Excurrent Duct Research Articles

Transforming Growth Factor Beta 1 Induces Tight Junction Disruptions and Loss of Transepithelial Resistance Across Porcine Vas Deferens Epithelial Cells1

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.

Prenatal development of the bovine epididymis: Light microscopical, glycohistochemical and immunohistochemical studies

Prenatal development of the epididymis was studied in bovine fetuses ranging from 10 to 90cm crown-rump length (CRL) (75–285 pcd). The studies aimed to apply both glycohistochemistry and immunohistochemistry for the detection of the differentiation of the developing prenatal epididymis. Both conventional histological and histochemical techniques were applied on paraffin sections of the epididymis from different fetal stages. Establishment of the urogenital junction between the extra-testicular rete testis and the mesonephric duct, via the growing efferent ductules (ductuli efferentes) was first evident in fetuses with 10cm CRL. At the fetal age of 110 pcd (24cm CRL), the mesonephric duct began to lengthen and coil forming three distinct regions (caput, corpus and cauda). In addition to the macroscopical modifications in the extra-testicular excurrent duct system, histological differentiation involved both the tubular epithelial and the peritubular mesenchymal cells. The epithelium lining the efferent ductules was differentiated into ciliated and non-ciliated columnar cells. The simple epithelium of the epididymal duct increased in height and developed stereocilia on the apical surface. Additionally, some basal cells first appeared at 185 pcd (56cm CRL), within the epithelium lining the cauda only. Lectin histochemistry (WGA, PNA, GSA-I) showed early immunostaining in epithelium of the efferent ductules and in peritubular mesenchymal structures. Immunoreactivity for different proteins (S-100, fibroblast growth factor-1 and factor-2, angiotensin converting enzyme, laminin, alpha-smooth muscle actin) was evident, both in the epithelial and in the peritubular mesenchymal cells as early as at 75 pcd. On the basis of our histochemical observations, we conclude that both glycohistochemistry and immunohistochemistry are useful tools to demonstrate that the differentiation in the peritubular structures and efferent ductular epithelium begins earlier than other components.

The cytoskeletal proteins in the contractile tissues of the testis and its excurrent ducts of the passerine bird, Masked Weaver ( Ploceus velatus)

The cellular composition of the testicular capsule, seminiferous peritubular tissue, the epithelia as well as periductal muscle cell layers of the excurrent ducts was studied, in sexually mature and active Masked Weaver ( Ploceus velatus) birds of the passerine family, Ploceidae. Ultrastructure of the contractile cells in the testicular capsule, peritubular and periductal tissues showed that these cells were smooth muscles of typical morphological characteristics. Variability in the immunohistochemical co-expression of microfilaments and intermediate filaments in the different tissues was evident. Actin and desmin proteins were co-expressed immunohistochemically in the testicular capsule and seminiferous peritubular smooth muscle layer. Actin was singly and very weakly expressed in the rete testis epithelium while cytokeratins and desmin were co-expressed in the epithelium of the excurrent ducts. The periductal muscle layer of all ducts of the epididymis, the ductus deferens as well as the seminal glomus, strongly co-expressed actin and desmin. Vimentin was absent in all cells and tissue types studied. There is clear evidence that the tissues of the male gonad and its excurrent ducts in the Masked Weaver, as has been reported for members of the Galloanserae and Ratitae, contain well-formed contractile tissues whose function would include the transportation of luminal through-flow from the testis into, and through, its excurrent ducts. The microtubule helix in the head and of the mid-piece, of elongating spermatids, as well as of the mature spermatozoa in the various excurrent ducts, including some spermatozoa in the seminal glomus, also co-expressed these three proteins.

Swine models of cystic fibrosis reveal male reproductive tract phenotype at birth.

Nearly all male cystic fibrosis (CF) patients exhibit tissue abnormalities in the reproductive tract, a condition that renders them azoospermic and infertile. Two swine CF models have been reported recently that include respiratory and digestive manifestations that are comparable to human CF. The goal of this study was to determine the phenotypic changes that may be present in the vas deferens of these swine CF models. Tracts from CFTR(-/-) and CFTR(ΔF508/ΔF508) neonates revealed partial or total vas deferens and/or epididymis atresia at birth, while wild-type littermates were normal. Histopathological analysis revealed a range of tissue abnormalities and disruptions in tubular organization. Vas deferens epithelial cells were isolated and electrophysiological results support that CFTR(-/-) monolayers can exhibit Na(+) reabsorption but reveal no anion secretion following exposure to cAMP-generating compounds, suggesting that CFTR-dependent Cl(-) and/or HCO(3)(-) transport is completely impaired. SLC26A3 and SLC26A6 immunoreactivities were detected in all experimental groups, indicating that these two chloride-bicarbonate exchangers were present, but were either unable to function or their activity is electroneutral. In addition, no signs of increased mucus synthesis and/or secretion were present in the male excurrent ducts of these CF models. Results demonstrate a causal link between CFTR mutations and duct abnormalities that are manifested at birth.

Postnatal Changes in the Expression of Claudin‐11 in the Testes and Excurrent Ducts of the Domestic Rabbit (Oryctolagus cuniculus domesticus)

We examined the expression of claudin-11 (CLDN11) in the testes and male reproductive tracts of rabbits. The rabbit CLDN11 cDNA sequences were nearly identical with human, mouse, and bovine CLDN11. The levels of CLDN11 mRNA and protein (22 kDa) were markedly increased in the testis during adult development. On postnatal day (PND) 10, CLDN11 was colocalized with ZO-1 at the lateral contacts between adjacent Sertoli cells and was perpendicular to the basal lamina. In adult testis on PND 180, CLDN11 was codistributed with ZO1, and the pattern of immunoreactivity consisted of wavy linear tracts parallel to the basal lamina, which was different according to the spermatogenic stage. These results suggest that CLDN11 participates in inter-Sertoli cell tight junctions (TJs) at the blood-testis barrier in adult rabbits. CLDN11 was also found in the basal regions of Sertoli cells adjacent to the basal lamina in adult testis, suggesting that CLDN11 also participates in the adhesion between Sertoli cells and the basal lamina. CLDN11 mRNA and protein expressions were decreased in the adult epididymis compared with those in immature animals. In adults, CLDN11 mRNA levels were relatively high in the efferent duct, followed by those in the vas deferens, proximal corpus, and distal cauda, although low levels were observed in the initial segment and caput. On PND 10, CLDN11 immunoreactivity was identified at the apicolateral contacts between adjacent epithelial cells in the epididymis and vas deferens. In adults, CLDN11 was found in the nonciliated cells in the efferent duct and at the lateral contacts in the epithelial cells in the epididymal segments. In the caput, CLDN11 was found at the apicolateral contacts between adjacent epithelial cells, but expression was weak to negligible in the corpus of the vas deferens. CLDN11 may play an important role in TJs and cell adhesion in immature rabbit excurrent duct epithelia. In adult rabbits, CLDN11 in efferent duct epithelium and epididymal epithelium may contribute to the specific environment for sperm maturation.

Gene Expression in the Epididymis of Normal and Vasectomized Men: What Can We Learn About Human Sperm Maturation?

Anatomically, the human epididymis is unusual when compared with the excurrent duct of other eutherian mammals. Furthermore, clinical observations suggest that it may not be as important for sperm maturation as is the case for laboratory animals. In contrast, hierarchical clustering of microarray data of epididymides from normal men revealed 2274 modulated qualifiers between the epididymal segments, 1184, 713, and 269 of them being highly expressed in the caput, corpus, and cauda, respectively. The organization of qualifiers according to their similarities by gene ontology indicated that caput transcripts are dedicated to cell-cell adhesion, whereas the corpus is characterized by genes involved in response to other organisms (ie, defense mechanisms) and the cauda transcriptome is specialized in muscle contraction and establishment of localization. A region-specific gene expression pattern thus characterizes the human epididymis as in animal models. In humans, vasectomies have consequences on the epididymal transcriptome. Cluster analysis revealed that 1363 genes are expressed in both normal and vasectomized epididymides, whereas 911 and 660 of them are specifically expressed in normal and vasectomized epididymides, respectively. Three of the affected genes are particularly interesting because of their involvement in sperm biochemical remodeling during epididymal transit: dicarbonyl/l-xylulose reductase, Niemann-Pick disease, type C2, and cysteine-rich secretory protein 1. In some vasovasostomized men, these modifications in gene expression induced by vasectomy are irreversible, thus affecting the biochemical parameters, and potentially, the function of their ejaculated sperm. This may explain the discrepancies between a surgically successful vasovasostomy and fertility recovery.

Morphological Changes of the Epididymis and Description of the Excurrent Ducts of Phrynops geoffroanus (Testudines: Chelidae) During the Reproductive Cycle

The seminal ducts (efferent ductule, epididymis, and deferent duct) in adults of Phrynops geoffroanus were examined using light microscopy. A series of tubules (efferent ductules) connect the testes to the epididymides. The efferent ductules are formed by a rete of small tubules of varying diameters, with simple columnar epithelium formed by the ciliated cells, nonciliated cells, and few basal cells. The epididymis is a simple, long and highly convoluted tubule that receives the efferent ductules throughout its extension. It is covered by a pseudostratified columnar epithelium with three cellular types: the principal cells, which are the most abundant, basal cells, and a small narrow cell. The histological differences in the epididymis region (cranial, medial, and caudal), as well as the differences in the epithelium throughout the reproductive cycle, are discussed. The deferent ducts consist of a low pseudostratified epithelium with two cellular types: the principal and basal cells. During the months analyzed, spermatozoa were stored in the epididymis, and deferent ducts were found.

The epididymis, cytoplasmic droplets and male fertility.

The potential of spermatozoa to become motile during post-testicular maturation, and the relationship between the cytoplasmic droplet and fertilizing capacity are reviewed. Post-testicular maturation of spermatozoa involves the autonomous induction of motility, which can occur in vivo in testes with occluded excurrent ducts and in vitro in testicular explants, and artefactual changes in morphology that appear to occur in the testis in vitro. Both modifications may reflect time-dependent oxidation of disulphide bonds of head and tail proteins. Regulatory volume decrease (RVD), which counters sperm swelling at ejaculation, is discussed in relation to loss of cytoplasmic droplets and consequences for fertility. It is postulated that: (i) fertile males possess spermatozoa with sufficient osmolytes to drive RVD at ejaculation, permitting the droplet to round up and pinch off without membrane rupture; and (ii) infertile males possess spermatozoa with insufficient osmolytes so that RVD is inadequate, the droplet swells and the resulting flagellar angulation prevents droplet loss. Droplet retention at ejaculation is a harbinger of infertility caused by failure of the spermatozoon to negotiate the uterotubal junction or mucous and reach the egg. In this hypothesis, the epididymis regulates fertility indirectly by the extent of osmolyte provision to spermatozoa, which influences RVD and therefore droplet loss. Man is an exception, because ejaculated human spermatozoa retain their droplets. This may reflect their short midpiece, approximating head length, permitting a swollen droplet to extend along the entire midpiece; this not only obviates droplet migration and flagellar angulation but also hampers droplet loss.

Vasectomy Affects Cysteine‐Rich Secretory Protein Expression Along the Human Epididymis and Its Association With Ejaculated Spermatozoa Following Vasectomy Surgical Reversal

The epididymis is essential for the acquisition of sperm fertilizing ability and forward motility. After vasectomy, the flux and composition of the epididymal fluid are modified, causing possible sequelae to the occluded excurrent duct. Some of these sequelae may not be reversible following vasovasostomy, affecting sperm physiology and their fertilizing ability. We previously demonstrated that the epididymal expression in men of a major glycoprotein secreted by the epididymis, cysteine-rich secretory protein 1 (CRISP1), and its encoding mRNA are affected by vasectomy. In this study we showed that following vasectomy, the increased level of CRISP1 is not due to a secretory defect but to its accumulation in the intraluminal compartment of the cauda epididymidis. Western blot analyses were performed to determine the amount of CRISP1 associated with spermatozoa of men who had undergone surgical vasectomy reversal. Spermatozoa of vasovasostomized men are characterized by a significant increase (P < .05) in CRISP1 levels when compared with normal donors. There was no linear correlation between CRISP1 levels and the period of time elapsed between vasectomy and vasovasostomy. CRISP1 was also present in seminal plasma of normal and vasovasostomized men, but not in vasectomized individuals. The soluble concentration of CRISP1 was significantly higher (P < .05) in seminal plasma of vasovasostomized men when compared with normal men. Knowing that one of the proposed functions of CRISP1 is to modulate sperm capacitation, we evaluated the level of tyrosine protein phosphorylation of 2 AXAP proteins of the fibrous sheath, p81 and p105. Spermatozoa of vasovasostomized men were characterized by a 50% increase of protein tyrosine phosphorylation when compared to spermatozoa of normal men (P < .05). Our results are discussed with regard to the fertilizing ability of ejaculated spermatozoa of some vasovasostomized men.

Follistatin Overexpression in the Testis and Epididymis of Transgenic Mice Causes Infertility Due to Defects in Fluid Resorption, Sperm Transit, and Morphology in Excurrent Ducts.

Follistatin Overexpression in the Testis and Epididymis of Transgenic Mice Causes Infertility Due to Defects in Fluid Resorption, Sperm Transit, and Morphology in Excurrent Ducts.

PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: A proteome-based model for sperm mobility phenotype1,2

Sperm mobility is defined as sperm movement against resistance at body temperature. Although all mobile sperm are motile, not all motile sperm are mobile. Sperm mobility is a primary determinant of male fertility in the chicken. Previous work explained phenotypic variation at the level of the sperm cell and the mitochondrion. The present work was conducted to determine if phenotypic variation could be explained at the level of the proteome using semen donors from lines of chickens selected for low or high sperm mobility. We began by testing the hypothesis that premature mitochondrial failure, and hence sperm immobility, arose from Ca(2+) overloading. The hypothesis was rejected because staining with a cell permeant Ca(2+)-specific dye was not enhanced in the case of low mobility sperm. The likelihood that sperm require little energy before ejaculation and the realization that the mitochondrial permeability transition can be induced by oxidative stress arising from inadequate NADH led to the hypothesis that glycolytic enzymes might differ between lines. This possibility was confirmed by 2-dimensional electrophoresis for aldolase and phosphoglycerate kinase 1. This outcome warranted evaluation of the whole cell proteome by differential detergent fractionation and mass spectrometry. Bioinformatics evaluation of proteins with different expression levels confirmed the likelihood that ATP metabolism and glycolysis differ between lines. This experimental outcome corroborated differences observed between lines in previous work, which include mitochondrial ultrastructure, sperm cell oxygen consumption, and straight line velocity. Although glycolytic proteins were more abundant within highly mobile sperm, quantitative PCR of representative testis RNA, which included mRNA for phosphoglycerate kinase 1, found no difference between lines. In summary, we propose a proteome-based model for sperm mobility phenotype in which a genetic predisposition puts sperm cells at risk of premature mitochondrial failure as they pass through the excurrent ducts of the testis. In other words, we attribute mitochondrial failure to sperm cell and reproductive tract attributes that interact to affect sperm in a stochastic manner before ejaculation. In conclusion, our work provides a starting point for understanding chicken semen quality in terms of gene networks.

Undernutrition during foetal to prepubertal life affects aquaporin 9 but not aquaporins 1 and 2 expression in the male genital tract of adult rats

Expression of aquaporin water channels (AQPs) in the male excurrent ducts, is of major importance for local water movements. To study the influence of pre- and postnatal undernutrition on AQP-expression in the adult male genital tract, 4 pregnant female rats were fed ad libitum (control group) and 4 with 33.5% of gestational feed requirements (underfed group). Feeding restriction of underfed group pups continued up to weaning (25 days of age), then all pups were fed ad libitum until slaughtered at 100 days of age. Epididymides were sampled and processed for aquaporin immunohistochemistry. Expression of AQP1 was similar either in the control and underfed groups of rats, strongly evidenced at the apical and lateral plasma membrane of the efferent ducts non-ciliated cells, in the smooth muscle cells surrounding epididymal duct and in blood vessel endothelium throughout the epididymis. AQP2-immunoreactivity was present in the corpus and cauda regions, strongly expressed in the principal cells of both groups of rats. In contrast, AQP9 expression was modified by early life undernourishment, as it was weakly evidenced at the microvilli in the principal cells and strongly diminished or completely lacked in the clear cells of the cauda, in underfed group epididymides. Since it is known that clear cells are involved in luminal fluid acidification, this function might be altered in adult animals, which were underfed during early life.

The Excurrent Ducts of the Testis of the Emu (Dromaius novaehollandiae) and Ostrich (Struthio camelus): Microstereology of the Epididymis and Immunohistochemistry of its Cytoskeletal Systems

The volumetric proportion of the various ducts of the epididymis of the emu and ostrich and the immunohistochemistry of actin microfilaments, as well as cytokeratin, desmin and vimentin intermediate filaments, were studied in the various ducts of the epididymis of the emu and ostrich. The volumetric proportions of various ducts, which are remarkably different from those of members of the Galloanserae monophyly, are as follows: the rete testis, 5.2 +/- 1.4% for the emu and 2.4 +/- 1.8% for the ostrich; efferent ducts, 14.2 +/- 2.3% (emu) and 11.8 +/- 1.8% (ostrich); epididymal duct unit, 25.8 +/- 5.8% (emu) and 26.1 +/- 4.1% (ostrich) and connective tissue and its content, 54.7 +/- 5.8% (emu) and 60.0 +/- 4.9% (ostrich). Unlike in mammals and members of the Galloanserae monophyly, only vimentin was immunohistochemically demonstrated in the rete testis epithelium of the emu, and none of the cytoskeletal protein elements in the ostrich rete testis. The epithelium of the efferent ducts of the emu co-expressed actin, cytokeratin and desmin in the non-ciliated type I cells, and vimentin in the ciliated cell component. The ostrich demonstrated only cytokeratin in this epithelium. The ratite epididymal duct unit is different from that of mammals in lacking actin (only weaky expression in the ostrich), desmin and cytokeratin, and a moderate/strong immunoexpression of vimentin in the basal cells and basal parts of the NC type III cell in the epididymal duct unit. Immunoexpression of the microfilaments and intermediate filaments varied between the two ratite birds, as has been demonstrated previously in birds of the Galloanserae monophyly, and in mammals.

An insight into testis and gubernaculum dynamics of INSL3 - RXFP2 signalling during testicular descent in the dog

Insulin-like 3 (INSL3) plays a prominent role in male development and is supposed to induce the growth of the gubernaculum testis (g.t.), thus being directly involved in testicular descent in humans and rodents. This happens through activation of the RXFP2 receptor (GREAT or LGR8). The INSL3-RXFP2 complex is reputed to play an additional paracrine role in the testis, possibly acting as part of an autocrine feedback loop. The present work provides evidence of the immunolocalisation of INSL3 in the Leydig cells of canine fetuses and of the expression of RXFP2 receptor in different tissues of the g.t. of the same specimens. RXFP2 was localised at the cell membrane of g.t. muscle and connective cells, as well as in the epithelial cells of the developing excurrent ducts. Notably, RXFP2 immunoreactivity of the g.t. was limited to fetuses at ~35-45 days of gestation, which is also the fetal period when the endocrine compartment of the dog testis is active endocrinologically, as confirmed by the anti-P450c17 and anti-INSL3 immunoreactivities of the fetal Leydig cells, and by anti-Müllerian hormone immunoreactivity of the Sertoli cells. The same immunoreactivities were also evaluated in the testes of cryptorchid dogs of different ages. RXFP2 immunoreactivity was absent from genital tracts of cryptorchid testes and g.t. remnants.

Reproductive Biology of Agkistrodon piscivorus Lacépède (Squamata, Serpentes, Viperidae, Crotalinae)

Aspects of the reproductive biology of male and female Agkistrodon piscivorus are described using histological techniques, reviewed, and compared with historical data on A. piscivorus. These include anatomical description at the macro and microscopic levels, and correlation of the male and female urogenital cycles to the reproductive life history of A. piscivorus. New anatomical descriptions and discussion of the efferent ducts, including the ductuli efferentes, proximal and distal ductuli epididymides, ductus deferens, and ampulla ductus deferens, are also presented here at the light and electron microscopy levels. Morphology of all distinct regions of the male and female urogenital systems are discussed and compared with historical investigations on other squamates. In comparison to other snakes, A. piscivorus possesses some unique reproductive characters, whereas others are more conserved. In terms of the reproductive cycle, the ability of males and females to store sperm allows the dissociation of reproductive event timing between the sexes. Thus, the only event that must be coordinated between the sexes is copulation, which is proposed to occur in the fall and the spring in A. piscivorus. In females, the atrophy and activity of the reproductive organs (e.g., secretory activity) varies concurrently with vitellogenesis and the mating seasons. In males, spermatogenesis peaks in the summer, independent of the mating season, except in Louisiana where a spring and fall peak of spermatogenesis occur, and where the spring and fall mating seasons overlap. Renal sexual segment hypertrophy in males peaks in the fall and spring in more southern populations (Alabama and Louisiana) concurrent with fall and spring mating seasons. In Georgia, only a fall peak is observed. Secretory activity of the male excurrent ducts also peaks during times of mating activity in one population studied (Louisiana).

Aquaporin 9 expression in the developing rat epididymis is modulated by steroid hormones

Fluid and solute transport across the epithelium of the male excurrent duct is important for sperm maturation and storage. Aquaporin 9 (AQP9), which allows permeation of water and neutral solutes, is abundant throughout the male reproductive tract, where it is expressed at the apical membrane of rat epididymal principal cells as early as at 1 week of age. We evaluated the effect of neonatal exposure to: 1) a GNRH antagonist (GNRHa); 2) diethylstilbestrol (DES); 3) ethinyl estradiol (EE); 4) DES plus testosterone (DES+TE); and 5) the anti-androgen flutamide on AQP9 expression in the epididymis of peripubertal rats. Control groups received the vehicle alone. In 25-day-old rats, quantification of the mean pixel intensity of immunofluorescence-stained sections showed a significant decrease in AQP9 staining in the apical membrane of epididymal principal cells after treatments with GNRHa, DES, or flutamide, compared to controls. These results were confirmed by western blotting. While EE induced a marked decrease in AQP9 levels by western blotting, the decrease in AQP9-associated fluorescence was not significant compared to controls. DES+TE-treated rats showed levels of AQP9 protein similar to controls, indicating maintenance of AQP9 expression by testosterone treatment in the presence of DES. Our data show that expression of AQP9 in the developing rat epididymis is downregulated by neonatal DES, GNRHa, EE, and flutamide, and that the effects mediated by estrogens can be prevented by testosterone administration.

Considerations in Evaluating Human Spermatogenesis on the Basis of Total Sperm per Ejaculate

Total number of sperm per ejaculate (TSperm) is an important measure for clinicians to provide advice to patient couples. However, TSperm per hour of abstinence (TSperm/h) is a better measure for epidemiologist-andrologist teams or clinicians to evaluate spermatogenesis because it is a rate function. This review looks at the interplay and impacts of rate of sperm accumulation in the excurrent duct system, abstinence interval, sexual arousal, and masturbation vs intercourse on observed TSperm. It also examines why and when TSperm/h might provide a meaningful quantitative evaluation of spermatogenesis (ie, rate of sperm production). There is no doubt that TSperm increases with longer abstinence, and in different men plateaus after 2-9 days. Clinicians wishing to maximize number of fully functional sperm available during intercourse, or for artificial insemination, might wish to recommend 6-7 days of abstinence. Diagnostically, the important feature is TSperm/h. After abstinence interval exceeds 64-72 hours, TSperm/h has started to decline in most nonoligozoospermic men as rate of sperm accumulation in the excurrent ducts approaches zero; apparently increasingly more sperm are voided in urine. Clinicians or epidemiologist-andrologist teams wishing to have optimal distinction among individuals with high, typical, or low sperm production (ie, normal or abnormal spermatogenesis) should accurately measure TSperm/h for samples provided after 42-54 hours' abstinence (never <or=36 or >64 hours). Longer abstinence intervals reward men with poor sperm production, because sperm accumulate in the excurrent ducts for 7 days or more of abstinence, and penalize men with good sperm production, because after 3 days or less of abstinence their excurrent ducts probably are full.

Phosphoglycerate Kinase 2 (PGK2) Is Essential for Sperm Function and Male Fertility in Mice1

Phosphoglycerate kinase 2 (PGK2), an isozyme that catalyzes the first ATP-generating step in the glycolytic pathway, is encoded by an autosomal retrogene that is expressed only during spermatogenesis. It replaces the ubiquitously expressed phosphoglycerate kinase 1 (PGK1) isozyme following repression of Pgk1 transcription by meiotic sex chromosome inactivation during meiotic prophase and by postmeiotic sex chromatin during spermiogenesis. The targeted disruption of Pgk2 by homologous recombination eliminates PGK activity in sperm and severely impairs male fertility, but does not block spermatogenesis. Mating behavior, reproductive organ weights (testis, excurrent ducts, and seminal vesicles), testis histology, sperm counts, and sperm ultrastructure were indistinguishable between Pgk2(-/-) and wild-type mice. However, sperm motility and ATP levels were markedly reduced in males lacking PGK2. These defects in sperm function were slightly less severe than observed in males lacking glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS), the isozyme that catalyzes the step preceding PGK2 in the sperm glycolytic pathway. Unlike Gapdhs(-/-) males, the Pgk2(-/-) males also sired occasional pups. Alternative pathways that bypass the PGK step of glycolysis exist. We determined that one of these bypass enzymes, acylphosphatase, is active in mouse sperm, perhaps contributing to phenotypic differences between mice lacking GAPDHS or PGK2. This study determined that PGK2 is not required for the completion of spermatogenesis, but is essential for sperm motility and male fertility. In addition to confirming the importance of the glycolytic pathway for sperm function, distinctive phenotypic characteristics of Pgk2(-/-) mice may provide further insights into the regulation of sperm metabolism.

Estrogen Sulfotransferase Is Highly Expressed Along the Bovine Epididymis and Is Secreted Into the Intraluminal Environment

Estrogen is found in high concentrations in the excurrent duct, where it regulates the expression of genes involved in water reabsorption. Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes specific sulfonation with a high affinity for estrogens. Because sulfated estrogens do not bind to estrogen receptors, they are considered to be hormonally inactive. EST may thus determine where along the male tract estrogenic environment predominates. Sulfotransferase activity increases along the epididymis and may also play a role in sperm physiology during the epididymal transit. Using a bovine model, we investigated the distribution of EST along the excurrent duct and the possibility that sterols associated with spermatozoa can be substrates of this enzyme. Reverse transcription polymerase chain reactions showed that mRNA encoding EST was expressed in the testis and all along the epididymis. A highly specific antiserum was raised against the bovine recombinant EST and used in Western blots and immunohistologic studies. Western blots of tissue homogenates showed that EST was localized all along the excurrent duct with a higher signal in the caput and corpus epididymidis. EST was detectable in the intraluminal compartment only in the caput epididymidis, where it was associated with epididymosomes and spermatozoa. EST was undetectable in different fractions of fluids collected in the cauda segment. In immunohistologic studies, EST was restricted to the acrosomal region of the caput, but not the cauda epididymal spermatozoa, and detectable in the cytoplasm of the epithelium bordering the lumen all along the epididymis as well as in the rete testis and vas efferens. This enzyme was also associated with the nucleus in the caput and corpus as well as with the apical membrane of the corpus epididymal epithelium. When recombinant EST was incubated in vitro in the presence of caput and cauda spermatozoa, it was able to add sulfate to sperm membrane cholesterol. Our study shows that EST is present in both the intracellular and intraluminal compartments of the epididymis, suggesting that this enzyme plays different roles along the excurrent duct.

The junctions between efferent ductules and epididymal duct in the boar.

Sperm transit through the male excurrent duct system is dependent on complete luminal patency. Since male excurrent ducts are derived embryologically from originally separate structures, the junctions between mesonephric tubules and the mesonephric duct are of major interest with respect to obstructive disorders. Therefore, we investigated the junctions between efferent ductules and epididymal duct in adult boars by means of serial semithin and ultrathin sections. Based on the anastomosing pattern and on the site of transition from low columnar, ciliated epithelium to high columnar, non-ciliated epithelium, three different types of anastomoses were identified, one of which was associated with luminal stenosis and sperm accumulation. The occurrence of end-to-side junctions with the epithelial transition being localized within the lumen of the tributary may thus impede normal sperm transport.