Increased serum levels of C21 steroids in female patients with multiple sclerosis.

Multiple sclerosis (MS) is one of the most common neurological diseases. This neurodegenerative autoimmune disease manifests as inflammatory and demyelinating impairment of the central nervous system (CNS). Although some studies demonstrated associations between altered steroidogenesis and pathophysiology of MS as well as the importance of steroids in the pathophysiology of MS, the knowledge concerning the steroid metabolome in female patients is limited. Hence, 51 steroids and steroid polar conjugates were measured in the serum of 12 women with MS, untreated with steroids and 6 age-corresponding female controls with the use of gas chromatography - mass spectrometry (GC-MS). The data were processed using age adjusted ANCOVA, receiver operating characteristics (ROC) analysis and orthogonal projections to latent structures (OPLS). Our data show higher levels of circulating C21 steroids including steroid modulators of ionotropic type A gamma-aminobutyric acid (GABA A) receptors and glutamate receptors. Furthermore, the levels of GABAergic androsterone and 5-androsten-3beta,7alpha,17beta-triol were also higher in the female MS patients. In conclusion, the data demonstrate higher levels of circulating C21 steroids and their polar conjugates and some bioactive C19 steroids in women with MS, which may influence neuronal activity and affect the balance between neuroprotection and excitotoxicity.

Chronic cigarette smoking alters circulating sex hormones and neuroactive steroids in premenopausal women.

Chronic smoking alters the circulating levels of sex hormones and possibly also the neuroactive steroids. However, the data available is limited. Therefore, a broad spectrum of free and conjugated steroids and related substances was quantified by GC-MS and RIA in premenopausal smokers and in age-matched (38.9+/-7.3 years of age) non-smokers in the follicular (FP) and luteal phases (LP) of menstrual cycle (10 non-smokers and 10 smokers, in the FP, and 10 non-smokers and 8 smokers in the LP). Smokers in both phases of the menstrual cycle showed higher levels of conjugated 17-hydroxypregnenolone, 5alpha-dihydroprogesterone, conjugated isopregnanolone, conjugated 5alpha-pregnane-3beta,20alpha-diol, conjugated androstenediol, androstenedione, testosterone, free testosterone, conjugated 5alpha-androstane-3alpha/beta,17beta-diols, and higher free testosterone index. In the FP, the smokers exhibited higher levels of conjugated pregnenolone, progesterone, conjugated pregnanolone, lutropin, and a higher lutropin/follitropin ratio, but lower levels of cortisol, allopregnanolone, and pregnanolone. In the LP, the smokers exhibited higher levels of free and conjugated 20alpha-dihydropregnenolone, free and conjugated dehydroepiandrosterone, free androstenediol, 5alpha-dihydrotestosterone, free and conjugated androsterone, free and conjugated epiandrosterone, free and conjugated etiocholanolone, 7alpha/beta-hydroxy-dehydroepiandrosterone isomers, and follitropin but lower levels of estradiol and sex hormone binding globulin (SHBG) and lower values of the lutropin/follitropin ratio. In conclusion, chronic cigarette smoking augments serum androgens and their 5alpha/beta-reduced metabolites (including GABAergic substances) but suppresses the levels of estradiol in the LP and SHBG and may induce hyperandrogenism in female smokers. The female smokers had pronouncedly increased serum progestogens but paradoxically suppressed levels of their GABA-ergic metabolites. Further investigation is needed concerning these effects.

Neuroactive steroids in periphery and cerebrospinal fluid.

Some peripheral steroids penetrate the blood-brain barrier (BBB), providing at least substances for the CNS steroid metabolome. That is why the predictive value of the peripheral steroids appears to be comparable with that of the cerebrospinal fluid (CSF) steroids. The concentrations of the CSF steroids are pronouncedly lower in comparison with the ones in circulation. The available data indicate that the levels of pregnenolone sulfate substantially increase in the rat brain tissue after the administration of pregnenolone into the circulation. In the human circulation there are about two orders of magnitude higher levels of pregnenolone sulfate compared to the free pregnenolone. Our data show insignificant correlation between CSF and serum pregnenolone, but a borderline one between CSF pregnenolone and serum pregnenolone sulfate. Therefore in humans, the circulating pregnenolone sulfate might be of an importance for pregnenolone concentration in the CNS. In contrast to free pregnenolone, dehydroepiandrosterone (DHEA) in the CSF correlates with both unconjugated and conjugated DHEA in the serum. These data as well as the low C17-hydroxylase-C17,20-lyase activity in the CNS might indicate that DHEA levels in the CNS are influenced by peripheral levels of DHEA and its sulfate. According to the information, available part of the neurosteroids may be synthesized de novo in the CNS, but substantial part of the steroid metabolites may be also synthesized in the CNS from the steroid precursors or directly transported through BBB from the periphery. The processes mentioned above may be complimentary in some cases. Brain synthesis may provide minimal level of neurosteroids, which are indispensable for the CNS functions. Thus, brain steroids of peripheral origin may reflect various physiological situations or even pathologies. This article is part of a Special Issue entitled: Neuroactive Steroids: Focus on Human Brain.

The identification and simultaneous quantification of neuroactive androstane steroids and their polar conjugates in the serum of adult men, using gas chromatography-mass spectrometry.

Certain androstane steroids (AS) modulate ionotropic receptors, as do the pregnane steroids. Whereas women produce significant amounts of neuroactive progesterone metabolites, the steroid neuromodulators in men originate mainly from the 3-oxo-4-ene C(19)-steroids, which are converted to their 3alpha- and 3beta-hydroxy-5alpha/5beta-reduced metabolites. The neuromodulating effects of AS prompted us to monitor circulating levels of the steroids to estimate metabolic pathways in the periphery that may influence brain concentrations of AS. Hence, the serum levels of 20 steroids and 16 steroid polar conjugates including 17-oxo- and 17beta-hydroxy-derivatives of 5alpha/beta-androstane-3alpha/beta-hydroxy-androstane steroids were quantified in 15 men (16-62 years of age) using GC-MS. The conjugated AS for the most part reached micromolar concentrations, these being two or three orders of magnitude higher than those of the free steroids. The ratios of conjugates to free steroids were one to two orders of magnitude higher than the values for the corresponding pregnane steroids. This data suggested that conjugation may considerably restrain the transport of free AS from the periphery into the central nervous system.

Circulating neuroactive C21- and C19-steroids in young men before and after ejaculation.

Twelve neuroactive and neuroprotective steroids, androgens and androgen precursors i.e. 3alpha,17beta-dihydroxy-5alpha-androstane, 3alpha-hydroxy-5alpha-androstan-17-one, 3alpha-hydroxy-5beta-androstan-17-one, androst-5-ene-3beta,17beta-diol, 3beta,17alpha-dihydroxy-pregn-5-en-20-one (17alpha-hydroxy-pregnenolone), 3beta-hydroxy-androst-5-en-17-one (dehydroepiandrosterone, DHEA), testosterone, androst-4-ene-3,17-dione (androstenedione), 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone), 3beta-hydroxy-pregn-5-en-20-one (pregnenolone), 7alpha-hydroxy-DHEA, and 7beta-hydroxy-DHEA were measured using the GC-MS system in young men before and after ejaculation provoked by masturbation. The circulating level of 17alpha-hydroxypregnenolone increased significantly, whereas the other circulating steroids were not changed at all. This fact speaks against the hypothesis that a drop in the level of neuroactive steroids, e.g. allopregnanolone may trigger the orgasm-related increase of oxytocin, reported by other authors.

[Proliferation and differentiation of regenerating Leydig cells following ethylene dimethane sulfonate exposure in rats]. / Proliferatsiia i differentsiatsiia regeneriruiushchikh kletok Leidiga krys posle vozdeistviia étandimethansul'fonatom.

Cell type of mammalian testis which is involved in the synthesis and secretion of testosterone and the maintenance of spermatogenesis are the fully differentiated interstitial Leydig cells (LC). Their ultrastructure possesses the typical characteristics of steroid-producing cells. It has been generally accepted that two waves of proliferation and differentiation can be discerned during the development of the Leydig cell population in the rodent and human testis. Treatment with ethane dimethane sulphonate (EDS) destroys selectively LC. A new LC population develops in the following weeks and this model has been used by us to study the proliferation and differentiation of new LC. Our results support the suggestion that the regeneration of a new LC population following EDS administration shows many similarities with the formation of the adult type LC in the prepubertal mammalian testis.

Isolation and immunocytochemical characterization of a library of monoclonal antibodies directed against rat testicular antigens.

OBJECTIVE: To characterize immunocytochemically the antigens recognized by monoclonal antibodies (Mabs) in the library we have accumulated and to reveal their spatiotemporal distribution in testicular tissue in the course of testis development. METHODS: Female BALB/c 2-month-old mice were immunized intraperitoneally with isolated immature Sertoli and germ cells obtained from 20 day old male Wistar rats. The obtained Mabs were characterized by its cell type-specific binding reaction using light immunocytochemistry (avidin-biotin-peroxidase complex technique, immunogold-silver staining, indirect immunofluorescence). RESULTS: On the basis of immunocystochemical results the selected Mabs were divided into four classes: 1. Mabs of class 1 recognized the differentiation specific antigens appearing during germ cell development, two of them revealing a stage-specific expression of nuclear antigens from preleptotene to early pachytene stage. Other Mabs of this class 1 detected the antigens in pachytene spermatocytes and acrosomes of round spermatids until their elongation; 2. the labeling of class 2 Mab was restricted only to Sertoli cell cytoplasm; 3. the binding of class 3 Mabs was observed in the cytoplasm of germ and Sertoli cells; 4. Mabs of class 4 reacted with antigens distributed in the cytoplasm of primary spermatocytes, Sertoli and Leydig cells. CONCLUSIONS: The Mabs from the library we have accumulated recognized the antigens in different cell types at various stages of testicular development and could be an useful tool for the characterization of cell- and development- specific molecules which may participate in germ cell differentiation and/or cell to cell interactions during testis development.

Stage-specific nuclear antigen is expressed in rat male germ cells during early meiotic prophase.

A germ cell nuclear antigen with approximately 44-kDa molecular weight was identified by a novel monoclonal antibody designated as Mab 2F2 from the library we have accumulated against rat testicular cells. In immature 20-day-old and adult rat testis the recognized antigen was expressed in the nuclei of early meiotic cells from preleptotene to early pachytene spermatocytes exhibiting a stage-specific appearance in the cycle of the seminiferous epithelium. The immunoreactivity was clearly associated with the meiotic chromosomes. The antigen was not detected in the late pachytene spermatocytes and more advanced stages of spermatogenesis. No labeling was observed in spermatogonia and somatic Sertoli and Leydig cells. The pattern of expression of the recognized antigen during early meiotic stages of spermatogenesis but not in mitotically dividing spermatogonia could strengthen its possible role in meiotic division.

Bradykinin stimulates prepubertal rat germ cell proliferation in vitro.

The effect of bradykinin on germ cell proliferation was studied by using in vitro organ cultures of testicular fragments from 3.5- and 4.5-day-old rats in the presence of 3H-thymidine. Bradykinin in tested concentrations of 10(-8), 10(-6) and 10(-4) M manifested a significant stimulation of rat prespermatogonial cell proliferation. The percentage of labeled germ cells increased up to 3-fold in comparison with the control value especially in 3.5-day-old rat testicular explants. Two kininase inhibitors: phosphoramidon and captopril were also used in combination with bradykinin. The percentage of labeled germ cells was slightly increased as compared to the samples treated with bradykinin only. Experiments with two agonists and two antagonists of B1 and B2 receptors for kinins showed that the rat prespermatogonial cell proliferation response to bradykinin is probably mediated via B2 receptors. The present findings suggest that bradykinin may be an important local testicular factor with paracrine or autocrine role in the regulation of spermatogonial cell proliferation and germ cell number.

A monoclonal antibody raised against rat ovarian antigen recognizes Leydig cell surface: an immunocytochemical study.

Using a monoclonal antibody (Mab) against constituents of rat ovarian granulosa cells, we found a 59-kDa protein located on the plasma membrane of a number of granulosa cells in follicles at different stages of development. This Mab (5G5) was found to bind to Leydig cells in rat male gonads. The localization of the antigen, recognized by Mab 5G5 in rat testis, was studied by light and electron microscopic immunocytochemistry (ABC method and IGS technique). Even though Sertoli cells in male gonads are regarded as the counterpart of granulosa cells in ovaries, the results of the experiments described here do not allow such an interpretation because staining with this antibody was restricted to the Leydig cell surface. The immunoreactivity in testicular sections from immature rats was similar to that found in adult testicular tissue. Our immunocytochemical results indicate that the plasma membrane of Leydig cells in rat male gonads shares certain biochemical and molecular properties with rat ovarian granulosa cells. On the basis of the immunocytochemical studies reported here, we suggest that the antigen recognized by Mab 5G5 may be common to all rat steroidogenic organs. Further studies are needed to establish the identity of the antigen in Leydig cells as well as its site of synthesis and its site of action. Thus, Mab 5G5 appears to hold significant potential as a powerful tool for future investigations.

Differential effects of prepubertal rat Sertoli cell secreted proteins on somatic testicular and nontesticular cells.

There is little information on the mitogenic and immunoregulatory activities of proteins, secreted by prepubertal Sertoli cells during the stage of meiosis initiation and before creation of the blood-testis barrier. We have previously demonstrated dose-dependent and age-related stimulation of BALB/c 3T3 fibroblasts and quiescent rat prespermatogonia (Kancheva et al., 1990) as well as inhibition of natural killer cell activity of mice, guinea pigs and human lymphocytes (Nikolova et al., 1992) by Sertoli cell-conditioned medium derived from 12-day-old rats. In the current study, using splenic lymphocytes stimulated by PHA, LPS and Con A, we have shown a dose-dependent inhibition of T and B lymphocyte proliferation by prepubertal Sertoli cell-secreted proteins (pSCSP). These results suggest that by the time the blood-testis barrier had been formed, Sertoli cell in rat testis had already synthesized immunoregulatory proteins. In addition we have found that pSCSP stimulate the proliferation of TM3 Leydig but not TM4 Sertoli cells. The differential effect of pSCSP is an expression of the different balance between growth factors secreted by Sertoli cells, which in turn is dependent on the requirements of the cell types at each stage of testicular development.

Species-specific effect of proteins secreted by cultured pre-pubertal rat Sertoli cells on natural killer cell activity.

The effect of proteins secreted by cultured pre-pubertal rat Sertoli cells (pSCP) on natural killer (NK) cell activity of rat, mice and guinea pig splenocytes and human peripheral blood lymphocytes was estimated. The results indicate that pSCP inhibited, in a dose-dependent manner, NK cell activity of mice, guinea pig and human lymphocytes but did not suppress lysis of YAC-1 target cells by rat NK cells. Species-specific differences in the effect of pSCP on NK cell activity probably result from differences in the binding of proteins within the effector cells. These data indicate that in the pre-pubertal period of gonadal development immature Sertoli cells synthesize a factor/s which might contribute to the maintenance of specific testis immunological environment.

Prepubertal rat Sertoli cells secrete a mitogenic factor(s) that stimulates germ and somatic cell proliferation.

Sertoli cells were isolated from prepubertal 6- and 12-day-old rats. The Sertoli cell-conditioned media (SCCM-6 and SCCM-12) can markedly stimulate the proliferation of somatic cells and quiescent rat prespermatogonia in a dose-dependent and an age-related manner. SCCM-12 stimulated cell proliferation of BALB/c 3T3 fibroblasts up to 7-fold over control values, but did not stimulate to the same degree the germ cell mitotic activity. SCCM-6 stimulated proliferation of prespermatogonia up to 5-10-fold over controls. The mitogenic factor(s) in SCCM-6 appears to be more specific to prespermatogonia than to somatic cells which is consistent with the in vivo stimulation of mitosis in germ cells 5-6 days after birth and with the action of 'mitosis inducing substance'. The mitogenic factor(s) appears to be protein with a molecular weight over 8000 and sensitive to heat and trypsin treatment. These results suggest that the different mitogenicity of prepubertal rat SCCM on germ and somatic cells may be due to secretion of multiple mitogens by Sertoli cells in an age-dependent manner.

An immunocytochemical study of the proliferating cell nuclear matrix antigen p125/6.5 during rat spermatogenesis.

In previous studies of proliferating mammalian cells a p125/6.5 nuclear matrix antigen displaying a marked increase in mitotic cells has been identified. This antigen was investigated by immunocytochemistry of cryosections of testes at different stages of postnatal development: newborn, 20 days after birth and sexually mature rats. In Sertoli cells, the distribution of the p125/6.5 antigen parallels [3H]thymidine incorporation: present in newborn and absent in sexually mature testes. The p125/6.5 antigen is present also in some prespermatogonia of the newborn rat testis, which do not incorporate [3H]thymidine. At later stages of development, the p125/6.5 antigen is present also in first meiotic prophase spermatocytes displaying an extrachromosomal nucleoplasmic distribution, while absent in spermatids and spermatozoa. These results show that the p125/6.5 antigen increases not only during mitosis, but also during meiosis. They suggest further that this antigen is characteristic of both proliferating cells and cells (prespermatogonia) committed to proliferation.

[Autoradiographic study of the proliferative activity of bone marrow, spleen and liver cells in healthy mice and in mice with Rauscher virus-induced erythroleukemia]. / Avtoradiograficheskoe izuchenie proliferativnoi aktivnosti kletok kostnogo mozga, selezenki i pecheni zdorovykh myshei i s éritroleikozom, indutsirovannym virusom Raushera.

Bone marrow, spleen and liver of healthy and erythroleukemic mice have been studied by autoradiography. A peripheral zone, where the most of cells were involved in mitosis, and a central one, where the mitotic index was lower, were established in the bone marrow. Normally a zone of high proliferative activity was found in the subcapsular region of the spleen. Boundaries of such zones in the bone marrow and spleen of erythroleukemic mice disappeared. Most of the cells forming the leukemia infiltrates in the liver are in the phase of DNA-synthesis.

Histoautoradiographic studies of 3H-thymidine labelled Erysipelothrix rhusiopathiae introduced into albino mice.

S-forms of E. rhusiopathiae were cultivated in a 3H-thimidine-containing medium. A suspension washed three times in succession was intravenously injected into albino mice. At certain intervals liver, spleen and kidney were examinated by routine autoradiogrphy. Intact labelled bacteria were found as early as 5 min after administration in the blood capillaries, around cells of the RES and in some nuclei of hepatocytes. Later they are detected in the cytoplasm of micro- and macrophages, around megacaryocytes and in some of their nuclei. After 6 h the labelled population sharply decreases due to a very active reproduction and mortality of the initial population. The localisation of bacteria in liver nuclei and the spreading of S-forms of E. rhusiopathiae in comparison with the avirulent L-forms therefrom are discussed.

Histoautoradiographic studies on the fate of 3H-thymidine labelled L-forms of E. rhusiopathiae in albino mice.

L-forms of Erysipelotrix rhusiopathiae labelled with 3H-thymidine were intravenously administered to albino mice. Autoradiographic studies of the liver, kidney and spleen were undertaken at periods ranging from 2 minutes to 15 days. On the second minute following the administration of the radioactive material whole labelled microorganisms and chains of silver grains were recovered in the examined organs. Up to the 15th minute labels were observed also in the cells of the RES. Following the 30th minute the silver grains were positioned at a characteristic site in the Golgi region of the hepatocytes. At the same time in the kidney they were localized in the glomerular space and in the lumen of the renal tubules, whereas in the spleen - mainly around the megakaryocytes. By the 15th day labelling gradually diminished, single silver grains being found over some nuclei of megakaryocytes, liver and kidney parenchymal cells. The present study throws light over some aspects of the interrelationship between the micro- and macroorganism concerning the mechanisms of desintegration, elimination and the uptaking of labelled microbial DNA.