The epididymis as a target for male contraceptive development.

The epididymis is an excellent target for the development of a male contraceptive. This is because the process of sperm maturation occurs in this organ; spermatozoa become motile and are able to recognise and fertilise an egg once they have traversed the epididymal duct. However, a number of attempts to interfere in sperm maturation and epididymal function or both have not been successful. The use of transgenic animals has proved useful in identifying a few epididymal targets but has yet to open the doors for drug development. Continuous focus on identifying additional epididymal targets and sperm-specific and epididymal-specific drugs is key to bringing a male contraceptive acting on the epididymis to the public.

We, the developing rete testis, efferent ducts, and Wolffian duct, all hereby agree that we need to connect.

BACKGROUND: The mechanisms by which the rete testis joins the efferent ducts, which joins the Wolffian duct during development, are not known. Mouse and chick models have been helpful in identifying genes that are important for the development of each part, but genes have not been identified as to those that play a role in the joining of each part. Clinical implications of the failure of the male reproductive tract to form a fully functional conduit for spermatozoa are not trivial. Epididymal disjunction, the failure of the efferent ducts to join the testis, is one of several epididymal anomalies that have been observed in some boys who were cryptorchid at birth. OBJECTIVE: A systematic review of studies focusing on the morphogenesis of the mesonephric duct and mesonephric tubules in different species, and identification of clinical issues should there be failure of these tissues to develop. DESIGN: PubMed and GUDMAP databases, and review of books on kidney development were searched for studies reporting on the mechanisms of morphogenesis of the kidney and epididymis. MAIN OUTCOMES MEASURE(S): Gaps in our knowledge were identified, and hypotheses coupled with suggestions for future experiments were presented. RESULTS: A total of 64 papers were identified as relevant, of which 53 were original research articles and 11 were book chapters and reviews covering morphogenesis and clinical issues. Investigators utilized multiple species including, human, mouse, chick, Xenopus, bovine, and sheep. CONCLUSION: Fundamental understanding of the morphogenesis of the male reproductive tract is limited, especially the morphogenesis of the rete testis and efferent ducts. Therefore, it is not surprising that we do not understand how each part unites to form a whole. Only one mechanism of joining of one part of the tract to another was identified: the joining of the Wolffian duct to the cloaca via controlled apoptosis.

Male sexual dysfunction in mice bearing targeted mutant alleles of the PEA3 ets gene.

PEA3, a member of the Ets family of transcriptional regulatory proteins, is expressed in a unique spatial and temporal pattern during mouse embryogenesis; its overexpression is positively correlated with HER2-mediated breast tumorigenesis in both humans and mice. To determine whether PEA3 plays a part in development and oncogenesis and to uncover its normal physiological role, we generated mice lacking functional PEA3 by gene targeting in embryonic stem cells. PEA3(-/-) mice arose from heterozygous crosses with the expected Mendelian frequency, revealing that PEA3 is dispensable for embryogenesis. PEA3 mutant mice displayed no overt phenotype and lived a normal life span. However, PEA3-deficient males failed to reproduce. PEA3 is expressed in several male sexual organs, but gross and histological analyses of the organs from PEA3(-/-) mice revealed no abnormalities. Spermatogenesis and spermiogenesis also appeared normal in mice homozygous for the PEA3 mutation, and their sperm were capable of fertilizing eggs in vitro. PEA3(-/-) males engaged in normal mating behavior, but they did not set copulatory plugs and sperm could not be detected in the uteri of females that had mated with PEA3(-/-) males. Erections could be evoked by abdominal pressure in PEA3-deficient male mice, and the results of in vitro experiments revealed that the corpus cavernosum isolated from PEA3 mutant males relaxed in response to acetylcholine. Therefore, the infertility of PEA3 mutant males involves either mechanisms proximal to the cavernosal smooth muscle or an ejaculatory dysfunction. However, PEA3 mutant mice are phenotypically distinguishable from other knockout mice with such deficits and thus provide a unique model for further investigation of male sexual dysfunction.

Male contraception: views to the 21st century, Bethesda, MD, USA, 9-10 September 1999.

For men who still wish to father children, the contraceptive options currently available are withdrawal and the condom. Although significant progress has been made on hormonal and vaccine-related approaches to male contraception, a marketed product is, at best, several years away. Therefore, the National Institute of Child Health and Human Development convened a workshop to discuss novel strategies for development of male contraceptives that focused on the testis and epididymis. Participants recognized that exploration of these new approaches will necessitate considerable investment of funds and research efforts.

Expression of multiple gamma-glutamyl transpeptidase messenger ribonucleic acid transcripts in the adult rat epididymis is differentially regulated by androgens and testicular factors in a region-specific manner.

Multiple gamma-glutamyl transpeptidase (GGT) messenger RNAs (mRNAsII-IV) are expressed in a region-specific manner in the rat epididymis. In the present study, we examined the role(s) of plasma testosterone (T) and testicular factors in regulating the region-specific pattern and quantity of GGT mRNAs expressed along the epididymal duct. Northern blot and ribonuclease protection analyses showed that bilateral orchiectomy for 1, 5, and 15 days dramatically reduced the expression of GGT mRNAsII-IV in the initial segment. Expression of GGT mRNAII and mRNAIII was maintained in the initial segment of orchiectomized animals receiving T implants that maintain normal serum T concentrations, but GGT mRNAIV expression remained low relative to sham-operated control values. Unilateral efferent duct ligation decreased GGT mRNAIV expression only in the initial segment. Hence, expression of GGT mRNAIV in the initial segment was not maintained by circulating T and required a factor(s) of testicular origin. In caput epididymidis, expression of GGT mRNAII and mRNAIII declined after orchiectomy and was not completely restored to control values in orchiectomized animals by plasma T alone, but also required a testicular factor(s). In contrast to the initial segment, expression of GGT mRNAIV in the corpus and cauda epididymidis did not require T and/or a testicular factor(s), as expression of this transcript remained unchanged in these regions after 1, 5, and 15 days of orchiectomy, orchiectomy and T replacement, and after unilateral efferent duct ligation. In the cauda epididymidis, expression of GGT mRNAII required circulating androgens and was unaffected by unilateral efferent duct ligation, whereas GGT mRNAIII expression was repressed by T. These data demonstrate that circulating T and a factor(s) of testicular origin differentially regulate the expression of each GGT mRNA in a region-specific manner.

The restricted penetration of iodinated rat FSH and LH into the seminiferous tubules of the rat testis.

The penetration of 125I-iodinated rat follicle-stimulating hormone (FSH; labelled by three different techniques) and luteinizing hormone (LH) through the walls of the seminiferous tubules of the rat testis has been studied by injecting the labelled hormone into rats with the efferent ducts of one testis ligated 16 h before the collection of samples of blood and tissues. The concentration of trichloracetic acid-precipitable and immunoprecipitable radioactivity was measured in blood plasma and rete testis fluid and calculated for the total secreted fluid retained in the testis by the ligature, and for the additional tubular fluid from the ligated testis, separated by centrifugation after decapsulating the testis and dispersing the cells. Very little intact hormone penetrated into the testicular fluids, even 16 h after injection of the labelled hormone, and the volume of distribution in the unligated testis of the trichloracetic acid-precipitable radioactivity was only slightly greater than that for markers known to be confined to the extracellular interstitial fluid. This suggests that the labelled hormones do not penetrate readily through the walls of the semiferous tubules into their lumina. Injected inorganic iodiide and trichloracetic acid-soluble 125I-circulating after the injection of iodinated hormones penetrated more rapidly into the tubules, but had not reached equilibrium between the testicular fluids and blood plasma 16 h after injection. Labelled FSH was reasonably stable in the circulation after injection, but 80% of the 125I was not protein-bound 16 h after injection of labelled LH.

Epididymal epithelium: its contribution to the formation of a luminal fluid microenvironment.

To understand the process of sperm maturation, an understanding of interactions between the spermatozoa with the luminal fluid microenvironment and with the epididymal epithelium is necessary. The composition of epididymal luminal fluid of several species is well documented but the manner by which the epididymis contributes to the formation of this specialized milieu is not so well understood. A major role played by the epididymis is to finely regulate the movement of molecules into and out of the lumen. This ensures that as spermatozoa progress along the duct they are exposed to a continually changing, but optimal environment necessary for their maturation and survival. This review focusses on our current understanding of the contributions of the epididymal epithelium to the formation of a specialized luminal fluid microenvironment. The role of the blood-epididymis barrier, the composition of the epididymal luminal fluid, the permeability properties of the epididymal epithelium, and recent studies on a number of luminal fluid proteins and expression of the genes which encode these proteins are discussed.

The testicular and epididymal luminal amino acid microenvironment in the rat.

Concentrations of amino acids were measured in arterial and testicular venous blood, and in fluids from the seminiferous tubule, rete testis, and the caput, corpus, and cauda epididymidis. There were no significant differences in the concentrations of any amino acids between arterial and testicular venous blood, whereas there were significant differences between arterial/venous blood and testicular interstitial fluid. The predominant amino acids measured within seminiferous tubule fluid (STF) and rete testis fluid (RTF) were glycine, alanine, glutamate, and glutamine. RTF contained approximately equal concentrations of basic and total amino acids, but 17 times higher acidic amino acids and 1.2 and 1.3 times lower uncharged polar and nonpolar amino acids, respectively, compared to STF. The concentration of total amino acids within caput fluid reached over 50 nmol/L, but then declined to approximately 50% and 0.1% of caput for corpus and cauda, respectively. The predominant amino acids measured within epididymal luminal fluids were glutamate and taurine; glutamate contributed to approximately 90% of the total amino acids measured in caput fluid. The presence of glutamate and taurine within the epididymal lumen is due primarily to a direct contribution from the epididymal epithelium, as measured using the split-drop stopped-flow microperfusion technique. Several other amino acids within the lumen also originate from the epididymal epithelium. Amino acids contribute approximately 20%, 9%, and 2% of the total osmolality of caput, corpus, and cauda fluid, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Stability and transcriptional regulation of gamma-glutamyl transpeptidase mRNA expression in the initial segment of the rat epididymis.

Gamma-glutamyl transpeptidase (GGT) is an enzyme believed to play a role in the protection of maturing spermatozoa in the epididymis. Our previous studies have shown that four GGT mRNAs (I-IV) transcribed from the single-copy rat GGT gene are differentially expressed and regulated in the rat epididymis. In particular, the normal expression of GGT mRNA(IV) in the epididymal initial segment is dependent upon the presence of testicular factors. The objective of this study was to test the hypothesis that the decreased expression of GGT mRNA(IV) in the initial segment following the in vivo removal of testicular factors by efferent duct ligation (EDL) is due to a decrease in stability and/or transcription rate. The stability of the GGT mRNAs was evaluated by measuring the rate of mRNA decay. These stability studies showed that GGT mRNA(IV) exhibited a rapid initial decay that slowed at later times to a decay rate similar to that of GGT mRNAs(II,III). The decay rates were not different following sham-operation or EDL, and thus the stability of GGT mRNAs were not influenced by the in vivo loss of testicular factors. Results of transcription analysis revealed that the transcription rate of GGT mRNA(IV) in the initial segment fell by approximately 68% following a 12-hour EDL. Additionally, secondary-structure models indicate two families of folding patterns for GGT mRNA(IV), which could be the reason for the two decay regimes detected in the stability study. Thus, the decreased expression level of GGT mRNA(IV) in the initial segment following the in vivo loss of testicular factors is a function of a decreased transcription rate and intricate decay kinetics.

Regional differences in luminal fluid polypeptides of the rat testis and epididymis revealed by two-dimensional gel electrophoresis.

Luminal fluid samples were collected by micropuncture of the seminiferous tubule, rete testis, and defined levels of the epididymal tubule. After removal of spermatozoa by centrifugation, the supernatant fluids were analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and an ultrasensitive silver staining procedure to define the sequential change in protein composition along the excurrent duct system. Fluid from each segment displayed a characteristic 2-D PAGE map composed of numerous polypeptides. Seminiferous tubule fluid contained a wide array of polypeptides, with most concentrated in the 45 Kd to 90 Kd range, but, in contrast, rete testis fluid lacked most of these polypeptides. The major complex of rete testis fluid comigrated with serum albumin and was present in all distal segments. Other major rete testis components were not noted distally. Fluid from the caput was characterized by new major components of 30 to 37 Kd, 28 to 30 Kd, 24 Kd, and 23 Kd, each of which consisted of multiple spots of apparent isoelectric variants; all except the 30 to 37 Kd complex were present in the fluid from more distal segments. Proceeding distally, there was a temporal appearance of new polypeptides, especially in the molecular weight range below 30 Kd. Two-dimensional PAGE analysis of detergent extracts of washed spermatozoa indicate that a specific subset of these fluid polypeptides are sperm associated.

Selective luminal absorption of L-carnitine from the proximal regions of the rat epididymis. Possible relationships to development of sperm motility.

The absorption of L-carnitine from the duct of the proximal regions of the rat epididymis was investigated using a stopped-flow, split-droplet microperfusion technique. L-carnitine was absorbed from the duct of the proximal caput epididymidis by a time-dependent and saturable transport system (Km = 25 micron; Vmax = 0.65 pmoles absorbed/min/mm3 tubular volume). Furthermore, absorption appeared to be primarily sodium-independent, although the existence of a minor sodium-dependent pathway cannot be ruled out. A similar transport system was not evident along the distal caput epididymidis, where absorption of L-carnitine was attributable to passive diffusion only. The inward and outward movement of L-carnitine across the epithelium of the proximal and distal caput epididymidis appears to be regulated so that the spermatozoa come into contact with high levels of L-carnitine in the distal caput region.

The male antifertility agents alpha chlorohydrin, 5-thio-D-glucose, and 6-chloro-6-deoxy-D-glucose interfere with sugar transport across the epithelium of the rat caput epididymidis.

The effects of the male antifertility agents alpha-chlorohydrin, 5-thio-D-glucose, and 6-chloro-6-deoxy-D-glucose on sugar transport (3H-3-O-methyl-D-glucose and 3H-2-deoxy-D-glucose) across the rat caput epithelium was studied in vivo and in vitro. The compound alpha-chlorohydrin reduced sugar transport uptake in vivo but not in vitro, whereas 5-thio-D-glucose and 6-chloro-6-deoxy-D-glucose were both effective in vivo and in vitro. The mechanism of action of these compounds on sugar movement across the caput epithelium is probably complex. Direct competition for the glucose carrier situated on the basolateral membrane and intratubular effects are suggested. Thirty-day injections of 5-thio-D-glucose or alpha-chlorohydrin did not have adverse effects on sugar transport or the permeability of the blood-testis and blood-epididymis barriers as assessed by an in vitro technique.

Changes in luminal fluid protein composition in the rat cauda epididymidis following partial sympathetic denervation.

Sympathetic denervation of the rat cauda epididymidis by surgical removal of the inferior mesenteric ganglion (IMG) results in an excessive accumulation of sperm in the cauda epididymidis as well as significant changes in cauda sperm motility and cauda epididymal gross histology. The objective of the present study was to determine if the cauda-specific changes in sperm storage, sperm motility, and epididymal histology following the loss of sympathetic innervation were accompanied by changes in the protein composition of epididymal fluid. One and 4 weeks after surgical IMG removal or sham operations, luminal fluid obtained from the caput and cauda epididymidis and cauda epididymal sperm-associated proteins were subjected to two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and silver-stained proteins were quantitated. One week after IMG removal, two cauda epididymal fluid (CEF) proteins (2 and 13) had increased 43% and 49%, respectively, whereas four CEF proteins (5, 8, 9, and 19) had decreased between 30% and 73% compared to controls. Four weeks after IMG removal, changes in CEF proteins observed 1 week following surgery were no longer present, but the staining intensities of three additional CEF proteins (11, 12, and 18) were reduced an average of 70% compared to control CEF proteins. By obstructing the cauda epididymidis, we confirmed that the changes in CEF protein composition observed following IMG removal were not the result of sperm accumulation but were due directly to the loss of innervation; the staining intensity of CEF protein 2 increased as a result of excessive sperm accumulation in the cauda epididymidis both in the presence and absence of innervation from the IMG. No significant changes in caput epididymal fluid proteins or cauda epididymal sperm-associated proteins were detected following IMG removal. These data show that the protein composition of rat CEF is significantly affected by the loss of sympathetic innervation and suggest that neuronal input may play an important role in the maintenance of epididymal function.

Oxidative stress differentially regulates the expression of gamma-glutamyl transpeptidase mRNAs in the initial segment of the rat epididymis.

Reactive oxygen species (ROS) have a powerful cytotoxic effect on spermatozoa and have been implicated in spermatozoal dysfunction and male infertility. gamma-Glutamyl transpeptidase (GGT) is essential to the metabolism of the antioxidant glutathione and, as such, is believed to be important in protecting spermatozoa against oxidative stress. The aims of this study were 1) to establish in vitro conditions in which ROS were generated and 2) to determine whether oxidative stress regulated the expression of GGT mRNAs I-IV in the initial segment of the epididymis. Initial segments were collected from adult male rats and incubated in culture media to which ROS-generating compounds, hypoxanthine and xanthine oxidase, were added. By 6.5 hours, incubation of tissue in high-oxidative stress conditions caused a 56% decrease in reduced glutathione concentration, a concomitant 240% increase in oxidized glutathione concentration, and a 25% decrease in adenosine triphosphate concentration. RNase protection analyses demonstrated an approximate 70% up-regulation of GGT mRNAs II-IV in a differential manner, depending on the concentration of oxidizing agents and the type of ROS generated. gamma-Glutamyl transpeptidase mRNA I was not expressed. These results support the hypothesis that expression of GGT mRNAs is regulated by oxidative stress in the initial segment of the rat epididymis.

Proteins in luminal fluid of the ram excurrent ducts: changes in composition and evidence for differential endocytosis.

Rete testis fluid (RTF) and luminal fluid collected by micropuncture at selected epididymal sites were analyzed to characterize the spectrum of proteins and to quantify the net gain or loss of total/bulk protein and androgen-binding protein (ABP) between successive regions within the ductus epididymidis. Based on one-dimensional SDS gel electrophoresis, the spectra of proteins in RTF and fluids from the proximal, central, and distal caput through proximal corpus epididymidis differed from each other. Concentrations of sperm, bulk protein, and ABP increased from the rete testis through the central caput epididymidis. Electron microscopic studies following intraluminal microinjections of RTF proteins conjugated to colloidal gold at specific sites in the excurrent ducts revealed that 145 times more protein-gold was endocytosed in the ductuli efferentes than in any of the four regions of the caput epididymidis. Thus, ductuli efferentes were the major extra-testicular site of endocytosis of bulk protein present in RTF; at least a portion of the uptake was specific. On a per sperm basis, the amount of protein present in the central caput epididymidis was less than 15% of that leaving the testis. Although most of the protein present in RTF (greater than or equal to 86 mg/d) must be absorbed in the ductuli efferentes and the initial segment of the epididymis and replaced by newly secreted proteins (greater than or equal to 34 mg/d), there was negligible loss of ABP in these regions. Net loss of ABP occurred primarily in the distal caput and proximal corpus epididymidis. These studies demonstrate that ABP is spared from endocytosis along with the bulk protein in RTF and conserved for functions in epididymal regions far distal to the site of bulk protein loss.

Transgenic technologies for the study of epididymal function.

Sperm mature and acquire the capacity for fertilization during their transit through the epididymis, however little is known of the molecular events that comprise sperm maturation. Recent advances in transgenic mouse technology hold promise for illumination of this process. Most of the existing infertile, transgenic mouse lines seem to have defects in epithelial structure or sperm transport rather than direct defects in the maturation of sperm. Temporally and spatially restricted targeted disruptions of epididymal specific genes should provide great insight into the epididymal contribution to sperm maturation.

Micropuncture and microanalytical studies of rhesus monkey and baboon epididymis and the human ductus deferens.

Using micropuncture and microanalytical techniques, we studied the microenvironment surrounding the maturing spermatozoa in different regions along the epididymis of the rhesus monkey (Macaca mulatta; 3 animals) and the baboon (Papio cynocephalus; 1 animal) and in the human ductus deferens. In the monkey and baboon, samples of luminal contents (luminal fluid and spermatozoa) of approximately 50 to 300 nanolitres were collected from several epididymal sites and the luminal fluid analyzed for inositol. Similarly, a sample of approximately 0.5 to 1.0 µl of luminal contents was collected from each human ductus deferens and the luminal fluid analyzed for sodium, potassium, chloride, inositol, carnitine, glycerophosphocholine (GPC), phosphocholine and total phosphate. Each analysis required the modification of standard methods to accommodate the very small sample volumes collected. We show that the microenvironment in the monkey, baboon, and human is different from those in other species with respect to the concentration of compounds estimated. In the luminal fluid of the human ductus deferens, the majority of the osmoticallyactive compounds are the inorganic ions which is in direct contrast to the rat, hamster, rabbit, ram and boar. In these species, organic compounds contribute significantly more to the osmolarity of the luminal fluid than do inorganic ions. Although the significance of these findings is unclear, a relationship seems to exist between the appearance of carnitine in the luminal fluid of the proximal caput epididymidis of the rat and the point where spermatozoa develop the potential for motility. These investigations also raise the question of which species most closely reflects the physiology of the reproductive system of man.

Sodium, potassium, and protein concentrations and 2D-SDS-page of epididymal luminal and ejaculated seminal fluids of the adult chimpanzee (Pan troglodytes).

The concentration of soluble protein and of sodium and potassium ions was estimated in chimpanzee caput epididymal luminal fluid, cauda epididymal luminal fluid, and ejaculated seminal fluid. Protein concentration was 48.5 ± 1.5 µg/µl in caput fluid, 26.8 ± 2.0 µg/µl in cauda fluid, and 53.0 ± 7.9 µg/µl in seminal fluid. Sodium concentration was 127.0 ± 7.0 mM in caput fluid, 34.5 ± 1.8 mM in cauda fluid, and 18.8 ± 1.8 mM in seminal fluid. Potassium concentration was 58.0 ± 0.0 mM in caput fluid, 56.8 ± 5.2 mM in cauda fluid, and 77.0 ± 1.7 mM in seminal fluid. Proteins in caput epididymal, cauda epididymal, and ejaculated seminal fluids, with approximate molecular weights (kDa) between <14.4 and 45.0 kDa and apparent isoelectric points (pIs) between 4.5 and 7.5, were resolved by two-dimensional SDS-polyacrylamide gel electrophoresis (2D-SDS-PAGE) and silver stained. In caput fluid, the most intensely stained polypeptides resolved between 14.4 and 21.5 kDa (pI 5.4-7.4). In cauda fluid, the number and intensity of stained components increased markedly, and the most intensely stained polypeptides resolved between 21.5 and 31.0 kDa (pI 5.7-7.3). In seminal fluid, polypeptides between <14.4 and 45.0 kDa (pI 5.7-7.4) appeared characteristically diffuse and distributed. These results demonstrate that the ions and the polypeptides in the luminal microenvironment change significantly along the epididymal duct of the male chimpanzee. © 1994 Wiley-Liss, Inc.