Ontogeny of TRα1 expression in the mouse testis and epididymis during postnatal development.

Thyroid hormone (T3 ) acts on the testis via thyroid hormone receptor alpha 1 (TRα1), though the cellular localization of TRα1 in testis remains controversial. Studies on the presence of TRα1 in the epididymis are also lacking. The present study, therefore, examined the cellular localization and expression pattern of TRα1 in testis and epididymis of Parkes mice during postnatal development. Immunohistochemical results showed localization of TRα1 in interstitial and tubular compartments of the testis all through the development. On postnatal day (PND) 14, only leptotene spermatocytes showed TRα1-immunoreactivity in the testis, while at PND 28, 42, and 90, a diverse staining pattern for TRα1 was seen in almost all the seminiferous tubules mainly in leptotene spermatocytes, round and elongating spermatids, and in Leydig cells. Further, qRT-PCR and immunoblot analyses showed that TRα1 was expressed in the testis at the transcript as well as protein level throughout the postnatal development. TRα1 was also seen in principal cells of the epididymis, with maximal expression at PND 90. TRα1 was also present in cauda epididymidal spermatozoa of adult mice at PND 90. The results suggest that TRα1 is expressed in the testis and epididymis and that it may help to regulate the spermatogenic process and male fertility.

Can maternal exposure to tamoxifen compromise sperm and behavioural parameters of male rat offspring?

Tamoxifen, a selective non-steroidal estrogen receptor modulator, is the standard adjuvant endocrine treatment for breast cancer. Since information on the risk of using tamoxifen during pregnancy is still scarce, this study evaluated whether the in utero and lactational treatment with this drug could compromise reproductive and behavioural parameters in male offspring. Pregnant Wistar rats were exposed to three doses of tamoxifen (0.12; 0.6; 3 µg/kg), by gavage, from gestational day 15 to lactational day 20. Tamoxifen exposure did not alter the anogenital distance in the male offspring; however, there was a significant increase in the body weight in the 0.12 µg/kg dose and a decrease in the 0.6 µg/kg dose. The male offspring treated with the highest dose exhibited a delay in the onset of puberty, evidenced by an increase in the age of preputial separation. Regarding sperm parameters, there was an increase in the sperm count in the cauda epididymis in the intermediate and highest dose groups, in addition to an increase in the number of static sperm and a decrease in the progressive sperm in the same groups. Moreover, an increase in the number of hyperplasia of the epithelial clear cells was observed in the epididymis. In conclusion, the present study demonstrated that maternal exposure to tamoxifen compromised the installation of puberty of the male offspring and the maturation of the epididymis, affecting sperm storage and motility in the adult life.

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.

Postnatal cadmium administration affects the presence and distribution of carbohydrates in the sperm membrane during maturation in the epididymis in adult Wistar rats.

Cadmium (Cd) is a heavy metal related to a decrease in sperm parameters. The transit of spermatozoa through the epididymis is necessary to generate changes in the sperm membrane, such as the assembly of various carbohydrates that are added to the spermatazoan's surface to prepare it for successful fertilisation of the oocyte. No studies have yet analysed whether Cd alters the presence and distribution of these carbohydrates. We aimed to evaluate the changes induced by Cd in the distribution pattern of N-acetylglucosamine, sialic acid, mannose and fucose on the sperm membrane in the epididymis (e.g. caput, corpus, cauda) and if it alters the epididymal epithelium. Male Wistar pups were treated with Cd doses (0.125, 0.25 and 0.5mg/kg) on postnatal days 1-49. At postnatal day 90, they were humanely killed, sperm samples were obtained from the epididymis and tissue samples were taken for histological analysis. Cd concentrations in the blood and epididymis increased in proportion to the dose administered and decreased the serum testosterone levels and sperm quality. Histological analysis revealed alterations in the epithelium in all Cd-treated groups. Cd altered the distribution patterns of carbohydrates and fluorescence indices. All these alterations affected the structure and functioning of sperm.

Estrogens and development of the rete testis, efferent ductules, epididymis and vas deferens.

Estrogen has always been considered the female hormone and testosterone the male hormone. However, estrogen's presence in the testis and deleterious effects of estrogen treatment during development have been known for nearly 90 years, long before estrogen receptors (ESRs) were discovered. Eventually it was learned that testes actually synthesize high levels of estradiol (E2) and sequester high concentrations in the reproductive tract lumen, which seems contradictory to the overwhelming number of studies showing reproductive pathology following exogenous estrogen exposures. For too long, the developmental pathology of estrogen has dominated our thinking, even resulting in the "estrogen hypothesis" as related to the testicular dysgenesis syndrome. However, these early studies and the development of an Esr1 knockout mouse led to a deluge of research into estrogen's potential role in and disruption of development and function of the male reproductive system. What is new is that estrogen action in the male cannot be divorced from that of androgen. This paper presents what is known about components of the estrogen pathway, including its synthesis and target receptors, and the need to achieve a balance between androgen- and estrogen-action in male reproductive tract differentiation and adult functions. The review focuses on what is known regarding development of the male reproductive tract, from the rete testis to the vas deferens, and examines the expression of estrogen receptors and presence of aromatase in the male reproductive system, traces the evidence provided by estrogen-associated knockout and transgenic animal models and discusses the effects of fetal and postnatal exposures to estrogens. Hopefully, there will be enough here to stimulate discussions and new investigations of the androgen:estrogen balance that seems to be essential for development of the male reproductive tract.

Cross-seeding between the functional amyloidogenic CRES and CRES3 family members and their regulation of Aß assembly.

Accumulating evidence shows that amyloids perform biological roles. We previously showed that an amyloid matrix composed of four members of the CRES subgroup of reproductive family 2 cystatins is a normal component of the mouse epididymal lumen. The cellular mechanisms that control the assembly of these and other functional amyloid structures, however, remain unclear. We speculated that cross-seeding between CRES members could be a mechanism to control the assembly of the endogenous functional amyloid. Herein we used thioflavin T assays and negative stain transmission electron microscopy to explore this possibility. We show that CRES3 rapidly formed large networks of beaded chains that possessed the characteristic cross-ß reflections of amyloid when examined by X-ray diffraction. The beaded amyloids accelerated the amyloidogenesis of CRES, a less amyloidogenic family member, in seeding assays during which beads transitioned into films and fibrils. Similarly, CRES seeds expedited CRES3 amyloidogenesis, although less efficiently than the CRES3 seeding of CRES. These studies suggest that CRES and CRES3 hetero-oligomerize and that CRES3 beaded amyloids may function as stable preassembled seeds. The CRES3 beaded amyloids also facilitated assembly of the unrelated amyloidogenic precursor Aß by providing a surface for polymerization though, intriguingly, CRES3 (and CRES) monomer/early oligomer profoundly inhibited Aß assembly. The cross-seeding between the CRES subgroup members is similar to that which occurs between bacterial curli proteins suggesting that it may be an evolutionarily conserved mechanism to control the assembly of some functional amyloids. Further, interactions between unrelated amyloidogenic precursors may also be a means to regulate functional amyloid assembly.

Delay in puberty indices of Wistar rats caused by Cadmium. Focus on the redox system in reproductive organs.

Puberty is a transitional period from juvenile stage to adulthood, followed by the functional maturation of gonads and reproductive organs. This period is sensitive to environmental pollutants like cadmium (Cd), a heavy metal that represents a serious health risk. Cd is an endocrine disruptor that interferes with reproduction by causing oxidative stress in the reproductive organs, affecting the sexual function and decreasing testosterone (T) levels. However, little research has been done on the effects of Cd on puberty markers and antioxidant systems. In this study, we evaluated the effects of Cd on puberty markers: preputial separation, testes descent and T levels, and the antioxidant activity (SOD, CAT, GSH/GSSG and TAC) in the seminal vesicles, testis and epididymis. Male Wistar pups were treated with 1 mg/kg Cd or saline solution by i.p. injection from day 1 to 35; the other treatment was administrated for 49 days. At the end of treatment, the animals were sacrificed, and the tissues of interest dissected, weighed and prepared for the respective assays. Cd treated rats from birth to puberty showed a delay onset in the puberty markers and a low weight in reproductive organs. Also, Cd induced differential effects on the redox system in reproductive organs and decreased T levels, these effects played a pivotal role in the delay of puberty markers onset (testes descent and preputial separation), affecting the development and sexual maturity of the male rats.

Expression profile of Toll-like receptor 4 in rat testis and epididymis throughout postnatal development.

Toll-like receptors (TLRs) belonging to pattern recognition receptors are involved in maintaining testicular and epididymal immune homeostasis. The purpose of the current study was to investigate TLR4 expression in rat testis and epididymis throughout postnatal development. Weak staining was detected in peritubular myoid cells and immature Sertoli cells while no staining was observed in gonocytes during prepubertal period. However, TLR4 expression began to appear in spermatocytes in pubertal period and gradually increased in spermatids. An intense staining was observed in steps 5-19 spermatids in post pubertal and mature periods. Similarly, TLR4 expression in the testes steadily increased from pubertal period to mature period. Puberty also caused a significant increase in TLR4 expression in epididymis. TLR4 expression in cauda epididymis was lower as compared to those of other epididymal segments. The majority of epididymal epithelial cells exhibited apical TLR4 expression, whereas basal cells showed intense intracytoplasmic immunoreaction. We detected an intense staining in epididymal smooth muscle cells. The expression levels of TLR4 showed dynamic changes in both spermatogenic cells, and entire testicular and epididymal tissues during postnatal development. These results suggest that TLR4 expression contributes not only to inflammation but also to the development of spermatogenic cells.

Testosterone promotes GPX5 expression of goat epididymal epithelial cells cultured in vitro.

Androgens are involved in maintaining epididymal structure and function. In the present study, primary culture of goat EECs and effect of testosterone on expression of glutathione peroxidase-5 (GPX5) in goat epididymal epithelial cells (EECs) were investigated. The EECs isolated from 12-mo-old goat caput epididymis were cultured with testosterone in vitro, and expression of glutathione peroxidase-5 (GPX5) and androgen receptors (ARs) was analyzed. Our results showed that testosterone effectively increased EEC proliferation activity, and EECs cultured with testosterone could maintain molecular markers for up to 12 passages. Compared with the control group, 100 nM testosterone significantly increased the mRNA and protein expression of GPX5 (P < 0.05) and ARs (P < 0.01 and P < 0.05, respectively) in EECs, and this effect was blocked by the AR blocker enzalutamide. In conclusion, testosterone can promote the expression of GPX5 in EECs by up-regulating AR expression. We established an effective culture system for goat EECs which can be for further investigation on the regulation of epithelial function.

Prenatal exposure to betamethasone causes intergenerational impairment of epididymal development in the rat.

BACKGROUND: Studies on epididymal toxicology are scarce. Betamethasone (BM) is a glucocorticoid used in clinical practice for antenatal therapy. We previously reported changes to testicular morphology, altered sperm quality, and fertility in adult rats following intrauterine administration of BM. OBJECTIVES: Given that high levels of corticosteroids during gestation lead to fetal androgen depletion, and the essential role of testosterone during epididymal development, here we investigated epididymal morphology and physiology in the F1 and F2 male offspring of female rats treated with BM during gestation. MATERIALS AND METHODS: Pregnant rats were randomly divided into two experimental groups: control (saline vehicle, n = 11) and BM-treated group (0.1 mg/kg betamethasone 21-phosphate disodium, n = 13). Rats received an intramuscular injection of vehicle or BM on gestational days 12, 13, 18, and 19. This encompasses the beginning of the critical window of male rat reproductive tract development. A subset of three males from each litter (n = 5 litters/group) was used: One rat per litter was euthanized at puberty, one was euthanized at adulthood, while the others were mated with a non-treated female to obtain the F2 generation. The same protocol described for the F1 was applied for F2, except for the mating protocol. RESULTS: In both F1 and F2 generations, prenatal BM exposure resulted in delayed differentiation of the cauda epididymal epithelium, characterized by increased cribriform appearance on PND 45, and displayed weaker or non-detectable Cx43 immunostaining. Furthermore, in the F1 generation only, immunostaining of TP63, a transcription factor expressed in basal cells, appeared more intense with a greater number of TP63-positive cells observed in the cauda epididymis. In adults, the epithelial area was reduced in the F1 BM rats. The contractile activity of isolated epididymal ducts was comparable between groups. DISCUSSION AND CONCLUSION: Prenatal BM exposure leads to intergenerational impairment in the development and structure of the rat epididymis.

Relevance of CRISP proteins for epididymal physiology, fertilization, and fertility.

BACKGROUND: The molecular mechanisms involved in the acquisition of mammalian sperm fertilizing ability are still poorly understood, reflecting the complexity of this process. OBJECTIVES: In this review, we describe the role of Cysteine RIch Secretory Proteins (CRISP1-4) in different steps of the sperm journey to the egg as well as their relevance for fertilization and fertility. MATERIALS AND METHODS: We analyze bibliography reporting the phenotypes of CRISP KO mice models and combine this search with recent findings from our team. RESULTS: Generation of individual KO for CRISP proteins reveals they are key mediators in different stages of the fertilization process. However, in spite of their important functional roles, KO males for each of these proteins remain fertile, supporting the existence of compensatory mechanisms between homologous CRISP family members. The development of mice lacking epididymal CRISP1 and CRISP4 simultaneously (DKO) revealed that mutant males exhibit an impaired fertility due to deficiencies in the sperm ability to fertilize the eggs in vivo, consistent with the proposed roles of the two proteins in fertilization. Interestingly, DKO males show clear defects in both epididymal epithelium differentiation and luminal acidification known to be critical for sperm maturation and storage. Whereas in most of the cases, these epithelium defects seem to specifically affect the sperm fertilizing ability, some animals exhibit a disruption of the characteristic immune tolerance of the organ with clear signs of inflammation and sperm viability defects. DISCUSSION AND CONCLUSION: Altogether, these observations confirm the relevance of CRISP proteins for male fertility and contribute to a better understanding of the fine-tuning mechanisms underlying sperm maturation and immune tolerance within the epididymis. Moreover, considering the existence of a human epididymal protein functionally equivalent to rodent CRISP1 and CRISP4, DKO mice may represent an excellent model for studying human epididymal physiology and pathology.

Tissue regeneration and the epididymal stem cell.

BACKGROUND: In most pseudostratified epithelia, basal cells represent a multipotent adult stem cell population. These cells generally remain in a quiescent state, until they are stimulated to respond to tissue damage by initiating epithelial regeneration. In the epididymis, cell proliferation occurs at a relatively slow rate under normal physiological conditions. Epididymal basal cells have been shown to share common properties with multipotent adult stem cells. The development of organoids from stem cells represents a novel approach for understanding cellular differentiation and characterization of stem cells. OBJECTIVE: To review the literature on tissue regeneration in the epididymis and demonstrate the presence of an epididymal stem cell population. METHODS: PubMed database was searched for studies reporting on cell proliferation, regeneration, and stem cells in the epididymis. Three-dimensional cell culture of epididymal cells was used to determine whether these can develop into organoids in a similar fashion to stem cells from other tissues. RESULTS: The epididymal epithelium can rapidly regenerate following orchidectomy or efferent duct ligation, in order to maintain epithelial integrity. Studies have isolated a highly purified fraction of rat epididymal basal cells and reported that these cells displayed properties similar to those of multipotent adult stem cells. In two-dimensional cell culture conditions, these cells differentiated into cells which expressed connexin 26, a marker of columnar cells, and cytokeratin 8. Furthermore, three-dimensional cell culture of epididymal cells resulted in the formation of organoids, a phenomenon associated with the proliferation and differentiation of stem cells in vitro. CONCLUSIONS: The rapid proliferation and tissue regeneration of the epididymal epithelium to preserve its integrity following tissue damage as well as the ability of cells to differentiate into organoids in vitro support the notion of a resident progenitor/stem cell population in the adult epididymis.

Effects of maternal inhalation of carbon black nanoparticles on reproductive and fertility parameters in a four-generation study of male mice.

BACKGROUND: Previous findings indicate that in utero exposure to nanoparticles may affect the reproductive system in male offspring. Effects such as decreased sperm counts and testicular structural changes in F1 males have been reported following maternal airway exposure to carbon black during gestation. In addition, a previous study in our laboratory suggested that the effects of in utero exposure of nanoparticles may span further than the first generation, as sperm content per gram of testis was significantly lowered in F2 males. In the present study we assessed male fertility parameters following in utero inhalation exposure to carbon black in four generations of mice. RESULTS: Filter measurements demonstrated that the time-mated females were exposed to a mean total suspended particle mass concentration of 4.79 ± 1.86 or 33.87 ± 14.77 mg/m3 for the low and high exposure, respectively. The control exposure was below the detection limit (LOD 0.08 mg/m3). Exposure did not affect gestation and litter parameters in any generation. No significant changes were observed in body and reproductive organ weights, epididymal sperm parameters, daily sperm production, plasma testosterone or fertility. CONCLUSION: In utero exposure to carbon black nanoparticles, at occupationally relevant exposure levels, via maternal whole body inhalation did not affect male-specific reproductive, fertility and litter parameters in four generations of mice.

Motile cilia of the male reproductive system require miR-34/miR-449 for development and function to generate luminal turbulence.

Cilia are cell-surface, microtubule-based organelles that project into extracellular space. Motile cilia are conserved throughout eukaryotes, and their beat induces the flow of fluid, relative to cell surfaces. In mammals, the coordinated beat of motile cilia provides highly specialized functions associated with the movement of luminal contents, as seen with metachronal waves transporting mucus in the respiratory tract. Motile cilia are also present in the male and female reproductive tracts. In the female, wave-like motions of oviductal cilia transport oocytes and embryos toward the uterus. A similar function has been assumed for motile cilia in efferent ductules of the male-i.e., to transport immotile sperm from rete testis into the epididymis. However, we report here that efferent ductal cilia in the male do not display a uniform wave-like beat to transport sperm solely in one direction, but rather exert a centripetal force on luminal fluids through whip-like beating with continual changes in direction, generating turbulence, which maintains immotile spermatozoa in suspension within the lumen. Genetic ablation of two miRNA clusters (miR-34b/c and -449a/b/c) led to failure in multiciliogenesis in murine efferent ductules due to dysregulation of numerous genes, and this mouse model allowed us to demonstrate that loss of efferent duct motile cilia causes sperm aggregation and agglutination, luminal obstruction, and sperm granulomas, which, in turn, induce back-pressure atrophy of the testis and ultimately male infertility.

New insights of polyamine metabolism in testicular physiology: A role of ornithine decarboxylase antizyme inhibitor 2 (AZIN2) in the modulation of testosterone levels and sperm motility.

The specific role of polyamines in the testis physiology is not fully understood. Antizymes (OAZs) and antizyme inhibitors (AZINs) are modulators of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis and polyamine uptake. Although the three known OAZs are expressed in the testis, only OAZ3 is testis specific and has been proven to have an essential role in male fertility. Regarding the two existing AZINs, AZIN2 is the most abundantly expressed member in this gonad. Whereas previous studies suggested that AZIN2 might participate in mouse spermatogenesis, immunohistological analysis of human testicular sections revealed that AZIN2 is also detected in the steroidogenic Leydig cells but not in the germinal epithelium. In the present study, we found a close ontogenic similarity in the mRNA levels of OAZs and AZINs between mice and rats, but an opposite expression pattern of ODC activity. Further analysis of AZIN2 and OAZ3 in the testis of mice with different alterations in spermatogenesis and fertility, induced either genetically or pharmacologically, corroborated that both AZIN2 and OAZ3 are mainly expressed in the haploid germinal cells. Finally, by using transgenic mice with a truncated Azin2 gene fused to the bacterial lacZ gene, we studied the expression of Azin2 in testes, epididymides and spermatozoa. AZIN2 was detected in spermatids and spermatozoa, as well as in Leydig cells, and in epithelial epidydimal cells. Azin2 knock-out male mice were fertile; however, they showed marked decreases in testicular putrescine and plasma and testicular testosterone levels, and a dramatic reduction in the sperm motility. These results suggest an important role for AZIN2 in testicular cells by modulating polyamine concentrations, testosterone synthesis and sperm function. Overall, our data corroborate the relevance of polyamine regulation in testis functions, where both AZIN2 and OAZ3 play fundamental roles.

Canonical Wnt/ß-Catenin Signaling Regulates Postnatal Mouse Epididymal Development But Does Not Affect Epithelial Cell Differentiation.

The epithelial lining of the epididymis establishes an optimal environment in which spermatozoa acquire the ability to fertilize an oocyte. This highly specialized organ develops from a simple embryonic tube known as the Wolffian duct (WD). How the simple columnar epithelium of WD acquires the complex features of the adult epididymal epithelium is currently unclear. During these first few weeks after birth, the epididymal epithelium undergoes major changes and by 5 weeks consists of four different cell types. The main objective of this study was to evaluate potential roles of Wnt signaling during postnatal epididymal development and differentiation. To analyze the activity of Wnt signaling during postnatal development, we evaluated the epididymis of TCFGFP mice, a Wnt reporter mouse model. Wnt signaling activity as indicated by green fluorescent protein expression was detected in the whole epididymis of TCFGFP mice during the first 2 weeks of life but was localized only to the caput region by 5 weeks of age. Using a genetic cell lineage tracing approach, we showed that all four of the epididymal epithelial cell types originated from the simple columnar epithelium of WD. To delineate the functional significance of epithelial Wnt signaling in epididymal development and differentiation, we generated a mouse model in which ß-catenin (Ctnnb1) was specifically ablated from the epididymal epithelium upon administration of doxycycline. Genetic suppression of epithelial Wnt/ß-catenin signaling inhibited epididymal development by affecting cell proliferation but had no effect on epithelial cell differentiation.

Ascorbic acid supplementation partially prevents the delayed reproductive development in juvenile male rats exposed to rosuvastatin since prepuberty.

Dyslipidemias are occurring earlier in the population due to the increase of obesity and bad eating habits. Rosuvastatin inhibits the enzyme HMG-CoA reductase, decreasing total cholesterol. Ascorbic acid is an important antioxidant compound for male reproductive system. This study aimed to evaluate whether ascorbic acid supplementation may prevent the reproductive damage provoked by rosuvastatin administration at prepuberty. Male pups were distributed into six experimental groups that received saline solution 0.9%, 3 or 10mg/kg/day of rosuvastatin, 150mg/day of ascorbic acid, or 150mg/day of ascorbic acid associated with 3 or 10mg/kg/day of rosuvastatin from post-natal day (PND) 23 until PND53. Rosuvastatin-treated groups showed delayed puberty installation, androgen depletion and impairment on testicular and epididymal morphology. Ascorbic acid partially prevented these reproductive damages. In conclusion, rosuvastatin exposure is a probable risk to reproductive development and ascorbic acid supplementation may be useful to prevent the reproductive impairment of rosuvastatin exposure.

Impact of male fertility status on the transcriptome of the bovine epididymis.

STUDY QUESTION: Can region-specific transcriptional profiling of the epididymis from fertile and sub-fertile bulls predict the etiology of fertility/sub-fertility in males? SUMMARY ANSWER: The highly regulated gene expression along the bovine epididymis is affected by the fertility status of bulls used for artificial insemination. WHAT IS KNOWN ALREADY: In mammals, sperm maturation and storage occur in the epididymis. Each epididymal segment has his own transcriptomic signature that modulates the intraluminal composition and consequently governs sequential modifications of the maturing male gamete. STUDY DESIGN, SIZE, DURATION: Epididymides from six Holstein bulls with documented fertility were used. These bulls were divided into two groups: high fertility (n = 3), and medium-low fertility (n = 3) and their epididymal transcriptomic profiles were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Bovine cDNA microarray probing and bioinformatic tools were used to identify genes that are differentially expressed in caput, corpus and cauda epididymidal tissues of bulls with the documented fertility index. MAIN RESULTS AND THE ROLE OF CHANCE: Hierarchical clustering and principal component analysis revealed a clear separation between caput, corpus and cauda epididymides. Some transcripts characterize a particular anatomical segment, whereas others are expressed in two out of three epididymal segments. Gene ontology analysis allowed deduction of specific functions played by each epididymal segment. The transcriptional profiles between fertile versus sub-fertile conditions clustered most closely in the corpus and cauda segments, whereas the profiles in the caput segment were distinct between fertile and sub-fertile bulls. Of the differently expressed genes, 10 (AKAP4, SMCP, SPATA3, TCP11, ODF1, CTCFL, SPATA18, ADAM28, SORD and FAM161A) were found to exert functions related to reproductive systems and 5 genes (DEAD, CYST11, DEFB119, DEFB124 and MX1) were found to be associated with the defense response. LARGE SCALE DATA: The GEO number for public access of bovine epididymis microarray data is GSE96602. LIMITATIONS, REASONS FOR CAUTION: Further work is required to link these modulations of epididymal functions with sperm fertilizing ability in order to understand the etiology of certain cases of idiopathic infertility in livestock and men. WIDER IMPLICATIONS OF THE FINDINGS: As fertility can be quantified in bulls used for artificial insemination, this species is a unique model to aid in the understanding of male fertility/sub-fertility in man. Our data provide a molecular characterization that will facilitate advances in understanding the involvement of epididymal physiology in sub/infertility etiology. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a grant to R.S. from the Natural Sciences and Engineering Research Council (NSERC) of Canada. C.L., A.A., E.C. and R.S. have no conflict of interest to declare. P.B. is R&D director at Alliance Boviteq Inc., a bovine artificial insemination company.

Junctional adhesion molecule A: expression in the murine epididymal tract and accessory organs and acquisition by maturing sperm.

STUDY QUESTION: Is junctional adhesion molecule A (JAM-A), a sperm protein essential for normal motility, expressed in the murine post-testicular pathway and involved in sperm maturation? SUMMARY ANSWER: JAM-A is present in the prostate and seminal vesicles and in all three regions of the epididymis where it is secreted in epididymosomes in the luminal fluid and can be delivered to sperm in vitro. WHAT IS KNOWN ALREADY: JAM-A shares with the plasma membrane Ca2+ATPase 4 (PMCA4, the major Ca2+ efflux pump in murine sperm) a common interacting partner, CASK (Ca2+/CaM-dependent serine kinase). JAM-A, like PMCA4, plays a role in Ca2+ regulation, since deletion of Jam-A results in significantly elevated intracellular Ca2+ levels and reduced sperm motility. Recently, PMCA4 was reported to be expressed in the epididymis and along with CASK was shown to be in a complex on epididymosomes where it was transferred to sperm. Because of the association of JAM-A with CASK in sperm and because of the presence of PMCA4 and CASK in the epididymis, the present study was performed to determine whether JAM-A is expressed in the epididymis and delivered to sperm during their maturation. STUDY DESIGN, SIZE, DURATION: The epididymides, prostate and seminal vesicles were collected from sexually mature C57BL/6J and Institute for Cancer Research mice and antibodies specific for JAM-A and Ser285 -phosphorylated JAM-A (pJAM-A) were used for the analysis. Tissues, sperm and epididymal luminal fluid (ELF) were studied. Epididymosomes were also isolated for study. Caput and caudal sperm were co-incubated with ELF individually to determine their abilities to acquire JAM-A in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sections of all three regions of the epididymis were subjected to indirect immunofluorescence analysis. Epididymal tissues, fluid, sperm, prostate and seminal vesicle tissues were analyzed for JAM-A and/or pJAM-A via western blotting analysis. The relative amounts of JAM-A and pJAM-A among epididymal tissues, ELF and sperm were detected by western blot via quantification of band intensities. Epididymosomes were isolated by ultracentrifugation of the ELF after it was clarified to remove cells and tissue fragments, and the proteins western blotted for JAM-A and pJAM-A, and exosomal biochemical markers. FACS analysis was used to quantify the amount of JAM-A present on caput and caudal sperm, as well as the amount of JAM-A acquired in vitro after their co-incubation with ELF. MAIN RESULTS AND THE ROLE OF CHANCE: Western blots revealed that JAM-A is expressed in all three regions of the epididymis, the prostate and seminal vesicles. As confirmed by indirect immunofluorescence, a western blot showed that JAM-A has a higher expression in the corpus and caudal regions, where it is significantly (P < 0.01) more abundant than in the caput. Both JAM-A and Ser285-phosphorylated JAM-A (pJAM-A) are secreted into the ELF where it is highest in the distal regions. In the ELF, both JAM-A and pJAM-A were detected in epididymosomes. Western blotting of sperm proteins showed a significant (P < 0.01) increase of JAM-A and pJAM-A in caudal, compared with caput, sperm. Flow-cytometric analysis confirmed the increase in JAM-A in caudal sperm where it was 1.4-fold higher than in caput ones. Co-incubation of caput and caudal sperm with ELF demonstrated ~2.3- and ~1.3-fold increases, respectively, in JAM-A levels indicating that epididymosomes transfer more JAM-A to caput sperm that are less saturated with the protein than caudal ones. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: First, although the ELF was clarified prior to ultracentrifugation for epididymosome isolation, we cannot rule out contamination of the epididymosomal proteins by those from epididymal epithelial cells. Second, the JAM-A detected in the prostate and seminal vesicles might not necessarily be secreted from those organs and may only be present within the tissues, where it would be unable to impact sperm in the ejaculate. WIDER IMPLICATIONS OF THE FINDINGS: Although performed in the mouse the study has implications for humans, as the highly conserved JAM-A is a signaling protein in human sperm. There is physiological significance to the finding that JAM-A, which regulates sperm motility and intracellular Ca2+, exists in elevated levels in the cauda where sperm gain motility and fertilizing ability. The study suggests that the acquisition of JAM-A in the epididymal tract is involved in the mechanism by which sperm gain their motility during epididymal maturation. This increased understanding of sperm physiology is important for aspects of ART. STUDY FUNDING AND COMPETING INTEREST(S): The work was supported by NIH-RO3HD073523 and NIH-5P20RR015588 grants to P.A.M.-D. The authors declare there are no conflicts of interests.

[Reaction of Reproductive System and Epididymal Spermatozoa .of Rats to Electromagnetic Radiation from Mobile Phone (1745 MHz) of Various Duration].

Changes in the weight of reproductive system organs, quantitative and qualitative indicators of the epididymal spermatozoa of rats on the Ist and 30th days after the end of an electromagnetic exposition from the mobile phone (1745 MHz, the 8 hour/day, power density 0.2-20 µW/cm²) of various duration (from 1 to 90 days) have been researched. A significant increase in the absolute and relative weight of the epididymis and seminal vesicles and the number of epididymal spermatozoa was revealed on the 1st day after electromagnetic expo- sure during 7 days. The increase in the exposure duration up to 14 days is accompanied by a fall of the above indicators, and after a longer exposure (30, 60 days) there is an increase of the absolute weight of the testes; in all other cases, significant deviations in terms of the weight of reproductive organs are not observed. Expo- sition of various duration at 1745 MHz, except for 7-day radiation exposure, had no significant effect on the amount of epididymal spermatozoa and DNA fragmentation in them; however, viability of mature male germ cells in irradiated animals decreases independently of the duration of exposure. The revealed disturbances of the researched indicators are mainly normalized in the remote period (on the 30th day) after electromagnetic radiation.