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ISI Document Delivery No.: IM1YR Times Cited: 0 Cited Reference Count: 25 Savchuk, I. Morvan, M. L. Antignac, J. P. Kurek, M. Le Bizec, B. Soder, O. Svechnikov, K. Frimurare Barnhuset Foundation; Kronprinsessan Lovisas Foundation; Sallskapet Barnavard; Stiftelsen Sigurd och Elsa Goljes Minne; Stiftelsen Olle Engkvist ByggmastareSwedish Research Council; Stiftelsen Gunvor och Josef Aners; Stiftelsen Jane och Dan Olssons; Stiftelsen Tornspiran This work supported by the Frimurare Barnhuset Foundation, Kronprinsessan Lovisas Foundation, the "Sallskapet Barnavard"; the "Stiftelsen Sigurd och Elsa Goljes Minne", the "Stiftelsen Olle Engkvist Byggmastare", the "Stiftelsen Gunvor och Josef Aners", the "Stiftelsen Jane och Dan Olssons" and the "Stiftelsen Tornspiran". 0 3 Elsevier science inc New york 1878-5867; International audience; Steroidogenic potential of the human fetal kidney (hFK) at the end of first trimester is poorly investigated. Little is known about the ontogeny of steroidogenic enzymes and activities of steroidogenic pathways in the hFK at early pregnancy. Our aim was to explore steroidogenesis and the expression of steroidogenic enzymes in the hFK at gestational weeks (GW) 9-12. Steroids in the hFK were analyzed by gas chromatography/coupled to tandem mass spectrometry. The expression of steroidogenic enzymes in the hFK at GW 9-12 was investigated by qPCR, automated Western blotting and immunohistochemistry. We observed that the hFK produced substantial amount of steroids of the Delta(5) and Delta(4) pathways and several steroid precursors in the biosynthesis of DHT via the backdoor pathway but not DHT itself. The levels of steroids and expression of relevant steroidogenic enzymes (e.g., CYP17A1, HSD3B1, HSD3B2, CYP11B1 and AKR1C4) we significantly higher in the hFK at GW11-12 compared to GW9. We also found the expression of sex steroid receptors (e.g., AR, ER alpha and ER beta) in the hFK at GW9-12. No sex dependent differences in the levels of all identified steroids and expression of steroidogenic enzymes in the hFK from male and female fetuses were found. Altogether, our data indicate that the hFK at early pregnancy is steroidogenic organ with potential to synthesize multiple steroids that may play an important role in the formation and development of this organ in humans.

International audience; The onset of steroidogenesis in human fetal testes (HFT) during the first trimester is poorly investigated. One important unresolved question is the ontogeny of steroidogenic enzymes and formation of steroidogenic pathways in the HFT at early pregnancy. Our aim was to explore steroidogenesis, the expression of steroidogenic enzymes and their maturation in the HFT at gestational weeks (GW) 8-12. Steroids in the HFT were analyzed by gas chromatography/coupled to tandem mass spectrometry. The expression of steroidogenic enzymes in the HFT at GW8-12 was investigated by qPCR, automated Western blotting and immunohistochemistry. We demonstrated that the HFT at GW8-9 produced low level of testosterone via the Delta(4) pathway and progesterone was the major steroid found in the testicular tissue. In contrast, more mature Leydig cells from the HFT at GW11-12 synthesized high levels of androgens via the Delta(5) pathway. We also observed a significant upregulation of the expression of StAR, CYP11A1, CYP17A1 and its accessory proteins, P450 oxidoreductase (POR) and cytochrome b5 in the HFT at GW11-12 compared to GW89. Altogether, our data suggest that that human fetal Leydig cells differentiate rapidly at the end of the first trimester by acquiring capacity to express high levels of steroidogenic enzymes and switch from the Delta(4) to the Delta(5) pathways to synthesize high levels of androgens due to maturation of the CYP17-POR-b5 complex.

Savchuk, I Morvan, M L Antignac, J P Gemzell-Danielsson, K Le Bizec, B Soder, O Svechnikov, K eng Ireland Mol Cell Endocrinol. 2018 Dec 5;477:148-155. doi: 10.1016/j.mce.2018.06.012. Epub 2018 Jun 19.; International audience; It is generally accepted that androgens produced by fetal Leydig cells (FLC) control proper masculinization of the male external genitalia. Here, we hypothesized that the human genital tubercle (GT) has potential to synthesize androgens independently of FLC at early pregnancy. We observed that human GT of both genders have capacity to synthesize steroids of the Delta(4), Delta(5) and alternative pathway of DHT synthesis including the androgen itself. The presence of steroids in the GT was associated with the expression of corresponding steroidogenic enzymes. Levels of steroids and the expression of steroidogenic enzymes were similar in the GT from male and female fetuses. In contrast to the GT, the human fetal testis synthesized DHT from testosterone but not via the alternative pathway. Our findings strongly suggest that the human GT at early pregnancy can synthesize DHT via the alternative pathway, which may play an important role in organogenesis of the urethra.

Obesity is a global health problem and impacts negatively on levels of testosterone and quality of sperm production. At present little is known about mechanisms that attenuate testicular function in obese males. Our study characterized testicular steroidogenesis and explored levels of relevant paracrine and hormonal factors in rats with short- and long-term obesity. We have found that obesity state increased serum levels of estradiol and leptin in both groups of obese rats and inhibited the expression of StAR and Cyp11a1 associated with low levels of intratesticular testosterone in rats with long-term obesity. Further, long-term obesity reduced the number of Leydig cells, increased the testicular levels of the proinflammatory adipocytokine TNFα and the number of testicular macrophages. All together, our data indicate that long-term obesity may cause chronic inflammation in the testis and negatively impacts on Leydig cell steroidogenesis.

Reproduction is an important target of obesity complications, including adverse effects on spermatogenesis and steroidogenesis. Adipocytokines are key mediators in various complications of obesity. Our aim was to study the potential of adipocytokines to affect Sertoli cell function, which is crucial for spermatogenesis, and possibly link these findings to the observed attenuation of spermatogenesis in obese males. Testicular biopsies were obtained from healthy donors. Highly purified adult human Sertoli cells (HSCs) were isolated by fluorescence-activated cell sorting. Cells were cultured and exposed to different concentrations of adipocytokines (10–1000ngmL−1) for 2–7 days. Expression of selected Sertoli cell genes was quantified by quantitative polymerase chain reaction. Long-term treatment (7 days) of HSCs with higher concentrations of chemerin, irisin, nicotinamide phosphoribosyltransferase (Nampt), resistin and progranulin significantly suppressed FSH receptor expression (by 79%, 83%, 64%, 71% and 26% respectively; P

Functional gonads are mandatory for sexual reproduction and survival of higher animal species. However, at the level of the individual subject, acquired or inherited gonadal dysfunction and infertility are not commonly associated with severe life-threatening phenotypes. Medical progress and increased societal interest have led to more prioritised agendas for reproductive health problems. Increasing attention is focused on disorders of sex development, fertility and sexual function. Despite this engagement, our understanding of the detailed molecular and cellular adverse events behind such problems is still incomplete. Critical early steps, such as determination of the gonads, occur at precise temporal windows of development. The sex chromosomes are obvious critical contributors, but many other human chromosomes also contribute to sex differentiation, engaging multiple genes and proteins. The aim of this review is to give an up-to-date and comprehensive summary of the events required for gonadal ontogenesis in the human male, from the stage of embryonic sex determination to postnatal maturation including puberty. The principal genes involved in these processes are tabulated and discussed. Morphological events relevant for human gonadal development are covered, in particular in connection with early germ cell maturation and spermatogenesis. Consequences of maldevelopment caused by, e.g. cryptorchidism, are discussed.

Phthalate esters are known to exert harmful effects on mammalian reproduction and fertility, but their potential adverse effects on the hormonal functions of the ovary have not yet been elucidated in detail. Here, we investigated the effects of di-(2-ethylhexyl) phthalate (DEHP) on the hypothalamic–pituitary–gonadal axis of young developing female rats, as well as on ex vivo steroidogenesis by granulosa cells (GCs) and secretion of LH by gonadotropes. Exposure of 20-day-old female rats to 500 mg DEHP by oral gavage once daily for 10 days reduced their serum levels of progesterone and estradiol, while tending to enhance levels of LH. Furthermore, primary cultures of GCs isolated from these rats exhibited an attenuated capacity to produce progesterone in response to stimulation by LH and FSH, as well as a lower degree of transport of endogenous cholesterol into mitochondria. Moreover, the abilityof primary cultures of pituitary cells isolated from DEHP-treated rats to produce and secrete LH in response to GnRH was significantly enhanced. In addition, 2-ethylhexanoic acid, a metabolite of DEHP, significantly potentiated GnRH-stimulated production of LH by cultures of pituitary cells isolated from untreated 20-day-old female rats. Together, these data indicate that DEHP exerts dual effects on the pituitary–gonadal axis, stimulating the hormonal function of the pituitary and, at the same time, by inhibiting steroidogenesis by GCs.

Aim: To reach consensus among specialists from the Nordic countries on the present state-of-the-art in treatment of undescended testicles. Methods: A group of specialists in testicular physiology, paediatric surgery/urology, endocrinology, andrology, pathology and anaesthesiology from all the Nordic countries met for two days. Before the meeting, reviews of the literature had been prepared by the participants. Recommendations: The group came to the following unanimous conclusions: (1) In general, hormonal treatment is not recommended, considering the poor immediate results and the possible long term adverse effects on spermatogenesis. Thus, surgery is to be preferred. (2) Orchiopexy should be done between 6 and 12 months of age, or upon diagnosis, if that occurs later. (3) Orchiopexy before age one year should only be done at centres with both paediatric surgeons/urologists and paediatric anaesthesiologists. (4) If a testis is found to be undescended at any age after 6 months, the patient should be referred for surgery—to paediatric rather than general surgeons/urologists if the boy is less than one year old or if he has bilateral or non-palpable testes, or if he has got relapse of cryptorchidism.

Interleukin-1α (IL-1α) plays an important role in the regulation of immune responses as well as in non-inflammatory events in different types of cells. Here we have investigated the involvement of the extracellular signal-regulated kinase (ERK) cascade in IL-1α-induced steroidogenesis by primary cultures of immature rat Leydig cells. Our findings indicate that protein kinase C functions as an upstream component of signal transduction from the IL-1 receptor type I (IL-1RI) to the ERK cascade. It was observed that IL-1α upregulated both steroidogenic acute regulatory (StAR) protein expression and its phosphorylation when compared with controls. Selective inhibition of these mitogen-activated protein kinases (MAPKs) by UO126 enhanced both the expression and phosphorylation of the StAR protein, but suppressed androgen production by the immature Leydig cells as well as dissipating the mitochondrial electrochemical potential (Ψm) in these cells. The evidence that water-soluble cholesterol but not 22R-hydroxycholesterol-stimulated steroidogenesis was inhibited by UO126 suggested that an intact Ψm across the inner mitochondrial membrane is required for cholesterol translocation and is positively regulated by the ERK cascade. We propose that activation of ERKs by IL-1α plays a dual role in the regulation of steroidogenesis in immature Leydig cells: these MAPKs downregulate StAR expression and phosphorylation, while at the same time they support an intact Ψm across the inner mitochondrial membrane, thereby promoting translocation of cholesterol into the mitochondria of the Leydig cell.

Procymidone is a fungicide with anti-androgenic properties, widely used to protect fruits from fungal infection. Thereby it contaminates fruit products prepared for human consumption. Genistein-containing soy products are increasingly used as food additives with health-promoting properties. Therefore we examined the effects of long-term dietary administration (3 months) of the anti-androgen procymidone (26.4 mg/animal per day) or the phytoestrogen genistein (21.1 mg/animal per day) to rats on the pituitary-gonadal axis in vivo, as well as on Leydig cell steroidogenesis and on spermatogenesis ex vivo. The procymidone-containing diet elevated serum levels of LH and testosterone and, furthermore, Leydig cells isolated from procymidone-treated animals displayed an enhanced capacity for producing testosterone in response to stimulation by hCG or dibutyryl cAMP, as well as elevated expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450 scc) and cytochrome P450 17α (P450c17). In contrast, the rate of DNA synthesis during stages VIII and IX of spermatogenesis in segments of seminiferous tubules isolated from genistein-treated rats was decreased without accompanying changes in the serum level of either LH or testosterone. Nonetheless, genistein did suppress the ex vivo steroidogenic response of Leydig cells to hCG or dibutyryl cAMP by down-regulating their expression of P450 scc. Considered together, our present findings demonstrate that long-term dietary administration of procymidone or genistein to rats exerts different effects on the pituitary–gonadal axis in vivo and on Leydig cell steroidogenesis ex vivo. Possibly as a result of disruption of hormonal feedback control due to its anti-androgenic action, procymidone activates this endocrine axis, thereby causing hyper-gonadotropic activation of testicular steroidogenesis. In contrast, genistein influences spermatogenesis and significantly inhibits Leydig cell steroidogenesis ex vivo without altering the serum level of either LH or testosterone.

Testes from rats of different maturational ages were explored for presence of paracrine sertoli cell growth factors. Pubertal and adult testes contained a 17 kDa protein, with potent stimulatory effect on immature Sertoli cell multiplication in vitro. The bioactivity of this protein was mimicked by rat interleukin-1 (IL-1) and neutralized by IL-1 receptor antagonist. A receptor-mediated action was further supported by the demonstration of IL-1 receptor type I mRNA and protein expression in the cultured sertoli cells and in intact immature rat testes. IL-1alpha showed higher efficacy in stimulating proliferation than IL-1beta and follicle-stimulating hormone (FSH), and displayed synergistic action in combination with FSH. As IL-1alpha is constitutively produced by the rat testis and IL-1beta readily inducible by proinflammatory stimuli, our results suggest that IL-1 may serve as a growth factor for Sertoli cells under physiological and pathophysiological conditions.

We evaluated small intestinal motility, secretion, and mucosal permeability in fasted Sprague–Dawley rats following administration of luminal Escherichia coli heatstable enterotoxin (500 ng ml −1 ) alone or in combination with intravenous endotoxin (100 μg kg −1 ). Systemic endotoxin alone, or in combination with enterotoxin, caused a disruption of the regular interdigestive myoelectric complex as well as induction of spike bursts and diarrhea. Enterotoxin alone produced only minimal effects on intestinal myoelectric activity. Exposure of the intestine to systemic endotoxin and luminal enterotoxin in combination, led to a 2-fold increase in intestinal secretion in comparison with controls. This effect was significantly greater than the increase observed with enterotoxin (64±51%, P P P

Tissue concentrations of malondialdehyde in the gastrointestinal tract of the rat were quantified as indicators of lipid peroxidation and tissue damage after challenge with tachykinins and after pretreatment with allopurinol. Neurokinin A, neurokinin B, and substance P given intravenously during 30 min increased the production of malondialdehyde in the stomach, duodenum, jejunum, and colon in a dose-dependent manner at doses from 100 to 400 pmol/kg/min (P < 0.05-0.01). However, the stomach seemed less responsive to the tachykinin challenge. For comparison, a similar dose-dependent increase of malondialdehyde was found in the liver and lung, with more pronounced effects of neurokinin B (P < 0.05-0.01). Pretreatment with allopurinol, 10 mg/kg, significantly reduced malondialdehyde responses to tachykinin challenge in intestinal tissues (P < 0.001). In conclusion, elaboration of malondialdehyde is suggested to reflect the ability of gastrointestinal tissues to react to tachykinins at high concentrations with liberation of free radicals as part of an inflammatory reaction.

Searching for useful markers of spermatogonial stem cells and their differentiation, we used rat testes from ages representing different stages of testicular maturation to investigate the expression profile of transcription factor activation protein-2γ (Ap-2γ). The immunohistochemical and immunocytochemical evaluation using Ap-2γ and promyelocytic leukemia zinc finger in combination with sorting of CD9 and CD90 positive cells (undifferentiated spermatogonia) by fluorescence-activated cell sorting was performed. Our experiments revealed that Ap-2γ is detectable in testes of late fetal age and up to 60 days postnatally and is expressed in gonocytes and spermatogonia from late fetal age throughout all maturational stages. Restricted nuclear expression of Ap-2γ to undifferentiated male germ cells was verified by coexpression of Ap-2γ with promyelocytic leukemia zinc finger in sections of paraffin-embedded testes as well as in cells sorted positive for CD9 and CD90 expression. Our study demonstrated clearly that nuclear expression of Ap-2γ is a useful marker for identifying undifferentiated male germ cells, although its functional role is yet to be fully explored.

We have investigated the proliferative response of thymocytes from different mouse strains to cytokines in vitro. Interleukin 2 (IL-2), IL-4 and IL-7 induced proliferation of thymocytes from NMRI/KI (a locally bred NMRI mouse strain), NMRI/H ('traditional' NMRI mice), C3H/HeJ and C3H/HeN mice. NMRI/KI thymocytes showed the most prominent proliferation in response to IL-1 alpha and IL-1 beta. IL-3, IL-5, IL-6, IL-8, IL-10, tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), inhibin and lipopolysaccharide (LPS) induced no thymocyte proliferation. Germfree NMRI/KI mouse thymocytes showed a significantly lower proliferation in response to IL-1 alpha and IL-1 beta than conventional mice. Rat tissues, previously shown to contain lymphocyte activating factors (LAFs), were also tested. Skin, tongue, esophagus, proventricular stomach, testis and placenta were all positive in the LAF assay utilizing NMRI/KI thymocytes, whereas none of the tissue extracts could induce proliferation in NMRI/H thymocytes. The higher cytokine responsiveness in conventional mice compared with germfree might suggest that exposure to microflora induces a higher state of activation of the immune system. The LAF assay, utilizing NMRI/KI thymocytes, is a highly sensitive IL-1 bioassay with a detection level of 1 pg/ml for IL-1 beta and 2 pg/ml for IL-1 alpha. The specificity of the assay is increased by utilizing NMRI/H mice to exclude the presence of IL-2, IL-4 and IL-7.

The in vitro effects of insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), truncated IGF-I, insulin, and human GH (hGH) on premitotic and premeiotic DNA synthesis of adult rat germ cells in vitro were investigated. Two-millimeter segments of seminiferous tubules from four different stages containing type A4-spermatogonia (stage I), type B spermatogonia (stage V), resting preleptotene spermatocytes (stage VIIa), and preleptotene spermatocytes in the S-phase (stage VIII-IX), respectively, were isolated by transillumination-assisted microdissection. They were cultured in serum-free medium at 34 or 37 C with and without growth factors, labeled for 4 h with tritiated thymidine, and harvested at 24, 48, and 72 h. Spontaneous progression of spermatogenesis was noted at both incubation temperatures, with a more rapid rate at 37 C. IGF-I significantly stimulated [3H]thymidine uptake in originally stage I and stage V tubule segments (type A4 and B spermatogonia, respectively) after 48 h of culture at 37 C. Improved maintenance of the DNA synthesis of stage VIII-IX tubules was found after 48 h at 37 C and 72 h at 34 C. Truncated IGF-I produced a similar response, but was more potent. IGF-II showed slight stimulation of stage V tubules after 72 h at both 34 and 37 C and maintenance of stage VIII-IX tubules after 48 h at 37 C and 72 h at 34 C. hGH was effective only at 34 C, showing slight stimulation of stage I tubule segments after 48 and 72 h of incubation. Insulin at high concentrations was effective only at 37 C and stimulated DNA synthesis in stages I, V, and VIIa after 48 h and stages V and VIIa after 72 h of incubation. It is concluded that IGFs stimulate premitotic DNA synthesis of rat germ cells in vitro and may also maintain premeiotic DNA synthesis. Whether the slight response to hGH is mediated via local production of IGF-I by the tissue cultures remains to be investigated. As IGF-I and IGF-II are locally produced in the testis, the present results suggest that these factors have a selective paracrine or autocrine role in the regulation of spermatogonial proliferation during spermatogenesis.

Sex steroids are crucial regulators of sexual differentiation and the proper development of secondary sex characteristics and patterns of sexual behavior. Since Leydig cells are the primary major producers of these steroid hormones, maintenance of the normal functions of these cells determines the reproductive capacity and fertility of males. The present minireview discusses recent findings concerning endocrine and paracrine regulation of the proliferation, differentiation and involution of human Leydig cells. The physiology and function of the two distinct fetal and adult populations of human Leydig cells are described, with particular focus on the paracrine environment that triggers their differentiation and functional maturation. The roles of established and more recently discovered paracrine regulators of this maturation, including insulin-like factor 3, platelet-derived growth factor-α, desert hedgehog, ghrelin and leptin are considered. A brief description of the origin, ontogenesis and functional markers of human fetal and adult Leydig cells is presented.

Levels of rat testicular interleukin-1-like factor (tIL-1) have been shown to correlate with DNA synthetic activity during the cycle of the rat seminiferous epithelium, suggesting its role as a spermatogonial or meiotic growth factor. To explore this further, a new in vitro model system was developed. Rat seminiferous tubule segments from stages I, V, Vila, and VIII IX of the cycle were isolated by transillumination-assisted microdissection, cultured in chemically defined serum-free medium supplemented with human recombinant IL-1α, and labeled with [3H]thymidine. During incubation, spontaneous progression of spermatogenesis was noted. Inactive stage Vila tubule segments differentiateddifferentiated to stage VIII and initiated DNA synthesis, and concomitantly started to secrete IL-1-like factor. DNA synthesis of stages VIII-IX ceased through differentiation of spermatocytes to leptotene- zygotene (stages XII-XIII of the cycle). IL-1α stimulated DNA synthesis significantly in spermatogonia of stage I. Meiot...

Lymphocyte activating factors (LAFs), e.g., interleukin-1 (IL-1) and IL-1-like factors, have previously been demonstrated outside the immune system in the skin, thymus epithelium, and the human and rat testis. We have studied the presence of LAFs in normal tissues of the adult rat, utilizing a highly IL-1 sensitive murine thymocyte proliferation assay. We have demonstrated high amounts of LAF activity in the tongue, esophagus, proventricular part of the stomach, and the liver. Some activity was also demonstrated in the duodenum, placenta, spleen, Peyer's patches, glandular stomach, and jejunum, but no bioactivity was present in other gastrointestinal, endocrine, lymphoid, or haematopoeitic tissues. We were also unable to detect any LAF activity in the reproductive organs (except for the testis), urinary tract, skeletal and muscular tissues, brain, eyes, salivary glands, or lung. In the esophagus the activity was mainly localized to the mucosa. The LAF activity in the skin was partly inhibited by treatment with a mixture of antibodies against human IL-1 alpha and IL-1 beta. Dose response curves and gel filtration on a Sephacryl S-200 column suggested the presence of a high molecular weight (90,000-100,000 Da) LAF inhibitory factor in the liver. In all positive tissues, the demonstrated LAFs had a molecular weight of 15,000-25,000 Da, as determined by Sephacryl S-200 gel filtration. Of the positive tissues, the skin, tongue, esophagus, and the proventricular part of the stomach all contain stratified squamous epithelium. It is tempting to suggest that the detected LAFs have a similar function in these barrier tissues, e.g., to serve as host defence factors, or, alternatively or additionally, as tissue growth factors.

Summary It has been shown previously that the intact adult rat testis produces large amounts of an interleukin-1 (IL-1)-like growth factor. The present study has investigated whether this testicular IL-1-like factor (tIL-1) is produced and secreted differentially by the fourteen stages of the seminiferous epithelial cycle in the rat testis. Seminiferous tubule segments representing defined stages were identified by tran-sillumination-assisted microscopy and isolated by microdissection. Pooled segments were either homogenized and extracted with aqueous buffer or incubated for 24 h to produce conditioned media (CM). The recovered material was then analysed for IL-1 bioactivity in a sensitive murine thymocyte proliferation assay. When divided into four stage groups, extracts of stages II-VI, IX-XII and XIII-I showed equally high IL-1 activity whereas stage group VII-VIII showed much lower activity. More detailed analysis with 10 different stage groups showed that tIL-1 activity was undetectable in extracts of substages VIIab and VIIcd. The same pattern was seen when CM from cultured tubular segments were analysed. Labelling of seminiferous tubules with tritiated thymidine in vitro and analysis by auto-radiography revealed that DNA-synthesizing spermatogonia were absent in sub-stages VIIb and VIIc and sparse in substages VIIa and VIId. The results show that tIL-1 activity is produced in a stage-dependent manner and suggest that tIL-1 might be involved in the regulation of spermatogonial proliferation in vivo.

The gene encoding choline acetyltransferase (ChAT; EC 2.3.1.6), the key enzyme in the synthesis of the neurotransmitter acetylcholine (ACh), is shown to be expressed in rat and human testes. High levels of two ChAT transcripts of 3.5 and 1.3 kilobases were detected by Northern blot analysis of adult rat testis RNA. A single ChAT mRNA species of 3.2 kilobases was detected in human testis. Cells responsible for the synthesis of ChAT mRNA in rat testis were localized by in situ hybridization in the middle part of the seminiferous epithelium, where the labeling was mostly found over spermatocytes and spermatids. Studies on the ontogeny of ChAT mRNA expression showed low levels in prepubertal rats with increasing levels as sexual maturation is reached. A peak of expression was seen at postnatal day 32, correlating with the onset of postmeiotic spermatogenesis. Results from surgical and pharmacological treatments suggest that androgens, as well as pituitary factors, could influence the relative levels of the two ChAT mRNAs detected in rat testis. Evidence for translation of the mRNA detected in the testis was obtained from the demonstration of ChAT-like immunoreactivity in ejaculated human spermatozoa. The staining was restricted to the postacrosomal region of the head, where the membrane of the sperm first fuses with that of the egg during fertilization, and to the annulus, a ring of dense material in the caudal end of the midpiece. Combined, these findings support the hypothesis that the neurotransmitter ACh is involved in reproductive function.

Neurokinin A (NKA)-immunoreactivities and substance P (SP)-immunoreactivities were found in picomolar amounts in colonic tissues and almost an order of magnitude higher amounts in vagal, pelvic, splanchnic, and lumbar colonic nerves of the cat. Continuous electric stimulation of the pelvic nerve at 4 Hz or intermittent electric burst stimulation of the pelvic nerve at 40 Hz during 1 second with 10-second rest periods produced a marked release of NKA-like immunoreactivity (NKA-LI) and SP-LI from the colon to blood (P less than 0.001). Reflex activation of the pelvic nerve by mechanical stimulation of the anus or rectal distension produced a less pronounced release of NKA-LI and SP-LI from the colon to blood (P less than 0.01). There was a simultaneous colonic contraction and vasodilation during each nerve stimulation. Reverse-phase high performance liquid chromatography showed presence of NKA, NKA oxide, NKA (3-10)/NKA (4-10), and neuropeptide K (NPK) in colonic tissues and release of all these molecular forms except NPK on nerve stimulation. Substance P and SP oxide were present both in colonic tissue extracts and in released material. Close intraarterial infusions of NKA, neurokinin B, SP, NPK, eledoisin, and physalaemin at doses of 0.1-100 pmol/min induced dose-dependent contractions of the proximal and distal colon (P less than 0.001) and vasodilatation (P less than 0.001), NKA being the most potent. The effects of the tachykinins were reduced after tetrodotoxin (P less than 0.05) and atropine (P less than 0.05) but unchanged after treatment with hexamethonium. Our findings indicate that tachykinins are released from the pelvic nerve to induce a nonadrenergic noncholinergic contraction and vasodilatation of the colon in the cat.

Aim: To correlate infant birth weight with maternal and infant biometric data, including the expression of placental IGF-I and IGF-II at birth, and levels of serum zinc and ferritin. Methods: The data consisted of observations from 89 women from Karachi, Pakistan. Placental and cord blood samples were taken immediately following delivery and were subsequently divided into two groups, small and large for gestational age (SGA and LGA). Results: The mean birth weight was 2.79 kg; the prevalence of SGA being 13.4% (≤10th percentile); the prevalence of LGA being 23.6% (≥90th percentile). Placental IGF-I and IGF-II mRNA expression was greater in the LGA group (p < 0.05). Furthermore, a significant correlation was noted between infant birth weight and maternal anthropometric parameters (p < 0.01). Cord zinc levels were also significantly higher in the LGA group (p < 0.05). Conclusion: Maternal anthropometry, along with placental IGF-I and IGF-II mRNA levels, correlated significantly with infant birth weight suggesting the importance of these growth factors for birth weight outcomes. The higher zinc levels in the LGA group also suggest the importance of this micronutrient in foetal growth. Our results suggest that growth problems have a multifactorial aetiology arising from within the infant rather than due to maternal constraint alone.

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.

During the past decade, considerable information concerning the effects of endocrine disrupters (EDCs) on animals and humans has been accumulated. These compounds of anthropogenic or natural origin mimic the action of sex hormones, thus disturbing the endocrine system. The present overview covers the different classes of EDCs, such as pesticides (that act as androgen receptor (AR) antagonists, i.e. anti-androgens), and phthalates and dioxins (which appear to inhibit fetal testosterone synthesis). The effects of these compounds on steroidogenesis by Leydig cells and reproductive development are reviewed and their possible key role in connection with increasing frequencies of abnormalities in reproductive development, such as hypospadias and cryptorchidism, is debated. Moreover, the influence of different classes of EDCs on ovarian and adrenal steroidogenesis is also described. In addition, putative interactions between EDCs and mediators of inflammation, that can potentially intensify the inflammatory process, are discussed.

Background and Aim: The phytoestrogen resveratrol is found in grapes, mulberries and peanuts, all of which are consumed regularly by humans. Resveratrol is also used in chemotherapy against cancer and aging and as a cardioprotectant. The aim of the present study was to characterize the effects of resveratrol on rat adrenal steroidogenesis and to study the underlying mechanism. Methods: Adrenocortical cells were isolated from the adrenal glands of normal male rats (in vitro) and from male rats administered resveratrol in their diet for 12 weeks (ex vivo). Cells from resveratrol-treated and non-treated rats were tested ex vivo for responsiveness to ACTH and cells from normal rats were tested in vitro for responsiveness to ACTH in the presence and absence of resveratrol. Corticosterone and progesterone production were measured by RIA and expression of steroidogenic enzymes analyzed by PAGE/Western blotting. Results: Corticosterone production was inhibited 47% by 50 µM resveratrol in vitro and 20% ex vivo, while progesterone production was elevated to 400% of the control value in in vitro experiments. Resveratrol treatment decreased adrenal cytochrome P450 c21-hydroxylase expression in vivo and cell culture conditions. No changes in cell viability or morphology were caused by exposure to resveratrol in both ex vivo and in vitro experiments. Conclusion: Resveratrol suppresses corticosterone production by primary rat adrenocortical cell cultures in vitro and ex vivo by inhibiting cytochrome P450 c21-hydroxylase.

We studied the involvement of the ERK cascade in human chorionic gonadotropin (hCG)-induced steroidogenesis by primary cultures of immature rat Leydig cells. Our findings indicate that protein kinase A and protein kinase C function as upstream kinases in connection with transduction of the signal from the gonadotropin receptor to the ERK cascade. These MAPKs enhance the stimulatory effects of hCG on the de novo synthesis of the steroidogenic acute regulatory protein and the activity of protein phosphatase 2A, which are associated with increased androgen production by the Leydig cell. Specific inhibition of ERK1/2 by Uo126 suppressed all of these cellular responses to hCG. In contrast, steroidogenesis from 22OHC (a cell-permeable form of cholesterol) is not inhibited by Uo126, suggesting that cholesterol delivery to mitochondria is being affected by this compound. We propose that the ERK cascade is an important part of the signal transduction pathway involved in the rapid hormonal responses of Leydig cells to trophic hormones. In hCG-activated Leydig cells, these MAPKs may play a role in controlling the biosynthesis of the steroidogenic acute regulatory protein as well as regulating protein phosphatase 2A activity, thereby governing cholesterol transport across the mitochondrial membrane.

We have investigated the involvement of the steroidogenic acute regulatory (StAR) protein in interleukin-1alpha (IL-1alpha)-induced steroidogenesis in immature (40-day-old) and adult Leydig cells in vitro. Further, IL-1alpha-mediated signaling pathway(s) controlling StAR expression in immature Leydig cells were also studied. IL-1alpha stimulated both androgen production and StAR protein expression in a dose- and time-dependent manner in immature but not adult Leydig cells. These effects of IL-1alpha were prevented by pretreatment of the cells with the specific inhibitors of the p38 MAP kinase, SB203580 and PD169316, suggesting that this kinase is an important part of IL-1alpha signaling in the immature Leydig cell. The present results suggest that IL-1alpha, which is constitutively produced by the rat testis from postnatal day 25, is an important paracrine regulator of postnatal Leydig cell maturation. Regulation of StAR protein expression is one of the possible mechanisms by which IL-1alpha contributes to the differentiation of immature Leydig cells into adult cells.

The number of Sertoli cells is positively correlated with the number of germ cells produced in the testis, but the regulation of Sertoli cell proliferation and final density is poorly understood. Using non-aggregated Sertoli cells from 8 to 9-day-old rat testes, highly enriched by lectin binding, we explored effects of Sertoli cell growth factor candidates in vitro. Proliferation was assessed by 3H-thymidine incorporation, bromodeoxyuridine labeling and supravital staining, and FSH was used as positive control. Transforming growth factor-alpha (TGF-alpha) was found to stimulate Sertoli cell proliferation in a dose-dependent manner. Epidermal growth factor (EGF) and betacellulin mimicked the effect, demonstrating specificity of the response as they share receptors with TGF-alpha. Insulin-like growth factor I and II, acidic and basic fibroblast growth factor and stem cell factor lacked significant stimulatory effects. We conclude that EGF/TGF-alpha is a growth factor for Sertoli cells in vitro, possibly contributing to paracrine regulation of Sertoli cell proliferation in vivo.

Interleukin-1 (IL-1), a multifunctional cytokine produced mainly by activated macrophages, is also produced in the intact testis. Rat testicular IL-1 was found to be identical to IL-1alpha, judged by immunoneutralization of the bioactive protein and sequence comparison of cloned rat testicular and macrophage pro-IL-1alpha cDNA. Testicular IL-1alpha mRNA was first demonstrated on postnatal day 15, and the corresponding bioactive protein from day 20. IL-1alpha mRNA was still low on day 20, but then increased rapidly in parallel with the bioactive protein to establish a plateau level from day 25. In adult testes, IL-1alpha mRNA and immunoreactive protein were low in stage VII of the seminiferous epithelial cycle, whereas other stages showed a clearly detectable expression. In the adult testis, the concentration of IL-1alpha was 75 pg/mg testicular protein (approximately 200 pM). In conclusion, production of testicular IL-1alpha is developmentally and stage-dependently regulated, probably at the transcriptional level, emphasizing an important paracrine role in testicular function.

A rat acute lymphoblastic leukaemia (ALL) model was used to study the mechanisms involved in the tendency to testicular relapse of ALL in boys. Previous studies have indicated that the infiltration and growth of leukaemic lymphoblasts in the testis are influenced by the same endocrine and paracrine control systems that regulate normal testicular function. In the present study the effects of aqueous extracts of scrotal, abdominal and estrogen-treated postpubertal rat testes on rat-leukaemic lymphoblast proliferation were evaluated. The effects of recombinant cytokines analogous to those observed in the testis on leukaemic cell DNA-synthesis were also evaluated since changes in the levels of these factors have been observed in association with cryptorchidism and low levels of gonadotropins. Transforming growth factor-beta 1 (TGF-beta1), significantly inhibited the proliferation of leukaemic rat lymphoblasts after 24 h of culture, whereas TGF-beta 2, interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6 or combinations of them were inactive. Extracts of estrogen-treated testes and abdominal testes of unilaterally cryptorchid animals inhibited leukaemic T-cell proliferation significantly more than extracts of normal testes. The inhibitory activity in abdominal testes could be neutralized by anti-TGF-beta 1 antibodies. These results suggest that testicular TGF-beta 1 may influence growth of leukaemic lymphoblasts in the testis but also that other as yet unknown, testicular factors are involved in the regulation of leukaemic cell function in the testis.

In the mammalian seminiferous epithelium, the cell associations follow each other in a wavelike fashion. The cell associations are characterized by constant cell composition and are identified usually by changing morphology of the acrosome system and of the nucleus in the early spermatids. Cells at DNA synthesis—that is, the six different classes of spermatogonia and preleptotene spermatocytes—are also accurately localized in defined stages of the cycle of the seminiferous epithelium.

The potential role of transforming growth factor beta (TGF beta) in the regulation of the immunological milieu of the testis was investigated. Antibodies neutralizing TGF beta reversed the previously observed suppression of rat peripheral blood lymphocyte proliferation induced by rat abdominal testis extract. Recombinant TGF beta 1 dose-dependently inhibited testicular interleukin-1-like factor-driven proliferation of murine thymocytes and ConA-stimulated rat peripheral blood mononuclear cells. Extracts of seminiferous tubules contained a M(r) approximately 25 K TGF beta-like growth inhibitor of the CLL-64 mink lung epithelial cell line. The present findings suggest an important role for TGF beta in testicular immunosuppression.

Summary Stage-specific DNA synthesis and interleukin-1 (IL-1) bioactivity were measured in the rat testis at 2, 6, 12 and 25 days after local irradiation with 3 Gy to investigate whether there was any correlation between germ cell DNA synthesis during repopulation and IL-1-like bioactivity in the seminiferous epithelium. DNA synthesis by intermediate and type-B spermatogonia was reduced significantly at 2, 6 and 12 days after irradiation in seminiferous tubules at stages II-III and IV-V. IL-1 bioactivity was increased significantly at 6 and 25 days after irradiation at stages II-VI during repopulation of spermatogonia. At 2, 6 and 25 days after irradiation at stages VIIa-c a significant increase in IL-1 bioactivity was observed that correlated with repair synthesis of DNA. Increased IL-1 bioactivity was also observed at stages IX-XII at 6 days post-irradiation. These observations support the concept that IL-1 is a spermatogonial growth factor which might also stimulate repair synthesis of DNA.

Rat seminiferous tubule segments in defined stages of the epithelial cycle were isolated by transillumination-assisted microdissection. The segments were cultured together with ConA-stimulated peripheral blood lymphocytes (PBL) and incorporation of 3H-labelled thymidine was measured. Tubule segments in stages II-VIII of the seminiferous epithelial cycle inhibited PBL proliferation significantly more than stages IX-I. Inhibition was lowest in stages IX-XII and increased progressively to reach a maximum in stages II-VIII. In a more detailed analysis, tubules in stages V and VI inhibited PBL proliferation significantly less than stage II tubules. No significant difference was observed between stages II and VII. The immunosuppressive activity had molecular weights of approximately 25 kDa and approximately 65 kDa in stage II-VIII seminiferous tubules. In stage II-VI seminiferous tubules activity was present also at approximately 10 kDa. The results suggest that the seminiferous tubules produce high-molecular weight immunosuppressive activity in a stage-dependent way. In addition to its contribution to the immunologically privileged milieu of the testis this activity may also be involved in the physiological regulation of DNA synthesis in the seminiferous epithelium.

Both intrinsic and extrinsic neurons of the gut respond to mechanical and chemical stimuli by the release of neurotransmitters. We summarize here some of our recent work on the role of vasoactive intestinal polypeptide (VIP), substance P (SP) and neurokinin A (NKA) in the secretory, motor and vascular effects of hydrochloric acid stimulation in the isolated rat duodenal loop and electrical nerve stimulation and mechanical stimulation of the cat colon. Isolated duodenal loops of conscious rats were perfused with isotonic saline, and challenged at hourly intervals with brief exposures to increasing concentrations of HCL. The concentrations of bicarbonate and prostaglandin E2 (PGE2) released from the duodenal mucosa were significantly augmented already by pH 5.0 whereas VIP was significantly augmented at pH 3.0 and the tachykinins SP and NKA at pH 2.0. Continuous electric stimulation of the pelvic nerve in cats at 4 Hz during 1 s with 10 s rest produced a marked release of NKA-LI and SP-LI from the colon to blood. Reflex activation of the pelvic nervae by mechanical stimulation of the anus or rectal distension produced a less pronounced release of NKA-LI and SP-LI from the colon to blood. There was a simultaneous colonic contraction and vasodilation during each nerve stimulation. Close intraarterial infusions of NKA, neurokinin B, SP, neuropeptide K (NPK), eledoisin and physalemin at doses of 0.1–100 pmol/min induced dosedependent proximal and distal colonic contractions and vasodilation, NKA being the most potent. The effects of the tachykinins were reduced after tetrodotoxin and atropine, but unchanged after treatment with hexamethonium. Taken together, the present findings indicate that peptides present in primary afferents including VIP, SP, and NKA are involved in physiological regulatory mechanisms including smooth muscle contraction, bicarbonate secretion and vasodilation in the gut.

The present data indicate that immune cells are regulated locally in the testis by Leydig cells, Sertoli cells and resident testicular macrophages. The effects of these cells are mediated by several peptide factors, including protectin, a group of high molecular weight testicular immunosuppressive factors, and testicular interleukin-1 alpha-like factor. The testicular interleukin-1 alpha-like factor is produced by Sertoli cells and is under hypophyseal control. Its synthesis starts at puberty concomitantly with the onset of spermatogenesis and it may act as a spermatogonial growth factor. Protectin, which is under hypophyseal control, may be involved in the mechanism of prolonged transplant survival in the testicular interstitial tissue. Its levels increase at puberty. Both the testicular interleukin-1 alpha-like activity and protectin may be important in testicular pathophysiology.

The testicular cells are regulated by factors produced locally in the testis. These factors include peptide growth factors, pro-opiomelanocortin derivatives, neuropeptides and steroids. Several agents able to affect steroido- and spermatogenesis can also affect leukocytes and many of the testis-regulating factors are produced by immune cells, suggesting that testicular cells and leukocytes may interact. In the present article, the effects of various testicular cell and leukocyte produced factors on steroido- and spermatogenesis are reviewed. The possibility that leukocytes may produce substances able to affect the testicular functions suggests that inhibition of immune system activation in the testis may be important also for reasons other than protection of autoantigenic germ cells from an autoimmune attack.

It has been suggested that tumor necrosis factor alpha (TNF alpha) may serve as an important antigen-independent host defense mechanism against parasitic organisms. Sera from 66 patients with leishmaniasis and 68 patients with leprosy, all from Ethiopia, were tested for TNF alpha using an enzyme-linked immunoassay. Sera from patients with the multi-parasitic/bacillary type of disease (visceral or diffuse cutaneous leishmaniasis and lepromatous leprosy), known to be associated with absent or low specific T cell response, contained significantly higher TNF alpha titers than those of patients with pauci-parasitic/bacillary disease (localized cutaneous leishmaniasis and nonlepromatous leprosy). High titers of TNF alpha in the absence of a functioning T cell response do not appear to confer resistance against Leishmania aethiopica and Mycobacterium leprae.

A number of inflammatory mediators and cytokines were injected locally into the testis of adult rats in order to test their ability to induce leukocyte accumulation and increased vascular permeability (as studied by a carbon labelling technique). Human recombinant interleukin-1 beta (IL-1 beta) caused increased vascular permeability and leukocyte migration. All the other factors studied--histamine, serotonin, bradykinin, interleukin-1 alpha (IL-1 alpha), and a partially purified interleukin-1 alpha-like factor (tIL-1) from rat testis--did not induce any acute signs of increased vascular permeability or inflammatory response after local injection. It is suggested that local production of IL-1 beta from testicular macrophages could be responsible for the inflammation-like changes that are seen in rat testes after treatment with human chorionic gonadotrophin.

Nerve growth factor (NGF) is synthesized in male germ cells. The NGF receptor (NGFR) mRNA was found in the Sertoli cells of rat testis. Hypophysectomy increased both NGFR mRNA in testis and the number of NGFR hybridizing cells in seminiferous tubules. This was suppressed by treatment with chorionic gonadotropin or testosterone, but not with follicle-stimulating hormone. The NGFR mRNA also increased after destruction of Leydig cells or blocking of the androgen receptor. This suggests that NGF produced by male germ cells regulates testicular function in an androgen-modulated fashion by mediating an interaction germ and Sertoli cells.

We have established a testis tissue culture model which enables studies of the effects of growth factors on germ cell proliferation in vitro. Segments of seminiferous tubules of adult rat testes representing defined stages of the seminiferous epithelial cycle with representative premitotic and premeiotic germ cells were identified by transillumination and prepared by microdissection. The tubule segments were incubated at 34°and 37°C for 24, 48, and 72 h with and without growth factors and the DNA synthesis was determined at the end of the incubation. Recombinant epidermal growth factor (EGF) was found to stimulate premeiotic DNA synthesis in a dose-dependent fashion whereas premitotic DNA synthesis was only slightly affected. This indicates that EGF is mainly a meitotic growth factor during spermatogenesis. Together with previous results demonstrating specific effects of interleukin-1α, nerve growth factor and insulin-like growth factors, the present finding form the basis of a hypothesis that mitotic and meiotic proliferation of germ cells is differentially regulated by locally produced growth factors acting in a paracrine fashion.

A method enabling the direct assay in a cell culture system of material separated by thinlayer chromatography (TLC) without prior elution of active material from the TLC sheet is described. After chromatographic development on a plastic cellulose sheet, small disks were punched out from the sheet and transferred to microwells used for cell culture. Lymphoid cells in culture medium were incubated with the disks and the DNA synthesis was measured by [3H]thymidine incorporation. The reliability of the method was tested with l -alanine and thymidine, which are known to enhance the DNA synthesis of lymphoid cells and to interfere with the uptake of labeled thymidine, respectively. The incorporation of [3H]thymidine was stimulated with disks containing l -alanine and inhibited with disks containing unlabeled thymidine, completely in agreement with the expected results. Elution experiments showed that more than 75% of l -alanine and thymidine on the TLC disks was eluted into the culture medium within 1 h. Elution of material of higher molecular weight was also demonstrated. The method was used for the separation and assay of a thymocyte-specific growth factor isolated from calf thymus and greatly facilitated the detection of the active material after TLC.

A growth factor for cultured guinea pig thymocytes was isolated from an extract of calf thymus. This thymocyte growth factor (TGF) was purified to apparent homogeneity employing reverse phase high performance liquid chromatography as the final step. TGF was characterized as an N-terminal blocked hydrophilic peptide with an apparent Mr of 1600. The amino acid composition revealed a high content of acidic amino acids but no apparent relationship with previously defined growth factors and thymic differentiation hormones. TGF was active in nanomolar concentration and stimulated the DNA synthesis and mitotic activity specifically in a subpopulation of thymocytes with immature cortical phenotype. The responsive thymocytes were recruited from G1 into S phase within 2 h after addition of TGF. In the absence of the growth factor, the target cells were blocked at a putative restriction point in G1, 1 1/2 h prior to the onset of the S phase. It is proposed that TGF is a progression growth factor participating in the regulation of the intense proliferation of immature thymocytes in the thymus cortex in vivo.

A method to detect single tumor necrosis factor alpha (TNF alpha)-producing cells, based on immunofluorescence staining, is presented. We have developed a rapid and simple procedure to permeabilize blood leukocytes for antibodies without causing cell aggregation. Using monoclonal as well as polyclonal TNF alpha-specific antibodies cytoplasmic TNF alpha could be detected in lipopolysaccharide (LPS)-stimulated monocytes in identical ways. In addition, one out of 15 tested fresh blood samples from healthy blood donors contained occasional TNF alpha-producing cells. We found a well-defined staining of the intracellular TNF alpha with a local, polar accumulation in a juxtanuclear position of the cell. This finding most probably indicated the presence of the monokine in the Golgi organelle because the sequential staining for TNF alpha and the Golgi zone by specific antibodies coincided. Thus, we believe the TNF alpha accumulation at this site reflects the production rather than cellular uptake of the cytokine, since the incubation of blood leukocytes with recombinant TNF alpha did not lead to any detectable staining. By performing two-color staining of cell surface antigens and cytoplasmic TNF alpha of LPS-stimulated blood leukocytes we could demonstrate that monocytes exclusively contributed to the TNF alpha synthesis. At the peak of the response, which occurred 2-3 h after LPS exposure, 50-60% of the blood monocytes produced TNF alpha. We noted a rapid decline in the number of TNF alpha-producing cells already 6 h after initiation of these cultures.

Testicular interstitial fluid (ISF) was collected by in vivo perfusion of rat testes and analyzed for the presence of interleukin-1 (IL-1) activity by utilizing a murine thymocyte proliferation assay. IS obtained from nine rats were all positive with dose-response curves of IL-1 activity similar to those produced by rat testicular aqueous extracts, rat macrophage IL-1 and human recombinant IL-1 alpha. Chromato-focusing of pooled ISF revealed a single peak of IL-1 activity with an estimated isoelectric point of 6.1-6.3. HPLC size exclusion chromatography demonstrated two active peaks with apparent molecular ratios Mr of 15,000-18,000 and 5000-7000, respectively. The molecular properties of the 15,000-18,000 Mr component are very similar to those of an IL-1-like factor previously isolated from seminiferous tubules. Our results indicate that the testicular IL-1-like factor is secreted by the seminiferous tubules into the interstitial tissue. Its function in the testicular interstitium is unknown but it might be relevant for the tendency to testicular relapse of childhood lymphocytic leukemia.

A thymic peptide previously found to recruit thymocytes from G1 into S phase has been purified from a crude thymic extract by subsequent steps of gel exclusion chromatography and reverse phase high performance liquid chromatography (HPLC). The purified material, which appeared homogeneous on thin-layer chromatography and HPLC, stimulated the DNA synthesis of cultured guinea pig thymocytes in a nanomolar concentration range. The amino acid composition revealed a high content of acidic amino acids and no apparent homology to previously defined growth factors and thymus differentiation hormones.

Thymocytes were separated according to increasing buoyant density into the three subpopulations la (25% of recovered cells), lb (20%) and II (55%), and according to binding to peanut agglutinin (PNA) into PNA+ (65 %) and PNA– cells (35 %). The frequency of PNA+ was 56% in la, 60% in lb and 66% in population II. Electronic cell volume determinations disclosed mean volumes of 160 fl for la, 130 fl for lb and 100 fl for population II. PNA+ and PNA– cells were very similar as regards cell volume. Thus, PNA+ and PNA– cells are remarkably uniformly distributed among cell categories of different density and cell volume. The rapidly cycling thymocytes, regarded as the most immature cells in the thymus, and the target cells for a thymocyte growth factor both belonged to the PNA+ cells of population la. The mitogen-responsive thymocytes also belonged to population la, but were PNA-. The largest subpopulation of thymocytes, apparently corresponding to the small, non-cycling cortical cells, were recovered as PNA+ cells of population II.

The intrathymic location of cells responding to the thymocyte growth peptide (TGP) was investigated in guinea pigs. Thymus cells were labelled by topical application of a fluorescein isothiocyanate solution, and the percentage of fluorescent cells analyzed by flow cytometry. Large cells forming rosettes with rabbit erythrocytes, known to respond to TGP, had a lower percentage of fluorescent cells than non-rosetting cells, which do not respond to TGP. If the TGP-responding cells are anatomically segregated in the guinea pig thymus, this might indicate that they are preferentially located in the deep, juxtamedullary part of the cortex, where one major proliferating compartment is situated.