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.

Cultures of GH3 cells contain both single and dual hormone secretors.

Analysis of GH3 cultures by fixed sequential plaque assays revealed the presence of cells that release GH only, as well as those that release both GH and PRL (mammosomatotropes). Chronic treatment of these cultures with 17 beta-estradiol and TRH (factors found to alter the amounts of GH and PRL secreted) caused shifts in the overall proportions of GH to PRL secreting cells. Each agent, however, influenced the cultures differently. Estradiol treatment resulted in cultures which contained only mammosomatotropes, whereas TRH treatment resulted in cultures that contained PRL-only cells in addition to the other two functional cell types present in control populations. These results indicate that factors which alter the amounts of GH and PRL secreted by GH3 cultures also change the types of secretors present. Moreover, the manner in which the proportions of dual hormone secretors changed in response to these factors supports the view that multipotential cells serve as a transitional cell type in a conversion from GH to PRL secretors.

Functional variations among prolactin cells from different pituitary regions.

Reverse hemolytic plaque assays were used to compare the responsiveness of cells from different pituitary regions to the modulatory effects of human pancreatic GH-releasing factor (GRF), TRH, and dopamine (DA). Tissues from the peripheral rim (outer zone) and the central region (inner zone) of adenohypophyses from day 10 lactating rats were dispersed with trypsin, and the cells were placed into culture. On the following day, these cells were subjected to GH plaque assays (conducted in the presence or absence of GRF) and PRL plaque assays (performed with or without TRH and DA). Cells from both zones responded similarly to GRF with a rapid acceleration of GH plaque formation. However, the rate of PRL plaque formation in response to TRH and DA differed between cells from these regions. For outer zone cells, plaque development increased greatly with TRH treatment, but was only moderately affected by DA. Plaque formation from inner zone cells was influenced slightly by TRH, but markedly inhibited by DA. These results suggest that PRL, but not GH, cells from these pituitary regions are differentially responsive to at least two hypothalamic secretagogues. We then performed fixed sequential plaque assays to determine whether the proportions of cells that released PRL only (classical mammotropes) or those that released both GH and PRL (mammosomatotropes) also differed between the inner and outer zones. Using this approach, we found that the outer zone contained a much larger proportion of dual hormone secretors than did the inner zone. These results, when taken together with the responsiveness differences discussed above, raise the possibility that the release of PRL from mammotropes and mammosomatotropes is regulated differently and that the ratio of these two cell types may dictate, in part, the manner in which a specific region of the pituitary responds to hypothalamic input.

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.

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.

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.

Functional maturation of somatotropes in fetal rat pituitaries: analysis by reverse hemolytic plaque assay.

Immunocytochemistry (ICC) and a reverse hemolytic plaque assay for GH were used to investigate the temporal relationships between the initiation of hormone storage and release by developing somatotropes and the onset of responsiveness of these cells to stimulatory and inhibitory secretagogues. Anterior pituitaries obtained from rats on days 18-21 of fetal development (pups were generally delivered on fetal day 22, which is equivalent to day 0 of neonatal life) were monodispersed with trypsin, cultured for 24 h, and then subjected to reverse hemolytic plaque assay and/or ICC for GH. GH-containing cells (determined by ICC) were extremely rare (less than 1%) in cultures derived from day 18 fetuses, but accounted for 22.4%, 25.2%, and 24.5% of all cells in cultures from day 19-21 fetuses, respectively. The proportion of GH-releasing cells, as determined in a long term (120-min incubation with antibody) plaque assay, was less than 1%, 22.4%, and 22.9% for days 18, 20, and 21, respectively, but only 13.6% for day 19 cells. Thus, many pituitary cells from day 19 fetuses contained, but did not release, GH. While GH-releasing factor (1-44) (1 X 10(-7) M) had no effect on the percentage of GH plaque-forming cells in long term incubations, it enhanced (by approximately the same degree in day 19-21 groups) the percentage of cells that formed plaques and the size of the plaques in short term (45-min) incubations with antibody. Somatostatin (1 X 10(-7) M) exerted inhibitory effects on these variables when tested in long term incubations, and age of the donor rats did not influence pituitary responsiveness to this secretagogue. These results suggest that the capacities of fetal somatotropes to store GH and release it under basal and regulated conditions are attained, in large part, within an extremely narrow time frame between days 18 and 19 of fetal development.

Identification by plaque assays of a pituitary cell type that secretes both growth hormone and prolactin.

Sequential application of reverse hemolytic plaque assays for GH and PRL revealed the presence of individual pituitary cells that released both hormones. These dual cells accounted for approximately one third of all GH and/or PRL secretors in 24-h pituitary cultures derived from male rats. Additional studies in which a different version of the plaque assay and double-staining immunocytochemistry were applied separately to dispersed pituitary cells from males yielded results that were virtually identical. These results suggest that mammosomatotropes, cells that secrete both GH and PRL, may exist in pituitaries of normal rats.

Ontogeny of prolactin cells in neonatal rats: initial prolactin secretors also release growth hormone.

Reverse hemolytic plaque assays and immunocytochemistry were used to monitor the ontogeny of individual hormone-secreting and hormone-containing cells in rats. Monodispersed anterior pituitary cells from fetal rats (sex unspecified) and neonatal rats of each sex were cultured for 24 h and then subjected to immunocytochemistry or plaque assays for PRL or GH. PRL secretors first appeared in appreciable numbers in cultures from 4-day-old animals, and by day 5, they accounted for 8-12% of all cells in culture. The percentage of GH secretors rose to a peak on day 5 (comprising approximately 40% of all cells), when the values were slightly higher than those observed previously in adults. The percentage of cultured cells from 4- to 5-day-olds that released PRL or GH was not influenced by the sex of the donor animal and was consistent with immunocytochemical estimates. Using a sequential plaque assay that enabled the detection of both GH and PRL release from the same cells, we found that of every 100 pituitary cells from 5-day-old males that released PRL and/or GH 62.5 released GH only, 1.7 released PRL alone, and the remaining 35.8 released both hormones. Almost identical proportions were found for females. These findings were confirmed using an additional variation of the plaque assay and by double staining immunocytochemistry. Taken together, these results indicate that mammosomatotropes, cells that release both GH and PRL, appear early in the neonatal development of both sexes and raise the possibility that PRL-secreting cells arise from GH-secreting cells.

Cultures of GH3 cells are functionally heterogeneous: thyrotropin-releasing hormone, estradiol and cortisol cause reciprocal shifts in the proportions of growth hormone and prolactin secretors.

Utilization of reverse hemolytic plaque assays revealed that cultures of GH3 cells are not functionally homogeneous, but contain approximately twice as many GH as PRL secretors. Chronic treatment of these cultures with TRH, estradiol, or cortisol (factors that induce reciprocal alterations in the amount of GH and PRL released by entire cultures of GH3 cells) caused reciprocal shifts in the proportions of GH and PRL cells, without influencing the combined percentages of hormone-secreting cell types present. These results indicate that alterations in hormone release caused by these modulatory factors may in part be due to changes in the ratios of cells committed to the secretion of each hormone. Moreover, the reciprocal nature of these shifts suggests that an interconversion of one cell type to another may have occurred.

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.

A novel bioassay for lactogenic activity: demonstration that prolactin cells differ from one another in bio- and immuno-potencies of secreted hormone.

A reverse hemolytic plaque assay for detecting casein release from individual mammary cells in culture was developed as a bioassay for PRL. Treatment with rat PRL caused dose-related increases in the percentage of mammary cells that released casein and the average size of casein plaques that formed. The assay exhibited exquisite sensitivity (156 fg rat PRL per assay slide) and could be used to evaluate the biopotency of PRL released from individual cells. By combining this "plaque bioassay" with a standard plaque assay for measuring the secretion of immunoreactive PRL, it was possible to compare the bio- and immuno-potencies of hormone released from the same pituitary cells. The results of three separate studies revealed major differences among PRL secretors in these potency estimates. Given the existence of PRL variants with different biological and immunological efficacies, these findings suggest that PRL cells differ from one another in the molecular form(s) of hormone released.

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.

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.

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.

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.

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.

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.

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.

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.