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Farmers are carving a new agricultural frontier from the forests in the Southeast Asian Massif (SAM) in the 21st century, triggering significant environment degradation at the local scale; however, this frontier has been missed by existing global land use and forest loss analyses. In this paper, we chose Thailand's Nan Province, which is located in the geometric center of SAM, as a case study, and combined high resolution forest cover change product with a fine-scale land cover map to investigate land use dynamics with respect to topography in this region. Our results show that total forest loss in Nan Province during 2001-2016 was 66,072 ha (9.1% of the forest cover in 2000), and that the majority of this lost forest (92%) had been converted into crop (mainly corn) fields by 2017. Annual forest loss is significantly correlated with global corn price (p < 0.01), re-confirming agricultural expansion as a key driver of forest loss in Nan Province. Along with the increasing global corn price, forest loss in Nan Province has accelerated at a rate of 2,616 ± 730 ha per decade (p < 0.01). Global corn price peaked in 2012, in which year annual forest loss also reached its peak (7,523 ha); since then, the location of forest loss has moved to steeper land at higher elevations. Spatially, forest loss driven by this smallholder agricultural expansion emerges as many small patches that are not recognizable even at a moderate spatial resolution (e.g. 300 m). It explains how existing global land use/cover change products have missed the widespread and rapid forest loss in SAM. It also highlights the importance of high-resolution observations to evaluate the environmental impacts of agricultural expansion and forest loss in SAM, including, but not limited to, the impacts on the global carbon cycle, regional hydrology, and local environmental degradation., (© 2018 John Wiley & Sons Ltd.)

Embryonic stem cells (ESCs) hold promise for the treatment of many medical conditions; however, their utility is limited by immune rejection. The objective of our study is to establish tolerance or promote engraftment of transplanted ESCs as well as mature cell populations derived from ESCs. Luciferase (luc(+))-expressing ESCs were utilized to monitor the survival of the ESCs and differentiated progeny in living recipients. Allogeneic recipients conditioned with fractioned total lymphoid irradiation (TLI) and anti-thymocyte serum (ATS) or TLI plus regulatory T cells (T(reg)) promoted engraftment of ESC allografts after transplantation. Following these treatments, the engraftment of transplanted terminally differentiated endothelial cells derived from ESCs was also significantly enhanced. Our findings provide clinically translatable strategies of inducing tolerance to adoptively transferred ESCs for cell replacement therapy of medical disorders.

Store-operated calcium (SOC) channels are vital for activation of the immune cells, and mutations in the channel result in severe combined immunodeficiency in human patients. In lymphocytes, SOC entry is mediated by the Orai1 channel, which is activated by direct binding of STIM1. Here we describe an alternative approach for identifying inhibitors of SOC entry using minimal functional domains of STIM1 and Orai1 to screen a small-molecule microarray. This screen identified AnCoA4, which inhibits SOC entry at submicromolar concentrations and blocks T cell activation in vitro and in vivo. Biophysical studies revealed that AnCoA4 binds to the C terminus of Orai1, directly inhibiting calcium influx through the channel and also reducing binding of STIM1. AnCoA4, unlike other reported SOC inhibitors, is a molecule with a known binding site and mechanism of action. These studies also provide proof of principle for an approach to ion channel drug discovery.

Acute graft-versus-host disease (GVHD) is induced by alloreactivity of donor T cells toward host antigens presented on antigen-presenting cells (APCs). Apoptotic cells are capable of inducing tolerance by altering APC maturation. Apoptosis can be induced by extracorporeal photopheresis (ECP). We demonstrate that the use of ECP as a prophylaxis prior to conditioning significantly improves survival (P < .0001) after bone marrow transplantation (BMT) by inhibiting the initiation phase of acute GVHD in a murine BMT model. ECP-treated autologous splenocytes resulted in immune tolerance in the host, including reduced dendritic cell activation with decreased nuclear factor-κB engagement, increased regulatory T-cell (Treg) numbers with enhanced expression of cytolytic T lymphocyte-associated antigen 4, potentiating their suppressive function. The protective effect required host production of interleukin-10 and host Tregs. Conventional T cells that entered this tolerant environment experienced reduced proliferation, as well as a reduction of tissue homing and expression of activation markers. The induction of this tolerant state by ECP was obviated by cotreatment with lipopolysaccharide, suggesting that the inflammatory state of the recipient prior to treatment would play a role in potential clinical translation. The use of prophylactic ECP may provide an alternative and safe method for immunosuppression in the bone marrow transplant setting., (© 2014 by The American Society of Hematology.)

Lag-3 has emerged as an important molecule in T cell biology. We investigated the role of Lag-3 in conventional T cell (Tcon) and regulatory T cell (Treg) function in murine GVHD with the hypothesis that Lag-3 engagement diminishes alloreactive T cell responses after bone marrow transplantation. We demonstrate that Lag-3 deficient Tcon (Lag-3(-/-) Tcon) induce significantly more severe GVHD than wild type (WT) Tcon and that the absence of Lag-3 on CD4 but not CD8 T cells is responsible for exacerbating GVHD. Lag-3(-/-) Tcon exhibited increased activation and proliferation as indicated by CFSE and bioluminescence imaging analyses and higher levels of activation markers such as CD69, CD107a, granzyme B, and Ki-67 as well as production of IL-10 and IFN-g early after transplantation. Lag-3(-/-) Tcon were less responsive to suppression by WT Treg as compared to WT Tcon. The absence of Lag-3, however, did not impair Treg function as both Lag-3(-/-) and WT Treg equally suppress the proliferation of Tcon in vitro and in vivo and protect against GVHD. Further, we demonstrate that allogeneic Treg acquire recipient MHC class II molecules through a process termed trogocytosis. As MHC class II is a ligand for Lag-3, we propose a novel suppression mechanism employed by Treg involving the acquisition of host MHC-II followed by the engagement of Lag-3 on T cells. These studies demonstrate for the first time the biologic function of Lag-3 expression on conventional and regulatory T cells in GVHD and identify Lag-3 as an important regulatory molecule involved in alloreactive T cell proliferation and activation after bone marrow transplantation.

To investigate the role of mast cells in hematopoietic cell transplantation, we assessed graft-versus-host disease (GVHD) in C57BL/6-Kit(W-sh/W-sh) recipients, which virtually lack mast cells, compared with C57BL/6 WT recipients. GVHD was severely exacerbated in C57BL/6-Kit(W-sh/W-sh) mice (median survival time = 13 vs 60 days in wild-type [WT] mice; P < .0001). The increased mortality risk in C57BL/6-Kit(W-sh/W-sh) hosts correlated with increased T-cell numbers in lymph nodes, liver, and gastrointestinal tract sites, as indicated by bioluminescence imaging (P < .001). We did not detect any deficit in the number or function of CD4(+)CD25(+) regulatory T cells (Tregs) in C57BL/6-Kit(W-sh/W-sh) mice. Furthermore, Tregs were equally effective at reducing GVHD in C57BL/6-Kit(W-sh/W-sh) recipients compared with WT recipients containing mast cells. Furthermore, we found that survival of C57BL/6-Kit(W-sh/W-sh) mice during GVHD was significantly improved if the mice were engrafted with bone marrow-derived cultured mast cells from WT C57BL/6 mice but not from interleukin (IL)-10-deficient C57BL/6 mice. These data indicate that the presence of mast cells can significantly reduce GVHD independently of Tregs, by decreasing conventional T-cell proliferation in a mechanism involving IL-10. These experiments support the conclusion that mast cells can mediate a novel immunoregulatory role during hematopoietic cell transplantation.

Regulatory T cell (Treg) immunotherapy is a promising strategy for the treatment of graft rejection responses and autoimmune disorders. Our and other laboratories have shown that the transfer of highly purified CD4(+)CD25(+)Foxp3(+) natural Treg can prevent lethal graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation across both major and minor histocompatibility barriers. However, recent evidence suggests that the Treg suppressive phenotype can become unstable, a phenomenon that can culminate in Treg conversion into IL-17-producing cells. We hypothesized that the intense proinflammatory signals released during an ongoing alloreaction might redirect a fraction of the transferred Treg to the Th17 cell fate, thereby losing immunosuppressive potential. We therefore sought to evaluate the impact of Il17 gene ablation on Treg stability and immunosuppressive capacity in a major MHC mismatch model. We show that although Il17 gene ablation results in a mildly enhanced Treg immunosuppressive ability in vitro, such improvement is not observed when IL-17-deficient Treg are used for GVHD suppression in vivo. Similarly, when we selectively blocked IL-1 signaling in Treg, that was shown to be necessary for Th17 conversion, we did not detect any improvement on Treg-mediated GVHD suppressive ability in vivo. Furthermore, upon ex vivo reisolation of transferred wild-type Treg, we detected little or no Treg-mediated IL-17 production upon GVHD induction. Our results indicate that blocking Th17 conversion does not affect the GVHD suppressive ability of highly purified natural Treg in vivo, suggesting that IL-17 targeting is not a valuable strategy to improve Treg immunotherapy after hematopoietic cell transplantation., (Copyright © 2013 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Previous work has demonstrated that both rapamycin (RAPA) and IL-2 enhance CD4⁺CD25⁺Foxp3⁺ regulatory T-cell (Treg) proliferation and function in vitro. We investigated whether the combination of RAPA plus IL-2 could impact acute GVHD induction after bone marrow transplantation (BMT). RAPA plus IL-2 resulted in improved survival and a reduction in acute GVHD lethality associated with an increased expansion of donor type CD4⁺Foxp3⁺ Tregs and reduced CD4⁺CD25⁻ conventional T cells (Tcons). RAPA plus IL-2, but not either drug alone, increased both expansion of donor natural Tregs and conversion of induced Tregs from donor CD25⁻ Tcons while IL-2 alone increased conversion of Tregs from CD25⁻ Tcon. RAPA plus IL-2 treatment resulted in less production of IFN-γ and TNF, cytokines known to be important in the initiation of acute GVHD. These studies indicate that the pharmacologic stimulation of T cells with IL-2 and the suppression of Tcon proliferation with RAPA result in a selective expansion of functional Tregs and suppression of acute GVHD.

CD4(+) natural killer T (NKT) cells, along with CD4(+)CD25(+) regulatory T cells (Tregs), are capable of controlling aberrant immune reactions. We explored the adoptive transfer of highly purified (> 95%) CD4(+)NKT cells in a murine model of allogeneic hematopoietic cell transplantation (HCT). NKT cells follow a migration and proliferation pattern similar to that of conventional T cells (Tcons), migrating initially to secondary lymphoid organs followed by infiltration of graft-versus-host disease (GVHD) target tissues. NKT cells persist for more than 100 days and do not cause significant morbidity or mortality. Doses of NKT cells as low as 1.0 × 10(4) cells suppress GVHD caused by 5.0 × 10(5) Tcons in an interleukin-4 (IL-4)-dependent mechanism. Protective doses of NKT cells minimally affect Tcon proliferation, but cause significant reductions in interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) production by donor Tcons and in skin, spleen, and gastrointestinal pathology. In addition, NKT cells do not impact the graft-versus-tumor (GVT) effect of Tcons against B-cell lymphoma-1 (BCL-1) tumors. These studies elucidate the biologic function of donor-type CD4(+)NKT cells in suppressing GVHD in an allogeneic transplantation setting, demonstrating clinical potential in reducing GVHD in HCT.

Embryonic stem cells (ESCs) are an attractive source for tissue regeneration and repair therapies because they can be differentiated into virtually any cell type in the adult body. However, for this approach to succeed, the transplanted ESCs must survive long enough to generate a therapeutic benefit. A major obstacle facing the engraftment of ESCs is transplant rejection by the immune system. Here we show that blocking leukocyte costimulatory molecules permits ESC engraftment. We demonstrate the success of this immunosuppressive therapy for mouse ESCs, human ESCs, mouse induced pluripotent stem cells (iPSCs), human induced pluripotent stem cells, and more differentiated ESC/(iPSCs) derivatives. Additionally, we provide evidence describing the mechanism by which inhibition of costimulatory molecules suppresses T cell activation. This report describes a short-term immunosuppressive approach capable of inducing engraftment of transplanted ESCs and iPSCs, providing a significant improvement in our mechanistic understanding of the critical role costimulatory molecules play in leukocyte activation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Natural killer (NK) cells suppress graft-versus-host disease (GVHD) without causing GVHD themselves. Our previous studies demonstrated that allogeneic T cells and NK cells traffic similarly after allogeneic bone marrow transplantation (BMT). We therefore investigated the impact of donor NK cells on donor alloreactive T cells in GVHD induction. Animals receiving donor NK and T cells showed improved survival and decreased GVHD score compared with controls receiving donor T cells alone. Donor T cells exhibited less proliferation, lower CD25 expression, and decreased interferon-gamma (IFN-gamma) production in the presence of NK cells. In vivo, we observed perforin- and Fas ligand (FasL)-mediated reduction of donor T cell proliferation and increased T cell apoptosis in the presence of NK cells. Further, activated NK cells mediated direct lysis of reisolated GVHD-inducing T cells in vitro. The graft-versus-tumor (GVT) effect was retained in the presence of donor NK cells. We demonstrate a novel mechanism of NK cell-mediated GVHD reduction whereby donor NK cells inhibit and lyse autologous donor T cells activated during the initiation of GVHD.

Based on their ability to control T-cell homeostasis, Foxp3(+)CD4(+)CD25(+) regulatory T cells (Tregs) are being considered for treatment of autoimmune disorders and acute graft-versus-host disease (aGVHD). When combining Tregs with the immunosuppressant rapamycin (RAPA), we observed reduced alloreactive conventional T-cell (Tconv) expansion and aGVHD lethality compared with each treatment alone. This synergistic in vivo protection was paralleled by intact expansion of polyclonal Tregs with conserved high FoxP3 expression. In contrast to Tconv, activation of Tregs with alloantigen and interleukin-2 preferentially led to signal transducer and activator of transcription 5 (STAT5) phosphorylation and not phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway activity. Expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a negative regulator of the PI3K/Akt/mTOR pathway, remained high in Tregs but not Tconv during stimulation. Conversely, targeted deletion of PTEN increased susceptibility of Tregs to mTOR inhibition by RAPA. Differential impact of RAPA as a result of reduced usage of the mTOR pathway in Tregs compared with conventional T cells explains the synergistic effect of RAPA and Tregs in aGVHD protection, which has important implications for clinical trials using Tregs.

The transporter associated with antigen presentation (TAP) is an ATP-binding cassette (ABC) protein which transports peptides for presentation to the immune system. TAP is composed of two half transporters, TAP1 (ABCB2) and TAP2 (ABCB3), which heterodimerize to function. In humans, the TAP family consists of TAP1, TAP2, and TAPL (ABCB9). While the TAP1-TAP2 complex is well characterized, TAPL's dimerization state and function are unknown. To identify interactions within the human TAP family, we adapted the dihydrofolate reductase protein-fragment complementation assay (DHFR PCA) to half ABC transporters. This assay has been shown to be suitable for the study of membrane-bound proteins in vivo [Remy, I., Wilson, I. A., and Michnick, S. W. (1999) Science 283, 990-993]. With this method, in vivo TAP1-TAP2 heterodimerization was confirmed, no homodimerizations were detected with TAP1 or TAP2, and TAPL did not show any interaction with TAP1 or TAP2. However, we found strong evidence that TAPL forms homodimers. These results provide evidence of a novel homomeric TAPL interaction and demonstrate that the DHFR PCA will be of general utility in studies of half ABC transporter interactions in vivo.

A high degree of binding of 5alpha-[3H]-androstenone was recorded in membrane-enriched fractions of porcine olfactory tissue. The specific (i.e. high affinity, low capacity) binding had a mean Ka approximately 2x10(8)M(-1). A Hill plot of the data showed a Hill coefficient of approximately 2, possibly suggesting co-operativity of binding, with binding constants increasing from 8x10(7) to 1.6x10(9)M(-1) with increasing substrate concentration. The level of specific binding of 5alpha-[3H]-androstenone was nearly 10-fold higher than in corresponding respiratory tissue preparations and was markedly reduced in the presence of excess (approximately 1 microM) unlabelled 5alpha-androstenone. Corresponding fractions derived from rat olfactory tissue showed only 25% of the binding recorded for the pig. After incubation of 5alpha-[3H]-androstenone with solubilised olfactory cilial tissue (porcine), gel filtration and chromatography on a typical "glycoprotein" column (Concanavalin A-Sepharose B) were performed. Specific binding was recorded only in fractions corresponding to glycoproteins with Mr of approximately 70-90 kDa. In a third series of experiments, fractions containing high concentrations of cilia, some still attached to the dendritic endings (as shown by electron microscopy) were obtained by a novel method involving stripping them off the nasal epithelium. The basal adenylate cyclase (AC) activity was very significantly (P<0.01) higher in olfactory, compared with respiratory, cilia; storage at -70 degrees C for 3 weeks greatly reduced AC activity. When fresh male and female porcine olfactory cilia preparations were incubated with 5alpha-androstenone plus GTP, AC activity was increased fourfold (P<0.01). However, responses of porcine respiratory cilia were not significant statistically, neither were changes in basal levels of AC activities in rat olfactory cilia.

International Olympic Committee accredited laboratories play a key role in upholding the principle of fair play and innate ability, as desired by the majority of sports competitors and spectators. Not only does doping damage the image of sport, but it can also be harmful to the individual. The great majority of samples test negative but, when an adverse finding is declared, the analytical data must be of a sufficiently high standard to withstand legal challenges by third parties. The most widely misused performance-enhancing drugs are the anabolic-androgenic steroids, commonly referred to as 'anabolic steroids'. This review attempts to address the complex issues concerning anabolic steroids in sport by considering the clinical, biochemical and analytical perspectives.

In on-going studies of 'classical' and ring B-unsaturated oestrogens in equine pregnancy, the products of metabolism of [2,2,4,6,6-2H(5)]-testosterone and [16,16,17-2H(3)]-5,7-androstadiene-3 beta,17 beta-diol with equine placental subcellular preparations and allantochorionic villi have been identified. Using mixtures of unlabelled and [2H]-labelled steroid substrates has allowed the unequivocal identification of metabolites by twin-ion monitoring in gas chromatography-mass spectrometry (GC-MS). Two types of incubation were used: (i) static in vitro and (ii) dynamic in vitro. The latter involved the use of the Oxycell cartridge (Integra Bioscience Systems, St Albans, UK) whereby the tissue preparation was continuously supplied with supporting medium plus appropriate cofactors in the presence of uniform oxygenation. [2H(5)]-Testosterone was converted into [2H(4)]-oestradiol-17 beta, [2H(4)]-oestrone and [2H(3)]-6-dehydro-oestradiol-17 alpha in both placental and chorionic villi preparations, but to a greater extent in the latter, confirming the importance of the chorionic villi in oestrogen production in the horse. On the basis of GC-MS characteristics (M(+) m/z 477/482 (as O-methyl oxime-trimethyl silyl ether), evidence for 19-hydroxylation of testosterone was found in static incubations, while the presence of a 6-hydroxy-oestradiol-17 alpha was recorded in dynamic incubations (twin peaks in the mass spectrum at m/z 504/507, the molecular ion M(+)). It was not possible to determine the configuration at C-6. The formation of small, but significant, quantities of [2H(4)]-17 beta-dihydroequilin was also shown, and a biosynthetic pathway is proposed. In static incubations of placental microsomal fractions, the 17 beta-dihydro forms of both equilin and equilenin were shown to be major metabolites of [2H(3)]-5,7-androstadiene-3,17-diol. Using static incubations of chorionic villi, the deuterated substrate was converted into the 17 beta-dihydro forms of both equilin and equilenin, together with an unidentified metabolite (base peak, m/z 504/506). The isomeric 17-dihydroequilins were also obtained using the dynamic in vitro incubation of equine chorionic villi, together with the 17 beta-isomer of dihydroequilenin. Confirmation of the identity of 17 beta-dihydroequilin and 17 beta-dihydroequilenin was obtained by co-injection of the authentic unlabelled steroids with the phenolic fraction obtained from various incubations. Increases in the peak areas for the non-deuterated steroids (ions at m/z 414 (17 beta-dihydroequilin) and 412 (17 beta-dihydroequilenin) (both as bis-trimethyl silyl ether derivatives) were observed. Biosynthetic pathways for formation of the ring B-unsaturated oestrogens from 5,7-androstadiene-3 beta,17 beta-diol are proposed.

The intracellular movements of pregnenolone in rat testes were investigated. Whole testes were incubated in the presence or absence of pregnenolone (2.5mM) in the medium for 120 min (in some studies 30, 60, and 90 min). The testes were homogenised, subcellular fractions prepared and analysed in quadruplicate for steroid content by gas chromatography-mass spectrometry with selected ion monitoring. Quantification of pregnenolone and 11 of its metabolites, obtained from non-incubated whole testes, provided values for endogenous amounts. Pregnenolone was the only steroid of quantitative importance found initially in the mitochondrial fraction but was subsequently found in the microsomal fraction, where metabolism occurred. Identification and quantification of metabolites indicated that both classical pathways for testosterone production were operating, with the 4-en-3-oxosteroid pathway predominating. By 120 min, virtually all pregnenolone metabolites, including pregnenolone itself, were found in the cytosol, consistent with an overall movement from mitochondria to endoplasmic reticulum to cytosol.

Oestradiene-3,17-diol and oestratriene-3,17-diol (or the diol of Heard's ketone (3-hydroxy-5(10),6,8-oestratriene-17-one) have been extracted on a large scale from pooled urines and allantoic fluid obtained from pregnant mares. Initial purification was achieved using column chromatography, and further purification by high performance liquid chromatography or silver nitrate (argentation) thin layer chromatography. The steroids were characterised using gas chromatography-mass spectrometry. Positions of the double bonds in ring B of oestradienediol were deduced on the basis of results of ultraviolet (UV) and nuclear magnetic resonance (NMR) spectroscopy, hydrogenation, and incubation studies with the enzyme 5-ene-3beta-hydroxysteroid dehydrogenase/steroid-4,5-isomerase. The reference steroid, 5,7-cholestadien-3beta-ol (7-dehydrocholesterol), with its conjugated double bond system, behaved entirely differently to oestradienediol, consistent with the latter having no conjugated system. These data, together with detailed results of NMR studies, have led us to designate the positions of the double bonds in oestradienediol as 5(10),7-. The instability of the dienediol became apparent when the steroid was converted to its bis-trimethylsilyl (TMS) ether. The phenomenon was exacerbated when derivatisation was performed at elevated temperatures or when the fraction containing the dienediol was stored at 4 degrees C prior to being derivatised. The facile oxidation product was shown to be 5(10),6, 8-oestratriene-3,17-diol, implying that the two steroids are related and, furthermore, that all the sites of unsaturation are in the B ring. Because of the facile oxidation of oestradienediol to oestratrienediol (the diol of Heard's ketone), we propose, that this, and by implication, Heard's ketone itself, are artefacts of the isolation procedures which were utilised in the original studies. A possible mechanism is proposed for the biosynthesis of 5, 7-oestradienediol from a ring-B unsaturated C(19) compound, involving C(19) demethylation without aromatisation.

Equine umbilicus was cannulated in utero and a series of cord plasma samples removed for analysis. After steroid extraction and derivatisation, gas chromatographic-mass spectrometric (GC-MS) analysis demonstrated large differences in steroid content between the plasma samples obtained from the umbilical artery and vein, the blood supplies leading to and from the placental surface, respectively. 3Beta-hydroxy-5,7-androstadien-17-one, dehydroepiandrosterone, pregnenolone, 3beta-hydroxy-5alpha-pregnan-20-one, 5-pregnene-3beta,20beta-diol and 5beta-pregnane-3beta,20beta-diol were identified as major constituents in extracts from umbilical arterial plasma samples, mostly as unconjugated steroids. Together with 5alpha-pregnane-3,20-dione, these steroids were identified in extracts from umbilical venous plasma samples but at significantly reduced levels to those determined in arterial plasma samples. Oestradiol-17alpha, dihydroequilin-17alpha and dihydroequilenin-17alpha were identified in extracts (mostly sulphate-conjugated) from both umbilical arterial and venous plasma samples, much larger amounts being detected in the plasma sampled from, rather than to, the placental surface. Equilin, equilenin, oestrone, oestradiol-17beta, dihydroequilin-17beta and dihydroequilenin-17beta were not detected in the present studies. Isomers of 5(10)-oestrene-3,17beta-diol together with 5(10),7-oestradiene-3,17beta-diol and its possible oxidative artifact, 5(10),7,9-oestratriene-3,17beta-diol, were tentatively identified only in sulphate-conjugated extracts from umbilical venous plasma samples. No glucuronic acid-conjugated steroids could be detected. The implications of this work in the elucidation of the biosynthetic pathways leading to both the formation of oestrogens and C18 neutral steroids at the placental surface are discussed.

The aim of this investigation was to study the effects of prostaglandins E1 and E2 (PGE1 and PGE2) on the accumulation, release and metabolism of C19 steroids by human gingival fibroblasts (HGF) and gingivae, due to their anabolic potential in inflammatory repair. For the accumulation studies, HGF were incubated with 14C-testosterone at timed intervals and the cell-digests analysed for label uptake. The release of 5 alpha-dihydrotestosterone (DHT) by HGF was studied by preincubating the cells with 14C-DHT and reincubating with cold steroid to quantify its release at timed intervals. For the metabolic studies, HGF/gingival tissue were incubated with 14C-testosterone and serial concentrations of PGE1 and PGE2 to study their effects on the synthesis of DHT. The incubations were terminated at 24 h and extracted metabolites were analysed and quantified. The accumulation of 14C-testosterone by human gingival fibroblasts was elevated 3-fold at 24 h by PGE1 (n = 3, p < 0.001; 1-way ANOVA). The release of 14C-DHT was enhanced nearly 2-fold by PGE1 (n = 3, p < 0.001), compared with controls. Both PGE1 and PGE2 caused 2-fold increases in DHT synthesis by HGF and 3-fold increases in 4-androstenedione formation (n = 4, p < 0.001). With the tissue incubations PGE1/PGE2 caused 3-4 fold increases in DHT synthesis (n = 5, p < 0.005; Wilcoxon signed rank statistic for paired observations). Direct stimulation of the accumulation/release and metabolism of these steroids by prostaglandins in gingivae may contribute to the anabolic potential of androgens in inflammatory periodontal disease.

The products of metabolism of the sulphates (0.5 micromol/l) of androsterone, dehydroepiandrosterone (DHA) and 5alpha-androst-16-en-3beta-ol have been investigated after incubation with 72 h cultures of human axillary bacterial isolates for 3 days at 37 degrees C. The medium used, tryptone soya broth (TSB), contained yeast extract and Tween 80. The isolates used were Coryneform F1 (known previously to metabolize testosterone and to be involved in under-arm odour (UAO) production, i.e. UAO +ve), Coryneform F46 (inactive in both the testosterone metabolism and UAO tests, i.e. UAO -ve) and Staphylococcus hominis/epidermidis (IIR3). Control incubations of TSB alone, TSB plus each of the steroid sulphates and TSB plus each of the bacterial isolates were also set up. After termination of reactions and addition of internal standards, 5alpha-androstan-3beta-ol and 5alpha-androstan-3-one (50 ng each), extracted and purified metabolites were subjected to combined gas chromatography-mass spectrometry with specific ion monitoring. Steroidal ketones were derivatized as their O-pentafluorobenzyl oximes; steroidal alcohols (only androst-16-enols in this study) were derivatized as their tert-butyldimethylsilyl ethers. Analysis was achieved by negative ion chemical ionization mass spectrometry for the pentafluorobenzyl oximes at [M-20]- and electron impact positive ion mass spectrometry for the tert-butyldimethylsilyl ethers at [M-57]+. The incubation broth contained two compounds which had gas chromatographic and mass spectrometric properties identical to those of DHA and 4-androstenedione. It was not possible, therefore, to show unequivocally that DHA sulphate (DHAS) was converted microbially into DHA, although this is implied by the finding of small quantities of testosterone and 5alpha-dihydrotestosterone in incubations with F1. With androsterone S, no free androsterone was recorded and only very small (5 pg or less) amounts of testosterone. Two odorous steroids, androsta-4,16-dien-3-one and 5alpha-androst-2-en-17-one (Steroid I) were formed (mean quantities 40 and 45 pg, respectively). The sulphate of 5alpha-androst-16-en-3beta-ol was metabolized with F1 into large quantities of the odorous steroids, 5alpha-androst-16-en-3-one and Steroid I. In addition, much smaller quantities of androsta-4,16-dien-3-one were formed. In contrast, incubations of DHAS with F46 resulted in no metabolites except, possibly, DHA, but the sulphate moiety of androsterone S was also cleaved to yield the free steroid together with large amounts of Steroid I. In incubations of DHAS and androsterone S with F1, no 16-unsaturated steroids were formed, although 5alpha-androst-16-en-3beta-yl S was de-sulphated and the free steroid further metabolized. No evidence was obtained for androst-16-ene metabolism in incubations with F46. In incubations with S. hominis/epidermidis (IIR3), androsterone S was converted into androsterone and, in high yield, to Steroid I plus some 5alpha-androst-16-en-3-one. Both DHAS and androsterone S were converted into androst-16-enols. Sulphatase activity was also manifested when 5alpha-androst-16-en-3beta-yl S was utilized as substrate with IIR3, large quantities of Steroid I and 5alpha-androst-16-en-3-one being formed, together with further metabolism of androst-16-enes. In view of the fact that both DHAS and androsterone S occur in apocrine sweat, the metabolism of these endogenous substrates by human axillary bacteria to several odorous steroids may have important implications in the context of human odour formation.

Homogenates of histologically normal human testis from three men were incubated separately with pregnenolone, 16-dehydropregnenolone, 5alpha-pregnane-3,20-dione, 3beta-hydroxy-5alpha-pregnan-20-one and androsta-5,16-dien-3beta-ol (androstadienol) in the presence of NADPH in a study of androst-16-ene and androgen biosynthesis. After the addition of internal standards and initial extraction and purification, metabolites were identified using gas chromatography-mass spectrometry (GC-MS) and monitoring selectively for three principal ions in each case at the appropriate GC retention time. Quantification was achieved by comparison with calibration lines for authentic steroids, together with the appropriate internal standards, prepared by monitoring three ion fragments for each analyte. In all experiments, androstadienol was found to be the major androst-16-ene metabolite of pregnenolone (seven times the control, i.e. endogenous, quantity; 19.8 +/- 3 ng/100 mg homogenate protein, mean +/- SEM, n = 9). Pregnenolone was also converted to androsta-4,16-dien-3-one (androstadienone) with three times the endogenous quantity (44 +/- 10 ng/100 mg homogenate protein, mean +/- SEM, n = 9) being formed. The formation of testosterone occurred only in trace amounts in the incubations of testis taken from one man (a 69-yr-old) but appreciable yields (six times endogenous levels 90 +/- 7 ng/100 mg homogenate protein, mean +/- SEM, n = 9) were found with testes from two younger men. Only traces of 5alpha-dihydrotestosterone were detected. Using androstadienol as the substrate, androstadienone was shown to be the major metabolite (approximately 10 times greater than control incubations) together with 5alpha-androst-16-en-3alpha- and 3beta-ols at approximately twice the endogenous quantities (5 ng/100 mg homogenate protein). In some incubations with androstadienol, 5alpha-androst-16-en-3-one (5alpha-androstenone) was formed (32 +/- 1 ng/100 mg homogenate protein/h; mean +/- SEM, n = 3); surprisingly, no endogenous 5alpha-androstenone could be detected. No evidence was obtained for the production of testosterone or 5alpha-DHT from androstadienol. Using cytosolic fractions of human testis, specific (displaceable) binding of 5alpha-androstenone was determined, with binding sites of approximately 200 fmol/mg tissue and a Ka of approximately 8 nmol/l.

Maternal endometrial and fetal allantochorionic tissues were separated manually from the placentae of seven healthy thoroughbred and three pony mares, ranging in gestational age from 100 to 318 days. The homogeneity of subcellular fractions prepared from these tissues was assessed initially using the marker enzymes, succinate dehydrogenase, NADPH cytochrome C reductase and lactate dehydrogenase for the mitochondrial, microsomal and cytosolic fractions, respectively. Light microscopy and histochemical analysis demonstrated that the separated fetal allantochorionic membrane, which is made up of allantoic and chorionic epithelia, contained no significant contamination of maternal tissues. The maternal endometrium, however, was found to contain appreciable amounts of fetal chorion torn off during the separation process. Tissue homogenates and subcellular fractions were incubated with testosterone together with [4-(14)C] and [(2)H5 or (2)H3] labelled analogues in either an NADPH (1 mM) or a NADPH-regenerating environment; control experiments (without additional cofactor) were also performed. After extraction of the tissue homogenates, neutral and phenolic (oestrogen) unconjugated steroids were separated by column chromatography. Radiolabelled studies revealed that in allantochorionic tissue incubations 67-77% of testosterone was converted to oestrogenic material, subcellular fractionation indicating that oestrogen production was largely confined to the microsomal fraction and time-course studies showing that the rate of formation appeared to be linear up to 90 min. In contrast, only 5-25% conversion occurred using maternal endometrial tissues, which could be accounted for by the contaminating presence of fetal chorion. No oestrogen production was detected in control incubations. These radiolabelled studies demonstrate that aromatase activity is located on the fetal allantochorionic surface and, together with the histochemical data, further delineate this activity to the chorion in mature equine placenta. Gas chromatographic-mass spectrometric (GC-MS) analysis of the phenolic extracts from allantochorionic tissue homogenate incubations indicated the presence of substrate-derived oestradiol-17beta (E2), 6-oxo-oestradiol-17beta (6-oxo-E2) and 6beta-hydroxyoestradiol-17beta (6beta-OH-E2). Whereas all three oestrogens were identified as metabolites from testosterone in incubations performed using allantochorionic tissue homogenates and post-mitochondrial suspensions (PMS), only E2 was identified from incubations performed using microsomal fractions prepared from this tissue. We conclude that both the microsomal and cytosol fractions are required for the conversion of E2 to the 6-oxygenated species in vitro. Using stable isotope-labelled substrates and GC-MS analysis the mechanism of formation of these metabolites from these in vitro incubation studies may be inferred. GC-MS analysis of the neutral extracts from allantochorionic tissue homogenate incubations confirmed the presence of small quantities of substrate-derived 5(10)-oestrenediols. No substrate-derived 5(10)-oestrene-3,17-diols were detected in extracts from microsomal preparations incubated in the absence of cytosol. These data suggest that demethylation of C19 steroids to produce C18 neutral steroids may require the synergistic action of enzymic activities that appear to reside both in the microsomal and cytosolic fractions of equine allantochorionic tissues.

This study examined the relationship between psychological mood, stress and oral hygiene behaviour in a group of 51 regular dental attenders. Subjects brought a saliva sample for cortisol radioimmunoassay, completed the Hospital Anxiety and Depression (HAD) Scale, were assessed for plaque and gingivitis, and were then instructed in toothbrushing. 5 weeks later, 47 subjects were given a full repeat examination. There was a slight reduction in plaque and gingivitis scores, but no change in mood as assessed by HAD Scale and salivary cortisol concentration. Mean anxiety scores were associated with gingivitis level, and mean depression scores with plaque. Neither mood nor cortisol were predictors of subsequent change in plaque or gingivitis.

The metabolism of varying quantities of pregnenolone has been studied in nuclei-free homogenates from Macaca fascicularis testes by using capillary gas chromatography, after derivatization of metabolites as O-methyl oximes/trimethylsilyl ethers. Evidence was obtained indicating that both pathways for testosterone biosynthesis were operating. 5-Androstene-3 beta, 17 beta-diol was formed in especially high quantities. Two 16-androstenes, namely 5,16-androstadien-3 beta-ol and 5 alpha-androst-16-en-3 beta-ol, were also quantitatively important as metabolites. Co-incubation of stored homogenates with relaxin resulted in 80-100% reduction of the formation of all metabolites quantified except for 5 alpha-androst-16-en-3-one, which was stimulated. Freezing the homogenates at -10 degrees C for 3 weeks resulted in marked 4- to 6-fold reduction in the yields of testosterone and of the 5-ene and 4-ene metabolites from pregnenolone.

The concentrations of five 16-androstene steroids were determined, by a GC-MS method, in freshly-produced apocrine sweat (adrenaline-induced), in 8 men and 2 women. The ranges of concentrations (nmol/microliter) in apocrine sweat were: 5 alpha-androst-16-en-3-one (5 alpha-A), 0.1-2.0 and 4,16-androstadien-3-one (androstadienone), 0-1.9, 5,16-Androstadien-3 beta-ol (androstadienol) was also found in 5 of the subjects (range 0.05-1.05). 5 alpha-Androst-16-en-3 alpha- or 3 beta-ols [3 alpha (beta)-androstenols] were only found in small amounts (< 0.1 nmol/microliters) in a few subjects. In the second study, prior to apocrine sweat collection (adrenaline injection), the axillary skin of 6 of the male subjects was washed with diethyl ether on an adjacent site of the axillary vault. The concentrations of 16-androstenes were compared in the ethereal extracts and apocrine sweat. The former contained detectable levels (pmol/cm2) of androstadienone (17.9 +/- 2.4), 3 alpha-androstenol (6.9 +/- 3.7), 3 beta-androstenol (1.8 +/- 1.0) and androstadienol (1.9 +/- 0.5) (means +/- SEM) in all 6 subjects. All but 1 subject also had 5 alpha-androstenone, the mean value for the others being 2.5 +/- 0.6. The axillary skin levels of 3 alpha- and 3 beta-androstenols, androstadienol and, in 3 subjects, androstadienone exceeded those in the apocrine sweat obtained from the same subjects, whereas levels of 5 alpha-androstenone in the skin extracts were all lower than in apocrine sweat samples, when related to the corresponding areas of skin sampled. The metabolism of 16-androstenes was studied in vitro in the presence of two aerobic coryneform bacteria, previously shown to metabolize testosterone as well as being capable of producing odour from extracts of axillary sweat in an odour-generation test. Although both coryneforms caused complex metabolic reactions and were capable of oxidation or reduction at C-3 and C-4, the overall direction favoured reduction. For example, large quantities of the more odorous 5 alpha-androstenone and 3 alpha-androstenol were formed from androstadienol and androstadienone. In contrast, strains of corynebacteria, unable to produce odour and incapable of metabolizing testosterone, were also unable to metabolize 16-androstenes.(ABSTRACT TRUNCATED AT 400 WORDS)

Using the rapid gas chromatographic steroid profiling technique, a number of metabolites of pregnenolone have been separated and quantified after incubation of this steroid with adult rat and neonatal porcine testicular homogenates. It was shown that the 5-ene-3 beta-hydroxy- and the 4-en-3-oxosteroid pathways for androgen biosynthesis were operating in both species, although the former pathway appeared to be more important in porcine testis. This tissue was characterised by the formation of several odorous, and pheromonal, 16-androstenes, which were quantitatively more important than the androgens. Three non-steroidal anti-inflammatory drugs (NSAIDS) caused dose-related inhibition of androgen and 16-androstene biosynthesis when co-incubated with pregnenolone. The order of potency was flurbiprofen > indomethacin > > > aspirin. The possibility that the NSAIDS may interfere with cytochrome P-450 is discussed, since several steroid-transforming enzymes, known to be dependent on this cytochrome for their activity, were markedly inhibited.

Human semen was examined for the presence of 16-androstenols, 16-androstenones and androgens. Extracts were analysed by gas chromatography-mass spectrometry after derivatization of steroids under study. In a qualitative study, 5 alpha-androst-16-en-3 alpha- and 3 beta-ols, 5,16-androstadien-3 beta-ol and 5 alpha-androstan-3 beta-ol were detected in a semen pool A. Hydroxyl groups were converted to tert-butyldimethylsilyl ethers, the ions selected for monitoring being [M-57]+, consistent with loss of the tert-butyl group. For a more detailed quantitative study, a second semen pool B was used. In this case, all hydroxyl groups were converted to trimethylsilyl ethers, while oxo groups were not derivatized. As with semen pool A, separation of steroids was achieved using capillary gas chromatography with appropriate temperature programming. Quantification was carried out by mass spectrometry using selected ion monitoring of two significant ions and appropriate internal standards. The following steroids were identified at the concentrations indicated: 5 alpha-androst-16-en-3 alpha- and 3 beta-ols and 5,16-androstadien-3 beta-ol (concentration range, 0.5-0.7 ng/ml). 5 alpha-Androst-16-en-3-one and 4,16-androstadien-3-one were also present at levels of 0.7-0.9 ng/ml. Two androgens, testosterone and 5 alpha-dihydrotestosterone were found at concentrations of 0.5 and 0.3 ng/ml, respectively. These data, showing the presence of 16-androstenes and androgens in human semen, appear to be consistent with testicular formation of these steroids. The possible significance of the odorous 16-androstenes is discussed.

The relationships between testicular 16-androstene and testosterone formation in young pigs and levels of 5 alpha-androstenone in fat exhibited at older ages were investigated. Testis tissue samples were taken from Large White male pigs at 75, 100 and 125 days of age (8 pigs per age group) and incubated with a mixture of [3H]progesterone and [14C]pregnenolone. 5 alpha-androstenone levels in fat were measured in all 24 pigs at 125, 145 and 170 days of age. Less androstadienone, more 3 alpha-androstenol and 11-fold more 3 beta-androstenol + androstadienol were synthesized from pregnenolone than from progesterone. 5 alpha-androstenone synthesis increased significantly between 100 and 125 days, whereas that of androstadienone declined between 75 and 100 days of age. Litter had a significant effect on all 16-androstene rates of formation. Fat 5 alpha-androstenone levels were positively correlated with 5 alpha-androstenone formation from both precursors. They were correlated with 3 beta-androstenol + androstadienol (positively) and testosterone (negatively) rates of synthesis from progesterone. It is concluded that in vitro measurements of 5 alpha-androstenone formation by testis tissue could be used as an early predictor of fat 5 alpha-androstenone levels exhibited at older ages.

The axillary microflora of 34 male subjects were studied in relation to their underarm odour intensity. The predominant groups of micro-organisms were aerobic coryneforms, Micrococcaceae and propionibacteria. There was no competition for habitat between these groups (Fisher's exact test P greater than 0.05). There was an association between the population density of aerobic coryneforms and the intensity of odour (Spearman, P = 0.001). Dominance of aerobic coryneforms within the axillary microflora was associated with high odour intensity (chi 2, P = 0.005). An in-vitro odour model was developed using a diethyl ether extract of axillary skin incubated with test bacteria. Underarm odour was produced exclusively by aerobic coryneform bacteria. Of aerobic coryneforms, 71.4% were odour producers and these were identified as Corynebacterium xerosis.

The known involvement of axillary microflora with under-arm odour (UAO) production led us to determine whether the odorous 16-androstene steroids are formed in the axilla by bacterial metabolism of an odourless precursor such as testosterone. Axillary bacteria from 34 men were selectively cultured for aerobic coryneform bacteria (ACB), Micrococcaceae and propionibacteria. Overnight suspensions of bacteria were incubated separately at 37 degrees C for two weeks with radiolabelled testosterone plus unlabelled testosterone (0.5 mg) and 0.5-mg quantities of 4,16-androstadien-3-one (androstadienone) and 5,16-androstadien-3 beta-ol (androstadienol). After extraction and purification by Sep-Pak cartridges and thin-layer chromatography, the eluted steroids were derivatised as the pentafluorobenzyl oximes (PFBO) and tert.-butyl dimethylsilyl (TBDMS) ethers. Saturated analogues were used as internal standards. Selected-ion monitoring electron-impact mass spectrometry was performed at the m/z corresponding to the M+.ion for the PFBO derivatives and the [M - 57]+ ion for the TBDMS ethers. Only ACB produced classical musk-like UAO (UAO + ve) in an in vitro odour-producing system with 29% being UAO -ve. ACB (UAO +ve) metabolised far more (p = 0.001) testosterone than ACB (UAO -ve), the principal metabolites being 5 alpha(beta)-dihydrotestosterone, 5 alpha(beta)-androstane-3,17-dione and 4-androstene-3,17-dione (4-androstenedione). No non-polar 16-androstenes were formed. Micrococcus luteus (ten strains) metabolised testosterone to 4-androstenedione only; propionibacterium spp. did not metabolise testosterone at all. However, incubation of 16-androstenes with ACB gave evidence for 4-ene-5 alpha(beta)-reduction, 3 alpha(beta)-oxido-reduction and epimerisation. In general the direction of transformations favoured formation of the more odorous 5 alpha-androst-16-en-3-one (5 alpha-androstenone) and 5 alpha-androst-16-en-3 alpha-ol (3 alpha-androstenol) from less odorous steroids. Such transformations, in vivo, would not require de novo synthesis of 5 alpha-androstenone or 3 alpha-androstenol and would be consistent with utilisation by ACB of 16-androstenes already present in small quantities in fresh apocrine secretions, which are odourless, to produce a more powerfully smelling mixture on the axillary skin surface.

Synopsis We have examined the relationship between human axillary skin microflora and underarm odour (UAO), in particular, the ability of cutaneous bacteria to transform steroids. A study was made of bacterial population density and odour intensity of the axillae of 34 normal male subjects. There was a statistically significant association between population density of aerobic coryneform bacteria and UAO intensity. No associations could be found between population densities of staphylococci, micrococci or propionibacteria and UAO intensity. An in vitro model for formation of UAO was developed, and used to test individual bacterial isolates. Only aerobic coryneforms could produce axillary odour in vitro, most notably C. xerosis. Many aerobic coryneforms could transform testosterone, the principal metabolites being 5alpha- and 5beta-DHT, androstenedione, and 5alpha- and 5beta-androstanedione. UAO positive coryneforms were more metabolically active than UAO negative bacteria. Micrococci also transformed testosterone to androstenedione, whilst staphylococci and propionibacteria could not metabolize it. A hypothesis for the role of aerobic coryneforms in the formation of human axillary odour is discussed.

Stable isotope gas chromatography/mass spectrometry has been used successfully in the elucidation of structures of urinary steroid metabolites in the horse and in the identification of metabolites isolated from in vivo perfusion and in vitro incubation studies using equine tissue preparations. Deuterium-labeled steroids, testosterone, dehydroepiandrosterone, and 5-androstene-3 beta,17 beta-diol have been synthesized by base-catalyzed isotope exchange methods and the products characterized by gas chromatography/mass spectrometry. [16,16(-2)H2]Dehydroepiandrosterone (plus radiolabeled dehydroepiandrosterone) was perfused into a testicular artery of a pony stallion and was shown to be metabolized into 2H2-labeled testosterone, 4-androstenedione, isomers of 5-androstene-3,17-diol, 19-hydroxytestosterone, and 19-hydroxy-4-androstenedione. In further studies, equine testicular minces have been incubated with 2H2-labeled and radiolabeled dehydroepiandrosterone and 5-androstene-3 beta, 17 beta-diol. The metabolites, whose identity was confirmed by stable isotope gas chromatography/mass spectrometry, proved the interconversion of the two substrates, as well as formation of testosterone and 4-androstenedione. The aromatization of dehydroepiandrosterone was also confirmed, together with the formation of an isomer of 5(10)-estrene-3,17-diol from both substrates showing 19-demethylation without concomitant aromatization. In studies of the feto-placental unit, the allantochorion was shown to aromatize [2H5]testosterone to [2H4]estradiol, the loss of one 2H from the substrate being consistent with aromatization of the A ring. The formation of 6-hydroxyestradiol was also confirmed in this study. The same technique has been valuable in determining the structure of two metabolites of nandrolone isolated from horse urine.(ABSTRACT TRUNCATED AT 250 WORDS)

Three cases of gingival overgrowth induced by cyclosporin and/or nifedipine have been reported. Patient A was under medication with cyclosporin plus nifedipine, patient B with nifedipine only and patient C with cyclosporin only. The significance of androgen metabolism in gingival tissue with respect to hyperplastic changes has been studied by several workers. Hence, we have investigated whether gingival tissue from the above patients showed significant metabolism of the androgen, testosterone, to its biologically-active form, 5 alpha-dihydrotestosterone (5 alpha-DHT). Radical gingivectomy was carried out in all 3 cases to remove the hyperplastic tissue. The excised tissue was incubated with labelled testosterone in order to study the extent of androgen metabolism. Healthy gingivae from males and females produced 5 alpha-DHT (22.4 +/- 7.7, s.e.m., n = 8). Very significantly higher values (p less than 0.001) were recorded for patients A, B and C (1139, 542 and 994 fmol/mg, respectively). These represented increases of 51-, 24- and 44.4-fold, respectively over control values. Corresponding production of 4-androstenedione from testosterone was 28 +/- 8.3, s.e.m., n = 8, fmol/mg. In patients A, B and C, 4-androstenedione production was elevated: 85, 901 and 113 fmol/mg, respectively, representing increases of 3-, 32- and 4-fold. Even the lower values of 85 and 113 fmol/mg were very highly significant (p less than 0.001) compared with control values. Although healthy female gingival tissue does not metabolize testosterone significantly, in the presence of inflammation the extent of 5 alpha-DHT formation is comparable to that of male samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Three mature Large White boars were anaesthetized and received [7(n)-3H]pregnenolone by continuous infusion into right and left spermatic arteries for up to 180 min. Spermatic venous blood flow was measured by separate timed collections of completely diverted outflow from each testis and blood not sampled was returned to the peripheral circulation. The total radioactivity in plasma from each testis increased markedly during the first 60 min of infusion to reach a plateau from 80 to 180 min. Radiolabelling of 5 alpha-androst-16-en-3-one, 5 alpha-androst-16-en-3 beta-ol and -3 alpha-ol showed similar patterns with ratios of mean radioactivity of 5:3:1 respectively between 80 and 180 min. In comparison, the amounts of tritiated 4,16-androstadien-3-one formed were very small. The radiolabelling of testosterone and 4-androstenedione occurred more rapidly than that of the 16-androstenes and reached maxima by 30 min. However the amounts were only one-fifth (testosterone) and one-tenth (4-androstenedione) those of the combined quantities of tritiated 16-androstenes. Addition of human chorionic gonadotrophin (hCG) to the infusate to one testis in each animal (so that 5000 i.u. hCG were delivered in 15-20 min) produced no change in the outputs of radiolabelled steroids although radioimmunoassay of spermatic venous plasma in samples from the third experiment showed a transient increase in the concentration of 4-androstene-3,17-dione during the hCG infusion. It is suggested the lack of response to hCG could be produced by saturation and down regulation of binding sites by the very high local concentrations of hCG.

The biosynthesis of 16-androstenes has been studied in neonatal porcine testicular microsomes using 17-hydroxypregnenolone and 16-dehydropregnenolone, separately, as substrates. The metabolites formed after microsomal incubation with these substrates were purified, derivatized as O-methyloxime-trimethylsilyl ethers and analysed by capillary gas chromatography-mass spectrometry. In the incubation of 17-hydroxypregnenolone with microsomes, 16-dehydropregnenolone was identified as an intermediate in the biosynthesis of 16-androstenes. Further microsomal incubation of 16-dehydropregnenolone has established the intermediary role of this steroid in the production of 16-androstenes.