Endocrine regulation of male fertility by the skeleton.

Interactions between bone and the reproductive system have until now been thought to be limited to the regulation of bone remodeling by the gonads. We now show that, in males, bone acts as a regulator of fertility. Using coculture assays, we demonstrate that osteoblasts are able to induce testosterone production by the testes, though they fail to influence estrogen production by the ovaries. Analyses of cell-specific loss- and gain-of-function models reveal that the osteoblast-derived hormone osteocalcin performs this endocrine function. By binding to a G protein-coupled receptor expressed in the Leydig cells of the testes, osteocalcin regulates in a CREB-dependent manner the expression of enzymes that is required for testosterone synthesis, promoting germ cell survival. This study expands the physiological repertoire of osteocalcin and provides the first evidence that the skeleton is an endocrine regulator of reproduction.

Proteomics Identifies Golgi phosphoprotein 3 (GOLPH3) with A Link Between Golgi Structure, Cancer, DNA Damage and Protection from Cell Death.

GOLPH3 is the first example of a Golgi resident oncogene protein. It was independently identified in multiple screens; first in proteomic-based screens as a resident protein of the Golgi apparatus, and second as an oncogene product in a screen for genes amplified in cancer. A third screen uncovered the association of GOLPH3 with the Golgi resident phospholipid, phosphatidyl inositol 4 phosphate (PI4P) to maintain the characteristic ribbon structure of the Golgi apparatus favoring vesicular transport of secretory proteins.

Clusterin in the mouse epididymis: possible roles in sperm maturation and capacitation.

Clusterin (CLU) is known as an extracellular chaperone for proteins under stress, thus preventing them from aggregation and precipitation. We showed herein that CLU, expressed by principal cells of the mouse caput epididymis, was present in high amounts in the lumen. In the cauda epididymis, CLU bound tightly to the sperm head surface and its amount on total sperm was similar to that in the bathing luminal fluid. In both immotile and motile caudal epididymal sperm, CLU was localized over the entire sperm head except at the convex ridge, although in the motile sperm population, the CLU immunofluorescence pattern was distinctively mottled with a lower intensity. However, when motile sperm became capacitated, CLU was relocalized to the head hook region, with immunofluorescence intensity being higher than that on the non-capacitated counterparts. Under a slightly acidic pH of the epididymal lumen, CLU may chaperone some luminal proteins and deliver them onto the sperm surface. Immunoprecipitation of epididymal fluid proteins indicated that CLU interacted with SED1, an important egg-binding protein present in a high amount in the epididymal lumen. In a number of non-capacitated sperm, fractions of SED1 and CLU co-localized, but after capacitation, SED1 and CLU dissociated from one another. While CLU moved to the sperm head hook, SED1 translocated to the head convex ridge, the egg-binding site. Overall, CLU localization patterns can serve as biomarkers of immotile sperm, and non-capacitated and capacitated sperm in mice. The chaperone role of CLU may also be important for sperm maturation and capacitation.

Implications of caveolae in testicular and epididymal myoid cells to sperm motility.

Seminiferous tubules of the testis and epididymal tubules in adult rodents are enveloped by contractile myoid cells, which move sperm and fluids along the male reproductive tract. Myoid cells in the testis influence Sertoli cells by paracrine signaling, but their role in the epididymis is unknown. Electron microscopy revealed that elongated myoid cells formed several concentric layers arranged in a loose configuration. The edges of some myoid cells in a given layer closely approximated one another, and extended small foot-like processes to cells of overlying layers. Gap junction proteins, connexins 32 and 43, were detected within the myoid cell layers by immunohistochemistry. These myoid cells also had caveolae that contained caveolin-1 and cavin-1 (also known as PTRF). The number of caveolae per unit area of plasma membrane was significantly reduced in caveolin-1-deficient mice (Cav1(-/-) ). Morphometric analyses of Cav1-null testes revealed an enlargement in whole-tubule and epithelial profile areas, whereas these parameters were slightly reduced in the epididymis. Although sperm are non-motile as they pass through the proximal epididymis, statistical analyses of cauda epididymidis sperm concentrations revealed no significant differences between wild-type and Cav1(-/-) mice. Motility analyses, however, indicated that sperm velocity parameters were reduced while beat cross frequency was higher in gametes of Cav1(-/-) mice. Thus while caveolae and their associated proteins are not necessary for myoid cell contractility, they appear to be crucial for signaling with the epididymal epithelium to regulate the proper acquisition of sperm motility. Mol. Reprod. Dev. 83: 526-540, 2016. © 2016 Wiley Periodicals, Inc.

Isolated Rat Epididymal Basal Cells Share Common Properties with Adult Stem Cells.

There is little information on the function of epididymal basal cells. These cells secrete prostaglandins, can metabolize radical oxygen species, and have apical projections that are components of the blood-epididymis barrier. The objective of this study was to develop a reproducible protocol to isolate rat epididymal basal cells and to characterize their function by gene expression profiling. Integrin-alpha6 was used to isolate a highly purified population of basal cells. Microarray analysis indicated that expression levels of 552 genes were enriched in basal cells relative to other cell types. Among these genes, 45 were expressed at levels of 5-fold or greater. These highly expressed genes coded for proteins implicated in cell adhesion, cytoskeletal function, ion transport, cellular signaling, and epidermal function, and included proteases and antiproteases, signal transduction, and transcription factors. Several highly expressed genes have been reported in adult stem cells, suggesting that basal cells may represent an epididymal stem cell population. A basal cell culture was established that showed that these basal cells can differentiate in vitro from keratin (KRT) 5-positive cells to cells that express KRT8 and connexin 26, a marker of columnar cells. These data provide novel information on epididymal basal cell gene expression and suggest that these cells can act as adult stem cells.

Antimicrobial host defence peptide, LL-37, as a potential vaginal contraceptive.

STUDY QUESTION: Does antimicrobial peptide, LL-37, inhibit sperm fertilizing ability? SUMMARY ANSWER: Our results indicate that LL-37 inhibits mouse and human sperm fertilizing ability. WHAT IS KNOWN ALREADY: LL-37, a cationic antimicrobial peptide, exerts its microbicidal effects through the disruption of microbial cytoplasmic membranes following its interaction with microbial surface anionic phospholipids. ALL-38 (an LL-37 close analogue: LL-37 + Ala at the N-terminus) is produced in the vagina 2-6 h post-intercourse from its precursor hCAP-18, a seminal plasma component. At this time, motile sperm have already swum into the uterine cavity, thus unexposed to ALL-38. Since sperm contain a substantial amount of acidic sulfogalactosylglycerolipid (SGG) on their surface, treatment of sperm with LL-37 may cause their membrane disruption in an analogous manner to that occurring on microbial membranes. STUDY DESIGN, SIZE AND DURATION: Mouse/human sperm treated (2-30 min) with LL-37 in a physiological concentration range (up to 10.8 µM) were assessed for SGG-dependent LL-37 binding, and parameters relevant to fertilizing ability, namely motility and intactness of the sperm acrosome and plasma membrane. Ability of mouse sperm to fertilize eggs in vitro was also evaluated. Each study was performed with greater than or equal to three different sperm samples. The efficacy of LL-37 to inhibit sperm fertilizing ability in vivo was determined in female mice (n = 26 each for LL-37 treatment and no treatment), using sperm retrieved from 26 males. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human sperm samples were donated by fertile men. LL-37 was chemically synthesized and was biotinylated for sperm binding studies. Sperm motility was assessed by videomicroscopy and the acrosomal status by Coomassie blue staining of acrosome-intact mouse sperm or the exposure of CD46, an inner acrosomal membrane protein, of acrosome reacted human sperm. Sperm membrane permeabilization/disruption was assessed by the loss of hypo-osmotic swelling response, an incorporation of Sytox Green (a membrane impermeable fluorescent DNA dye), and electron microscopy. Mouse IVF was scored by the presence of two pronuclei in eggs 6 h post-insemination. Ability of mouse sperm to fertilize eggs in vivo was determined by the pregnancy outcome of female mice injected transcervically with sperm with or without LL-37. MAIN RESULTS AND THE ROLE OF CHANCE: Biotinylated LL-37 bound to both mouse and human sperm and the binding was partially dependent on sperm surface SGG. Mouse and human sperm became immotile and underwent a premature acrosome reaction upon treatment with LL-37 at 3.6 and 10.8 µM, respectively. The initial action of LL-37 on both mouse and human sperm appeared to be through permeabilization/disruption of sperm surface membranes evidenced by the loss of hypo-osmotic swelling response, Sytox Green staining and electron microscopy revealing ultrastructural damage. Mouse sperm treated with 3.6 µM LL-37 lost the ability to fertilize eggs both in vitro and in vivo. All 26 female mice inseminated with sperm and LL-37 did not become pregnant. No apparent damage to the reproductive tract was observed as revealed by histological characterization in LL-37-inseminated mice and these females resumed fecundity following mating with fertile males. LIMITATIONS, REASONS FOR CAUTION: Direct demonstration that LL-37 treated human sperm fail to fertilize eggs was limited by legal restrictions on obtaining human eggs for such use. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal selective inhibitory effects of LL-37 on sperm fertilizing ability in mice without apparent impairment to the female reproductive tract. LL-37 is therefore a promising candidate to be developed into a vaginal contraceptive with microbicidal activity. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Grand Challenges Explorations grant from the Bill & Melinda Gates Foundation (OPP1024509), Canadian Institutes of Health Research (MOP119438 & CCI82413) and International Collaboration and Exchanges NSFC of China (No.30611120525). There are no competing interests to declare.

Stability of DNA methylation patterns in mouse spermatogonia under conditions of MTHFR deficiency and methionine supplementation.

Little is known about the conditions contributing to the stability of DNA methylation patterns in male germ cells. Altered folate pathway enzyme activity and methyl donor supply are two clinically significant factors that can affect the methylation of DNA. 5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key folate pathway enzyme involved in providing methyl groups from dietary folate for DNA methylation. Mice heterozygous for a targeted mutation in the Mthfr gene (Mthfr(+/-)) are a good model for humans homozygous for the MTHFR 677C>T polymorphism, which is found in 10% of the population and is associated with decreased MTHFR activity and infertility. High-dose folic acid is administered as an empirical treatment for male infertility. Here, we examined MTHFR expression in developing male germ cells and evaluated DNA methylation patterns and effects of a range of methionine concentrations in spermatogonia from Mthfr(+/-) as compared to wild-type, Mthfr(+/+) mice. MTHFR was expressed in prospermatogonia and spermatogonia at times of DNA methylation acquisition in the male germline; its expression was also found in early spermatocytes and Sertoli cells. DNA methylation patterns were similar at imprinted genes and intergenic sites across chromosome 9 in neonatal Mthfr(+/+) and Mthfr(+/-) spermatogonia. Using spermatogonia from Mthfr(+/+) and Mthfr(+/-) mice in the spermatogonial stem cell (SSC) culture system, we examined the stability of DNA methylation patterns and determined effects of low or high methionine concentrations. No differences were detected between early and late passages, suggesting that DNA methylation patterns are generally stable in culture. Twenty-fold normal concentrations of methionine resulted in an overall increase in the levels of DNA methylation across chromosome 9, suggesting that DNA methylation can be perturbed in culture. Mthfr(+/-) cells showed a significantly increased variance of DNA methylation at multiple loci across chromosome 9 compared to Mthfr(+/+) cells when cultured with 0.25- to 2-fold normal methionine concentrations. Taken together, our results indicate that DNA methylation patterns in undifferentiated spermatogonia, including SSCs, are relatively stable in culture over time under conditions of altered methionine and MTHFR levels.

Effects of Heparan sulfate acetyl-CoA: Alpha-glucosaminide N-acetyltransferase (HGSNAT) inactivation on the structure and function of epithelial and immune cells of the testis and epididymis and sperm parameters in adult mice.

Heparan sulfate (HS), an abundant component of the apical cell surface and basement membrane, belongs to the glycosaminoglycan family of carbohydrates covalently linked to proteins called heparan sulfate proteoglycans. After endocytosis, HS is degraded in the lysosome by several enzymes, including heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), and in its absence causes Mucopolysaccharidosis III type C (Sanfilippo type C). Since endocytosis occurs in epithelial cells of the testis and epididymis, we examined the morphological effects of Hgsnat inactivation in these organs. In the testis, Hgsnat knockout (Hgsnat-Geo) mice revealed statistically significant decrease in tubule and epithelial profile area of seminiferous tubules. Electron microscopy (EM) analysis revealed cross-sectional tubule profiles with normal and moderately to severely altered appearances. Abnormalities in Sertoli cells and blood-testis barrier and the absence of germ cells in some tubules were noted along with altered morphology of sperm, sperm motility parameters and a reduction in fertilization rates in vitro. Along with quantitatively increased epithelial and tubular profile areas in the epididymis, EM demonstrated significant accumulations of electrolucent lysosomes in the caput-cauda regions that were reactive for cathepsin D and prosaposin antibodies. Lysosomes with similar storage materials were also found in basal, clear and myoid cells. In the mid/basal region of the epithelium of caput-cauda regions of KO mice, large vacuolated cells, unreactive for cytokeratin 5, a basal cell marker, were identified morphologically as epididymal mononuclear phagocytes (eMPs). The cytoplasm of the eMPs was occupied by a gigantic lysosome suggesting an active role of these cells in removing debris from the epithelium. Some eMPs were found in proximity to T-lymphocytes, a feature of dendritic cells. Taken together, our results reveal that upon Hgsnat inactivation, morphological alterations occur to the testis affecting sperm morphology and motility parameters and abnormal lysosomes in epididymal epithelial cells, indicative of a lysosomal storage disease.

SYPL1 defines a vesicular pathway essential for sperm cytoplasmic droplet formation and male fertility.

The cytoplasmic droplet is a conserved dilated area of cytoplasm situated at the neck of the sperm flagellum. Viewed as residual cytoplasm inherited from late spermatids, the cytoplasmic droplet contains numerous saccular elements as its key content. However, the origin of these saccules and the function of the cytoplasmic droplet have long been speculative. Here, we identify the molecular origin of these cytoplasmic droplet components by uncovering a vesicle pathway essential for formation and sequestration of saccules within the cytoplasmic droplet. This process is governed by a transmembrane protein SYPL1 and its interaction with VAMP3. Genetic ablation of SYPL1 in mice reveals that SYPL1 dictates the formation and accumulation of saccular elements in the forming cytoplasmic droplet. Derived from the Golgi, SYPL1 vesicles are critical for segregation of key metabolic enzymes within the forming cytoplasmic droplet of late spermatids and epididymal sperm, which are required for sperm development and male fertility. Our results uncover a mechanism to actively form and segregate saccules within the cytoplasmic droplet to promote sperm fertility.

Arylsulfatase A deficiency causes seminolipid accumulation and a lysosomal storage disorder in Sertoli cells.

Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of male germ cells. During spermatogenesis, apoptosis occurs in >50% of total germ cells. Sertoli cells phagocytose these apoptotic germ cells and degrade their components using lysosomal enzymes. Here we demonstrated that SGG was a physiological substrate of Sertoli lysosomal arylsulfatase A (ARSA). SGG accumulated in Sertoli cells of Arsa(-/-) mice, and at 8 months of age, this buildup led to lysosomal swelling and other cellular abnormalities typical of a lysosomal storage disorder. This disorder likely compromised Sertoli cell functions, manifesting as impaired spermatogenesis and production of sperm with near-zero fertilizing ability in vitro. Fecundity of Arsa(-/-) males was thus reduced when they were older than 5 months. Sperm SGG is known for its roles in fertilization. Therefore, the minimal sperm fertilizing ability of 8-month-old Arsa(-/-) males may be explained by the 50% reduction of their sperm SGG levels, a result that was also observed in testicular germ cells. These unexpected decreases in SGG levels might be partly due to depletion of the backbone lipid palmitylpalmitoylglycerol that is generated from the SGG degradation pathway in Sertoli cells and normally recycled to new generations of primary spermatocytes for SGG synthesis.

Mice lacking the USP2 deubiquitinating enzyme have severe male subfertility associated with defects in fertilization and sperm motility.

The ubiquitin-proteasome system plays an important role in spermatogenesis. However, the functions of deubiquitinating enzymes in this process remain poorly characterized. We previously showed that the deubiquitinating enzyme USP2 is induced in late elongating spermatids. To identify its function, we generated mice lacking USP2. Usp2 -/- mice appeared normal, and the weights of major organs, including the testis, did not differ from wild type (Usp2 +/+). However, although the numbers of testicular spermatids and epididymal spermatozoa were normal in Usp2 -/- males, these animals had a severe defect in fertility, yielding only 12% as many offspring as Usp2 +/+ littermates. Spermatogenesis in Usp2 -/- mice was morphologically normal except for the presence of abnormal aggregations of elongating spermatids and formation of multinucleated cells in some tubules. The epididymal epithelium was morphologically normal in Usp2 -/- mice, but some abnormal cells other than sperm were present in the lumen. Usp2 -/- epididymal spermatozoa manifested normal motility when incubated in culture media, but rapidly became immotile when incubated in PBS in contrast to Usp2 +/+ spermatozoa, which largely maintained motility under this condition. Usp2 -/- and +/+ spermatozoa underwent acrosome reactions in vitro with similar frequency. In vitro fertilization assays demonstrated a severe defect in the ability of Usp2 -/- spermatozoa to fertilize eggs. This could be bypassed by intracytoplasmic sperm injection or removal of the zona pellucida, which resulted in fertilization rates similar to that of Usp2 +/+ mice. We demonstrate for the first time, using mouse transgenic approaches, a role for the ubiquitin system in fertilization.

Characterization of pannexin1 and pannexin3 and their regulation by androgens in the male reproductive tract of the adult rat.

Pannexins (Panxs) are channel-forming proteins that have homology to the invertebrate gap junction proteins, the innexins. These proteins form membrane channels implicated in ATP release. To evaluate the role of Panxs in the male reproductive tract, we investigated the distribution and regulation of Panx1 and 3 in the testis, efferent ducts (ED), and epididymis of adult rats. In the testis, Panx1 localized to the basal compartment of the seminiferous epithelium, while Panx3 was expressed in Leydig cells. In the ED, both Panxs were expressed in the apical region of ciliated cells. In the epididymis, Panx1 was detected at the base of the epithelium, at times encompassing basal cells, while Panx3 was restricted to the apical plasma membrane of principal cells. Panx3 immunoreactions were high throughout the entire epididymis while Panx1 was high in all regions except the initial segment. Multiple transcripts for Panx1 were identified, and sequence analysis indicated that alternative splicing might account for them. Orchidectomy resulted in the expression of multiple immunoreactive Panx1 bands, and these appeared to be androgen-repressed throughout the epididymis. Panx3 levels in all epididymal regions were also androgen-repressed. Deglycosylation experiments indicated that some Panx1 species were due to glycosylation, but this did not account for all Panx1 immunoreactive species. In summary, Panxs expressed in the epididymis and regulated by both alternative splicing events and androgens. These proteins may play a role in ATP secretion into the epididymal lumen and basal extracellular spaces for functions involving sperm transport and maturation.

Castration causes an increase in lysosomal size and upregulation of cathepsin D expression in principal cells along with increased secretion of procathepsin D and prosaposin oligomers in adult rat epididymis.

In the epididymis, lysosomal proteins of the epithelial cells are normally targeted from the Golgi apparatus to lysosomes for degradation, although their secretion into the epididymal lumen has been documented and associated with sperm maturation. In this study, cathepsin D (CatD) and prosaposin (PSAP) were examined in adult epididymis of control, and 2-day castrated rats without (Ct) and with testosterone replacement (Ct+T) to evaluate their expression and regulation within epididymal epithelial cells. By light microscope-immunocytochemistry, a quantitative increase in size of lysosomes in principal cells of Ct animals was noted from the distal initial segment to the proximal cauda. Androgen replacement did not restore the size of lysosomes to control levels. Western blot analysis revealed a significant increase in CatD expression in the epididymis of Ct animals, which suggested an upregulation of its expression in principal cells; androgens restored levels of CatD to that of controls. In contrast, PSAP expression in Ct animals was not altered from controls. Additionally, an increase in procathepsin D levels was noted from samples of the epididymal fluid of Ct compared to control animals, accompanied by an increased complex formation with PSAP. Moreover, an increased oligomerization of prosaposin was observed in the epididymal lumen of Ct rats, with changes reverted to controls in Ct+T animals. Taken together these data suggest castration causes an increased uptake of substrates that are acted upon by CatD in lysosomes of principal cells and in the lumen by procathepsin D. These substrates may be derived from apoptotic cells noted in the lumen of proximal regions and possibly by degenerating sperm in distal regions of the epididymis of Ct animals. Exploring the mechanisms by which lysosomal enzymes are synthesized and secreted by the epididymis may help resolve some of the issues originating from epididymal dysfunctions with relevance to sperm maturation.

Alterations in the human blood-epididymis barrier in obstructive azoospermia and the development of novel epididymal cell lines from infertile men.

Post-testicular sperm maturation requires a specific luminal environment in the epididymis that is created, in part, by the blood-epididymis barrier. There is limited information on gene expression in the epididymis of infertile obstructive azoospermia (OA) patients due to the difficulty in obtaining tissues. The objectives of this study were to determine if epididymal tight junction proteins are altered in OA and to develop cell lines that could serve to elucidate alterations in the epididymis of infertile men. Epididymal claudin (CLDN) 1, CLDN4, and CLDN10 mRNA levels were altered in OA downstream from the obstruction site. Epithelial cell lines derived from the caput epididymidis of one OA patient were developed (infertile human caput epididymal cell line [IHCE]). IHCEs were composed of homogenous populations of diploid cells that ultrastructurally resembled in vivo principal cells. The cells expressed cytokeratin, SPAG11B, CLDN2, CLDN3, desmoplakin, and vimentin. However, the cells did not express several other epididymal markers (CRISP1, SPINLW1, NPC2, CD52, or DCXR) or junctional proteins (CDH1, CDH2, CLDN1, CLDN4, CLDN7, or CLDN8). Further studies using IHCE1 and transepithelial resistance indicated that the cells were unable to form tight junctions. Microarray analyses comparing gene expression in IHCE1 and a recently developed fertile human caput epididymal cell line revealed differential expression of genes encoding junctional proteins, cell junction regulators, and epididymal proteins. Together, these data indicate that epididymal cellular junctions appear to be altered in OA.

Assessing the role of claudins in maintaining the integrity of epididymal tight junctions using novel human epididymal cell lines.

The epididymis is responsible for posttesticular sperm maturation. Sperm maturation is dependent on the luminal microenvironments along the epididymis. Though the role of the epididymis is well established, the molecular and cellular mechanisms responsible for sperm maturation remain to be elucidated, particularly in the human, as limited biological tools exist. We have established the first stable epithelial cell lines transformed with SV40 large T antigen (LTAg) from two regions of the human adult epididymis. The cell lines are composed of homogenous populations of diploid principal cells that possess ultrastructural characteristics similar to those of human principal cells in vivo. These cells express transcripts for adherens (cadherins CDH1 and CDH2) and tight (claudins CLDN1, CLDN2, CLDN3, CLDN4, CLDN7, and CLDN8) junctions as well as desmosomes (desmoplakin, DSP). Transepithelial resistance (TER) measurements in fertile human caput epididymal cell line 1 (FHCE1) as well as the immunolocalization of tight junctional protein 1 (TJP1), occludin, and CLDN1 indicate that these cells form functional tight junctions. Furthermore, knockdown of CLDN1, CLDN3, CLDN4, or CLDN7 using specific siRNAs resulted in significant decreases in TER, suggesting that these CLDNs are essential for the barrier function of the blood-epididymis barrier. Disruption of CLDN1, CLDN3, CLDN4, and CLDN7 could, therefore, lead to epididymal dysfunction, resulting in male infertility.

Dysregulation of WNT/CTNNB1 and PI3K/AKT signaling in testicular stromal cells causes granulosa cell tumor of the testis.

Synergistic effects of dysregulation of the WNT/CTNNB1 and phosphatidylinositol 3-kinase (PI3K)/AKT pathways are thought to be important for the development and progression of many forms of cancer, including the granulosa cell tumor of the ovary. Sustained WNT/CTNNB1 signaling in Sertoli cells causes testicular degeneration and the formation of foci of poorly differentiated stromal cells in the seminiferous tubules in mice. To test if concomitant dysregulation of the WNT/CTNNB1 and PI3K/AKT pathways could synergize to cause testicular cancer, Pten(tm1Hwu/tm1Hwu);Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) mice that express a dominant, stable CTNNB1 mutant and lack the expression of phosphatase and tensin homolog (PTEN) in their Sertoli cells were generated. These mice developed aggressive testicular cancer with 100% penetrance by 5 weeks of age, and 44% of animals developed pulmonary metastases by 4 months, whereas Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) controls were phenotypically normal. Surprisingly, the tumors could not be classified as Sertoli cell tumors, but rather bore histologic and ultrastructural characteristics of granulosa cell tumors of the testis (GCTT). Pten(tm1Hwu/tm1Hwu);Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) testicular tumors did not express CYP17, CYP19, germ cell nuclear antigen, estrogen receptor 1 or progesterone receptor, but expressed the early granulosa cell markers WNT4 and FOXL2, confirming the diagnosis of GCTT. Immunohistochemical analyses of Pten(tm1Hwu/tm1Hwu);Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) GCTT demonstrated a tumor marker profile similar to that reported in human GCTT. Immunoblotting analyses revealed high levels of phosphorylation of AKT and the PI3K/AKT signaling effector FOXO1A in Pten(tm1Hwu/tm1Hwu);Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) GCTT, suggesting the involvement of FOXO1A in the mechanism of GCTT development. Together, these data provide the first insights into the molecular etiology of GCTT and the first animal model for the study of GCTT biology.

Membrane domain specificity in the spatial distribution of aquaporins 5, 7, 9, and 11 in efferent ducts and epididymis of rats.

Water content within the epididymis of the male reproductive system is stringently regulated to promote sperm maturation. Several members of the aquaporin (AQP) family of water channel-forming integral membrane proteins have been identified in epididymal cells, but expression profiling for this epithelium is presently incomplete, and no AQP isoform has yet been identified on basolateral plasma membranes of these cells. In this study, we explored AQP expression by RT-PCR and light microscopy immunolocalizations using peroxidase and wide-field fluorescence techniques. The results indicate that several AQPs are coexpressed in the epididymis including AQP 5, 7, 9, and 11. Immunolocalizations suggested complex patterns in the spatial distribution of these AQPs. In principal cells, AQP 9 and 11 were present mainly on microvilli, whereas AQP 7 was localized primarily to lateral and then to basal plasma membranes in a region-specific manner. AQP 5 was also expressed regionally but was associated with membranes of endosomes. Additionally, AQPs were expressed by some but not all basal (AQP 7 and 11), clear (AQP 7 and 9), and halo (AQP 7 and 11) cells. These findings indicate unique associations of AQPs with specific membrane domains in a cell type- and region-specific manner within the epididymis of adult animals.

Alterations in the testis of hormone sensitive lipase-deficient mice is associated with decreased sperm counts, sperm motility, and fertility.

Hormone-sensitive lipase (HSL, Lipe, E.C.3.1.1.3) functions as a triglyceride and cholesteryl esterase, supplying fatty acids, and cholesterol to cells. Gene-targeted HSL-deficient (HSL(-/-)) mice reveal abnormal spermatids and are infertile at 24 weeks after birth. The purpose of this study was to follow the evolution of spermatid abnormalities as HSL(-/-) mice age, characterize sperm motility in older HSL(-/-) mice, and determine if mice expressing a human testicular HSL transgene (HSL(-/-)ttg) produce normal motile sperm. In situ hybridization indicated that HSL is expressed exclusively in steps 5-16 spermatids, but not in Sertoli cells. In HSL(-/-) mice, abnormalities were evident in step 16 spermatids at 5 weeks after birth, with defects progressively increasing in spermatids with age. The defects included multinucleation of spermatids, abnormal shapes and a reduction of elongating spermatids. In older HSL(-/-) mice, sperm counts appeared reduced by 42%, but this value was lower because samples were compromised by the presence of small degenerating germ cells in addition to sperm, both of which appeared of similar size and density. Sperm motility was dramatically reduced with only 11% classified as motile in HSL(-/-) mice compared to 76-78% of sperm in wild-type and HSL(-/-)ttg mice. Sperm morphology, counts, and motility were normal in HSL(-/-)ttg mice, as was their fertility. Collectively, the data indicate that HSL deficiency results in abnormal spermatid development with defects arising at 5 weeks of age and progressively increasing at later ages. HSL(-/-) mice also show a dramatic reduction in sperm counts and motility and are infertile.

Presence of aberrant epididymal tubules revealing undifferentiated epithelial cells and absence of spermatozoa in a combined neuraminidase-3 and -4 deficient adult mouse model.

Mammalian neuraminidases are responsible for the removal of sialic acids from glycoproteins and glycolipids and function in a variety of biological phenomena such as lysosomal catabolism and control of cell differentiation and growth. Disruption of Neu3 and Neu4 genes has led to the generation of a mouse model revealing severe neurological disorders. In this study a morphological analysis was performed on the epididymis of 3 month-old neu3-/-neu4-/- mice as compared with wild type animals. In neu3-/-neu4-/- mice the majority of tubules of the main epididymal duct were large and lined by differentiated epithelial cells, but revealing lysosomal abnormalities in principal and basally located cells. Of particular note was the presence of aberrant epididymal tubules (ATs) juxtaposed next to the main tubules. ATs were small and of different shapes. Layers of myoid cells encased ATs, which they shared with those of the main tubules, but no interstitial space existed between the two. While some ATs were a dense mass of cells, others revealed a distinct lumen devoid of spermatozoa. The latter revealed an undifferentiated epithelium consisting of cuboidal cells and basal cells, with junctional complexes evident at the luminal front. The absence of spermatozoa from the lumen of the ATs suggests that they were not in contact with the main duct, as also implied by the undifferentiated appearance of the epithelium suggesting lack of lumicrine factors. Despite the presence of ATs, the main duct contained ample spermatozoa, as the neu3-/-neu4-/- mice were fertile. Taken together the data suggest that absence of Neu3 and Neu4 leads to defects in cell adhesion and differentiation of epithelial cells resulting in aberrant tubular offshoots that fail to remain connected with the main duct. Hence Neu3 and Neu 4 play an essential role in the guidance of epithelial cells during early embryonic formation.