L-Arginine supplementation prevents allodynia and hyperalgesia in painful diabetic neuropathic rats by normalizing plasma nitric oxide concentration and increasing plasma agmatine concentration.

PURPOSE: Neuropathic pain is a common diabetic complication. It is characterized by symptoms of spontaneous and stimulus-evoked pain including hyperalgesia and allodynia. L-Arginine is a common precursor of many metabolites of biological interest, in particular, nitric oxide (NO), ornithine, and hence polyamines. In central nervous system, NO, glutamate, and polyamines share an N-methyl-D-aspartate (NMDA) receptor-mediated effect. We hypothesized that a variation in arginine metabolism caused by diabetes may contribute to development and maintenance of neuropathic pain and to the worsening of clinical and biological signs of diabetes. METHODS: We examined whether oral L-arginine supplementation (2.58 ± 0.13 g/l in drinking water for 3 weeks) could improve the development of neuropathic pain and the clinical, biological, and metabolic complications of diabetes in streptozocin (STZ)-induced diabetic (D) rats. RESULTS: STZ administration induced classical symptoms of type 1 diabetes. Diabetic rats also displayed mechanical hypersensitivity, tactile, and thermal allodynia. Plasma citrulline and NO levels were increased in diabetic hyperalgesic/allodynic rats. L-Arginine supplementation failed to reduce hyperglycaemia, polyphagia, and weight loss. Moreover, it abolished hyperalgesia and allodynia by normalizing NO plasma concentration and increasing plasma agmatine concentration. CONCLUSIONS: L-Arginine supplementation prevented the development of mechanical hyperalgesia, tactile, and thermal allodynia in painful diabetic neuropathy with concomitant reduction of NO and increased agmatine production, offering new therapeutic opportunities for the management of diabetic neuropathic pain.

Prostasomes: inhibitors of capacitation and modulators of cellular signalling in human sperm.

Seminal fluid inhibits sperm capacitation mainly because of its high cholesterol content. Prostasomes are the main source of cholesterol in seminal fluid. They are known to have numerous protective properties and are able to transfer proteins and lipids to spermatozoa, but their impact on capacitation and acrosome reaction (AR) is not yet well understood. The aim of this study was to determine the effects of prostasomes on human sperm capacitation and AR. After 80% Percoll selection, freshly ejaculated human spermatozoa were incubated for 3 h under capacitating conditions with prostasomes, phosphodiesterase inhibitor 3-iso-butyl-methylxantine (IBMX), or a combination of prostasomes and IBMX. Physiological concentration of prostasomes significantly decreased tyrosine phosphorylation levels of human sperm capacitation markers P110 and P80 (p < 0.01), and the proportions of capacitated (p < 0.05) and acrosome-reacted spermatozoa (p < 0.05). Prostasomes significantly increased the proportion of spermatozoa that did not incorporate propidium iodide and significantly attenuated the effect of IBMX on P110 tyrosine phosphorylation. Prostasomes had no effect on the pH(i) increase associated with capacitation. They significantly increased intracellular cAMP concentration ([cAMP](i)) and, when prostasomes and IBMX were present together, [cAMP](i) was further increased. To our knowledge, this is the first study to show clearly that prostasomes inhibit capacitation and spontaneous AR.

[Role of reactive oxygen species (ROS) on human spermatozoa and male infertility]. / Rôles des dérivés actifs de l'oxygène (DAO) sur les spermatozoïdes humains et infertilité masculine.

Pro- and antioxidant are balanced in the sperm environment. Reactive Oxygen Species (ROS) are essential to the acquisition of fertilizing ability and contribute to chromatin condensation, membrane remodeling and activation of intracellular signaling pathways, during epididymal maturation, capacitation, and acrosome reaction. However, endogenous and exogenous factors can upset that balance by stimulating ROS production and/or decreasing antioxidant defenses, a situation called oxidative stress. Oxidative stress is described as a major cause of male infertility. It induces membranes and nucleus alterations, resulting in loss of mobility and decline in sperm fertilizing ability. Those are risk factors for low fertility, abnormalities of preimplantation development, and miscarriages. Various methods exist for measuring the pro- and antioxidants status of sperm, yet are little used in routine for diagnostic purposes. Meanwhile, many studies have shown the beneficial effects of oral antioxidants supplementation, or addition to semen freezing medium, to prevent in vivo and limit in vitro the deleterious effects of ROS, respectively.

Leptin: a potential regulator of polymorphonuclear neutrophil bactericidal action?

It is well known that leptin, the ob gene product, is involved in the regulation of food intake and thermogenesis. Recent studies also demonstrate that leptin may be able to modulate functions of cells involved in nonspecific immune response such as phagocytosis and secretion of cytokines by macrophages. This and the prominent implication of polymorphonuclear neutrophils (PMNs) in infectious response suggested a possible role of leptin as a modulator of PMN functions. We detected a leptin receptor on the PMN membrane by immunocytochemistry with an anti-leptin receptor. Using chemiluminescence we then demonstrated that leptin enhances oxidative species production by stimulated PMNs. These results show for the first time that a functional leptin receptor is present on PMNs and that leptin may be able to influence their oxidative capacity.

The gonadotrope polypeptide (GP 87) released from pituitary cells under luteinizing hormone-releasing hormone stimulation is a secretogranin II form.

Cultured gonadotrope cells from 14 day old female rat pituitaries have been shown to release a highly acidic protein when incubated with LHRH: the gonadotrope polypeptide (GP 87). Moreover, a new tyrosine-sulfated acidic protein, secretogranin II (Sg II), clearly distinct from the chromogranin species, was described in the secretory granule matrix of endocrine cells secreting peptide hormones by the regulated pathway. Recently, the release of Sg II from female rat pituitary stimulated by LHRH was demonstrated in vitro. Several physicochemical (Mr; pI) and biological (cellular localization in the pituitary; LHRH-stimulated release) properties are common to Sg II and GP 87. To verify if these 2 polypeptides are similar or distinct components, other physicochemical characteristics (heat-stability, sulfation, phosphorylation) were compared using isotope incorporation followed by either 1- or 2-dimensional polyacrylamide gel electrophoresis and autoradiography. Furthermore, the similarity of GP 87 to Sg II was studied by immunoblotting on nitrocellulose sheets following electrophoresis of intracellular and secreted proteins. Antisera raised against bovine Sg II (extracted from whole pituitaries) and against rat GP 87 (released into the medium of cultured pituitary cells stimulated by LHRH) were used. The overall data presented here suggest that GP 87 is the Sg II form contained in, and released by, gonadotrope cells.

Peptides co-released with luteinizing hormone by perifused pituitary cell aggregates.

The proteins co-released with gonadotropins were analyzed using perifusion of pituitary cell aggregates from 14-day-old female rats, after a pre-labeling period with [35S]methionine. Radioimmunoassays of hormones and electrophoretic analysis were performed on each 4 min effluent. Gonadotropin-releasing hormone (GnRH) pulses increased significantly (P less than 0.01) the release of several proteins (Mr range from 140,000 to 28,000). The main stimulation appeared for -1, a 87 kDa species, previously characterized as gonadotrope polypeptide 87 (GP87) in monolayer cultures and identified as a secretogranin II (SgII) form; -2, a second species of 80 kDa designated as B2. Secretory patterns of radiolabeled GP87 and B2 paralleled the luteinizing hormone (LH) ones. The release of these species was -1, GnRH dose dependent; -2, monophasic for short pulses but complex when the duration of GnRH pulses increased to 16 min, suggesting different pools of GP87 and B2 as for LH; -3, induced by thyrotropin-releasing hormone (TRH). A slight output was also elicited by corticotropin-releasing factor (CRF) and growth hormone-releasing hormone (GHRH), but this release was partly impaired in the presence of a potent anti-GnRH ([Ac-D-(2)-NAL1,pF-D-Phe2,D-Trp3,D-Arg6]-LAF) suggesting a non-specific effect of these two factors. GP87/SgII thus appeared mainly associated with the release of hormonal glycoproteins. In conclusion, perifusion of pituitary cell aggregates allows a precise minute-to-minute kinetic analysis of the various proteins co-released with hormones. The similar timing in output of LH, GP87 and B2 suggests that these three proteins co-exist in the same secretory granules inside gonadotropes.

LHRH promotes the synthesis and release of a 87,000 Da protein (GP-87) by enriched gonadotrophs.

Protein secretion by cultured pituitary cells from 14-day-old female rats was estimated using [35S]methionine incorporation followed by either one- or two-dimensional electrophoresis and autoradiography. Stimulation of total cells or gonadotrophs by LHRH promoted the synthesis and release of a specific polypeptide (apparent molecular weight 87,000, pI = 4.6). Silver staining of cellular proteins from both gonadotroph-enriched and gonadotroph-depleted populations prepared by centrifugal elutriation revealed a high concentration of this polypeptide in the gonadotrophs and a very low level in the other cell population. This species was thus called Gonadotrope Polypeptide GP-87. Release of labeled GP-87 by gonadotrophs was both time dependent (up to 4 h) and LHRH dose dependent (from 10(-9) M to 10(-7) M) as was the release of LH. Attempts to precipitate GP-87 from the incubation medium with anti-LH antiserum were unsuccessful suggesting that GP-87 is not a 'big' form of LH. TRH neither stimulated the release of GP-87 from gonadotrophs nor from lactotrophs though it did stimulated PRL release. From these data, we conclude that gonadotrophs in culture synthesize a specific polypeptide (GP-87), LHRH stimulates both the synthesis and release of GP-87, and the pituitary cell response is peptide specific. The LHRH-induced synthesis and release of GP-87 could be an important step in the molecular processes that regulate gonadotrophin secretion.

Steroid receptors and regulation of LH and secretogranin II (GP87) secretion in pituitary cell aggregates.

The gonadotrope polypeptide (GP87), a secretogranin II form, has previously been found to be co-released with luteinizing hormone (LH) by rat gonadotrope cells under luteinizing hormone-releasing hormone (LHRH) stimulation. Nothing is hitherto known concerning its function and regulation. In the present paper, the modulatory effects of steroids on GP87 release were investigated using cultures of rat pituitary cell aggregates. As steroid effects are dependent on the presence of receptors, we firstly demonstrated that the steroid receptors are maintained with their typical characteristics in this culture system. In steroid-free medium, the estrogen receptor concentration increased significantly (P < 0.01) within 7 days in culture (646 +/- 122 fmol/mg DNA, n = 3) when compared to the level in freshly dispersed cells (355 +/- 59 fmol/mg DNA, n = 4) suggesting that cells can synthesize the estrogen receptor and that this cell culture system is suitable for studying regulatory effects of steroids. Pituitary cell aggregates from 14 day-old female rats were maintained for 24 h in the absence or presence of 10 nM estradiol (E(2)) or 50 nM 5?-dihydrotestosterone (DHT) and incubated for 2.5 h with [(35)S] methionine. Perifusion was initiated and 4 min pulses of 20 nM LHRH occurred every 1 h. The effects of steroids were investigated on LH, total and labeled GP87. The results indicated that: (1) DHT pretreatment reduced the amplitude of LHRH-stimulated GP87 and LH release responses; (2) by contrast, E(2) pretreatment enhanced the effects of LHRH on total or labeled GP87 release whereas the LH release was not significantly modified; and (3) when E(2) treatment started only 60 min before the first LHRH pulse, no differences between responses of E(2)-treated and control aggregates were observed. The present data show for the first time that sex steroids can modulate the GP87 release through a direct effect on the pituitary. E(2) could either stimulate the GP87 synthesis or increase its intracellular trafficking. That LH and GP87 do not strictly exhibit the same release response further suggests either some heterogeneity of GP87 localization in gonadotrope sub-types or within secretory granules.

Quantitative analysis of desmosterol, cholesterol and cholesterol sulfate in semen by high-performance liquid chromatography.

A simple, rapid and accurate method to separate and quantify cholesterol, desmosterol and cholesterol sulfate in human spermatozoa and seminal plasma (SP) is described. This high-performance liquid chromatographic procedure is based on reversed-phase chromatography on a Inertsil ODS2 5 microm silica column with a binary gradient of mixtures of chloroform-methanol and chloroform-methanol-water as the mobile phase at a flow-rate of 0.25 ml/min. Sterols are separated with good resolution and high reproducibility. The eluted sterols are quantified using a light-scattering (mass) detector. As little as 64, 64 and 68 pmol of cholesterol, desmosterol and cholesterol sulfate, respectively, can be quantified under these conditions. Cholesterol is the predominant sterol both in spermatozoa (107+/-7 nmol/10(8) spermatozoa) and SP (0.83+/-0.10 micromol/ml) whereas the concentrations of desmosterol were 38+/-6 nmol/10(8) in spermatozoa and 0.18+/-0.02 micromol/ml in SP. Cholesterol sulfate represents about 6% of total cholesterol in the spermatozoa and SP. In conclusion, this method offers interesting perspectives for the quantitative analysis of these sterols not only in semen, but also in other biological samples.

Impact of maternal hyperlipidic hypercholesterolaemic diet on male reproductive organs and testosterone concentration in rabbits.

The concept of Developmental Origins of Health and Disease initially stemmed from the developmental programming of metabolic diseases. Reproductive functions and fertility in adulthood may also be programmed during foetal development. We studied the impact of dietary-induced maternal hyperlipidaemia and hypercholesterolaemia (HH), administered at 10 weeks of age and throughout the gestation and lactation, on male reproductive functions of rabbit offspring. Male rabbits born to HH dams and fed a control diet had significantly lighter testes and epididymes compared with rabbits born to control dams at adulthood. No significant changes in sperm concentration, sperm DNA integrity and sperm membrane composition were observed, but serum-free testosterone concentrations were decreased in HH males. This study confirms the importance of maternal metabolic status for the development of male reproductive organs.

Liver X Receptor activation downregulates AKT survival signaling in lipid rafts and induces apoptosis of prostate cancer cells.

Cholesterol is a structural component of lipid rafts within the plasma membrane. These domains, used as platforms for various signaling molecules, regulate cellular processes including cell survival. Cholesterol contents are tightly correlated with the structure and function of lipid rafts. Liver X receptors (LXRs) have a central role in the regulation of cholesterol homeostasis within the cell. Therefore, we investigated whether these nuclear receptors could modulate lipid raft signaling and consequently alter prostate cancer (PCa) cell survival. Treatment with the synthetic LXR agonist T0901317 downregulated the AKT survival pathway and thus induced apoptosis of LNCaP PCa cells in both xenografted nude mice and cell culture. The decrease in tumor cholesterol content resulted from the upregulation of ABCG1 and the subsequent increase in reverse cholesterol transport. RNA interference experiments showed that these effects were mediated by LXRs. Atomic force microscopy scanning of the inner plasma membrane sheet showed smaller and thinner lipid rafts after LXR stimulation, associated with the downregulation of AKT phosphorylation in these lipid rafts. Replenishment of cell membranes with exogenous cholesterol antagonized these effects, showing that cholesterol is a key modulator in this process. Altogether, pharmacological modulation of LXR activity could thus reduce prostate tumor growth by enhancing apoptosis in a lipid raft-dependent manner.

Kinetics of occurrence of some features of apoptosis during the cryopreservation process of bovine spermatozoa.

BACKGROUND: Cryopreservation/thawing of bovine spermatozoa induces a reduction in cell viability and is possibly associated with a form of programmed cell death that we previously named 'apoptosis-like phenomenon'. METHODS: In this study, we specified, by flow cytometry, the moment of appearance of some characteristics of apoptosis during the cryopreservation process. We also studied the presence and/or activation in bovine sperm cells of specific proteins involved in somatic cell apoptosis by western blot and fluorimetry. RESULTS: A decrease of the mitochondrial membrane potential (DeltaPsim) was detectable 5 min after sperm dilution in the cryopreservation medium, caspase activation after 3 h of equilibration and an increase in plasma membrane permeability after the complete process of cryopreservation/thawing. The presence of the pro-apoptotic factor Bax, a protein that facilitates the formation of mitochondrial pores, was observed in bovine spermatozoa, but the anti-apoptotic factor Bcl-2 was not detectable. Moreover, it was observed that bovine spermatozoa contain cytochrome c and apoptosis-inducing factor (AIF), two proteins usually released from the mitochondria during the apoptotic process. Activated caspase-9, involved in the mitochondrial pathway, was detected in bovine spermatozoa but not caspase-3 and -8. CONCLUSIONS: The early features of apoptosis appear as ordered events during the cryopreservation/thawing process of bovine sperm cells. Bovine spermatozoa contain the machinery necessary to proceed to apoptosis involving especially the mitochondrial pathway.

Study of two markers of apoptosis and meiotic segregation in ejaculated sperm of chromosomal translocation carrier patients.

BACKGROUND: To try to explain the infertility of chromosomal translocation carrier patients, we compared the expression of two markers of apoptosis in the sperm of patients and of fertile donors, and we studied the meiotic segregation in the ejaculated sperm of these translocation carriers. METHODS: Twenty semen samples of translocation carriers, [reciprocal (n=14) and Robertsonian translocations (n=6)], were compared with the semen samples of donors (n=20). Different tests were applied: annexin V binding assay; terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL); and fluorescence in situ hybridization (FISH). RESULTS: The annexin V binding assay in sperm of patients with chromosomal translocation (n=17) showed a significantly increased proportion of sperm with externalized phosphatidylserine (PS) than in the control group (n=20, P

Separation and quantification of cholesterol and major phospholipid classes in human semen by high-performance liquid chromatography and light-scattering detection.

A high-performance liquid chromatographic method coupled with light-scattering detection for the separate and accurate quantification of cholesterol and main phospholipid classes was applied to human spermatozoa and seminal plasma (SP). This method is based on normal-phase chromatography with silica gel as stationary phase and a ternary gradient with hexane, mixtures of chloroform-methanol and water as mobile phase. Lipids are separated with a good resolution and a high reproducibility. About 5 x 10(6) spermatozoa or 25 microl of seminal plasma are sufficient to accurate quantitative analysis of phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidycholine (PC), sphingomyelin (SM) and cholesterol. PC is the predominant phospholipid class in spermatozoa (102+/-8 nmol/10(8) spermatozoa) whereas SM is the major in the SP (163+/-6 nmol/ml). Both in spermatozoa and SP, PI is the minor class of the phospholipids (12+/-1 nmol/10(8) spermatozoa and 24+/-2 nmol/ml). In conclusion, this method offers interesting perspectives for analysis of sperm lipid composition in semen samples with low quantities of spermatozoa.

Characterization of a monoclonal antibody to a human intra-acrosomal antigen that inhibits fertilization.

Among monoclonal antibodies (mAb) selected after the immunization of mice with human ejaculated spermatozoa, mAb I9G9 (IgG1 kappa) was found by immunoperoxidase staining to label most of the acrosome of human spermatozoa permeabilized with methanol-acetone. The antigen was poorly expressed on the surface of fresh ejaculated sperm, but was detectable on most viable sperm after 5-h incubation in medium containing human serum albumin (HSA) followed by 30-min incubation with the calcium ionophore A23187. This treatment resulted in acrosomal loss. Immunoelectron microscopy labeling with I9G9 mAb localized the antigen within the acrosome. Immunocytochemistry on testis sections showed that antigen was located in the round spermatids within the adluminal compartment of the seminiferous epithelium. Western blotting of sperm extract proteins showed that sperm intra-acrosomal (SIAA) recognized by I9G9 mAb had a polymorphism of immunogenic peptides from 16 to 35 kDa. Most of the antigenic peptides possessed an isoelectric point of approximately 5. When spermatozoa were treated with a series of protease inhibitors, the polymorphism of immunogenic peptides was reduced, suggesting that the multiple form of the antigen was due, at least in part, to proteolytic processing. In the testis, only a single peptide band of 35 kDa was detected with mAb I9G9. Studies of human tissue specificity by Western blotting showed that the epitope recognized by I9G9 mAb was present solely in ejaculated spermatozoa and the testis. I9G9 mAb did not agglutinate or immobilize sperm but inhibited the penetration of zona-free hamster ova by human sperm.

Membrane fluidity and lipid content of human spermatozoa selected by swim-up method.

In this work, we examined whether spermatozoa (spz) from normospermic fertile patients and selected by a swim-up (S-U) procedure had a particular membrane fluidity related to their maturity and their lipid content as compared with the sperm cells from the whole ejaculate (total sperm). Swim-up selected sperm had a reduced cytoplasmic space as revealed by a lower creatine kinase (CK) activity compared with total sperm (2 +/- 1 vs. 12 +/- 5 mUI/10(7) spz, p < 0.05). The cholesterol (Chol) and total phospholipid (PL) contents were significantly lower in S-U selected sperm than in total sperm (0.72 +/- 0.08 vs. 1.20 +/- 0.30 nmol/10(6) spz for Chol and 1.77 +/- 0.17 vs. 2.78 +/- 0.50 nmol/10(6) spz for PL, p < 0.05) and such a decrease was observed for the three major membrane PL: phosphatidylethanolamine (PE), phosphatidylcholine (PC) and sphingomyelin (SM). However, these decreases were not associated with a change in either Chol/PL or PC/(PC + PE) molar ratios. Membrane fluidity estimated by fluorescence polarization remained comparable between the S-U sperm fraction and total sperm (fluorescence polarization anisotropy, r, which is inversely proportional to the fluidity: 0.235 +/- 0.006 vs. 0.230 +/- 0.005). The sperm membrane fluidity obtained in normospermic patients was compared with abnormospermic ones (oligoasthenoteratospermia). In abnormospermic patients, the membrane fluidity was decreased in migrated spermatozoa compared with total sperm (anisotropy: 0.210 +/- 0.010 vs. 0.250 +/- 0.013, p < 0.01). Our data suggest that the S-U method selected a subpopulation of mature spermatozoa characterised by a low content of Chol and PL, likely related to a reduced membrane area. The fact that Chol/PL and PC/(PC + PE) molar ratios were unchanged shows a maintenance of the membrane quality. This was confirmed by the fluorescence anisotropy measurement showing no difference in plasma membrane fluidity between S-U selected sperm and total sperm. In abnormal semen the migrated spermatozoa had a lower fluidity compared with total sperm suggesting a defective sperm function. These results bring new elements characterizing the S-U selected spermatozoa.

Cholesterol, phospholipids and markers of the function of the accessory sex glands in the semen of men with hypercholesterolaemia.

The effect of hypercholesterolaemia on the cholesterol and phospholipid content of spermatozoa and seminal plasma was studied. Testosterone and specific markers of the accessory sex glands were also measured. Semen samples from 11 hypercholesterolaemic patients (plasma cholesterol > 6.42 mmol/l, plasma triglycerides < 2 mmol/l) were compared with those of 11 normocholesterolaemic controls (plasma cholesterol < 5.14 mmol/l, plasma triglycerides < 2 mmol/l). Cholesterol, phospholipids and the molar ratio of cholesterol: phospholipids were not significantly different between the two groups of patients either in spermatozoa or in seminal plasma. In hypercholesterolaemic patients the total amount of carnitine in the ejaculate was significantly higher, but there were no significant differences in the levels of acid phosphatase or fructose. There were no significant differences in seminal plasma levels of testosterone in the two groups of subjects. These results suggest that hypercholesterolaemia has no effect on cholesterol and phospholipid levels in spermatozoa and does not cause gross modification of the secretory function of the accessory sex glands.

Improvement of motility and fertilization potential of postthaw human sperm using glutamine.

The effectiveness of three amino acids, glutamine, proline, and histidine, and one amino acid-related compound, betaine, in preserving human sperm diluted v/v in a basal medium (BM) containing 14% glycerol during the freeze-thaw (FT) process was studied. At 80 mM in BM, only glutamine improved the 5- to 60-min postthaw total and progressive motilities and velocity. The presence of glutamine at 80 mM is not sufficient to achieve lower concentrations of the toxic agent glycerol in FT medium. Glutamine may therefore have a mechanism of protection on human spermatozoa that is independent from that of glycerol. The zona-free hamster egg penetration test showed that the percentage of eggs penetrated was significantly greater when spermatozoa were frozen-thawed with 80 mM glutamine in BM. Consequently, the presence of glutamine at 80 mM in a glycerol-FT medium maintains human sperm motility and fertilizing ability during the FT process.

Binding of estradiol and 5 alpha-androstane-3 beta-17 beta-diol by the male rat enriched gonadotrope cells.

Dispersed pituitary cells from 42-day old male rats were separated using centrifugal elutriation. Based on LH and PRL cellular contents, fractionated cells were pooled into two fractions: "Lactotrope++ population" and "gonadotrope++ population". Estradiol and 5 alpha-androstane-3 beta, 17 beta-diol binding was measured in these fractions. Results revealed that: (1) The steroid receptors are not destroyed by cell dispersion and elutriation. (2) The estradiol receptor content is higher in gonadotrope++ cells than in lactotrope++ cells. (3) The number of binding sites for the two steroids changes in the different fractions: whereas it is exactly similar in "lactotrope++ population", it is much higher for estradiol than for 5 alpha-androstane-3 beta, 17 beta-diol in "gonadotrope++ population". These results suggest two different species--or conformations--of receptor binding sites for estradiol in the male rat pituitary; the first one could link both steroids, the second one would be specific for estradiol.