The results of study 1 are presented first, followed by those obtained in study 2. Study 1 examined the effect of neonatal treatment with DES, ethinyl estradiol (EE), or GnRHa on the number of cells in the epididymis that were rich with H+-ATP ase at 25 days of age. The number of cells positive for H+-ATPase was then quantified and calculated as the number of positive cells per millimeter of epididymal tubular basal membrane.
Representative images illustrating the number of H+-ATPase-rich cells in the cauda epididymis of control and treated animals are shown in Figure 1. Panels A and B illustrate H+-ATPase immunostaining within control epididymides. Both images demonstrate that H+-ATPase is principally expressed in the apical pole of a subpopulation of epithelial cells. In control animals, a large number of H+-ATPase-positive cells were detected at Postnatal Day 25. However, after neonatal administration of either GnRHa, DES (Fig. 1, C and D, respectively), or EE (data not shown), the number of cells expressing H+-ATPase was much smaller.
Alternatively, a commercially available p-actin monoclonal antiserum (Sigma) was added to the membrane at a dilution of 1:8000 and incubated overnight. The membranes were washed at least four times for 15 min in TBST. The second antibody was then added to the membrane and incubated for 1 h (for H+-ATPase, we used a rabbit anti-chicken immunoglobulin G conjugated to horseradish peroxidase [Sigma]; for p-ac-tin, we used a goat anti-mouse immunoglobulin G conjugated to peroxidase) before being thoroughly washed in TBST.
Whole epididymides from each treatment group were powdered in a porcelain mortar with a pestle under liquid nitrogen. The powder was scraped into an Eppendorf tube and stored on dry ice. Protein was extracted by the addition of 200 pl of cold extraction buffer (10 mM Hepes pH 7.9, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM PMSF and 1X Complete protease inhibitor cocktail [Roche, Lewes, U.K.]). The tissue remained on ice for 15 min before adding 25 pl of 10% Nonident P-40 (Sigma). The tube was then vortexed three times for 10 sec. The tube was then centrifuged at 12 000 X g for 1 min at 4°C, the supernatant was decanted, and 100-pg aliquots were frozen on dry ice before being stored at —40°C.
Control incubations were performed using antibodies that had been preabsorbed with an excess of the immunizing peptide (11 amino acids that correspond to the C-terminus of the 56 kDa, B1 subunit of the H+-ATPase) prior to the first incubation step.
Quantification of H+-ATPase Immunoexpression
Study 1. Black-and-white photographs were taken of the whole epididymis of each animal. The number of H+-ATPase positive cells on each photograph was counted and the basement membrane of the associated tubule was drawn around using a Wacom graphics tablet (Vancouver, WA). The mean number of immunopositive cells per millimeter of epididymal tubular basal membrane was calculated for each animal.
Plasma testosterone levels were measured using an ELISA adapted from an earlier radioimmunoassay method as detailed elsewhere. The limit of detection was ~12 pg/ml.
The PLP-fixed epididymides of control and treated 25-day-old rats were cryoprotected by immersion in 30% sucrose in PBS for at least 4 h. The entire epididymis (head, body, and tail) was then mounted in OTC compound Tissue-Tek (Miles Inc., Elkhart, IN) and frozen at —29°C in a Reichert Frigocut cryostat (Reichert Jung, Derry, NH). Sections were cut at 4 pm, picked up onto Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA), and kept at 4°C.
5. Subcutaneous injection of 10 mg/kg of a long-acting gonadotropin-releasing hormone antagonist (GnRHa; Antarelix, Europeptides, Ar-genteuil, France) in 5% mannitol administered on Postnatal Days 2
5. This regimen has been shown to abolish gonadotropin secretion until Postnatal Days 15-20 and to lower testosterone levels;
Animals, Treatments, Sample Collection, and Processing
Wistar rats were bred and maintained in the Medical Research Council animal facility (Edinburgh). All animal studies were performed under license from and according to the legal requirements of the United Kingdom Home Office. Rats were administered one of the following treatments postnatally. In our studies the day of birth was assigned as Day 1. Based on the nomenclature below, the rats treated in a preliminary study (study 1) received either treatment 1, 2, 3, or 5. In a follow-up study (study 2) a cohort of rats was treated with one of the six regimens listed below.
It is well known that testosterone is required in order to stabilize the Wolffian duct prior to its differentiation into the epididymis, vas deferens, and seminal vesicles. Testosterone exerts its effects via activation of the androgen receptor (AR), as does the 5a-reduced androgen, dihydrotestosterone (DHT). It is less clear whether the metabolites of testosterone, namely, estradiol or the major metabolite of DHT, 5a-androstane-3a, 17p-diol, have any role to play in epididymal differentiation or maturation.
These proton-secreting cells are often referred to as ‘‘mitochondria-rich cells’’.
Previous studies have localized H+-ATPase to specific epithelial cell types in all regions of the epididymis; these are apical (or narrow) cells within the initial segment region, and clear (or light) cells within the caput, corpus, and cauda regions of the epididymis. The timing of postnatal differentiation of narrow and clear cells has been assessed primarily by electron microscopy, and they are fully differentiated around the time of sexual maturation (~42 days) in the rat.
The major function of the epididymis is to maintain a lumenal fluid environment, which is optimal for the maturation and, in many animals, the storage of spermatozoa. The ability of epididymal epithelial cells to alter the ionic composition of the lumenal fluid has been measured, some of the major transport proteins involved in this process have been identified, and their sites of expression have been characterized. Transepithelial transport of water and various anions and solutes take place in the epididymis.