Epididymides Of Mice Research Articles

Hippo kinases MST1 and MST2 control the differentiation of the epididymal initial segment via the MEK-ERK pathway.

Although the roles of the Hippo pathway in organogenesis and tumorigenesis have been well studied in multiple organs, its role in sperm maturation and male fertility has not been investigated. The initial segment (IS) of the epididymis plays a critical role in sperm maturation. IS differentiation is governed by ERK1/2, but the mechanisms of ERK1/2 activation in IS are not fully understood. Here we show that double knockout (dKO) of mammalian sterile 20-like kinases 1 and 2 (Mst1 and Mst2), homologs of Hippo in Drosophila, in the epididymal epithelium led to male infertility in mice. Sperm in the cauda epididymides of mutant mice were immotile with flagellar angulation and severely disorganized structures. Loss of Mst1/2 activated YAP and increased proliferation and cell death in all the segments of epididymis. The mutant mice showed substantially suppressed MEK/ERK signaling in the IS and failed IS differentiation. Deletion of Yap restored the reduced MEK/ERK signaling, and partially rescued the defective IS differentiation and fertility in Mst1/2 dKO mice. Our results demonstrate that YAP inhibits the MEK/ERK pathway in IS epithelial cells, and MST1/2 control IS differentiation and fertility at least partially by repressing YAP. Taken together, the Hippo pathway is essential for sperm maturation and male fertility.

Infection of epididymal epithelial cells and leukocytes drives seminal shedding of Zika virus in a mouse model.

While primarily a mosquito-borne virus, Zika virus (ZIKV; genus Flavivirus in the Flaviviridae family) is capable of being sexually transmitted. Thirty to fifty percent of men with confirmed ZIKV infection shed ZIKV RNA in their semen, and prolonged viral RNA shedding in semen can occur for more than 6 months. The cellular reservoir of ZIKV in semen is unknown, although spermatozoa have been shown to contain ZIKV RNA and antigen. Yet, spermatozoa are not a requisite for sexual transmission, as at least one case of ZIKV sexual transmission involved a vasectomized man. To determine the cellular reservoirs of ZIKV in semen, an established animal model of sexual transmission was used. The majority of virus detected in the seminal fluid of infected mice during the peak timing of sexual transmission was from the supernatant fraction, suggesting cell-free ZIKV may be largely responsible for sexual transmission. However, some ZIKV RNA was cell-associated. In the testes and epididymides of infected mice, intracellular staining of ZIKV RNA was more pronounced in spermatogenic precursors (spermatocytes and spermatogonia) than in spermatids. Visualization of intracellular negative strand ZIKV RNA demonstrated ZIKV replication intermediates in leukocytes, immature spermatids and epididymal epithelial cells in the male urogenital tract. Epididymal epithelial cells were the principal source of negative-strand ZIKV RNA during the peak timing of sexual transmission potential, indicating these cells may be the predominant source of infectious cell-free ZIKV in seminal fluid. These data promote a more complete understanding of sexual transmission of ZIKV and will inform further model development for future studies on persistent ZIKV RNA shedding.

Alteration of transporter activities in the epididymides of infertile initial segment-specific Pten knockout mice.

A fully functional initial segment, the most proximal region of the epididymis, is important for male fertility. Our previous study generated a mouse model to investigate the importance of initial segment function in male fertility. In that model, phosphatase and tensin homolog (Pten) was conditionally removed from the initial segment epithelium, which resulted in epithelial de-differentiation. When spermatozoa progressed through the de-differentiated epithelial duct, they developed angled flagella, suggesting compromised sperm maturation, which eventually resulted in male infertility. To understand the molecular mechanisms, by which PTEN regulates epididymal sperm maturation, we compared the transcriptome profile of the initial segment between controls and initial segment-specific Pten knockouts and revealed that water, ion, and organic solute transporter activities were one of the top molecular and cellular functions altered following loss of Pten. Alteration in protein levels and localization of several transporters following loss of Pten were also observed by immunofluorescence analysis. Epithelial cells of the initial segment from knockouts were more permeable to fluorescein isothiocyanate-dextran (4000 Da) compared to controls. Interestingly, conditional deletion of Pten from other organs also resulted in changes in transporter activity, suggesting a common role of PTEN in regulation of transporter activity. Taken together, our data support the hypothesis that loss of Pten from the initial segment epithelium results in changes in the transporting and permeability characteristics of the epithelium, which in turn altered the luminal fluid microenvironment that is so important for sperm maturation and male fertility.

The expression characteristics of FAM71D and its association with sperm motility.

What are the features of FAM71D (Family with sequence similarity 71, member D) expression and is there an association between FAM71D expression and sperm motility? FAM71D, a novel protein exclusively expressed in the testis, is located in sperm flagella and is functionally involved in sperm motility. Some testis-specific proteins have been reported as potential diagnostic biomarkers to evaluate the spermatogenesis process and sperm quality. We have identified a novel testis-specific protein, FAM71D, through microarray data analysis, yet little is known about its expression and function. FAM71D mRNA and protein expression was quantified during mouse testis development. Its localization in germ cells was detected by dual-labeled immunostaining in testis sections and sperm smears. The clinical significance was assessed by comparing FAM71D expression in spermatozoa from normozoospermic controls and asthenozoospermic patients. Testes were dissected from C57BL/6 J male mice at postnatal ages of 1, 2, 3, 4, 6, 8 weeks and 6 months, and sperm was collected from cauda epididymides of adult mice by the swim-up method. Human spermatozoa were isolated from 100 human semen samples by density gradient Percoll centrifugation. RT-qPCR and western blot were performed to semi-quantify the expression of FAM71D in mouse testis, and in the ejaculated spermatozoa of normozoospermic controls and asthenozoospermic patients. Immunofluorescence staining was used to detect the localization of FAM71D. Co-immunoprecipitation assay was performed to evaluate the interaction between FAM71D and calmodulin. An antibody blocking assay was employed to assess the role of FAM71D in sperm motility. Our results showed that FAM71D was exclusively expressed in the testis in an age-dependent manner. FAM71D expression exhibited dynamic change in the cytoplasm of spermatids during spermiogenesis and was finally retained in sperm flagella. FAM71D could interact with calmodulin. Use of anti-FAM71D antibody on sperm significantly decreased sperm motility. Expression level of FAM71D was markedly reduced in the ejaculated spermataozoa of asthenozoospermic patients (P < 0.05), and this was correlated with sperm progressive motility (r = 0.7435, P < 0.0001). N/A. The sample size was limited and it is necessary to verify the correlation of FAM71D expression with sperm motility in larger cohorts. Furthermore, our results were descriptive and follow-up studies would be needed to elucidate the detailed role of FAM71D in sperm motility. This is the first systematic study to document the expression of endogenous FAM71D and a function for FAM71D in sperm motility. It provides new insights into our understanding of sperm motility regulation and causes of male infertility. This study was funded by the National Natural Science Foundation of China, Guangdong Natural Science Foundation and the Shenzhen Project of Science and Technology. The authors have no competing interests.

Abnormal spermatogenesis and male infertility in testicular zinc finger protein Zfp318-knockout mice.

Zfp318, a mouse gene with a Cys2/His2 zinc finger motif, is mainly expressed in germ cells in the testis. It encodes two alternative transcripts, which regulate androgen receptor-mediated transcriptional activation or repression by overexpression of them. However, the role of Zfp318 is still obscure invivo, especially in spermatogenesis. To elucidate the role of Zfp318 during gamete production, we established a knockout mouse line. Zfp318-null male mice exhibited infertility, whereas Zfp318-null female mice displayed normal fertility. ZFP318 was expressed during multiple stages of spermatogenesis, from spermatocytes to round spermatids. The nuclei of secondary spermatocytes showed high levels of expression. Histological analysis and quantitative analysis of DNA content showed decreased numbers of both spermatids in the seminiferous tubules andmature spermatozoa in the epididymides of Zfp318-null mice. These results suggest that Zfp318 isexpressed as a functional protein in testicular germ cells and plays an important role in meiosis during spermatogenesis.

Xenogeneic and endogenous spermatogenesis following transplantation of rat germ cells into testes of immunocompetent mice

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, and are characterised by their ability to self-renew and to produce differentiated progeny that form spermatozoa. It has been demonstrated that rat spermatogenesis can occur in the seminiferous tubules of congenitally immunodeficient recipient mice after transplantation of rat SSCs. However, the testis is often viewed as an immune-privileged site in that autoimmunogenic antigens on germ cells do not normally elicit an immune response in situ. In the present study, we tried to transplant rat SSCs into immunocompetent mice after depletion of their own germ cells by means of busulfan. The results showed that some transplanted SSCs could undergo complete spermatogenesis in recipient mouse testes, the rat spermatozoa being detected in 7 of 28 recipient epididymides. A significant increase in mouse spermatozoa was also noted in all 28 epididymides of recipient mice regardless of whether rat spermatozoa were concurrently present or not. These results suggest that transplanted rat SSCs can be tolerated in the testes of immunocompetent mice and that the transplantation of rat SSCs stimulates endogenous spermatogenesis in the recipient mice.

Members of the murine Pate family are predominantly expressed in the epididymis in a segment-specific fashion and regulated by androgens and other testicular factors

BackgroundSpermatozoa leaving the testis are not able to fertilize the egg in vivo. They must undergo further maturation in the epididymis. Proteins secreted to the epididymal lumen by the epithelial cells interact with the spermatozoa and enable these maturational changes, and are responsible for proper storage conditions before ejaculation. The present study was carried out in order to characterize the expression of a novel Pate (prostate and testis expression) gene family, coding for secreted cysteine-rich proteins, in the epididymis.MethodsMurine genome databases were searched and sequence comparisons were performed to identify members of the Pate gene family, and their expression profiles in several mouse tissues were characterized by RT-PCR. Alternate transcripts were identified by RT-PCR, sequencing and Northern hybridization. Also, to study the regulation of expression of Pate family genes by the testis, quantitative (q) RT-PCR analyses were performed to compare gene expression levels in the epididymides of intact mice, gonadectomized mice, and gonadectomized mice under testosterone replacement treatment.ResultsA revised family tree of Pate genes is presented, including a previously uncharacterized Pate gene named Pate-X, and the data revealed that Acrv1 and Sslp1 should also be considered as members of the Pate family. Alternate splicing was observed for Pate-X, Pate-C and Pate-M. All the Pate genes studied are predominantly expressed in the epididymis, whereas expression in the testis and prostate is notably lower. Loss of androgens and/or testicular luminal factors was observed to affect the epididymal expression of several Pate genes.ConclusionsWe have characterized a gene cluster consisting of at least 14 expressed Pate gene members, including Acrv1, Sslp1 and a previously uncharacterized gene which we named Pate-X. The genes code for putatively secreted, cysteine-rich proteins with a TFP/Ly-6/uPAR domain. Members of the Pate gene cluster characterized are predominantly expressed in the murine epididymis, not in the testis or prostate, and are regulated by testicular factors. Similar proteins are present in venoms of several reptiles, and they are thought to mediate their effects by regulating certain ion channels, and are thus expected to have a clinical relevance in sperm maturation and epididymal infections.

Alterations in the Testis and Epididymis Associated With Loss of Function of the Cystatin-Related Epididymal Spermatogenic (CRES) Protein

Cystatin-related epididymal spermatogenic protein (CRES) or cystatin 8 (Cst8 gene) is a member of the cystatin superfamily of cysteine protease inhibitors. It differs from typical cystatins because it lacks consensus sites for cysteine protease inhibition and exhibits reproductive-specific expression. In the present study, we examined CRES expression within the testes, efferent ducts, and epididymides of normal mice by light microscope immunolocalization. Alterations to these tissues in male mice lacking the Cst8 gene (Cst8(-/-2)) were also characterized by histomorphometry and electron microscopy. In the normal testis, CRES was localized exclusively in mid and late elongating spermatids. In the efferent ducts, CRES was localized to the apical region of the epithelial cells suggestive of localization in the endosomes. In the initial segment of the epididymis, principal cells showed supranuclear and luminal reactions. In the cauda region, CRES was present exclusively as aggregates in the lumen and was detected in clear cells. Compared with wild-type mice (Cst8(+/+)), older (10-12 months) Cst8(-/-) mice had modest but statistically significant reductions in tubular, epithelial, and/or luminal profile areas in the testis and epididymis. By electron microscopy, some Cst8(-/-) tubules in the testis were normal in appearance, but others showed a vacuolated seminiferous epithelium, degenerating germ cells, and alterations to ectoplasmic specializations. In the epididymal lumen, abnormally shaped sperm heads and tails were noted along with immature germ cells. In addition, principal cells contained numerous large irregularly shaped lysosomes suggestive of disrupted lysosomal functions. In both the testis and epididymis, however, these abnormalities were not apparent in younger mice (4 months), only in the older (10-12 months) Cst8(-/-) mice. These findings suggest that the altered testicular and epididymal histology reflects a cumulative effect of the loss of CRES and support a role for CRES in maintaining the normal integrity and function of the testis and epididymis.

Effect of 1-p-Chlorobenzyl-1H-Indazol-3-Carboxylic Acid (AF 1312/TS) on the Sex Organs of the Male Laboratory Mouse

Oral administration (500 mg/kg body weight/day, for 28 days) of 1-p-chlorobenzyl-1H-indazol-3-carboxylic acid (AF 1312/TS) induced suppression of spermatogenesis without adversely affecting the Leydig cells. The seminiferous tubules were almost depopulated and contained only Sertoli cells and spermatogonia. The drug also induced significant decrease in the levels of testicular RNA and protein; DNA level, however, remained unaltered. Regressive histological changes were also noted in the epididymides of drug-treated mice. Cauda epididymidal spermatozoa from drug-treated mice were immotile and fragmented. AF 1312/TS caused no alteration in the level of fructose in the seminal vesicle. The drug-induced alterations were reversible, and 56 days after drug withdrawal the reproductive organs returned to their pretreatment state. The results suggest that AF 1312/TS selectively suppresses spermatogenesis without interfering with the endocrine functions of the testis.

The Flagellar Protein RSPH1 Is a Candidate Regulator of the Testis and Sperm-Restricted Protein Phosphatase, PP1gamma2.

The protein RSPH1 (also known as TSGA2, MEICHROACIDIN, RSP44) is present in the sperm flagellum. Previous analysis has suggested that various isoforms of the protein are expressed differentially in testis and epididymis. Targeted disruption of Rsph1 impairs spermiogenesis resulting in a male sterility phenotype with remarkable similarity to the one observed in Protein Phosphatase 1 gamma (PP1gamma)-null mice. The initial goal of this study was to confirm that two different isoforms of the protein, resulting from alternate splicing, are expressed in testis and epididymis respectively. Testes and epididymides of PP1gamma-null mice, both nearly devoid of spermatozoa, were used for this study. Western blot analyses and fluorescence immuno-cytochemistry were performed with antibodies raised against synthetic peptide antigens corresponding to the N- and C- termini of mouse RSPH1. Column purification, GST pull-down, and tap-tagged fusion proteins expressed transgenically in the testes were used to isolate the binding partners of RSPH1 in testis and sperm extracts. Our results confirmed that RSPH1 is expressed in the mouse epididymis as a 30-kDa protein compared to the 48-kDa testis isoform and that expression of the 30-kDa, N-terminally truncated, isoform is restricted to the corpus and caudal regions of the epididymis.The 30-kDa isoform of RSPH1 appears to be incorporated into maturing spermatozoa during their passage through the epididymis. Protein micro-sequencing, co-elution during column chromatography, and co-immunoprecipitation show that in testis, RSPH1 is bound to 14-3-3 and PPP1R11, proteins known to regulate the catalytic activity of the protein phosphatase, PP1gamma2. Taken together our results suggest that one role of RSPH1 could be to regulate the activity of the testis- and sperm-restricted protein phosphatase, PP1gamma2. (poster)

Changing P2X receptor localization on maturing sperm in the epididymides of mice, hamsters, rats, and humans: a preliminary study

To study using immunohistochemistry the localization of P2X receptor subtypes on the head of immature sperm in the human, mouse, hamster, and rat caput epididymidis. Basic research. University-based hospital. Three human epididymides were obtained from patients undergoing orchidectomy for metastatic prostate cancer. P2X(1), P2X(2), P2X(3), and P2X(4) receptor immunolocalization on sperm. In the present study, P2X(1,2, and 3) receptor localization was immunohistochemically demonstrated on the head of immature sperm in the human, mouse, hamster, and rat caput epididymidis. P2X(4) receptor immunostaining was also observed on the head of sperm in the caput epididymidis of mice, hamsters, and humans, but not rats. There was a subsequent loss of receptor staining on sperm in the cauda epididymidis, except in humans where staining of P2X(4) receptors persisted. Comparision with peanut agglutinin (PNA) binding studies suggested the P2X receptors were located on the acrosome membrane. P2X(5-7) receptors were examined but found to be absent. The change in localization of receptor subtypes is coincidental with the functionally essential morphologic and maturational changes seen in sperm as they travel through the epididymis, and is suggestive of a role for purinergic signaling in sperm maturation and possibly fertility.

Sperm from Hyh Mice Carrying a Point Mutation in αSNAP Have a Defect in Acrosome Reaction

Hydrocephalus with hop gait (hyh) is a recessive inheritable disease that arose spontaneously in a mouse strain. A missense mutation in the Napa gene that results in the substitution of a methionine for isoleucine at position 105 (M105I) of αSNAP has been detected in these animals. αSNAP is a ubiquitous protein that plays a key role in membrane fusion and exocytosis. In this study, we found that male hyh mice with a mild phenotype produced morphologically normal and motile sperm, but had a strongly reduced fertility. When stimulated with progesterone or A23187 (a calcium ionophore), sperm from these animals had a defective acrosome reaction. It has been reported that the M105I mutation affects the expression but not the function of the protein. Consistent with an hypomorphic phenotype, the testes and epididymides of hyh mice had low amounts of the mutated protein. In contrast, sperm had αSNAP levels indistinguishable from those found in wild type cells, suggesting that the mutated protein is not fully functional for acrosomal exocytosis. Corroborating this possibility, addition of recombinant wild type αSNAP rescued exocytosis in streptolysin O-permeabilized sperm, while the mutant protein was ineffective. Moreover, addition of recombinant αSNAP. M105I inhibited acrosomal exocytosis in permeabilized human and wild type mouse sperm. We conclude that the M105I mutation affects the expression and also the function of αSNAP, and that a fully functional αSNAP is necessary for acrosomal exocytosis, a key event in fertilization.

Neutral α-glucosidase activity in mouse: a marker of epididymal function?

Neutral alpha-glucosidase (NAG) activity is considered a functional epididymal marker in several species. Unlike the rat, no NAG activity has been detected in mice. The aims of the present study were to evaluate NAG secretory activity (the supernatant of the incubated tissue) in mouse epididymis and to determine whether it could be used as a functional epididymal marker. Epididymides (whole or in parts) were incubated in the presence or absence of testosterone (10(-5) m) and secretory NAG activity was compared with known positive controls. Furthermore, we compared enzyme activity in epididymides from well-fed and undernourished mice (50% food restriction for 21 days), a model that alters the epididymal maturation processes. Spectrophotometric analysis revealed NAG activity in mouse epididymis (22.6 +/- 3.7 mU g(-1) tissue; n = 4), being higher in the caput. NAG activity was statistically higher in the caput than in the corpus and in the cauda. No significant differences existed between the caput NAG activity and complete epididymis NAG activity. In undernourished mice, we confirmed changes in epididymal maturation observed previously (i.e. increased number of immature spermatozoa and diminution of the sperm concentration). Concordantly, the epididymides of undernourished mice exhibited decreased enzyme secretory activity, which increased to values similar to those seen in controls following incubation in the presence of testosterone (22.5 +/- 2.6, 12.5 +/- 1.0 and 22.4 +/- 3.7 mU g(-1) tissue, n = 9 in control (n = 7), undernourished (n = 9) and undernourished + testosterone groups (n = 9), respectively). In conclusion, NAG activity was detected in mouse epididymis. Although the present study supports the possibility of using NAG as an epididymal marker, more studies are necessary to effectively prove that NAG activity can be used as an epididymal marker.

The Region-Specific Functions of Two Ubiquitin C-Terminal Hydrolase Isozymes along the Epididymis

We previously showed that gad mice, which are deficient for ubiquitin C-terminal hydrolase L1 (UCH-L1), have a significantly increased number of defective spermatozoa, suggesting that UCH-L1 functions in sperm quality control during epididymal maturation. The epididymis is the site of spermatozoa maturation, transport and storage. Region-specific functions along the epididymis are essential for establishing the environment required for sperm maturation. We analyzed the region-specific expression of UCH-L1 and UCH-L3 along the epididymis, and also assessed the levels of ubiquitin, which has specificity for UCH-L1. In wild-type mice, western blot analysis demonstrated a high level of UCH-L1 expression in the caput epididymis, consistent with ubiquitin expression, whereas UCH-L3 expression was high in the cauda epididymis. We also investigated the function of UCH-L1 and UCH-L3 in epididymal apoptosis induced by efferent duct ligation. The caput epididymides of gad mice were resistant to apoptotic stress induced by efferent duct ligation, whereas Uchl3 knockout mice showed a marked increase in apoptotic cells following ligation. In conclusion, the response of gad and Uchl3 knockout mice to androgen withdrawal suggests a reciprocal function of the two UCH enzymes in the caput epididymis.

The occurrence of strongylid nematodes in the epididymides of wood mice

The recent discovery of a larval nematode in the epididymides of free-living wood mice (Apodemus sylvaticus) suggests a sexual transmission of these parasites. They have been placed within the bursate nematodes (order Strongylida) through 18S rDNA analysis, suggesting that they are undetermined metastrongyloid nematodes. The possibility that these parasites are transmitted sexually opens an intriguing field of research because sexually transmitted metazoan parasites are known to occur mainly in invertebrates, whereas in vertebrates sexually transmitted parasites are usually microparasites such as viruses, bacteria and protozoa.

Relief effect of vitamin A on the decreased motility of sperm and the increased incidence of malformed sperm in mice exposed neonatally to bisphenol A.

Administration of 50 microg of bisphenol A (BPA) for the first 5 days after birth resulted in a decrease in the percentage of moving sperm, and an increase in the incidence of malformed sperm, in the epididymides of mice at 10 weeks of age, although no marked changes were found in the testicular histology between BPA-treated and vehicle-treated control mice. The deteriorating effects of 50 microg of BPA were ameliorated by the concurrent administration of 100 IU of retinol acetate (RA). Neonatal treatment with 0.5 microg of BPA for 5 days resulted in an increase in the incidence of malformed sperm, whereas the BPA effect became more severe in mice nursed by mothers fed a vitamin A-deficient (VAD) diet only a few days before and after parturition. On the other hand, neonatal treatment with 20 microg of estrogen for the first 5 days after birth resulted in an increase in the number of estrogen receptor alpha (ERalpha)-positive cells in the epithelium of the vas deferens, whereas only a few epithelial cells showed weak ERalpha-positive signals in the vehicle-treated control mice at 18 days after birth. This increase, however, was suppressed by the concurrent administration of RA. Although five daily treatments with 50 microg BPA led to no significant increase in the number of ERalpha-positive cells, it may have been due to the weak estrogenic activity of BPA, as discussed. These findings clearly showed that in mice, neonatal exposure to a relatively large dose of BPA causes damage to the motility and morphology of sperm, but the BPA effect is, to some extent, inhibited by a supplement of VA, and enhanced under a VAD condition.

Gene therapy and the germ line.

Gene therapy and the germ line.

I. Abnormalities in cells of the testis, efferent ducts, and epididymis in juvenile and adult mice with beta-hexosaminidase A and B deficiency.

Beta-hexosaminidase (Hex) is a lysosomal enzyme that exists as two major isoenzymes: Hex A (subunit structure, alphabeta) and Hex B (betabeta). The presence of Hex in the testis and epididymis suggests important roles for the enzyme and its substrates in male fertility and reproductive functions. Disruption of the Hexb gene encoding the beta-subunit of Hex has led to the generation of a mouse model of human Sandhoff disease that survives to adulthood, enabling us to analyze the effects of Hex A and Hex B deficiency on epithelial cellular morphology of the male reproductive tract. At 1 and 3 months of age, the testes, efferent ducts, and epididymides of Hex-deficient (Hexb -/-) and wild-type (Hexb +/+) mice were perfuse fixed and analyzed by routine light and electron microscopy (LM and EM, respectively) as well as with immunocytochemistry employing antibodies to lysosomal proteins. In the testis, the morphological appearance and topographical arrangement of the cell types of the seminiferous epithelium of Hexb -/- mice were similar to those of wild-type animals at both ages. Both Sertoli and germ cells appeared to be unaffected. However, at both ages, myoid cells and macrophages showed an increased number of lysosomes in their cytoplasm as compared with the number seen in controls. The epithelial cells of the efferent ducts also showed an accumulation of lysosomes that increased with age as compared with controls. Principal cells of the entire epididymis revealed an increase in the size and number of lysosomes at 1 month of age as compared with those of controls, and by 3 months, these lysosomes often filled the supranuclear and basal regions of the cells. Narrow cells of the distal initial segment and intermediate zone, normally slender cells showing several lysosomes, became greatly enlarged and entirely filled with lysosomes in Hexb -/- mice. Clear cells of the caput, corpus, and cauda regions also showed a progressive increase in the size and number of lysosomes with age as compared with controls; the clear cells of the mutant mice were often enlarged and at times bulged into the lumen. Some basal cells of each epididymal region in Hexb -/- mice were similar to controls at 1 and 3 months, showing few lysosomes, while others showed an accumulation of lysosomes. Lysosomes of all affected epithelial cells were of varying sizes, but many large ones were present, apparently resulting from lysosomal fusion. Although pale stained, their identification as lysosomes was confirmed by EM immunocytochemistry with anti-cathepsin D and anti-Hex A antibodies. Predominantly in the proximal initial segment, large, pale cellular aggregates were noted in the LM analysis at the base of the epithelium, which by EM analysis were identified as belonging to two different cell types, narrow cells and halo cells. Taken together, these data reveal an increase in the size and number of lysosomes in all epithelial cell types lining the efferent ducts and entire epididymis as well as in myoid cells and macrophages of the testis. In the light of data showing epididymal defects restricted predominantly to the initial segment in Hexa -/- (Hex A-deficient) mice, our data on the Hexb -/- mice demonstrate a major role for Hex that can be fulfilled by either Hex A or Hex B in the epididymis.

Major differences between the testis and epididymis in the induction of granulomas in response to extravasated germ cells. I. A light microscopical study in mice.

A spermatic granuloma is a chronic inflammatory lesion which surrounds extravasated spermatozoa. Clinically, the lesion develops in the interstitial spaces of the epididymis and vas deferens, and only exceptionally in the testis itself. In the present study, murine testes and epididymides were injured using a needle and the histological appearances of these organs was then compared. Traumatic injury induced extravasation of germ cells in both testes and epididymides. A few days later, spermatic granulomas consistently formed in the epididymides, however, such lesions were not induced in the testes. To examine the possibility that epididymal spermatozoa have inherently greater ability to form spermatic granulomas than do testicular germ cells, isolated epididymal spermatozoa or testicular germ cells were locally injected into the testes and epididymides of recipient mice. Spermatic granulomas readily formed in the epididymides after local injection of either epididymal spermatozoa or testicular germ cells. In contrast, such lesions did not form in the testes even when epididymal spermatozoa were injected. Therefore, this study suggests that the microenvironment of the testicular interstitium, rather than the extravasated components from the ruptured seminiferous tubules, is the main factor determining the limited formation of spermatic granulomas in the testis.

Spermatic granulomata are experimentally induced in epididymides of mice receiving high-dose testosterone implants. I. A light-microscopical study.

It is known that a spermatic granuloma is induced by the inflammatory reaction following leakage of spermatozoa outside the germ cell ducts and is the main clinical complication of vasectomy. In the present study, we found that spermatic granulomata were experimentally induced in the epididymides of mice treated with high-dose testosterone. Testosterone powder (0.02, 0.2, or 2 mg per gram body weight) was implanted into ICR male mice, which were then killed from 7 to 63 days after the treatment for histological examination at the light-microscopic level. The results showed that the testis exhibited little or no degenerative change; however, the epididymides were frequently affected by spermatic granulomata after day 35 in mice implanted with high-dose testosterone (2 but not 0.2 or 0.02 mg per gram body weight). Observation of the early histological changes revealed that the ductal epithelium of the epididymides became vacuolated around day 25. Thereafter, the basement membrane of the epididymal ducts was ruptured after day 30, followed by leakage of spermatozoa into the adjacent interstitial tissue. The extravasated spermatozoa were then surrounded by macrophages (= epithelioid cells) and lymphocytes, resulting in the formation of a spermatic granuloma. In contrast, other mice treated with the same dose of deoxycorticosterone or estradiol did not show the induction of spermatic granulomata. Therefore, this study demonstrated that a spermatic granuloma is specifically formed in the epididymis by testosterone and that the lesion is started by vacuolation of the epididymal duct epithelium.