Immunolocalization of Aquaporins 1 and 9 in the Ram Efferent Ducts and Epididymis

Aquaporins (AQPs) are essential membrane protein channels for the transport of water across membranes. Fluid movement in the epididymis is important for modulation of the luminal environment, in which sperm mature and reside. This study was designed to understand the morphology and localization of AQPs in ram efferent ducts (ED) and epididymis. For this purpose, the epididymis of seven animals were removed for histologic and immunohistochemical analyses. AQP1 immunoreactivity was observed in the apex of the ED, and AQP9 was found adjacent to the nuclei of the epithelial cells of the ED. The epithelial lining of ram epididymis is pseudostratified columnar and presents principal, basal, apical and narrow cells. In the initial segment (IS), a moderate reaction for AQP1 was observed in the apical cytoplasm of epithelial cells. An intense reactivity for AQP1 was noted over the microvilli of principal cells and in spermatozoa in the caput. In the corpus and cauda, AQP1 was noted only over the endothelial cells of vascular channels located in intertubular spaces. A weak‐to‐moderate reaction for AQP9 was observed in the nuclei of epithelial cells in the IS, caput and corpus of the epididymis. In the cauda, an intense reaction to AQP9 was observed in the epithelial border. In the IS, caput and corpus, the reactivity for AQP9 differed from those observed in domestic animals. The cauda showed a pattern similar to that previously described. These results indicate that AQPs 1 and 9 have reversed locations and roles in rams, suggesting activity variations related with fluid and solute absorption throughout the epididymis.     

绵羊附睾褪黑素受体1的表达与定位

为研究褪黑素受体1（MT1）在不同年龄绵羊附睾中的表达模式，选用幼龄绵羊（2~3月龄）、青年绵羊（6~8月龄）和成年绵羊（2~3岁）的附睾，采用实时荧光定量PCR和免疫组化技术检测不同年龄绵羊附睾各部位MT1基因mRNA的表达量和MT1的分布情况。结果表明：幼龄绵羊附睾尾MT1基因的转录水平极显著高于附睾头和附睾体（P<0.01）；青年绵羊附睾体和附睾尾MT1基因的转录水平显著高于附睾头（P<0.05）；成年绵羊附睾尾MT1基因的转录水平显著高于附睾头和附睾体（P<0.05），附睾各部位MT1基因的转录水平随年龄增长呈明显降低趋势；定位结果显示，MT1在各年龄组绵羊附睾的各个部位均有分布，且主要分布在附睾上皮细胞中。综合上述结果，不同年龄绵羊附睾的不同部位均有MT1表达和分布，并随年龄增长各部位的表达量降低，相同年龄绵羊附睾尾MT1的表达水平较高。     

Localization of Oestrogen Receptors in the Epididymis During Sexual Maturation of the Domestic Cat

Oestrogens are involved in regulation of spermatogenesis and sperm maturation and are essential for male fertility. To study the role of oestrogens on epididymal function in the domestic cat, we analyzed the localization patterns of oestrogen receptors (ERs) within the epididymis of juvenile, pubertal and adults using immunohistochemistry. Cat epididymal tissues obtained during routine castrations were fixed in chilled Bouin's solution and processed for immunohistochemistry with ER-specific antibodies. For a certain receptor type, ER localization was influenced by donor age. In the juvenile epididymis, ERα was localized in the nuclei of epithelial cells of efferent ducts and undifferentiated epithelium of the ductus epididymis. During puberty, ERα localization in the undifferentiated epithelium of the epididymis shifted from the nuclei to the cytoplasm and plasma membrane. Oestrogen receptor-α level was highest in the pubertal and adult epididymis, especially within the cytoplasm and in plasma membranes of caput epithelial cells. This finding was suggestive of a role in fluid reabsorption within the efferent ducts and the epididymis. In corpus and cauda regions, ERα was less abundant, suggesting a minor role for oestrogens in sperm storage areas. Interestingly, localization of ERβ was neither influenced by age nor location within the epididymis and was ubiquitous throughout. Results demonstrate that oestrogen actions within the epididymis may be predominantly mediated through ERα during sexual maturation in the domestic cat.     

The Ductus Epididymis of the Alpaca: Immunohistochemical and Lectin Histochemical Study

Our objective was to characterize epithelial cells lining the epididymal duct (caput, corpus, cauda) of the alpaca using AE1/AE3 cytokeratin antibodies and a battery of different lectins: Con-A, UEA-I, LTA WGA, GSA-II, GSA-IB4, SBA, PNA, ECA, DBA, MAL-II and SNA. Sialidase digestion and deglycosylation pre-treatments were also employed. The principal cells (PCs) along the epididymis showed differences in immunostaining patterns toward keratin antibodies. Lectin histochemistry demonstrated variations in the content and distribution of glycosidic residues of glycoconjugates in different epididymal regions. In particular, staining of the Golgi zone in the epithelial PCs was interpreted as evidence for synthesis and secretion of O- and N-linked oligosaccharides. In the caput, the apical mitochondria-rich cells contained mainly β-GalNAc, subterminal α-GalNAc, α-Gal and Neu5Ac α2,3Gal residues. Conversely, in the corpus they were particularly rich in α-GalNac and β-Gal-(1–3)- d -GalNAc linked to sialic acid moieties. Basal cells mainly expressed β-GalNAc and α-Gal in the caput, α-Gal in the corpus and α-Fuc and β-GalNAc in the cauda. The differences in immunostaining patterns and in lectin histochemistry in the alpaca epididymis reported in this investigation seem to be related to regional differences in function.     

Immunolocalization of insulin-like growth factor-I (IGF-I) and its receptors (IGF-IR) in the equine epididymis

Insulin-like growth factor plays a paracrine/autocrine role in regulating testicular function in the stallion, but its presence in the equine epididymis remains unknown. The aim of this study was to test the hypothesis that insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR) are localized in the caput, corpus, and cauda of the epididymis in an age-dependent manner. Immediately after castration, epididymal tissue was fixed, paraffin-embedded, and processed for immunohistochemistry (IHC). Western blot was also performed using equine epididymal extracts to verify the specificity of the antibodies against IGF-I and IGF-IR. Immunolabeling of IGF-I was observed in the cytoplasm of principal and basal cells in the caput, corpus, and cauda at the pre-pubertal (3–7 months), pubertal (12–18 months), post-pubertal (2–4 years), and adult stages (4.5–8 years). Immunolabeling of IGF-IR was observed in the cytoplasm of principal cells in all regions of the epididymis in each age group. Immunolabeling of IGF-IR was also detected in the cytoplasm of basal cells from animals of all ages. Bands observed by Western blot corresponded to the molecular weights of IGF-I and IGF-IR, ~23 kDa and 95 kDa, respectively. These results suggest that IGF-I might function as an autocrine and/or paracrine factor during the development, maintenance and/or secretions of the stallion epididymis.     

Histological Characteristics of Adult Yak Epididymis in Different Breeding Seasons

To compare the histological changes of the adult yak's epididymis in different breeding seasons,six adult yak testis in breeding season and nine adult yak testis in breeding interval were collected for structure investigation by HE staining,Masson's and Gomori's histochemistry methods,and IPP (Image-Pro Plus) statistics method was used to quantitative statistics.The results showed that comparing with the adult yak in the breeding interval,the epididymis ducts of adult yak in the breeding season were covered with the columnar ciliated epithelium.The collagen and reticular fiber in cauda epididymis were obviously more abundant than caput and corpus epididymitis.And the thickness of columnar epithelium cells in caput and corpus epididymitis,the length of the cilia in caput,and also the internal and external lumen diameter of caput and corpus epididymitis were all significantly increased in the breeding season (P<0.05),but the external lumen diameter of the cauda epididymis had no significant differences (P>0.05).In conclusion,the research showed that the distribution of collagen and reticular fiber in adult yak's epididymis interstitial were similar,and they were more rich in cauda epididymis,which might relate to the capacity and the sperm transport;The changes of the epithelial thickness,the length of cilia,the internal and external lumen diameter were close related to the different breeding seasons,and it might be a common phenomenon in plateau mammals that the enlargement and reduction of the epididymal duct in different breeding seasons.     

Expression of Occludin in Testis and Epididymis of Wild Rabbits, Lepus sinensis coreanus

Tight junctions (TJs) in inter-Sertoli junctional areas and epididymal epithelia build up the blood–testis barrier (BTB) and the blood–epididymal barrier (BEB), respectively. In this study, the expression of occludin, an integral member of the TJs, was examined in testis and different regions of epididymis of Lepus sinensis coreanus , an Korean wild rabbit species. In testis, intense occludin immunoreactivity was found in the basally located inter-Sertoli junctional area together with diffused immunoreactivity of occludin in the cytoplasm of Sertoli cells. It can be suggested that occludin is one of the robust elements of BTB in seminiferous tubules of rabbit testis. In proximal and distal caput epididymis, occludin immunoreactivity was found in the lateral as well as apical contacts of epithelial cells. In corpus epididymis, intense occludin immunoreactivity was found in the basolateral as well as apical contacts of epithelial cells together with cytoplasmic signal. In cauda epididymis, occludin immunoreactivity in luminal epithelia was relatively strong but largely found in the cytoplasm. This suggests that intriguing regulatory mechanisms differentially recruit occludin to the TJ in the different regions of epididymal epithelia. The differences in the subcellular localization as well as expression levels of occludin among the epididymal segments may reflect differential paracellular permeability of epithelia along the epididymal tubules and be correlated with sperm maturation in rabbit. In Western blot, a major form of occludin was MW 62 kDa together with small fragments of MW 34–39 kDa in testis and epididymis, suggesting the peptide cleavage of occludin. This is the first report on the molecular nature of TJs in a wild rabbit testis and epididymis.     

Comparative rna-seq analysis of region-specific miRNA expression in the epididymis of cattleyak

The epididymis is the site of post-testicular sperm maturation, which constitutes the acquisition of sperm motility and the ability to recognize and fertilize oocytes. The role of miRNA in male reproductive system, including the control of different steps leading to proper fertilization such as gametogenesis, sperm maturation and maintenance of male fertility where the deletion of Dicer in mouse germ cells led to infertility, has been demonstrated. The identification of miRNA expression in a region-specific manner will therefore provide valuable insight into the functional differences between the regions of the epididymis. In this study, we employed RNA-seq technology to explore the expression pattern of miRNAs and establish some miRNAs of significant interest with regard to epididymal sperm maturation in the CY epididymis. We identified a total of 431 DE known miRNAs; 119, 185 and 127 DE miRNAs were detected for caput versus corpus, corpus versus cauda and caput versus cauda region pairs, respectively. Our results demonstrate region-specific miRNA expression in the CY epididymis. The GO and KEGG enrichment for the predicted target genes indicated the functional values of miRNAs. Furthermore, we observed that the expression of miR-200a was downregulated in the caput, compared with cauda. Since the family of miR-200 has previously been suggested to contribute to the distinct physiological function of sperm maturation in epididymis of adult rat, we speculate that the downregulation of miR-200a in CY caput epididymis may play an important role of sperm maturation in the epididymis of CY. Therefore, our findings may not only increase our understanding of the molecular mechanisms regulated by the miRNA functions in region-specific miRNA expression in the CY epididymis, it could provide a valuable information to understand the mechanism of male infertility of CY.     

不同繁殖季节成年牦牛附睾组织特征比较

为比较不同繁殖季节成年牦牛附睾组织结构特征变化,应用苏木精-曙红常规染色、Masson's和Gomori's特殊组织化学染色方法比较不同繁殖季节牦牛（6头繁殖期成年牦牛和9头繁殖间期成年牦牛）附睾的组织结构特点并用IPP图像分析软件进行定量分析。结果显示,与繁殖间期相比,繁殖期附睾尾间质胶原纤维和网状纤维较附睾头及附睾体明显增多;附睾头和附睾体柱状上皮厚度显著增加（P<0.05）,附睾头纤毛长度增加显著（P<0.05）;附睾头和附睾尾管腔内径及外径显著增高（P<0.05）;但是附睾尾管腔外径繁殖期与繁殖间期相比差异不显著（P>0.05）。本研究结果表明,成年牦牛附睾管外围网状纤维与胶原纤维分布一致,二者在附睾尾较为丰富,可能与其较强的收缩能力及精子运输有关;柱状纤毛上皮高度及纤毛长度、管腔内径与外径的变化与其所处的不同繁殖季节密切相关,附睾管腔的膨大和回缩亦可能是高原地区季节性繁殖的哺乳动物中一种普遍存在的现象。     

Expression of lactoferrin in the boar epididymis: effects of reduced estrogen

Lactoferrin is regulated by estrogen in the female reproductive tract and evidence in immature mice suggests that it may be estrogen regulated in males as well. The estrogen regulation of lactoferrin in the epididymis of the boar, a high estrogen-producing male, is unknown. This study was designed to test the hypothesis that lactoferrin expression in the boar epididymis is regulated by estrogen. Twenty-one littermate pairs of boars were treated with vehicle or Letrozole, an aromatase inhibitor, from 1 week of age until castration at 2 through 8 months. Epididymal tissue was collected at castration and fixed for immunolocalization of lactoferrin. Epididymal and testicular tissues were also collected from five mature boars (1-2.5 years) and fixed for immunocytochemistry (ICC). Lactoferrin was localized in the principal cell cytoplasm of the caput, corpus and cauda of developing boars but only in the corpus and cauda of mature boars. Basal cells were negative for lactoferrin. Sperm in the corpus and cauda was also positive for lactoferrin. The efferent ducts and testes were negative for lactoferrin. Intensity of lactoferrin immunostaining increased with age in the corpus and cauda regardless of treatment. Reduced endogenous estrogen in the epididymis during development did not affect the intensity of immunostaining between control and Letrozole-treated animals. Lactoferrin expression in the epididymis of the developing boar does not appear to be regulated by estrogen.     

RBP4基因在绵羊性成熟前后附睾中表达的研究

试验旨在研究视黄醇结合蛋白4（retinol binding protein 4,RBP4）基因在绵羊性成熟前和性成熟后附睾中的表达水平,探讨其在绵羊生殖机能调控方面的作用。以绵羊性成熟前和性成熟后附睾为材料,采用实时荧光定量PCR方法检测RBP4基因mRNA在绵羊性成熟前、后附睾中表达量的差异,采用免疫组化技术检测RBP4基因在绵羊性成熟前、后附睾头和附睾尾中蛋白的表达水平。结果显示,RBP4基因在绵羊附睾中性成熟前的表达量为1.3368,性成熟后的表达量为0.6450,虽然二者间的表达量差异不显著（P>0.05）,但性成熟前的表达量高于性成熟后的表达量。RBP4蛋白在性成熟前、后附睾头上皮层、平滑肌层、组织间质中均有阳性表达,主要位于上皮细胞质、平滑肌细胞质;在附睾尾上皮层、组织间质中有阳性表达,主要位于上皮细胞质。推测RBP4基因可能与附睾主细胞分泌功能和附睾头处的收缩功能有关,预示RBP4可能参与附睾微环境的调控,有利于精子成熟、运动和储存。     

Histochemical detection of glycoconjugates in the male reproductive system of the horse

Lectins are glycoproteins of plant and animal origin that have the ability to bind specific carbohydrate residues of cell glycoconjugates, particularly in terminal positions. In this study, the binding of lectins, Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), Bandeiraea simplicifolia BS-1 (isolectin B4), Triticum vulgaris (WGA), Arachis hypogaea (PNA), and Ulex europaeus (UEA-I), was studied in the reproductive systems of male thoroughbred horses.DBA was detected in the stereocilia of the caput and corpus epididymis, and in the vas deferens. It was weakly detected in connective tissue of the corpus epididymis. Strong SBA staining was seen in epithelial cells in the testis, stereocilia of the corpus and cauda epididymis, and in the vas deferens. There were intense positive reactions for isolectin B4 in interstitial cells in all tissue and serosa of the vas deferens. PNA staining was seen only in stereocilia in the caput and corpus epididymis, and in the vas deferens. Strong WGA staining was seen throughout the testis, except in Sertoli cells, stereocilia, and connective tissue. UEA-I was detected in secondary spermatids, stereocilia, and epithelial cells of the cauda epididymis. These results show that degenerating cells in the testis, epididymal tubules, and vas deferens have differential affinities for lectins, and suggest that lectins play a role in the reproductive system of the horse. The heterogeneity of the lectin staining pattern in the reproductive tubules of adult horses suggests that the carbohydrate composition of each cell type is region specific.     

牦牛GPX5基因CDS区的克隆和表达研究

GPX5作为谷胱甘肽过氧化物酶的成员之一,表达于牦牛附睾之中,其主要作用为抗氧化应激。本实验旨在通过分子技术对牦牛GPX5基因进行克隆,分析其生物信息学的特征和附睾组织的表达特点。结果显示:牦牛GPX5基因CDS区有630 bp、209个氨基酸被编码,所编码蛋白带正电,具有亲水性且不稳定,无跨膜域却存在信号肽;GPX5基因在附睾头的表达高于附睾的颈和尾(P<0.01)。牦牛附睾中的GPX5能有效抵抗氧化应激以保护精子。     

免疫细胞在牦牛附睾和输精管的分布规律

旨在研究免疫细胞在健康雄性牦牛附睾和输精管的分布。采用免疫组织化学和实时荧光定量(qRT-PCR)方法对幼龄(5~6月龄)及成年(3~4岁)牦牛附睾(头、体、尾)和输精管中CD68⁺巨噬细胞、CD3⁺ T淋巴细胞、CD79α⁺ B淋巴细胞、IgA⁺和IgG⁺浆细胞的分布特征及其表面标志分子的表达水平进行研究。结果显示：CD68⁺巨噬细胞、CD3⁺ T淋巴细胞、CD79α⁺ B淋巴细胞、IgA⁺和IgG⁺浆细胞主要分布在附睾管和输精管的上皮和间质;另外,CD68和CD3 mRNA和蛋白水平在各年龄组牦牛附睾头和附睾体显著高于附睾尾和输精管(P < 0.05),而CD79α、IgA和IgG mRNA和蛋白水平在附睾尾和输精管显著高于附睾头和附睾体(P < 0.05);此外,在成年牦牛附睾和输精管CD3、CD79α、IgA、IgG、CD68 mRNA和蛋白水平均显著高于幼龄牦牛(P < 0.05)。综上提示,牦牛附睾头可能主要是细胞免疫发生的位点,而附睾尾和输精管则主要进行体液免疫应答,此外,成年牦牛附睾和输精管的局部免疫可能更完善,以上数据为进一步研究高原牦牛局部生殖免疫和病理提供了形态学资料。     

Resorption of the Element Zinc from Spermatozoa by the Epididymal Epithelium

In this study, elimination of the element zinc from spermatozoa during epididymal maturation was investigated. Testes and epididymides from 40 bulls were collected; epididymal fluid was flushed, pooled, labelled with 0.5 MBq ⁶⁵Zn²⁺ per sample and proteins were separated on a Sephacryl S‐200 HR and zinc chelate column chromatography. To follow the resorption of zinc in the epididymal epithelial lining, an autometallographic technique (AMG) was performed in tissue from caput, corpus, cauda and vas deferens. The results showed a zinc‐binding protein fraction with an apparent molecular weight of 150–160 kDa, which was enriched after chelate column chromatography. Specific labelling of ⁶⁵Zn was about five times higher in the caput than in the cauda epididymidis. AMG revealed no detectable zinc in the caput, but a significant increase of zinc resorption from the corpus to the cauda and vas deferens. Controls showed that the detectable zinc was located within the principal cells. In conclusion, our study proves that zinc present in the sperm flagellum starts to be mobilized in the caput epididymidis and is resorbed by the epididymal epithelium as from the corpus. This zinc elimination is a mandatory step in sperm maturation to obtain motility.     

Estrogen and androgen receptor expression in relation to steroid concentrations in the adult boar epididymis

The steroid hormone regulation of the epididymis in a high estrogen producing animal like the boar is not currently understood. To test the hypothesis that the boar epididymis is an estrogen and androgen responsive tissue, the presence of estrogen and androgen receptors, in conjunction with steroid hormone concentrations were investigated in the boar epididymis. Epididymal (caput, corpus, cauda) and testicular samples of boars (1–2.5 years; n = 5) were collected for immunolocalization of estrogen receptor alpha (ER), estrogen receptor beta (ERβ) and androgen receptor (AR). Concentrations of testosterone, estradiol and estrogen conjugates (EC) in the tissue were also determined. AR and ERβ were localized in the principal and basal cells of all three epididymal regions. ER was localized in the principal cells of the caput, some cells of the corpus and was not present in the cauda. Testosterone (p < 0.0001), estradiol (p < 0.0001) and EC (p < 0.005) were significantly lower in the epididymis compared with the testis. The epididymal regions were not significantly different from each other for testosterone (p > 0.15) or estradiol (p > 0.09). EC were significantly higher in the corpus than either the caput (p = 0.003) or cauda (p = 0.002). These results suggest that the boar epididymis is responsive to both estrogens and androgens and that both steroid hormones are important for proper epididymal function. Since testosterone and estradiol concentrations are similar throughout the epididymis, regional differences in steroid hormone regulation are likely due to differences in receptor expression.     

精子受精抗原-1(FA-1) mRNA在绵羊睾丸和附睾中的表达

本研究通过RT-PCR检测了该基因在绵羊睾丸和附睾中的表达情况。首先,提取绵羊睾丸组织、附睾头、体、尾部组织总RNA,以此为模板,反转录合成cDNA,自行设计引物,PCR扩增出462 bp的目的DNA。然后,将目的片断克隆入T载体,通过菌液PCR和重组质粒酶切,鉴定重组质粒中的目的DNA。再经序列分析鉴定目的片断。同时,以β-actin为内参照物,进行RT-PCR半定量分析,比较精子受精抗原(FA-1)在睾丸、附睾头、体、尾组织中的表达量。结果表明,FA-1在绵羊睾丸和附睾中均表达。     

Epididymis of Viscacha (Lagostomus maximus maximus): A Morphological Comparative Study in Relation to Sexual Maturity

The morphological variations and the androgen receptor (AR) expression were studied in viscacha epididymis in relation to sexual maturity. The animals were divided into immature, pre‐pubertal and adult, according to their corporal weight and testicular histology. The epididymides were studied by light microscopy, immunohistochemistry for AR and morphometric analysis. In pre‐pubertal and adult animals, four well‐differentiated segments (initial, caput, corpus and cauda) were observed, while in immature animals, three segments were identified (initial‐caput segment, corpus and cauda). In each segment, the structural parameters and the relative cell distribution were different between the groups. The serum testosterone levels of pre‐pubertal and adults showed a very significant increase related to sexual maturity. The AR expression in epithelial and fibromuscular stromal cells was different between the groups. In conclusion, the present work demonstrates that the morphological characteristics of the viscacha epididymis vary while sexual maturity is reached, the development of initial and caput is subsequent to corpus and cauda development and the androgens might play an important role during this process.