Dale B. Hales

Role of cytokines in testicular function

Inflammatory disease has been established to affect male reproductive function and fertility. Relevant inflammatory diseases include general and chronic infectious diseases as well as localized acute or chronic infections of the male genitourinary tract. Male accessory gland infections account for almost 15% of all cases of male infertility seen in infertility clinics while fertility usually is not a clinical objective among patients with acute systemic infections such as Gramnegatives sepsis. Infections of the male accessory glands frequently are associated with increased counts of white blood cells in semen and elevated levels of proinflammatory cytokines in semen and the testis. There is a mounting body of evidence that demonstrates the importance of cytokines and chemokines in the regulation of testicular and glandular function during pathophysiological states as well as under normal physiological conditions when cytokines act as growth and differentiation factors. The purpose of this review is to examine the role of cytokines in the regulation of steroidogenesis and spermatogenesis in the testis under physiological and pathophysiological conditions and considers clinical investigations that help to improve the evaluation and treatment of male infertility.

Histopathology of ovarian tumors in laying hens: a preclinical model of human ovarian cancer.

The high mortality rate due to ovarian cancer (OVCA) is attributed to the lack of an effective early detection method. Because of the nonspecificity of symptoms at early stage, most of the OVCA cases are detected at late stages. This makes the access to women with early-stage disease problematic and presents a barrier to development and validation of tests for detection of early stage of OVCA in humans. Animal models are used to elucidate disease etiologies and pathogenesis that are difficult to study in humans. Laying hen is the only available animal that develops OVCA spontaneously; however, detailed information on ovarian tumor histology is not available. The goal of this study was to determine the histological features of malignant ovarian tumors in laying hens. A total of 155 young and old (1-5 years of age) laying hens (Gallus domesticus) were selected randomly and evaluated grossly and microscopically for the presence of ovarian tumors. Histological classification of tumors with their stages and grades was determined with reference to those for humans. Similar to humans, all 4 types including serous, endometrioid, mucinous, and clear cell or mixed carcinomas were observed in hen ovarian tumors. Some early neoplastic as well as putative ovarian lesions were also observed. Similarities in histology, metastasis, and stages of hen OVCA to those of humans demonstrate the feasibility of the hen model for additional delineation of the mechanism underlying ovarian carcinogenesis, preclinical testing of new agents for the prevention, and therapy of this disease.

Epithelial ovarian cancer experimental models

Epithelial ovarian cancer (OvCa) is associated with high mortality and, as the majority (>75%) of women with OvCa have metastatic disease at the time of diagnosis, rates of survival have not changed appreciably over 30 years. A mechanistic understanding of OvCa initiation and progression is hindered by the complexity of genetic and/or environmental initiating events and lack of clarity regarding the cell(s) or tissue(s) of origin. Metastasis of OvCa involves direct extension or exfoliation of cells and cellular aggregates into the peritoneal cavity, survival of matrix-detached cells in a complex ascites fluid phase and subsequent adhesion to the mesothelium lining covering abdominal organs to establish secondary lesions containing host stromal and inflammatory components. Development of experimental models to recapitulate this unique mechanism of metastasis presents a remarkable scientific challenge, and many approaches used to study other solid tumors (for example, lung, colon and breast) are not transferable to OvCa research given the distinct metastasis pattern and unique tumor microenvironment (TME). This review will discuss recent progress in the development and refinement of experimental models to study OvCa. Novel cellular, three-dimensional organotypic, and ex vivo models are considered and the current in vivo models summarized. The review critically evaluates currently available genetic mouse models of OvCa, the emergence of xenopatients and the utility of the hen model to study OvCa prevention, tumorigenesis, metastasis and chemoresistance. As these new approaches more accurately recapitulate the complex TME, it is predicted that new opportunities for enhanced understanding of disease progression, metastasis and therapeutic response will emerge.

Bacterial endotoxin lipopolysaccharide and reactive oxygen species inhibit Leydig cell steroidogenesis via perturbation of mitochondria.

Chronic inflammatory disease and acute infection are well known to inhibit gonadal steroidogenesis. Previous studies have demonstrated that immune activation in response to lipopolysaccharide (LPS) results in reductions in serum testosterone, and this is a direct effect on the Leydig cell. We hypothesize that during the early onset of LPS endotoxemia in vivo, testicular macrophages produce reactive oxygen species (ROS) leading to perturbation of Leydig cell mitochondria and an inhibition in steroidogenesis. To investigate the mechanism of LPS inhibition of Leydig cell steroidogenesis, alterations in mitochondria and markers of oxidative stress were assessed in vivo and in Leydig cell primary culture. After a single injection of mice with LPS, serum testosterone was significantly decreased within 2 h. LPS injection of mice resulted in significant reductions in steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydogenase-Δ4-Δ5 isomerase (3β-HSD) proteins. LPS significantly increased lipid peroxidation of Leydig cell membranes, indicating that LPS results in oxidative damage in vivo. Mitochondria in Leydig cells isolated from LPS-injected mice were disrupted and showed a marked reduction in the mitochondrial membrane potential (Δψm). Similar to the effects of LPS, treatment of Leydig cells with hydrogen peroxide acutely inhibited steroidogenesis, reduced StAR and 3β-HSD protein levels, and disrupted Δψm. These results suggest that LPS acutely inhibits Leydig cell function by ROS-mediated disruption of Leydig cell mitochondria. Taken together, these results demonstrate the necessity of having respiring mitochondria with an intact Δψm to facilitate StAR function and Leydig cell steroidogenesis. The acute effects of LPS demonstrate how sensitive Leydig cell mitochondrial steroidogenesis is to inflammation-induced oxidative stress.

Steroidogenic acute regulatory protein in the rat brain: cellular distribution, developmental regulation and overexpression after injury.

The central nervous system synthesizes steroids which regulate the development and function of neurons and glia and have neuroprotective properties. The first step in this process involves the delivery of free cholesterol to the inner mitochondrial membrane where it can be converted into pregnenolone. This delivery is mediated by steroidogenic acute regulatory protein (StAR). Here, we present a detailed analysis of the distribution of StAR expression in neurons and glia, in the developing, adult and aged male rat brain. Immunohistochemical analysis revealed that StAR is widely distributed throughout the brain, although in each brain area it is restricted to very specific neuronal and astroglial populations. In most regions expressing StAR, immunoreactivity appeared at P10 and the levels of expression then either increased or remained constant until adulthood. In 2‐year‐old rat brains, StAR immunoreactivity was increased compared to young adults. StAR was expressed in the subventricular zone of the adult brain, in proliferating cells which incorporate BrdU as well as in germinal layers in the developing brain. These findings indicate that StAR expression is developmentally regulated and that StAR may play some function in regulating cell proliferation in the brain. Furthermore, StAR mRNA and protein levels were acutely and transiently increased in the hippocampus following excitotoxic brain injury induced by the administration of kainic acid. This raises the possibility that the up‐regulation of StAR expression and the subsequent modifications in steroidogenesis may be part of the mechanisms used by the brain to cope with neurodegeneration.

IMMUNE ENDOCRINE INTERACTIONS AND LEYDIG CELL FUNCTION: THE ROLE OF CYTOKINES

Inflammatory disease is known to affect male reproductive function and fertility. Male accessory gland infections (MAGI) account for almost 15% of all cases of male infertility seen in infertility clinics. Infections of the male accessory glands are associated with increased counts of white blood cells in semen and elevated levels of pro‐inflammatory cytokines in the semen and the testis. Numerous studies have underscored the importance of cytokines in the regulation of testicular and glandular function during pathophysiological events as well as under normal physiological conditions when cytokines act as growth and differentiation factors. The purpose of this paper is to particularly review the role of cytokines in the regulation of Leydig cell function in the testis primarily under pathophysiological conditions, and also considers clinical investigations that help to improve the evaluation and treatment of male infertility.

Studies on the Onset of Leydig Precursor Cell Differentiation in the Prepubertal Rat Testis

Leydig cells of the adult rat testis differentiate postnatally from spindle-shaped cells in the testis interstitium during the neonatal-prepubertal period. Which spindle-shaped cell types are the precursor for Leydig cells and the stimulus for initiation of their differentiation are, however, two unresolved issues. In the present study, our objectives were to identify unequivocally which spindle-shaped cells are the precursors to Leydig cells and to test whether the initiation of their differentiation into Leydig cells depends on LH. Testes from fifteen groups of Sprague-Dawley rats (n = 4 per group) from 7-21 days of age were fixed in Bouin solution and embedded in paraffin. Immunoexpression of 3beta-hydroxysteroid dehydrogenase (3betaHSD), cytochrome P450 side-chain cleavage (P450(scc)), 17alpha-hydroxylase cytochrome P450 (P450(c17)), and LH receptors (LHR) in interstitial cells (other than fetal Leydig cells) was observed using the avidin biotin method. Of all spindle-shaped cell types in the testis interstitium, only the peritubular mesenchymal cells showed positive immunolabeling for all three steroidogenic enzymes, beginning from the 11th postnatal day. All three enzymes were expressed simultaneously in these cells, and their numbers increased significantly thereafter. Immunoexpression of LHR in a few of these cells was just evident for the first time on postnatal Day 12 (i.e., after acquiring the steroidogenic enzyme activity). Their numbers gradually increased with time. The number of immunolabeled cells per 1000 interstitial cells (excluding fetal Leydig cells and capillary endothelial cells) was not significantly different for the three steroidogenic enzymes tested at all ages; however, a lower value was observed for LHR at each time-point. Based on these observations, we suggest that 1) the precursor cell type for the adult generation of Leydig cells in the postnatal rat testis is the peritubular mesenchymal cells, 2) precursor cells acquire 3beta-HSD, P450(scc), and P450(c17) enzyme activity simultaneously during Leydig cell differentiation, and 3) onset of precursor cell differentiation during Leydig cell development does not depend on LH.

Diametric effects of bacterial endotoxin lipopolysaccharide on adrenal and Leydig cell steroidogenic acute regulatory protein.

Immune activation results in the activation of adrenal steroidogenesis and inhibition of gonadal steroidogenesis. Previous studies indicated that these effects were caused primarily by activation and suppression of the secretion of ACTH and LH, respectively. However, other evidence indicated a direct effect of the immune system on the gonads. In this study, serum testosterone, quantitated by RIA after lipopolysaccharide injection, showed a significant decrease within 2 h. Parallel measurement of serum LH showed no change. There were no differences in LH receptor or cAMP produced in Leydig cells between vehicle- and lipopolysaccharide-injected mice. The 30-kDa form of the steroidogenic acute regulatory (StAR) protein was quantitated, by Western blot, in Leydig cells and was found to decrease in a time-dependent manner. No change in StAR protein messenger RNA (mRNA) was detected by Northern analysis during this time, nor were any changes found in the levels of mRNA for the steroidogenic enzymes P450scc, 3β-...

Mitochondrial function in Leydig cell steroidogenesis.

Abstract: The first and rate‐limiting step in the biosynthesis of steroid hormones is the transfer of cholesterol into mitochondria, which is facilitated by the steroidogenic acute regulatory (StAR) protein. Recent studies of Leydig cell function have focused on the molecular events controlling steroidogenesis; however, few studies have examined the importance of the mitochondria. The purpose of this investigation was to determine which aspects of mitochondrial function are necessary for Leydig cell steroidogenesis. MA‐10 tumor Leydig cells were treated with 8‐bromo‐cAMP (cAMP) and site‐specific mitochondrial disrupters, pro‐oxidants, and their effects on progesterone synthesis, StAR expression, mitochondrial membrane potential (ΔΨm) and ATP synthesis were determined. Dissipating ΔΨm with CCCP inhibited progesterone synthesis, even in the presence of newly synthesized StAR protein. The electron transport inhibitor antimycin A significantly reduced cellular ATP, inhibited steroidogenesis, and reduced StAR protein expression. The F0/F1 ATPase inhibitor oligomycin reduced cellular ATP and inhibited progesterone synthesis and StAR protein expression, but had no effect on ΔΨm) Disruption of pH with nigericin significantly reduced progesterone production and StAR protein, but had minimal effects on ΔΨm. Sodium arsenite at low concentrations inhibited StAR protein but not mRNA expression and inhibited progesterone without disrupting ΔΨm. The mitochondrial Ca2+ inhibitor Ru360 also inhibited StAR protein expression. These results demonstrate that ΔΨm, ATP synthesis, ΔpH and [Ca2+]mt are all required for steroid biosynthesis, and that mitochondria are sensitive to oxidative stress. These results suggest that mitochondria must be energized, polarized, and actively respiring to support Leydig cell steroidogenesis and alterations in the state of mitochondria may be involved in regulating steroid biosynthesis.

Influence of urogenital infection on sperm function.

Male accessory sex gland infections are considered to be hazards to male fertility. Various pathophysiologic concepts have evolved from experimental and clinical studies that begin to explain the effects of bacteria and immunologic events on spermatozoa. Recent studies have identified and evaluated mediators that are responsible for specific molecular processes in infections that particularly affect the function of spermatozoa.