Epididymal Sections Research Articles

HACE2 upregulation and participation of macrophages and clear cells in the immune response of epididymis to SARS-CoV-2 in K18-hACE2 mice.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus caused the coronavirus disease 2019 pandemic, and the prevalence of deaths among men is higher than among women. The epididymis, divided into caput, corpus, and cauda, shows a region-specific immunity. The K18-hACE2 mouse expresses human angiotensin-converting enzyme 2 (hACE2), the receptor that allows SARS-CoV-2 infection. However, studies using this transgenic mouse to evaluate the impact of this viral infection in epididymis have not yet been performed. We evaluated the expression of hACE2 in the epididymis of SARS-CoV-2-infected K18-hACE2 mice, and assessed the epididymal immune response, focusing on F4/80+ mononuclear phagocytes and tumor necrosis factor-alpha expression. The following analyses were performed in the epididymal sections of infected mice: epithelial height and duct diameter, birefringent collagen, Terminal deoxynucleotidyl Transferase-mediated dUTP Nick End Labelling, immunoreactions for detection of hACE2, spike, FGF, V-ATPase, F4/80, tumor necrosis factor-alpha, and iNOS. Viral particles were identified under electron microscopy. hACE2, Rigi, Tgfb1 and Tnfa expression were also evaluated by real-time quantitative polymerase chain reaction. All epididymal regions expressed hACE2, which increased in all epididymal regions in the infected mice. However, the caput appeared to be the most infected region. Despite this, the caput region showed minimal changes while the cauda showed significant epithelial changes associated with increased iNOS immunoexpression. The F4/80+ mononuclear phagocyte area increased significantly in both stroma and epithelium. In addition to the epithelial and stromal mononuclear phagocytes, tumor necrosis factor-alpha was also detected in clear cells, whose cytoplasm showed a significant increase of this cytokine in the infected animals. The K18-hACE2 mouse is a useful model for evaluating the impact of SARS-CoV-2 infection in the epididymis. The infection induced hACE2 upregulation, favoring the virulence in the epididymis. The epididymal regions responded differentially to infection, and the activation of F4/80+ mononuclear phagocytes associated with the increased tumor necrosis factor-alpha immunolabeling in clear cells indicates a role of clear cells/mononuclear phagocytes immunoregulatory mechanisms in the epididymal immune response to SARS-CoV-2 infection.

Application of Lectins for Detection of Glycomic Differences in the Epididymal Duct in Water Buffalo (Bubalus bubalis) and Dromedary Camel (Camelus dromedarius)

The present study was conducted to detect the differences in glycohistochemical features in the epididymal duct of the dromedary camel and the water buffalo. Epididymal sections (caput, corpus and cauda) from both species were stained with fluorescein isothiocyanate (FITC) conjugated lectins. Binding sites for five lectins (DBA, GSA-1, HPA, PNA and WGA) have been found in both species. The binding sites of different lectins showed significant variations in the pattern of distribution in both a species. This included both species-specific and region-specific order. Additionally, only three (GSA-1, PNA and WGA) out the five lectins studied exhibited binding sites in all epididymal regions in both species. The other two lectins (DBA and HPA) followed the same order recorded for the other three (GSA-1, PNA and WGA) in buffalo, but failed to show any binding sites in cauda epididymis in camel. In conclusion, the variable regional and species-specific distribution features of lectins revealed that both species have diverse glycomic characteristics that may be related to their different reproductive patterns. However, the glycome-associated functional capacities remain obscured and need further profound investigations.

Environmental Effects Of Monosodium Glutamate On (NF-κB) Levels In The Male Reproductive System Of Rats

Monosodium glutamate (MSG) has been identified as a food additive that Adversely affects MSG use on the reproductive efficiency by several mechanisms, this study was carried out to determine its role in stimulating the immune response in the reproductive system by estimating levels of NF-κB in serum and of its expression in reproductive organs. Thirty sex males rats were used in this study each included 12 male rats. Male rats A and B are receiving 60 and 120 mg/kg for 28 days respectively male rats of the control group were left without treatment for the period of the experiment. The levels of Nuclear factor-kappa NF-κB in serum and tissue fluid of testes and epididymis were measured. histological changes in addition to detection NF-κB expression were studying in testes and epididymis. Results of the present study recorded the toxic effect of MSG as it caused an elevation in the levels of NF-κB in serum, testicular, and epididymis tissue fluid. histological alternation in the reproductive organs was observed to represent detachment and vacuolation of the seminal epithelium, degeneration of spermatogenesis edema in a lumen in testicular tissues. Epididymal sections appeared to sever necrosis, degeneration in the epithelial layer and stereocilia, and the lumen of the epididymis. Also there was an increasing density of NFκB protein immunoreactivity in both testis and epididymis.

MORPHOLOGICAL EVALUATION OF CISPLATIN-INDUCED TESTICULAR DAMAGE IN WISTAR RATS

Twenty-one Wistar rats weighing 200 - 250 mg were categorized into three treatment groups- the control, saline and the cisplatin groups, each comprising of seven rats per group, to assess the effects of cisplatin-induced testicular damage in Wistar rats. The control group received feed mash and water ad libitum, the saline group received equal volumes of physiological saline intraperitoneally with normal feeds and water, while the cisplastin group received 8 mg/kg body weight of cisplastin only for five days and had normal feeds and water for twelve weeks before sacrifice. The effects of these treatments on total sperm count, sperm motility, percentage live:dead sperm and on testicular and epididymal morphology of the Wistar rats were assessed. The results revealed total sperm count in the cisplatin group as significantly different from control (p<0.001), and the motile sperm count in the cisplatin group was also significantly different from control (p<0.001). Also, there was a greater percentage of dead sperm and more morphologically abnormal forms in the cisplatin group, compared to control. The testicular and epididymal sections of the saline treatment showed normal progression of the spermatogenic germ cell series and normal testicular interstitium. The sections of the testes from the cisplatin group had seminiferous tubules with atrophic changes, detached basement membrane from the seminiferous epithelium, inflammed interstitium with oedema, paucity of Leydig cells, vascular congestion and haemorrhage. The epididymis showed lumen with empty or scanty spermatozoa, necrosis and haemorrhage. These findings affirms the reality of severe gonadotoxicity associated with use of cisplatin as chemotherapeutic agent, especially in the male.

Cellular Changes and Effect on DNA Integrity of the Epididymal Cells of Swiss Albino Mice Post Exposure to High Dose of Cyclophosphamide: A Histological and TUNEL Assay Study

Background: Many studies done in past literature focuses on gonadal toxicity of Cyclophosphamide, but very less studies are done on the epididymis post exposure to this drug. Hence a sincere attempt is made in present study to look into the cellular changes in the epididymis through histology and TUNEL Assay. Subjects & Methods: Healthy male Swiss albino adult mice, were obtained from Central Animal Research Facility, Manipal were used for the study. The adult as well as prepubertal male mice were divided into control and test groups. In test group, the mice were injected with Cyclophosphamide at variable doses. From each group certain no. of mice was sacrificed on different days. A control group was kept for each intervals. The epididymal tissue was then extracted and used in histological and Tunnel Assay study. Results: Even though there was no significant change in the number of basal cells, the apical cells decreased marginally at higher doses of CP. A TUNEL assay was performed in the epididymal sections to understand its effect on the DNA integrity. Up to 100 mg/kg, CP did not induce any significant increase in the TUNEL positive tubules. However, at 200 mg/kg dose and above, there was an increase in the number of tubules with TUNEL positive cells indicating its DNA damaging effect. However, this increase was statistically not significant Conclusion: The present study highlights the need for looking into the cellular damage of the cells after exposure to one of the commonly used anticancer drug- cyclophosphamide. Keywords: Epididymal Histology, Tunnel Assay, Cyclophosphamide, Swiss Albino mice.

Determination of osteopontin and antioxidant concentrations from epididymal fluids in mongrel dogs

The extracellular matrix phosphoprotein, Osteopontin (OPN) has been found in tissue fluids and organs, including the male reproductive tract. The vesicular gland and the ampullary region in the male reproductive system have been documented to secrete OPN, a 55 kDa fertility associated protein. The objective of this study was to quantify OPN and antioxidant concentrations in different epididymal sections in mongrel dogs. Current results showed a significantly (p<0.01) higher concentration of OPN in cauda epididymal fluid than in the caput. Glutathione peroxidase (GPx) and Catalalse (CAT) concentrations were also higher in the cauda epididymal segment than in the caput. To the author’s best knowledge, this is the first report on quantification of OPN levels in the epididymal fluid harvested from the different sections of epididymis. Further studies should focus on correlation and association of this protein from the different epididymal segments with seminal attributes.

The possible protective role of zinc oxide nanoparticles (ZnONPs) on testicular and epididymal structure and sperm parameters in nicotine-treated adult rats (a histological and biochemical study).

Exposure to nicotine in smoking contributes to most unexplained male infertility but the mechanisms remain to be fully elucidated. Zinc (Zn) is an essential trace element in normal growth, development and reproduction. Zinc oxide nanoparticles (ZnONPs) are well-known antioxidants. Therefore, this work was designed to investigate the potential ability of ZnONPs to protect testis and epididymis in nicotine-treated rats. Forty adult male Wistar albino rats were divided into control group and two experimental groups (treated and supplemented rats). In the treated group, rats received nicotine at a dose of 1mg/kg/day orally for 30days. Rats in the supplemented group received ZnONPs (10mg/kg/day) with nicotine (1mg/kg/day), orally for the same period. Testicular and epididymal sections were stained with H&E to assess the histological changes. Negrosin-eosin staining of epididymal sperms was performed to assess their viability and morphological changes. Serum testosterone, FSH and LH levels were assessed. Also, oxidative stress parameters and semiquantitative real-time PCR for steroidogenic enzymes were measured. Morphometric studies of both organs were statistically analyzed. Mild to severe testicular and epididymal structural changes together with sperm morphological abnormalities were detected in nicotine-treated rats. Biochemical results also showed a decrease in all measured parameters except for an increased malondialdehyde (MDA) level that meant deterioration of their reproductive function. On the other hand, ZnONP supplementation in the last group showed an obvious improvement in all investigated parameters.

Normal Fertility Requires the Expression of Carbonic Anhydrases II and IV in Sperm

HCO3− is a key factor in the regulation of sperm motility. High concentrations of HCO3− in the female genital tract induce an increase in sperm beat frequency, which speeds progress of the sperm through the female reproductive tract. Carbonic anhydrases (CA), which catalyze the reversible hydration of CO2 to HCO3−, represent potential candidates in the regulation of the HCO3− homeostasis in sperm and the composition of the male and female genital tract fluids. We show that two CA isoforms, CAII and CAIV, are distributed along the epididymal epithelium and appear with the onset of puberty. Expression analyses reveal an up-regulation of CAII and CAIV in the different epididymal sections of the knockout lines. In sperm, we find that CAII is located in the principal piece, whereas CAIV is present in the plasma membrane of the entire sperm tail. CAII and CAIV single knockout animals display an imbalanced HCO3− homeostasis, resulting in substantially reduced sperm motility, swimming speed, and HCO3−-enhanced beat frequency. The CA activity remaining in the sperm of CAII- and CAIV-null mutants is 35% and 68% of that found in WT mice. Sperm of the double knockout mutant mice show responses to stimulus by HCO3− or CO2 that were delayed in onset and reduced in magnitude. In comparison with sperm from CAII and CAIV double knockout animals, pharmacological loss of CAIV in sperm from CAII knockout animals, show an even lower response to HCO3−. These results suggest that CAII and CAIV are required for optimal fertilization.

Profiling of relaxin and its receptor proteins in boar reproductive tissues and spermatozoa

BackgroundRelaxin levels in seminal plasma have been associated with positive effects on sperm motility and quality, and thus having potential roles in male fertility. However, the origin of seminal relaxin, within the male reproductive tract, and the moment of its release in the vicinity of spermatozoa remain unclear. Here, we assessed the longitudinal distribution of relaxin and its receptors RXFP1 and RXFP2 in the reproductive tract, sex accessory glands, and spermatozoa of adult boars.MethodsSpermatozoa were harvested from three fertile boars and reproductive tract (testes and epididymis) and sex accessory gland (prostate and seminal vesicles) tissues were collected post-mortem from each boar. Epididymis ducts were sectioned into caput, corpus, and cauda regions, and spermatozoa were mechanically collected. All samples were subjected to immunofluorescence and/or western immunoblotting for relaxin, RXFP1, and RXFP2 detection. Immunolabeled-spermatozoa were submitted to flow cytometry analyses and data were statistically analyzed with ANOVA.ResultsBoth receptors were detected in all tissues, with a predominance of mature and immature isoforms of RXFP1 and RXFP2, respectively. Relaxin signals were found in the testes, with Leydig cells displaying the highest intensity compared to other testicular cells. The testicular immunofluorescence intensity of relaxin was greater than that of other tissues. Epithelial basal cells exhibited the highest relaxin immunofluorescence intensity within the epididymis and the vas deferens. The luminal immunoreactivity to relaxin was detected in the seminiferous tubule, epididymis, and vas deferens ducts. Epididymal and ejaculated spermatozoa were immunopositive to relaxin, RXFP1, and RXFP2, and epididymal corpus-derived spermatozoa had the highest immunoreactivities across epididymal sections. Both vas deferens-collected and ejaculated spermatozoa displayed comparable, but lowest immunofluorescence signals among groups. The entire sperm length was immunopositive to both relaxin and receptors, with relaxin signal being robust in the acrosome area and RXFP2, homogeneously distributed than RXFP1 on the head of ejaculated spermatozoa.ConclusionsImmunolocalization indicates that relaxin-receptor complexes may have important roles in boar reproduction and that spermatozoa are already exposed to relaxin upon their production. The findings suggest autocrine and/or paracrine actions of relaxin on spermatozoa, either before or after ejaculation, which have possible roles on the fertilizing potential of spermatozoa.

Effects of CAII/CAIV double knockout on fertility

Bicarbonate (HCO3‐) is an essential factor to modulate sperm motility. Female genital tract fluids are rich in HCO3‐ and contribute to an increase of sperm beat frequency thus supporting sperm to reach the site of fertilization. Carbonic anhydrases (CA) catalyze the reversible hydration of CO2 to HCO3‐ and are important candidates in the regulation of both the HCO3‐ homeostasis in sperm and the composition of the male and female genital tract fluids. We show that CAII and CAIV are localized in epithelial cells lining the epididymal duct and that they appear with the onset of puberty. Gene expression analyses with tissue from respective knockout animals reveal an up‐regulation of CAII and CAIV in the different epididymal sections, indicating a compensatory mechanism for the gene defect. In sperm, the presence of CAII is restricted to the principal piece, whereas CAIV is detectable in the plasma membrane of the entire sperm tail. CAII and CAIV single knockout animals display an impaired HCO3‐ homeostasis resulting in significantly diminished sperm motility, velocity and response to HCO3‐. Enzyme activity tests show that CAII and CAIV account for nearly 95% of the total CA activity in sperm. We generated CAII/CAIV double knockout animals which display a significant reduction in sperm motility resulting in sub‐fertility. Interestingly, biochemically created CAIV deficient sperm of CAII knockout animals show a lower response to HCO3‐ as compared to sperm from double knockout animals. From these results we conclude that both CAII and CAIV play a key role for a successful fertilization.

Histometric analysis of vampire bat (Desmodus rotundus) testicles treated with coumestrol by oral route

The vampire bat (Desmodus rotundus) is a hematophagous species, and is considered the main transmitter of rabies to cattle in Latin America. The purpose of the present study was to evaluate the effect of coumestrol (COU; 5 µg per g body weight, administered orally for 30 d) on the histoarchitecture of vampire bat testicles. A total of 10 male vampire bats were divided randomly into two groups: COU-treated and control (N = 5 per group). Testis samples were obtained for histometric evaluation. The numbers of epididymal sections containing spermatozoa for each bat were counted by light microscopy and fluorescent signal detection was performed by confocal microscopy. We found significant group differences in germinal epithelium thickness, seminiferous tube diameter, intertubular space, number of spermatogenia, Sertoli cell counts, spermatid cell counts, Leydig cell counts, and the number of epididymal sections with spermatozoa. Based on our results, we conclude that COU, when administered orally for 30 d, can modify the histoarchitecture of vampire bat testicular tissue in a manner that would be expected to impair fertility.

Mixed testicular atrophy related to atherosclerosis: first lessons from the ApoE−/−/ LDL receptor−/− double knockout mouse model

Age-related testicular changes are associated with declining spermatogenesis and testosterone levels. A relationship to atherosclerosis has never been investigated systematically. The ApoE(-/-)/LDL receptor(-/-) double knockout mouse model, providing a remarkable homology to human atherosclerosis, is an ideal tool to investigate spermatogenetic alterations in this context. Testes (n = 10) from ApoE(-/-)/LDL receptor(-/-) double knockout mice at the age of 80 weeks were perfused in vivo with contrast agent, harvested and scanned with micro-CT at (4.9 μm³) voxel size. Testes (n = 8) of C57/BL mice at the same age served as controls. Testis volume (mm³) and total vascular volume fraction (mm³) were quantified using micro-CT. Serum testosterone levels were determined. Testicular histology and epididymal sections were analysed for tubular structure, spermatogenetic scores and sperm count. The expression of protamine 2 as a marker for elongated spermatids, inflammation markers (CD4, F4/80) and hypoxia inducible factor 1 alpha (HIF1 alpha) were investigated using immunohistochemistry. ApoE(-/-)/LDL receptor(-/-) double knockout mice exhibit diminished testis and vascular volume fraction with respect to that of controls (p < 0.001). These findings were associated with a reduction of testosterone levels (p < 0.001). Mixed atrophy was present in 41% of the seminiferous tubuli in ApoE(-/-)/LDL receptor(-/-) double knockout mice at the age of 80 weeks. Sperm counts from the epididymis demonstrated a significant decrease in ApoE(-/-)/LDL receptor(-/-) double knockout mice (p < 0.001). In addition, sperm specific protamine 2 expression was decreased in testicular tissue and epididymis of ApoE(-/-)/LDL receptor(-/-) double knockout mice compared with that of control mice. Peritubular inflammatory infiltration and the expression of the hypoxia related marker was observed. Mixed testicular atrophy in ApoE(-/-)/LDL receptor(-/-) double knockout mice is linked to reduced testis volume, vascular volume fraction and low testosterone serum levels, suggesting a direct relation between atherosclerosis and disturbed spermatogenesis.

Dynamics of zonula occludens-2 expression during preimplantation embryonic development in the hamster

The objective was to study the expression of zonula occludens-2, a tight junction protein, during preimplantation hamster embryonic development, to predict its possible localization, source, and roles in trophectoderm differentiation and blastocyst formation in this species. Comparison of zonula occludens-2 expression pattern between the hamster and mouse preimplantation embryos from the zygote up to the blastocyst stage was also an objective of this study. Zonula occludens-2 localization was noted in nuclei of blastomeres in all stages of hamster and mouse embryonic development. Compared to mice, where zonula occludens-2 was first localized in the interblastomere membrane at the morula stage, hamster embryos had membranous zonula occludens-2 localization from the 2-cell stage onwards. Based on combined results of immunolocalization study in parthenogenic embryos and ovarian and epididymal sections, and quantitative PCR done in oocytes and all developmental stages of preimplantation embryos, perhaps there was a carry-over of zonula occludens-2 proteins or mRNA from the dam to the embryo. Based on these findings, we inferred that maternally derived zonula occludens-2 was involved in nuclear functions, as well as differentiation of blastomeres and blastocoel formation during preimplantation embryonic development in the hamster.

Identification of Spermatozoa in Archived Testicular Cancer Specimens: Implications for Bench Side Sperm Retrieval at Orchiectomy

To evaluate the patterns of spermatogenesis in the normal testicular parenchyma of primary testicular cancers and estimate the feasibility of sperm retrieval at the time of radical orchiectomy. We reviewed the archived histologic sections of 39 consecutive patients who had undergone radical orchiectomy for primary testicular cancer at 3 university-affiliated hospitals. We examined all areas of normal (noncancerous) testicular parenchyma to evaluate the level of spermatogenesis and presence of mature spermatozoa in these sections. A minimum of 100 seminiferous tubules were scored per case. We also evaluated the epididymal tubules for the presence of mature spermatozoa. A review of the clinical chart was performed to evaluate the relationship between clinical data and histologic findings. In nearly 40% of the cases evaluated (15/38), the predominant histologic pattern was full spermatogenesis. Mature spermatozoa were identified in nearly 80% (30/38) of the testicular histologic sections and in 50% (14/28) of the evaluable epididymal sections. Clinical stage (presence of extranodal disease) and tumor marker levels were related to the probability of identifying mature spermatozoa in the testis. The data suggest that sperm retrieval at the time of radical orchiectomy is a feasible fertility option, with a 40% probability of recovering spermatozoa by random biopsy of the noncancerous testicular parenchyma and an 80% probability of recovering spermatozoa with a more extensive dissection. In 50% men, spermatozoa may be recovered by epididymal aspiration alone.

Lactate dehydrogenase activity of rat epididymis and spermatozoa: effect of constant light.

During its passage through the epididymis, the gamete undergoes a process of "maturation" leading to the acquisition of its fertilizing ability. The epididymis displays regional variations in the morphology and metabolic properties of its epithelium which are relevant for the progressive development of mature sperm characteristics. The epididymis has spontaneous peristaltic contractions and receives sympathetic innervation that is modulated by melatonin, a hormone synthesized and released by the pineal gland. Constant lighting disrupts melatonin synthesis and secretion. We have studied the effect of constant light on lactate dehydrogenase (LDH; EC 1.1.1.27) and its isozyme C4 activities and protein content in whole epididymis, epididymal tissue and in spermatozoa from caput and cauda segments. Animals were exposed from birth to an illumination schedule of 14 h light:10 h dark (group L:D). At 60 days of age one group of animals was submitted to constant light over 50 days (group L:L). In order to test the fertilizing ability, the rats of each group were mated with soliciting estrous females. The percentage of pregnancies in females mated with males maintained in L:L was remarkably lower than those in females mated with males maintained in the L:D photoperiod (44% and 88% respectively). Constant light increased protein concentration and LDH activity in caput as well as in cauda of total epididymis. On the contrary, in epididymal tissue, the protein content decreased in both epididymal sections compared with controls. When enzymatic activity was expressed in Units per spermatozoa, constant light induced a significant reduction of total LDH and LDHC4 in caput and cauda spermatozoa while LDH activity of epididymal tissue was not affected. In spite of the decrease in LDH per sperm cell when rats were exposed to constant light, in total epididymis (epididymis tissue plus sperm cells content) and in spermatozoa, values of enzyme activities expressed per weight unit were higher than those of controls. This is explained by the increase in the amount of stored spermatozoa, both in caput and cauda, produced by exposure of animals to constant light. Our results confirm that in rats, chronic exposure to constant light promotes a reduction of fertilizing ability and indicates that continuous lighting reduces the total LDH and LDHC4 activities, possibly due to moderate aging of spermatozoa within the duct by lengthening of the sperm transit through the epididymis.

β-Defensin 22 is a major component of the mouse sperm glycocalyx

Surface components of sperm isolated from the cauda epididymides were stabilized by whole sperm fixation for immunization of rabbits. The resulting immunoglobulins (Igs) recognized a single protein of 130 kDa (non-reduced) or 54-57 kDa (reduced) on western blots of cauda sperm. Igs recognized the same 54-57 kDa protein band on whole tissue blots of the corpus and cauda epididymidis and vas deferens. No immunoreactive bands were detected on blots of the prostate, seminal vesicles, testes, caput epididymis, or any of various non-reproductive tissues. Removal of sperm from the vas deferens prior to blotting eliminated the detection of the sperm antigen. Antibodies raised to synthetic peptides, identical in amino acid sequence to two unique spans of DEFB22, recognized the same 130/54-57 kDa antigen on western blots of both caudal sperm and the purified antigen isolated with the anti-sperm Ig. From indirect immunofluorescence, both the anti-sperm and anti-peptide Igs appeared to localize to the entire sperm surface, a pattern confirmed at the ultrastructural level. Real-time PCR identified the corpus epididymides as the major site of expression of DEFB22, with negligible expression in the testes, caput epididymides, and vas deferens. Immunostaining of epididymal sections showed DEFB22 being released into the lumen at the distal caput/proximal corpus, with sperm becoming intensely coated with DEFB22 as they reached the distal corpus. Most uterine sperm recovered from mice 4 h following copulation exhibited DEFB22 coating the entire sperm surface. By contrast, some sperm recovered from the oviduct and cumulus extracellular matrix showed loss of DEFB22 from the sperm head.

Early puberty in local Naga boar of India: Assessment through epididymal spermiogram and in vivo pregnancy

Male Naga pig of India, a miniature breed is known for its meat quality and early puberty. No scientific efforts were made to verify the farmers’ view that this breed reaches puberty at around 2 months of age. A preliminary study was, therefore, conducted with the objectives: (a) to find out the age at puberty based on mature spermiogram and in vivo pregnancy and (b) to record the sperm morphology in different parts of the epididymis. Animals were selected from two different age groups: group I aged 53 days and 2.4 kg and group II of 85 days and 3.0 kg. Semen samples collected from different sections of epididymis were analyzed for sperm motility, live spermatozoa, and morphological abnormalities. Motility increased ( P < 0.01) and live spermatozoa and total morphological abnormalities decreased ( P < 0.001) from caput through cauda epididymis in both the groups. Sperm motility, live spermatozoa and morphologically normal spermatozoa in each section of the epididymis were higher ( P < 0.01) in group II than I. Boars with >60% progressive motility, >70% live spermatozoa, <15% total morphological abnormalities and <10% abnormal acrosomes in cauda epididymal spermatozoa were considered mature spermiogram. As per this definition, pigs of group II had only mature spermiogram. In vivo pregnancy confirmation indicated that Naga boar could impregnate female as early as 90 days of age. In conclusion, Naga boar attained puberty by not later than 3 months with 3.0 kg, which is the lowest body weight at puberty in this species reported so far, as reflected by mature epididymal spermiogram and in vivo pregnancy confirmation.

Spermatozoa modulate epididymal cell proliferation and protein secretion in vitro

Normal epididymal function, such as protein expression and secretion, is primarily regulated by testicular androgens and temperature. However, the role of spermatozoa in this critical process has never been studied. In order to determine whether sperm itself could regulate epididymal function, we have developed a cell culture system of bovine epididymal cells to study the interactions between spermatozoa and the epididymal epithelium. Primary cells from caput, corpus, and cauda epididymal tissues were cultured in the presence of androgens at 32 degrees C (scrotal) and 37 degrees C (abdominal). Newly synthesized proteins were metabolically labeled with (35)S-methionine after sperm co-incubation and the pattern of secreted proteins was analyzed by two-dimensional polyacrylamide gel electrophoresis. Proliferation rate, protein secretion rate and electrophoretic patterns of secreted proteins were evaluated 48 hr post-co-incubation. Incubation at 32 degrees C indicated that spermatozoa stimulation increases the level of protein secretion of cultured cells from all epididymal sections while it slightly decreases proliferation of corpus cells. At 37 degrees C, spermatozoa co-incubation significantly decreases the protein secretion rate of cultured cells from all epididymal sections. Independently of cell incubation temperature, spermatozoa stimulation induces both an increase in the intensity of radiolabeled proteins and the appearance of new secreted proteins of caput cells without affecting the protein pattern of corpus or cauda cells. Incubation at 37 degrees C, however, greatly modifies the pattern of proteins expressed at 32 degrees C by cauda cells. Taken together, these results support the hypothesis that spermatozoa themselves affect epididymal cell function, most importantly for caput epididymides.

Site-specific changes in zinc levels in the epididymis of rats exposed to ionizing radiation

The epididymis is an accessory sex organ that plays an important role in sperm maturation and storage. Trace elements, such as copper (Cu), manganese (Mn), zinc (Zn) and selenium (Se), have a pivotal role in spermatogenesis. We studied the effects of radiation on trace element levels in the epididymis in male Wistar rats using inductively coupled plasma–mass spectrometry (ICP–MS). We determined trace element concentration in segment-dissected specimens and used micro-PIXE analysis to determine Zn in epididymal sections in situ. Zn concentrations in the caput and cauda epididymis of control rats were 37.7 ± 6.5 μg/g wet weight and 18.7 ± 4.1 μg/g wet weight, respectively. At 6 h after irradiation at a single dose of 5Gy, the Zn level decreased by 33% in the caput epididymis, whereas the level did not change in the cauda segment. Similar results were obtained for Se, but not both Cu and Mn. PIXE spot analysis revealed that Zn in the lumen of the epididymal tubules decreased after irradiation.

Identification of the bull sperm p80 protein as a PH‐20 ortholog and its modification during the epididymal transit

We have identified an 80 kDa protein in ejaculated bull spermatozoa (p80) which is found in acrosomal and post-acrosomal areas of the head. It has a hyaluronidase activity and shares homologies with PH-20, a sperm surface glycoprotein involved in sperm-egg interaction. The aim of the present study was to characterize bull sperm p80 protein at the nucleic and amino acid levels to determine whether it is the bovine PH-20 ortholog. The complete nucleotide sequence determined by RT-PCR, 3' and 5' RACE show that bull p80, displays identity with the PH-20 nucleotide and amino acid sequences. Messenger RNA and protein expressions determined by Northern blot and immunohistochemistry revealed that the protein is testicular (expressed in spermatocytes and spermatids). The localization of p80 on spermatozoa, determined by indirect immunofluorescence using a monoclonal antibody, shows the protein in acrosomal and post acrosomal areas of the head with an increase in the signal intensity as sperm progress through the epididymis. Post-translational modifications of the protein were investigated during the epididymal maturation by Western blot on protein extracts from sperm collected in the caput, corpus and cauda portions of bull epididymis. Glycolysation status of sperm p80 protein on proteins from ejaculated and epididymidal sperm was investigated. Result show that the glycosylation status is modified as spermatozoa migrate through the epididymis. Hyaluronidase activity evaluated in protein extracts from spermatozoa of the three different epididymal sections revealed that the activity is higher at pH 7 than 4 and is not affected by epididymal maturation. These data strongly suggest that p80 is the bovine PH-20.