Developmental changes in inhibin alpha and inhibin/activin betaA and betaB mRNA levels in the gonads during post-hatch prepubertal development of male and female chickens

Dimeric inhibins and activins are barely detectable in the plasma during prepubertal development of male and female chickens. This may be misconstrued to indicate that the proteins are not produced in the gonads and have no functional significance during this period. Very few studies have actually determined the mRNA expression profile of the inhibin and activin subunits in the gonads prior to puberty in order to establish their secretion at the local level and postulate potential roles for the inhibin and activins at this developmental stage. In this study, the expression of the mRNA for the alpha-, betaA-, and betaB-subunits was determined in the ovary and testis of chickens during prepubertal development. Gene expression was determined at 3, 5, 6, 8, 10, 12, 16, and 18 weeks of age by RT-PCR. Messenger RNA level was quantified by competitive RT-PCR at 3, 6, 12, and 18 weeks of age in order to detect any changes with development, suggest potential relationship to the profile of dimeric inhibins and activins reported previously and to suggest potential paracrine and endocrine roles for them. The results show that all the inhibin/activin subunit mRNAs are expressed in the testis of the chicken throughout the period of prepubertal development up to 18 weeks of age. However, in the ovary, only the betaA- and betaB-subunits were detected at all ages whereas the alpha-subunit mRNA could only be detected just before puberty. Quantification of the mRNA levels showed variation of each subunit with age. These temporal changes suggest relationship with paracrine functional role in the ovary or the testis. Quantitative changes in expression levels also suggests that there may be some relationship between mRNA levels and the type and amount of dimeric inhibins and activins produced at any developmental stage. There are major differences between the male and female gonads in the timing of the expression of different subunits. In conclusion, the expression of the mRNA subunits in the testis and ovary suggests that inhibins and activins are being produced but may be principally involved in autocrine/paracrine function within the gonads.     

Expression of serpinb6 serpins in germ and somatic cells of mouse gonads

The serpin superfamily of serine protease inhibitors is implicated in the regulation of numerous physiological processes. In mice, Spi3/Serpinb6 has a broad tissue distribution. We have investigated the expression of Serpinb6 family members in embryonic and adult gonads. In male and female mice, Spi3/Serpinb6 and NK13/Serpinb6b were expressed in developing gonads and in both somatic and germ cells of adult gonads. By contrast, gonadal expression of Spi3C/Serpinb6c was sexually dimorphic and restricted to male germ cells and female somatic cells. These observations raise the question of the possible role(s) of the Serpinb6 family members in gonad development, gametogenesis, and/or fertilization.     

Role of colony-stimulating factor-1 in reproduction and development

The CSF-1 null mouse, osteopetrotic, has provided a powerful model in which to study the biological functions of CSF-1. In this review, I will describe our studies that have used this mouse model to determine the impact of a lack of CSF-1 on developmental processes and in reproduction. A role for CSF-1 in reproduction was originally suggested by the sex steroid hormone-regulated uterine epithelial synthesis of CSF-1 and the expression of its receptor in trophoblast and decidual cells. Studies on the fertility of CSF-1 deficient osteopetrotic mice (csfm^op/csfm^op) mice confirmed this suggestion and in addition revealed an unexpected function for CSF-1 in male fertility. In both sexes, CSF-1 appears to regulate gonadal steroidogenesis, probably through its action on macrophages that are abundant throughout the ovary and testis. In the female, CSF-1 affects ovulation in vivo and in vitro, and impacts the preimplantation embryo, increasing both its rate of development and the number of trophectodermal cells in the blastocyst. CSF-1 also has a role in mammary gland development during pregnancy, since at mid-gestation in csfm^op/csfm^op mice, ductal branching is impaired, and after partiturition, there is a failure to switch to lactation. The relative failure of csfm^op/csfm^op mice to respond to external stimuli also suggested a role for CSF-1 in the brain. CSF-1 mRNA is expressed in a regional specific manner in the brain through development whilst the CSF-1 receptor is expressed throughout the brain in microglia. CSF-1 is neurotrophic in embryonic neuronal cultures and its absence in csfm^op/csfm^op mice results in severe electrophysiological abnormalities in the cortex. This suggests that CSF-1 is a neurotrophic factor acting through the microglia. The pleiotropic roles for CSF-1 in reproduction and in the brain suggest that CSF-1 exerts many of its action through the trophic activities of cells of the mononuclear phagocytic lineage. Mol Reprod Dev 46:54–61, 1997. © 1997 Wiley-Liss, Inc.     

Role of light in the mediation of acute effects of a single afternoon melatonin injection on steroidogenic activity of testis in the rat

Young adult male rats, maintained either in an LD 12: 12 or in continuous illumination (LL) for one week, were given a single injection of 25 μg melatonin/100 g body wt or ethanolic-saline (control) at 17.00 h. Animals from each group were sacrificed at 11.00 h on the following day. The activity of two important steroidogenic enzymes, 17β-hydroxysteroid dehydrogenase (17β-HSD) and Δ⁵-3β-hydroxysteroid dehydrogenase (Δ⁵-3β-HSD), and serum concentrations of testosterone, were measured following highly specific and sensitive spectrophotometric techniques and RIA, respectively. A significant decrease in the activity of both the steroidogenic enzymes was noted in the testes of melatonin-treated rats maintained under normal light-dark schedules, but this response was found to be lacking in the LL rats. However, no significant changes in the level of serum testosterone were noted in either group of melatonin-treated rats from the values in respective groups of ethanolic saline-administered LD and LL rats. Exposure of ethanolic saline-injected rats to continuous light also did not cause any change in the steroidogenic activity of the testis from those in LD rats. The study indicates that continuous light as such does not affect the endocrine function of testis but abolishes suppressive effects of melatonin on the steroidogenic activity of the testis in rat.     

Two messenger RNA isoforms of the gonadotrophin-releasing hormone receptor,generated by alternative splicing and/or promoter usage,are differentially expressed in rainbow trout gonads during gametogenesis

The recent cloning of a gonadotrophin-releasing hormone receptor (GnRH-R) cDNA from rainbow trout showed that it contains several in-frame ATG codons, one of which, ATG2, corresponds to that found in other species. However, an upstream codon, ATG1, could give rise to a protein with a larger extracellular domain. Using S1 nuclease assay and a method combining primer extension and RACE-PCR, we characterized a second population of mRNA, termed mRNA-2, with a distinct 5'untranslated region and lacking ATG1. The genomic origin of the two mRNAs was determined by establishing the complete gene structure, which shows, for the first time in a vertebrate species that an alternative splicing and promoter usage generate two GnRH-R mRNA variants whose 5' extremities are encoded by two different exons. The analysis of the tissue distribution indicated that mRNA-2 presents a broader pattern of expression and is detected at higher levels than mRNA-1. Interestingly, it was found that those two mRNAs are differentially expressed in male and female gonads during gametogenesis. In particular, the variations of mRNA-1 levels parallel those of sGnRH expression during spermatogenesis, indicating that tissue-specific processing of the GnRH-R mRNA may underlie the effects of GnRH as a paracrine/autocrine regulator of gonadal functions.     

Effects of the aromatase inhibitor fadrozole on plasma sex steroid secretion,spermatogenesis and epididymis morphology in the lizard,Podarcis sicula

Recently, increasing importance has been attached to the role of estrogens and their receptors in male reproduction, since they have been found to be abundant in the male reproductive tract. In the lizard, Podarcis sicula, a seasonal breeder, estrogens seem to be involved in the regulation of testicular activity. Particularly, it has been hypothesized that the block of spermatogenesis and the complete regression of the epididymis and other secondary sexual characters (SSCs) in autumn might be due to high estrogen levels. To investigate the role of estrogens in the reproductive process of male lizards, we utilized Fadrozole ((AI) [4-(5,6,7,8-tetrahydroimidazole [1,5-a] pyridin-5-yl)-benzonitrile monohydrochloride] (CGS 16949A)), a nonsteroidal inhibitor of aromatase, the enzyme involved in the aromatization of androgens to estrogens, evaluating its effects on plasma sex-hormone release, spermatogenesis and epididymis morphology. For this purpose, adult male lizards, captured during the autumnal recrudescence, were intraperitoneally injected with 0.5 microg and 5 microg/g/body weight of AI for 15 and 30 days. In the animals treated with the higher AI dose, estrogen levels decreased if compared to the control groups, whereas androgen levels increased. Furthermore, histologic sections of testes and epididymes showed that the 30-day treatment with AI-induced spermatogenesis resumption with release of sperms into the large lumen of the seminiferous tubules, and the epididymes appeared more developed with moderately secreting columnar canal cells. Therefore, it is proposed that failure of spermatogenesis in autumn might be due to high estrogen levels.     

Effect of extracellular matrix proteins on in vitro testosterone production by rat Leydig cells

The aim of this study was to detect the effect of extracellular matrix (ECM) proteins on rat Leydig cell shape, adhesion, expression of integrin subunits and testosterone production, in vitro. Leydig cells isolated from adult rats were cultured on plates uncoated or coated with different concentrations of laminin-1, fibronectin, or type IV collagen in the presence or absence of hCG for 3 or 24 hr. A significant increase of cell adhesion and of alpha3, alpha5, and beta1 integrin subunit expression was observed when cells were cultured on ECM proteins, compared to those grown on uncoated plates. Leydig cells cultured on glass coverslips coated with ECM proteins for 24 hr exhibited elongated shapes with long cell processes (spreading), while cells cultured on uncoated plates showed few cell processes. A significant decrease in testosterone production was observed when basal and hCG-stimulated Leydig cells were cultured for 3 or 24 hr on plates coated with type IV collagen (12 and 24 microg/cm(2)) compared to uncoated plates. A significant though a slighter decrease in testosterone production was also observed in cells cultured on plates coated with fibronectin (12 and 24 microg/cm(2)), compared to uncoated plates. Laminin-1 did not modify testosterone production under basal or hCG stimulated conditions. These results suggest that ECM proteins are able to modulate Leydig cell steroidogenesis, in vitro.     

Cloning and expression analysis on a homolog of spermatogonial stem-cell renewal factor in Fenneropenaeus chinensis

The spermatogonial stem-cell renewal factor (SSRF) was named since its function in spermatogonial mitosis was reported in Japanese eel. Our previous study showed that a homolog of SSRF was highly expressed in the ovary of triploid shrimp, but not expressed in the ovary of diploid shrimp. To understand the function of SSRF in shrimp, the full-length cDNA of ssrf gene was cloned from Chinese shrimp Fenneropenaeus chinensis (Fcssrf) and its expression was analyzed. The full length of Fcssrf cDNA was 2588?bp and it contained an open reading frame encoding 450 amino acids. The predicted tertiary structure of FcSSRF was very similar to that of SSRF/eSRS34 from Anguilla japonica and TP/PD-ECGF from Homo sapiens. RT-PCR analysis showed that the Fcssrf was highly expressed in nerve, testis, hepatopancreas, gill, and stomach rather than in ovary. Expression of Fcssrf mRNA was not detected during embryonic stages and larval stages, from the nauplii to the post-larvae stage, in diploid, and triploid shrimp. However, it began to be expressed in juvenile stages (June–September) in diploid and triploid shrimp. Immunohistochemical analyses showed that FcSSRF was identified in both the diploid testis and triploid ovary. We inferred that the Fcssrf might be related to testis development.     

New host,geographic records,and histopathologic studies of Angiostrongylus spp (Nematoda: Angiostrongylidae) in rodents from Argentina with updated summary of records from rodent hosts and host specificity assessment

To date, 21 species of the genus Angiostrongylus (Nematoda:Angiostrongylidae) have been reported around the world, 15 of which are parasites ofrodents. In this study, new host, geographic records, and histopathologic studies ofAngiostrongylus spp in sigmodontine rodents from Argentina, withan updated summary of records from rodent hosts and host specificity assessment, areprovided. Records of Angiostrongylus costaricensis fromAkodon montensis andAngiostrongylus morerai fromsix new hosts and geographical localities in Argentina are reported. The gross andhistopathologic changes in the lungs of the host species due to angiostrongylosis aredescribed. Published records of the genus Angiostrongylus fromrodents and patterns of host specificity are presented. IndividualAngiostrongylusspecies parasitise between one-19 different hostspecies. The most frequent values of the specificity index (STD) were between 1-5.97.The elevated number of host species (n = 7) of A. morerai with a STD= 1.86 is a reflection of multiple systematic studies of parasites from sigmodontinerodents in the area of Cuenca del Plata, Argentina, showing that an increase insampling effort can result in new findings. The combination of low host specificityand a wide geographic distribution of Angiostrongylus spp indicatesa troubling epidemiological scenario although, as yet, no human cases have beenreported.     

Arginine vasopressin causes morphological changes suggestive of fluid transport in rat choroid plexus epithelium

Summary The experiments described herein use an in vitro preparation of choroid plexus to demonstrate that it is a vasopressin-responsive organ by morphologic criteria. Choroid plexus from rats was incubated for one hour in graded concentrations of arginine vasopressin (AVP). Within physiologic range of molar concentration, incubation in vasopressin induced a decrease in basal and lateral spaces in choroid plexus epithelial cells as well as an increase in number of dark cells. The number of cells with basal spaces decreased significantly from 82.7±9.2 in control tissue to 19±18 in tissue incubated in 10^-12 M AVP; similarly, the number with lateral cellular spaces decreased from 20±8.8 to 7.6±2.2 cells in 10^-10 M AVP. Dark cells increased in number from 3.8±2.6 in control conditions to 49±4 with 10^-9 M vasopressin. These data suggest important effects of arginine vasopressin in cerebrospinal fluid (CSF) on choroid plexus, compatible with enhanced fluid transport across choroid epithelial cells.     

Effect of green tea (Camellia sinensis L.) extract on morphological and functional changes in adult male gonads of albino rats

Green tea, prepared from the steamed and dried leaves of the shrub Camellia sinensis, is known for its antioxidant and anti-carcinogenic effects. However, its effects on male gonadal functions have not been explored adequately and the present investigation has been undertaken to evaluate the effect of green tea extract on gonads of adult male albino rats. Results of in vivo studies showed that green tea extract (GTE) at mild (1.25 g%, identical to 5 cups of tea/day), moderate (2.5 g%, identical to 10 cups of tea/day) and high (5.0 g%, identical to 20 cups of tea/day) doses, for a period of 26 days, altered morphology and histology of testis and accessory sex organs. A significant dose-dependent decrease in the sperm counts, inhibited activities of testicular delta(5)3beta-and 17beta-hydroxysteroid dehydrogenase (delta5-3beta3-HSD and 17beta3-HSD respectively) and decreased serum testosterone level were noticed. Significant increase in serum LH level was observed after moderate and high doses; serum FSH level also increased but not significantly. Histopathological examination showed inhibition of spermatogenesis evidenced by preferential loss of matured and elongated spermatids. Results of this study showed that GTE at relatively high dose may cause impairment of both the morphological and normal functional status of testis in rodents and thus its consumption at relatively high doses raises concern on male reproductive function in spite of its other beneficial effects.     

Effects of IL-18 on the proliferation and steroidogenesis of bovine theca cells: Possible roles in the pathogenesis of polycystic ovary syndrome

Interleukin 18 (IL-18) is a pleiotropic pro-inflammatory cytokine and is associated with arrested follicle development and anovulation which are the typical pathological changes of PCOS. Theca cells (TCs) have a key role in follicular growth and atresia. But whether IL-18 can directly affect ovarian TCs function is unknown. Therefore, the objective of this study was to determine the effect of IL-18 on proliferation and steroidogenesis of bovine TCs and to explore the biological effect of IL-18 on folliculogenesis. This work revealed that at 300-1000 pg/mL, IL-18 led to a time- and dose-dependently increase in cell proliferation (P < .05). IL-18 increased 17-hydroxyprogesterone (17OHP4) and androstenedione (A2) secretion with up-regulation of key steroidogenesis-related genes CYP11A1 and CYP17A1 (P < .05). Furthermore, our data demonstrated that the IL-18R protein is predominantly expressed in small-follicle (3-6 mm) TCs than large follicles (8-22 mm) by immunohistochemistry. We also found that the stimulation effects of IL-18 on TCs can be reversed with the addition of IL-18BP as early as at 4 hours of culture and reached the peak at 16 hours. We conclude that IL-18 appears to target TCs in bovine, and suggest an important role for this cytokine in ovarian function. Present findings further validate potential effects of IL-18 in the conditions associated with follicular dysplasia and excessive growth of ovarian TCs (such as PCOS). But additional research is needed to further understand the mechanism of action of IL-18 in theca cells as well as its precise role in folliculogenesis.     

Ecological causes of morphological evolution in the three‐spined stickleback

The central assumption of evolutionary theory is that natural selection drives the adaptation of populations to local environmental conditions, resulting in the evolution of adaptive phenotypes. The three‐spined stickleback (Gasterosteus aculeatus) displays remarkable phenotypic variation, offering an unusually tractable model for understanding the ecological mechanisms underpinning adaptive evolutionary change. Using populations on North Uist, Scotland we investigated the role of predation pressure and calcium limitation on the adaptive evolution of stickleback morphology and behavior. Dissolved calcium was a significant predictor of plate and spine morph, while predator abundance was not. Stickleback latency to emerge from a refuge varied with morph, with populations with highly reduced plates and spines and high predation risk less bold. Our findings support strong directional selection in three‐spined stickleback evolution, driven by multiple selective agents.     

Metabolic diversion of the phenylpropanoid pathway causes cell wall and morphological changes in transgenic tobacco stems

Studies involving transgenic plants with modifications in the lignin pathway reported to date, have received a relatively preliminary characterisation in relation to the impact on vascular integrity, biomechanical properties of tissues and carbon allocation to phenolic pools. Therefore, in this study transgenic tobacco plants (Nicotiana tabacum cv XHFD 8) expressing various levels of a bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) gene have been characterised for cell wall and related morphological changes. The HCHL enzyme converts p-coumaroyl-CoA to 4-hydroxybenzaldehyde thereby rerouting the phenylpropanoid pathway. Plants expressing high levels of HCHL activity exhibited reduced lignin deposition, impaired monolignol biosynthesis and vascular integrity. The plants also exhibited reduction in stem toughness concomitant with a massive reduction in both the cell wall esterified and soluble phenolics. A notable result of redirecting the carbon flux was the wall-bound accretion of vanillin and vanillic acid, probably due to the shunt pathway. Intracellular accumulation of novel metabolites such as hydroxybenzoic and vanillic acid derivatives also occurred in the transgenic plants. A line with intermediate levels of HCHL expression conferred correspondingly reduced lignin deposition, toughness and phenolics. This line displayed a normal morphology but distorted vasculature. Coloration of the xylem has been previously attributed to incorporation of alternative phenolics, whereas results from this study indicate that the coloration is likely to be due to the association of low molecular weight phenolics. There was no evidence of increased growth or enhanced cellulose biosynthesis as a result of HCHL expression. Hence, rerouting the phenylpropanoid biosynthetic pathway quantitatively and qualitatively modifies cell wall-bound phenolics and vascular structure.     

Androgen‐metabolizing enzymes show region‐specific changes across the breeding season in the brain of a wild songbird

The Lapland longspur (Calcarius lapponicus) is an arctic‐breeding songbird that shows rapid behavioral changes during a short breeding season. Changes in plasma testosterone (T) in the spring are correlated with singing but not territorial aggression in males. Also, T treatment increases song but not aggression in this species. In contrast, in temperate‐zone breeders, song and aggression are highly correlated, and both increase after T treatment. We asked whether regional or temporal differences in androgen‐metabolizing enzymes in the longspur brain explain hormone‐behavior patterns in this species. We measured the activities of aromatase, 5α‐reductase and 5β‐reductase in free‐living longspur males. Aromatase and 5α‐reductase convert T into the active steroids 17β‐estradiol (E₂) and 5α‐dihydrotestosterone (5α‐DHT), respectively. 5β‐Reductase deactivates T via conversion to 5β‐DHT, an inactive steroid. We examined seven brain regions at three stages in the breeding season. Overall, aromatase activity was high in the hypothalamus, hippocampus, and ventromedial telencephalon (containing nucleus taeniae, the avian homologue to the amygdala). 5β‐Reductase activity was high throughout the telencephalon. Activities of all three enzymes changed over time in a region‐specific manner. In particular, aromatase activity in the rostral hypothalamus was decreased late in the breeding season, which may explain why T treatment at this time does not increase aggression. Changes in 5β‐reductase do not explain the effects of plasma T on aggressive behavior. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 176–188, 1999