Maturing the sperm: unique mechanisms for modifying integral proteins in the sperm plasma membrane.

The epididymis has been understudied, in part due to its cancer resistance and the development of effective technologies for sperm injection and in vitro fertilization. However, it is worthy of study because--absent advanced reproductive technology--its proper function is essential for conceiving children: sperm leaving the testis are immature and nonfertile. Epididymal functions can be divided into several general categories (1) concentration of sperm; (2) functional maturation; (3) storage in a quiescent state until ejaculation; (4) removal of degenerating sperm; (5) provision of appropriate conditions for survival; (6) transport by the myoid cells; (7) protection; (8) maintenance of the blood epididymal barrier. In the past decade investigators have focused on those maturational changes of the integral proteins of the sperm plasma membrane which are directly related to sperm-ova interactions. It has traditionally been thought that changes in the sperm plasma membrane proteins were limited to simple binding or removal of proteins or interactions with the proteases, glycosylases and glycotransferases present. However, the epididymis can also release secretory products in bulk through apical blebs and inject integral membrane proteins with epididymosomes which fuse with the plasma membrane. The epididymis also activates and cleaves enzymes present on the sperm surface (e.g., germ cell angiotensin converting enzyme), thus enabling them to modify proteins on the sperm membrane. Aside from the need to understand epididymal function relative to the sperm, basic science on epididymal physiology is warranted because it may help us understand the functioning of androgens, protection of tissues from oxidative damage, and resistance to cancer and benign hyperplasic growth.

Gene expression in the LNCaP human prostate cancer progression model: progression associated expression in vitro corresponds to expression changes associated with prostate cancer progression in vivo.

Identification of the genes involved in prostate cancer (PCa) progression to a virulent and androgen-independent (AI) form is a major focus in the field. cDNA microarray was used to compare the gene expression profile of the indolent, androgen sensitive (AS) LNCaP PCa cell line to the aggressively metastatic, AI C4-2. Thirty-eight unique sequences from a 6388 cDNA array were found differentially expressed (> or =2-fold, 95% CI). The expression of 14 genes was lower in C4-2 than in LNCaP cells, while the reverse was true for 24 genes. Twelve genes were validated using Q-PCR, Western blotting and immunohistochemistry (IHC) of LNCaP and C4-2 xenograft. Q-PCR showed that 10 of 12 (83.3%) genes had similar patterns of expression to the array (LNCaP>C4-2: TMEFF2, ATP1B1, IL-8, BTG1, BChE, NKX3.1; LNCaP

The trigger-maintenance model of persistent mild to moderate hyperoxaluria induces oxalate accumulation in non-renal tissues.

Persistent mild to moderate hyperoxaluria (PMMH) is a common side effect of bariatric surgery. However, PMMH's role in the progression to calcium oxalate (CaOx) urolithiasis and its potential effects on non-renal tissues are unknown. To address these points, a trigger + maintenance (T + Mt) model of PMMH was developed in rats (Experiment 1). The trigger was an i.p. injection of PBS (TPBS) or 288 µmol sodium oxalate (T288). Maintenance (Mt) was given via minipumps dispensing PBS or 7.5-30 µmol potassium oxalate/day for 28 days. Urinary oxalate ranged from 7.7 ± 0.8 µmol/day for TPBS + MtPBS to 18.2 ± 1.5 µmol/day for T288 + Mt30 (p ≤ 0.0005). All rats receiving T288 developed CaOx nephrocalcinosis, and many developed 'stones'. This was also true for Mt doses that did not elevate urinary oxalate above that of TPBS + MtPBS (p > 0.1) and for rats that did not have a detectable surge in urinary oxalate post T288. When TPBS was administered, CaOx nephrocalcinosis did not develop regardless of the Mt dose even if urinary oxalate was elevated compared to TPBS + MtPBS (p ≤ 0.0005). One of the risks associated with PMMH is oxalate accumulation within tissues. Hence, in a second set of experiments (Experiment 2) different doses of oxalate (Mt0.05, Mt15, Mt30) labeled with (14)C-oxalate ((14)C-Ox) were administered by minipump for 13 days. Tissues were harvested and (14)C-Ox accumulation assessed by scintillation counting. (14)C-Ox accumulated in a dose dependent manner (p ≤ 0.004) in bone, kidney, muscle, liver, heart, kidney, lungs, spleen, and testis. All these tissues exhibited (14)C-Ox concentrations higher (p ≤ 0.05) than the plasma. Extrapolation of our results to patients suggests that PMMH patients should take extra care to avoid dietary-induced spikes in oxalate excretion to help prevent CaOx nephrocalcinosis or stone development. Monitoring for oxalate accumulation within tissues susceptible to damage by oxalate or CaOx crystals may also be required.

Partitioning of 14C-oxalate excretion in rats during a persistent oxalate challenge.

This study was done to resolve published discrepancies in oxalate excretion between humans and rats and to characterize oxalate partitioning in rats during persistent severe hyperoxaluria, such as that seen in many bariatric patients. Osmotic minipumps dispensing 360 micromole/day KOx + 3.9 +/- 0.14 microCi/day (14)C-oxalate were implanted subcutaneously. All excreta were collected. Rats were killed on day 13 and carcasses were dissected, ground, dissolved in HCl, and subjected to scintillation counting, and 92.1 +/- 3.9% of total oxalate administered was recovered. This was partitioned among the skin complex (38.2 +/- 7.7%), carcass complex (24.5 +/- 5.9%), and excreta (29.5 +/- 1.9%). The distribution of oxalate in the skin and carcass complexes led us to infer that only 29.5 +/- 1.9% of the administered oxalate entered the circulation. Of the circulated oxalate, 98.4 +/- 0.4% was excreted (total urine 78.9 +/- 1.7%; raw feces 21.0 +/- 1.7%). Thus, most oxalate that does enter the circulation is promptly excreted in rats, as in humans. Consequently, even after a large, persistent oxalate challenge, very little oxalate had accumulated in the internal organs, muscle, and skeleton. Unlike humans, however, rats excrete a significant fraction of oxalate in the feces.

Intraprostatic lymphocyte profiles in aged wistar rats with estradiol induced prostate inflammation.

PURPOSE: We report LS phenotypes in aged Wistar rats (Charles Rivers, Wilmington, Massachusetts) with EPI. MATERIALS AND METHODS: EPI was induced in 36 to 40-week-old male rats by castration with subcutaneous injections of 17beta-estradiol (0.25 mg/kg daily) plus dihydrotestosterone propionate (2.5 mg/kg daily) in sesame oil for 30 days. Controls were sham castrated, aged rats that were injected with sesame oil. Prostate, spleen and blood LSs in aged and young (10 to 12-week-old) rats were identified by flow cytometry in a cluster of differentiation system. RESULTS: All prostates in 6, 17beta-estradiol plus dihydrotestosterone propionate treated rats and in 3 of 7 controls (43%) showed inflammation foci. All studied LSs in Aged-SPIs and Aged-EPIs were similar. Blood LSs in Aged-SPIs, Aged-EPIs, Aged-NPIs and Youngs showed no differences. Levels of lymphocytes bearing the natural killer marker were decreased, and levels of total T and CD4(+) T cells were increased in prostates with age. Intraprostatic and splenic levels of CD4(+) natural killer T cells were down-regulated significantly in Aged-SPIs and Aged-EPIs compared to those in Aged-NPIs and Youngs. Levels of CD45RC(+)CD4(+)alphabetaTCR(+) T cells were decreased 2-fold in the spleen and up-regulated 2-fold in the prostate of Aged-SPIs and Aged-EPIs compared to those in Aged-NPIs and Youngs. CONCLUSIONS: Similar LS features in Aged-SPIs and Aged-EPIs may indicate that the EPI model is appropriate for studies of the immune aspects of prostate inflammation. Imbalance between suppressive CD4(+) natural killer T cells and autoreactive CD45RC(+)CD4(+)alphabetaTCR(+) T cells in Aged-SPIs and Aged-EPIs may suggest their role in prostate inflammation in this model.

Continuous infusion of oxalate by minipumps induces calcium oxalate nephrocalcinosis.

It is hypothesized that oxalate plays an active role in calcium oxalate (CaOx) nephrocalcinosis and oxalate driven nephrolithiasis by interacting with the kidney. We developed an adjustable, nonprecursor, continuous infusion model of hyperoxaluria and CaOx nephrocalcinosis to investigate this hypothesis. Minipumps containing PBS or KOx (60-360 micromol/day; n = 5-7/dose) were implanted subcutaneously in male Sprague-Dawley rats on D0 and D6. Rats were killed on D13. Oxalate excretion and CaOx crystalluria were monitored by 20+4 h urine collections. Localization and content of intrarenal crystals were determined on frozen sections using polarization and microFTIR. Oxalate excretion was significantly elevated in all KOx rats (P < or = 0.005). CaOx crystalluria was most persistent in the 240-360 micromol/day KOx rats, but even 60 micromol/day KOx rats showed sporadic crystalluria. One hundred percent of KOx rats had CaOx nephrocalcinosis as confirmed by microFTIR. Most crystals were localized to the lumens of the corticomedullary collecting ducts. A few crystals are localized just under the papillar urothelium. The minipump model is the first model of hyperoxaluria to provide continuous infusion of oxalate. It permits control of the levels of hyperoxaluria, crystalluria and CaOx nephrocalcinosis. The level of sustained hyperoxaluria and CaOx nephrocalcinosis induced by treatment with 360 micromol/day KOx for 13D models the conditions frequently observed in jejunoileal bypass patients. Adjustments in the length of treatment and level of hyperoxaluria may allow this model to also be used to study the oxalate driven CaOx-nephrolithiasis common in patients with hyperoxaluria due to other causes.

The healthy rat prostate contains high levels of natural killer-like cells and unique subsets of CD4+ helper-inducer T cells: implications for prostatitis.

PURPOSE: Chronic nonbacterial prostatitis spontaneously develops in aged Lewis and Wistar rats but not in Sprague-Dawley rats. We report the unique profile of lymphocyte subsets present in the healthy Sprague-Dawley rat prostate. MATERIALS AND METHODS: We compared enzymatic and mechanical methods of intraprostatic lymphocyte isolation in healthy 8 to 10-week-old Sprague-Dawley rats. Mechanical isolation was chosen because of its superior preservation of surface antigens. Intraprostatic lymphocyte subsets were analyzed by flow cytometry. RESULTS: Levels of prostatic alphabetaTCR+ T cells were similar and levels of prostatic B cells were decreased 5 to 10-fold compared with those found in other tissues (p

A simple procedure for isolating microgram quantities of biologically active bikunin from human urine.

OBJECTIVE: To report a simple, relatively rapid protocol to isolate biologically active bikunin from human urine using ion-exchange-trypsin affinity chromatography. Bikunin is a protease inhibitor which has been shown to play a role in various processes, including inhibition of calcium oxalate crystallization, the regulation of proliferation and modulation of carcinogenesis. The unavailability of the purified protein has hampered studies on bikunin's expanding role in these processes. MATERIALS AND METHODS: Female human urine was dialysed (15 kDa threshold) and crudely fractionated with a double-saturated ammonium sulphate precipitation. The first precipitation was with 35% saturated ammonium sulphate, and the supernatant was harvested, and the second with 90% saturated ammonium sulphate, and the precipitate collected. The protein mixture was then passed over Sepharose SP-fast-flow cation exchange and Sepharose Q-fast-flow anion exchange columns connected in series. The final purification was with a trypsin-affinity column which selectively bound bikunin. RESULTS: This procedure could recover 1 microg of bikunin per 2 mL of urine, and the final product was essentially free of contaminating inter-alpha-trypsin inhibitor heavy chains or bikunin-heavy chain conjugates. Product purity was confirmed by two-dimensional polyacrylamide gel electrophoresis combined with silver staining or Western blot. All isolations contained the 17 kDa minimally glycoslyated/sulphated form of bikunin and the 28 kDa form of bikunin. Some preparations also contained 33-48 kDa forms of bikunin. The protein cores of all three proteins were confirmed to be bikunin by mass spectrometry and Western blot. Harvested bikunin retained its trypsin inhibitory activity (L-benzoylarginine-p-nitroanilide assay). Preparations containing the 33-45 kDa form had two to three times more trypsin inhibitory activity than preparations without this band. CONCLUSIONS: This novel ion exchange-trypsin affinity chromatography protocol uses only two chromatographic steps. The product consists of three isomers of biologically active bikunin, free of contaminating heavy chains or bikunin-heavy chain conjugates. The ready availability of purified bikunin should facilitate future studies of bikunin's emerging role in urolithiasis, proliferation and carcinogenesis.

Decreased renal expression of the putative calcium oxalate inhibitor Tamm-Horsfall protein in the ethylene glycol rat model of calcium oxalate urolithiasis.

PURPOSE: Tamm-Horsfall protein is believed to inhibit calcium oxalate crystallization, aggregation or adhesion to the renal epithelium. We determined whether ethylene glycol induced urolithiasis changes the expression of renal and urinary Tamm-Horsfall protein. For comparison the expression of another calcium oxalate inhibitor, osteopontin, was also analyzed. MATERIALS AND METHODS: Male rats were treated with 0.75% ethylene glycol plus an AIN-76 diet (Dyets, Bethlehem Pennsylvania) (ethylene glycol group) or standard rat chow and water (control group) for up to 8 weeks (6 per group for 8 weeks and 3 per group for 3 days to 6 weeks). Kidneys and urine (8 weeks only) were harvested and analyzed by Northern and Western blot analysis, and immunohistochemistry. RESULTS: Tamm-Horsfall protein message and protein (membrane bound form) were decreased, while those of osteopontin were increased in the kidneys of rats treated with ethylene glycol for 8 weeks. As judged by immunochemistry Tamm-Horsfall protein and osteopontin were consistently present in a few tubules in rats in the ethylene glycol and control groups, respectively. In urine expression of the free form of Tamm-Horsfall protein (approximately 75 kDa.) was decreased but detectable in ethylene glycol treated rats. Although readily detected in tissue, osteopontin was not detected in the urine of control or ethylene glycol treated rats. In the time course experiment Tamm-Horsfall protein did not decrease until 4 weeks, when calcium oxalate crystals were detectable in the kidneys of treated rats. In contrast, osteopontin was increased, although inconsistently, beginning at 3 days. CONCLUSIONS: Unlike other calcium oxalate inhibitors, such as osteopontin, renal message and protein for Tamm-Horsfall protein was decreased in ethylene glycol treated rats. Tamm-Horsfall protein expression did not decrease until aggregates of crystals had been deposited in the kidneys, while osteopontin expression began to increase almost immediately. Comparisons of the data on kidneys and urine obtained by RNA or protein blot analysis and immunochemistry underscore the need to examine tissue and urine by multiple techniques to obtain the most accurate assessment of how protein expression is changed by a given treatment.

Minipump induced hyperoxaluria and crystal deposition in rats: a model for calcium oxalate urolithiasis.

PURPOSE: Unraveling the mechanisms leading to clinically active calcium oxalate (CaOx) stone disease and the development of effective medical therapies to treat it have been hampered by the lack of appropriate animal models. To address this problem we developed a model of hyperoxaluria and calcium oxalate crystal deposition by implanting osmotic minipumps subcutaneously into male rats, that is minipump induced hyperoxaluria and crystal deposition in rats. MATERIALS AND METHODS: Male Harlan-Sprague Dawley rats (225 to 290 gm) were implanted subcutaneously with 1-week 2 ml osmotic minipumps containing 1.5 M potassium oxalate (360 microM KOx/24 hours, [KOx-trt], 11) or phosphate buffered saline (PBS-trt, 9) on days 1 and 7. The 24-hour urine collections were performed on days 0, 4, 7, 11 and 14. Data were analyzed by ANOVA and Tukey's HSD. Urinary crystals were analyzed by light microscopy. Kidneys were harvested on day 14 and processed for light and polarizing microscopy, and RNA analysis.RESULTS Mean overall creatinine excretion +/- SEM (PBS-trt 107 +/- 7 and KOx-trt 123 +/- 6 microM/24 hours, p >0.07) and day 14 serum creatinine (PBS-trt 83 +/- 4 and KOx-trt 83 +/- 5 microM, p >or=0.9) were similar in the 2 treatment groups. Overall urinary volume (PBS-trt 11.3 +/- 0.8 and KOx-trt 18.0 +/- 1.5 ml/24 hours, p