Activation of transcription factor NF-kappaB by growth inhibitory cytokines in vulvar carcinoma cells.

Numerous investigations show that cytokines have a significant role in the regulation of cell growth. There is also increasing evidence for the role of transcription factors in cytokine-mediated growth-regulation of cancer cells. Our previous data demonstrate that several cytokines are able to inhibit DNA synthesis of vulvar carcinoma cells. The aim of this study was to investigate the effect of growth-inhibitory cytokines on the binding activity of transcription factor AP-1 and NF-kappaB in two vulvar carcinoma cell lines UM-SCV-6 and UM-SCV-1A in vitro. The effects of interferon gamma (IFN-gamma), interleukins 10 (IL-10) and 13 (IL-13), transforming growth factor beta(1) (TGF-beta(1)) and tumor necrosis factor alpha (TNF-alpha) on the DNA binding proteins were studied by electrophoretic mobility shift assay (EMSA). Our results showed that NF-kappaB and AP-1 were constitutively activated in both cell lines. The binding activity of NF-kappaB was found to be stimulated by TNF-alpha in both vulvar carcinoma cell lines while no effect on AP-1 was found by any of the cytokines. The binding activity of NF-kappaB was decreased by IL-10 and IL-13 in UM-SCV-1A cells suggesting that the pathway by which TNF-alpha activates NF-kappaB differs from that activated by interleukins.

Regulation of UT-OC-3 ovarian carcinoma cells by cytokines: inhibitory effects on cell proliferation and activation of transcription factors AP-1 and NF-kappaB.

The present study was designed to investigate the growth regulatory effects of cytokines in UT-OC-3 ovarian cystadenocarcinoma cells in vitro. The effects of interleukin-6 (IL-6), interferons alpha (IFN-alpha) and gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor alpha (TNF-alpha), and transforming growth factor beta1 (TGF-beta1) were investigated by (125)I-deoxyuridine ((125)IUdR) incorporation assay. In order to understand better the molecular mechanisms of the observed effects, the activation of DNA-binding proteins was studied by electrophoretic mobility shift assay. In addition, cellular DNA was tested by fragmentation analysis to determine if the most growth inhibitory cytokines are able to induce programmed cell death (apoptosis). After 48h in culture, TGF-beta1, TNF-alpha, IFN-alpha and IL-6 showed a clear inhibitory effect on (125)IUdR incorporation (P<0.005), and IFN-gamma and GM-CSF caused even more significant inhibition (P<0.001). IFN-alpha and IFN-gamma were both growth inhibitory after 72h in culture (P<0.005). Similarly, GM-CSF induced a slight inhibition (P<0.05), whereas TGF-beta1 and TNF-alpha almost blocked DNA synthesis (P<0.001) after 72h. IL-6 had no statistically significant effect on cell proliferation after 72h. Transcription factors AP-1 and NF-kappaB were both constitutively expressed in UT-OC-3 cells. The binding activity of AP-1 was found to be stimulated by the growth inhibitory cytokines, TGF-beta1 and TNF-alpha, and the binding of NF-kappaB was stimulated by TNF-alpha. Apoptosis does not seem to be induced by any of these cytokines in the UT-OC-3 ovarian cancer cell model.

Activin B: detection by an immunoenzymometric assay in human serum during ovarian stimulation and late pregnancy.

A recently developed immunoenzymometric assay for activin B has been characterized further by measurement during ovarian stimulation and pregnancy. The assay is based on a monoclonal anti-peptide antibody, anti-betaB(101-115). In addition to quantitative analyses, the antibody has been used for immunohistochemical localization of the activin betaB-subunit in human term placenta. Serum samples obtained from patients suffering from tubal factor infertility who were admitted for in-vitro fertilization (IVF) treatment protocols or from patients with proven fertility who were admitted for laparoscopic tubal ligation were collected. The aim was to correlate serum activin B concentrations with other parameters during IVF and with phases of the menstrual cycle. Serum samples obtained from healthy pregnant volunteers were studied to correlate activin B concentrations with clinical parameters. During the IVF treatment protocols, activin B was detectable in all patients studied, and a significant negative correlation was observed between serum activin B and oestradiol concentrations. On the other hand, no significant difference was observed in activin B concentrations when serum samples obtained from patients at different phases of the menstrual cycle were compared, and low concentrations of activin B were observed in the samples obtained from these patients. During pregnancy, a positive correlation was observed between serum activin B concentrations and gestational age. In immunohistochemical analyses of human placental tissue obtained from healthy parturients, the activin betaB-subunit was present in trophoblast, amniotic epithelial and Hofbauer cells. The results suggest a potential clinical application in female reproductive medicine for serum activin B measurements.

Inhibitory effects of cytokines on ovarian and endometrial carcinoma cells in vitro with special reference to induction of specific transcriptional regulators.

In the present study, we have investigated the effects of interferons-alpha (IFN-alpha) and -gamma (IFN-gamma), interleukin-10 (IL-10) and -13 (IL-13), transforming growth factor-beta1 (TGF-beta1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha) on cell proliferation and induction of transcription factors AP-1 and NF-kappaB in UM-EC-3 human endometrial adenocarcinoma cells and UT-OC-5 ovarian carcinoma cells in vitro. In addition, cellular DNA was extracted to study if any of these factors is able to induce apoptosis. In UM-EC-3 cell line DNA synthesis was inhibited by GM-CSF, IL-10, IL-13, TGF-beta1, IFN-alpha, and IFN-gamma after 48 and 72 h in culture, whereas TNF-alpha had no significant effect on cell proliferation in any of the experiments. The inhibition of DNA synthesis was similarly observed in UT-OC-5 ovarian carcinoma cells by IL-10, TNF-alpha, and IFN-gamma after 48 and 72 h, whereas IFN-alpha had no statistically significant effect. An inhibitory effect of GM-CSF was observed only after 48 h and TGF-beta after 72 h in culture, respectively. Transcription factors AP-1 and NF-kappaB were both constitutively active in UM-EC-3 and UT-OC-5 cells. The binding activity of AP-1 was found to be stimulated by all growth-inhibitory cytokines studied in both cell lines, whereas the specific binding activity of NF-kappaB was affected moderately only by TNF-alpha in UT-OC-5 ovarian carcinoma cells. No signs of DNA fragmentation typical of apoptosis were observed in any of these studies.

Regulation of proliferation of UM-SCV-1A and UM-SCV-6 vulvar carcinoma cells by cytokines.

The biology and pathogenesis of vulvar carcinoma are poorly understood at present. In order to understand this disease better, we have used recently developed squamous cell carcinoma lines of the vulva as models. Two cell lines originating from two individuals (UM-SCV-1A and UM-SCV-6) were cultured in vitro in 10% fetal calf serum. The effects of interleukins 10 and 13, interferons alpha and gamma, granulocyte/macrophage-growth-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF alpha), and transforming growth factor beta (TGFbeta) on the proliferation of the cells was investigated by using radioactively labelled uridine as tracer. In addition, an investigation on the molecular structure of extracted cellular DNA was carried out to investigate whether programmed cell death (apoptosis) would be inducible by any of the factors. In UM-SCV-1A cells, interleukin-10 (IL-10) and interleukin-13 (IL-13) caused an approximately 12-fold decrease in DNA synthesis in cells cultured for 72 h (P<0.001), while GM-CSF had no significant effect. TGFbeta showed a significant inhibitory effect on deoxyuridine incorporation (P<0.001), which was 2.0- and 4.2-fold at 48 h and 72 h, respectively. TFG alpha showed a 1.2-fold inhibitory effect on DNA synthesis at 48 h (P<0.01) and a 1.5-fold inhibition at 72 h (P<0.05). Interferon gamma (IFNgamma) showed an inhibitory effect on DNA synthesis (1.3-fold; P<0.01). In UM-SCV-6 cells, both IL-10 and IL-13 showed inhibitory effects on deoxyuridine incorporation (1.3- and 1.4-fold at 48 h, respectively; P<0.001) that were even more pronounced at 72 h (2.4- and 2.5-fold respectively; P<0.001). IFNgamma caused a 3.6-fold inhibition of DNA synthesis by UM-SCV-6 cells at 72 h (P<0.001). Both TFGbeta and TNF alpha inhibited uridine incorporation (3.0- and 1.6-fold at 48 h, respectively; 2.7-fold at 72 h for both factors). GM-CSF inihibited DNA synthesis by UM-SCV-6 cells 1.3- 2.0-fold at 48 h and 72 h, respectively. In dose/response analyses, the effect of INF alpha on DNA synthesis was inhibitory in both cell lines at 48 h, while stimulatory effects were observed at 72 h. Electrophoretic analyses of DNA isolated from cells cultured in the presence or absence of different factors did not reveal DNA fragmentation. All cytokines, with the exception of IFN alpha, showed inhibitory effects on DNA synthesis by vulvar carcinoma cells. Of the factors studied, the recently described interleukins 10 and 13 showed potent inhibition of cell growth, encouraging further investigation on the molecular mechanisms of the observed inhibition. Apoptosis does not seem to be induced in the two vulvar carcinoma cell lines by any of the cytokines studied.

Gonadotropins and ovarian gonadotropin receptors during the perimenopausal transition period.

As an early sign of perimenopause, the menstrual cycles become irregular. Associated with this irregularity, serum gonadotropin levels increase. There are studies correlating these changes to ovarian follicular morphology and number, but no reports are available, to our knowledge, correlating the increases in gonadotropin levels to respective cellular receptors. In fact, the regulation of gonadotropin receptors is poorly understood in the human. The action of gonadotropins on gonadal function is mediated through specific binding of the hormones to receptors located on the surface of target cells followed by activation of intracellular second messenger systems. In the ovary, the target cells for FSH are granulosa cells of ovarian follicles, while the action of LH is mediated through binding of the ligand to theca, granulosa and luteal cells. The cell-surface receptors for gonadotropins are located respectively on these cell types. The molecular nature of FSH receptors and LH receptors has been characterized in many species, and their structure is well-known both on the protein and nucleic acid level. Despite the vast amount of studies in rodents, there is limited information available on the regulation of gonadotropin receptors in the human. In a recent study, serum LH and FSH levels were correlated to respective receptor levels in ovarian tissue in order to better understand gonadotropin action and the physiology of the menopause in the human. High serum gonadotropin levels in perimenopause suggested the existence of low ovarian gonadotropin receptor levels. In most patients who are still experiencing cyclic menstrual bleeding no FSH receptors were detected possibly due to severe disturbances in follicular FSH receptor synthesis prior to ovarian follicle exhaustion.

Gonadotropins and gonadotropin receptors during the perimenopause.

Twenty-two perimenopausal patients (aged 47-56 years) admitted for elective abdominal hysterectomy and salpingo-oophorectomy were selected to understand better the clinical significance of increasing gonadotropin levels as an indicator of target organ responsiveness. Prior to anesthesia, blood was drawn from the patients for subsequent analyses of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH) and 17beta-estradiol (E2) levels. Ovarian tissue was obtained during surgery and frozen at -70 degrees C for subsequent analyses for FSH and LH receptor content. The phase of the menstrual cycle of the patients or postmenopause was determined by serum gonadotropin and E2 levels and histological evaluation of the endometrium. Patients with no detectable FSH receptors showed significantly higher serum FSH and LH levels (4.7- and 4.3-fold, respectively) when compared to patients with detectable FSH receptors; FSH receptors were present in 27% of the patients, LH receptors were present in 68% of the patients and a negative correlation was found between serum LH levels and ovarian LH receptors. In postmenopausal patients, neither FSH receptors nor LH receptors were detectable. High serum gonadotropin levels in perimenopausal patients thus suggest the existence of low or undetectable ovarian gonadotropin receptor levels.

Effects of recombinant human FSH in immature hypophysectomized male rats: evidence for Leydig cell-mediated action on spermatogenesis.

The mode of FSH actions within the testis was studied in immature hypophysectomized male rats by treatment with recombinant human FSH (recFSH, Org 32489). To elucidate the involvement of Leydig cells and androgens in the maintenance of spermatogenesis in FSH-treated hypophysectomized rats further, the recFSH treatment was given both alone and after destruction of Leydig cells with ethane-1,2-dimethane sulphonate (EDS). Three days after hypophysectomy (at 31 days of age) the rats were given one i.p. injection of vehicle or EDS and, 4 days later, they were implanted with osmotic minipumps releasing either 0.9% (w/v) NaCI or 1 IU recFSH/day. Recombinant FSH alone increased testicular weights 2.5-fold in 7 days (P < 0.01). The effect of FSH was similar in EDS-pretreated rats (P < 0.01). Testicular testosterone increased from 6.5 +/- 1.6 to 16.9 +/- 5.3 (S.E.M.) pmol/g tissue (P < 0.05) and serum testosterone from 0.12 +/- 0.02 to 0.22 +/- 0.03 nmol/l (P < 0.05) when the rats were treated with recFSH. EDS alone did not affect testicular testosterone but, when combined with recFSH, it totally abolished the stimulatory effect of FSH on testosterone. Testicular binding of 125I-labelled iodo human chorionic gonadotrophin (hCG) and 125I-labelled iodo recFSH was increased 2.5- and 2.1-fold respectively with recFSH treatment (P < 0.01). EDS, either alone or with FSH, abolished specific testicular hCG binding (P < 0.01), but had no effect on that of recFSH. However, FSH increased its own receptors only in animals not treated with EDS.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal control of apoptotic cell death in the testis: gonadotropins and androgens as testicular cell survival factors.

Although the requirement for pituitary gonadotropins during testicular cell differentiation is well documented, the possible role of FSH and LH in regulating testicular cell survival has not been studied. Using a quantitative autoradiographic method for the detection of internucleosomal DNA fragmentation, a hallmark feature of apoptosis, the hormonal control of apoptotic cell death was studied in testicular cells collected from immature rats after hypophysectomy. After surgery, animals were treated with daily injections of 20 IU long-acting FSH agonist (FSH-CTP) or 50 IU human CG (hCG) for 2 days. Hypophysectomy decreased testis weight by 25%, but treatment with FSH-CTP or hCG prevented the effect of hypophysectomy. Testes of intact animals contained predominantly high-mol wt DNA, whereas hypophysectomy increased DNA cleavage into low-mol wt (< 15 kilobases) ladders characteristics of apoptosis. In contrast, treatment with FSH-CTP or hCG inhibited hypophysectomy-induced apoptotic DNA cleavage by 84% and 51%, respectively. Hypophysectomy-induced DNA fragmentation was found in both interstitial cells and seminiferous tubules. Similar to whole testis, treatment with FSH-CTP suppressed hypophysectomy-induced apoptosis by over 90% in seminiferous tubules and interstitial cells. In contrast, hCG treatment was less effective in preventing hypophysectomy-induced DNA cleavage (46% suppression in tubules and 77% suppression in interstitial cells). Furthermore, testosterone replacement also suppressed hypophysectomy-induced DNA fragmentation by 75% in the whole testis tissue, 64% in tubules, and 55% in interstitial cells. To further study the role of gonadotropins, intact animals were treated with a potent GnRH antagonist (Azaline B, 10 microgram/day) to decrease serum gonadotropin levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of testicular messenger ribonucleic acid for luteinizing hormone receptor in the rat: developmental regulation of multiple transcripts during postnatal life.

On the basis of earlier observations of changing testicular LH receptor levels during postnatal development of the rat, we analyzed the levels of LH receptor mRNA transcripts in testes of rats at ages 5-70 days. Extracted testis RNA, prepared for Northern blotting, was hybridized with a specific LH receptor cRNA probe derived from subcloned cDNA corresponding to the extracellular domain of the receptor. Six LH receptor mRNA transcripts with molecular sizes of 7.8, 7.0, 4.2, 2.5, 1.8, and 1.2 kb were identified. Of these, the 1.2- and the 1.8-kb mRNA transcripts presumably code for truncated forms of LH receptor. At 5 days, only the 1.8- and the 4.2-kb mRNA transcripts were observed. Additional 7.0- and 1.2-kb transcripts appeared at 10 and 15 days, respectively. From the age of 25 days through adulthood, all six mRNA transcripts were observed. Densitometric analyses revealed that the amounts of the 7.0- and 1.8-kb mRNA transcripts correlated well with LH receptor levels, while the 4.2-kb transcript showed high levels earlier in life with poor correlation to LH receptor number. Because the 1.8 kb receptor transcript lacked transmembrane domains, the present results suggest the 7.0-kb LH receptor transcript as the likely candidate to encode the functional receptor. These data provide the basis for future analyses of the molecular regulation of LH receptor expression.

Stimulatory effects of recombinant follicle-stimulating hormone on Leydig cell function and spermatogenesis in immature hypophysectomized rats.

Although earlier reports suggest a stimulatory effect of FSH on Leydig cell function, controversy exists due to unavailability of FSH preparations free of contaminating LH. Recent availability of recombinant human FSH preparations made it possible to reinvestigate this question. Immature male rats were hypophysectomized (21-22 days old at surgery) and implanted with osmotic minipumps releasing 8 IU recombinant FSH or 18 IU purified human pituitary FSH (hpFSH)/day, whereas control animals received vehicle alone. After 7 days of treatment, testicular weight increased in the recombinant FSH and hpFSH-treated animals to values 2.3- and 2.5-fold those of controls, respectively. Analyses of the steroidogenic capacity of Leydig cells in testes of rats treated with recombinant FSH or hpFSH also revealed 2.9- and 3.8-fold androgen production in vitro compared to controls. In these rats recombinant FSH and hpFSH increased the LH receptor number in testicular homogenate by 50% and 70%, respectively. The increase in LH receptor number was associated with increases in the LH receptor mRNA levels. In hypophysectomized control rats, small seminiferous tubules contained spermatogonia and zygotene/early pachytene spermatocytes. In contrast, treatment with either FSH preparation enhanced the progression of meiosis, as evidenced by large number of pachytene spermatocytes and appearance of round spermatids. The present results show that LH-free recombinant FSH, like purified pituitary FSH, is capable of increasing the LH receptor content and steroidogenic responsiveness of Leydig cells through paracrine mechanisms together with a stimulatory effect on spermatogenesis. These observations suggest that prepubertal elevation of FSH secretion may be important for increasing Leydig cell steroidogenic capacity and spermatogenic progression.

Differential response of luteinizing hormone receptor and steroidogenic enzyme gene expression to human chorionic gonadotropin stimulation in the neonatal and adult rat testis.

To study further the functional differences of the fetal-neonatal and adult growth phases of Leydig cells, neonatal (5-day-old) and adult (60-day-old) male rats were challenged with a 600 IU/kg injection of human CG (hCG). Certain Leydig cell responses were monitored 1, 2, and 3 days after the hCG injection. The down-regulation of LH receptors and blockage of the 17-hydroxylase/C17-20 lyase step in adult testis, and the absence of these responses in neonatal testis were confirmed. Novel data were obtained on concomitant responses of LH receptor and steroidogenic enzyme messenger RNAs (mRNAs). The LH receptor mRNA was increased 4-5-fold by 2 days after hCG injection in the neonatal testis (P less than 0.05), but in the adult was decreased during all 3 days by 50% (P less than 0.05). The mRNA level of the cytochrome P450 for cholesterol side chain cleavage responded similarly at both ages, with a 180-260% increase during 2 and 3 days (P less than 0.05-0.01). In contrast, the 17-hydroxylase/17,20-lyase cytochrome P450 mRNAs displayed opposite responses, increasing 4.5-fold in 2 days (P less than 0.01) in the neonates, but decreasing by 80% in 1 day in the adults (P less than 0.01). No response of the aromatase cytochrome P450 mRNA to hCG stimulation was found at either age studied. These results demonstrate that the functional differences of the neonatal and adult Leydig cells to high gonadotropic stimulation occur at the level of expression of specific genes, including those of the LH receptor and the 17-hydroxylase/17,20-lyase cytochrome P450. Although aromatization of testicular androgens has been suggested to mediate the blockade of the 17-hydroxylase/C17-20 lyase step in adult testes, altered steady state levels of aromatase mRNA are not involved in this response. LH receptor mRNA decreases in adult rat testis in response to treatment with high levels of hCG. Thus, this phenomenon of down-regulation of membrane receptors includes a decreased LH receptor mRNA as well as cellular internalization of the existing receptors.

A rat seminiferous epithelial factor that inhibits Leydig cell cAMP and testosterone production: mechanism of action, stage-specific secretion, and partial characterization.

To elucidate further the seminiferous tubule-Leydig cell interaction we studied the effect of spent medium from 20 h rat seminiferous tubule cultures (STCM) on cyclic adenosine monophosphate (cAMP) and testosterone (T) production of Percoll-purified Leydig cells. 8% of STCM inhibited the human chorionic gonadotropin (hCG)-stimulated cAMP production by 30% in a 3 h, and 33% of STCm by 60% in a 20 h incubation. Likewise, a 40% decrease was found in the presence of 8% of STCM in the hCG-stimulated T production in a 3 h incubation. A similar inhibitory activity could be demonstrated in steroid-free rat interstitial fluid. STCM did not affect the viability of the Leydig cells (90-95% after a 20 h incubation) as judged by trypan blue exclusion and the adenosine triphosphate (ATP) content of the cells. Heating (80 degrees C for 10 min) abolished the inhibiting activity, and fractionation with Millipore Ultrafree filters showed that the inhibiting factor had an Mr of 30,000-100,000. When media from different stages of the seminiferous epithelial cycle were analyzed, only stages IX-I showed inhibition of T production (P less than 0.05). The cAMP production inhibiting activity was present in all stages, but stages IX-I showed significantly (P less than 0.05) greater inhibition than stages II-VI. STCM (16%) also inhibited cholera toxin- and forskolin-stimulated cAMP formation (approximately 50 and 60%, respectively; P less than 0.01), and the inhibitory activity persisted after a 3 h preincubation of Leydig cells with 100 micrograms/l pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of urokinase- and tissue-type plasminogen activator gene expression in the rat seminiferous epithelium.

The secretion of plasminogen activator by seminiferous tubules at defined stages of the epithelial cycle is influenced both by neighboring spermatogenic cells and by hormones. We have used cRNA probes for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators to analyze their mRNA levels in different stages of the epithelial cycle. Urokinase-type PA mRNA was most abundant in stages VII-VIII, while tPA mRNA levels showed smaller variations between the different stages. Both FSH and (Bu)2cAMP increased the steady-state level of tPA mRNA and tPA production without affecting those of uPA in stages VII-IX in vitro, whereas retinoic acid treatment selectively increased the concentration uPA mRNA and uPA production in stages II-VI. The results show that the expression of the uPA and tPA genes is differentially regulated in specific stages of the rat seminiferous epithelium.

Immunohistochemical localization of urokinase-type plasminogen activator in Sertoli cells and tissue-type plasminogen activator in spermatogenic cells in the rat seminiferous epithelium.

The occurrence of plasminogen activators of the urokinase-type (u-PA) and tissue-type (t-PA) at various stages of the epithelial cycle was studied immunohistochemically in rat seminiferous tubule segments. u-PA immunoreactivity was detected exclusively at stages VII and VIII in Sertoli cells, displaying a distinct granular cytoplasmic staining. t-PA immunoreactivity was found during mid- and late pachytene and diakinesis (stages VII-XIII) in spermatogenic cells, displaying a granular cytoplasmic staining with maximal intensity in stages IX-XIII. The specificity of the stainings was supported by staining controls, including absorption of the antibodies with purified preparations of the activators. It was also supported by zymographic studies of the occurrence of u-PA and t-PA in extracts of tubular segments at different stages of the cycle, isolated by transillumination-assisted microdissection. The possible functions of the two types of plasminogen activators in the seminiferous epithelium are discussed.

Types I and IV collagenolytic and plasminogen activator activities in preovulatory ovarian follicles.

During ovulation, enzymatic degradation of the extracellular matrix occurs within and around the graafian follicles. In this study, the activities of several different proteolytic enzymes were measured in the culture media of follicles taken from pregnant mare serum gonadotropin (PMSG)-primed immature rats. At 52 h after PMSG, the follicles were cultured for 2 to 15 h in media with or without human chorionic gonadotropin (hCG). Type I collagenase activity in hCG-stimulated follicles gradually increased within 6 h to 3.3-fold above that of the controls. Relatively pure populations of granulosa cells produced type I collagenase to a similar extent. Likewise, type IV collagenase increased 3.8-fold by 6 h after exposure of the follicles to hCG. In contrast, plasminogen activator activity increased by 3.9-fold at 2 h after hCG, but was negligible at 4, 6, and 15 h after incubation. These results suggest that plasminogen activator may activate both type I and type IV collagenase in hCG-stimulated ovulatory follicles.

Stage-specific regulation of plasminogen activator secretion in the rat seminiferous epithelium.

The cyclic secretion of plasminogen activator (PA) by Sertoli cells in stages VII and VIII of the rat seminiferous epithelial cycle is influenced by hormones and adjacent spermatogenic cells. To understand this interaction more in detail, we have analyzed the effects of FSH, (Bu)2cAMP, testosterone, insulin, and retinoic acid (RA) on staged seminiferous tubule segments in vitro. FSH stimulated stages VIIcd to XI of the cycle; similar results were obtained with (BU)2cAMP. RA stimulated PA secretion in stages I-VIIab, but testosterone and insulin had no effect in any stage. The secreted PA was mainly of the urokinase type, although small amounts of the tissue-type PA were found after stimulation by FSH and cAMP. These results suggest that spermatogenic cells modify the responsiveness of Sertoli cells to hormonal stimulation. Stages I-VIIab are sensitive to stimulation by RA whereas stages VIIcd-XI are preferentially stimulated by FSH and (Bu)2cAMP.

Local synthesis of plasminogen by the seminiferous tubules of the testis.

Plasminogen activation occurs through conversion of plasminogen to plasmin by plasminogen activators. In adult mammals, liver has been the only known site for plasminogen synthesis. Seminiferous tubules secrete plasminogen activator, but are behind a barrier that excludes the entrance of many macromolecules to this tissue. Therefore, it became of interest to study the existence of plasminogen in this system. After metabolic labeling, a 90 kDa lysine-binding polypeptide was found. This protein could be immunoblotted by anti-plasminogen antibody, and was shown to possess urokinase-dependent proteolytic activity. The findings suggest that plasminogen is synthesized by seminiferous tubules. We propose that local plasminogen synthesis may occur in tissues which are separated by specific anatomical barriers.

Testicular plasminogen activators during postnatal development in the rat.

In the seminiferous epithelium, Sertoli cells secrete plasminogen activator (PA) under regulation of follicle stimulating hormone, cyclic AMP and neighbouring spermatogenic cells. Recent observations suggest that preleptotene spermatocytes upon their release from the basement membrane of the seminiferous tubule are important regulators of PA secretion. To study further the role of PA's in the seminiferous tubules, we have analyzed the endogenous levels and secretion rates of PA at various ages during postnatal development, and performed biochemical analyses of the types of PA in the testis and spent media from seminiferous tubular cultures. Cyclic secretion of PA started at the age of 28 days, and from 40 days onwards, the high secretion rates were localized in stages VII and VIII of the cycle of the seminiferous epithelium. The secreted PA is most obviously of the urokinase type; both urokinase-type and tissue-type PA-like activities were found in seminiferous tubular homogenates. The increase in testicular PA levels concomitant to the onset of meiosis in the epithelium was due to the urokinase-type PA-like activity.