The role of stallion seminal proteins in fertilisation

Seminal plasma proteins are secretory proteins originating mainly from the epididymis and the accessory sex glands. They are involved in the remodelling of the sperm surface which occurs during sperm transit through the male genital tract and continues later at ejaculation. During this process, collectively called post-testicular sperm maturation, the spermatozoa acquire the ability to fertilise an egg. Seminal plasma proteins have been shown to contribute to early and central steps of the fertilisation sequence, e.g. the establishment of the oviductal sperm reservoir, modulation of capacitation and gamete interaction. The major equine seminal plasma proteins belong to three protein classes, which contain widely occurring protein modules. Fn-2 type proteins are characterised by two or four tandemly arranged Fn-2 modules and have been implicated in the modulation of sperm capacitation. Multiple members of the cysteine-rich secretory proteins (CRISP) have been identified in the male genital tract of a number of species. CRISP proteins have been shown to be involved in various functions related to sperm-oocyte fusion, innate host defense function and ion channel blockage. Spermadhesins occur only in ungulate species. Their carbohydrate- and zona pellucida-binding properties would suggest a role of these proteins in gamete recognition. The major proteins of equine seminal plasma have been isolated and characterised regarding their expression along the male genital tract, protein structure and their functions.     

Structural and molecular characterization of equine sperm-binding fibronectin-II module proteins

Phospholipid-binding proteins in the male genital tract are characterized by differing numbers Fn-2 modules (B-domain) carrying N-terminal extensions (A-domain) of variable length. In the stallion, three different proteins were identified, SP-1, SP-2, and EQ-12. SP-1 and SP-2 of the AA'BB'- and ABB'-type, respectively, are major proteins of the seminal plasma. Here we report the cDNA sequences of SP-1, and of a new member of the SP-2 family (SPnew) and the partial characterization of their iso- and glycoforms. The phosphorylcholine (PC)-binding ability of the long Fn-2 protein, EQ-12, with four tandemly arranged Fn-2 modules was determined by PC-affinity chromatography. Expression patterns of EQ-12, and the SP-proteins were studied by means of RT-PCR, Northern blot analysis and immunological approaches indicating differential expression along the male reproductive tract. The vast majority of the short SP-1 and SP-2 proteins are produced by the ampulla whereas EQ-12 originates from the epididymis. Indirect immunofluorescence microscopy of sperm isolated from different regions of the epididymis and Western blot analysis indicate that both, the long and the short Fn-2 proteins associate to the sperm surface during post-testicular maturation. Sperm binding of Fn-2 proteins at the post-acrosome and midpiece was at first detected in the corpus epididymis. Enhanced fluorescence intensity after ejaculation point to an increased number of molecules bound to the sperm surface. The function of these proteins is discussed in regard to their structure-function relationships.     

Autoradiographic investigation of sperm transit through the male mouse genital tract after tritiated thymidine incorporation

The transit of spermatozoa in the genital tract of the male mouse was investigated by quantitative light microscopic autoradiography after intraperitoneal injection of tritiated thymidine. Transit duration in the caput and the corpus of the epididymis was shown to be 3 days; the total duration of transit in the genital tract was 5 days. These findings indicate that the time required for the transit of spermatozoa in the epididymal caput and corpus was comparable to that calculated in other mammals studied. However, the duration of sperm storage in the epididymal cauda appeared to be shorter than that previously reported for rodents.     

Induction of epididymal boar sperm capacitation by pB1 and BSP-A1/-A2 proteins, members of the BSP protein family

A family of proteins designated BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa, collectively called BSP (bovine seminal plasma) proteins, constitute the major protein fraction of bull seminal plasma. BSP proteins can stimulate sperm capacitation by inducing cholesterol and phospholipid efflux from sperm. Boar seminal plasma contains one homologous protein of the BSP family, named pB1; however, its physiological role is still unknown. In the current study, we report a novel method to purify pB1 from boar seminal plasma by chondroitin sulfate B-affinity chromatography and reverse-phase-high performance liquid chromatography. We also studied the effect of pB1, BSP-A1/-A2, and whole boar seminal plasma on boar sperm capacitation. Boar epididymal sperm were washed, preincubated in noncapacitating medium containing pB1 (0, 2.5, 5, 10 or 20 microg/ml), BSP-A1/-A2 (0 or 20 microg/ml) proteins, or whole seminal plasma (0, 250, 500, or 1000 microg/ml), then washed and incubated in capacitating medium. Acrosomal integrity was assessed by chlortetracycline staining. The status of sperm capacitation was evaluated by the capacity of sperm to undergo the acrosome reaction initiated by the addition of the calcium ionophore, A23187. The pB1 and BSP-A1/-A2 proteins increased epididymal sperm capacitation as compared with control (sperm preincubated without proteins). This effect reached a maximum level at 10 microg/ml pB1 and at 20 microg/ml BSP-A1/-A2 (2.3- and 2.2-fold higher than control, respectively). Whole boar seminal plasma did not induce sperm capacitation. In addition, pB1 bound to boar epididymal sperm and was lost during capacitation. These results indicate that BSP proteins and their homologs in other species induce sperm capacitation in a similar way.     

Expression of scatter factor/hepatocyte growth factor is regionally correlated with the initiation of sperm motility in murine male genital tract: Is scatter factor/hepatocyte growth factor involved in initiation of sperm motility?

Based upon findings that the scatter factor/hepatocyte growth factor (SF/HGF) has strong mitogenic and motogenic properties, and that the sperm cell acquires its fertilizing capacity and motility in the distal parts of mammalian epididymis, the present study was conducted to investigate the role of SF/HGF in initiation of sperm cell motility. This was investigated by determining the expression of SF/HGF in various regions of the murine male genital tract by scatter and cell tracking assays using MDCK epithelial cells, Western blot procedure, and the immunohistochemical procedure using paraffin sections of various regions of the male genital tract. The findings from all these assays indicate that SF/HGF is differentially expressed in various parts of the male genital tract with slight or no expression in the testes, caput epididymis, and vas deferens, and with the highest expression in cauda and corpus (distal) epididymis followed by expression in the corpus (proximal) epididymis. This region-specific SF/HGF expression pattern coincides with the pattern of acquiring the fertilizing capacity and motility by the sperm cell during its transit through the male genital tract. However, wherever SF/HGF was expressed in the male genital tract, its molecular weight was slightly higher (Mr, 82 kD), compared to the SF/HGF expressed in various other somatic tissues (Mr, 78 kD), indicating that the genital tract SF/HGF may be a different molecular species that shares some immunoreactive epitopes with the somatic cell SF/HGF. Incubation of immotile sperm from caput epididymis with the purified human placental SF/HGF of 78 kD initiated motility in 5–15% of sperm population. These results strongly suggest that the SF/HGF-like activity is expressed in the male genital tract in a region-specific manner, and this activity may have a role in initiation of sperm motility acquired during its transit through the epididymis in mammals. © 1994 Wiley-Liss, Inc.     

Human sperm proteins from testicular and epididymal origin that participate in fertilization: modulation of sperm binding to zona-free hamster oocytes, using monoclonal antibodies.

In order to identify human sperm surface proteins involved in the gamete recognition process, mouse monoclonal antibodies were directed against human spermatozoa and screened with live spermatozoa by enzyme-linked immunosorbent assay (ELISA). Immunoperoxidase staining of human testis showed the early presence of four corresponding proteins on germinal cells, while six were detected primarily in testis fluid. The presence of 17 proteins was evidenced in the epididymis. Eight were detected with a decreasing gradient from the beginning to the end of the organ, including vasa efferentia for three of them. The other nine were observed in only one defined segment, usually the caput epididymis, which was found to be the most active region. Comparison of spermatozoa patterns from testis, vasa efferentia, and the three regions of epididymis pointed out a progressive coating. By contrast, three antibodies displayed a migration of spermatozoa surface domains in the course of epididymal transit. Six antibodies were found to inhibit human spermatozoa adherence to zona-free hamster oocytes, while nine promoted it. Molecular weights of antigens corresponding to nine of the antibodies ranged from 11 to 215 kDa. No correlation could be established with previously described human proteins. These observations emphasize the role of epididymis in human sperm maturation.     

Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies

The synthesis and secretion of proteins in the different regions of the human epididymis were studied in vitro. Epididymal tissues obtained from patients undergoing castration for prostatic carcinoma or from cadavers were incubated in the presence of [35S]methionine, and the resulting radiolabeled proteins were analysed on SDS-PAGE. The corpus region was found to be the most active segment in total protein synthesis. Significant qualitative and quantitative changes were observed in the pattern of proteins secreted from the different epididymal regions. To establish those epididymal proteins that interact with maturing sperm, the secreted products were immunoreacted with antibodies raised against a Triton X-100 extract of ejaculated human sperm heads. The antibodies react mainly with the head region of ejaculated spermatozoa as judged by indirect immunofluorescence. Protein A-gold labeling of freeze-fracture images showed gold particle distribution on the sperm plasma membrane. Western blot analysis of the secreted proteins revealed four bands (66, 37, 32, and 29 kDa) in the proximal regions and six additional bands (80, 76, 48, 27, 22, and 17 kDa) in the distal part of the epididymis. Immunoprecipitation of the secreted proteins with these antibodies revealed six radioactive bands of 170, 80, 76, 60, 48, and 37 kDa, which indicates that certain proteins of epididymal origin bind to the sperm plasma membrane.     

On the diversity of sperm basic proteins in the vertebrates: V. Cytochemical and amino acid analysis in Squamata, Testudines, and Crocodylia

The variability of sperm basic proteins in representatives of three reptilian orders, Squamata, Testudines, and Crocodylia, has been examined by cytochemistry, acid-urea polyacrylamide gel electrophoresis, and amino acid analysis of amidoblack-stained bands. Snakes contain type 3B intermediate sperm basic proteins by cytochemical criteria. Electrophoresis of basic proteins from epididymis chromatin as well as from testis and ductus deferens cell suspensions shows two fast-moving bands in the vicinity of herring protamine. These proteins are triprotamines containing about 27 mol % arginine, along with lysine and histidine. Lizards have type 1 protamines in their sperm nuclei cytochemically and also show a two-banded electrophoretic pattern similar to that of snakes. However, these proteins are triprotamines, similar to those in snakes with 25 mol % arginine. It may be that these are testis-specific proteins of the spermatid stage in lizards since a cytochemical transition can be observed from type 3A intermediate proteins in spermatids of testis to type 1 protamine in mature sperm of ductus deferens. Turtles contain type 3A intermediate sperm basic proteins cytochemically and basic proteins from epididymis chromatin display both a prominent band and a minor band close to, but slightly slower than, the two bands for snakes and lizards. Amino acid analysis of these bands shows that these basic proteins are also triprotamines but with a higher level of arginine, about 48 mol %, than that in snake and lizard sperm proteins. Basic proteins from epididymis chromatin of a single Mississippi alligator show three main bands moving close to the bands of snakes, lizards, and turtles. These proteins have amino acid compositions typical for triprotamines, with 28-39 mol % arginine. The data indicate that the sperm basic proteins of representatives of 25 species in three reptilian orders are very similar, in contrast to the diversity of sperm protein types found in frogs (Kasinsky, Huang, Kwauk, Mann, Sweeney, and Yee: J. Exp. Zool., 203:109-126, '78; Kasinsky, Huang, Mann, Roca, and Subirana: J. Exp. Zool., 234:33-46, '85a). This appears to be part of a macroevolutionary trend from diversity of sperm basic proteins in frogs to relative constancy in reptiles (Kasinsky, Mann, Pickerill, Gutovich, and Byrd, Jr.:J. Cell Biol., 91:1879, '81; Kasinsky, Mann, Lemke, and Huang: In: Chromosomal Proteins and Gene Expression, Plenum Press, New York, pp. 333-352, '85b). We present the hypothesis that one factor for such a trend resides in the fact that fertilization is internal in reptiles but external in anurans.     

Aquaporin 9 expression along the male reproductive tract

Fluid movement across epithelia lining portions of the male reproductive tract is important for modulating the luminal environment in which sperm mature and reside, and for increasing sperm concentration. Some regions of the male reproductive tract express aquaporin (AQP) 1 and/or AQP2, but these transmembrane water channels are not detectable in the epididymis. Therefore, we used a specific antibody to map the cellular distribution of another AQP, AQP9 (which is permeable to water and to some solutes), in the male reproductive tract. AQP9 is enriched on the apical (but not basolateral) membrane of nonciliated cells in the efferent duct and principal cells of the epididymis (rat and human) and vas deferens, where it could play a role in fluid reabsorption. Western blotting revealed a strong 30-kDa band in brush-border membrane vesicles isolated from the epididymis. AQP9 is also expressed in epithelial cells of the prostate and coagulating gland where fluid transport across the epithelium is important for secretory activity. However, it was undetectable in the seminal vesicle, suggesting that an alternative fluid transport pathway may be present in this tissue. Intracellular vesicles in epithelial cells along the reproductive tract were generally poorly stained for AQP9. Furthermore, the apical membrane distribution of AQP9 was unaffected by microtubule disruption. These data suggest that AQP9 is a constitutively inserted apical membrane protein and that its cell-surface expression is not acutely regulated by vesicular trafficking. AQP9 was detectable in the epididymis and vas deferens of 1-wk postnatal rats, but its expression was comparable with adult rats only after 3--4 wk. AQP9 could provide a route via which apical fluid and solute transport occurs in several regions of the male reproductive tract. The heterogeneous and segment-specific expression of AQP9 and other aquaporins along the male reproductive tract shown in this and in our previous studies suggests that fluid reabsorption and secretion in these tissues could be locally modulated by physiological regulation of AQP expression and/or function.     

Changes in the morphology of spermatozoa during their passage through the genital tract in dairy bulls with normal and impaired spermat ogenesis

An investigation was conducted on changes in sperm morphology along the excurrent duct of bulls. The study comprised 20 bulls with proven fertility and normal semen picture, and 10 bulls with pathologic semen. The morphology of spermatozoa was evaluated on ejaculates and on postslaughter collected contents from the excurrent duct. The incidence of pathologic sperm heads decreased along the duct in both groups of bulls. The main decrease generally occurred in caput epididymidis. In bulls with pathologic semen, the decrease continued in lower regions of epididymis and was deferens. The rate and pattern of sperm removal seem to primarily depend on the quality of spermatozoa entering the excurrent duct. The removal was clearly selective and is assumed to be associated with phagocytosis of spermatozoa mainly in the efferent ductules and proximal part of caput epididymis. Proximal cytoplasmic droplets were present on almost all sperm in the efferent ductules. The incidence decreased during passage along the genital tract. Migration of cytoplasmic droplets from a proximal to a distal position took place between regions C and D of the caput epididymidis. The incidence of middle-piece abnormalities decreased during passage along the genital tract, while the incidence of sperm tail abnormalities increased in the corpus and cauda epididymidis in all bulls.     

Zona pellucida protein binding ability of porcine sperm during epididymal maturation and the acrosome reaction

In many mammals, the first interaction between gametes during fertilization occurs when sperm contact the zona pellucida surrounding the egg. Although porcine sperm first contact the zona pellucida via their plasma membrane, the regions of the sperm surface that display zona receptors have not been determined. We have used the Alexa 488 fluorophore conjugated to solubilized porcine zona pellucida proteins to observe zona receptors on live boar sperm. Zona proteins bound live, acrosome-intact sperm on the anterior portion of the sperm head, concentrated in a thin band over the acrosomal ridge. When sperm membranes were permeabilized by fixation or acrosome reactions induced by the ionophore A23187, zona binding was extended to a broad area covering the entire acrosomal region. Zona binding proteins were present in the acrosomes of sperm from all regions of the epididymis. In contrast, zona binding sites were found on the plasma membrane of most sperm from the corpus and cauda epididymis, but on only 6% of caput epididymal sperm. In conclusion, acrosome-intact boar sperm exhibit concentrated zona protein binding over the acrosomal ridge and acquire this binding in the corpus region of the epididymis, correlating with the developmental stage at which sperm gain the ability to fertilize oocytes.     

Changes in the motility patterns of spermatozoa from the rabbit epididymis as assessed by computer-aided sperm motion analysis

Sperm maturation in the epididymis includes changes in their potential for motility that enables spermatozoa to reach the egg and penetrate its investments. The motility characteristics of spermatozoa from the testis, the epididymis, and vas deferens of the rabbit were investigated by computer-assisted sperm analysis (CASA). Various forms of motility were displayed by sperm from different regions of the epididymis released into incubation medium Testicular sperm were motile, although nonprogressive. The maximum percentage motility was expressed by sperm in the proximal cauda epididymidis, and forward progression was developed by spermatozoa from the distal caput. Once forward progression was established, the curvilinear velocity was about the same for sperm from all regions of the tract, whereas straight-line velocity increased between the mid-corpus and cauda and paralleled the decline in lateral displacement of the head. The maintenance of motility in vitro was best maintained by sperm from the distal regions of the tract although sperm from the distal caput maintained motility better than sperm from the proximal and midcorpus regions. Analysis of the motile sperm cells revealed several types of trajectories (“irregular,” “small circular,” “large circular and arcs,” “jagged” and “straight-line”) that were analyzed by discriminant analysis using the variables generated by CASA. Accuracy of classification varied from 70% to 96%, depending on the type of track. The classification function was then applied to the changes that occurred during incubation and showed that irregular trajectories gave way to small and then large circular tracks and progressive forms as sperm matured. © 1996 Wiley-Liss, Inc.     

Molecular cloning and characterization of three novel lysozyme-like genes, predominantly expressed in the male reproductive system of humans, belonging to the c-type lysozyme/alpha-lactalbumin family

Lysozymes, especially c-type lysozymes, are well-recognized bacteriolytic factors widely distributed in the animal kingdom and play a mainly protective role in host defense. The relatives of c-type lysozymes, alpha-lactalbumins, however, are only found in mammalian milk and possess a distinct biological function. These two proteins, having similar amino acid sequences, gene structure, and dimensional conformation, belong to the c-type lysozyme/alpha-lactalbumin family. Using human lysozyme as an information probe, we cloned four human cDNAs encoding homologues of human lysozyme; these were named LYZL2, LYZL4, LYZL6, and SPACA3 by the HUGO Gene Nomenclature Committee. Of these four, SPACA3 has been reported to code an intra-acrosomal sperm protein SLLP1. To our knowledge, the other three are reported here for the first time. Using Northern blot hybridization, including 16 different human tissues, we found that these four lysozyme-like genes were all highly expressed in the testis/epididymis. Further analysis of one, LYZL4, by in situ hybridization revealed that its mRNA was only detected in the epithelium of human epididymis, most abundantly in the caput, suggesting that LYZL4 plays a physiological role in male reproduction. By sequence analysis, we found that two essential catalytic residues of the human lysozyme were conserved in LYZL2 and LYZL6, whereas one site in LYZL4 and two sites in SPACA3 were replaced. The LYZL2, LYZL4, LYZL6, and SPACA3 genes were mapped to human chromosome 10p11.23, 3p21.33, 17q11.2, and 17q12, respectively, and displayed a similar genomic structure. Our data suggest that these four lysozyme-like genes, which have arisen from a common progenitor gene, play a major role in human reproduction.     

In vitro Capacitation of Ejaculated Rabbit Spermatozoa

Prior to fertilization mammalian spermatozoa undergo physiological changes in the female reproductive tract. These changes are collectively known as capacitation. In essence capacitation is a further differentiation that the sperm cell must acquire beyond the maturational changes that it undergoes in the epididymis. Although capacitation in vitro has been easily achieved in rodents, its accomplishment in the rabbit is inconsistent and difficult. We report here successful in vitro capacitation of ejaculated rabbit sperm, assessed by in vitro and in vivo fertilization of rabbit ova. Sperm were used from pooled ejaculates collected from bucks of proven fertility since sperm collected from individual bucks resulted in significant differences in fertilization levels. Conditions favoring in vitro capacitation were: (1) extended incubation time of 12 h, (2) addition of 20% heated rabbit serum to incubation medium, and (3) an atmosphere of 5% CO2, 8% O2, and 87% N2 during incubation. In vitro capacitation of sperm under these conditions resulted in 67% fertilization compared to 89% for control sperm capacitated for 13 h in the uterus.     

Novel sperm-binding proteins of epididymal origin contain four fibronectin type II-modules

Novel fibronectin type II (Fn2)-module proteins were cloned from human and canine epididymal cDNA libraries. cDNA sequences predicted a highly conserved protein family, related but not homologous to ungulate seminal plasma proteins (approximately 50% sequence identity), and the first known examples of proteins with four tandemly arranged Fn2-domains. By Northern blot and in situ hybridization analyses the encoding mRNAs were shown to be abundant products of the epididymal duct epithelium, but not detectable in other tissues. Homologous mRNAs were identified in the epididymides of various mammals, representing members of this novel protein family of epididymal origin. Within the Fn2-module-encoding stretches, species homologues displayed >85% sequence identity, but showed high variability at their predicted N-termini. An antipeptide antiserum in Western blot analyses detected 30-35 kDa immunoreactive protein bands in epididymal tissue, cauda epididymidal fluid, and sperm membrane protein preparations. The tandem arrangement of increasing numbers of Fn2-modules might functionally correspond to the tendency to form oligomers that has been described for lipid-binding proteins.     

Identification of two novel polycystic kidney disease-1-like genes in human and mouse genomes

Mutations to the prototypical members of the two general classes of polycystins, polycystin-1 encoded by PKD1 and polycystin-2 encoded by PKD2, underlie autosomal-dominant polycystic kidney disease. Here we report the identification of a pair of genes homologous to PKD1 from both the human and mouse genomes. PKD1L2 and PKD1L3 are located on human chromosome 16q22-q23 and mouse chromosome 8 and are alternatively spliced. The human and mouse forms of PKD1L2 are highly conserved, with each one consisting of 43 exons and approximately 2,460 codons. PKD1L3 shows regional sequence divergence, with the mouse form having two additional exons and a much larger exon 5. The predicted protein products of PKD1L2 and PKD1L3 contain the combination of GPS and PLAT/LH2 domains that uniquely define them as polycystin-1 family members. They are predicted to have 11 membrane-spanning regions with a large extracellular domain consistent with the proposed receptor function of this protein family. PKD1L2 and PKD1L3 contain strong ion channel signature motifs that suggest their possible function as components of cation channel pores. Polycystin-1-related proteins may not only regulate channels, but may actually be part of the pore-forming unit.     

人新基因hbrp的染色体定位及其对TPK活性的抑制作用（简报）

hbrp(Human BSP-Related Protein)是我们实验室最近在睾丸组织中克隆的一个人与BSP（bovine seminal plasma)蛋白相关的新基因。为了将有关该新基因信息与现有人类基因组转录图相整合,我们应用荧光原位杂交(fluorescent in situ hybridizationFISH)法进行了该基因的人染色体基因定位,结果成功地将hbrp基因定位在人19号染色体长臂1区3带上。hbrp基因是在对BSP蛋白功能的研究过程中发现并克隆的,其同源性分析发现,与其序列最相近     

SPAM1 (PH-20) protein and mRNA expression in the epididymides of humans and macaques: utilizing laser microdissection/RT-PCR

Background  

The Sperm Adhesion Molecule 1 (SPAM1) is an important sperm surface hyaluronidase with at least three functions in mammalian fertilization. Previously our laboratory reported that in the mouse, in addition to its expression in the testis, Spam1 is synthesized in the epididymis where it is found in membranous vesicles in the principal cells of the epithelium in all three regions. Since SPAM1 is widely conserved among mammals the aim of the study was to determine if its expression pattern in the epididymis is conserved in rodents and primates.     

Members of the Toll-like receptor family of innate immunity pattern-recognition receptors are abundant in the male rat reproductive tract

Protecting developing and maturing spermatozoa and reproductive tissues from microbial damage is an emerging aspect of research in reproductive physiology. Bacterial, viral, and yeast infections of the testis and epididymis can hinder maturation and movement of spermatozoa, resulting in impaired fertility. Toll-like receptors (TLRs) are a broad family of innate immunity receptors that play critical roles in detecting and responding to invading pathogens. Objectives of this study were to determine if organs of the rat male reproductive tract express mRNAs for members of the TLR family, to characterize expression patterns for TLRs in different regions of the epididymis, and to determine if TLR adaptor and target proteins are present in the male reproductive tract. Messenger RNA for Tlr1-Tlr9 was abundantly expressed in testis, epididymis, and vas deferens, as determined by RT-PCR, while Tlr10 and Tlr11 were less abundantly expressed. Tlr mRNA expression showed no region-specific patterns in the epididymis. Immunoblot analysis revealed relatively equal levels of protein for TLRs 1, 2, 4, and 6 in testis, all regions of the epididymis and vas deferens, and lower levels of TLRs 3, 5, and 9-11. TLR7 was primarily detected in the testis. The TLR adapter proteins, myeloid differentiation primary response gene 88 and TLR adaptor molecule 1, as well as v-rel reticuloendotheliosis viral oncogene homolog and NFKBIA, were prominent in testis, epididymis, and vas deferens. The abundant expression of a majority of TLR family members together with expression of TLR adaptors and activation targets provides strong evidence that TLRs play important roles in innate immunity of the male reproductive tract.