Biochemical characterization of integral membrane heparan sulfate proteoglycans in Sertoli cells from immature rat testis

(35)S-Radiolabeled cultured Sertoli cells from immature rat testis were extracted with detergent and the different proteoheparan sulfate (HSPG) forms of the extract were discriminated and quantified on the basis of their high anionic charge, hydrodynamic size, lipophilic properties, susceptibility to trypsin and phosphatidylinositol phospholipase C (PI-PLC). Trypsin released 50% of total cellular HSPG corresponding to 80% of total hydrophobic HSPG. Trypsin-accessible HSPG were presumed to be integral membrane species. Trypsin-resistant HSPG, probably intracellular, distributed into non-lipophilic (37.5%) and lipophilic (12.5%) populations. Biochemical analysis of PG copurified with plasma membrane confirmed the existence of hydrophobic HSPG integrated into this structure. Among hydrophobic HSPG accessible to trypsin, 35% were PI-PLC released and radiolabeled by [(3)H]inositol indicating that about one third of integral membrane HSPG were intercalated into the plasma membrane through a phosphatidylinositol anchor (glypican type). PI-PLC-resistant forms represented HSPG inserted into the membrane through a hydrophobic segment of the core protein (syndecan type). No lipophilic PG was present in other cell compartments (culture medium, cell periphery, extracellular matrix). (125)I-Iodinated hydrophobic HSPG were deglycanated and submitted to SDS-polyacrylamide gel electrophoresis. In the glypican family, a core protein (64--65 kDa) was detected, whereas in the syndecan family, bands of 60 and 68 kDa were observed which may correspond to self-association of different core proteins. In Sertoli cell, specific functional attributes of different integral membrane HSPG forms remain to be investigated.     

Regulation of proteoglycan and hyaluronan synthesis by elevated level of intracellular cyclic adenosine monophosphate in peritubular cells from immature rat testis

The effects of an increase in intracellular cAMP concentration on proteoglycan (PG) synthesis by peritubular (PT) cells from immature rat testis were investigated. In the presence of dBcAMP for 72 h, the [³H]-hexosamine incorporation in secreted PG and in cellassociated PG was reduced, whereas [³⁵S]-sulfate radioactivity was enhanced in secreted PG and not affected in cell-associated PG. Cholera toxin and IBMX, known to generate high intracellular cAMP levels, induced similar changes. Cyclic AMP did not alter PG protein moiety synthesis but enhanced PG turnover. Cholera toxin and dBcAMP profoundly modified PG characteristics: (1) Apparent molecular weight of PG was increased. (2) This was due to an increase in glycosaminoglycans (heparan sulfate (HS) and chondroitin sulfate (CS)) length. (3) The number of glycosaminoglycan chains was presumably reduced. (4) Heparan sulfate and chondroitin sulfate chains of medium and cell layer-associated PG appeared oversulfated. (5) The pattern of cell layer associated PG was modified with a decrease in HSPG and a correlative increase in CSPG. Cholera toxin and dBcAMP also dramatically stimulated hyaluronan synthesis by possible phosphorylation induced activation of hyaluronan synthase(s).     

In vitro action of PG F2 alpha on progesterone and cAMP synthesis in small bovine luteal cells

The action of prostaglandin F2 alpha (PG F2 alpha) on incubated small bovine luteal cells in the presence or in the absence of bovine luteinizing hormone (LH) or dibutyryl cyclic adenosine monophosphate (db cAMP) was investigated. In the absence of LH and db cAMP, PG F2 alpha stimulated progesterone synthesis at concentrations of 10 ng/ml and 100 ng/ml but had no effects at concentrations below 1 ng/ml. PG F2 alpha partially inhibited the LH or db cAMP stimulated progesterone synthesis. This inhibition was maximal for PG F2 alpha concentrations around 100 pg/ml whereas distinctly higher or lower concentrations were without effect. At the concentration of 100 pg/ml, PG F2 alpha partially inhibited the LH induced cAMP accumulation. These results demonstrate an "in vitro" action of PG F2 alpha on bovine luteal cells. They indicate that the luteolytic action of PG F2 alpha in the bovine species could involve, as already suggested for the rat, both an inhibition of the LH induced synthesis of cAMP and an inhibition of the action of cAMP.     

Changes of progesterone content of rat uterine flushings in relation to serum concentrations of progesterone during the oestrous cycle.

Uterine fluid was collected from four-day cyclic rats at each stage of the oestrous cycle and assayed for progesterone and protein content. Progesterone was determined by radioimmunoassay either after ethanol (or 2.5% NaOH) denaturation of proteins from uterine flushings ('total' progesterone) or without protein denaturation ('ether-extractable' progesterone). The amount of 'ether-extractable' progesterone in the lumen was constant from metoestrus to pro-oestrus (340 pg per uterus) but lower in oestrus (200 pg per uterus). However, 'total' progesterone content of uterine fluid was subject to cyclic variations and was highest in dioestrus (890 pg per uterus) and lowest in oestrus (350 pg per uterus), in contrast to serum progesterone which is lowest in dioestrus and highest in oestrus. Protein content of uterine flushings peaked to 780 micrograms per uterus in pro-oestrus then fell to about 140 micrograms per uterus until the end of the oestrous cycle. Changes in protein content of the lumen were followed by qualitative variations since the mean amount of 'bound' progesterone ('total' progesterone minus 'ether-extractable' progesterone) released per milligram of denatured lumen protein rose from 1.8 pmol in pro-oestrus to 18.2 pmol in dioestrus. The changes of luminal 'bound' progesterone during the oestrous cycle suggest that progesterone binding to luminal proteins could be an important modulator of progesterone action in rat uterus. Moreover, the variations in progesterone content of the lumen, irrespective of serum progesterone concentrations, are consistent with the hypothesis that progesterone synthesis occurs in the uterus.     

Human chorionic gonadotropin affects tissue levels of progesterone and cyclic adenosine 3',5'-monophosphate in the metestrus rat uterus in vitro

The effect of human chorionic gonadotropin (hCG) on in vitro progesterone (P) level in the uterus was investigated by using short-term incubations of uterine tissue taken from 4-day cyclic rats at different stages of the estrous cycle. Control incubations resulted in a decrease of endogenous P content in uteri removed from rats in proestrus (PRO), estrus (EST), and metestrus (MET): -25% (n = 6), -60% (n = 6), and -45% (n = 8), respectively. The amount of P found after incubation of MET tissue in the presence of hCG was significantly higher (p less than 0.001) than that found after control incubations. The hCG effect was dose-dependent and was not observed with PRO or EST tissue. Although the mean P level found in MET tissue after incubation with 10 IU/ml hCG was not significantly different from the mean level found in unincubated tissue (1562 +/- 341 vs. 1470 +/- 174 pg/mg protein, n = 8), an obvious synthesis was observed in two experiments. It thus seems likely that observed hCG effect would involve a de novo P synthesis rather than a decrease of P catabolism. Furthermore, hCG induced a dose-dependent increase of cyclic adenosine 3', 5'-monophosphate (cAMP) uterine levels in MET tissue. N,O'-dibutyryl cyclic AMP [Bu)2 cAMP) at 5 mM induced a significative increase (p less than 0.01) of P uterine level in EST and MET tissue compared to control incubations, but had no effect on PRO tissue. Our results suggest the progressive maturation throughout the rat estrous cycle of a luteinizing hormone/hCG- and cAMP-dependent process able to regulate uterine P content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of cell membrane proteoglycans impairs FSH receptor/Gs coupling and ERK activation through PP2A-dependent mechanisms in immature rat Sertoli cells

Background

During the pre-pubertal life, the cessation of Sertoli cell proliferation and the onset of differentiation are associated with a shift in the FSH-mediated signaling leading to inhibition of the ERK-mitogenic pathway and to a concomitant sensitization of cAMP/PKA pathway.

Methods

To highlight the role of cell proteoglycans (PGs) in the shift of FSH signaling, both FSH-induced cAMP production and ERK1/2 inactivation were studied in untreated and sodium chlorate PG-depleted cultured Sertoli cells from 20 day-old rats.

Results

Depletion of cell membrane PGs by sodium chlorate reduced FSH-, but not cholera toxin-stimulated cAMP production as well as basal ERK phosphorylation through an okadaic acid (OA)-sensitive mechanism. Involvement of PP2A was further substantiated by a marked decrease in membrane- associated PP2A activity under SC conditions and by the OA-induced restoration of PKA-dependent ERK inactivation in SC-treated cells.

Conclusions

In 20-day-old rat Sertoli cells, transmembrane cell PGs, through tethering/activation of PP2A activity exerts regulatory control on both FSH receptor/Gs coupling and ERK phosphorylation.

General significance

Besides their antiproliferative roles, cell PGs such as syndecan-1, could be involved in the increase in cAMP response to FSH occurring in Sertoli cells at the time of transition between proliferative and differentiated states.