Fra1 activity in the frog, Rana esculenta, testis: a new potential role in sperm transport

Using an anti-Fos family member antibody, we have previously described in Rana esculenta testis the presence of a nuclear, 43 kDa protein that we hypothesized to be Fra1. With the assistance of an antibody against Fra1 that does not cross-react with other Fos family members, here we report data on Fra1 expression, localization, and putative activity in Rana esculenta testis during its annual reproductive cycle. Western blot analysis confirms that the nuclear, 43 kDa protein is Fra1. Immunocytochemistry validates the Western blot results and shows cytoplasmic and nuclear immunostaining of Fra1 in peritubular myoid cells, efferent ducts, and blood vessels. We report for the first time in a vertebrate, experimental evidence showing that the expression of Fra1 is related to peritubular myoid cells during sperm transport from the tubular compartment to efferent ducts.     

脂多糖调控对大鼠心脏微血管内皮细胞的转录组分析

目的探索脂多糖(LPS)对大鼠心脏微血管内皮细胞(rCMECs)转录组的调节作用。方法对照组(正常培养rCMECs),LPS组(100 ng/mL LPS处理6 h的rCMECs),每组进行3个生物学重复转录组测序。得到差异基因后,使用实时定量PCR对部分差异基因mRNA的表达进行验证。分别对上调和下调基因进行GO和KEGG富集,并对差异基因进行共表达网络分析。采用独立t检验进行统计学分析。结果LPS处理后,265个基因表达上调,118个基因的表达下调。前10个最显著上调基因为:Mt2a、Cyp7b1、Sod2、Icam1、Ccl2、AC128848.1、Mt1、Cebpd、Serpinb2和Tnfrsf11b。前10个最显著下调基因为:Cavin2、Ankrd1、Edn1、Prss35、Lmod1、Dhrs3、Ttc22、Sema6a、Map2k3和Sema7a。定量PCR的结果表明Mt2a、Sod、Ccl2、Cxcl1、Icam1和Vcaml基因的表达得到了上调(P均<0.01);而Cavin2、Ankrd1、Edn1和Prss35基因表达下调(P均<0.05)。GO和KEGG富集的结果表明,上调基因与内皮细胞对炎性免疫细胞的趋化作用和黏附作用密切相关;而下调基因则是与钙离子信号和G蛋白相关通路以及内皮通透性增加有关。此外,差异基因进行共表达网络分析发现Sod2处于核心位置,提示其可能与LPS诱导的rCMECs的各种变化密切相关。结论LPS调控了rCMECs中大量与炎性免疫细胞进入心肌组织相关基因的表达。     

Rat Sertoli cell extracellular matrix regulates glycosaminoglycan synthesis by peritubular myoid cells in vitro

We have previously reported metabolic cooperation between Sertoli and peritubular myoid cells in terms of synthesis of one of the main testicular extracellular matrix (ECM) constituents, glycosaminoglycans (GAG). This study concerns Sertoli cell ECM-peritubular myoid cell interactions in terms of GAG synthesis. We have examined the responses of hormones and other regulatory agents such as a combination of follicle-stimulating hormone (FSH), insulin, retinol, and testosterone (FIRT) on peritubular myoid cells, and tested if Sertoli cell ECM or serum factor substitute for the stimulation by FIRT. Testicular peritubular myoid cells cultured on Sertoli cell ECM showed significant increases in the levels of cell- and ECM-associated GAG over that when cultured on uncoated plastic. This indicates a specific cell-substratum interaction between Sertoli cell ECM and peritubular myoid cells in the testis in terms of GAG synthesis. Moreover, in terms of cell-associated GAG synthesis, peritubular myoid cells cultured on Sertoli cell ECM or on plastic in the presence of serum substituted for the stimulatory response of FIRT on peritubular myoid cells cultured on uncoated plastic. The data are discussed in relation to the possible role of cell-substratum interaction in maintaining peritubular myoid cell functions. © 1993 Wiley-Liss, Inc.     

Calcyclin is differentially expressed in rat testicular cells

Immature rat Sertoli cells aggregate and form tubule-like structures when cultured on a monolayer of peritubular myoid cells. In this study, differential gene expression of monocultures and direct cocultures of peritubular cells and Sertoli cells were examined. One of the cDNA clones isolated showed high homology to calcyclin and a microvascular differentiation gene, CEC5, which was reported to be highly homologous to CASK, a membrane-associated guanylate kinase homolog. Sequencing and mRNA analysis of rat calcyclin demonstrated that the gene was differentially expressed and was found only in peritubular cells and cocultures with increased levels. In contrast, CASK was expressed by Sertoli cells, peritubular cells, and cocultures, whereas CEC5 was never found in the testicular somatic cells. Our findings point to a paracrine regulation of calcyclin expression in testicular peritubular fibroblasts which seems to be related to tubular growth.     

大鼠和小鼠睾丸表皮生长因子表达的免疫组织化学定位观察

为了了解大鼠和小鼠睾丸是否产生ＥＧＦ及其细胞定位,本实验用ＥＧＦ单克隆抗体对大鼠和小鼠睾丸进行了免疫细胞化学定位研究,结果显示：（１）出生后,大鼠和小鼠睾丸即开始产生ＥＧＦ,分泌活动主要位于睾丸间质细胞。（２）至性成熟期,少数精原细胞、精母细胞及个别圆形精子细胞和管周肌样细胞也产生ＥＧＦ,使生精小管尤其是血睾屏障管腔小室侧的ＥＧＦ分泌增加。（３）在本实验中,睾丸支持细胞未见明显ＥＧＦ阳性染色。结果表明,大鼠和小鼠睾丸是可以产生ＥＧＦ的,间质细胞是其主要的ＥＧＦ分泌细胞。进入性成熟期后,少数精原细胞、精母细胞及个别圆形精子细胞和管周肌样细胞也产生ＥＧＦ。大鼠和小鼠睾丸在发育过程中ＥＧＦ分泌量呈上升趋势,至性成熟期达分泌高峰     

Ultrastructure of peritubular tissue in association with tubular hyalinization in human testis.

Testicular peritubular tissue, also known as the tunica propria, surrounds the seminiferous tubules and is responsible for contractile, paracrine and transport functions. The aim of the present report is to describe the pathomorphology of peritubular tissue in association with tubular hyalinization in human testis. Twenty-seven testicular biopsies from 21 subfertile and infertile men were studied with the electron microscope. Biopsies from five patients showed complete or nearly complete tubular hyalinization. In addition to changes described earlier, the following new ultrastructural features were observed: 1. loss of polarity and configuration of myoid cells; 2. protrusion of myoid cells towards the tubule and evagination of basal lamina surrounding the tubule towards the interstitial direction leading to 'bridge' formation. These 'bridges' of myoid cells often created completely separated small compartments within the tunica propria; 3. vacuolization and fragmentation of myoid cell nuclei; 4. a balloon-like swelling of myoid cell containing phagolysosomes and lipid droplets. We conclude that disorganization and loss of vital functions of the extracellular matrix and myoid cells contribute to the pathogenesis of tubular hyalinization.     

Dax1 regulates testis cord organization during gonadal differentiation

Mutations of the DAX1 nuclear receptor gene cause adrenal hypoplasia congenita, an X-linked disorder characterized by adrenal insufficiency and hypogonadotropic hypogonadism. Targeted deletion of Dax1 in mice also reveals primary testicular dysgenesis, which is manifest by obstruction of the rete testis by Sertoli cells and hyperplastic Leydig cells, leading to seminiferous tubule dilation and degeneration of germ cells. Because Dax1 is expressed early in gonadal development, and because Sertoli and Leydig cells are located ectopically in the adult, we hypothesized that these testis abnormalities are the result of an early defect in testis development. In Dax1(-/Y) males, the gonad develops normally until 12.5 dpc. However, by 13.5 dpc, the testis cords are disorganized and incompletely formed in Dax1-deficient mice. The number of germ and Sertoli cells is unchanged, and the expression of Sertoli-specific markers appears to be normal. However, the number of peritubular myoid cells, which normally surround the testis cords, is reduced. BrdU labeling of peritubular myoid cells is low, consistent with decreased proliferation. The basal lamina produced by peritubular myoid and Sertoli cells is disrupted, leading to open and incompletely formed testis cords. Leydig cells, which normally reside in the peritubular space and extend from the coelomic surface to the dorsal surface of the gonad, are restricted to the coelomic surface of Dax1-deficient testis. We conclude that Dax1 plays a crucial role in testis differentiation by regulating the development of peritubular myoid cells and the formation of intact testis cords. The developmental abnormalities in the Dax1-deficient testis lay the foundation for gonadal dysgenesis and infertility in adult mice and, potentially in humans with DAX1 mutations.     

On the character of the monolayer outgrowth and the fate of the peritubular myoid cells in cultured mouse testis

Postnatal differentiation of the peritubular myoid cells in mouse testis is hormone dependent. In order to analyse the differentiation of the peritubular tissue, an attempt was made to develop an experimental model system utilizing an in vitro method. Fragments obtained from adult, 7- or 10-day-old mice, were cultured in McCoy's modified 5a medium for 9–19 days. The fragments and monolayers that grew from them were examined with the electron microscope at the end of the culture period. Monolayers originating from either mature or immature testicular expiants were comparable in appearance. They were composed of spindle-shaped cells that contained abundant profiles of granular endoplasmic reticulum and free ribosomes, as well as arrays of 40–60 Å thick filaments and associated dense bodies. In these respects they resembled smooth muscle cells in culture, in developmental, and in pathological conditions. Examination of the peritubular tissue in the testicular explants indicated that the monolayer of myoid cells originated from the fibroblasts rather than the peritubular myoid cells. Peritubular cells in explants from mature rats retained their myoid features at the end of the culture period but myoid cell differentiation failed to progress in expiants obtained from immature animals. Additional work is necessary in order to establish the suitability of these preliminary culture attempts to support normal development before conclusions may be drawn concerning the role of hormones in myoid cell differentiation. The role of microfilaments as a contractile organelle of cells is discussed.     

Dhh signaling pathway regulates reconstruction of seminiferous tubule-like structure

The reconstruction of a tubule-like structure in vitro has provided a promising system to analyze factors that drive morphogenesis and the underlying mechanisms. In this study, we took advantage of the inhibitor cyclopamine and a smoothened agonist to detect the role of the Dhh signaling pathway in the reconstructed tubule-like structure. Sertoli cells did not show polarity, rounded peritubular myoid cells and scattered Leydig cells were observed, combined with less laminin and lower proliferation of Leydig, peritubular myoid, germ, and Sertoli cells. However, in the presence of SAG, elongated peritubular myoid cells gathered at the bottom of polarized Sertoli cells, and most of the Leydig cells gathered at the outer part of the elongated peritubular myoid cells. Moreover, SAG promoted the secretion of laminin, assisting in the formation of the basal membrane and promoting the proliferation of Leydig, peritubular myoid, and germ cells. The level of Gli1 was significantly downregulated when treated with cyclopamine, whereas it was significantly upregulated when treated with SAG. These results indicate that the Dhh signaling pathway regulates the reconstruction of tubule-like structures by regulating the expression of Gli1.     

Immunolocalization of estrogen receptor alpha, estrogen receptor beta and androgen receptor in the pre-, peri- and post-pubertal stallion testis

In various species, androgens and estrogens regulate the function of testicular Leydig, Sertoli, peritubular myoid, and germ cells by binding to their respective receptors and eliciting a cellular response. Androgen receptor (AR) is expressed in Sertoli cells, peritubular myoid cells, Leydig cells and perivascular smooth muscle cells in the testis depending on the species, but its presence in germ cells remains controversial. Two different estrogen receptors have been identified, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), and their localization and function in testicular cells varies depending on the species, developmental stage of the cell and type of receptor. The localization of AR in an immature and mature stallion has been reported but estrogen receptors have only been reported for the mature stallion. In the present study, the localizations of AR and ERα/ERβ were investigated in pre-pubertal, peri-pubertal and post-pubertal stallions. Testes were collected by routine castration from 21 horses, of light horse breeds (3 months-27 years). Animals were divided into the following age groups: pre-pubertal (3-11 months; n=7), peri-pubertal (12-23 months; n=7) and post-pubertal (2-27 years; n=7). Testicular tissue samples were fixed and embedded, and the presence of AR, ERα and ERβ was investigated by immunohistochemistry (IHC) using procedures previously validated for the horse. Primary antibodies used were rabbit anti-human AR, mouse anti-human ERβ and rabbit anti-mouse ERα. Sections of each region were incubated with normal rabbit serum (NRS; AR and ERα) or mouse IgG (ERβ) instead of primary antibody to generate negative controls. Androgen receptors were localized in Leydig, Sertoli and peritubular myoid cells of all ages. Estrogen receptor alpha was localized in Leydig and germ cells of all ages but only in pre- and peri-pubertal Sertoli cells and post-pubertal peritubular myoid cells. Estrogen receptor beta was localized in Leydig and Sertoli cells of all ages but in only pre-pubertal germ cells and absent in peritubular myoid cells of all ages. Taken together, the data suggest that estrogen regulates steroidogenesis by acting through ERα and ERβ in the Leydig cells and promotes gametogenesis by acting through ERβ in the Sertoli cells and ERα in the germ cells. In contrast androgen receptors are not found in germ cells throughout development and thus are likely to support spermatogenesis by way of a paracrine/autocrine pathway via its receptors in Leydig, Sertoli and peritubular myoid cells.     

Pleiotropic actions of melatonin in testicular peritubular myoid cells of immature Syrian hamsters

BackgroundPeritubular myoid cells are emerging as key regulators of testicular function in adulthood. However, little is known about the role of testicular peritubular myoid cells (TPMCs) in the development of the male gonad. We found that, compared to testes of young adult hamsters, gonads of 21 day-old animals show increased melatonin concentration, seminiferous tubular wall thickening and a heterogeneous packaging of its collagen fibers thus raising the question whether melatonin may be involved in the regulation of TPMCs.MethodsWe established primary cultures of TPMCs from immature hamsters (ihaTPMCs), which we found express melatonergic receptors.ResultsExogeneous melatonin decreased the levels of inflammatory markers (NLRP3 inflammasome, IL1β) but increased the expression of cyclooxygenase 2 (COX2, key enzyme mediating prostaglandin synthesis) and of the glial cell line-derived neurotrophic factor (GDNF) in ihaTPMCs. Melatonin also stimulated ihaTPMCs proliferation and the expression of extracellular matrix proteins such as collagen type I and IV. Furthermore, collagen gel contraction assays revealed an enhanced ability of ihaTPMCs to contract in the presence of melatonin.ConclusionMelatonin regulates immune and inflammatory functions as well as contractile phenotype of the peritubular wall in the hamster testis.General significanceIf transferable to the in vivo situation, melatonin-dependent induction of ihaTPMCs to produce factors known to exert paracrine effects in other somatic cell populations of the gonad suggests that the influence of melatonin may go beyond the peritubular wall and indicates its contribution to testicular development and the establishment of a normal and sustainable spermatogenesis.     

Establishment of a peritubular myoid-like cell line and interactions between established testicular cell lines in culture

Established cell lines and primary cultures derived from somatic cells of the testis have been used to study cell-cell interactions. Primary cultures of Sertoli cells or Sertoli-derived cell lines from the mouse (TM4) and rat (TR-ST) will aggregate when plated on monolayers of primary cultures of peritubular myoid cells or a rat (TR-M) cell line which has many properties of peritubular myoid cells. Time-lapse cinematography and scanning and transmission electron microscopy reveal that Sertoli cells formed aggregates after 1 day in coculture, display surface activity and move on the monolayer. When these aggregates touch one another, they rapidly combine. By the 4th day of culture, spherical aggregates are composed of 50 to 200 cells. They do not display surface activity or movement on the myoid monolayer. On the 5th and 6th day of culture most spherical aggregates have flattened to form dome-shaped aggregates in close association with the monolayer. Cells in the aggregates are characterized by long microvilli and some ruffles. In large aggregates, cells sometimes form close associations within the aggregates although junctions are seldom observed. Sertoli-derived cell lines will not aggregate on monolayers of Leydig-derived (TM3) or testicular endothelial-derived (TR-1) cell lines. Neither TM3 nor TR-1 cells will aggregate when plated on myoid monolayers. The TR-M cells produced an extensive extracellular matrix beneath the cells which contains collagen, an amorphous globular material resembling elastin and a fibrous noncollagenous component. Sertoli cells plated on this matrix will not aggregate. Thus the aggregation of Sertoli cells on myoid cell monolayers is cell type, but not species dependent and not determined solely by extracellular matrix components produced by TR-M cells.