bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of raloxifene on salivary sex steroid concentrations in premenopausal women
Authors: Chatterton Robert T , Zujewski JoAnne , Mateo Esnar T , Eng-Wong Jennifer , Jordan V Craig
Published: December 2006

Raloxifene is a selective oestrogen receptor modulator used clinically for the treatment and the prevention of osteoporosis in postmenopausal women. The drug has been evaluated in the Study of Tamoxifen and Raloxifene as an agent to reduce breast cancer incidence in postmenopausal women at high risk. However, about 30% of women who develop breast cancer do so in their premenopausal years. In this pilot study, salivary oestradiol and progesterone were determined throughout the menstrual cycle for a total of 22 subjects, 14 of whom completed pre- and postraloxifene (60 mg daily) salivary collections. The mean concentration of oestradiol during the menstrual cycle when subjects were taking raloxifene was significantly greater (P<0.001) than during baseline cycles. Neither salivary progesterone and cortisol nor menstrual cycle length were affected by raloxifene treatment. These data demonstrate that raloxifene administered to premenopausal women increases the concentration of oestradiol that diffuses into the salivary glands, and which presumably represents the concentration available to other organs as well. The results reflect increases in serum oestradiol reported earlier.
The cholinergic influence on the mesenteric ganglion affects the liberation of ovarian steroids and nitric oxide in oestrus day rats: characterization of an ex vivo system
Authors: Orozco Adriana Vega , Sosa Zulema , Fillipa Verónica , Mohamed Fabian , Rastrilla Ana María
Published: December 2006

The axons that constitute the ovarian nervous plexus originate mostly in the principal neurons of the superior mesenteric ganglion (SMG) that is part of the sympathetic ganglionic chain and exhibits cholinergic receptors. In order to observe the effect of acetylcholine, the main neurotransmitter in the ganglionic transmission, the purpose of the present work was: first, to standardize an integrated ex vivo superior mesenteric ganglion-ovarian nervous plexus-ovary (SMG-ONP-O) system in oestrus day rats; secondly, to determine if the ganglionic cholinergic stimulus modifies the release of nitric oxide and steroids in the ovary compartment in the absence of humoral factors; and thirdly, to investigate if there are differences in the responses between the left and right ovaries caused by the neural stimulus. The ex vivo experimental left and right systems were developed and standardized. The systems were incubated in Krebs–Ringer phosphate buffer in a Dubnoff metabolic shaker. The progesterone release was determined to standardize the incubation times, obtaining different responses between the left and right systems, which shows that both systems have their own autonomic tone. Non-specific stimulation with KCl in the ganglion compartment provoked different responses in terms of release of progesterone and oestradiol. Progesterone decreased in the left and right systems. However, oestradiol diminished at short times and increased at 60 and 120 min in the left ovary, whereas it increases at 30 and 60 min in the right ovary. These different responses show the sensitivity and viability of both systems. When acetylcholine was used in the ganglion compartment, the release of nitric oxide, progesterone, androstenedione and oestradiol was evaluated. The liberation of nitrite increased at 15, 30 and 60 min in the left system and decreased in the right system at 120 min. Progesterone showed a decrease in its release at 15, 30 and 120 min and androstenedione at 15 min in the left ovary compartment. In the right ovary, only progesterone decreased in relation to the control at 120 min while androstenedione did not show significant changes. Oestradiol showed an increase in the left ovary compartment at all the studied times, while in the right ovary it did not show any changes. These results indicate that the neural stimulus from the superior mesenteric ganglion through the ovarian nervous plexus is one of the factors modulating the secretory activity of the ovarian steroids and nitric oxide. The system is viable and also shows a different sensitivity of the left ovary in relation to the right one at least in this cycle stage, characterized by marked irrigation and profound structural changes in the ovary.
Expression and functional role of hepatocyte growth factor and its receptor (c-met) during fetal mouse testis development
Authors: Ricci G , Catizone A , Galdieri M
Published: December 2006

The hepatocyte growth factor (HGF) is a pleiotropic cytokine able to regulate different cellular functions. HGF action is mediated by its receptor, c-met, a glycoprotein with tyrosine kinase activity. We previously demonstrated that c-met is expressed in the newly formed seminiferous cords of the mice embryonic testes and that HGF acts as a morphogenetic factor. In this paper, we report that at 15.5 days post-coitum (dpc) c-met is expressed in the testicular cords, whereas at 18.5 dpc c-met expression is almost exclusively localized in the interstitial tissue of the testis in particular in the fetal Leydig cells. In addition, we demonstrate that HGF gene is expressed during the fetal period of testis development, heavily detectable in the interstitial compartment of 18.5 dpc testes. Interestingly, HGF is not expressed in the Leydig cells that, as above reported, express the HGF receptor. Looking for the functional role of HGF on Leydig cells, we evaluated the amount of testosterone secreted by testes isolated from 18.5 dpc embryos and cultured in the presence of HGF. The results of the in vitro organ culture show that, at this age, HGF increases the amount of testosterone secreted in the culture medium. On the contrary, HGF does not modulate the amount of testosterone secreted by testes isolated from 15.5 dpc embryos. In conclusion, we report that HGF is produced in the interstitial compartment of the developing testis but not by the Leydig cells. Conversely, the HGF receptor c-met is expressed in the Leydig cells and HGF modulates Leydig cell function during the late period of prenatal development.
Phenotypic and global gene expression profile changes between low passage and high passage MIN-6 cells
Authors: O’Driscoll Lorraine , Gammell Patrick , McKiernan Eadaoin , Ryan Eoin , Jeppesen Per Bendix , Rani Sweta , Clynes Martin
Published: December 2006

The long-term potential to routinely use replacement β cells/islets as cell therapy for type 1 diabetes relies on our ability to culture such cells/islets, in vitro, while maintaining their functional status. Previous β cell studies, by ourselves and other researchers, have indicated that the glucose-stimulated insulin secretion (GSIS) phenotype is relatively unstable, in long-term culture. This study aimed to investigate phenotypic and gene expression changes associated with this loss of GSIS, using the MIN-6 cell line as model. Phenotypic differences between MIN-6(L, low passage) and MIN-6(H, high passage) were determined by ELISA (assessing GSIS and cellular (pro)insulin content), proliferation assays, phase contrast light microscopy and analysis of alkaline phosphatase expression. Differential mRNA expression was investigated using microarray, bioinformatics and real-time PCR technologies. Long-term culture was found to be associated with many phenotypic changes, including changes in growth rate and cellular morphology, as well as loss of GSIS. Microarray analyses indicate expression of many mRNAs, including many involved in regulated secretion, adhesion and proliferation, to be significantly affected by passaging/ long-term culture. Loss/reduced levels, in high passage cells, of certain transcripts associated with the mature β cell, together with increased levels of neuron/glia-associated mRNAs, suggest that, with time in culture, MIN-6 cells may revert to an early (possibly multi-potential), poorly differentiated, ‘precursor-like’ cell type. This observation is supported by increased expression of the stem cell marker, alkaline phosphatase.
Dynamin II interacts with the cadherin- and occludin-based protein complexes at the blood–testis barrier in adult rat testes
Authors: Lie Pearl P Y , Xia Weiliang , Wang Claire Q F , Mruk Dolores D , Yan Helen H N , Wong Ching-hang , Lee Will M , Cheng C Yan
Published: December 2006

In adult rat testes, blood–testis barrier (BTB) restructuring facilitates the migration of preleptotene spermatocytes from the basal to the adluminal compartment that occurs at stage VIII of the epithelial cycle. Structural proteins at the BTB must utilize an efficient mechanism (e.g. endocytosis) to facilitate its transient ‘opening’. Dynamin II, a large GTPase known to be involved in endocytosis, was shown to be a product of Sertoli and germ cells in the testis. It was also localized to the BTB, as well as the apical ectoplasmic specialization (apical ES), during virtually all stages of the epithelial cycle. By co-immunoprecipitation, dynamin II was shown to associate with occludin, N-cadherin, zonula occludens-1 (ZO-1), β-catenin, junctional adhesion molecule-A, and p130Cas, but not nectin-3. An in vivo model in rats previously characterized for studying adherens junction (AJ) dynamics in the testes by adjudin (formerly called AF-2364, 1-(2,4-dichlorobenzyl)-1H-indazole-3-car-hohydrizide) treatment was used in our studies. At the time of germ cell loss from the seminiferous epithelium as a result of adjudin-induced AJ restructuring without disrupting the BTB integrity, a significant decline in the steady-state dynamin II protein level was detected. This change was associated with a concomitant increase in the levels of two protein complexes at the BTB, namely occludin/ZO-1 and N-cadherin/β-catenin. Interestingly, these changes were also accompanied by a significant increase in the structural interaction of dynamin II with β-catenin and ZO-1. β-Catenin and ZO-1 are adaptors that structurally link the cadherin- and occludin-based protein complexes together at the BTB in an ‘engaged’state to reinforce the barrier function in normal testes. However, β-catenin and ZO-1 were ‘disengaged’ from each other but bound to dynamin II during adjudin-induced AJ restructuring in the testis. The data reported herein suggest that dynamin II may assist the ‘disengagement’ of β-catenin from ZO-1 during BTB restructuring. Thus, this may permit the occludin/ZO-1 complexes to maintain the BTB integrity when the cadherin/catenin complexes are dissociated to facilitate germ cell movement.
Transient receptor potential vanilloid channels functioning in transduction of osmotic stimuli
Authors: Liedtke Wolfgang
Published: December 2006

In signal transduction of metazoan cells, ion channels of the family of transient receptor potential (TRP) have been identified to respond to diverse external and internal stimuli, amongst them osmotic stimuli. This review will highlight findings on the TRPV subfamily, both vertebrate and invertebrate members. Out of the six mammalian TRP vanilloid (TRPV) channels, TRPV1, TRPV2, and TRPV4 were demonstrated to function in transduction of osmotic stimuli. TRPV channels have been found to function in cellular as well as systemic osmotic homeostasis in vertebrates. Invertebrate TRPV channels, five in Caenorhabditis elegans and two in Drosophila, have been shown to play a role in mechanosensation, such as hearing and proprioception in Drosophila and nose touch in C. elegans, and in the response to osmotic stimuli in C. elegans. In a striking example of evolutionary conservation of function, mammalian TRPV4 has been found to rescue osmo-and mechanosensory deficits of the TRPV mutant strain osm-9 in C. elegans, despite not more than 26% orthology of the respective proteins.
Human neurons express type I GnRH receptor and respond to GnRH I by increasing luteinizing hormone expression
Authors: Wilson Andrea C , Salamat M Shahriar , Haasl Ryan J , Roche Kelly M , Karande Anjali , Meethal Sivan Vadakkadath , Terasawa Ei , Bowen Richard L , Atwood Craig S
Published: December 2006

Gonadotropin-releasing hormone receptor I (GnRHR I) has been localized to the limbic system of the rat brain, although the functional consequences of GnRH signaling through these receptors is unknown. In this paper, we characterize the expression of GnRHR I in the human hippocampus and cortex, and the functionality of GnRHR I in human neuroblastoma cells. Robust GnRHR I immunoreactivity was detected in the cell body as well as along the apical dendrites of pyramidal neurons in the CA2, CA1, and end plate, but was clearly lower in the subiculum of the hippocampus. Immunolabeling was also evident in cortical neurons, including those located in the entorhinal cortex and occipitotemporal gyrus but was not observed within the granular layer of the dentate gyrus. No differences in immunohistochemical staining were observed between control and Alzheimer’s disease brain. GnRHR I mRNA and protein (mature, immature, and other variant) expression was detected in human neuroblastoma cells (M17, SH-SY5Y) and rat embryonic primary neurons and varied with differentiation and GnRH treatment. Since GnRHR I was expressed by extrapituitary cells, and hypothalamic GnRH I secretion markedly increases post-menopause/andropause, we treated human M17 neuroblastoma cells cultured in serum-free conditions with GnRH I for 6 h and measured LH expression. M17 neuroblastoma cells express LHβ mRNA, while immunoblot analysis indicated the presence of three LH variants (approximately 30, 47, and 60 kDa) that were upregulated by low concentrations of GnRH I, but down-regulated at higher GnRH I concentrations. LH expression was also found to increase in differentiating embryonic rat primary cortical neurons. Our results demonstrate that neurons expressing GnRHR I are functional, responding to GnRH I by upregulating LH production. Post-reproductive surges in GnRH I secretion may explain the accumulation of LH in pyramidal neurons of the aged human and rat.
Chronic local inflammation in mice results in decreased TRH and type 3 deiodinase mRNA expression in the hypothalamic paraventricular nucleus independently of diminished food intake
Authors: Boelen A , Kwakkel J , Wiersinga W M , Fliers E
Published: December 2006

During illness, changes in thyroid hormone metabolism occur, known as nonthyroidal illness and characterised by decreased serum triiodothyronine (T3) and thyroxine (T4) without an increase in TSH. A mouse model of chronic illness is local inflammation, induced by a turpentine injection in each hind limb. Although serum T3 and T4 are markedly decreased in this model, it is unknown whether turpentine administration affects the central part of the hypothalamus–pituitary–thyroid axis (HPT-axis). We therefore studied thyroid hormone metabolism in hypothalamus and pituitary of mice during chronic inflammation induced by turpentine injection. Using pair-fed controls, we could differentiate between the effects of chronic inflammation per se and the effects of restricted food intake as a result of illness. Chronic inflammation increased interleukin (IL)-1β mRNA expression in the hypothalamus more rapidly than in the pituitary. This hypothalamic cytokine response was associated with a rapid increase in local D2 mRNA expression. By contrast, no changes were present in pituitary D2 expression. TSHβ mRNA expression was altered compared with controls. Comparing chronic inflamed mice with pair-fed controls, both preproTSH releasing hormone (TRH) and D3 mRNA expression in the paraventricular nucleus were significantly lower 48 h after turpentine administration. The timecourse of TSHβ mRNA expression was completely different in inflamed mice compared with pair-fed mice. Turpentine administration resulted in significantly decreased TSHβ mRNA expression only after 24 h while later in time it was lower in pair-fed controls. In conclusion, central thyroid hormone metabolism is altered during chronic inflammation and this cannot solely be attributed to diminished food intake.
Intracellular gonadotropin-releasing hormone receptors in breast cancer and gonadotrope lineage cells
Authors: Sedgley Kathleen R , Finch Ann R , Caunt Christopher J , McArdle Craig A
Published: December 2006

Gonadotropin-releasing hormone receptors (GnRHRs) are expressed in gonadotropes and several extra-pituitary sites. They are assumed to be cell surface proteins but the human (h) GnRHR lacks features favoring plasma membrane localization and receptor location varies with cell type. When expressed in mammary (MCF7) cells, cell surface hGnRHR binding was much lower than that of mouse and sheep GnRHRs (type I GnRHRs without C-terminal tails), Xenopus (X) and marmoset type II GnRHRs (type II GnRHRs with C-tails) or chimeric receptors (type I GnRHRs with added XGnRHR C-tails). hGnRHR binding was higher in αT4 (gonadotrope-derived) cells and was increased less by C-tail addition. Whole cell levels of tagged human, Xenopus and chimeric GnRHRs were comparable (Western blotting) and confocal microscopy revealed that the hGnRHR is primarily intracellular (distribution similar to the endoplasmic reticulum marker, calreticulin), whereas most XGnRHR is at the plasma membrane, and adding the C-tail increased cell surface hGnRHR levels. A membrane-permeant antagonist increased cell surface hGnRHR number (>4-fold, t½ = 4 h) and also increased hGnRHR signaling and hGnRHR-mediated inhibition of proliferation. A more rapid increase in hGnRHR binding occurred when the temperature was raised from 4 to 37 °C (>5-fold, t½ = 15 min) and this effect was prevented by mutation to prevent signaling. Thus, cell surface GnRHR expression depends on receptor and cell type and the hGnRHR is primarily an intracellular protein that traffics to the cell surface for signaling in MCF7 cells. Manipulations favoring such trafficking may facilitate selective targeting of extra-pituitary GnRHRs.
Urinary Prostate‐Specific Antigen Is a Noninvasive Indicator of Sexual Development in Male Children
Authors: Sato Itaru , Yoshikawa Atsuko , Fugimoto Masatoshi , Shimizu Keiko , Ishiwari Atsuya , Mukai Toshiji , Iwamoto Teruaki
Published: December 2006

ABSTRACT: Testicular androgen induces the synthesis of prostate specific antigen (PSA) in acinar epithelial cells of the prostate. We examined PSA activity in urine from 136 male children from birth up to 17 years of age. We detected PSA at various intervals in early infant urine over a period of 1–4 months. During this period, urinary secretion of testosterone (T) gradually declined, accompanied by 1 or more surges of T prior to a transient increase in PSA in urine from full‐ and preterm infants (67%, n = 6). Although mean urinary T concentrations during elevations of PSA in preterm infants were 3.1 and 5.6 times greater than in full‐term infants and adults, the overall mean urinary PSA concentration of full and preterm infants was just 45% and 18% that of adults, respectively. PSA was not detected in children aged 0.3 to 9 years, after which a gradual increase in urinary PSA activity was observed after 10 years of age. Urinary PSA activity was markedly persistent after Tanner stage III pubertal development. To our knowledge, this is the first study to demonstrate an induction of PSA during early infancy by bioactive T in normally developing human males. We conclude that urinary PSA is a non‐invasive, useful indicator for developmental studies from neonatal and adolescent males, which can be measured with a confirmatory semiquantitative PSA assay.
Relative Levels of Semen Platelet Activating Factor‐Receptor (PAFr) and Ubiquitin in Yearling Bulls With High Content of Semen White Blood Cells: Implications for Breeding Soundness Evaluation
Authors: Sutovsky P. , Plummer W. , Baska K. , Peterman K. , Diehl J. R. , Sutovsky M.
Published: December 2006

ABSTRACT: High content of the platelet activating factor (PAF) and its plasma membrane receptor (PAFr) in semen is thought to benefit fertility in farm animals and humans. We used flow cytometric, biochemical, and immunocytochemical analysis to examine PAFr levels alone (Trial 1, n = 156 bulls) or in a dual assay with sperm defect marker ubiquitin (UBI; Trial 2, n = 88 bulls), in semen samples from 160 yearling bulls undergoing Breeding Soundness Evaluations (BSE). In both trials, we observed increased PAFr levels in semen samples with high content of white blood cells (WBC). Consequently, PAFr levels within such semen samples correlated negatively with several subjective parameters of BSE, including palpation, satisfaction of evaluation, and scrotal circumference. Due to a high WBC content, increased semen sample dilution had to be applied for microscopic evaluation. There was a negative correlation between semen PAFr and conventional sperm morphology, while the increased levels of PAFr correlated positively with sperm UBI content. Immunofluorescence microcopy revealed high expression of PAFr on the surface of leukocytes and morphologically normal spermatozoa, while reduced immunoreactivity was observed in defective spermatozoa immunoreactive to anti‐UBI antibodies. A single PAFr band of appropriate mass was observed in Western blots of ejaculated spermatozoa, while testicular and epididymal spermatozoa also displayed several larger bands indicative of posttranslational processing or modification. Collectively, these data suggest that high levels of semen PAFr in young bulls are indicative of semen contamination with WBC. In the future, objective protein marker‐based semen analyses in young bulls will likely require additional parameters distinguishing between marker expression in the spermatozoa and in the contaminating WBC. While identification of high sperm PAFr levels may support fertility, this assay alone is not reliable, due to the expression of PAFr in WBC that contaminate semen samples.
Prolonged Histamine Deficiency in Histidine Decarboxylase Gene Knockout Mice Affects Leydig Cell Function
Authors: Mondillo Carolina , Falus András , Pignataro Omar , Pap Erna
Published: December 2006

ABSTRACT: The present study focuses on histaminergic regulation of Leydig cell physiology, since limited information is available so far. To evaluate the dependency of Leydig cells on histamine (HA), we performed experiments using highly purified Leydig cells in culture, isolated from wild type (WT) and histidine decarboxylase (Hdc) gene knockout (HDC KO)—so HA‐deprived—mice. HDC KO Leydig cells showed lower basal and human choriogonadotropin (hCG)‐induced testosterone production compared to WT Leydig cells, presumably due to altered P450scc gene (Cyp11a1) expression levels. Moreover, in HDC KO cells, hCG did not increase basal expression levels of HA H1 and H2 receptor genes, while the hormone showed a significant inducing effect in WT cells. Based on these findings, we propose that prolonged HA deficiency in HDC KO mice affects various aspects of Leydig cell physiology, most importantly the response to hCG, providing definite evidence that HA plays a role as direct modulator of Leydig cell function and steroid synthesis in the testis. Also, the results presented herein constitute the first molecular evidence for the expression of HA H1 and H2 receptor subtypes in isolated Leydig cells.
Induction of Oxidative Stress by Organic Hydroperoxides in Testis and Epididymal Sperm of Rats In Vivo
Authors: Kumar Thimmappa R. , Muralidhara
Published: December 2006

ABSTRACT: The present study describes the extent and pattern of oxidative stress induction in testis and epididymal sperm of rats following in vivo exposure to repeated sublethal doses of 2 model pro‐oxidants, namely, t‐butyl hydroperoxide (tbHP) and cumene hydroperoxide (cHP). Single sublethal (1/40, 1/20, and 1/10 LD50) doses of hydroperoxides (HP) administered intraperitoneally to male rats (CFT‐Wistar strain) failed to induce any significant increase in malondialdehyde or reactive oxygen species (ROS) levels in testis or epididymal sperm. However, repeated doses for 1 or 2 weeks induced a marked dose‐related enhancement of lipid peroxidation (LPO) and ROS levels in both testis and epididymal sperm. Further evidence, such as significant perturbations in both enzymic and nonenzymic antioxidants and enhanced levels of protein carbonyls in testis, suggested induction of oxidative stress. In testis, moderate depletion in reduced glutathione levels and marked diminution in ascorbic acid and α‐tocopherol content were accompanied by increased activities of various antioxidant enzymes, namely glutathione peroxidase, glutathione‐S‐transferase, and catalase, in both the HP treatments. Furthermore, significant alterations in the specific activities of testicular enzymes such as LDH‐X, G‐6‐PDH, and SDH indicated altered testicular physiology. Both HP at higher doses induced significant DNA damage (determined by fluorimetric analysis of DNA unwinding assay) in testis and epididymal sperm. Increased total iron levels in testis of HP‐treated rats are indicative of the possible involvement of iron‐mediated free radical reactions in this model. These findings provide an account of early oxidative damage in testis and epididymal sperm following short‐term exposure to HP in vivo, and this model is being further exploited for understanding the consequences of chronic oxidative stress‐mediated alterations for the physiology of male reproductive system and its implications for fertility.
Hyalp1 in Murine Sperm Function: Evidence for Unique and Overlapping Functions With Other Reproductive Hyaluronidases
Authors: Miller Kimberly A. , Shao Minghai , Martin‐Deleon Patricia A.
Published: December 2006

ABSTRACT: While Sperm adhesion molecule 1 (SPAM1) is the highly conserved mammalian sperm hyaluronidase (hyase), multiple hyases are present in the mouse testis. In this study we show that one of the murine hyases, Hyalp1, which is predominantly expressed in the testis in a 24‐kd isoform has neutral enzymatic activity. On sperm, Hyalp1 is localized on the plasma membrane of the anterior head and was shown to have neutral hyase activity for an isoform of ∼55–56 kd, contributing modestly to the overall neutral hyase activity. This activity is associated with in vitro cumulus penetration, since antibody inhibition of Hyalp1 significantly (P = .034) retarded the rate of penetration of wild‐type (WT) sperm. Antibody‐inhibited Spam1 null sperm were more severely retarded (P = 4.2 × 10−19), suggesting an up‐regulation of Hyalp1 in these mice. A functionality test of the hyaluronic acid (HA) receptor domain identified in the N‐terminus by in silico analysis revealed that sperm Hyalp1 is significantly (P = .006) involved in the progesterone‐induced HA‐enhanced acrosome reaction. Finally, developmental reverse transcription polymerase chain reaction (RT‐PCR) shows that testicular transcripts of Hyalp1 are detected as early as 6 days postparturition, similar to transcripts for Spam1, suggesting that the gene might also play a role in the developing testes prior to spermiogenesis. Taken together, the findings reveal that Hyalp1 likely has a unique function in the adult testis, and redundant overlapping ones with Spam1 and may compensate for it in Spam1 null mice.
Serotonin Concentration, Synthesis, Cell Origin, and Targets in the Rat Caput Epididymis During Sexual Maturation and Variations Associated With Adult Mating Status: Morphological and Biochemical Studies
Authors: Jiménez‐Trejo Francisco , Tapia‐Rodríguez Miguel , Queiroz Daniel B. C. , Padilla Patricia , Avellar Maria Christina W. , Manzano Patricia Rivas , Manjarrez‐Gutiérrez Gabriel , Gutiérrez‐Ospina Gabriel
Published: December 2006

ABSTRACT: The caput epididymis of some mammals contains large quantities of serotonin whose origin, targets, and physiological variations have been poorly studied. We combined morphological and biochemical techniques to begin approaching these aspects of serotonin in the rat caput epididymis. Serotonin immunostaining was detected in mast, epithelial, and neuroendocrine cells. Epithelial cells displayed immunoreactivity to 5HT1A, 5HT2A, and 5HT3 serotonin receptors. Endothelial and mast cells labeled positive for 5HT1B serotonin receptors and spermatozoa displayed 5HT2A and 5HT3 serotonin receptor immunoreactivity. Epithelial, endothelial, and mast cells stained positive for serotonin transporters. Only epithelial cells showed tryptophan hydroxylase immunoreactivity; this enzyme catalyzes the limiting step in the serotonin synthetic pathway. In addition, Western blot analyses of caput homogenates documented the presence of 2 protein bands (∼51 kd and ∼48 kd) that were immunoreactive for tryptophan hydroxylase. Chromatographic analyses documented the presence of tryptophan hydroxylase in the caput, and showed that both its activity and serotonin availability increased with sexual maturation and decreased following p‐chlorophenylalanine treatment, an inhibitor of tryptophan hydroxylase activity. Interestingly, serotonin concentration and tryptophan hydroxylase activity tended to be higher in breeding males than in those with no mating experience. We think that these results support the existence of a local serotoninergic system in the rat caput epididymis that might regulate some aspects of male reproductive function.
The Advancement of Pure Local Anesthesia for Penile Surgeries: Can an Outpatient Basis Be Sustainable?
Authors: Hsu Geng‐Long , Hsieh Cheng‐Hsing , Chen Heng‐Shuen , Ling Pei‐Ying , Wen Hsien‐Sheng , Liu Li‐Jen , Chen Cheng‐Weng , Chua Ceferino
Published: December 2006

ABSTRACT: Although topical anesthetic blockage for penile surgeries has been substantially reported in the medical literature, its methodology, reliability, and reproducibility have not been consistent. We report on several methods of topical blocks for local anesthesia in patients with indications for penile surgeries. From March 1993 to March 2003, a total of 1131 men, ages 19 to 87, underwent penile surgeries in which 165, 203, 708, 45, and 10 patients received penile implantation, modified Nesbit procedure, venous surgery, venous patches, and arterial revascularization respectively, under pure local anesthesia on an outpatient basis. They were categorized into the implant, Nesbit, venous, patch, and arterial groups respectively. Proximal dorsal nerve blockage, peripenile infiltration, and topical injection, although challenging, were sufficient local anesthesia for patients in the last 4 patient groups. A new method of crural blockade, however, was also required for optimal anesthesia of the cavernous nerve for implantation purposes. The anesthetic effects and postoperative results were satisfactory. Common immediate side effects included puncture of the corpus spongiosum or the deep dorsal vein as well as the innominate vessel, subcutaneous ecchymosis, transient palpitations, and acceptable low level of pain. There were no significant late complications. In the implant group, however, 6.1% of patients (10/165) had experienced pain over the perineum for 1 to 2 weeks postoperatively. Overall there were statistical differences in scoring between the 5 groups in which the implant group stood out when a visual analog scale of 100 mm was used. Topical nerve blockades proved to be reliable, simple, and safe, with minimal complications. They offer the advantages of less morbidity, reduced effects of anesthesia, protection of privacy, and a rapid return to preoperative daily activity.
A Stereological Analysis of the Response of Spermatogenesis to an Acute Inflammatory Episode in Adult Rats
Authors: Liew Seng H. , Meachem Sarah J. , Hedger Mark P.
Published: December 2006

ABSTRACT: Male fertility is inhibited by inflammatory disease, but the mechanisms responsible are poorly defined. The effects of acute systemic inflammation induced by a single IP injection of lipopolysaccharide (LPS) on spermatogenic function in adult male rats were investigated using detailed stereological analysis. The earliest effect observed was a significant maturational delay of meiosis during the leptotene/zygotene phase (at stages IX—XIII) within 24 hours. This was followed within 6 days by an increase in premature release of these cells and the adjacent, more luminally located generation of round spermatids from the seminiferous epithelium. An increase in germ cell apoptosis within stages IX—XIII also occurred at this time. These data indicate that the initial effects of acute inflammation on the seminiferous epithelium are most pronounced on stages IX—XIII. The effects were not consistent with a loss of hormonal regulation, suggesting that a direct effect of inflammation on the function of the Sertoli cell during this critical stage of meiosis is involved. In the longer term, however, the consequences of this acute inflammatory episode were relatively minor: within 28 days there had been a compensatory increase in the efficiency of the seminiferous epithelium, restoring the spermatogenic capacity of the testis towards preinflammation levels.
Apoptosis and Molecular Pathways in the Seminiferous Epithelium of Aged and Photoinhibited Syrian Hamsters (Mesocricetus auratus)
Authors: Morales Eva , Ferrer Concepcion , Zuasti Adelina , Garcia‐Borron Jose C. , Canteras Manuel , Pastor Luis M.
Published: December 2006

ABSTRACT: Aging and short photoperiod exposure induce germ cell apoptosis in the Syrian hamster; however, the specific germ cells affected and the molecular pathways triggered have not been elucidated. We analyzed germ cell apoptosis and the expression of the Fas/Fas‐L system, Bcl‐2 family, and p53 in aged and photoinhibited hamsters and compared with those young maintained in natural photoperiod. Aging increased apoptosis in spermatogonia and spermatocytes, but in photoinhibited hamster testes only an increase in apoptotic spermatocytes was observed. Apoptosis was higher in aged hamsters in stages I—IV, V—VI and VII—VIII. Aging increased apoptosis of spermatogonia in stages I—IV and V—VI. Apoptotic pachytene spermatocytes were significantly higher in stages I—IV, V—VI, and VII—VIII in aging. Apoptotic preleptotene and pachytene spermatocytes were higher in aging, but no differences were observed in leptotene‐zygotene. Fas‐L was expressed by Sertoli cells, of young, aged, and photoinhibited hamsters. Bcl‐xL was strongly expressed in germ cells on young hamsters and slightly in aging and after short photoperiod exposure. Spermatocytes of photoinhibited hamsters were intensively stained with Fas, Bax, Bcl‐xs/L, and p53. In conclusion, aging increases apoptosis in spermatogonia and spermatocytes, depending on the stage of the seminiferous epithelium cycle, whereas after a short photoperiod exposure only an increase in apoptotic spermatocytes is observed. The results suggest that Fas, Bcl‐xL, Bax, and p53 participate in germ cell apoptosis induction after short photoperiod exposure, whereas only Bcl‐xL is involved in aging.
Association of Long Polyglycine Tracts (GGN Repeats) in Exon 1 of the Androgen Receptor Gene With Cryptorchidism and Penile Hypospadias in Iranian Patients
Authors: Radpour Ramin , Rezaee Mina , Tavasoly Ali , Solati Sanaz , Saleki Ahmad
Published: December 2006

ABSTRACT: Hypospadias (a urethral orifice located along the ventral side of the penis) and cryptorchidism (failure of the testes to descend into the scrotal sacs) are the 2 most common congenital malformations in males, affecting 0.3%–0.7% and 2%–4%, respectively, at birth. To study the association of CAG/GGN trinucleotide repeats in the androgen receptor gene with cryptorchidism and hypospadias in an Iranian population, we performed a case‐control study of 76 cryptorchid and 92 hypospadiac (divided into subgroups of glanular, penile, and penoscrotal hypospadias) Iranian males. The length of the CAG/GGN repeat segment was evaluated by using polymerase chain reaction (PCR) sequencing in exon 1 and PCR—single‐strand conformation polymorphism (PCR‐SSCP) in exons 2–8. There were no significant differences in CAG lengths between the cases and controls, but GGN numbers were found to be significantly higher (median, 24 vs 22) among both subjects with penile hypospadias (P = .018) and those with a history of cryptorchidism (P = .001) compared with controls. In addition, the GGN numbers among subjects with penile hypospadias were significantly different compared with the 2 other subgroups of hypospadias (P = .001). We were able to identify 12 different CAG alleles and 8 different GGN alleles in the cryptorchid group. Although further studies are needed to elucidate the possible role of specific CAG/GGC combinations as a susceptible factor, our data suggested the possible association between polyglycin tract polymorphism in androgen receptor gene and cryptorchidism.
Effect of Seminal Plasma Fractions From Entire and Vasectomized Rams on the Motility Characteristics, Membrane Status, and In Vitro Fertility of Ram Spermatozoa
Authors: Ghaoui Racha El‐Hajj , Gillan Lindsay , Thomson Peter C. , Evans Gareth , Maxwell W. M. Chis
Published: December 2006

ABSTRACT: Whole seminal plasma from ram semen collected before and after vasectomy was separated into 2 fractions, supernatant and pellet of vesicles, and their protein profiles characterized by one‐dimensional (1D) gel electrophoresis. The effects of autologous whole seminal plasma and these fractions on motility characteristics (assessed subjectively and by computer‐assisted sperm analysis), membrane status (assessed by chlortetracycline staining patterns), and in vitro fertility (assessed by fertilization success and timing of fertilization events) of washed frozen‐thawed ram spermatozoa were studied. Regardless of vasectomy, whole seminal plasma and supernatant displayed similar protein patterns. These fractions, when included in the postthaw buffer, improved the motility characteristics (59.6% ± 6.21% and 39.6% ± 6.21% vs 31.7% ± 6.46% and 15.5% ± 6.46% total motility) and membrane integrity (36.6% ± 8.52% and 31.2% ± 8.19% vs 30.3% ± 11.49% and 21.6% ± 10.28% B staining pattern [characteristic of capacitated acrosome‐intact cells] for whole seminal plasma and supernatant vs control at 3 and 6 hours of postthaw incubation, respectively) of frozen‐thawed spermatozoa and improved their ability to fertilize in vitro—matured oocytes compared with control buffer without seminal plasma fractions (25.3%, 47.4%, and 37.4% vs 12.3%, 20.2%, and 20.5% oocytes fertilized for spermatozoa incubated with supernatant vs control at 2, 6, and 18 hours after insemination, respectively). Vesicles were absent from semen collected after vasectomy. Pellets of vesicles collected before vasectomy had no effect on spermatozoa at their normal protein concentration but marginally improved both motility characteristics and in vitro fertility, possibly due to contamination from supernatant proteins, when their concentration in the postthaw medium was increased by threefold. It was concluded that the vesicle‐free supernatant fraction of seminal plasma, but not the seminal plasma membrane vesicles, improved the function and fertility of frozen‐thawed ram spermatozoa when added to the postthaw medium.
Molecular Mechanisms of Insulin Resistance in Humans and Their Potential Links With Mitochondrial Dysfunction
Authors: Morino Katsutaro , Petersen Kitt Falk , Shulman Gerald I.
Published: December 2006

Recent studies using magnetic resonance spectroscopy have shown that decreased insulin-stimulated muscle glycogen synthesis due to a defect in insulin-stimulated glucose transport activity is a major factor in the pathogenesis of type 2 diabetes. The molecular mechanism underlying defective insulin-stimulated glucose transport activity can be attributed to increases in intramyocellular lipid metabolites such as fatty acyl CoAs and diacylglycerol, which in turn activate a serine/threonine kinase cascade, thus leading to defects in insulin signaling through Ser/Thr phosphorylation of insulin receptor substrate (IRS)-1. A similar mechanism is also observed in hepatic insulin resistance associated with nonalcoholic fatty liver, which is a common feature of type 2 diabetes, where increases in hepatocellular diacylglycerol content activate protein kinase C-ε, leading to reduced insulin-stimulated tyrosine phosphorylation of IRS-2. More recently, magnetic resonance spectroscopy studies in healthy lean elderly subjects and healthy lean insulin-resistant offspring of parents with type 2 diabetes have demonstrated that reduced mitochondrial function may predispose these individuals to intramyocellular lipid accumulation and insulin resistance. Further analysis has found that the reduction in mitochondrial function in the insulin-resistant offspring can be mostly attributed to reductions in mitochondrial density. By elucidating the cellular and molecular mechanisms responsible for insulin resistance, these studies provide potential new targets for the treatment and prevention of type 2 diabetes.
Fatty Acid Signaling in the β-Cell and Insulin Secretion
Authors: Nolan Christopher J. , Madiraju Murthy S.R. , Delghingaro-Augusto Viviane , Peyot Marie-Line , Prentki Marc
Published: December 2006

Fatty acids (FAs) and other lipid molecules are important for many cellular functions, including vesicle exocytosis. For the pancreatic β-cell, while the presence of some FAs is essential for glucose-stimulated insulin secretion, FAs have enormous capacity to amplify glucose-stimulated insulin secretion, which is particularly operative in situations of β-cell compensation for insulin resistance. In this review, we propose that FAs do this via three interdependent processes, which we have assigned to a “trident model” of β-cell lipid signaling. The first two arms of the model implicate intracellular metabolism of FAs, whereas the third is related to membrane free fatty acid receptor (FFAR) activation. The first arm involves the AMP-activated protein kinase/malonyl-CoA/long-chain acyl-CoA (LC-CoA) signaling network in which glucose, together with other anaplerotic fuels, increases cytosolic malonyl-CoA, which inhibits FA partitioning into oxidation, thus increasing the availability of LC-CoA for signaling purposes. The second involves glucose-responsive triglyceride (TG)/free fatty acid (FFA) cycling. In this pathway, glucose promotes LC-CoA esterification to complex lipids such as TG and diacylglycerol, concomitant with glucose stimulation of lipolysis of the esterification products, with renewal of the intracellular FFA pool for reactivation to LC-CoA. The third arm involves FFA stimulation of the G-protein–coupled receptor GPR40/FFAR1, which results in enhancement of glucose-stimulated accumulation of cytosolic Ca2+ and consequently insulin secretion. It is possible that FFA released by the lipolysis arm of TG/FFA cycling is partly “secreted” and, via an autocrine/paracrine mechanism, is additive to exogenous FFAs in activating the FFAR1 pathway. Glucose-stimulated release of arachidonic acid from phospholipids by calcium-independent phospholipase A2 and/or from TG/FFA cycling may also be involved. Improved knowledge of lipid signaling in the β-cell will allow a better understanding of the mechanisms of β-cell compensation and failure in diabetes.
β-Cell Lipases and Insulin Secretion
Authors: Fex Malin , Lucas Stephanie , Winzell Maria Sörhede , Ahrén Bo , Holm Cecilia , Mulder Hindrik
Published: December 2006

Lipids have been implicated in β-cell stimulus-secretion coupling. Thus, lipases in β-cells would be required to generate coupling factors from intracellular lipids. Indeed, we found that glucose stimulates lipolysis in rodent islets and clonal β-cells. Lipolysis and diglyceride lipase activity in islets are abolished by orlistat, a pan-lipase inhibitor. Moreover, orlistat dose-dependently inhibits glucose- and forskolin-stimulated insulin secretion, while leaving glucose oxidation and the rise in ATP-to-ADP ratio intact. In an effort to identify β-cell lipase(s), we found that hormone-sensitive lipase (HSL), the rate-limiting enzyme for acylglyceride hydrolysis in adipocytes, is active in rodent β-cells. To further address the role of HSL, a global and β-cell–specific inactivation, respectively, of the lipase has been created in mice. Whereas our line of HSL null mice is moderately glucose intolerant due to reduced peripheral insulin sensitivity, it exhibits normal islet metabolism and insulin secretion. Preliminary analysis of the β-cell–specific HSL knockout has revealed no evidence for disturbed islet function. Thus, studies of ours and others indicate that there is a complex lipid regulatory component in β-cell stimulus-secretion coupling. The role of HSL and other lipases needs to be further clarified to provide a balanced view of the role of lipids and lipolysis in β-cells.
Amino Acid Metabolism, β-Cell Function, and Diabetes
Authors: Newsholme Philip , Brennan Lorraine , Bender Katrin
Published: December 2006

Specific amino acids are known to acutely and chronically regulate insulin secretion from pancreatic β-cells in vivo and in vitro. Mitochondrial metabolism is crucial for the coupling of amino acid and glucose recognition to exocytosis of insulin granules. This is illustrated by in vitro and in vivo observations discussed in the present review. Mitochondria generate ATP, which is the main coupling messenger in insulin secretion, and other coupling factors, which serve as sensors for the control of the exocytotic process. Numerous studies have sought to identify the factors that mediate the key amplifying pathway over the Ca2+ signal in nutrient-stimulated insulin secretion. Predominantly, these factors are nucleotides (ATP, GTP, cAMP, and NADPH), although metabolites have also been proposed, such as long-chain acyl-CoA derivatives and glutamate. This scenario further highlights the importance of the key enzymes or transporters, e.g., glutamate dehydrogenase, the aspartate and alanine aminotransferases, and the malate-aspartate shuttle in the control of insulin secretion. In addition, after chronic exposure, amino acids may influence gene expression in the β-cell, which subsequently alters levels of insulin secretion. Therefore, amino acids may play a direct or indirect (via generation of putative messengers of mitochondrial origin) role in insulin secretion.
Interleukin-6 Regulation of AMP-Activated Protein Kinase
Authors: Ruderman Neil B. , Keller Charlotte , Richard Ann-Marie , Saha Asish K. , Luo Zhijun , Xiang Xiaoqin , Giralt Mercedes , Ritov Vladimir B. , Menshikova Elizabeth V. , Kelley David E. , Hidalgo Juan , Pedersen Bente K. , Kelly Meghan
Published: December 2006

Interleukin (IL)-6 is a pleiotropic hormone that has both proinflammatory and anti-inflammatory actions. AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that among its other actions responds to decreases in cellular energy state by enhancing processes that generate ATP and inhibiting others that consume ATP but are not acutely necessary for survival. IL-6 is synthesized and released from skeletal muscle in large amounts during exercise, and in rodents, the resultant increase in its concentration correlates temporally with increases in AMPK activity in multiple tissues. That IL-6 may be responsible in great measure for these increases in AMPK is suggested by the fact it increases AMPK activity both in muscle and adipose tissue in vivo and in incubated muscles and cultured adipocytes. In addition, we have found that AMPK activity is diminished in muscle and adipose tissue of 3-month-old IL-6 knockout (KO) mice at rest and that the absolute increases in AMPK activity in these tissues caused by exercise is diminished compared with control mice. Except for an impaired ability to exercise and to oxidize fatty acids, the IL-6 KO mouse appears normal at 3 months of age. On the other hand, by age 9 months, it manifests many of the abnormalities of the metabolic syndrome including obesity, dyslipidemia, and impaired glucose tolerance. This, plus the association of decreased AMPK activity with similar abnormalities in a number of other rodents, suggests that a decrease in AMPK activity may be a causal factor. Whether increases in IL-6, by virtue of their effects on AMPK, contribute to the reported ability of exercise to diminish the prevalence of type 2 diabetes, coronary heart disease, and other disorders associated with the metabolic syndrome remains to be determined.