Rete Testis Fluid Proteins Research Articles

Proteome of the rete testis fluid from tropically-adapted Morada Nova rams

The rete testis has a close relationship with sperm development and may have other functions besides serving as an intercalated channel. The aim of this study was to identify and characterize the proteins of rete testis fluid (RTF) from tropically-adapted Morada Nova rams. Testicles obtained from six Morada Nova rams were dissected and the head of the epididymis was separated to access the efferent ducts. Rete testis fluid was obtained by gentle massage of the testis. The fluid was centrifuged to remove cell debris and sperm. RTF samples (containing 400μg protein) were separated by 2-D SDS-PAGE and gels, analyzed using PDQuest software (Bio Rad, USA). Proteins were identified using tandem mass spectrometry. Gene ontology and protein network were analyzed using the software tool for searching annotations of proteins (STRAP) and STRING database. Gels had, on average, 227±13.5 spots and 51% of the proteins were found above 40kDa, corresponding to 65% of the intensity of all spots detected. Based on gene ontology analysis, the most common biological processes associated with RTF proteins were regulation (24.3%) and cellular process (23.3%). Binding (27.3%) and catalytic activity (19.3%) corresponded to the most frequent molecular functions. Albumin, clusterin, serotransferrin, immunoglobulin gamma-1 chain and alpha-2-HS-glycoprotein were the most abundant proteins in the ram rete testis fluid. In conclusion, proteins identified in the ram rete testis fluid are linked to several physiological processes associated with sperm protection and spermatogenesis.

On the regulation of Crisp-1 mRNA expression and protein secretion by luminal factors presented in vivo by microperfusion of the rat proximal caput epididymidis.

Synthesis and secretion of certain epididymal proteins are regulated by lumicrine factors from the testis or from upstream regions of the excurrent ducts. Cysteine-rich secreted protein-1 (Crisp-1) is a major androgen regulated protein in the epididymal lumen fluid of the rat and other species. Previous research has demonstrated that disturbance of the luminal microenvironment through obstruction of the tract reduces Crisp-1 synthesis and secretion. The present study was undertaken to determine the influence of the luminal microenvironment on rat proximal caput epididymal Crisp-1 secretion into lumen fluid and on Crisp-1 gene expression in the same tubules. Western blot analysis demonstrated that Crisp-1 protein concentrations were reduced from control levels by perfusion with artificial caput fluid containing no testicular factors and were not increased by perfusion with fluids containing rete testis fluid proteins. Crisp-1gene expression was also reduced by perfusion with artificial caput fluid and not increased by perfusion with rete testis fluid proteins. Perfusion with artificial caput fluid containing 5alpha-dihydrotestosterone did increase one Crisp-1 transcript. This study demonstrates that intraluminal testicular proteins are not important co-regulators with androgens of Crisp-1gene expression or resulting Crisp-1 secretion into the rat proximal caput tubule lumen in vivo.

Central Effect of Rete Testis Fluid, Inhibin 32K, and Follicular Fluid on Plasma Gonadotropin Concentrations in Sheep: Inhibin is not the Rete Testis Fluid Protein Able to Suppress Luteinizing Hormone Pulses1

We have previously shown that peripheral administration of rete testis fluid (RTF) proteins was able to suppress LH pulses through the suppression of LHRH pulses. This activity was named "LHRH Statin." The aims of the present work were to analyze LH inhibition after an intracerebroventricular injection of RTF and to determine whether inhibin is the factor responsible for this inhibition. Castrated rams (experiment 1) or ewes (experiment 2) received an intracerebroventricular injection of RTF, purified bovine inhibin 32K, bovine follicular fluid, or human serum albumin as control. Animals were bled every 15 min for 5 h before injection and for 7 h after injection. LH mean levels were significantly lowered (p < 0.01) only in the RFT-treated groups. FSH levels were not affected irrespective of group, source, or dose of inhibin. These experiments show first, that protein(s) present in ovine RTF can suppress LH secretion in sheep; second, that bovine follicular fluid or purified bovine inhibin 32K have no effect on LH secretion. Furthermore, the results suggest that centrally administered inhibin has no effect on FSH secretion under our experimental conditions. Together, these experiments clearly demonstrate that inhibin 32K does not exert any "LHRH statin" activity.

Proteins in luminal fluid of the ram excurrent ducts: changes in composition and evidence for differential endocytosis.

Rete testis fluid (RTF) and luminal fluid collected by micropuncture at selected epididymal sites were analyzed to characterize the spectrum of proteins and to quantify the net gain or loss of total/bulk protein and androgen-binding protein (ABP) between successive regions within the ductus epididymidis. Based on one-dimensional SDS gel electrophoresis, the spectra of proteins in RTF and fluids from the proximal, central, and distal caput through proximal corpus epididymidis differed from each other. Concentrations of sperm, bulk protein, and ABP increased from the rete testis through the central caput epididymidis. Electron microscopic studies following intraluminal microinjections of RTF proteins conjugated to colloidal gold at specific sites in the excurrent ducts revealed that 145 times more protein-gold was endocytosed in the ductuli efferentes than in any of the four regions of the caput epididymidis. Thus, ductuli efferentes were the major extra-testicular site of endocytosis of bulk protein present in RTF; at least a portion of the uptake was specific. On a per sperm basis, the amount of protein present in the central caput epididymidis was less than 15% of that leaving the testis. Although most of the protein present in RTF (greater than or equal to 86 mg/d) must be absorbed in the ductuli efferentes and the initial segment of the epididymis and replaced by newly secreted proteins (greater than or equal to 34 mg/d), there was negligible loss of ABP in these regions. Net loss of ABP occurred primarily in the distal caput and proximal corpus epididymidis. These studies demonstrate that ABP is spared from endocytosis along with the bulk protein in RTF and conserved for functions in epididymal regions far distal to the site of bulk protein loss.

The recovery of the testis over 8 weeks after short-term dosing with ethylene glycol monomethyl ether: Histology, cell-specific enzymes, and rete testis fluid protein

Ethylene glycol monomethyl ether (EGME) has been found to affect meiotic spermatocytes, spermatids, other stages of spermatocytes, and spermatogonia, depending on the dose used. These studies, which examine testicular recovery from EGME treatment, analyzed tissues from rats treated for 5 days with 0, 50, 100, or 200 mg EGME/kg/day and sacrificed at eight subsequent weekly intervals; some epididymal sperm parameters of these animals have been described. Histologically, the testes of the low-dose group showed very mild changes, while the 100- and 200-mg/kg groups showed widespread damage and cell death which recovered somewhat during the course of the study. There was no treatment-related effect on seminal vesicle or prostate weights. Rete testis fluid protein levels were changed only in the high-dose group, when protein levels rose to a maximum of sixfold the control values at Week 4; by Week 6, there was no difference between groups. Changes in cell-specific enzyme activities were dose dependent and generally mirrored changes in the number of germ cells in the testis.

The Recovery of the Testis over 8 Weeks after Short-Term Dosing with Ethylene Glycol Monomethyl Ether: Histology, Cell-Specific Enzymes, and Rete Testis Fluid Protein

The Recovery of the Testis over 8 Weeks after Short-Term Dosing with Ethylene Glycol Monomethyl Ether: Histology, Cell-Specific Enzymes, and Rete Testis Fluid Protein

Variability in the apparent molecular weight of inhibin.

An in vitro bioassay based on suppression of GnRH-stimulated FSH secretion by pituitary cells in culture was used to monitor inhibin activity after dialysis, gel filtration or polyacrylamide gel electrophoresis of protein preparations from a variety of gonadal secretions and extracts under native and dissociating conditions. The suggestion that inhibin is a peptide of molecular weight less than 5000 was not confirmed. Although some fractions of low molecular weight suppressed FSH secretion, the amount of activity was low and the dose response curves were not parallel with a standard preparation of inhibin. Under most conditions, inhibin eluted with an apparent molecular weight of about 90 000. However, gel filtration of rete testis fluid protein in 1 M acetic acid resulted in elution of inhibin activity with a lower apparent molecular weight and with polyacrylamide gel electrophoresis in 0.1% (w/v) sodium dodecylsulfate, the apparent molecular weight was 30 000. It is concluded that inhibin is a protein which tends to aggregate and coelute with larger molecules.

Factors affecting the entry of testosterone into the lumen of the cauda epididymidis of the anaesthetized rat.

When [3H]testosterone was infused into the general circulation of the rat, perfusion of a length of the cauda epididymidis (17 +/- 1.0 (s.e.m.) cm, n = 36) with perfusates of varied composition revealed a low entry of radioactivity (1--10% plasma levels; 10 exps) with protein-free perfusates, and a greater entry (15--48%; 10 exps) when the perfusate contained bovine serum albumin (38 mg/ml). When the perfusate contained ovine or rat testicular fluid, or rat epididymal fluid at protein concentrations of 3 mg/ml or less, the entry of radioactivity into the epididymis was greater than when the perfusate contained 3 mg BSA/ml. The addition of ovine rete testis fluid protein (3 mg/ml to BSA (38 mg/ml) in the perfusate increased the uptake of radioactivity (58--106%; 6 exps). Radioactivity in blood was principally associated with testosterone (90, 95% total blood activity, 2 rats), whereas both [3H]testosterone (37, 41% total perfusate activity) and [3H]dihydrotestosterone (42, 63% total perfusate activity) was present in BSA-containing perfusates. The proportion of dihydrotestosterone appeared to increase when the perfusate contained protein of testicular origin.

Studies on Purification of Inhibin from Ovine Testicular Secretions Using an In Vitro Bioassay

An in vitro bioassay based upon inhibition of follicle stimulating hormone (FSH) secretion by rat pituitary cells in culture was used to investigate preliminary steps in the purification of inhibin from ovine rete testis fluid (RTF) and testicular lymph (OTL). Since androgens also suppress gonadotrophin levels in this assay, steroids were removed from test substances. Charcoal adsorption and ethanol precipitation were found to be unsatisfactory because of large losses of inhibin activity. Using RTF protein as the starting material a two‐fold purification was achieved by precipitation with 50% ammonium sulphate. Chromatography of the ammonium sulphate precipitate on DEAE Sephadex resulted in further purification and separation of inhibin from testosterone.

Inhibin: Evidence for Its Existence, Methods of Bioassay and Nature of the Active Material

Results are presented on the changes in serum FSH, LH and testosterone levels and in testis weight after efferent duct ligation, X‐irradiation or heating the testes, or injections of antiserum to testosterone. These results can only be explained by the existence of a feed‐back substance “inhibin”, which is produced by the seminiferous tubules and which regulates the secretion of FSH by the pituitary. Several bioassay methods have been developed for the detection of inhibin‐like activity and have been used to follow the purification of this activity, mainly from ram rete testis fluid proteins. The active material exhibits an apparent heterogeneity, with apparent molecular weights of 90 000, 20 000 and &lt; 5000.