SLC9A3 Protein Is Critical for Acrosomal Formation in Postmeiotic Male Germ Cells.

Solute carrier family 9 isoform 3 (SLC9A3), a Na⁺/H⁺ exchanger, regulates the transepithelial absorption of Na⁺ and water and is primarily expressed on the apical membranes of the intestinal epithelium, renal proximal tubule, epididymis, and vas deferens. Loss of the Slc9a3 allele in mice enhances intestinal fluid and causes diarrhoea as a consequence of diminished Na⁺ and HCO3- absorption. Hence, the loss also causes male infertility and reveals the abnormal dilated lumen of the rete testis and calcification in efferent ductules. However, whether loss of Slc9a3 alleles also disrupts mammalian spermatogenesis remains unknown. First, through immunoblotting, we determined that SLC9A3 is highly expressed in the murine testis compared with the small intestine, epididymis, and vas deferens. During murine spermatogenesis, SLC9A3 is specifically expressed in the acrosome region of round, elongating, and elongated spermatids through immunostaining. Furthermore, SLC9A3 signals are enriched in the acrosome of mature sperm isolated from the vas deferens. In Slc9a3 knockout (KO) mice, compared with the same-aged controls, the number of spermatids on the testicular section of the mice progressively worsened in mice aged 20, 35, and 60 days. Sperm isolated from the epididymis of Slc9a3 KO mice revealed severe acrosomal defects. Our data indicated that SLC9A3 has a vital role in acrosomal formation during spermiogenesis.

Plasticity of basal cells during postnatal development in the rat epididymis.

Our previous study has shown that basal cells sense luminal factors by forming a narrow body projection that can cross epithelial tight junctions. As a first step toward characterizing the structural plasticity of basal cells, in this study, we followed their appearance and morphology in the rat epididymis and vas deferens (VD) during postnatal development and examined their modulation by androgens in adulthood. Immunofluorescence labeling for cytokeratin 5 showed that basal cells are absent at birth. They progressively appear in a retrograde manner from the VD and cauda epididymis to the initial segments during the postnatal weeks PNW1-3. At the onset of differentiation, basal cells are in contact with the lumen and their nucleus is located at the same level as that of adjacent epithelial cells. Basal cells then position their nucleus to the base of the epithelium, and while some are still in contact with the lumen, others have a 'dome-shaped' appearance. At PNW5-6, basal cells form a loose network at the base of the epithelium, and luminal-reaching basal cells are rarely detected. The arrival of spermatozoa during PNW7-8 did not trigger the development of projections in basal cells. However, cells with a narrow luminal-reaching projection began to reappear between PNW8 and PNW12 in the corpus and the cauda. Treatment with flutamide from PNW10 to PNW12 significantly reduced the number of luminal-reaching basal cell projections. In summary, basal cells exhibit significant structural plasticity during differentiation. Fewer apical-reaching projections were detected after flutamide treatment in adulthood, indicating the role of androgens in the luminal-sensing function of basal cells.

Developmental changes in methylation of spermatogenesis-specific genes include reprogramming in the epididymis.

We have determined the status of DNA methylation at specific sites in three spermatogenesis-specific genes, Pgk-2, ApoA1 and Oct-3/4, throughout the development and differentiation of male germ cells in the mouse. We observed a specific demethylation event in the Pgk-2 gene in prospermatogonia at about the time of birth, about 10 days before the onset of transcription which first occurs in primary spermatocytes. All three genes were unmethylated in adult spermatogenic cells in the testis, but were remethylated in mature spermatozoa in the vas deferens. Surprisingly, we found that this remethylation is part of the process of sperm maturation which occurs in the epididymis.

Spatial and temporal evolution of imposex in dogwhelk Nucella lapillus (L.) populations from North Wales, UK.

Nucella lapillus imposex and organotin tissue contamination were assessed, during 2006, at twenty sites in North Wales, between Anglesey and Shell Island on the Lleyn Peninsula. Vas Deferens Sequence Index (VDSI), Relative Penis Size Index (RPSI) and the percentage of affected females (%I) were used to assess imposex levels which varied between 0.5 and 3.8 for VDSI, 0.0 and 11.5% for RPSI and 49 and 100% for %I. Tributyltin (TBT) and triphenyltin (TPT) concentrations in whole tissues varied between 0.8 and 39 and 0.4 and 2.1 ng Sn/g dry weight, respectively. TBT represented the higher fraction of butyltin compounds in the tissues, suggesting that TBT inputs continue to occur. Comparisons with nineteen years of data collected during previous studies demonstrated that there had been a significant reduction in imposex levels over the last two decades following the introduction of legislative restrictions in the U.K. regarding the use of organotin based antifouling paints.

Exposure to 9-cis retinoic acid induces penis and vas deferens development in the female rock shell, Thais clavigera.

To clarify how tributyltin (TBT) and triphenyltin (TPT) interact with the retinoid X receptor (RXR) to induce growth of male sex organs in female gastropods, we treated female rock shells (Thais clavigera) with three different concentrations (0.1, 1, or 5 microg/g wet wt) of 9-cis-retinoic acid (9CRA) or with a single concentration (1 microg/g wet wt) of TBT, TPT, or fetal bovine serum (as a control). The effects of each treatment were measured as the incidence of imposex, the length of the penis-like structure, and the vas deferens sequence (VDS) index. 9CRA induced imposex in a dose-dependent manner; imposex incidence was significantly higher in the rock shells that received 1 (P < 0.05) or 5 microg (P < 0.001) 9CRA than in the controls. After 1 month, the rock shells treated with 5 microg 9CRA exhibited substantial growth of the penis-like structure that was not as evident in the other treated shells. The length of the structure differed between the 0.1- and 5-microg 9CRA treatment groups (P < 0.05) but not between the 1- and 5-microg 9CRA treatment groups (P > 0.05). Compared with the control, the VDS index increased significantly in the 1- (P < 0.05) and 5-microg (P < 0.001) 9CRA groups. The penis-like structures behind the right tentacle in female rock shells treated with 5 microg 9CRA were essentially the same as the penises and vasa deferentia of normal males and of TBT-treated or TPT-treated imposexed females. These results further support the hypothesis that imposex in gastropods could be mediated by RXR.

Histological structure and age-related changes in the luminal diameter of the excurrent duct system of guinea cocks (Numida meleagris) and associated changes in testosterone concentrations.

As little information is available on the reproductive system of guinea fowl (Numida meleagris), a study was conducted on 49 male guinea fowl to document the histological structure and developmental changes in the luminal diameter of the ducts within the excurrent duct system and associated changes in concentrations of testosterone. Age-related changes were analyzed using the Kruskal-Wallis test and medians separated by the Mann-Whitney U-test. Tubuli recti were clearly visible in the guinea fowl and the rete testes were both intracapsular and extracapsular. Regardless of age, the luminal diameter of the proximal ductuli efferentes was the largest, while that of the connecting duct was the smallest. The luminal diameter of all ducts within the epididymal region increased (P < 0.001) monthly until 20 wk of age, and then increased marginally every month thereafter. Peripheral testosterone concentrations also peaked at 20 wk of age and declined thereafter. In adult birds, the ductus deferens enlarged posteriorly, from an average of about 279 µm cranially to 678 µm caudally. Peripheral testosterone concentrations strongly and positively correlated with the luminal diameter of ducts within the excurrent duct system. The pattern of increase in the luminal diameter of all ducts followed the pattern of testosterone secretion in these birds, which indicates that testosterone concentrations may be closely related to the development of the excurrent duct system in male guinea fowl.

Étant donné le peu d'informations disponibles sur le système reproducteur de la pintade (Numida meleagridis), une étude a été menée sur 49 pintades mâles afin de documenter la structure histologique et les changements développementaux dans le diamètre de la lumière des tubes à l'intérieur du système de tubes excréteurs et les changements associés dans les concentrations de testostérone. Les changements associés à l'âge ont été analysés par le test de Kruskal-Wallis et les médianes séparées par le test de U de Mann-Whitney. Les tubes droits étaient clairement visibles chez les pintades et les rete testis étaient intracapsulaires et extracapsulaires. Indépendamment de l'âge, le diamètre de la lumière des canaux efférents était le plus large, alors que celui du canal connecteur était le plus petit. Le diamètre de la lumière de tous les canaux à l'intérieur de la région de l'épididyme a augmenté (P < 0,001) mensuellement jusqu'à 20 semaines d'âge, et augmenta par la suite de manière marginale à chaque mois. Les concentrations périphériques de testostérone ont également atteint un pic à 20 sem d'âge et ont décliné par la suite. Chez les oiseaux adultes, le canal déférent s'élargissait postérieurement, d'une moyenne d'environ 279 µm cranialement jusqu'à 678 µm caudalement. Les concentrations périphériques de testostérone corrélaient fortement et positivement avec le diamètre de la lumière des canaux dans le système de tubes excréteurs. Le patron de l'augmentation de la lumière de tous les canaux suivait le patron de sécrétion de testostérone chez ces oiseaux, ce qui indique que les concentrations de testostérone pourraient être intiment associées au développement du système de tubes excréteurs chez la pintade mâle.(Traduit par Docteur Serge Messier).

Transcriptomic Analysis of Male Black Tiger Shrimp (Penaeus monodon) After Polychaete Feeding to Enhance Testicular Maturation.

To reveal molecular mechanism of how polychaetes enhanced reproductive maturation in the male black tiger shrimp (Penaeus monodon), transcriptomic profiles of male reproductive organs (testes and vas deferens) between polychaete-fed and commercial pellet-fed male brooders were compared using cDNA microarray. The overall profiles were distinguishingly different between the two feed groups as well as between testes and vas deferens. Additionally, six of 11 differentially expressed gene identified by the microarray (HNRPUL1 and GCP4 in testes, MAT2B, CDC16, and CSN5 in vas deferens, and SLD5 in both organs) were validated by quantitative real-time PCR (qPCR) and found to exhibit significantly higher expression levels in polychaete-fed shrimp than those in commercial pellet-fed shrimp. From microarray and qPCR results, the differentially expressed transcripts in both testes and vas deferens between different feeds belonged to DNA replication and microtubule nucleation pathways. Interestingly, while the transcripts involved in nutrient uptake and nucleotide biosynthesis were increased only in testes, those involved in protein refolding and apoptosis were increased only in vas deferens. These findings suggest that polychaetes may enhance spermatogenesis by increasing spermatogonia proliferation in testes and by regulating mature spermatozoa in vas deferens.

Bi-species imposex monitoring in Galicia (NW Spain) shows contrasting achievement of the OSPAR Ecological Quality Objective for TBT.

Imposex is decreasing worldwide after the total ban on tributyltin (TBT) from antifouling paints. In order to assess improvement in the NE Atlantic, the OSPAR Convention designed an Ecological Quality Objective (EcoQO) based on the VDSI (vas deferens sequence index, an agreed measure of imposex) in the rock snail Nucella lapillus; wherever this is not available, the mud snail Nassarius reticulatus was proposed as a proxy. We determined VDSI in Galician populations of rock (n≥34) and mud (n≥18) snails at regular intervals from pre-ban times until 2009 and 2011, respectively. While imposex in the former started decreasing in 2006 and by 2009 the EcoQO had been met in the area, VDSI in the latter was not significantly reduced until 2011 and values contradict such an achievement. This suggests that the OSPAR imposex bi-species scheme may not be of direct application in the current post-ban scenario.

Age-associated and tissue-specific expression of osteopontin in male Hu sheep reproductive tract.

Osteopontin (OPN) is indispensable in mammalian reproduction, but the role of OPN in male reproductive tract and fertility remains unclear. The objective of this study is to elucidate the function of OPN by unveiling the localization and expression of OPN in the reproductive tract (testis, epididymis, and ductus deferens) of male Hu sheep in different ages (10-days, 4-months, and 8-months). To accomplish this, the localization, mRNA and protein expression patterns of OPN in all samples were investigated. Immune staining showed that OPN was present in the testicular interstitium of prepubertal Hu sheep testis (10-days and 4-months group), while it was immunostained in acrosomes of spermatids nearby adluminal compartment of seminiferous tubules in sexual maturity Hu sheep testis (8-months group). The localization of OPN in epididymis gradually changed from the loose connective tissue to the apical region of principal cells (pseudostratified columnar epithelium) with growing (10-days to 8-months). In addition, increase trend was observed in the mRNA expression levels of OPN with growing in the same reproductive tissues (P<0.05). Furthermore, two different OPN isoforms of 30kDa and 34kDa were detected in the reproductive tract of male Hu sheep by western blot. Immunofluorescence detection showed that OPN was localized in the cauda epididymal spermatozoa. These results suggested that the expression of OPN might be closely related to spermatogenesis and spermatozoa function in Hu sheep. This will be helpful for us to understand how OPN regulate the high reproductive capacity in Hu sheep.

Imposex levels in the dogwhelk Nucella lapillus (L.)--continuing improvement at high latitudes.

In 1990, restrictions on the use of tributyltin (TBT)-based antifouling paints were implemented in Iceland. A previous study showed that the level of imposex in the dogwhelk, Nucella lapillus, in Icelandic waters had decreased significantly between 1992 and 1998. In this study, we repeated the survey on imposex in N. lapillus at 33 locations from the Icelandic coast in 2003. The results indicated that both Vas Deferens Sequence Index (VDSI) and Relative Penis Size Index (RPSI) had further declined in 13 locations since 1998. Among these 13 sites, RPSI was reduced to zero in five cases. While improvements from 1992/1993 to 1998 were seen in reduced levels of imposex near both large and small harbours, the pattern from 1998 to 2003 was somewhat different, with improvement mainly observed near smaller harbours. No significant changes in imposex levels near larger harbours occurred over this period. Although the imposex levels still remain high near the large harbour complexes in Reykjavík and Hafnarfjördur, it is evident that regulations, including the use of less toxic antifouling paints and community action, have lead to substantial improvements in the marine environment of Iceland. International Maritime Organisation's ban on the application of TBT after 2003 is apparently necessary to allow further improvements in larger harbours. The environmental effects of new antifoulants replacing TBT need to be further evaluated.

Developmental and hormonal regulation of specific proteins in mouse vas deferens and seminal vesicle.

This paper is concerned with hormonal regulation of the developmental pattern of major proteins of the mouse vas deferens (mouse vas deferens protein: MVDP, 34.5 kD) and seminal vesicle (15.5, 120 and 140 kD) whose expression is regulated by testosterone at adulthood. The ontogeny of these proteins, studied by SDS-polyacrylamide gel electrophoresis, appeared to be uncoordinated. MVDP was not accumulated until animals were 20 days old and its concentration increased sharply from 20 to 30 days of age. In seminal vesicle, the 15.5 kD protein did not accumulate before day 30 whereas 120 and 140 kD proteins appeared and accumulated between 30 and 40 days. In 30-day-old mice castrated at birth or treated with cyproterone acetate over 29 days, MVDP levels were not abolished and were similar to those measured in 20-day-old males. Testosterone administration, from 1 to 10 days of age, did not induce precocious expression of MVDP. These results suggest that the neonatal expression of MVDP is independent of androgens. In seminal vesicle, the first expression of the 3 proteins studied was dependent upon testicular androgens as shown by neonatal castration and injection experiments. The marked increase in the levels of the 4 proteins studied, during sexual maturation, was not associated with quantitative or qualitative changes in tissular androgen concentrations, suggesting that other factors may be necessary for protein expression. Whereas thyroxine may induce a precocious accumulation of MVDP, prolactin had no stimulatory effect on the accumulation of proteins from vas deferens and seminal vesicle. The results suggest that during sexual maturation gene activation by androgens was progressive.

Age-, cell- and region-specific immunoexpression of estrogen receptor alpha (but not estrogen receptor beta) during postnatal development of the epididymis and vas deferens of the rat and disruption of this pattern by neonatal treatment with diethylstilbestrol.

This study in rats sought to 1) characterize immunoexpression of estrogen receptor alpha (ERalpha) and ERss in the efferent ducts, epididymis, and vas deferens during postnatal development; 2) establish whether ER expression changed after neonatal treatment with diethylstilbestrol (DES); and 3) determine whether ER changes coincided with abnormal epididymal/vas development. Rats were administered 10 microg DES or vehicle on days 2, 4, 6, 8, 10, and 12 and were sampled on days 10, 18, 25, 35, and 90+. At all ages, ERalpha was immunoexpressed intensely in the efferent ducts. On day 10, immunoexpression of ERalpha was absent from the epididymis and vas, but was detectable on day 18 in epithelial cells in the caput, corpus, and proximal cauda. Epithelial expression of ERalpha was absent from the distal cauda and in the proximal and distal vas was confined to a band of periductal stromal cells. Thus, on day 18, the site of ERalpha expression delineated the epididymis-vas boundary. On days 25-35, epithelial expression of ERalpha was absent, but stromal expression persisted in the vas and distal cauda. In adults, immunoexpression of ERalpha in the epididymis and vas was absent. In contrast, ERbeta was immunoexpressed in epithelial cells and some stromal cells in the efferent ducts, epididymis, and vas at all ages. In the vas, stromal expression of ERalpha and ERbeta was in different layers. DES treatment caused 1) underdevelopment of the epididymal duct and reduced epithelial height in epididymis and vas; 2) coiling of the extraepididymal vas; 3) thickening of the periductal actin-free stromal layer in the distal cauda and vas; and 4) reduced cell proliferation on day 18 in the epididymis and vas, based on incorporation of bromodeoxyuridine, especially in the epithelium. These changes coincided with abnormalities in cell- and region-specific immunoexpression of ERalpha, but not ERbeta. Thus, in DES-treated rats on day 18, epithelial expression of ERalpha occurred in all regions of the epididymis and vas instead of being confined to the caput, corpus, and proximal cauda as in controls. Similarly, stromal ERalpha expression in the vas of DES-treated rats was not confined to a periductal layer as in controls, but occurred diffusely in the muscle layer. It is suggested that 1) estrogens play a role in peripubertal development of the epididymis and vas; 2) the cellular site of expression of ERalpha either plays a role in or reflects demarcation of the epididymal/vas boundary; and 3) blurring of this boundary in DES-treated rats coincides with altered ERalpha immunoexpression.

Induction of reproductive tract developmental abnormalities in the male rat by lowering androgen production or action in combination with a low dose of diethylstilbestrol: evidence for importance of the androgen-estrogen balance.

This study tested the hypothesis that testis/reproductive tract abnormalities induced in the rat by neonatal treatment with diethylstilbestrol (DES) result from disturbance of the androgen-estrogen balance. Male rats were treated neonatally with a dose of DES (0.1 micro g) that induced either no or small effects on its own or with a dose (10 micro g) that induced major reproductive tract abnormalities. To allow quantification, the abnormalities chosen for study were distension of the rete testis and efferent ducts and reduction in epithelial cell height in the efferent ducts and vas deferens. To alter the androgen-estrogen balance, other rats were treated with DES (0.1 micro g) in combination with a treatment to suppress either androgen production [GnRH antagonist (GnRHa)] or androgen action (flutamide); other rats were treated with GnRHa or flutamide alone. Testosterone levels were measured to verify the effects of treatment. Combined administration of DES (0.1 micro g) plus GnRHa or flutamide induced significantly greater distension/overgrowth of the rete testis and efferent ducts (ED) and a reduction in epithelial cell height of the ED than did DES (0.1 micro g) administered alone. Neither GnRHa nor flutamide affected rete or ED distension when administered alone, but both significantly reduced ED epithelial cell height. Neonatal treatment with bisphenol-A (100 micro g) with or without GnRHa had no significant effect on any of these parameters. In contrast to the ED, a reduction in cell height of the vas deferens was induced to an equal extent by DES (10 micro g), DES (0.1 micro g) with GnRHa, and GnRHa alone, suggesting greater sensitivity of this tissue to both androgen and estrogen action. The induction of major abnormalities in rats treated with DES (10 micro g) was coincident with loss of androgen receptor immunoexpression in affected tissues. Reduced androgen receptor immunoexpression was also induced by combined treatment with DES (0.1 micro g) plus GnRHa or flutamide, whereas treatment with any of these compounds alone had no or only minor effects. These findings suggest that reduced androgen action sensitizes the reproductive tract to estrogens, demonstrating that the balance in action between androgens and estrogens, rather than their absolute levels, may be of fundamental importance in determining normal or abnormal development of some regions of the male reproductive tract.

Comparison of the effects of the 5 alpha-reductase inhibitor finasteride and the antiandrogen flutamide on prostate and genital differentiation: dose-response studies.

Studies were performed to compare the effects of 5 alpha-reductase inhibition and antiandrogen receptor blockade on differentiation of male internal and external genital structures and prostate in the rat. Dose-response studies were performed on male rats treated in utero during the period of sexual differentiation with either the potent 5 alpha-reductase inhibitor finasteride or the antiandrogen flutamide. The treated animals were raised to adulthood and killed, and genital structures were evaluated. Treatment with the 5 alpha-reductase inhibitor finasteride at a dose of 25 mg/kg.day resulted in significant feminization of the external genitalia. There was no further feminization of the genitalia at doses up to 300 mg/kg.day. Wolffian ductal differentiation occurred at all doses evaluated. Seminal vesicle weight, however, significantly decreased at 25 mg/kg.day, but without a further decrease at higher doses of the 5 alpha-reductase inhibitor. Vas deferens and epididymal weights were unchanged at all doses evaluated. There was a significant decrease in prostate size at 25 and 50 mg/kg.day, with no further decrease at higher doses. In flutamide-treated animals, complete feminization of the genitalia occurred at 24 mg/kg.day in all animals. At 18 mg/kg.day, Wolffian ductal differentiation occurred, but seminal vesicle weight was decreased. At dosages of 100, 200, and 300 mg/kg.day flutamide, the vas deferens was absent unilaterally or bilaterally, with small remnants of epididymal head and tail present. At dosages of 24 mg/kg.day and above, the prostate was absent. Studies with the 5 alpha-reductase inhibitor finasteride demonstrate the dependency of prostate and male external genital differentiation on dihydrotestosterone (DHT). However, unlike androgen receptor blockade with flutamide, finasteride did not totally abolish prostate differentiation or completely feminize the external genitalia, despite increasingly higher doses. Since there is no evidence of multiple 5 alpha-reductase isoenzymes to date in the rat, these results suggest that testosterone (T) can compensate for DHT to some degree at the level of the androgen receptor. Wolffian differentiation, however, was not affected by inhibition of DHT, demonstrating its T dependency, but seminal vesicle growth was impaired. Thus, inhibition of 5 alpha-reductase activity limits seminal growth potential in adulthood. Studies with the antiandrogen flutamide show that at doses significantly above that required to completely block prostate differentiation and cause genital feminization, Wolffian ductal differentiation is significantly impaired. Thus, higher doses of flutamide are needed to block the paracrine effect of T on the Wolffian ducts.

Maturational and maintenance effects of testosterone on terminal axon density and neuropeptide expression in the rat vas deferens.

Testosterone causes growth of many pelvic ganglion cells at puberty and their maintenance during adulthood. Here we have focused on two populations of pelvic ganglion cells that project to the rat vas deferens: noradrenergic neurons that innervate the smooth muscle and synthesize neuropeptide Y, and cholinergic neurons that primarily innervate the mucosa and contain vasoactive intestinal peptide. We have assessed the muscle innervation after pre- or postpubertal castration, using immunohistochemistry to determine axon density and radioimmunoassay to quantify levels of neuropeptides in tissue extracts. Our results show that androgen deprivation in each period causes substantial effects. Noradrenergic axons in the muscle increase in density after castration, partly due to organ size being smaller than age-matched controls. However, when corrected for target size, there is an overall decrease in total number of axons. This implies that androgen exposure at puberty has a direct effect on neurons to ensure that the adult pattern of innervation is attained, and that this is not simply by matching terminal field to target size. Similar effects of pre- and postpubertal castration imply that continued exposure to testosterone is necessary to maintain normal target innervation. Castration in both time periods increased the density of axons containing vasoactive intestinal peptide, however the effects of castration on the total number of these axons in the muscle were more variable. The concentration of vasoactive intestinal peptide increased substantially following either pre- or postpubertal castration although absolute amounts per vas deferens were decreased. Effects on neuropeptide Y concentration were less pronounced but the total amount per vas deferens was decreased after pre- or postpubertal castration. Our study shows that the action of testosterone (or a metabolite) on a pelvic ganglion cell soma is likely to reflect a change in its terminal field, but that these effects are not mediated simply by testosterone influencing the size of its target organ.

Abolition of the nerve-induced responses of the isolated vas deferens of the young rat by agents blocking -adrenoceptors.

The mechanical responses of isolated fieldstimulated vasa deferentia from rats 3-10 days old were abolished by low doses of drugs blocking alpha-adrenoceptors. In preparations from older animals this effect was absent or there was a potentiation of the responses. The effect of atropine also changed during postnatal development. In the early period a moderate (10-40%) decline of the nerve-induced responses was generally observed, but later even high concentrations of atropine were without effect.

Postnatal development of delta-opioid receptor subtypes in mice.

1. The density and affinity of binding sites for the delta-selective opioid ligands [3H]-[D-Ala2, Asp4]deltorphin (DELT-I), [3H]-[D-Ala2Glu4]-deltorphin (DELT-II), [3H]-[D-Pen2,D-Pen5]enkephalin (DPDPE), and [3H]-naltrindole (NTI) were determined in whole brain from 10, 15, 25 and 60 day-old C57BL mice. 2. At all ages, the analyses of the homologous displacement curves, gave best fits to single rather than to multiple site models. The binding capacity (Bmax) labelled by [3H]-NTI was about one half that labelled by [3H]-DELT-I, [3H]-DELT-II and [3H]-DPDPE. In 25 and 60 day-old mouse brain the DPDPE Bmax was 25% less than the deltorphin-II Bmax. 3. In saturation experiments, specific binding of [3H]-DELT-I on adult mouse brain homogenates was best fitted by a two-site model (34%, high affinity site, Kd = 1.08 nM and 66% low affinity sites, Kd = 39.9 nM). 4. DPDPE produced a biphasic inhibition of specific [3H]-DELTI-I binding, from 15 days of age onwards. The relative percentage of high and low affinity sites was 72% and 28% in 15 day-, 65% and 35% in 25 day- and 30% and 70% in 60 day-old mice. 5. In adult mouse brain labelled with [3H]-DELT-I, DELT-II recognized 71% of high-affinity and 29% of low-affinity sites DELT-I and DPDPE produced monophasic inhibition of specific [3H]-DELT-II binding to brain homogenates of adult mice. 6. These data suggest that a sub-population of delta-sites (probably the delta 2-subtype), recognized by DELT-I, with high affinity for DELT-II and low affinity for DPDPE develops from 25 days onward. 7. In electrically stimulated mouse vas deferens (MVD) the rank order of potency of the three delta-agonists was: DELT-I > DELT-II > DPDPE in 10 day-old mice: and DELT-I- DELT-II > DPDPE, from 25 days onward. During this time, the potency of DELT-II increased about 15 fold whereas the potency of DELT-I and DPDPE increased only 5 times. The higher efficacy of DELT-II could depend on receptor maturation towards the delta 2-subtype.

Post-natal development of functional neurotransmission in rat vas deferens.

Responses of the rat vas deferens to drugs and to field stimulation were examined in sexually immature rats. The vasa from immature rats often exhibited spontaneous contractions and displayed greater sensitivity to the contractile effects of alpha-adrenoceptor agonists. The responses of the vasa from immature rats to single pulse field stimulation lacked the adrenergic component of the response although the non-adrenergic component was present. The responses were antagonized by alpha 2-adrenoceptor agonists. In the presence of cocaine, an adrenergic component of the response did appear. During trains of pulses the pre- and postjunctional effects of adrenergic transmission which are found in adult rats were absent in vasa from immature rats. Electron microscopic studies showed no qualitative differences in adrenergic innervation in vasa from immature and adult rats. It is concluded that a state of 'pre-innervation supersensitivity' associated with a lack of functional adrenergic transmission exists in the vas deferens of immature rats. The supersensitivity disappears and functional transmission develops during the period in which testosterone secretion increases in the rat. The reason for the lack of functional transmission at a time when the innervation appears to be morphologically mature is not clear but may be due to the noradrenaline release mechanism not being fully operative.

Characterization and ontogeny of P1-purinoceptors on rat vas deferens.

1. The P1-purinoceptors which mediate the inhibition by adenosine of nerve-mediated contraction of the rat vas deferens have been investigated by use of the agonists N6-cyclopentyladenosine (CPA) and 5'-N-ethylcarboxamidoadenosine (NECA) and the A1-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). The ontogeny of the responses to adenosine and to the two co-transmitters which induce the contractions in this tissue, adenosine 5'-triphosphate (ATP) and noradrenaline (NA), have also been studied. 2. The order of potency for the adenosine agonists in inhibiting the nerve-mediated contractions was CPA = NECA > adenosine. Micromolar concentrations of DPCPX were required to antagonize the inhibition by adenosine and NECA of nerve-mediated responses, whereas the inhibitory effect of CPA was antagonized by nanomolar concentrations of the antagonist. 3. NECA and adenosine inhibited contractions induced by ATP (10 microM) or by NA (10 microM), NECA being at least ten fold more potent than adenosine, whereas CPA was inactive. Micromolar concentrations of DPCPX were required to antagonize the effect of adenosine on the contractions induced by ATP (10 microM). 4. Nerve-stimulated contractions could be observed in neonatal tissues from day 15 and increased with age, and could be inhibited by adenosine from this time, the potency of adenosine decreasing with age. Responses to ATP also appeared at day 15 and increased with age up to day 25, while responses to NA were present from day 10 (the earliest day tested) and decreased with age. 5. These results show that the rat vas deferens contains both prejunctional Al-receptors and postjunctional A2-receptors, and that adenosine acts on the latter populations to inhibit nerve-mediated contractions.The high potency of adenosine in the neonate and the parallel development of responses to ATP and to nerve-mediated contractions support suggestions that purinergic responses may be particularly important in neonatal tissues.

Androgen dependence during development of the mouse vas deferens protein mRNA.

The mRNA encoding a major protein of the mouse vas deferens (MVDP) was first detected in 10-day-old males and its concentration increased sharply between 10 and 20 days, reaching adult levels at 40 days. This increase was not associated with an increase in tissular androgen concentrations. In 30-day-old mice castrated at birth or treated with cyproterone acetate over 29 days, MVDP mRNA levels were not abolished and were similar to those measured in 10- and 20-day-old controls. These results suggest that the neonatal expression of MVDP gene is independent of androgens. In addition, precocious accumulation of MVDP mRNA could be induced by injection of excess amounts of androgens in 20- but not in 10-day-old animals. The prepubertal increase in MVDP mRNA levels is androgen-dependent but other factors may be necessary for MVDP expression.