Comparison of lectin-staining pattern in testis and epididymis of gerbil, guinea pig, mouse, and nutria

The testis and epididymis of gerbil, guinea pig, nutria, and mouse were studied after staining with seven rhodamine-conjugated lectins to disclose the distribution of glycoproteins with different sugar residues. In the testis, the lectins showed a variable affinity for Leydig cells, tubular basement membrane, cytoplasm, acrosome, and plasma membrane of maturing spermatids as well as for Sertoli cell extensions. During acrosomal development, the staining pattern showed characteristic changes with different lectins indicating a gradual processing of the glycoprotein components. The staining in the Sertoli cell extensions displayed a cyclic change linked with the release of spermatozoa. A nuclear staining was prominent in zygotene and pachytene spermatocytes in the mouse, weak in the nutria, but absent in gerbil and guinea pig. The principal cells of epididymis showed a lectin-stained Golgi region as well as a similar staining in the apical surface, microvilli, and tubular contents. This staining was most prominent in the caput/corpus regions with some interspecies differences indicating the epididymal areas active in secretion. Narrow cells active in absorption of testis-derived material were lectin-positive in the initial segment of mouse, gerbil, and nutria epididymis. Large light cells with a strong affinity for some lectins were found in the proximal cauda of gerbil and guinea-pig epididymis. In the nutria, corresponding cells were arranged as islands within the low epithelium. The distal cauda of mouse, gerbil, and nutria was the site for lectin-stained light cells interspersed among the low principal cells. It is concluded that the high and low light cells may be active in the absorption and phagocytosis of residual bodies/cytoplasmic droplets and surplus epididymal secretory material, respectively. Thus, labeled lectins formed a useful tool in the analysis of glycoprotein distribution, processing, secretion, absorption, and degradation in the male reproductive tissues.     

Characterization of the glycoconjugates of boar testis and epididymis

Lectin histochemistry was used to perform in situ characterization of the glycoconjugates present in boar testis and epididymis. Thirteen horseradish peroxidase- or digoxigenin-labelled lectins were used in samples obtained from healthy fertile boars. The acrosomes of the spermatids were stained intensely by lectins with affinity for galactose and N-acetyl-galactosamine residues, these being soybean, peanut and Ricinus communis agglutinins. Sertoli cells were stained selectively by Maackia ammurensis agglutinin. The lamina propria of seminiferous tubules showed the most intense staining with fucose-binding lectins. The Golgi area and the apical part of the principal cells of the epididymis were stained intensely with many lectins and their distribution was similar in the three zones of the epididymis. On the basis of lectin affinity, both testis and epididymis appear to have N- and O-linked glycoconjugates. Spermatozoa from different epididymal regions showed different expression of terminal galactose and N-acetyl-galactosamine. Sialic acid (specifically alpha2,3 neuraminic-5 acid) was probably incorporated into spermatozoa along the extratesticular ducts. These findings indicate that the development and maturation of boar spermatozoa are accompanied by changes in glycoconjugates. As some lectins stain cellular or extracellular compartments specifically, these lectins could be useful markers in histopathological evaluation of diseases of boar testis and epididymis.     

Lectin-binding pattern as tool to identify and enrich specific primary testis cells of the tilapia (Oreochromis niloticus) and medaka (Oryzias latipes)

Cell type-specific lectin binding is a useful tool for the analysis of developing systems. We describe the binding pattern of 21 different fluorescein isothiocyanate (FITC)-labelled lectins to the testis of two model teleost species, the medaka (Oryzias latipes) and the tilapia (Oreochromis niloticus). The analysis of the binding pattern was carried out on tissue sections (medaka and tilapia) and using primary culture cells (only tilapia). Lectin binding was studied by confocal microscopy and for histological analysis some sections were, in addition, stained with bodipy to gain additional information concerning the cytological organization of the cystic mode of spermatogenesis in fish. The observed differences in lectin staining of different cell types in primary cultures were quantified by flow cytometry. Only few lectins bound specifically to haploid cells while the reaction to diploid or tetraploid cells was generally stronger. However, the extracellular material around the haploid spermatids and spermatozoa in spermatocysts showed a strong staining reaction with several lectins (e.g., Phaseolus vulgaris Erythro agglutinin). The apparent differences in the cellular lectin-binding pattern can be used to identify particular cell types, to monitor their differentiation in vitro or to enrich particular cell types from heterogeneous cultures using magnetic beads coated with anti-FITC antibodies. Using the latter approach, we show that it is possible to enrich for gonial cells and at the same time deplete the preparation for haploid cells and Sertoli cells.     

Lectin binding sites on head structures of the spermatid and spermatozoon of the mosquito Culex quinquefasciatus (Diptera, Culicidae).

The presence of intranuclear and acrosomal lectin binding sites in spermatids and spermatozoa of the mosquito Culex quinquefasciatus was analysed. Direct and indirect lectin-gold techniques were used on LR White-embedded cells. The nuclear compartment was the structure most intensely labelled. Early spermatid nucleus showed moderate labelling for peanut agglutinin (PNA), Griffonia simplicifolia IB4 (GS-IB4) and Ricinus communis agglutinin (RCA), and light labelling for the other lectins tested. The sperm nucleus was intensely labelled by all lectins. The acrosome, an enzyme-containing structure, was labelled by some lectins. The anterior acrosomal region was labelled by PNA, while the proximal acrosomal region was labelled by PNA and G. simplicifolia II (GS II) lectins, and showed the presence of fucose residues with the use of Ulex europaeus I (UEA-I) lectin. The spermatozoa stored in the spermatheca showed the same pattern of labelling as that observed in spermatozoa localized in testis and seminal vesicles for all lectins tested. Carbohydrate residues in the nuclear compartment may be involved with the process of chromatin condensation. In the acrosomal region these residues may play a role in the process of sperm-oocyte interaction.     

Sertoli cell expression of galectin-1 and -3 and accessible binding sites in normal human testis and Sertoli cell only-syndrome.

Galectins are vertebrate lectins interacting with beta-galactosides and derivates thereof such as blood group A, B and H determinants. The expression of gelectin-1 and -3 and galectin-specific binding sites by human Sertoli cells was analyzed in normal human testis and Sertoli cell only-syndrome (SCOS). Staining intensity was scored semiquantitatively on a 4-grade scale. Sertoli cells in normal testes displayed a moderate cytoplasmic and weak nuclear staining for galectin-1-specific binding sites. Galectin-3-specific binding sites were expressed in Sertoli cells less intensely than accessible ligands for galectin-1 (mean score 2.25 for galectin-1 and 1.50 for galectin-3). Germ cells were only weakly reactive. Tubular walls were negative for both classes of galectin-specific binding sites. In SCOS, galectin-1 binding was moderate to strong and more pronounced than galectin-3 binding by Sertoli cells (mean scores 4.00 and 2.25). Tubular walls were negative for galectin-staining. The ratio for galectin-1-/galectin-3-specific binding (staining score ratio) was 1.50 form normal testis and 1.78 for SCOS disclosing a relative increase of galectin-3 binding sites in the latter. Staining with galectin-1- and -3-specific antisera showed a strong cytoplasmic galectin-1 immunoreactivity in Sertoli cells of normal and SCOS testis (score 4.00 for both). Anti-galectin-3 did not stain Sertoli cells or germ cells in normal testis. Only Leydig cells were labeled (score 3.00). In SCOS a weak to moderate nuclear staining of Sertoli cells was noted (score 2.00). Galectin-3 expression and galectin-1-specific binding sites were found to be increased in Sertoli cells of SCOS. This modulation of reactivity can have implications for Sertoli cell interactions with galectin-reactive extracellular matrix components like laminin and for anti-apoptotic effects.     

Lectin binding sites on head structures of the spermatid and spermatozoon of the mosquito Culex quinquefasciatus (Diptera,Culicidae)

The presence of intranuclear and acrosomal lectin binding sites in spermatids and spermatozoa of the mosquito Culex quinquefasciatus was analysed. Direct and indirect lectin-gold techniques were used on LR White-embedded cells. The nuclear compartment was the structure most intensely labelled. Early spermatid nucleus showed moderate labelling for peanut agglutinin (PNA), Griffonia simplicifolia IB4 (GS-IB4) and Ricinus communis agglutinin (RCA), and light labelling for the other lectins tested. The sperm nucleus was intensely labelled by all lectins. The acrosome, an enzyme-containing structure, was labelled by some lectins. The anterior acrosomal region was labelled by PNA, while the proximal acrosomal region was labelled by PNA and G. simplicifolia II (GS II) lectins, and showed the presence of fucose residues with the use of Ulex europaeus I (UEA-I) lectin. The spermatozoa stored in the spermatheca showed the same pattern of labelling as that observed in spermatozoa localized in testis and seminal vesicles for all lectins tested. Carbohydrate residues in the nuclear compartment may be involved with the process of chromatin condensation. In the acrosomal region these residues may play a role in the process of spermoocyte interaction.     

小鼠精子表面Con A结合糖复合物的形成与变化

用辣根过氧化物酶标记的ＣｏｎＡ（伴刀豆素Ａ）对小鼠睾丸与附睾切片,以及对取自附睾和子宫（交配后）内的精子涂片进行了标记,旨在认识精子在发生、成熟和获能过程中表面糖复合物的形成与变化。本研究表明,睾丸内的生精细胞和支持细胞均呈ＣｏｎＡ标记阳性。附睾的输出小管和附睾管上皮细胞,ＣｏｎＡ标记呈中度至强阳性,有部位的差别。附睾头和附睾尾内精子表面的标记无明显差别,标记位置均主要在顶体区和尾部。精子在子宫内存留１．５小时后,顶体后区出现中度阳性标记,但存留３小时和６小时后,顶体和顶体后区的标记均减弱或消失。这些结果提示,（１）精子发生期即可合成ＣｏｎＡ结合糖复合物,（２）精子在附睾成熟过程中表面的ＣｏｎＡ结合糖复合物无明显变化,（３）精子获能后顶体后区出现的ＣｏｎＡ结合糖复合物可能与受精能力有关。     

Lectin staining of rat testis and epididymis after ligation of excurrent ducts at different levels

Seven rhodamine-conjugated lectins (PNA, RCA I, SBA, Con A, WGA, UEA I, and DBA; see Table 1) were utilized in studying the staining pattern of glycoproteins in rat testis and epididymis after ligation of ductuli efferentes (DE), corpus epididymidis (CE), and vas deferens (VD) for various time periods. Ductuli efferentes ligation caused a widening of seminferous tubules and detachment of spermatids with formation of multinuclear cells. These cells acquired a strong affinity for all lectins. Corpus epididymidis ligation also caused degeneration of spermatids with increased lectin staining in some tubules, but after 7 days another cell population close to the periphery of seminiferous tubules showed an increased nuclear affinity for some lectins followed by a clear degeneration and strong cytoplasmic staining with all lectins. Vas deferens ligation caused no degenerative changes in testicular spermatids. However, the peripheral cell population showed degenerative changes similar to those found after CE ligation. In both cases this was coincident with the formation of spermatic granulomas at the site of ligation. Ductuli efferentes ligation caused a gradual decrease of intratubular content in caput epididymidis, while the contrary was true after CE ligation. The latter was associated with intratubular accumulation of lectin-positive swollen cells and sperm aggregates as well as an increased lectin staining of narrow cells in initial segment and light cells in distal caput. After VD ligation an increased staining of light cells was initially found in distal cauda and distal caput, but, concomitant with distension of the tubules this reaction decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comprehensive profiling of accessible surface glycans of mammalian sperm using a lectin microarray

It is well known that cell surface glycans or glycocalyx play important roles in sperm motility, maturation and fertilization. A comprehensive profile of the sperm surface glycans will greatly facilitate both basic research (sperm glycobiology) and clinical studies, such as diagnostics of infertility. As a group of natural glycan binders, lectin is an ideal tool for cell surface glycan profiling. However, because of the lack of effective technology, only a few lectins have been tested for lectin-sperm binding profiles. To address this challenge, we have developed a procedure for high-throughput probing of mammalian sperm with 91 lectins on lectin microarrays. Normal sperm from human, boar, bull, goat and rabbit were collected and analyzed on the lectin microarrays. Positive bindings of a set of ~50 lectins were observed for all the sperm of 5 species, which indicated a wide range of glycans are on the surface of mammalian sperm. Species specific lectin bindings were also observed. Clustering analysis revealed that the distances of the five species according to the lectin binding profiles are consistent with that of the genome sequence based phylogenetic tree except for rabbit. The procedure that we established in this study could be generally applicable for sperm from other species or defect sperm from the same species. We believe the lectin binding profiles of the mammalian sperm that we established in this study are valuable for both basic research and clinical studies.     

Functional relationships of the mammalian testis and epididymis

The development of knowledge in four areas of research in male reproductive physiology of particular interest is reviewed. The concept of the blood-testis barrier (BTB), which arose following dye exclusion from the seminiferous tubules, has now been established as the differential transfer from interstitial fluid to tubular and rete testis fluids of molecules of physiological importance. The composition of fluid collected mostly from the rete testis of several species, not only reflects the nature of the barrier, but also the secretory capacity of the Sertoli cell. The functional significance of the transfer of molecules into testicular fluid and the composition of the fluid flowing into the epididymis are discussed. Sertoli cells establish the structural basis of the BTB during puberty and divide the seminiferous epitheliuym into basal and adluminal compartments. The Sertoli cell is the prime target for follicle stimulating hormone (FSH). The responses evoked by FSH are discussed, including special mention of androgen binding protein (ABP) and the protein hormone, 'inhibin', with FSH-suppressing properties. The control of FSH in the lamb is mentioned including new evidence to support a tubular source of a feedback agent with significance during the impuberal stage. Finally, some of the biochemical properties of the epididymis and its fluid contents are reviewed and the epididymal sperm are identified as the site of the antifertility action of the 6-chloro-6-deoxy sugars.     

Exposure in utero to di(n-butyl) phthalate alters the vimentin cytoskeleton of fetal rat Sertoli cells and disrupts Sertoli cell-gonocyte contact

Di(n-butyl) phthalate (DBP) is commonly used in personal care products and as a plasticizer to soften consumer plastic products. Male rats exposed to DBP in utero have malformations of the male reproductive tract and testicular atrophy characterized by degeneration of seminiferous epithelium and decreased sperm production. In the fetal testis, in utero exposure to DBP reportedly resulted in reduced testosterone levels, Leydig cell aggregates, and multinucleated gonocytes (MNG). We investigated whether exposure in utero to DBP affects rat fetal Sertoli cells and compromises interactions between Sertoli and germ cells in the developing testis. Histological examination showed that MNG occurred at low frequency in the normal fetal rat testis. Exposure in utero at the dose level of DBP above estimated environmental or occupational human exposure levels significantly increased the number of these abnormal germ cells. Postnatally, MNG exhibited aberrant mitoses and were detected at the basal lamina. MNG were not apoptotic in the fetal and postnatal rat testes, as indicated by TUNEL. Sertoli cells in DBP-exposed fetal testis had retracted apical processes, altered organization of the vimentin cytoskeleton, and abnormal cell-cell contacts with gonocytes. The effect of DBP on Sertoli cell morphology at the level of light microscopy was reversed after birth and cessation of exposure. Our data indicate that fetal Sertoli cells are targeted by exposure in utero to DBP and suggest that abnormal interactions between Sertoli and germ cells during fetal life play a role in the development of MNG.     

Immunologically reactive albumin-like protein in human testis and seminal plasma

An immunologically reactive albumin-like protein (albumin) was localized, by an immunostaining technique, in the testis of infertile men (normal spermatogenesis, obstructive azoospermia) at the level of the Sertoli cells and in some cells of the germinal epithelium (secondary spermatocytes and early spermatids). No positive reaction was detectable in prepubertal testis. In vasectomized men, mean seminal albumin values were drastically reduced (by about 80%) in comparison to fertile controls, indicating a probable testicular origin. Mean seminal albumin values were also decreased in patients affected by azoospermia due to a seminiferous tubular lesion (about 40%) and in oligozoospermic patients (about 30%). In the same seminal samples transferrin, an index of Sertoli cell function, was also measured. Albumin and transferrin results were well correlated in the seminal plasma of each group (with the exception of vasectomized subjects), including a group of men with abnormally high concentrations of seminal transferrin. A weak correlation was found between seminal albumin and sperm count. We suggest that the presence of albumin in the human adult testis and in seminal plasma could be related to its ability to transport androgens.     

Active cell aggregation by immature rat Sertoli cells in primary culture: a role for cell surface glycoproteins.

Follicle stimulating hormone (FSH) stimulates “colony formation” by immature rat Sertoli cells in primary culture. “Colony formation” involves cell aggregation. Consequently, the involvement of cell surface glycoproteins in cell aggregation was investigated by treatment of dissociated 10-day rat testis cells with sodium metaperiodate, glucosamine, various lectins, tunicamycin, and puromycin. Treatment of control cultures with 5 μM glucosamine stimulated cell aggregation; however, glucosamine did not affect FSH-stimulated cultures. Treatment of dissociated testis cells with 5 μM sodium metaperiodate, 10 μg/ml castor bean agglutinin (ricin), or 2.5 μg/ml horseshoe crab agglutinin inhibited FSH stimulation of cell aggregation. A similar inhibition of cell aggregation was observed following addition of 10 μg/ml puromycin or tunicamycin to culture media from 0- to 18-hours incubation. Treatment with soybean agglutinin, concanavalin A, or wheat germ agglutinin had no effect. The galactose-specific lectins, Ricin, Ricinus communis agglutinin I, and Bendeirea simplicifolia agglutinin, inhibit the FSH stimulation of ³H-aminoacid incorporation as well as cell aggregation in 24-hour cultres. The inhibition of cell aggregation by sodium metaperiodate treatment was reversed with 5 μM sodium borohydride reduction. Sodium metaperiodate treatment did not alter cell viability (as assayed with trypan blue dye exclusion), did not alter cell attachment, nor significantly decrease ¹²⁵I-FSH binding by cultured testis cells. The results suggest that FSH stimulation of cell aggregation by immature rat Sertoli cells requires cell surface glycoprotein interactions. Furthermore, the specificity of lectin inhibition suggests that glycoproteins with terminal galactose and sialic acid residues are required for the FSH induction of cell aggregation.     

Xenografting of adult mammalian testis tissue

Xenografting of testis tissue from immature males from several mammalian species to immunodeficient mouse hosts results in production of fertilization-competent sperm. However, the efficiency of testis tissue xenografting from adult donors has not been critically evaluated. Testis tissue xenografting from sexually mature animals could provide an option to preserve the genetic material from valuable males when semen for cryopreservation cannot be collected. To assess the potential use of this technique for adult individuals, testes from adult animals of six species (pig, goat, cattle, donkey, horse and rhesus monkey) were ectopically grafted to host mice. Grafts were recovered and analyzed at three time points: less than 12 weeks, between 12 and 24 weeks and more than 24 weeks after grafting. Histological analysis of the grafts revealed effects of species and donor tissue maturity: all grafts from species with greater daily sperm production (pig and goat) were found to have degenerated tubules or grafts were completely degenerated. None of the xenografts from mature adult bull and monkeys contained differentiated spermatogenic cells when examined more than 12 weeks post-grafting but tubules with Sertoli cells only remained. In grafts from a young adult bull, Sertoli cells persisted much longer than with the mature adult grafts. In grafts from a young adult horse, spermatogenesis proceeded to meiosis. In grafts from a young adult donkey and monkey, however, complete spermatogenesis was found in the grafts. These results show that testis tissue grafts from mature adult donors did not support germ cell differentiation but seminiferous tubules with Sertoli cells only survived in some species. The timing and progression of tubular degeneration after grafting of adult testis tissue appear to be related to the intensity of spermatogenesis at the time of grafting. Testis tissue from sub-adult donors survives better as xenograft than tissue from mature adult donors, and complete spermatogenesis can occur albeit with species-specific differences.     

Assessing acrosomal status of bovine sperm using fluoresceinated lectins

Binding of 12 lectins to bull sperm was analyzed to select a lectin that bound preferentially to the acrosomal region. Peanut agglutinin (PNA) and Pisum sativum agglutinin (PSA) were suitably specific for intracellular, acrosome-associated glycoconjugates. Peanut agglutinin exhibited almost no detectable binding to sperm surface receptors, but intense binding to the area of the acrosome anterior to the equatorial segment. In contrast, PSA bound intensely to anterior and equatorial acrosomal regions, and weakly to the other regions of the sperm. Acrosomal labeling by both lectins decreased when sperm were induced to acrosome-react with calcium ionophore. To determine if these lectins could be used to assess acrosomal status, we compared the percentage of acrosome-reacted sperm that were detected by staining with naphthol yellow and erythrosin B with the percentage that were detected by lectin labeling. The incidence of reacted sperm detected by PSA labeling was not significantly different from that detected by naphthol yellow/ erythrosin B (P = 0.46). The incidence of reacted sperm detected by PNA was correlated with the incidence detected by naphthol yellow/erythrosin B, but was significantly lower (P = 0.003). We conclude that labeling permeabilized sperm with fluoresceinated PSA can serve as a rapid assay for acrosomal status.     

Effect of trypsinization on lectin binding to germ cells from ICR and T/t6 mice

Flow cytometry was used to quantify the binding of fluorescein isothiocyanate (FITC)-labeled lectins to testis cells from ICR and T/t6 mice before and after trypsin treatment. Soybean agglutinin, wheat germ agglutinin, and concanavalin A bound well to testis cells of both mouse strains. Limax flavus agglutinin (LFA) bound very slightly and Ulex europeas agglutinin (UEA) did not bind at all. Trypsinization increased binding of soybean agglutinin and decreased binding of wheat germ agglutinin in both mouse strains, providing evidence for masked carbohydrate-binding sites on the surface of germ cells. It did not affect binding of the other lectins. Trypsin treatment was an attempt to increase lectin binding, particularly the binding of LFA and UEA to the reported T/t-specific carbohydrates, sialic acid, and L-fucose, respectively. These studies indicate that the T/t6 locus alleles do not alter the surface carbohydrate content of testis cells sufficiently to be detected by lectin-binding differences.     

Glycoconjugates of the human sperm surface: Distribution and alterations that accompany capacitation in vitro

We have studied changes in the binding of fluoresceinated lectins to human sperm during in vitro capacitation. We first determined the surface labeling pattern of viable sperm obtained by the swim-up procedure. Sperm were labeled with 100 μg/ml FITC-conjugated lectin at 4°C for 30 min. We simultaneously used Hoechst stain 33258 as a supravital stain to help differentiate surface from intracellular lectin labeling. Of 14 lectins studied, six (phytohemagglutinin-E, concanavalin A, Ricinus communis agglutinin-I, and the lectins of wheat germ, Lens culinaris, and Pisum sativum) bound to the entire surface of sperm, sometimes with minor local heterogeneity. Three lectins (from peanut, Maclura pomifera, and soybean) usually bound in a punctate manner, with more label on the tail than on the head. Five lectins (Ulex europaeus, Dolichos biflorus, Helix pomatia, and Vicia villosa lectins, and lectin II of Griffonia simplicifolia) bound very poorly or not at all to the sperm surface. Sperm were also inspected for changes in surface lectin binding patterns after 0, 5, and 23 hr of incubation in a capacitating medium. Two lectins showed reproducible changes. The labeling by Maclura pomifera agglutinin decreased by 5 hr in eight of ten experiments, and among sperm labeled with concanavalin A, the incidence of sperm with a highly fluorescent anterior margin of the sperm head increased by about 3.5-fold between 0 and 5 hr. The labeling pattern of the other lectins did not change.     

Relationship of intratesticular testosterone content of stallions to age, spermatogenesis, Sertoli cell distribution and germ cell-Sertoli cell ratios

Testes were obtained from 47 1-20-year-old stallions during the natural breeding season. Total testicular testosterone and testosterone/g testis increased with age (P less than 0.005), and total testicular testosterone was associated with larger testis size (P less than 0.05). Neither testosterone per gram nor per paired testes were related to total Sertoli cell number (P greater than 0.05), but greater testosterone per paired testes was associated with fewer Sertoli cells per unit of seminiferous tubule length (P less than 0.005) or basement membrane area (P less than 0.02) and with a higher number of germ cells supported per Sertoli cell (P less than 0.05). Although values for testosterone per gram and per paired testes were unrelated (P greater than 0.10) to sperm production/g testis or to the yield of spermatids/spermatogonium, testosterone per paired testes was positively related to sperm production per paired testes (P less than 0.05). It is concluded that intratesticular testosterone increases with age, is related in a positive manner to quantitative rates of sperm production, and can account for some of the differences in sperm production among individual stallions within a single breeding season.     

Rai14 (Retinoic Acid Induced Protein 14) Is Involved in Regulating F-Actin Dynamics at the Ectoplasmic Specialization in the Rat Testis*

Rai14 (retinoic acid induced protein 14) is an actin binding protein first identified in the liver, highly expressed in the placenta, the testis, and the eye. In the course of studying actin binding proteins that regulate the organization of actin filament bundles in the ectoplasmic specialization (ES), a testis-specific actin-rich adherens junction (AJ) type, Rai14 was shown to be one of the regulatory proteins at the ES. In the rat testis, Rai14 was found to be expressed by Sertoli and germ cells, structurally associated with actin and an actin cross-linking protein palladin. Its expression was the highest at the ES in the seminiferous epithelium of adult rat testes, most notably at the apical ES at the Sertoli-spermatid interface, and expressed stage-specifically during the epithelial cycle in stage VII-VIII tubules. However, Rai14 was also found at the basal ES near the basement membrane, associated with the blood-testis barrier (BTB) in stage VIII-IX tubules. A knockdown of Rai14 in Sertoli cells cultured in vitro by RNAi was found to perturb the Sertoli cell tight junction-permeability function in vitro, mediated by a disruption of F-actin, which in turn led to protein mis-localization at the Sertoli cell BTB. When Rai14 in the testis in vivo was knockdown by RNAi, defects in spermatid polarity and adhesion, as well as spermatid transport were noted mediated via changes in F-actin organization and mis-localization of proteins at the apical ES. In short, Rai14 is involved in the re-organization of actin filaments in Sertoli cells during the epithelial cycle, participating in conferring spermatid polarity and cell adhesion in the testis.     

Distribution of the microtubule-dependent motors cytoplasmic dynein and kinesin in rat testis.

To examine the possible role of microtubule-based transport in testicular function, we used immunofluorescent techniques to study the presence and localization of the microtubule mechanoenzymes cytoplasmic dynein (a slow-growing end-directed motor) and kinesin (a fast-growing end-directed motor) within rat testis. Cytoplasmic dynein immunofluorescence was observed in Sertoli cells during all stages of spermatogenesis, with a peak in apical cytoplasm during stages IX-XIV. Cytoplasmic dynein immunofluorescence was also localized within Sertoli cells to steps 9-14 (stages IX-XIV) germ cell-associated ectoplasmic specializations. In germ cells, cytoplasmic dynein immunofluorescence was observed in manchettes of steps 15-17 (stages I-IV) spermatids, and small, hollow circular structures were seen in the cytoplasm of step 17 and step 18 spermatids during stages V and VI. Kinesin immunofluorescence was observed in manchettes of steps 10-18 spermatids (stages X-VI). The stage-dependent apical Sertoli cell cytoplasmic dynein immunofluorescence, in conjunction with the previously reported orientation of Sertoli cell microtubules (slow-growing ends toward the lumen) and peak secretion of androgen-binding protein and transferrin, is consistent with the hypothesis that cytoplasmic dynein is involved in Sertoli cell protein transport and secretion. Further, the localization of cytoplasmic dynein and kinesin to manchettes is consistent with current hypotheses concerning manchette function.