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The major challenge of follicular fluid proteomic analysis is the presence of high-abundance proteins that originate from plasma. These proteins can prevent the detection of lower abundant ones, produced locally by follicle cells and that may have important roles in follicular activity. In this study, the novel technology called hexapeptide ligand library was evaluated to enrich the low-abundance proteins in follicular fluid of human (HFF), porcine (PFF) and equine (EFF) prior 2D-PAGE. Our results showed that the new strategy enabled detection of many new protein spots, increased resolution and highly improved the intensity of low-abundance proteins by 2D-PAGE., (© 2010 Blackwell Verlag GmbH.)

The platypus epididymal proteome is being studied because epididymal proteins are essential for male fertility in mammals and it is considered that knowledge of the epididymal proteome in an early mammal would be informative in assessing the convergence and divergence of proteins that are important in the function of the mammalian epididymis. Few of the epididymal proteins that have been identified in eutherian mammals were found in platypus caudal epididymal fluid, and the major epididymal proteins in the platypus (PXN-FBPL, SPARC and E-OR20) have never been identified in the epididymis of any other mammal.

The final stages of sperm differentiation occur outside the gonad, in the epididymal tubule. These last maturation steps, essential to the quality of spermatozoa, are not under the genomic control of the germ cells. A series of sequential interactions with the epididymal fluid, mostly specific proteins present in the lumen of different regions, are believed to induce the final steps of sperm maturation. In order to provide the luminal changes required for this maturation to occur, the epithelium may resort to two basic mechanisms: absorption and secretion. Far from being a uniform channel, the epididymal duct is a canal with highly specialized regional differentiation of its epithelial ultrastructure and its secretory and absorptive functions. This review focuses on the ultrastructural characteristic of the epithelial cells, their specific secretory activity according to the epididymal regions and their eventual role in sperm maturation of the boar. The chronology of the changes that occur in and on the sperm and in the surrounding environment are described. Relationships between the highly regionalized epididymal activities, sperm characteristics linked to their survival and fertility potential are also presented in this review.

Proteins present in and secreted into the lumen of various regions of the stallion epididymis were characterized qualitatively and quantitatively by two-dimensional electrophoresis. Using this proteomic approach, 201 proteins were found in the lumen and 117 were found that were secreted by the epithelium in various parts of the organ. Eighteen proteins made up 92.6% of the total epididymal secretory activity, lactoferrin (41.2%) and clusterin (24.8%) being the most abundant. Procathepsin D, HE1/CTP (cholesterol transfer protein), GPX (glutathione peroxidase), beta-N-acetyl-hexosaminidase, and PGDS (prostaglandin D2 synthase) were the other major compounds secreted. The most abundant proteins found in the luminal fluid were albumin and the secreted proteins: lactoferrin, PGDS, GPX, HE1/CTP, and hexosaminidase. Three main secretory epididymal regions were identified from the protein pattern, i.e., regions E0-E2, E3-E5, and E6-E9. Region E0-E2 was characterized by the secretion of clusterin (53%), PGDS (44%), and GPX (6%). Region E3-E5 had the highest number of secreted proteins, the highest protein concentrations (60-80 mg/ml), and the highest spermatocrit value (85%). Lactoferrin (60% in E4), clusterin (29% in E3), hexosaminidase (10% in E3), and procathepsin D (6.9% in E4) were the most abundant proteins in this region. Region E6-E9, in which few region-specific secreted compounds were found, was characterized by a high quantity of lactoferrin in the luminal fluid (2-14 mg/ml). Comparison between the secretion of the major proteins and their concentrations in the lumen throughout the organ showed that the behavior of each protein is specific, in particular for the three isoforms of clusterin.

The number of proteins secreted by the boar epididymis increased progressively from 1 mo of age to the adult period. The first specific secretory activity was revealed at 2 mo in the distal caput (hexosaminidase, clusterin, and lactoferrin) and in the corpus (train O/HE1). Train A and glutathione peroxidase specific to the proximal caput, and trains E and M specific to the corpus, appeared at 4 mo. At 5 mo, secretion of procathepsin L occurred in the middle caput and that of mannosidase and E-RABP in the distal caput. Approximately 48% of all the proteins secreted in the adult boar epididymis were dependent on the presence of androgens, either stimulated (33.6%) or repressed (14.4%); 47% were modulated by other factors, and 5% were unregulated. In the proximal caput, 50% of the specific secreted proteins were controlled essentially by factors emanating from the testis. In more distal regions, two proteins secreted in the corpus were regulated by factors from the anterior regions. The regionalization of the secretory activity of the epididymal epithelium resulted in a specific regulation for each protein, which was modulated according to the region of expression and influenced by either testicular or epididymal factors that remain to be identified.

The synthesis and secretion of proteins by the boar genital tract were studied in vitro by incubating epididymal tissues with [35S]methionine and cysteine. Characterization of the major neosynthesized proteins was performed electrophoretically by one- and two-dimensional PAGE analysis, and an epididymal protein cartography was established. Some of the proteins secreted were found to be unregionalized. Polarization studies of the secretions in the epididymal tubule were carried out by in vitro incubation of isolated tubules, and most of these unregionalized proteins were found not to be secreted in the epididymal lumen. Inside the epididymal lumen, protein secretion was highly regionalized, and electrophoresis analysis detected few proteins secreted at all points along the organ. A total of 146 epididymal proteins, covering 220 spots, were found to be secreted by the epididymis. The distal caput showed the highest number of spots, the lowest number of proteins secreted being found in the proximal caput and cauda. Most of the epididymal proteins analyzed are highly polymorphic in terms of both isoelectric point and molecular mass. The presence and importance of the different compounds in the various regions of the epididymis were established. Several distinct secretory regions of the epididymis can be determined by the presence of major characteristic proteins. The concentrations of a given protein in the fluids of various regions were not related to the respective secretion intensity of that protein. Identification of some major epididymal proteins was accomplished by N-terminal amino microsequencing and by the use of specific antisera. Of the various major proteins, clusterin, glutathione peroxidase, retinol-binding protein, lactoferrin, EP4, beta-N-acetyl-hexosaminidase, alpha-mannosidase, and procathepsin L were identified and localized along the organ. Several polypeptides found in this study remain unidentified.

The involvement of proteins extracted from spermatozoa of fertile semen in sperm-zona binding was examined under hemizona assay conditions. One droplet of a suspension of spermatozoa was exposed to sperm proteins and then tested for zona binding, while a parallel semen suspension droplet incubated with culture medium served as a control. The reliability of the test was increased by relating the number of spermatozoa bound to each inseminated hemizona to the surface area of the hemizona and expressed as the binding index. For spermatozoa incubated with extracted proteins, the binding index was greater than (P = 0.001) that of controls (125.2 +/- 45.1 versus 63.6 +/- 29.2, respectively). As a first control, two other protein sources (fetal calf serum and human follicular fluid) were tested in the hemizona assay. No significant differences were found in zona binding for other protein-exposed spermatozoa compared with controls. As a second and reverse control, exposure of one hemizona to sperm proteins before insemination with untreated spermatozoa induced a marked decrease (P = 0.0003) in sperm binding, compared with that of the matched hemizona not exposed to sperm proteins (control) (3.4 +/- 1.4 versus 74.5 +/- 6.8, respectively). Taken together, these findings confirm the involvement of extracted sperm proteins in sperm-zona interactions. Therefore, in the cases in which fertilization in vitro fails because of a lack of sperm-zona binding, incubation of deficient spermatozoa with proteins extracted from spermatozoa of fertile ejaculates should restore their ability to interact with the oocyte and, thus, should enhance the prognosis for in vitro fertilization.

We used two-dimensional gel electrophoresis to analyse the 35S-labelled proteins secreted in vitro by the epithelium of the epididymis. Polypeptides with molecular weight ranging from 10 to > 200 kDa and isoelectric points from < 4 to 9 were observed. These compounds showed high degree of polymorphism. Some were secreted in distinct zones while others were present in the caput and the cauda epididymis.

Ram "rete testis" fluid (RTF) routinely collected throughout the year has been used as a source of inhibin. The mean flow rate and mean concentration of spermatozoa in the fluid remained constant during the first 12 days of cannulation. More than 50 castrated or cryptorchid rams have been treated with low doses of steroid-free RTF over a 25-h blood sampling period. Human serum albumin was injected as a control. RTF depressed both FSH and LH plasma levels although the pattern was different for each hormone. There was no change in prolactin secretion. LH secretion was affected first while FSH remained unchanged in castrated and in cryptorchid rams. Thereafter, the maximum depression of FSH plasma levels occurred at a time when LH started to return or had returned to preinjection levels in the cryptorchid and castrated animals respectively. In the cryptorchid rams, RTF suppressed pulsatile LH secretion which was present before treatment but in the castrated animals, RTF lowered LH plasma levels which were constant and showed no pulsatile changes before treatment. Both FSH and LH inhibitory activities have been found in all active fractions obtained by purification of RTF. These activities are papain-sensitive and active fractions have a high apparent molecular weight (greater than or equal to 100 000) as shown by gel filtration and ultrafiltration. These and other results in the literature have lead to a re-definition of inhibin as a protein factor of gonadal origin able to depress plasma levels of FSH and LH, even at low doses.