Developmental expression of the glutathione S-transferase Yo subunit in the rat testis and epididymis using light microscope immunocytochemistry

Background: Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione with various toxic electrophilic compounds. GSTs are composed of several classes based on the degree of sequence homology of their subunits. The Yo subunit, a member of the mu class, is expressed at high levels in the testis and epididymis. The purpose of this study was to immunolocalize the GST-Yo in these tissues during development. Methods: The testes and epididymides of rats aged 7, 15, 21, 28, 39, 42, 45, 49, and 56 days were fixed in Bouin's fixative, and immunostained for light microscopic analysis. Results: In the testis the cytoplasm of all germ cells was unreactive until day 39. At that time, step 18 spermatids appeared moderately reactive, while the few observed step 19 spermatids were intensely reactive as were their residual bodies. The presence of residual bodies indicates that spermiation takes place as early as day 39; however, the number of step 19 spermatids is low at this age. A progressive increase in the size of the tubule and number of elongating spermatids was seen between days 42 and 49. In addition, by day 49, a weak staining was observed in steps 12–15, moderate in steps 16–17, and intense in steps 18–19 spermatids. In terms of the intensity of staining, cell types stained, size of the tubules, and number of elongating spermatids, no difference was noted between day 49, 56, and adult animals. Thus Yo protein expression in germ cells reached maturity by day 49. The epithelial cells of the rete testis were intensely reactive at day 7 and remained so throughout development. In contrast, while the epithelial cells of the efferent ducts at day 7 were intensely reactive, they were weakly reactive by day 39 and remained so at later ages. Along the entire epididymis, the columnar epithelial cells showed a moderate apical/supranuclear reaction from day 7 to 28. By day 39 principal cells of the initial segment became weakly reactive, while those in the caput and corpus were moderately stained, a situation seen at later ages including adults. Only by day 49 did principal cells of the proximal cauda become moderately stained as seen in adult animals. Thus the expression of the Yo protein in the principal cells of the proximal cauda may be regulated by different factors than those of the caput and corpus epididymidis. Alternatively, the expression of the Yo subunit in principal cells of the proximal cauda may develop later since this region would be the last to receive luminally derived testicular products. In the initial segment, the decrease in staining of principal cells at day 39 may be due to an inhibiting factor emanating from the testis. Spermatozoa appeared in the lumen of each epididymal region well after the expression of Yo had reached its adult staining pattern indicating that they are not a factor. Conclusions: Overall these results suggest that the expression of GST-Yo in the various cells of the testis and epididymis are controlled by different factors during postnatal development. © 1994 Wiley-Liss, Inc.     

Developmental expression of sulfated glycoprotein-2 in the epididymis of the rat

Background: Sulfated glycoprotein-2 (SGP-2), also designated as clusterin, is a protein secreted by the epididymis and which binds to spermatozoa. In adult rats it is secreted at high levels by principal cells of the distal initial segment, intermediate zone and caput epididymidis, and at relatively lower levels by principal cells of the corpus and cauda epididymidis. The objective of this study was to correlate the developmental events in the maturation of the epididymis with the timing of SGP-2 expression in order to evaluate the testicular or epididymal factors which may regulate it. Methods: Our approach was to follow and compare the developmental expression of SGP-2 by immunocytochemistry in normal untreated control rats and rats whose efferent ducts were ligated on day 15 and examined at different postnatal ages thereafter. Results: In control animals, SGP-2 expression in principal cells of the distal initial segment, intermediate zone, and caput and distal cauda epididymidis, as characterized in normal 90-day-old adult animals, was attained between postnatal days 39 and 49. However, only by postnatal day 56 did SGP-2 display in the corpus and proximal cauda the characteristic secretory pattern found in adult rats. In contrast, in efferent duct ligated rats examined at postnatal day 64, SGP-2 was absent in principal cells of the corpus and proximal cauda epididymidis but continued to be secreted by the distal initial segment, intermediate zone, and caput and distal cauda epididymidis. Furthermore, unlike the case in control rats, SGP-2 was secreted at high levels by the principal cells of the proximal initial segment. Thus during normal postnatal development, in the proximal initial segment, the production of SGP-2 is suppressed by luminal factors originating from the testis, while in the distal initial segment, intermediate zone, and caput epididymidis, it is unaffected by these factors. On the other hand, the production of SGP-2 in the corpus and proximal region of the cauda epididymidis is normally stimulated by luminal factors originating from the testis, while in the distal cauda, it is unaffected by these factors. Conclusions: Our results thus show a differential regulation of SGP-2 expression in principal cells of the proximal versus distal regions of the epididymis and even within subdivisions of each region. In some regions of the epididymis, SGP-2 production appears to be unaffected by luminal factors originating from the testis, while in other regions it is either inhibited or stimulated by these factors. © 1994 Wiley-Liss, Inc.     

Developmental expression of immobilin in the rat epididymis

Background: Immobilin is a protein secreted by principal cells of the distal initial segment, intermediate zone and caput epididymidis of adult rats, which serves to immobilize spermatozoa. In the distal cauda, epithelial clear cells are involved in its endocytosis. The objective of this study was to correlate the developmental events in the maturation of the epdidymis with the timing of immobilin secretion and endocytosis in order to evaluate the testicular or epididymal factors which may influence or regulate immobilin expression. Methods: Our approach was to follow and compare the developmental expression of immobilin by light microscope immunocytochemistry in control and efferent duct ligated rats of different postnatal ages. Results: Coincident with the morphological maturation of the principal cells by postnatal day 39, immobilin displayed the characteristic secretory immunostaining pattern found in adults. This adult-like expression occurred despite the absence of spermatozoa in the lumen but was coincident with high levels of circulating and luminal androgens. In contrast, immobilin secretion in rats whose efferent ducts were ligated at day 15 was weak to non-existent in the principal cells of the caput epididymidis at day 28 and remained so into adulthood, indicating that principal cells of this region of the epididymis are dependent either directly or indirectly upon testicular factors present in the lumen for immobilin expression. However, secretion of immobilin in the principal cells of the distal initial segment was unaffected by ligation and unlike the case in control rats high levels of immobilin also continued to be secreted into adulthood by the principal cells of the proximal initial segment. Thus in the distal initial segment immobilin secretion is not regulated by luminal factors originating from the testis, while in the proximal initial segment the normal suppression of immobilin that occurs by postnatal day 39 is. Despite ligation, endocytosis of immobilin by clear cells of the distal cauda epididymidis occurred by day 49, indicating that luminal testicular factors are not essential for stimulating the uptake of immobilin by these cells. Conclusions: The results taken together suggest that there are stimulatory and inhibitory luminal testicular factors involved in the regional development of immobilin secretion in the epididymis. There are also immobilin secreting regions in the epididymis, whose secretory development is independent of luminal testicular factors. © 1994 Wiley-Liss, Inc.     

环磷酰胺对大鼠睾丸和附睾的影响

目的探讨抗肿瘤药物所致大鼠睾丸和附睾损害,为寻找男性不育症中药治疗的研究提供理论依据。方法选用16只15周龄SD大鼠随机分为对照组和实验组,每组8只;实验组腹腔注射环磷酰胺20mg·kg-1·d-1,连续5d,用药2个月后,应用HE染色法研究大鼠睾丸、附睾远期组织学变化,用原位缺口末端标记法(TUNEL方法)检测生精细胞凋亡。结果实验组大鼠体重、睾丸和附睾重量均显著减轻(P<0.01),睾丸生精小管直径缩小、间距增宽、生精上皮变薄、生精细胞层次和数量减少、生精小管腔多未见精子形成,实验组睾丸生精小管直径、面积、生精上皮细胞数、均显著低于对照组(P<0.01);实验组生精细胞凋亡增多,与对照组比较,生精细胞显著凋亡(P<0.01);附睾管管腔内腔内精子稀少,含有大量脱落细胞,管壁变薄。结论环磷酰胺对大鼠睾丸、附睾远期损害明显,促进生精细胞凋亡。     

Gene expression in the efferent ducts,epididymis, and vas deferens during embryonic development of the mouse

The tissues of the male reproductive tract are characterized by distinct morphologies, from highly coiled to un‐coiled. Global gene expression profiles of efferent ducts, epididymis, and vas deferens were generated from embryonic day 14.5 to postnatal day 1 as tissue‐specific morphologies emerge. Expression of homeobox genes, potential mediators of tissue‐specific morphological development, was assessed. Twenty homeobox genes were identified as either tissue‐enriched, developmentally regulated, or both. Additionally, ontology analysis demonstrated cell adhesion to be highly regulated along the length of the reproductive tract. Regulators of cell adhesion with variable expression between the three tissues were identified including Alcam, various cadherins, and multiple integrins. Immunofluorescence localization of the cell adhesion regulators POSTN and CDH2 demonstrated cell adhesion in the epithelium and mesenchyme of the epididymis may change throughout development. These results suggest cell adhesion may be modulated in a tissue‐specific manner, playing an important role in establishing each tissue's final morphology. Developmental Dynamics 239:2479–2491, 2010. © 2010 Wiley‐Liss, Inc.     

Expression pattern of galectin-1 and galectin-3 in rat testes and epididymis during postnatal development

Galectins are a family of lectins-binding beta-galactosides involved in a variety of extracellular and intracellular processes, thereby contributing to homeostasis, cell adhesion, cellular turnover, and immunity. This study aimed to determine the localization and expression of galectin-1 (Gal-1) and galectin-3 (Gal-3) in the testis and epididymis of rats at postnatal [(prepubertal (day 5), pubertal (day 20), postpubertal (day 50) and mature (day 70)] periods by using immunohistochemistry and Western blotting. Gal-1 and Gal-3 were differentially expressed in different types of cells in the testis and epididymis during postnatal development. While we detected Gal-1 expression in some spermatogenic cells and Leydig cells in the testis, not in the epididymal epithelium, Gal-3 was expressed in Sertoli cells, peritubular myoid cells, Leydig cells, smooth muscles and interstitial CD68-positive macrophages. Epithelial cells of the corpus and cauda epididymis showed an intense Gal-3 expression. Gal-1 expression was higher in the testis than in the epididymis on days 50 and 70. The expression of Gal-3 in the testis increased from the prepubertal to mature period. While the expression difference of Gal-3 was not statistically significant in the testis and epididymis until puberty, Gal-3 expression in the postpubertal and mature periods was higher in the epididymis. The expression of Gal-3 in the corpus and cauda epididymis was higher than that in the caput epididymis. In conclusion, our findings suggest that puberty has potential regulatory effect on the expression of galectins in testis and epididymis of rats. Gal-1 and 3 may play a role in the development of the reproductive system and the preservation of the immune-privileged environment in the testis, due to their pro-apoptotic and anti-apoptotic functions. The presence of intense expression of Gal-3 in the corpus and cauda epididymis may contribute to the maturation and storage of spermatozoa.     

己烯雌酚对幼年香猪睾丸与附睾雌激素受体和一氧化氮合酶表达和分布的影响

目的 探讨己烯雌酚（DES）对幼年香猪睾丸和附睾中雌激素受体（ERs）和内皮型一氧化氮合酶（eNOS）表达的影响,及DES对诱导雄性动物生精异常的作用机制。 方法 将20只20～25日龄幼年雄性香猪分成４组,对照组只注射生理盐水,实验组经腹腔每天注射DES(实验1组：0.03mg/kg;实验2组：0.3mg/kg;实验3组：3mg/kg),连续注射9d。最后１次注射24h后手术取出左侧睾丸和附睾组织经中性多聚甲醛固定,石蜡包埋,常规制备5μm石蜡切片,采用免疫组织化学SP法检测睾丸和附睾组织中ERα、ERβ和eNOS的表达。 结果 ERα只在输出小管上皮细胞胞核中表达;ERβ在输出小管上皮细胞、间质细胞及附睾管上皮细胞胞核中均有表达,在睾丸生精小管细胞胞质中也有少量表达。DES能够使ERα在输出小管中的表达率显著下降（P<0.05）。随着DES用量的增多,ERβ在输出小管和附睾管中的表达率显著升高（P<0.05）,当DES用量为3mg/kg时ERβ在输出小管和附睾管中的表达率明显高于用量0.03mg/kg和0.3mg/kg（P<0.05）。但0.03mg/kg用量的DES却使睾丸生精小管中ERβ的表达率显著性下降（P<0.05）,并在0.3mg/kg和3mg/kg用量时在睾丸生精小管中未能检测到ERβ受体的表达。eNOS在睾丸生精小管细胞和输出小管上皮细胞中都有表达,DES能促进eNOS在睾丸生精小管细胞中表达（P<0.05）而降低其在输出小管细胞中表达（P<0.05）。结论 DES明显影响ERs和eNOS在睾丸和附睾中的表达。     

Ultrastructure and immunocytochemical characteristics of cells in the octopus cell area of the rat cochlear nucleus: comparison with multipolar cells

Cells in the octopus cell area of the rat ventral cochlear nucleus have been connected to the monaural interpretation of spectral patterns of sound such as those derived from speech. This is possible by their fast onset of firing after each octopus cell and its dendrites have been contacted by many auditory fibres carrying different frequencies. The cytological characteristics that make these large cells able to perform such a function have been studied with ultrastructural immunocytochemistry for glycine, GABA and glutamate, and compared to that of other multipolar neurons of other regions of the ventral cochlear nucleus. Cells in the octopus cell area have an ultrastructure similar to large-giant D-multipolar neurons present in other areas of the cochlear nucleus, from which they differ by the presence of a larger excitatory axo-somatic synaptic input and larger mitochondria. Octopus cells are glycine and GABA negative, and glutamate positive with different degree. Large octopus cells receive more axo-somatic boutons than smaller octopus cells. Fusiform octopus cells are found sparsely within the intermediate acoustic striae. These cells are large to giant excitatory neurons (23-35 microm) with 62-85% of their irregular perimeter covered with large axo-somatic synaptic boutons. Most boutons contain round vesicles and are glycine and GABA negative but glutamate positive. The latter excitatory boutons represent about 70% of the input to octopus cells. Glycine positive boutons with flat and pleomorphic vesicles account for 9-10% of the input while GABA-ergic boutons with pleomorphic vesicles represent about 20% of the synaptic input. Other few, multipolar cells within the rat octopus cell area are surrounded by more inhibitory than excitatory terminals which contain flat and pleomorphic vesicles, a feature distinctive from that of true octopus cells. The latter resemble multipolar cells seen outside the octopus cell area that project to the contralateral inferior colliculus and cochlear nucleus. Based on this study, two types of large multipolar cells are present in the octopus cell area: 1) those that receive about 70% of axo-somatic R boutons and stain more intensely for glutamate may correspond to pure onset neurons (Oi); 2) those with less than 33% of R axosomatic boutons, with less immunoreactivity to glutamate and sometimes glycine positive may represent the onset chopper neurons (Oc). In the octopus cell area the first type appears more prevalent. The present study suggests that octopus cells are a special type of excitatory D-multipolar neuron confined to the octopus cell area and mainly innervated by glutamatergic cochlear nerve terminals.     

Immunocytochemical and ultrastructural heterogeneities of normal and glibenclamide stimulated pancreatic beta cells in the rat

When studied morphologically in semi-thin sections in the rat in vivo, pancreatic beta cells displayed heterogeneous immunoreactivities for insulin and amylin, depending on the islet size and the intra-islet position of the beta cells. In larger islets, cortical beta cells (beta cells with contacts with all islet cell types and with the exocrine parenchyma) which are located in the periphery were more densely immunostained for insulin and amylin than medullary beta cells (beta cells with contacts only with other beta cells) which are located in the centre of the islet. Ultrastructurally, these findings were accompanied by differences in the number of secretory granules and mitochondria. Beta cells in small islets and at extra-islet sites exhibited a dense immunoreactivity. After administration of glibenclamide, immunoreactivities for insulin and amylin were diminished in a time-dependent manner, decreasing first in medullary and thereafter in cortical beta cells of larger islets. Ultrastructurally, the beta cells exhibited the typical signs of stimulation. A minority of beta cells in small islets and all beta cells in extra-islet locations remained unchanged. Thus pancreatic beta cells under basal and stimulatory conditions in vivo exhibit heterogeneity in hormone content and in ultrastructural features. These differences may represent the basis for a functional heterogeneity of the insulin secretory response of the individual beta cell both in vivo and in vitro in states of normal and impaired insulin secretion. As heterogeneity was observed only among beta cells in islets, while single beta cells surrounded by acinar cells exhibited no changes in insulin immunoreactivity, interactions between beta cells as well as between beta cells and other endocrine cells may be critical for expression of heterogeneity within the beta cell population.     

Changes in the arrangement of actin filaments in myoid cells and sertoli cells of rat testes during postnatal development and after experimental cryptorchidism

Background: Abundant actin filaments are present in myoid cells and Sertoli cells in the testis. In the adult rat, the filaments form a lattice arrangement within the myoid cell, and show a hexagonal pattern in the basal junctional regions of Sertoli cells. Methods: Isolated seminiferous tubules and frozen sections were prepared from juvenile to adult Wistar rat testes, stained with FITC-conjugated phalloidin, and observed by confocal microscopy. Unilateral cryptorchidism was induced in adult rats, and seven days later, their testes were also examined. Results: In the myoid cell, parallel actin filaments running circularly around the seminiferous tubules were observed at 15 and 20 days of age. Then, at 30 days, actin filaments arranged longitudinally along the tubular long axis appeared in addition to the circular bundles. A lattice arrangement of actin-filament bundles in myoid cells became obvious at 40 days, when elongated spermatids are found in the tubule. Actin filaments in the basal junctional regions of Sertoli cells did not acquire the hexagonal pattern seen in the adult testis until 30 days of age. In the cryptorchid testes, the arrangement of actin filaments in the both cells showed a remarkable change compared to the control testis; the filaments became thinner and disrupted. Conclusions: A lattice arrangement of the actin filaments in the myoid cell appear at around 30 days, before the completion of spermatogenesis. A hexagonal pattern of the filaments in the junctional regions of Sertoli cells has already developed at this age. Cryptorchidism affects the actin filaments of the both cells. © 1995 Wiley-Liss, Inc.     

Localization of orexin B and orexin-2 receptor in the rat epididymis

The peptides orexin A (OXA) and orexin B (OXB) derived from the proteolytic cleavage of a common precursor molecule, prepro-orexin, were originally described in the rat hypothalamus. Successively, they have been found in many other brain regions as well as in peripheral organs of mammals and other less evolved animals. The widespread localization of orexins accounts for the multiple activities that they exert in the body, including the regulation of energy homeostasis, feeding, metabolism, sleep and arousal, stress, addiction, and cardiovascular and endocrine functions. Both OXA and OXB peptides bind to two G-coupled receptors, orexin-1 (OX1R) and orexin-2 (OX2R) receptor, though with different binding affinity. Altered expression/activity of orexins and their receptors has been associated with a large number of human diseases. Though at present evidence highlighted a role for orexins and cognate receptors in mammalian reproduction, their central and/or local effects on gonadal functions remain poorly known. Here, we investigated the localization of OXB and OX2R in the rat epididymis. Immunohistochemical staining of sections from caput, corpus and cauda segments of the organ showed intense signals for both OXB and OX2R in the principal cells of the lining epithelium, while no staining was detected in the other cell types. Negative results were obtained from immunohistochemical analysis of hypothalamic and testicular tissues from OX2R knock-out mice (OX2R^?/?) and OX1R/OX2R double knock-out (OX1R^?/?; OX2R^?/?) mice, thus demonstrating the specificity of the rabbit polyclonal anti-OX2R antibody used in our study. On contrary, the same antibody clearly showed the presence of OX2R in sections from hypothalamus and testis of normal mice and rats which are well known to express the receptor. Thus, our results provide the first definite evidence for the immunohistochemical localization of OXB and OX2R in the principal cells of rat epididymis.     

甘氨酸受体α_1亚基在乳鼠心肌细胞的表达及损害性因素对其表达的影响

目的:研究甘氨酸受体α1亚基(GlyRα1)在乳鼠心肌细胞中的表达以及脂多糖(LPS)、缺氧/复氧(H/R)、异丙肾上腺素(ISO)和高糖(HG)对其表达的影响。方法:体外培养乳鼠心肌细胞,Western blotting方法检测心肌细胞上GlyRα1的表达;心肌细胞分别用LPS、H/R、ISO以及HG处理24 h,采用CCK-8试剂检测细胞活力,Western blotting方法检测心肌细胞上GlyRα1的表达。结果:Western blotting方法检测到乳鼠心肌细胞上GlyRα1的表达;LPS(20 mg/L)、ISO(100μmol/L)以及HG(25mmol/L)处理心肌细胞24 h与心肌细胞H/R 3 h对心肌细胞存活率无明显影响;LPS组、H/R 3 h组以及ISO组心肌细胞上GlyRα1表达均高于对照组(P0.01),而HG组心肌细胞上GlyRα1表达低于对照组(P0.01)。结论:乳鼠心肌细胞上存在GlyRα1,并且一定浓度的LPS、ISO与一定时间的H/R均可上调乳鼠心肌细胞GlyRα1的表达,而HG可下调乳鼠心肌细胞GlyRα1的表达。     

Quantitative analysis of GABA-like-immunoreactive and parvalbumin-containing neurons in the CA1 region of the rat hippocampus using a stereological method,the disector

The numerical density of neurons in the CA1 region of the rat dorsal hippocampus has been estimated by a stereological method, the disector, using pairs of video images of toluidine blue-stained, plastic-embedded, 0.5-m-thick sections, 3 m distant from each other. The chemical properties of those disector-counted cells were further analyzed by postembedding immunocytochemical methods on adjacent, semithin sections using antibodies against gamma-aminobutyric acid (GABA) and a specific calcium-binding protein, parvalbumin (PV). The density of neurons in the CA1 region was 35.2 × 10³/mm³; numerical densities in the stratum oriens (SO), stratum pyramidale (SP), and strata radiatum-lacunosum-moleculare (SRLM) were 11.3 × 10³/mm³, 272.4 × 10³/mm³, and 1.9 × 10³/mm³, respectively. The numerical densities of GABA-like immunoreactive (GABA-LIR) and PV-immunoreactive (PV-IR) neurons were 2.1 × 10³/mm³ and 1.1 × 10³/mm³, respectively, which were 5.8% and 3.2% of all neurons, respectively. In the CA1 region only about 60% of PV-positive neurons were GABA-LIR. However, taking the previous observation into consideration that almost all hippocampal PV-positive neurons were immunoreactive for the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), neurons that were immunoreactive to either GABA or PV or both (GABA+ and/or PV + neurons) were regarded as a better representative of GABAergic neurons in this region; thus, the numerical density of these GABA + and/or PV + neurons was 2.5 × 10³/mm³ and they were 7.0% of all neurons in the CA1 region. Lamellar analysis showed that the numerical densities of GABA+ and/or PV+, GABA-LIR, and PV-IR neurons were highest in the SP, where they were 8.2 × 10³/mm³, 6.2 × 10³/mm³, and 5.4 × 10³/mm³, respectively. The results of the present study indicate that the proportions of GABAergic neurons and a subpopulation of them, PV-containing GABAergic neurons, to other presumable non-GABAergic neurons are far smaller in the CA1 region of the hippocampus than in several neocortical regions previously reported.     

The expression pattern of PARP-1 and PARP-2 in the developing and adult mouse testis

Although the importance of the PARP family members in the adult testis has already been acknowledged, their expression in the developing testis has not been addressed. We performed immunohistochemistry by using PARP-1 and PARP-2 antibodies on the developing mouse testis at embryonic day (E) 15.5, E17.5, postnatal day (PN) 0, PN3, PN9, PN20 and adult. Our results showed that at embryonic and early postnatal days, the expression of PARP-1 was in the nuclei of gonocytes and spermatogonia. PARP-1 was positive in interstitial cells with nuclear localization at all studied ages. At embryonic and early postnatal days, the expression of PARP-2 was in the cytoplasm of gonocytes and spermatogonia. During the progress of spermatogenesis, PARP-2 was localized in the cytoplasm of pre-leptotene spermatocytes on PN9, in the cytoplasm of pachytene spermatocytes on PN15 and in the cytoplasm of round spermatids on PN20. In the adult, PARP-2 staining can still be observed in the cytoplasm of spermatogonia, but to a much lesser degree than in the round and elongating spermatids. For all the studied ages, PARP-2 was positive in Sertoli cells and interstitial cells with cytoplasmic localization. Our results indicate that PARP proteins are present in germ and somatic cells during testis development in mice.     

Central distribution of efferent and afferent components of the pudendal nerve in rat

Summary Central distribution of efferent and afferent components of the pudendal nerve was examined in the rat by the horseradish peroxidase (HRP) method after HRP application to the central cut end of the pudendal nerve. The pudendal motoneurons were located in the dorsolateral, dorsomedial and lateral groups at L5 and L6. Each of the dorsolateral and dorsomedial groups constituted a slender longitudinal cell column. Pudendal motoneurons in the lateral group were scattered at L5, rostrodorsally to the dorsolateral group. The neurons in the dorsolateral and lateral groups were labelled with HRP applied to the nerve branch innervating the ischiocavernosus and sphincter urethrae muscles. The neurons in the dorsomedial group were labelled with HRP applied to the branch supplying the sphincter ani externus and bulbospongiosus muscles. Some dendrites of pudendal motoneurons in the dorsomedial group extended to the contralateral dorsomedial group. These crossing dendrites were observed not only in male rats but also in female. The average number of the pudendal motoneurons in the dorsolateral and dorsomedial groups were larger in male rats than in female. A few neurons of the intermediolateral nucleus at upper L6 were also labelled with HRP applied to the dorsalis penis (clitoridis) nerve. Axon terminals of the pudendal nerve were distributed, bilaterally with an ipsilateral predominance, to the gracile nucleus, as well as to the dorsal horn and dorsal commissural gray from L4 to S2. A few labelled axons were seen in the intermediolateral nucleus at L6 and S1. Axon terminals from the dorsalis penis nerve were distributed more medially in the dorsal horn than those from the perinealis nerve.     

Developmental changes in transmission of respiratory rhythm in the rat

We used cross-correlation to examine the short time-scale synchronisation of left and right phrenic nerve discharges in in-situ preparations of rats over a range of ages, to investigate the development of respiratory rhythm transmission to phrenic motoneurones. We found central peaks in the cross-correlograms, indicative of short time-scale synchronisation, at all ages (2-41 days), whose half-amplitude widths varied inversely with age (40-1.8 ms). In 10 preparations < or =5-days-old the central peaks were unaffected by a mid-sagittal section from C3 to C6. Carbenoxalone (CBX), a gap junction blocker, and its inactive analogue glycyrrhzic acid (GZA), eliminated central peaks in preparations younger than 12 days but not in older preparations. We concluded that in rats older than approximately 12 days short time-scale synchronisation is produced by bilaterally-projecting axons of medullary pre-motor neurones, whereas in younger rats it is due to pre-synaptic synchronisation of left and right medullary pre-motor neurones. While the latter mechanism may be gap junction connections, these experiments cannot unequivocally demonstrate it.     

Analysis of RhoE expression in the testis,epididymis and ductus deferens,and the effects of its deficiency in mice

Rho proteins are a large family of GTPases involved in the control of actin cytoskeleton dynamics, proliferation and survival. Rnd1, Rnd2 and RhoE/Rnd3 form a subfamily of Rho proteins characterized by being constitutively active. The role of these proteins has been studied during the last years in several systems; however, little is known about their expression and functions in the reproductive organs. In this work we analysed the localization and the effect of RhoE deficiency in the testes using mice lacking RhoE expression (RhoE gt/gt), and our research shows some unexpected and relevant results. First, we have observed that RhoE is only expressed in Leydig cells within the testicular parenchyma and it is absent of seminiferous tubules. In addition, RhoE is expressed in the excurrent ducts of the testis, including the ductuli efferentes, epididymis and ductus deferens. Moreover, the testes of postnatal 15‐day‐old RhoE null mice are smaller, both in absolute values and in relation to the body weight. Furthermore, the dimensions of their seminiferous tubules are also reduced compared with wild‐types. In order to study the role of RhoE in the adult, we analysed heterozygous animals as RhoE null mice die early postnatally. Our results show that the testes of adult RhoE heterozygous mice are also smaller than those of the wild‐types, with a 17% decrease in the ratio testis weight/body weight. In addition, their seminiferous tubules have reduced tubular diameter (12%) and a thinner epithelial wall (33%) that appears disorganized and with a swollen lumen. Finally, and probably as a consequence of those alterations, the sperm concentration of heterozygous animals was found to be lower than in the wild‐types. These results indicate that accurate levels of RhoE in the testes are necessary for a correct development and function of male gonads, and suggest novel and unexpected roles of Rnd GTPases in the reproductive physiology.     

Cytology, synaptology and immunocytochemistry of commissural neurons and their putative axonal terminals in the dorsal cochlear nucleus of the rat

The first binaural integration within the auditory system responsible for sound localization depends upon commissural neurons that connect the two symmetrical cochlear nuclei. These cells in the deep polymorphic layer of the rat dorsal cochlear nucleus were identified with the electron microscope after injection of the retrograde tracer, Wheat Germ Agglutinin conjugated to Horseradish Peroxydase, into the contralateral cochlear nucleus. Commissural neurons are multipolar or bipolar with an oval to fusiform shape. Few commissural neurons, most inhibitory but also excitatory, connect most of the divisions of the rat cochlear nuclei. The most common type is a glycinergic, sometimes GABAergic, moderately large cell. Its ergastoplasm is organized into peripheral stacks of cisternae, and few axo-somatic synaptic boutons are present. Another type of commissural neuron is a medium-sized, spindle-shaped cell, glycine and GABA-negative, with sparse ergastoplasm and synaptic coverage. A giant, rare type of commissural neuron is glycine-positive and GABA-negative, with short peripheral stacks of ergastoplasmic cisternae. It is covered with synaptic boutons, many of which contain round synaptic vesicles. Another rare type of commissural neuron is a moderately large cell, oval to fusiform in shape, immunonegative for both glycine and GABA, and contacted by many axo-somatic boutons. It contains large dense mitochondria and numerous dense core vesicles of peptidergic type. Some labelled boutons, mostly inhibitory and probably derived from commissural neurons, contact pyramidal, cartwheel, giant and tuberculo-ventral neurons. The prevalent inhibition of electrical activity in a cochlear nucleus observed after stimulation of the contralateral cochlear nucleus may be due to commissural inhibitory terminals which contact excitatory neurons such as pyramidal and giant cells. Other inhibitory commissural terminals which contact inhibitory neurons such as cartwheel and tuberculo-ventral neurons, may explain the stimulation of electrical activity in the DCN after contralateral stimulation.