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.

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.

Regulation of gene transcription in the epididymis.

The epididymis exhibits region-specific as well as cell-specific patterns of gene expression within the epithelium. The spatial and temporal patterns of gene expression originate during development and are critical to the formation and maintenance of a fully functional epididymis. Despite the number of mechanisms reported to contribute to the regulation of eukaryotic gene expression, little is known about the specific mechanisms involved in the control of epididymal gene expression. This review will outline some of the cis-DNA elements and associated transcription factors that have been identified in the epididymis, in addition to discussing the potential role of co-regulator molecules and changes in chromatin structure as critical control points of gene expression. Although gene expression can be controlled at several points, discussion will focus on gene regulation at the transcriptional level. The role of post-transcriptional control, with particular attention to mRNA stability, will also be discussed.

Resting (basal) secretion of proteins is provided by the minor regulated and constitutive-like pathways and not granule exocytosis in parotid acinar cells.

Resting secretion of salivary proteins by the parotid gland is sustained in situ between periods of eating by parasympathetic stimulation and has been assumed to involve low level granule exocytosis. By using parotid lobules from ad libitum fed rats stimulated with low doses of carbachol as an in vitro analog of resting secretion, we deduce from the composition of discharged proteins that secretion does not involve granule exocytosis. Rather, it derives from two other acinar export routes, the constitutive-like (stimulus-independent) pathway and the minor regulated pathway, which responds to low doses of cholinergic or beta-adrenergic agonists (Castle, J. D., and Castle, A. M. (1996) J. Cell Sci. 109, 2591-2599). The protein composition collected in vitro mimics that collected from cannulated ducts of glands given low level stimulation in situ. Analysis of secretory trafficking along the two pathways of resting secretion has indicated that the constitutive-like pathway may pass through endosomes after diverging from the minor regulated pathway at a brefeldin A-sensitive branch point. The branch point is deduced to be distal to a common vesicular budding event by which both pathways originate from immature granules. Detectable perturbation of neither pathway in lobules was observed by wortmannin addition, and neither serves as a significant export route for lysosomal procathepsin B. These findings show that parotid acinar cells use low capacity, high sensitivity secretory pathways for resting secretion and reserve granule exocytosis, a high capacity, low sensitivity pathway, for massive salivary protein export during meals. An analogous strategy may be employed in other secretory cell types.

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.

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.

Involvement of polyomavirus enhancer activator 3 in the regulation of expression of gamma-glutamyl transpeptidase messenger ribonucleic acid-IV in the rat epididymis.

Gamma-glutamyl transpeptidase (GGT) mRNA-IV and polyomavirus enhancer activator 3 (PEA3) mRNA are highly expressed in the initial segment of the rat epididymis, and both are regulated by testicular factors. PEA3 protein in rat initial segment nuclear extracts has been shown to bind to a PEA3/Ets binding motif, which is derived from the partially characterized GGT mRNA-IV promoter region. This suggests that PEA3 may be involved in regulating transcription from the rat GGT mRNA-IV gene promoter in the initial segment. Using DNA oligonucleotide primers and DNA sequencing analysis, an approximately 1500-basepair (bp) DNA sequence at the 5' region of the promoter was obtained. Using transient transfection, PEA3 activated transcription of the rat GGT mRNA-IV promoter only in cultured epididymal cells from the rat initial segment, but not in Cos-1 or NRK-52E cells. Promoter deletion analysis indicated that a PEA3/Ets binding motif between nucleotides -22 and -17 is the functional site for PEA3 to activate transcription of GGT promoter IV and that an adjacent Sp1 binding motif is also required to maintain promoter IV activity in epididymal cells. Transcriptional activation of promoter IV was shown to be epididymal cell-specific and PEA3-specific. In addition, PEA3 may act as a weak repressor for transcription of promoter IV, probably using a PEA3/Ets binding motif(s) distal to the transcription start site. A model of how PEA3 is involved in the regulation of transcription of GGT promoter IV in epididymal cells is proposed.

Differential induction of gamma-glutamyl transpeptidase in primary cultures of rat and mouse hepatocytes parallels induction during hepatocarcinogenesis.

In carcinogen-treated rats, gamma-glutamyl transpeptidase (GGT) is induced in preneoplastic liver lesions and liver tumors. However, in mice, GGT is rarely detected during hepatocarcinogenesis. Data in this study reveal that GGT is not induced in mouse hepatocytes when they are maintained in vitro under the same conditions that induce GGT activity in primary cultures of rat hepatocytes. GGT activity in rat hepatocytes increased 20-fold during the first 7 days in culture, but there was no induction of GGT in primary cultures of mouse hepatocytes. Comparison of intracellular glutathione levels in rat and mouse liver cells showed that the glutathione level was higher in the mouse liver cells than the rat. Blocking glutathione synthesis with buthionine sulfoximine reduced the intracellular glutathione concentration in mouse liver cells but did not trigger an induction of GGT. Analysis of the GGT mRNA in primary cultures of rat hepatocytes showed that only GGT mRNA(III) is induced. This is the same GGT mRNA species present in preneoplastic hepatic lesions and liver tumors in the rat (1-3). Therefore activation of promoter III in the GGT gene is responsible for induction of GGT in both hepatocytes in vitro and liver tumors in vivo. These data show that primary cultures of rat and mouse hepatocytes provide a model system with which to study interspecies differences in the regulation of this enzyme and to better understand the role of GGT in normal and neoplastic processes.

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.

Regulation of gamma-glutamyl transpeptidase catalytic activity and protein level in the initial segment of the rat epididymis by testicular factors: role of basic fibroblast growth factor.

The objective of this study was to test the hypothesis that gamma-glutamyl transpeptidase (GGT) catalytic activity and protein level in the initial segment are regulated by testicular factors. In the rat epididymis, levels of GGT catalytic activity were initial segment > caput > corpus = cauda. GGT catalytic activity and protein level in the initial segment decreased after efferent duct ligation (EDL) for 3 days, but were recovered when initial segment pieces were incubated with ovine or rat rete testis fluid (oRTF or rRTF, respectively). Factors responsible for the recovery were shown to be greater than 10 kDa and protein-like, but these factors were not androgen-binding protein or testosterone in oRTF. Further experiments were designed to test the hypothesis that growth factors within oRTF and rRTF regulate GGT catalytic activity and GGT protein level. Basic fibroblast growth factor (bFGF) but not epidermal growth factor recovered GGT catalytic activity and protein level in the initial segment following 3-day EDL. Western blot analyses also revealed that bFGF-like proteins were present in rRTF, epididymal luminal fluid, and rat initial segment homogenate, and that the level of bFGF-like proteins declined in the initial segment following 3-day EDL. Using a bFGF monoclonal antibody, a small amount of bFGF-like proteins was found to be also present in oRTF. Our studies suggest that bFGF is one of the testicular factors involved in the regulation of epididymal GGT catalytic activity and protein level. Since decreased GGT catalytic activity caused by 3-day EDL in the rat initial segment was also recovered by a tumor promoter, phorbol 12-O-tetradecanoylphorbol 13-acetate, it is possible that a signal transduction pathway is involved in the regulation of GGT catalytic activity and GGT protein level by testicular factors. Western blot analyses also indicated that the 43-kDa bFGF-like protein in the lumen of the rat epididymis originates from the testis, is concentrated in the initial segment, and is reabsorbed by the epididymal epithelia from proximal to distal epididymal regions.

Testicular regulation of epididymal gene expression.

Normal epididymal function is regulated by androgens and testicular factors. Our studies have been directed towards identifying testicular factors that regulate the function of the initial segment and the mechanisms by which this is achieved. The initial segment appears to be critical for normal sperm maturation in view of recent gene knock-out studies. Previous and ongoing studies from this and other laboratories have shown that the expression of several genes including proenkephalin, cystatin-related epididymal specific (CRES), 5 alpha-reductase and gamma-glutamyl transpeptidase (GGT) within the initial segment is highly dependent upon the presence of testicular factors. A lumicrine mechanism of regulation of these genes is proposed. The regulation of gamma-glutamyl transpeptidase (GGT) is described as a model enzyme for studying the role and identification of testicular factors. GGT appears to play an important role in the protection of spermatozoa from oxidative stress. Multiple GGT mRNAs (II-IV) are expressed within the epididymis, but GGT mRNA IV is the only form that is highly expressed in the initial segment, especially within zone 1A, and is regulated by testicular factors. Testicular factors control this transcript by regulating both its rate of transcription and its stability. Evidence is presented to suggest that basic fibroblast growth factor (bFGF) is a candidate testicular factor that regulates GGT activity in the epididymis. Basic FGF may regulate gene expression in the epididymis via the ras-raf-MAPK second messenger pathway and by members of the Ets transcription family.

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.

Identification, expression, and regulation of the transcriptional factor polyomavirus enhancer activator 3, and its putative role in regulating the expression of gamma-glutamyl transpeptidase mRNA-IV in the rat epididymis.

Gamma-glutamyl transpeptidase (GGT) mRNA-IV is highly expressed in the initial segment of the rat epididymis and is regulated by testicular factors. The promoter region for GGT mRNA-IV contains five conserved polyomavirus enhancer activator 3 (PEA3)-binding motifs (5'-AGGAAG-3'). We hypothesize that PEA3 is present in the rat epididymis and is regulated by one or more testicular factors. Western blot analyses showed that a 62-kDa protein was detected in the nuclear extract from the rat initial segment at higher levels than in the distal epididymal regions. Electrophoretic mobility shift assays (EMSAs) showed that the nuclear extract specifically bound to the PEA3 motif, forming a DNA-protein complex. This complex contained the 62-kDa PEA3 protein as demonstrated by EMSAs and Southwestern analyses. Northern blot analyses and RNase protection analyses showed that PEA3 mRNA was predominantly expressed in the initial segment as compared to the distal epididymal regions and was under the regulation of testicular factors. These results suggest that PEA3 could be involved in the regulation of expression of the rat GGT mRNA-IV gene in response to testicular factors in the initial segment.

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.

Expression of E-selectin mRNA during ischemia/reperfusion injury.

To better understand molecular and cellular processes involved in tissue inflammation, we have examined expression of endothelial leukocyte adhesion molecule 1 (E-selectin) mRNA in adult male rats after ischemia/reperfusion (I/R) injury and after intravenous injection of lipopolysaccharide (LPS). The polymerase chain reaction was used to generate a rat E-selectin cDNA fragment by using heart total RNA from rats exposed to LPS. This partial cDNA fragment spanned sequences from complement repeat region-5 to the second cytoplasmic tail domain. Comparison of the predicted amino acid sequence from the rat cDNA fragment to mouse and human E-selectin protein sequences showed significant conservation. The rat E-selectin cDNA fragment was used as a probe to examine the regulation of E-selectin mRNA expression by Northern blot analysis. As previously described in other animal species, E-selectin mRNA expression was induced after intravenous injection of LPS. In contrast, ischemia did not induce E-selectin mRNA expression, except in the setting of I/R injury. I/R injury triggered expression of E-selectin mRNA in the kidney. These experiments represent a first in vivo examination of E-selectin mRNA expression after I/R injury and constitute an initial step in characterizing a model system for investigating inflammation in the setting of acute ischemic injury.

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.