The role of calcium in signal transduction processes in Sertoli cells

Sertoli cells play a pivotal role in regulation and maintenance of spermatogenesis. They are hormonally regulated predominantly by follicle-stimulating hormone (FSH) and testosterone (T). Although FSH and T have distinct mechanisms of action they act synergistically in promoting spermatogenesis. Stimulation of freshly isolated Sertoli cells with FSH evokes a prompt rise in cytosolic calcium which is quantitatively reproduced by cAMP. The cytosolic calcium response to FSH in Sertoli cells is predominantly attributable to serial signaling after the generation of endogenous cAMP. Calcium homeostasis of Sertoli cells may also be regulated by cAMP-independent metabolism. Vasoactive testicular paracrine hormones such as angiotensin II (AII) and vasopressin acting via inositol triphosphate generation induce cytosolic calcium rise predominantly derived from the thapsigargin-sensitive endoplasmic reticulum. Investigations involving androgens action on cytosolic calcium reveal a common mechanism of action between the peptide and steroid regulators of Sertoli cell function, indicating that cytosolic calcium ions may represent a unifying biochemical mechanism that could explain the synergism of FSH and T. Androgens rapidly and specifically increase cytosolic calcium, consistent with a plasma membrane site of action. This argues for the possible existence of a short term non-genomic signaling pathway in hormonal regulation of Sertoli cell function in addition to the classical longer term, slower genomic response.     

Rat Sertoli cell aromatase cytochrome P450: Regulation by cell culture conditions and relationship to the state of cell differentiation

Summary Primary cultures of immature rat Sertoli cells in plastic dishes are highly responsive to follicle stimulating hormone (FSH) and its second messenger, cAMP, in metabolizing testosterone to estradiol, thus indicating the presence of an active, hormone-regulated aromatase cytochrome P450 (P450arom). However, in vivo studies indicated that P450arom is FSH-responsive only in very young animals, where the cells have not yet differentiated, but they lose this ability later on in development. Sertoli cells grown on Matrigel (a reconstituted basement membrane), laminin (a basement membrane component), or in bicameral chambers coated with Matrigel, assume structural and functional characteristics more similar to that of in vivo differentiated Sertoli cells. When the cells were cultured on laminin or Matrigel, the FSH- and cAMP-induced estradiol production was greatly reduced by 30 and 60%, respectively. When Sertoli cells were cultured in bicameral chambers coated with Matrigel, no induction of testosterone aromatization by FSH or cAMP was observed. However, FSH-induced cAMP formation was greater when the cells were cultured on basement membrane or in the chambers than on plastic dishes. These results suggest that culture conditions favoring the assumption by Sertoli cells of a phenotype closer that of the differentiated cells in vivo (tall columnar and highly polarized) suppress the induction of P450arom by FSH and cAMP. We then examined the mechanism(s) by which cell phenotype affects p450arom activity. Northern blot analyses of Sertoli cell RNA revealed one major band of 1.9 Kb and two minor bands of 3.3 and 5.2 Kb. However, there were no changes at the level of the expression of P450arom messenger RNA under the different culture conditions. No differences were found in P450arom enzymatic activity measured by the³H₂O release method in microsomes prepared from Sertoli cells cultured under the various conditions. Similarly, no differences were observed in the amount of protein detected by immunoblot analysis of Sertoli cell extracts using an antiserum raised against the human placental enzyme. Recombination experiments using microsomes from cells cultured on plastic or in the chambers and cytosol from control or FSH-treated cells cultured on plastic also proved inadequate in inducing P450arom activity. These data suggest that: a) P450arom activity could be used as a specific marker for Sertoli cell differentiation, and b) the differentiation process in Sertoli cells is associated with specific changes in the microenvironment or the regulation of P450arom, or both, that rendered the enzyme insensitive to FSH or cAMP induction.     

Voltage-gated calcium channels in rat Sertoli cells.

We have studied Ca2+ voltage-gated channels of immature rat Sertoli cells by measuring intracellular Ca2+ concentration and its variation following administration of various agents in fura-2-loaded, confluent monolayers in culture. Our findings indicate that the basal Ca2+ intracellular level is about 100 nM, a value that falls within the range found in most eukaryotic cells. The intracellular Ca2+ level is rapidly increased by fetal bovine serum through release of intracellularly stored Ca2+ and opening of membrane cation channels. Substantial Ca2+ influx in rat Sertoli cells seems to be mediated by voltage-gated cation channels, which are sensitive to nifedipine, nicardipine, and omega-conotoxin. To investigate whether FSH, which controls several morphological and biochemical events of prepubertal Sertoli cells, modified Ca2+ influx in this cell type, we analyzed the cell response to acute FSH administration. The results show that, although not influencing the basal concentration of Ca2+, FSH decreases intracellular calcium influx induced by membrane depolarization. Similar data were also obtained by adding dibutyryl cAMP to the external medium and by increasing endogenous cAMP.     

Hormonal stimulation alters the type of plasminogen activator produced by Sertoli cells

Primary cultures of immature rat Sertoli cells, maintained in serum-free medium, secrete two types of plasminogen activator (PA). When cultured under basal conditions, the preparations predominantly produce PA having a relative molecular weight (Mr) of 45,000 to 48,000. This PA activity is inactivated by antiserum against urokinase-type PA. When Sertoli cells are stimulated by follicle-stimulating hormone (FSH) or by dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP), PA secretion is increased. The PA produced under these conditions has an Mr of 70,000, and is inactivated by antiserum against tissue-type PA but not by antiserum against urokinase-type PA. We conclude that, under basal conditions, Sertoli cells primarily secrete PA having the characteristics of urokinase-like PA (mu PA), and that Sertoli cells stimulated by FSH or by dbcAMP predominantly produce PA having the properties of tissue-type PA (tPA). Segments of adult rat seminiferous tubules, at defined stages of the cycle of the seminiferous epithelium, also produce and secrete two types of PA into the medium when maintained in organ culture. Segments at all stages examined release primarily mu PA in preparations cultured under basal conditions. In contrast, segments cultured in the presence of FSH synthesize larger amounts of PA, predominantly of the tPA type. An additional protease, which is independent of plasminogen, is secreted by tubule segments stimulated by FSH. The activity of this novel protease is not detectable in cultures maintained under basal conditions. We discuss the data in relation to the possible role of proteases in the restructuring of the seminiferous tubule during spermatogenesis.     

Cyclic nucleotide phosphodiesterases in somatic and germ cells of mouse seminiferous tubules

The distribution of phosphodiesterase forms in somatic and germ cells, and their variations during testicular development and germ cell differentiation have been investigated. Seminiferous tubules from immature mice and Sertoli cells in culture possessed two enzyme activities which were comparable to forms described for different tissues and species: (a) a calcium-calmodulin-dependent enzyme with high affinity for guanosine 3',5'-(cyclic)-monophosphate (cGMP), and (b) a calcium-calmodulin-independent enzyme with high affinity for adenosine 3',5'-(cyclic)-monophosphate (cAMP) the activity of which increased in cultured Sertoli cells after treatment with FSH or dibutyryl cAMP. Seminiferous tubules from adult animals and germ cells at the meiotic and post-meiotic stage of differentiation possessed two enzyme forms that could be distinguished from those present in somatic cells of the seminiferous tubules: (a) a calcium-calmodulin-dependent form with high affinity for both cAMP and cGMP, similar to forms described in other tissues from different species, and (b) a calcium-calmodulin-independent phosphodiesterase with high affinity for cAMP and present only in post-meiotic cells, previously identified also in germ cells of the rat.     

Regulation of angiotensin II receptors in cultured rat ovarian granulosa cells by follicle-stimulating hormone and angiotensin II

The regulation of ovarian granulosa cell angiotensin II (Ang-II) receptor formation and progesterone secretion by follicle-stimulating hormone (FSH) and Ang-II was studied in cultured cells prepared from hypophysectomized, diethylstilbestrol-treated immature rats. Ang-II receptors (estimated by the specific cell binding of the Ang-II receptor antagonist 125I-[Sar1,Ile8]Ang-II) were present on freshly prepared granulosa cells and increased by over 2-fold (to 2150 binding sites/cell; KD = 0.5 nM) when cultured in serum-free medium for 48 h. FSH prevented the normal increase in Ang-II receptor expression. Maximal FSH-dependent decrease in Ang-II receptors and increase in progesterone secretion occurred at 100 ng/ml FSH. The inhibitory effect of FSH on granulosa cell Ang-II receptor content was partially mimicked by the cAMP analogue 8-bromo-cAMP, since 8-bromo-cAMP suppressed (by 96%) Ang-II receptor content to a greater extent than FSH (by 60%). Granulosa cell Ang-II receptor content was not modified by progesterone or 17 beta-estradiol, but was decreased by testosterone (by 35%). Ang-II also produced a decrease in granulosa cell Ang-II receptor content, but did not modify progesterone secretion or aromatase activity. The effect of Ang-II on granulosa cell Ang-II receptor content was mimicked by the Ca2+ ionophore A23187, but not by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, suggesting that an elevation of cytosolic Ca2+ may be important for the homologous down-regulation of the Ang-II receptor. These data show homologous and heterologous down-regulation of granulosa cell Ang-II receptors. If these regulatory mechanisms exist in the FSH-sensitive healthy follicle, our findings suggest that in the process of maturation, healthy and dominant follicles may become decoupled from angiotensinergic influences.     

Purification of ovine transferrin and study of the hormonal control of its secretion in enriched cultures of ovine Sertoli cells.

Ovine transferrin (o-transferrin) was purified from sheep serum by fractionated precipitation with ammonium sulphate, ion-exchange chromatography on DEAE trisacryl and finally by affinity chromatography on Affigel blue to remove albumin. Ovine transferrin was identified by its apparent molecular weight in sodium dodecyl sulphate polyacrylamide gel electrophoresis and by its N-terminal amino-acid sequence. The procedure presented in this report permits the preparation of highly purified o-transferrin with a good recovery (52% of initial total immunoactivity). An antiserum against o-transferrin was then raised in rabbits, using this highly purified preparation. A specific radioimmunoassay was set up using 125I-labelled o-transferrin. Its detection threshold (4 ng/ml) was low enough to measure o-transferrin in spent culture media of ovine Sertoli cells, which ranged between 15 and 600 ng/ml. Sheep seminiferous tubule cells, containing approximately 80% Sertoli cells, were cultured at a high density (1.5 x 10(6) cells/cm2) on a thin layer of reconstituted basement membrane. Kinetic studies showed that basal daily secretion of o-transferrin was reduced by half (-49%) between Day 1 and Day 2 of culture, and progressively decreased thereafter. Under FIRT (500 ng ovine follicle-stimulating hormone (FSH)/ml + 10 micrograms insulin/ml + 500 ng retinol/ml + 5 x 10(-7) mol/l testosterone) stimulation, the ratio of stimulated to basal secretions increased 11-fold between Day 1 (1.1) and Day 6 (12). When 10% fetal calf serum was added, mean o-transferrin secretion was a third of that in serum-free medium, suggesting that fetal calf serum contains factors that inhibit secretion of ovine Sertoli cell transferrin. In the presence of serum, the ratio of FIRT-stimulated to basal secretions doubled between Day 1 (1.0) and Day 4-6 (2.0). Between Days 2 and 4 of culture, insulin had a slight stimulatory effect on o-transferrin secretion (128% of control at 10 micrograms insulin/ml), as well as epidermal growth factor (124% of control at 50 ng/ml). Testosterone at up to 5 x 10(-7) mol/l had no effect; 500 ng retinol/ml doubled o-transferrin secretion (218% of control) as did 500 ng FSH/ml (220% of control). A combination of retinol and FSH increased the secretion 4-fold, indicating that maximal stimulation of o-transferrin secretion by ovine Sertoli cells requires the combined actions of mechanisms dependent and independent of cAMP.     

Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates. Our observations should facilitate efforts to achieve a differentiated functional state of Sertoli and peritubular cells in culture as well as to select secretory proteins for assessing their possible biological role in testicular function.     

Regulation of glypican-1, syndecan-1 and syndecan-4 mRNAs expression by follicle-stimulating hormone, cAMP increase and calcium influx during rat Sertoli cell development.

In seminiferous tubules, Sertoli cells provide structural and nutritional support for the developing germinal cells. Cell- to-cell signaling and cell adhesion require proteoglycans expressed at the cell membrane. A preliminary biochemical and structural approach indicated that cell surface proteoglycans are mostly heparan sulfate proteoglycans (HSPG). Glypican-1, syndecans-1 and -4 were identified using a molecular approach. Their differential regulation was demonstrated in immature rat Sertoli cells. Follicle-stimulating hormone (FSH) is the main regulator of Sertoli cell function. Signal transduction triggered by FSH involves both an increased intracellular cAMP synthesis and a calcium influx. This study demonstrates that FSH, through its second messengers (increase in intracellular cAMP and intracellular calcium), downregulated the glypican-1 mRNA expression in Sertoli cells from 20-day-old rats. On the other hand, syndecan-1 mRNA expression is not modulated by FSH as it would result from the antagonistic effects of increased intracellular cAMP and intracellular calcium levels. Finally, syndecan-4 mRNA expression is not regulated by this pathway. The present study was extended during Sertoli cell development. Indeed, Sertoli cells undergo extensive changes during the postnatal period both in structure and function. These important transformations are critical for the establishment of spermatogenesis and development of the adult pattern of testicular function. Our data indicated that the regulation of HSPG mRNA expression is HSPG-specific and depends on the Sertoli cell developmental stage.     

Transglutaminase 2 activity promotes membrane resealing after mechanical damage in the lung cancer cell line A549

Transglutaminase is a Ca2+-dependent enzyme catalyzing protein cross-linking reactions. We investigated the contribution of this enzyme to the resealing of the injured plasma membrane in animal cells, using a lung cancer-derived cell line, A549. After mechanical injury by razor-scratching, the level of membrane resealing was estimated by differential incorporation of dextrans labeled with two distinct fluorescent dyes. The recovery level was decreased in the presence of excess primary amine, as a competitive inhibitor of transglutaminase. We established a cell line that stably expresses shRNA (short hairpin RNA) to specifically inhibit the expression of TGase 2 (tissue-type isozyme of transglutaminase) and confirmed the suppressed resealing level in the cell. Furthermore, additional expression of TGase 2 rescued the ability for membrane resealing. These results show that, after mechanical damage, this enzyme appeared to contribute to membrane resealing.     

Acute effects of adenosine triphosphates, cyclic 3',5'-adenosine monophosphates, and follicle-stimulating hormone on cytosolic calcium level in cultured immature rat Ssertoli cells.

The ability of ATP and FSH to induce intracellular calcium [Ca(2+)](i) changes in Sertoli cells is imperfectly understood and reports are conflicting. We have applied the single-cell microfluorometry technique with the calcium probe indo-1 to investigate [Ca(2+)](i) in individual cultured Sertoli cells. When cells were exposed to ATP, cAMP, and FSH, a fast and biphasic increase in [Ca(2+)](i) was obtained in 100%, 70%, and 56% of cells, respectively. Caffeine did not activate Ca(2+) mobilization, while thapsigargin suppressed the peak response. External calcium free-EGTA buffer suppressed the plateau phase, while blockers of voltage-operated Ca(2+) channels did not abolish the response to cAMP and ATP. We conclude that the three messengers mobilized Ca(2+) from intracellular thapsigargin-sensitive stores, which induced a subsequent Ca(2+) influx from the extracellular medium by a voltage-independent Ca(2+) entry. The well-documented mechanisms by which these messengers act on cells support the idea that they release Ca(2+) from smooth endoplasmic reticulum by two different pathways, or that FSH and cAMP first release ATP, which then acts on cells. Among the cells, 77% and 80% responded, respectively, to FSH and cAMP by a delayed long-lasting decrease in [Ca(2+)](i) that was never recorded in the presence of ATP. This suggests that FSH and cAMP also promote a slow redistribution of [Ca(2+)](i) from the exchangeable pool to the bound nonexchangeable pools. Involvement of voltage-operated and voltage-independent calcium channels in the response of Sertoli cells to ATP, FSH, and cAMP is discussed.     

A novel hypothalamic peptide, pituitary adenylate cyclase activating peptide, modulates Sertoli cell function in vitro.

Pituitary adenylate cyclase-activating peptide (PACAP), a novel hypothalamic peptide that has been shown to exist in several tissues including the testis, was examined for its effects on cultured rat Sertoli cells. PACAP stimulates cAMP accumulation in Sertoli cells cultured from 15-day-old rats in the presence or absence of methylisobutylxanthine, a phosphodiesterase inhibitor, and in the presence of pertussis toxin, a blocker of the adenylate cyclase inhibitory pathway. Maximal stimulation, which is 20-40% of that attainable with FSH, occurs at PACAP concentrations of 10 nM: the ED50 is approximately 100 pM. The ability of PACAP to stimulate Sertoli cell cAMP declines with increasing age of donor animals (15-60 days of age) in a fashion similar to the FSH effect. PACAP stimulation of Sertoli cell cAMP accumulation is additive with submaximal, but not maximal, concentrations of FSH or forskolin. PACAP also stimulates the secretion of lactate, estradiol, and inhibin in a concentration-dependent manner. The stimulation of Sertoli cell cAMP accumulation by PACAP is not altered by a vasoactive intestinal peptide antagonist, and vasoactive intestinal peptide alone does not stimulate cAMP accumulation, indicating that PACAP is not acting via vasoactive intestinal peptide receptors. Further experiments are needed to determine whether PACAP is synthesized within the testis and if so, in which cell types; however, the present data clearly demonstrate that PACAP can modulate Sertoli cell function in vitro.     

Stimulatory effect of Sertoli cell culture medium on the FSH release by pituitary halves in vitro

The influence of the medium collected from cultured rat Sertoli cells on the spontaneous and LHRH-stimulated release of gonadotropins by incubated rat pituitary halves was examined. The homogeneity of the cultured population of Sertoli cells taken from 20-day-old rats ranged up to 98%. The cells in culture responded to FSH stimulation with characteristic morphological changes and with increased secretion of estradiol-17 beta. The hemi-pituitaries obtained from sexually mature male rats were incubated for 5 hours in the presence of Sertoli cell culture medium (SCCM) or its fractions obtained by use of ultrafiltration. The SCCM fraction deprived of MW less than 10 kD compounds exhibited a typical inhibin-like activity, whereas crude SCCM as well as its low-molecular-weight fraction stimulated the basal FSH release to about 150% and 175% of the control values, respectively. These fractions exerted an inhibitory effect on the LHRH-stimulated secretion of both LH and FSH. It is concluded that Sertoli cells cultured in chemically defined medium release, apart from inhibin, a non-steroidal, heat-labile substance of MW less than 10 kD which stimulates the basal secretion of FSH and LH and inhibits the LHRH-stimulated secretion of both gonadotropins from incubated rat hemi-pituitaries.     

Expression and function of class B scavenger receptor type I on both apical and basolateral sides of the plasma membrane of polarized testicular Sertoli cells of the rat

Class B scavenger receptor type I (SR-BI), a multiligand membrane protein, exists in various organs and cell types. In the testis, SR-BI is expressed in two somatic cell types: Leydig cells and Sertoli cells. Unlike interstitially localized Leydig cells, Sertoli cells present within the seminiferous tubules keep contact with spermatogenic cells and form the tight junction to divide the seminiferous epithelium into the basal and adluminal compartments. In this study, the expression and function of SR-BI in rat Sertoli cells were examined with respect to dependency on the spermatogenic cycle, the plasma membrane polarity, and the pituitary hormone follicle-stimulating hormone (FSH). When the expression of SR-BI was histochemically examined with testis sections, both protein and mRNA were already present in Sertoli cells during the first-round spermatogenesis and continued to be detectable thereafter. The level of SR-BI mRNA expression in Sertoli cells was lower at spermatogenic stages I-VI than at other stages. SR-BI was present and functional (in mediating cellular incorporation of lipids of high density lipoprotein) at both the apical and basolateral surfaces of polarized Sertoli cells. Finally, SR-BI expression at both the protein and mRNA levels was stimulated by FSH in cultured Sertoli cells. These results indicate that SR-BI functions on both the apical and basolateral plasma membranes of Sertoli cells, and that SR-BI expression in Sertoli cells changes during the spermatogenic cycle and is stimulated, at least in cultures, by FSH.     

Rapid signaling responses in Sertoli cell membranes induced by follicle stimulating hormone and testosterone: calcium inflow and electrophysiological changes

This minireview describes the rapid signaling actions of follicle stimulating hormone (FSH) and testosterone in immature Sertoli cells mainly related to Ca(2+) inflow and the electrophysiological changes produced by hormones. The rapid membrane actions of FSH occur in a time frame of seconds to minutes, which include membrane depolarization and the stimulation of (45)Ca(2+) uptake. These effects can be prevented by pertussis toxin (PTX), suggesting that they are likely mediated by Gi-protein coupled receptor activation. Furthermore, these effects were inhibited by verapamil, a blocker of the L-type voltage-dependent Ca(2+) channel (VDCC). Finally, FSH stimulation of (45)Ca(2+) uptake was inhibited by the (phosphoinositide 3-kinase) PI3K inhibitor wortmannin. These results suggest that the rapid action of FSH on L-type Ca(2+) channel activity in Sertoli cells from pre-pubertal rats is mediated by the Gi/Gβγ/PI3Kγ pathway, independent of its effects on insulin-like growth factor type I (IGF-I). Testosterone depolarizes the membrane potential and increases the resistance and the (45)Ca(2+) uptake in Sertoli cells of the seminiferous tubules of immature rats. These actions were nullified by diazoxide (K(+)(ATP) channel opener). Testosterone actions were blocked by both PTX and the phospholipase C (PLC) inhibitor U73122, suggesting the involvement of PLC - phosphatidylinositol 4-5 bisphosphate (PIP2) hydrolysis via the Gq protein in the testosterone-mediated pathway. These results indicate that testosterone acts on the Sertoli cell membrane through the K(+)(ATP) channels and PLC-PIP2 hydrolysis, which closes the channel, depolarizes the membrane and stimulates (45)Ca(2+) uptake. These results demonstrate the existence of rapid non-classical pathways in immature Sertoli cells regulated by FSH and testosterone.     

The role of calcium in follicle-stimulating hormone signal transduction in Sertoli cells.

Sertoli cells are hormonally regulated by follicle-stimulating hormone (FSH) acting upon a G-protein-linked cell surface FSH receptor. FSH increases intracellular cyclic AMP but the involvement of other signal transduction mechanisms including intracellular calcium in FSH action are not proven. Using freshly isolated rat Sertoli cells we measured cytosolic free ionized calcium levels by dual-wavelength fluorescence spectrophotometry using the calcium-sensitive fluorescent dye Fura2-AM. The cytosolic calcium concentration in unstimulated Sertoli cells was 89 +/- 2 nM (n = 151 experiments) and was markedly increased by either calcium channel ionophores (ionomycin, Bay K8644) or plasma membrane depolarization consistent with the presence of voltage-sensitive and -independent calcium channel in Sertoli cell membranes. Ovine FSH stimulated a specific, sensitive (ED50, 5.0 ng of S-16/ml), and dose-dependent (maximal at 20 ng/ml) rise in cytosolic calcium commencing within 60 s to reach levels of 192 +/- 31 nM after 180 s and lasting for at least 10 min. The effect of FSH was replicated by forskolin, cholera toxin, and dibutyryl cyclic AMP, suggesting that cyclic AMP may mediate the FSH-induced rise in cytosolic calcium. The FSH-induced rise in cytosolic calcium required extracellular calcium and was abolished by calcium channel blockers specific for dihydropyridine (verapamil, nicardipine), nonvoltage-gated (ruthenium red) or all calcium channels (cobalt). Thus FSH action on Sertoli cells involves a specific, rapid, and sustained increase in cytosolic calcium which requires extracellular calcium and involves both dihydropyridine-sensitive, voltage-gated calcium channels and voltage-independent, receptor-gated calcium channels in the plasma membranes of rat Sertoli cells. The replication by cyclic AMP of the effects of FSH suggests that calcium may be a signal-amplification or -modulating mechanism rather than an alternate primary signal transduction system for FSH in Sertoli cells.     

FSH,EGF,胰岛素促进小鼠卵母细胞体外减数分裂恢复机制的研究

FSH、EGF和胰岛素均对体外培养的小鼠卵母细胞的减数分裂的恢复起促进作用,而FSH的促进作用滞后,但作用后使卵丘细胞扩散。三者的促进作用似受卵巢颗粒细胞内游离钙和cAMP的调节。EGF和胰岛素可使培养的颗粒细胞内的cAMP水平降低;同时FSH使单个卵丘细胞内的游离Ca~(2 )水平降低,而胰岛素无影响。所以FSH、EGF和胰岛素诱发卵母细胞成熟的机制不同:EGF通过细胞内Ca~(2 )的升高和cAMP水平的下降促使卵母细胞的减数分裂恢复;FSH降低卵丘细胞内Ca~(2 )的水平,但由于卵丘细胞与卵母细胞之间的联系被打断,最终使GVBD发生;而胰岛素的作用只涉及胞内cAMP的变化。