exploration of the temporal relationship between receptor display and migration of the testes in developing rats. In male rat pups, serum RLF concentrations were high at day 2 before parturition (2.4 ng ml(-1)) and decreased sharply just before parturition. Thereafter, males and females had the same low serum concentrations until RLF concen-trations began to increase in males only, starting at day 10 after parturition and continuing until adult RLF concentrations (0.6 ng ml(-1)) were reached on day 39 after parturition. The testicles are descending into the scrotum during this phase of increasing RLF concentrations and are descended fully by day 19-21 after parturition, before adult hormone concentrations are established. The high prenatal serum RLF concentration coincides with high expression of RLF receptors in the gubernaculum tissue. Competitive binding of RLF per mg of membrane protein prepared from rat gubernacula at various developmental stages showed no increase in receptor density as sexual maturity was reached. Gubernaculum cells in primary culture showed an increased uptake of 5-bromo-2'-deoxyuridine in the presence of RLF compared with controls. These studies demonstrate that the synthetic RLF is biologically active and indicate that the cryptorchid phenotype INSL3(-/-) is a direct consequence of defective gubernaculum growth, caused by the absence of RLF during early phases of development.

Pulsatile exocytosis is functionally associated with GnRH gene expression in immortalized GnRH-expressing cells.

Pulsatile release of GnRH is essential for proper reproductive function, but little information is available on the molecular processes underlying this intermittent activity. Recently, GnRH gene expression (GnRH-GE) episodes and exocytotic pulses have been identified separately in individual GnRH-expressing cells, raising the exciting possibility that both activities are linked functionally and are fundamental to the pulsatile process. To explore this, we monitored GnRH-GE (using a GnRH promoter-driven luciferase reporter) and exocytosis (by FM1-43 fluorescence) in the same, living GT1-7 cells. Our results revealed a strong temporal association between exocytotic pulses and GnRH-GE episodes. To determine whether a functional link existed, we blocked one process and evaluated the other. Transcriptional inhibition with actinomycin D had only a modest influence on exocytosis, suggesting that exocytotic pulse activity was not dictated acutely by episodes of gene expression. In contrast, blockage of exocytosis with anti-SNAP-25 (which obstructs secretory granule fusion) abolished GnRH-GE pulse activity, indicating that part of the exocytotic process is responsible for triggering episodes of GnRH-GE. When taken together, our findings suggest that a careful balance is maintained between release and biosynthesis in GT1-7 cells. Such a property may be important in the hypothalamus to ensure that GnRH neurons are in a constant state of readiness to respond to changes in reproductive function.

Synchronized exocytotic bursts from gonadotropin-releasing hormone-expressing cells: dual control by intrinsic cellular pulsatility and gap junctional communication.

Periodic secretion of GnRH from the hypothalamus is the driving force for the release of gonadotropic hormones from the pituitary, but the roles of individual neurons in the context of this pulse generator are not known. In this study we used FM1-43 to monitor the membrane turnover associated with exocytosis in single GT1-7 neurons and found an intrinsic secretory pulsatility (frequency, 1.4 +/- 0.1/h; pulse duration, 17.3 +/- 0.6 min) that, during time in culture, became progressively synchronized among neighboring cells. Voltage-gated calcium channels and gap junctional communication each played a major role in synchronized pulsatility. An L-type calcium channel inhibitor, nimodipine, abolished synchronized pulsatility. In addition, functional gap junction communication among adjacent cells was detected, but only under conditions where pulsatile synchronization was also observed, and the gap junction inhibitor octanol abolished both without affecting pulse frequency or duration. Our results, therefore, provide strong evidence that the GnRH pulse generator in GT1-7 cells arises from a single cell oscillator mechanism that is synchronized through network signaling involving voltage-gated calcium channels and gap junctions.

Development of a destabilized firefly luciferase enzyme for measurement of gene expression.

Firefly luciferase is used widely as a reporter enzyme for studies of gene regulation and expression. The recent development of new technologies that combine luciferase reporter technology and digital imaging microscopy has enabled multiple measurements of gene expression in the same living cell. Although this approach has already provided new insights about expression dynamics, its future utility is limited by the three- to four-hour half-life of firefly luciferase in mammalian cells. Because of this, rapid increases or decreases in gene expression may not be detected, owing to the accumulation of residual luciferase. Accordingly, the goal of the present study was to develop a luciferase reporter with a reduced functional half-life. This was accomplished by adding a synthetic fragment to the firefly luciferase-coding sequence that encoded the proteolytic "PEST" signal from mouse ornithine decarboxylase. When placed under the control of estrogen response elements and expressed in human breast cancer T-47D cells, the modified luciferase protein (LUCODC-DA) displayed a functional half-life of 0.84 h compared to 3.68 h for the wild-type enzyme. As anticipated, the overall rate of photonic emissions in cells expressing the destabilized luciferase was about sevenfold lower than that of their wild-type counterparts, presumably because of the reduction of steady-state luciferase accumulation. Even so, the photonic activity derived from LUCODC-DA was still sufficient to enable real-time measurements of gene expression in single living cells.

The relationship between pulsatile secretion and calcium dynamics in single, living gonadotropin-releasing hormone neurons.

It is well established that pulsatile release of GnRH regulates the reproductive axis, but little is known about the mechanisms underlying this pulsatility. Recent findings that GT1 cells, a line derived from the mouse embryonic hypothalamus, release GnRH in a pulsatile manner indicates that this rhythmic activity is an intrinsic property of GnRH neurons. In several attempts to uncover the intracellular basis for this pulsatile phenomenon, it was revealed that intracellular calcium concentrations change in a rhythmic fashion in GnRH neurons and that cellular depolarization, which triggers a secretory event, is associated with profound calcium changes in the cells. These findings raised the intriguing possibility that periodic alterations in intracellular calcium concentrations may underlie the phenomenon of pulsatile secretion in GnRH neurons. To address this, we first adapted the use of FM1-43 fluorescence to monitor changes of secretion in individual GT1-7 cells and then combined this approach with simultaneous measurement ofintracellular free calcium ([Ca2+]i, fura 2 method). In initial validation experiments, we found that stimulation of exocytosis with K+ (75 mM) or N-methyl-D-aspartate (NMDA, 100 microM) predictably evoked dynamic increases of both FM1-43 and fura 2 fluorescence. Later measurement of calcium dynamics and exocytotic activity in unstimulated cells revealed that [Ca2+]i underwent transitions from quiescence to high oscillatory behavior, and that these shifts were frequently associated with exocytotic events. Moreover, these calcium oscillatory transitions and associated changes in secretory activity occurred synchronously among most adjacent cells and at a frequency similar to that reported for pulsatile release of GnRH by entire cultures of GnRH neurons. Taken together, these results indicate that the intrinsic secretory pulsatility of GnRH neurons appears to be a consequence of coordinated, periodic changes in the pattern of calcium oscillations within individual cells.

Sertoli cell expression of the cystic fibrosis transmembrane conductance regulator.

Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been associated with a number of male reproductive problems, including testis abnormalities and a reduction in germ cell quality and number. To establish at least one site of functional CFTR expression in the testis, we subjected cultured Sertoli cells to analysis of message, protein, and channel activity for CFTR. With reverse transcription-polymerase chain reaction, we obtained evidence for the presence of CFTR RNA when CFTR primers were used with RNA from cultured Sertoli cells. Western analysis performed with both anti-R and anti-C domain CFTR antibodies revealed immunoreactive material in extracts from primary Sertoli cell cultures that seemed consistent with CFTR previously identified in other cells and tissues. This led us to perform more detailed studies using the whole cell arrangement of the patch-clamp technique. Application of the membrane-soluble cAMP analog, 8-chlorophenyl-thio-cAMP, resulted in the activation of a Cl- current that displayed a permeability sequence of Br- > I- > or = Cl- and was blocked by diphenylamine-2-carboxylate and glibenclamide. In addition, a 13-pS conductance Cl- channel was measured in excised membrane patches exposed to the catalytic subunit of protein kinase A. When taken together, our findings of evidence of CFTR message, immunoreactive material that appeared consistent with CFTR, and Cl- channels with properties similar to those reported for CFTR provide strong evidence that Sertoli cells express a functional CFTR-like protein. The presence of CFTR in these cells may be needed to maintain the specific nutritional and fluid balance in the seminiferous tubule that is vital for normal spermatogenesis.

Chronic administration of the environmental pollutant 4-tert-octylphenol to adult male rats interferes with the secretion of luteinizing hormone, follicle-stimulating hormone, prolactin, and testosterone.

4-Tert-octylphenol (OP) is a prevalent environmental pollutant that has been shown to exert both toxic and estrogenic effects on mammalian cells. The effects of OP on the reproductive system of adult male vertebrates are virtually unknown. In the present study, we investigated the effects of chronic exposure to OP on reproductive hormone secretion in the adult male rat and compared the results qualitatively with those observed in other male rats treated chronically with estrogen. We injected corn oil vehicle or OP (20 or 80 mg) or estradiol valerate (EV; 0.8 or 8 microg) in oil s.c. into 2-mo-old male rats thrice weekly for either 1 or 2 mo. The 80-mg dosage of OP and one or both dosages of EV had the following effects: decreased anterior pituitary gland (APG) and serum LH and FSH concentrations; increased APG and serum prolactin (PRL) concentrations; increased APG/body weight ratios; decreased serum testosterone concentrations; decreased hematocrit; and decreased food consumption and body weight gain. To evaluate the response of the hypothalamus-APG to gonadal removal, we orchidectomized some of the rats after the end of treatment and decapitated them 3 wk later. In orchidectomized controls, serum LH and FSH concentrations rose markedly and serum PRL concentrations decreased. Similar changes were seen in orchidectomized rats treated previously with 20 or 80 mg OP. Moreover, there were no differences in mean serum LH, FSH, or PRL concentrations between controls and rats treated previously with either dosage of OP at 3 wk after orchidectomy. The results demonstrate that chronic administration of OP to adult male rats can adversely affect the secretion of reproductive hormones and strongly suggest that OP exerts these effects by acting like an estrogen. The opposite changes in LH, FSH, and PRL secretion observed after cessation of treatment with OP and orchidectomy suggest that chronic treatment with OP under the conditions of the present study did not result in any significant permanent deleterious effects on gonadotrophs or lactotrophs or the hypothalamic neurons controlling the secretion of the gonadotropins or PRL.

Chronic administration of 4-tert-octylphenol to adult male rats causes shrinkage of the testes and male accessory sex organs, disrupts spermatogenesis, and increases the incidence of sperm deformities.

The environmental toxicant 4-tert-octylphenol (OP) has been shown to exert estrogenic effects on mammalian cells in culture. Recent findings from our laboratories demonstrate clearly that OP administration disrupts reproductive hormone secretion in the adult male rat, quite likely as a result of estrogenic action. In the present study, we investigated the impact of these or other OP-induced changes on male reproductive tissues. Adult male rats were injected with OP (20 or 80 mg) or estradiol valerate (EV; 0.8 or 8 microg) s.c. in oil three times a week for either 1 or 2 mo. We found that an 80-mg dosage of OP for 2 mo or an 8-microg dosage of EV for 1 or 2 mo greatly reduced sperm numbers and adversely influenced the sizes, weights, and histological structures of the testes, epididymides, ventral prostate glands, seminal vesicles, and coagulating glands. The 80-mg dosage of OP for 1 mo reduced epididymal tubule size to a lesser extent than after 2 mo of treatment. Otherwise, treatment with 80 mg OP for 1 mo, 20 mg OP for 1 or 2 mo, or 0.8 microg EV for 1 mo had little or no effect on the histology of the tissues we examined. Additional evaluation of sperm morphology revealed marked increases in the proportions of head and tail abnormalities from animals that had received 80 mg of OP or 8 microg of EV for 1 mo and 20 mg of OP for 2 mo. The head abnormalities consisted mainly of pin heads, detached heads, and the absence of hooks, while tail abnormalities included mainly broken, coiled, and bent tails. Our results clearly demonstrate that OP can severely reduce the size and/or function of all of the male gametogenic and accessory reproductive organs studied. Moreover, the similarity of these cell and tissue changes between rats treated with OP and those treated with EV further suggests that OP may exert its action in an estrogenic-like manner.

Sertoli cells in culture release a pregnancy specific beta1 glycoprotein-like substance.

The possibility that pregnancy-specific beta1 glycoprotein (PSG) is released by Sertoli cells was investigated by using reverse hemolytic plaque assays which enable the visualization of release from individual cells in culture. We found that the proportions of cells releasing PSG increased gradually in 3-day old cultures prepared from animals of increasing age (10-, 20-, and 40-day old animals). The gradual appearance of PSG-releasing cells during this period differed markedly from that of transferrin (TF)-releasing cells, suggesting that the age-related development of PSG-releasing cells is regulated in a specific manner. PSG cells were also found in Sertoli cell cultures prepared from stage-associated seminiferous tubule segments of adult rat testes. The percentages of PSG plaque-forming cells differed from one stage-associated culture to another with maximal proportions associated with stages III-V and XIII, and minimal proportions found in stages VII, and IX-XI. The abundance of Sertoli cells that released PSG from stage to stage differed markedly from those that released TF indicating that modulatory processes specific for PSG are also present in the adult testis. Finally, PSG cells were also identified immunocytochemically in cultures prepared from staged tubule segments. The proportion of PSG staining cells from stage to stage were found to be virtually indistinguishable from those identified with plaque assays. When taken together, these results show clearly that Sertoli cells in culture release a PSG-like molecule. Moreover, this release appears to be controlled in an age-related and stage-dependent manner, suggesting that Sertoli cells may be central to PSG function in the testis.

Placental cell release of transferrin: analysis by reverse haemolytic plaque assay.

Recent demonstrations of transferrin (TF) mRNA in placental tissue raised the possibility that the placenta may serve as an extra-hepatic source of this iron-binding protein during development. In this study, we first confirmed these findings using cRNA probes for TF, and then adapted the TF reverse haemolytic plaque assay for use with rat placental cells to identify the location and functional characteristics of cells secreting this product. We found TF releasing cells in placentae (day 19-21) with greater proportions present in cultures from basal than labyrinth zone regions. These cells appeared quite stable as evidenced by observations that fresh populations and 24, 48 and 72 h cultures from the same placental regions all contained similar percentages of secretors. The rate of TF plaque formation was greatly enhanced in the presence of tumour necrosis factor (0.1 ng/ml) for basal zone and yolk sac cells, but not for labyrinth zone cells, suggesting a potent but regionally specific modulation of TF release. When taken together, these findings demonstrate clearly that TF is released from cells of the placenta. Moreover, the regional differences in frequency and modulation of these cells suggests that the release of placental TF is a dynamic and carefully controlled process.

Interleukin-6 secretion from rat Leydig cells in culture.

Recent evidence indicates that interleukin-6 (IL-6) acts on Sertoli cells to modulate secretory function. IL-6 is also detected in medium bathing tissue or cells from the seminiferous tubule, suggesting a testicular regulatory role. Because other cytokines found to be active in testicular function have more than one site of production, we examined whether Leydig cells may serve as an alternate source of IL-6. Purified Leydig cells were cultured with or without modulatory substances, and the medium was subjected to the 7TD1 bioassay for IL-6. Northern analysis using an IL-6 cDNA probe was performed on companion cell preparations. Incubation with either hCG or IL-1 beta increased the levels of bioactive IL-6 released into the medium and IL-6 mRNA detected in the cells in a dose-related manner. When used together, these agents had an additive stimulatory influence on both the release of IL-6 bioactivity and the amount of IL-6 mRNA. Our results demonstrate that IL-6 is secreted from enriched preparations of Leydig cells and that its release is under the control of at least two modulators of testicular function. Identification of interstitial cells as a site of IL-6 production coupled with reports of IL-6 release and action in seminiferous tubular cell preparations suggest that IL-6 may serve a role in signal integration or communication from one testicular location to another.

Single and dual hormone secretors in GH3 cultures respond differently to hypothalamic factors.

Recent studies using both normal and tumoral pituitary cell cultures have demonstrated that growth hormone (GH) and prolactin (PRL) secreting populations contain cells which release either one or both of these hormones. In order to determine whether these two cell types can be differentially regulated by hypothalamic factors we performed the following study employing plaque assays for GH and PRL. Using cultures of GH3 cells, a rat tumor cell line which contains both of these cell types, we found that the hypothalamic factors vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) when used together had a greater influence on plaque formation than when each was used individually. This suggested that cells were present in culture that responded to one peptide but not the other. Estradiol-treated cultures (which contain only dual-secreting cells) were then evaluated for VIP and TRH responsiveness and found to respond to TRH but not VIP. Finally, we assessed the peptide sensitivity of cultures that were exposed to a conjugate of VIP and the A-chain of ricin (a potent cytotoxin). In addition to eliminating VIP-responsive cells, this treatment markedly reduced the proportions of cells secreting GH-only while having no appreciable influence on dual-hormone secretors. When taken together, our findings indicate that single and dual secretors respond differently to at least two hypothalamic secretagogues and suggest that regulatory differences between these cell types may be important in the control of GH and PRL secretion.

Effects of interleukin-6, interleukin-2, and tumor necrosis factor alpha on transferrin release from Sertoli cells in culture.

Recent studies demonstrate that several cytokines are potent modulators of steroid release from the testis. In an attempt to determine whether these agents may influence other types of secreted substances, we used plaque assays to measure the effect of interleukin-6 (IL-6), interleukin-2 (IL-2), and tumor necrosis factor alpha on transferrin (TF) release from Sertoli cells in culture. Because Sertoli cells from different parts of the tubule respond differently to modulatory factors, we used cultures obtained by microdissection from stages III-V, VII, IX-XI, and XIII of the cycle of the seminiferous epithelium. Our results revealed that each agent increased the rate of TF plaque formation from cultures of IX-XI, and XIII staged segments but not from those staged III-V and VII. Moreover, IL-6, but not the other cytokines, modified the response of Sertoli cells to another regulator, FSH. This was evidenced by our findings that pretreatment with IL-6 for 1 h resulted in FSH-induced increases in the rate of plaque formation for cells from IX-XI segments, in addition to those segments which are normally responsive without pretreatment (III-V and VII segments). Further experiments revealed that IL-6 also had a chronic influence on the proportion of TF secretors present in certain staged cultures. Treatment for 24 h with IL-6 markedly reduced the percentage of TF secretors in cultures from stage XIII segments and resulted in a slight increase in TF cells for stage VII cultures. However, no chronic influences in TF secretors were detected with either IL-2 or tumor necrosis factor alpha treatment. Our results demonstrate very clearly that certain cytokines acting in a stage specific manner have acute and/or chronic influences on the release of TF from Sertoli cells. These findings, when viewed in light of reports of the presence of these factors in the testis, suggest strongly that cytokines or cytokine-like substances, by modulating the release of Sertoli cell substances, may play an important role in testis function.

Sertoli cell function varies along the seminiferous tubule: the proportion and response of transferrin secretors differ between stage-associated tubule segments.

Recent studies from our laboratory demonstrated clearly that only a portion of all Sertoli cells secrete transferrin (TF). These findings raised the possibility that differences in the functional type of Sertoli cells from one location to another may account in part for the stage-related variation in TF release along the seminiferous tubule. In order to address this, Sertoli cells derived from tubule segments corresponding to stages III-V, VII, IX-XI, and XIII of the seminiferous epithelial cycle were subjected to reverse hemolytic plaque assays to determine whether the proportion of TF cells present in those segments were similar or different. We found 21.4 +/- 1.8%, 20.3 +/- 2.0%, 48.3 +/- 2.5%, and 49.2 +/- 3.2% of all cells secreted TF in III-V, VII, IX-XI, and XIII staged segments, respectively. Results obtained from immunocytochemical staining of cells from different sections agreed well with those obtained with plaque assays, indicating that we had detected most, if not all, TF cells in these cultures. In additional experiments, we found that cultured cells from stage III-V and VII responded to FSH or isoproterenol with a large increase in the rate of TF plaque formation, whereas cells from IX-XI and XIII segments appeared to be unaffected. In contrast, bovine fibroblast growth factor caused a marked increase in the rate of TF plaque formation with IX-XI cells and only a slight increase with cells from III-V staged segments. Thus, the manner in which Sertoli cells respond to several modulatory agents appears not only to be stage-dependent, but also to be specific to the agent in question. When taken together, our observations demonstrate that cultured TF secretors obtained from different staged segments of the seminiferous tubule differ in proportion and responsiveness. These findings, when viewed in light of reports of a constant number of Sertoli cells along the seminiferous tubule, suggest that Sertoli cells may acquire and lose the ability to secrete TF or respond to modulation as the seminiferous cycle progresses.

The suckling stimulus increases the responsiveness of mammotropes located exclusively within the central region of the adenohypophysis.

Studies from several groups, including our own, have shown that the suckling stimulus increases the responsiveness of pituitary cells to PRL-releasing stimuli. These findings, when viewed in light of differences in PRL cell responsiveness from one pituitary region to another, raised the possibility that suckling may influence responsiveness of cells in only a specific portion of the gland rather than in the entire pituitary. To address this issue, we evaluated cell responsiveness by performing plaque assays [with and without TRH, Angiotensin II (AII), and dopamine] on cells from two different regions of pituitaries from suckled and nonsuckled rats. These pituitary regions consisted of the inner zone, which is a central area proximate to the neurointermediate lobe, and an outer zone, which encompasses the remaining peripheral area of the anterior lobe. We found that inner zone cells from nonsuckled animals were highly responsive to dopamine and relatively unresponsive to TRH and AII. However, after suckling, a complete shift occurred with inner zone cells becoming sensitive to TRH and AII and resistant to dopamine. In contrast to these inner zone alterations, outer zone cells did not change after suckling, but remained responsive to TRH and AII and unresponsive to dopamine. Our results demonstrate clearly that suckling-induced alterations in PRL cell responsiveness to certain modulatory agents can be attributed to a discrete subpopulation of cells located in a specific region of the pituitary.

Fibroblast growth factor modulates the release of transferrin from cultured Sertoli cells.

The acute and chronic effects of basic fibroblast growth factor (bFGF) on transferrin (TF) secretion from Sertoli cells were investigated by using reverse hemolytic plaque assays which enabled the visualization of release from individual cells in culture. We found that acute treatment with bFGF stimulates the release of TF from some but not al Sertoli cells in cultures obtained from 20-day-old rats. Chronic treatment with this growth factor resulted in increases in overall cell number in cultures from animals of each age tested (8-20 days of age). In contrast, this long-term treatment decreased markedly the proportions of Sertoli cells that secreted TF but only in cultures from 10-day-old animals. When taken together, these findings of acute and chronic influences of bFGF on TF secreting cells support the possibility that bFGF not only contributes to the modulation of the day-to-day release of certain substances from Sertoli cells, but may also influence development of the portions of the cell population that secrete these substances.

Sertoli cells in culture are heterogeneous with respect to transferrin release: analysis by reverse hemolytic plaque assay.

It has been reported that not all Sertoli cells store the same product or respond morphologically to secretagogue stimulation. The following studies were performed to determine whether functional differences among these cells are also present with respect to the secretion of a product. Sertoli cells obtained from 18- to 20-day-old rats were cultured for 3 days and then subjected to reverse hemolytic plaque assays for transferrin (TF). Release of TF could be detected from only 62.7 +/- 0.47% (mean +/- SE; n = 4 experiments) of all cells in culture. Results obtained from immunocytochemical staining of different batches of cells from the same dispersions agreed quite closely with these plaque assay values, indicating that not all Sertoli cells in culture contain or secrete TF. Differences in the basal rate of TF release were observed among these secretors, as evidenced by a gradual appearance of plaques over an 8-h period. Addition of FSH, cAMP, or isoproterenol to the assay incubation mixture resulted in an acceleration in the rate of plaque formation. Although approximately twice as many secretors could be identified after 0.5 and 1 h of incubation in the presence of these agents than in their absence, it still required at least 4 h for the remainder of the TF cells to form plaques. This would indicate that only a portion of all TF secretors respond acutely to these modulators. Thus, our observations that not all Sertoli cells secrete TF, and those that do release this substance respond differently to at least three stimulatory agents demonstrate clearly that Sertoli cells are heterogeneous with respect to TF release. Moreover, these findings raise the possibility that differences in the functional capacity of individual cells may be an important factor contributing to the modulation of Sertoli cell secretion.