Spermatogonial quantity in human prepubertal testicular tissue collected for fertility preservation prior to potentially sterilizing therapy.

STUDY QUESTION: Does chemotherapy exposure (with or without alkylating agents) or primary diagnosis affect spermatogonial quantity in human prepubertal testicular tissue? SUMMARY ANSWER: Spermatogonial quantity is significantly reduced in testes of prepubertal boys treated with alkylating agent therapies or with hydroxyurea for sickle cell disease. WHAT IS KNOWN ALREADY: Cryopreservation of spermatogonial stem cells, followed by transplantation into the testis after treatment, is a proposed clinical option for fertility restoration in children. The key clinical consideration behind this approach is a sufficient quantity of healthy cryopreserved spermatogonia. However, since most boys with malignancies start therapy with agents that are not potentially sterilizing, they will have already received some chemotherapy before testicular tissue cryopreservation is considered. STUDY DESIGN, SIZE, DURATION: We examined histological sections of prepubertal testicular tissue to elucidate whether chemotherapy exposure or primary diagnosis affects spermatogonial quantity. Quantity of spermatogonia per transverse tubular cross-section (S/T) was assessed in relation to treatment characteristics and normative reference values in histological sections of paraffin embedded testicular tissue samples collected from 32 consecutive boy patients (aged 6.3 ± 3.8 [mean ± SD] years) between 2014 and 2017, as part of the NORDFERTIL study, and in 14 control samples (from boys aged 5.6 ± 5.0 [mean ± SD] years) from an internal biobank. PARTICIPANTS/MATERIALS, SETTING, METHODS: Prepubertal boys in Sweden, Finland and Iceland who were facing treatments associated with a very high risk of infertility, were offered the experimental procedure of testicular cryopreservation. Exclusion criteria were testicular volumes >10 ml and high bleeding or infection risk. There were 18 patients with a diagnosis of malignancy and 14 patients a non-malignant diagnosis. While 20 patients had the testicular biopsy performed 1-45 days after chemotherapy, 12 patients had not received any chemotherapy. In addition, 14 testicular tissue samples of patients with no reported testicular pathology, obtained from the internal biobank of the Department of Pathology at Karolinska University Hospital, were included as control samples in addition to reference values obtained from a recently published meta-analysis. The quantity of spermatogonia was assessed by both morphological and immunohistochemical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: The main finding was a significant reduction in spermatogonial cell counts in boys treated with alkylating agents or with hydroxyurea for sickle cell disease. The mean S/T values in boys exposed to alkylating agents (0.2 ± 0.3, n = 6) or in boys with sickle cell disease and exposed to hydroxyurea (0.3 ± 0.6, n = 6) were significantly lower (P = 0.003 and P = 0.008, respectively) than in a group exposed to non-alkylating agents or in biobank control samples (1.7 ± 1.0, n = 8 and 4.1 ± 4.6, n = 14, respectively). The mean S/T values of the testicular tissue samples included in the biobank control group and the patient group exposed to non-alkylating agents were within recently published normative reference values. LIMITATIONS, REASONS FOR CAUTION: Normal testicular tissue samples included in this study were obtained from the internal biobank of Karolinska University Hospital. Samples were considered normal and included in the study if no testicular pathology was reported in the analysed samples. However, detailed information regarding previous medical treatments and testicular volumes of patients included in this biobank were not available. WIDER IMPLICATIONS OF THE FINDINGS: This study summarizes, for the first time, spermatogonial quantity in a prepubertal patient cohort just before and after potentially sterilizing treatments. Boys facing cancer and cytotoxic therapies are regarded as the major group who will benefit from novel fertility preservation techniques. There are no previous reports correlating spermatogonial quantity to cumulative exposure to alkylating agents and anthracyclines (non-alkylating agents) and no information about the timing of cytotoxic exposures among this particular patient cohort. For prepubertal boys in whom fertility preservation is indicated, testicular tissue should be obtained before initiation of chemotherapy with alkylating agents, whilst for those with sickle cell disease and treated with hydroxyurea, this approach to fertility preservation may not be feasible. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from The Swedish Childhood Cancer Foundation (PR2016-0124; TJ2016-0093; PR2015-0073, TJ2015-0046) (J.-B.S. and K.J.), the Jane and Dan Olssons Foundation (2016-33) (J.-B.S.), the Finnish Cancer Society (K.J.), the Foundation for Paediatric Research (J.-B.S.), Kronprinsessan Lovisas Förening För Barnasjukvård/ Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation (J.-B.S.), the Väre Foundation for Paediatric Cancer Research (K.J.) and the Swedish Research Council (2012-6352) (O.S.). R.T.M. was supported by a Wellcome Trust Fellowship (09822). J.P.A.-L. and M.K. were supported by the ITN Marie Curie program 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Clinical and biological markers of premature aging after autologous SCT in childhood cancer.

The aim of this study was to analyze the prevalence of frailty and physical health limitations among long-term survivors of high-risk neuroblastoma (HR NBL) and to investigate whether frail health is associated with variables of cardiovascular function, markers of inflammation and telomere length. A national study cohort of 19 (median age 22, range 16-30 years) long-term (>10 years) HR NBL survivors was studied and the findings were compared with 20 age- and sex-matched controls. Frailty was defined as ⩾3 of the following conditions: low muscle mass, low energy expenditure, slow running and weakness. The prevalence of frailty was significantly higher among the HR NBL survivors 9/19 (47%) than among the controls (0%). Thirteen (68%) of the survivors reported significant physical health limitations in vigorous activities, as opposed to none of the controls. The HR NBL survivors had significantly shorter telomere length and higher serum levels of high sensitivity C-reactive protein than did the controls. Frail health and poor physical functioning are prevalent among HR NBL survivors and suggest premature aging. Survivors with gonadal damage, very low fat mass percentage, low glycosylated hemoglobin A1c and increased common carotid artery intima-media thickness may be more prone to early aging after high dose therapy.

Prepubertal onset of obesity negatively impacts on testicular steroidogenesis in rats.

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.

In vitro differentiation of rat spermatogonia into round spermatids in tissue culture.

STUDY QUESTION: Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? SUMMARY ANSWER: We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. WHAT IS KNOWN ALREADY: Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. LIMITATIONS, REASONS FOR CAUTION: The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. WIDER IMPLICATIONS OF THE FINDINGS: The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests.

Insulin-Like Peptide 5 Interacts with Sex Hormones and Metabolic Parameters in a Gender and Adiposity Dependent Manner in Humans.

Insulin-like peptide 5 (INSL5) is a gut hormone produced by L-cells in the colorectal epithelium and may play a role in the regulation of metabolic processes. The biological role of INSL5 is poorly investigated and nothing is known about the role of this hormone in obese and lean humans. Two cohorts were analyzed in the study. In the first cohort (n=76) the relationship between serum levels of INSL5 and different metabolic and hormonal parameters in obese and lean men and women were investigated. In the second cohort 14 male subjects underwent bariatric surgery. Circulating levels of INSL5 were then measured before and after interventions.We report for the first time that circulating INSL5 interacts with multiple metabolic and hormonal variables in lean and obese men and women and is affected by bariatric surgery. Serum levels of INSL5 negatively correlated with testosterone and blood lipids but positively with cortisol in obese men. In contrast to males, obese women had a strong negative correlation of plasma levels of INSL5 with C-reactive protein (CRP). We observed that adipose tissue loss after bariatric surgery significantly reduced serum levels of INSL5 in obese men with and without Type 2 Diabetes Mellitus (T2DM) that was associated with the restoration of circulating levels of testosterone. All together, our data demonstrated that INSL5 may interact with some metabolic parameters in obese humans and this process is dependent of gender and obesity state.

Derivation and characterization of a cytocompatible scaffold from human testis.

STUDY QUESTION: Is it possible to derive a scaffold from human testis for the purpose of tissue engineering and regenerative medicine? SUMMARY ANSWER: We developed a method to produce a cytocompatible decellularized testicular matrix (DTM) while maintaining the native tissue-specific characteristics and components. WHAT IS KNOWN ALREADY: The potential benefits of tissue-specific scaffolds consisting of naturally-derived extracellular matrix (ECM) have been demonstrated using a wide variety of animal and human tissue sources. However, so far, testis scaffolds have never been considered for constructive remodelling purposes. STUDY DESIGN, SIZE, DURATION: Human cadaveric testicular tissue was exposed for 24 or 48 h to 1% Triton X-100 and/or 1% sodium dodecyl sulphate (SDS). Acellular samples were used for further scaffold characterization purposes. PARTICIPANTS/MATERIALS, SETTING, METHODS: The extent of decellularization was evaluated by histology. Confirmation of cell removal in DTM was done by a DNA quantification technique. Retention of testicular tissue-specific characteristics was evaluated by mass spectrometry, immunohistochemistry, Alcian blue staining and scanning electron microscopy. Soluble toxicity and testicular cell attachment was assessed to check the cytocompatibility of DTM scaffolds. MAIN RESULTS AND THE ROLE OF CHANCE: Histological analysis showed that DTM could be obtained by mechanical agitation in 1% SDS for 24 h. The resulting DTM was found to be clear of cells while retaining the typical three-dimensional structure and the major components of the native tissue scaffold, including collagen type I and IV, fibronectin, laminin and glycosaminoglycans. In addition, using proteomic analysis, we revealed numerous additional ECM proteins in DTM, indicating its complex nature. The mass spectrometry data were deposited to the ProteomeXchange with identifier PXD001524. Importantly, we demonstrated that DTM scaffolds are not cytotoxic, as evidenced by MTT assay not showing an aberrant fibroblast proliferation activity after indirect exposure, and support testicular cell attachment and infiltration. LIMITATIONS, REASONS FOR CAUTION: The functionality of human testicular cells in DTM needs to be investigated. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that the insights into the molecular composition of the testicular ECM provide new clues for the unravelling of its important yet poorly understood role in regulating testicular function, and DTM-based bioscaffolds are promising components for the development of human in vitro spermatogenesis as a treatment for various types of male fertility disorders.

Boys with undescended testes: endocrine, volumetric and morphometric studies on testicular function before and after orchidopexy at nine months or three years of age.

CONTEXT: A randomized controlled study was conducted comparing the outcome of surgery for congenital cryptorchidism at 9 months or 3 yr of age. OBJECTIVE: The aim of the study was to investigate whether surgery at 9 months is more beneficial than at 3 yr and to identify early endocrine markers of importance for testicular development. PATIENTS AND METHODS: A total of 213 biopsies were taken at orchidopexy, and the number of germ and Sertoli cells per 100 seminiferous cord cross-sections and the surface area of seminiferous tubules and interstitial tissue were analyzed. Inhibin B, FSH, LH, and testosterone were determined. Testicular volume was assessed by ultrasonography and by a ruler. RESULTS: The number of germ and Sertoli cells and testicular volume at 9 months were significantly larger than at 3 yr. The intraabdominal testes showed the largest germ cell depletion at 3 yr. At both ages, testicular volume correlated to the number of germ and Sertoli cells. None of the hormones measured during the first 6 months of life (LH, FSH, testosterone, and inhibin B) could predict the number of germ or Sertoli cells at either 9 or 36 months of age, nor could hormone levels predict whether spontaneous descent would occur or not. CONCLUSION: Morphometric and volumetric data show that orchidopexy at 9 months is more beneficial for testicular development than an operation at 3 yr of age. Testicular volume was furthermore shown to reflect germ cell numbers in early childhood, whereas endocrine parameters could not predict cellular structure of the testis or its spontaneous descent.

Ontogenesis of Ap-2γ expression in rat testes.

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.

Placental IGF-I, estrogen receptor, and progesterone receptor expression, and maternal anthropometry in growth-restricted pregnancies in the Swedish population.

BACKGROUND/AIMS: Fetal growth restriction is a complex problem of pregnancy arising from multiple etiologies. Key regulatory elements of growth are the insulin-like growth factor (IGF) axis, and estrogen and progesterone receptors. The aims were to determine the relations of expression of IGF-I, estrogen receptors α and ß (ERα and ERß, respectively), and progesterone receptor (PR), with maternal anthropometry, focusing on birth weight outcomes. METHODS: Placental samples were obtained from 33 patients following delivery. mRNA expression was determined by a solution hybridization technique. Samples were divided into normal control (NC) and growth-restricted (GR) groups. RESULTS: IGF-I expression was lower in the GR as compared to the NC group. PR levels correlated positively with IGF-I expression, infant anthropometry, and gestational age (GR). ERα correlated positively with PR expression (NC), and maternal BMI at delivery (GR). ERß correlated positively with maternal delivery weight and gestational age (NC). CONCLUSION: The differences in placental expression of IGF-I emphasize its key role in birth weight outcomes. We further suggest the importance of PR expression in the pathogenesis of intrauterine growth restriction, as there were direct correlations of PR expression with both IGF-I expression and infant anthropometric parameters, as well as gestational age.

The influence of di-(2-ethylhexyl) phthalate on steroidogenesis by the ovarian granulosa cells of immature female rats.

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 ability of 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.

Nordic consensus on treatment of undescended testes.

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.

Induction of steroidogenesis in immature rat Leydig cells by interleukin-1alpha is dependent on extracellular signal-regulated kinases.

Interleukin-1alpha (IL-1alpha) 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-1alpha-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-1alpha 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 (Psim) in these cells. The evidence that water-soluble cholesterol but not 22R-hydroxycholesterol-stimulated steroidogenesis was inhibited by UO126 suggested that an intact Psim 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-1alpha 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 Psim across the inner mitochondrial membrane, thereby promoting translocation of cholesterol into the mitochondria of the Leydig cell.

Phytoestrogen resveratrol suppresses steroidogenesis by rat adrenocortical cells by inhibiting cytochrome P450 c21-hydroxylase.

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 microM 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.

Extracellular signal-regulated kinases are involved in the acute activation of steroidogenesis in immature rat Leydig cells by human chorionic gonadotropin.

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.

Molecular identity, expression and functional analysis of interleukin-1alpha and its isoforms in rat testis.

Interleukin-1alpha (IL-1alpha) is a proinflammatory cytokine that has also been found to act as a paracrine mediator involved in the regulation of testicular functions. The present review provides an overview of the role of IL-1alpha in testicular physiology. Bioactive IL-1alpha isolated from adult rat testis was found to consist of three distinct immunoreactive protein species with apparent sizes of 45, 24 and 19 kDa. These isoforms showed bioactivity in a thymocyte proliferation and steroidogenesis assays with different biopotencies. The background of the molecular heterogeneity and processing, secretion and regulation of the isoforms of testicular IL-1alpha are discussed. All three isoforms have been found to be secreted into the testis tubular lumen and interstitial space. We have provided evidence that IL-1alpha is a paracrine factor that may be of importance in, e.g., the regulation of Leydig cell steroidogenesis. Pathophysiologically, testicular IL-1alpha may contribute to testicular relapse of acute lymphocytic leukemia in boys.

Inhibition of macrophage proinflammatory cytokine expression by steroids and recombinant IL-10.

Chronic lung disease (CLD) of prematurity is a prolonged respiratory failure in very-low-birth-weight neonates. Proinflammatory cytokines have been implicated in the development of CLD. Steroids have been shown to produce some improvement in neonates with this disease. The purpose of this study was to evaluate the downregulation of these proinflammatory cytokines by dexamethasone, budesonide and recombinant IL-10 (rIL-10) in order to elucidate the mechanism of the clinical benefit of steroids in babies. Our results showed that dexamethasone, budesonide and rIL-10 significantly inhibited both IL-6 and TNF-alpha production in the THP-1 cell line stimulated by lipopolysaccharide and Ureaplasma urealyticum antigen. Similar effects were found in macrophages from tracheobronchial aspirate fluid from newborn infants. In the rat alveolar macrophage cell line, steroids inhibited IL-6 and TNF-alpha production, while rat rIL-10 did not significantly decrease production. In conclusion, steroids and human rIL-10 were able to downregulate proinflammatory cytokine production, which may explain the beneficial effect of steroids and suggests that rIL-10 could be tried as an anti-inflammatory agent in neonates with a high risk of CLD.

Amifostine protects against early but not late toxic effects of doxorubicin in infant rats.

The improved prognosis and increased expected lifetime among long-term survivors of childhood malignancies have made these patients especially sensitiveto the late toxicity of cancer therapy and prone to secondary malignancies. Recently, new strategies aiming to protect against cancer treatment toxicity have been developed, including the drug amifostine (Ethyol), which is suggested to protect normal tissues from the toxic effects of radiation and cytotoxic agents. In the present study, the possible protective effect of amifostine against toxicity induced by a single injection of doxorubicin (3 mg/kg) in immature rats was evaluated. Specifically, we evaluated the protection against long-term toxicity and the effects of amifostine on growing immature tissues. Amifostine (50-200 mg/kg) given 15 min before doxorubicin had a significant protective effect against doxorubicin-induced early alopecia in young rats. Significant protection against cataract formation was obtained by the use of low-dose amifostine (50 mg/kg). However, amifostine did not protect young rats against the late toxic effect of doxoubicin on linear growth, body weight, plasma leptin levels, and heart or testicular tissue. Worrisome, and in contrast to earlier studies in adult rats, an increased doxorubicin toxicity actually was observed and mortality was increased when the higher doses of amifostine (100-200 mg/kg) were used. The present results suggest that more data from growing immature animal models are needed to analyze the safety of amifostine treatment and its mechanisms of action before wider clinical use of this drug in pediatric cancer patients is recommended.

Age-dependent stimulation of Leydig cell steroidogenesis by interleukin-1 isoforms.

Different isoforms of testicular interleukin-1 (IL-1) were analysed to determine whether there were differences in the ability to modulate rat Leydig cell steroidogenesis in vitro. Rat 17K IL-1alpha and IL-1beta, 32K IL-1alpha precursor (32proIL-1alpha) and a 24K splice variant (24proIL-1alpha) stimulated testosterone production by Leydig cells from 40- but not 80-day-old rats. The potency of the isoforms was IL-1alpha>IL-1beta>32proIL-1alpha>24proIL-1alpha, IL-1alpha being 50-fold more potent than IL-1beta. IL-1 receptor antagonist reversed the effects and IL-1 receptor type I mRNA was expressed by the responding Leydig cells, indicating a receptor mediated action. Inhibition of PKA and Ca(2+) channels abolished IL-1-induced steroidogenesis, while inhibition of PKC had no significant effect. Except for 24proIL-1alpha which was stimulatory, all IL-1 isoforms suppressed hCG-driven testosterone production. This inhibitory effect was abolished by androstendione, suggesting that P450c17 was suppressed by IL-1. Our results indicate that IL-1 plays a paracrine role in the regulation of Leydig cell steroidogenesis.

Endotoxin-induced interleukin 1 expression in testicular macrophages is accompanied by downregulation of the constitutive expression in Sertoli cells.

Interleukin-1alpha (IL-1alpha) is constitutively produced by Sertoli cells in adult rat testes. We demonstrate here that adult rats initiate expression of IL-1alpha and IL-1beta in testicular macrophages and decrease plasma testosterone by 60%, 2 h after administration of endotoxin. The macrophage activation was accompanied by downregulation of IL-1alpha mRNA expression in Sertoli cells. Despite increased tissue concentrations of IL-1alpha and IL-1beta immunoreactive protein, the level of bioactive IL-1 in the testis remained unchanged. Testes from prepubertal rats responded similarly to endotoxin, but lacked constitutive expression of IL-1alpha. We conclude that endotoxin-induced inflammation involves the testis by local macrophage activation and cytokine secretion. The paracrine mechanisms regulating IL-1 bioactivity in the testis are unknown but may represent a means to protect germ cells from noxious effects of inflammation.

Intratesticular transplantation of testicular cells from leukemic rats causes transmission of leukemia.

A rat T-cell leukemia model was used to study the safety of germ cell transplantation as a mean of preventing infertility in males undergoing gonadotoxic cancer treatment. Donor germ cells were harvested from the testes of terminally ill leukemic rats and were either used directly or cryopreserved and thawed before transplantation by rete testis microinjection. All rats transplanted with testicular cells from leukemic donors developed signs of terminal rat T-cell leukemia, whereas control animals remained healthy. Cryopreservation of the donor germ cells caused a 3- to 6-day delay in the terminal phase of leukemia. When a known number of leukemic cells were mixed with germ cells and microinjected into the testis, the rate of appearance of terminal leukemia was directly related to the number of transferred leukemic lymphoblasts. As few as 20 leukemic cells were able to cause a cancer relapse resulting in terminal leukemia 21 days after transplantation in three of five transplanted animals. Our results demonstrate that germ cell transplantation with the presently used techniques is not safe enough for clinical use. Improved methods for purging testicular specimens of cancer cells or totally new approaches with transient xenogenetic host models to detect contamination of malignant cells must be developed before this technique can be offered to patients without fear of disease relapse.