Epididymal compounds and their influence on the metabolism and survival of spermatozoa

Epididymal fluid, which is derived from testicular fluid, contains several unusual compounds. Little information is available on the composition of the testicular fluid of primates, but the fluid of the ram, bull, boar, and rat contains high concentrations of inositol and certain amino acids. Analyses have been made of epididymal fluid collected from the cauda epididymis of the Rhesus monkey and several nonprimate species (e.g., ram, bull, dog, stallion, rabbit, guinea pig, rat, and hamster), but similar information on the human is lacking. Cauda epididymal fluid appears to be similar in composition from one mammalian species to another. However, the epididymal plasma differs considerably from blood, lymph, and other extracellular fluids. The environment of spermatozoa in the epididymis is, therefore, highly specialized, and presumably in some way contributes to the prolonged survival of spermatozoa in this organ, and provides substrates for the metabolism of the spermatozoa. The chief characteristics of the cauda epididymal plasma are the low concentration of inorganic ions and the high levels of several unusual organic constituents namely, glycerylphosphorylcholine, carnitine, sialic acid, amino acids, glycosidases, and phosphatases. At least one antifertility compound, namely, orally administered α-chlorohydrin, appears to be concentrated in the epididymis. Studies on laboratory animals, domestic species, and man, suggest that it inhibits enzymes of the glycyolytic pathway in spermatozoa, and this may be the basis for its antifertility activity.     

Characteristics of artificially ejaculated and ductus deferens semen in the guinea-fowl

This study was conducted to determine whether the ductus deferens semen of the guinea-fowl is diluted with a fluid at ejaculation and to locate the region ejecting the fluid.Some of the ejaculated semen, which was collected by the lumbar massage method, gave a slight positive aldose reaction when examined by chromatography in contrast to a negative aldose reaction in the ductus deferens semen.No significant differences were found in the pH value and concentration of spermatozoa between the ejaculated and ductus deferens semen. However, the volume of the ejaculate was significantly (P<0.05) greater than that of the semen contained in the receptacular region which corresponds to the receptacle of the ductus deferens in cocks and drakes.These results suggest that a fluid containing aldose may be added in small amounts to the ductus deferens semen during natural ejaculation. It is probably a transudate from the tissue in the vicinity of the papilla of the ductus deferens.     

Microanatomy of the testes and testicular ducts of the butterfly lizard,Leiolepis ocellata Peters, 1971 (Reptilia: Squamata: Agamidae) during the active reproductive period

The microanatomy of the testes and testicular ducts (rete testis, ductuli efferentes, ductus epididymis and ductus deferens) of Leiolepis ocellata (Agamidae) was investigated using light microscopy including histochemistry. Each testis contains seminiferous tubules and interstitial tissues. The former house spermatogenic cells (spermatogonia A & B, preleptotene, primary and secondary spermatocytes, spermatids (steps 1–8) and spermatozoa) and Sertoli cells, while the latter comprise peritubular and intersitial tissues. The rete testis is an anastomosing duct, having intratesticular and extratesticular portions. The proximal region of ductuli efferentes has wider outer ductal and luminal diameters than those of the distal region. The convoluted ductus epididymis is subdivided into four regions (initial segment, caput, corpus and cauda), based on the ductal diameter, epithelium characteristics and cell components. The ductus deferens has the greatest diameter and is divided into the ductal and ampulla ductus deferens. The ductal portion is subdivided into the proximal and distal regions, based on the epithelium types and ductal diameters. The ampulla ductus deferens is a fibromuscular tube, having numerous mucosal folds projecting into the lumen. Spermiophagy is detectable in the ductus epididymis and ductus deferens. The present results contribute to improved fundamental knowledge on the microanatomy of the reptilian reproductive system.     

Effects of oxytocin and vasopressin on sperm transport from the cauda epididymis in sheep

This study was performed to determine whether oxytocin or vasopressin affect the transport of spermatozoa from the epididymis of rams in vivo. Under general anaesthesia, cannulae were inserted into each ductus deferens and passed into the cauda epididymis of 24 Oxford Down cross rams and the luminal fluid was collected at 10 min intervals for 2-3 h. Animals were divided into seven groups and received either (i) 2 ml 0.9% saline, (ii) 10 micrograms oxytocin, (iii) 100 micrograms oxytocin, (iv) 100 micrograms oxytocin antagonist, (v) 300 micrograms oxytocin antagonist followed by 100 micrograms oxytocin, (vi) 100 micrograms vasopressin, or (vii) 100 micrograms vasopressin followed by 100 micrograms oxytocin, all by i.v. injection. The mass of fluid and number of spermatozoa in each 10 min sample was measured and the motility of the spermatozoa was assessed. Treatment with saline did not affect the mass or the number of spermatozoa in the fluid collected. Oxytocin at 10 micrograms significantly increased both the output of fluid and the number of spermatozoa by twofold. Oxytocin at 100 micrograms produced a greater increase in both fluid output and the number of spermatozoa within 10 min of administration of the peptide. Treatment with oxytocin antagonist had no immediate effect, but subsequently caused a significant reduction in both fluid output and the number of spermatozoa. Pretreatment with oxytocin antagonist inhibited the stimulatory effect of oxytocin. Vasopressin did not increase the number or concentration of spermatozoa in the fluid and appeared to decrease fluid output. No significant changes in the morphology or motility of the spermatozoa collected was observed in any of the samples. These data demonstrate that oxytocin has specific actions on the epididymis to increase sperm transport. They indicate that local oxytocin may be involved in regulating basal contractility of the cauda epididymidis and that augmentation by the peptide in the peripheral circulation, as occurs around the time of ejaculation, may promote a significant increase in the transport of spermatozoa into the vas deferens and ejaculate.     

Ultrastructure of the male reproductive system and spermatophore formation in Labidocera aestiva (Crust.: Copepoda)

Summary The male reproductive system of Labidocera aestiva produces a flask-shaped spermatophore connected to a chitin-like coupling apparatus. As immature spermatozoa leave the anterior region of the testis, they pass through the lumen of a long, sinuous duct composed of a ductus deferens and seminal vesicle. Ultrastructural examination of the ductus deferens reveals a highly glandular, columnar epithelium. The cells contain arrays of rough endoplasmic reticulum and abundant, well-developed Golgi complexes. This region produces and releases into the lumen, a flocculent substance and two granular secretions that constitute the seminal fluid. In its terminal part, the ductus deferens synthesizes another secretion that forms the spermatophore wall enclosing the spermatozoa and seminal fluid. Final synthesis of the spermatophore wall occurs within the thin-walled seminal vesicle, although this region functions primarily as a storage organ. Contiguous to the seminal vesicle is an elongate, highly glandular spermatophore sac. The chitin-like coupling apparatus, which functions to attach the spermatophore to the female, is formed in the anterior region of the sac by secretions from eight cell types. The posterior region of the sac stores the flask-shaped spermatophore and produces secretions that aid ejaculation of the entire spermatophore complex.Contribution No. 236, Harbor Branch Foundation, Inc.     

Presence of Platelet-Activating Factor and Its Receptor in Baboon (Papio spp) Spermatozoa

Platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF] is a novel potent signaling phospholipid which has unique pleiotropic biological properties in addition to platelet activation. PAF has been detected in the spermatozoa in several species. The concentration of PAF is inversely related to human spermatozoal quality. PAF is present in squirrel monkey (a seasonal breeder) spermatozoa and is significantly higher during the breeding season. PAFs mechanism of action is a receptor-mediated event. There is no report on the presence of PAF or the PAF-receptor in nonhuman Old World primate spermatozoa. Therefore, the primary objective of this study was to determine if PAF is present in the spermatozoa from baboons, which are year-round breeders. A secondary objective was to determine the presence and localization of the PAF-receptor in spermatozoa. We extracted endogenous lipids from mature hybrid baboon (Papio spp) epididymal spermatozoa and assayed them for the presence of PAF by [ ¹²⁵ I]-radioimmunoassay. We also exposed baboon spermatozoa to PAF-receptor antibody followed by FITC-conjugated antibody. PAF was in all samples assayed (mean: 2.29 (±0.63) pmol/10 ⁶ spermatozoa). Baboon spermatozoa possess PAF-receptors most prevalently along the neck and midpiece regions. The data demonstrates that PAF and its receptor are present in baboon spermatozoa. Additional studies will elucidate the role of PAF in spermatozoal function.     

Effect on turkey spermatozoa of a frothy fluid derived from the cloaca of a male turkey

The influence on turkey spermatozoa of a frothy fluid derived from the cloacal region of a male turkey was investigated. The frothy fluid was collected from the turkey tom during mounting, and semen for the experiment was obtained from the ductus deferens removed after necropsy. Spermatozoa diluted with frothy fluid were examined for motility, viability, and fertilizing capacity and compared with semen diluted with phosphate buffer or undiluted ductal semen. The life-span of spermatozoa suspended in frothy fluid was slightly prolonged during in vitro storage as compared with the undiluted semen or the semen diluted with phosphate buffer; however, a rapid increase of the number of deformed spermatozoa during storage was observed in the semen diluted with frothy fluid. The fertilizing ability of spermatozoa was not influenced by the dilution with frothy fluid when the diluted spermatozoa were inseminated intravaginally immediately after the dilution. On the contrary, when spermatozoa suspended in frothy fluid were preserved at 0 C for 24 h, their fertilizing capacity decreased drastically, probably due to the increased number of abnormal spermatozoa during in vitro preservation.     

Concentrations of carnitine, glutamate and myo-inositol in epididymal fluid and spermatozoa from boars

This study examines the concentrations of carnitine, glutamate and myo-inositol in fluid and spermatozoa from six epididymal regions. Samples were taken from six post-pubertal boars, and the sperm concentration, the protein concentration in epididymal fluid and the concentrations of carnitine, myo-inositol and glutamate in the epididymal fluid and spermatozoa were analysed. In epididymal fluid the concentration of myo-inositol decreased in a proximo-distal direction, whereas intraluminal concentrations of L-carnitine and L-glutamate increased distally. As changes in the concentration of these solutes did not parallel changes in sperm concentration, this may reflect secretion or absorption of theses solutes. The sperm content of inositol fell as they moved from the distal caput whereas glutamate content increased from the distal caput to more distal regions and carnitine content remained unchanged during epididymal transit. This is the first attempt to elucidate the changes in the content of glutamate and inositol in epididymal spermatozoa of mammals and in the fluid from different epididymal regions of boars.     

Peptidase activities in the semen from the ductus deferens and uterus of the neotropical rattlesnake Crotalus durissus terrificus

To understand the role of peptidases in seminal physiology of Crotalus durissus terrificus, intra- and inter-seasonal activity levels of acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive neutral (APN-PI), cystyl (CAP), dipeptidyl-IV (DPPIV), type-1 pyroglutamyl (PAP-I) and prolyl-imino (PIP) aminopeptidases as well as prolyl endopeptidase (POP) were evaluated in soluble (SF) and/or membrane-bound (MF) fractions of semen collected from the ductus deferens of the male reproductive tract and from the posterior portion of the uterus. Seminal APB, PIP and POP were detected in SF, while other peptidases were detected in SF and MF. Only the convoluted posterior uterus in winter and autumn had semen. Relative to other examined peptidases, in general, APN-PI, APN-PS and APB activities were predominant in the semen from the uterus and throughout the year in the semen from the ductus deferens, suggesting their great relevance in the seminal physiology of C. d. terrificus. The levels of peptidase activities in the ductus deferens semen varied seasonally and were different from those of semen in the uterus, suggesting that their modulatory actions on susceptible peptides are integrated to the male reproductive cycle events and spermatozoa viability of this snake.     

Regional differences in the metabolism of testosterone in vitro in the epididymis & ductus deferens of the adult rhesus monkey Macaca mulatta.

Regional differences in the metabolism of testosterone in vitro in the epididymis (1.5 cm segments) and ductus deferens (2 cm segments) were investigated in the adult rhesus monkey, Macaca mulatta. The conversion of tritiated testosterone to its 5alpha-reduced derivatives increased from the proximal to the distal regions of the epididymis. The total amount of 5alpha-reduced products in the subsequent segments of the epididymis was highest in the intact monkey and on the intact side of the unilaterally castrated monkey and was followed in decreasing order by the castrated side of the hemiscastrate and least in the bilateral castrate. Hormonal requirements for maintenance of the biological functions of the epididymis and maturation of spermatozoa in the epididymis are discussed.     

Transforming growth factor beta 1 induces tight junction disruptions and loss of transepithelial resistance across porcine vas deferens epithelial cells

Epithelial cells lining the male excurrent duct contribute to male fertility by employing a number of physiological mechanisms that generate a luminal microenvironment conducive to spermatozoa maturation and storage. Among these mechanisms, male duct epithelia establish intercellular tight junctions that constitute a barrier to paracellular diffusion of water, solutes, large molecules, and cells. Mechanisms regulating the male duct epithelial barrier remain unidentified. Transforming growth factor beta (TGFB) is a regulatory cytokine present in high concentrations in human semen. This study examined whether TGFB has any effects on epithelial function exhibited by primary cultures of porcine vas deferens epithelia. TGFB1 exposure caused a 70%-99% decrease in basal transepithelial electrical resistance (R(TE), a sensitive indicator of barrier integrity), while a significant decrease in anion secretory response to forskolin was detected at the highest levels of TGFB1 exposure employed. SB431542, a selective TGFB receptor I (TGFBR1) inhibitor, prevented decreases in barrier function. Results also demonstrated that TGFB1 exposure modifies the distribution pattern of tight junction proteins occludin and claudin 7. TGFBR1 is localized at the apical border of the native porcine vas deferens epithelium. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) 11 (also known as p38-MAPK) did not alter the effect of TGFB1 on R(TE) significantly. These data suggest that epithelia lining the vas deferens are subject to disruptions in the physical barrier if active TGFB becomes bioavailable in the luminal fluid, which might be expected to compromise fertility.     

Expression and activity of gamma-glutamyl transpeptidase in the rat epididymis.

Following Northern analysis, GGT mRNA was found predominantly within the caput epididymides and kidney. The size of mRNAs for kidney, caput, corpus, and ductus deferens were 2.2, 2.3, 2.2, and 2.3 kb, respectively, whereas cauda showed a doublet of 2.2 and 2.3 kb. GGT transpeptidation and hydrolytic activity within epididymal luminal fluids collected by micropuncture showed caput = corpus greater than cauda and corpus greater than caput greater than cauda, respectively. Caput luminal GGT transpeptidation activity was significantly inhibited by serine-borate and was optimal at pH 8.0. The calculated Km and Vmax values for hydrolysis of GSH by caput luminal GGT were 0.06 microM and 2.19 nmoles/min/microliters luminal fluid at pH 8.5 compared to 0.49 microM and 0.49 nmoles/min/microliters luminal fluid, respectively, at the physiological pH 6.5 of caput fluid. These studies would suggest that the epididymis can control the activity of luminal GGT by pH. Lower Km (0.12 microM) and higher Vmax (1.13 nmoles/min/microliters luminal fluid) values were also calculated when GSSG was used compared to GSH. Results from Triton X-114 partitioning experiments suggest that luminal GGT probably exists in both membrane bound and nonmembrane bound forms. Western blot analysis of proteins within epididymal luminal fluids revealed both subunits of GGT in all epididymal regions studied. However, two lower molecular bands, approximately 22 kDa and 21 kDa, were also observed in cauda fluid. It is suggested that as GGT is transported along the epididymal duct it undergoes degradation, which accounts for its loss of activity in the distal epididymal regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructure and histochemistry of the male reproductive system of the genus Callinectes Stimpson, 1860 (Brachyura: Portunidae)

We described the ultrastructure and histochemistry of the reproductive system of five Callinectes species, and evaluate the seasonal variation in weight of the reproductive system and hepatopancreas by comparing annual changes of somatic indices. The somatic indices changed little throughout the year. In Callinectes, spermatogenesis occurs inside the lobular testes and, within each lobule, the cells are at the same developmental stage. Spermatogenesis and spermiogenesis follow the same development pattern in all Callinectes studied. Mature spermatozoa are released into the seminiferous ducts through the collecting ducts. Cells of the vas deferens are secretory as evidenced by rough endoplasmic reticulum, Golgi complex, and secretory vesicles that produce the seminal fluid. The anterior vas deferens shows two portions: proximal and distal. In proximal portion (AVDp), spermatozoa are clustered and embedded in an electron-dense, basophilic glycoproteinaceous secretion Type I. In the distal portion (AVDd), the spermatophore wall is formed by incorporation of a less electron-dense glycoproteinaceous secretion Type II. The secretion Type I change to an acid polysaccharide-rich matrix that separates the spermatophores from each other. The median vas deferens (MVD) stores the spermatophores and produces the granular glycoproteinaceous seminal fluid. The posterior vas deferens (PVD) has few spermatophores. Its epithelium has many mitochondria and the PVD seminal fluid changes into a liquid and homogeneous glycoprotein. Many outpocketings in the PVD and MVD help to increase the fluid production. Overall, the reproductive pattern of Callinectes is similar to other species that produce sperm plugs. The secretions of AVD, MVD, and PVD are responsible for the polymerization that forms the solid, waxy plug in the seminal receptacle. The traits identified here are common to all Portunidae species studied so far.     

Comparative biochemistry of mammalian epididymal plasma.

1. A comparison has been made of the chemical composition of epididymal plasma from the cauda epididymidis of nine mammalian species. 2. Results have shown that epididymal plasma contains low concentrations of inorganic ions and high levels of several unusual organic constituents, among which may be mentioned glycerylphosphorylcholine, hypotaurine, carnitine and several glycosidases and phosphatases. 3. The influence of this milieu on the motility and survival of spermatozoa is discussed.     

Immunohistochemical localization of prostaglandin H synthase in the epididymis and vas deferens of the mouse

Prostaglandins (PGE2, PGF2 alpha) in the excurrent ducts of the male reproductive tract appear to be both modulators of ductal contractility for transport of spermatozoa and factors involved in the regulation of sperm maturation. To identify the tissue sites for the production of prostaglandins (PGs) in the excurrent ductal system, we have employed an immunohistochemical technique to localize prostaglandin H (PGH) synthase in the epididymis and vas deferens of the mouse. A mouse monoclonal antibody to PGH synthase was used and was shown to be specific for the mouse enzyme by Western blot analysis. In sexually mature mice, PGH synthase was primarily localized to the epithelium of the epididymis and vas deferens. Within the epididymal epithelium, immunoactivity appeared in all cell types of the initial segment, in a subpopulation of cells with predominantly apically oriented nuclei in the caput and corpus, and in low levels in the cauda. PGH synthase reactivity was the most intense in the epithelial cells of the vas deferens. PGH synthase was not detected in smooth muscle cells, spermatozoa, or luminal fluid. This study suggests that the epithelium of the excurrent ductal system of the mouse is the major site for PG production. The regionalization of PGH synthase to cells in the epididymis thought to be involved in the absorption of luminal fluid suggests that PGs may play a role in fluid and ion transport.     

Effects of temperature on the immobilization and the initiation of motility of spermatozoa in the male reproductive tract of the domestic fowl, Gallus domesticus

1. The motility of undiluted fowl spermatozoa taken from testis, epididymis and ductus deferens was negligible at 40 degrees C, around the normal avian body temperature. 2. The immobilization was not permanent and motility was restored by decreasing the temperature to 30 degrees C or by suspending in a NaCl/TES buffer with 2 mM Ca2+, 2 mM HCO3- or 10% seminal plasma at 40 degrees C. 3. Demembranated spermatozoa taken from testis, epididymis and ductus deferens were also immotile at 40 degrees C. However, these spermatozoa were restored the motility at 30 degrees C except testicular spermatozoa. 4. These results suggest that the capacity of movement of fowl spermatozoa can be readily obtained from testis, but that these spermatozoa are immotile due to temperature-dependent immobilization in the male reproductive tract. 5. Furthermore, it is possible that changes in environmental temperature at ejaculation are one of the important exogenous physiological factors of the initiation of fowl sperm motility.     

Role of androgens in survival of spermatozoa in epididymis of tammar wallaby (Macropus eugenii).

Studies of undiluted micropuncture samples of luminal fluid from the cauda epididymidis of the tammar indicated that spermatozoa are immotile in situ and spontaneously activate during collection or subsequent incubation in vitro. The suppression of sperm motility was related to the androgen status of the tammars and when this was increased by the use of Silastic implants of testosterone propionate, the spontaneous activation of samples was delayed for up to 2 h during incubation in vitro. Spermatozoa survived for up to 9 weeks when isolated in the cauda epididymidis between ligatures around the ductus. However, even after isolation for 3 weeks their viability was reduced compared with samples from the contralateral, unligated duct. Isolation of a length of ductus between ligatures also reduced the concentration of spermatozoa in the lumen of the duct and reduced the concentration of some proteins in the epididymal plasma. However, it did not affect the electrophoretic pattern of detergent extracts of spermatozoa. A study of the effects of orchidectomy and testosterone therapy indicated that sperm survival in the epididymis is androgen dependent. Orchidectomy reduced the concentration of spermatozoa in the luminal fluid and the volume of luminal fluid, and resulted in an increase in the concentration and a change in the electrophoretic pattern of protein in the fluid. The effects of orchidectomy were reduced or prevented by testosterone therapy. It is concluded that the cauda epididymidis of the tammar is at least as well adapted for sperm storage as it is in the eutherian mammals that have been studied.     

Microphotometric measurement of some enzymatic activities in rabbit spermatozoa during epididymal maturation

Cytochemical quantitative measurements of isocitrate dehydrogenase (ICDH), malate dehydrogenase (MDH), cytochrome oxidase, lactate dehydrogenase (LDH), glucose 6-phosphate dehydrogenase (G6PDH) and glutamate dehydrogenase (GLDH) activities were made on rabbit spermatozoa collected from the testis, the different epididymal sites and the ductus deferens. These measurements were made on individual spermatozoa (at least 100 spermatozoa for each site under consideration) using a Vickers M 85 scanning microdensitometer.The activity patterns of the enzymes involved in the tricarboxylic acid cycle (ICDH, MDH) and in the respiratory chain (cytochrome oxidase) both showed a progressive decrease in the intramitochondrial oxidative metabolism from the testis to the ductus deferens. This was in contrast to LDH activity which represents the anaerobic glycolysis pathway rather than the activity of intramitochondrial LDH. The G6PDH activity could be related to those membrane modifications which the male gamete undergoes during its epididymal maturation. Potential GLDH activity was relatively intense in the spermatozoa from the testis and from the initial and distal segments of the genital tract, suggesting an intramitochondrial synthesis of enzymes such as cytochrome oxidase or ATPase.The quantitative variations of the enzymatic activities occurring during the transit of spermatozoa along the male genital tract suggested the existence of different specific interactions between the spermatozoon and the epididymal microenvironment.     

Sperm morphology and arrangement along the male reproductive tract of the butterfly Euptoieta hegesia (Insecta: Lepidoptera)

Summary

The present study was undertaken to describe the morphological and organizational modifications that occur in apyrene and eupyrene spermatozoa along the male adult reproductive tract of the butterfly, Euptoieta hegesia. Testis, vas deferens, vesicula seminalis and ductus ejaculatorius were studied by transmission electron microscopy. In the testis, both sperm types are organized into cysts; apyrene sperm are devoid of extracellular structures while eupyrene ones have lacinate and reticular appendages. In the testis basal region, both sperm pass through an epithelial barrier and lose their cystic envelope. The eupyrene morphological and organizational modifications are more drastic than the apyrene ones. From the vas deferens to the ductus ejaculatorius, apyrene sperm are dispersed in the lumen and acquire several concentric layers that are formed by the folding of their abundant cell membrane. The apyrene distribution observed here suggests that their functions include eupyrene transportation. Eupyrene sperm, however, remain aggregated along the tract. In the vas deferens, they are covered by a filamentous material that develops into a homogeneous matrix surrounding the spermatozoa coat in the vesicula seminalis and the ductus ejaculatorius. Eupyrene sperm undergo complex morphological changes that include the loss of lacinate appendages and the formation of a dense and heterogeneous extracellular coat. The formation of the matrix and the coat in eupyrene extratesticular sperm is related to the loss of lacinate appendages. These changes are in general extracellular and are probably important for sperm maturation.     

Ultrastructure of sperm storage and male genital ducts in a male holocephalan, the elephant fish, Callorhynchus milii.

In chondrichthyes, the process of spermatogenesis produces a spermatocyst composed of Sertoli cells and their cohort of associated spermatozoa linearly arrayed and embedded in the apical end of the Sertoli cell. The extratesticular ducts consist of paired epididymis, ductus deferens, isthmus, and seminal vesicles. In transit through the ducts, spermatozoa undergo modification by secretions of the extratesticular ducts and associated glands, i.e., Leydig gland. In mature animals, the anterior portion of the mesonephros is specialized as the Leydig gland that connects to both the epididymis and ductus deferens and elaborates seminal fluid and matrix that contribute to the spermatophore or spermatozeugmata, depending on the species. Leydig gland epithelium is simple columnar with secretory and ciliated cells. Secretory cells have periodic acid-Schiff positive (PAS+) apical secretory granules. In the holocephalan elephant fish, Callorhynchus milii, sperm and Sertoli cell fragments enter the first major extratesticular duct, the epididymis. In the epididymis, spermatozoa are initially present as individual sperm but soon begin to laterally associate so that they are aligned head-to-head. The epididymis is a highly convoluted tubule with a small bore lumen and an epithelium consisting of scant ciliated and relatively more secretory cells. Secretory activity of both the Leydig gland and epididymis contribute to the nascent spermatophores, which begin as gel-like aggregations of secretory product in which sperm are embedded. Fully formed spermatophores occur in the ductus. The simple columnar epithelium has both ciliated and secretory cells. The spermatophore is regionalized into a PAS+ and Alcian-blue-positive (AB+) cortex and a distinctively PAS+, and less AB+ medulla. Laterally aligned sperm occupy the medulla and are surrounded by a clear zone separate from the spermatophore matrix. Grossly, the seminal vesicles are characterized by spiral partitions of the epithelium that project into the lumen, much like a spiral staircase. Each partition is staggered with respect to adjacent partitions while the aperture is eccentric. The generally nonsecretory epithelium of the seminal vesicle is simple columnar with both microvillar and ciliated cells.