The reproductive system of male mice homozygous for a mutation in the estrogen receptor (ER) gene (ER knock-out; ERKO) appears normal at the anatomical level. However, these males are infertile, indicating an essential role for ER-mediated processes in the regulation of male reproduction. Adult ERKO male mice have significantly fewer epididymal sperm than heterozygous or wild-type males. Although spermatogenesis is occurring in some seminiferous tubules of 3- to 5-month-old ERKO males, other tubules either have a dilated lumen and a disorganized seminiferous epithelium with few spermatogenic cells or lack a lumen and contain mainly Sertoli cells. There are no obvious differences in seminiferous tubules at 10 days of age between wild-type and ERKO mice, but the lumen in ERKO males is dilated in all seminiferous tubules by 20 days. However, spermatogenesis progresses and similar numbers of sperm are present in the cauda epididymis of ERKO and wild-type males until 10 weeks of age. Disruption of spermatogenesis and degeneration of the seminiferous tubules become apparent after 10 weeks in the caudal pole of the testis and progresses in a wave to the cranial pole by 6 months. However, the seminal vesicles, coagulating glands, prostate, and epididymis do not appear to be altered morphologically in ERKO mice. Serum testosterone levels are somewhat elevated, but LH and FSH levels are not significantly different from those in wild-type males. Sperm from 8- to 16-week-old mice have reduced motility and are ineffective at fertilizing eggs in vitro. In addition, ERKO males housed overnight with hormone-primed wild-type females produce significantly fewer copulatory plugs than do heterozygous or wild-type males. These results suggest that estrogen action is required for fertility in male mice and that the mutation of the ER in ERKO males leads to reduced mating frequency, low sperm numbers, and defective sperm function.

Mass spectrometry can be used to map the distribution of targeted compounds in tissue, providing important molecular information in many areas of biological research. Matrix assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS) is well suited for the analysis of tissue samples with a spatial resolution of about 30 microm for compounds in a mass range from 1000 to over 50 000 Da. Direct analysis of tissue sections requires spotting or coating of the tissue with a matrix compound typically sinapinic acid or other cinnamic acid analogs. A raster of this sample by the laser beam and subsequent mass analysis of the desorbed ions can record molecular intensities throughout the section. The overall process is illustrated by profiling and imaging of mouse epididymis sections where protein activity changes markedly throughout the section.

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.

Regional differences in the proximal part of mouse epididymis were reported to provide a morphological baseline for studies on functional zonation of this part that is critical in sperm maturation. Macroscopical, histological, ultrastructural, and histochemical observations permitted us to subdivide this part into five segments, characterized by epithelial height, nuclear position, cytological and histochemical features of principal cells. Segment I corresponded to the initial segment previously described in rodents. Segment II differed from segment I by endoplasmic reticulum (ER) and dictyosomes aspect in principal cells, apical alkaline phosphatase and Ca2+-dependent ATPase activities. Segment III was characterized by spermatozoa package, high content of cells in multivesicular bodies, mitochondria shape, complex interdigitating membranes, and strong periodic acid-Schiff (PAS)-positive cell border. Segments IV and V presented the same cytological features but differed by their esterase activity. In the principal cells of each segment, dense spherical concretions were scattered in ER caveolae. Cells with apical nuclei were classified into two groups. The cells of the first group presented the same morphological and histochemical features as the adjacent principal cells and were scattered in the five segments ("apical cells"). The cells of the second group differed from the others by their goblet shape, a dense cytoplasm, and a high mitochondria succinate-D activity. They presented different cytological and histochemical features depending on their localization in segments I ("narrow cells"), II ("prominent cells"), or III, IV, V ("mitochondria goblet-cells"). The possible relationships between epithelium structure and epididymal functions were herein discussed.

Sertoli cells express functional receptors for FSH, one of the two pituitary hormones that regulate spermatogenesis in mammals. We recently produced genetic mutant (FORKO) mice that lack FSH receptor, in order to examine the effects on testicular function and fertility. Mutant males exhibited weight loss of testis, epididymis, and seminal vesicle as well as low levels of testosterone. Except for reduced seminiferous tubular diameter, no gross changes were apparent upon histological examination. Analysis of testicular germ cells by flow cytometry revealed a significant increase in the percentage of 2C cells (spermatogonia and non-germ cells) and a significant decrease in the percentage of HC cells (elongated spermatids) of FORKO males. The absolute number of homogenization-resistant elongated spermatids was also significantly reduced in the mutant males. A 2-fold increase in c-kit-positive 2C cells was recorded in the mutant males. Elongated spermatids of FORKO males showed a dramatic increase in propidium iodide binding suggesting reduced nuclear compaction. The increase in size of the sperm head in mutants, as well as susceptibility to dithiothreitol-induced decondensation, suggests the inadequate condensation of sperm chromatin. Sperm chromatin structure assay, a technique that reflects DNA stability, revealed that sperm from FORKO males are susceptible to acid denaturation, indicating the poor quality of sperm. These data allow us to conclude that genetic disruption of FSH receptor signaling in the rodent induces major changes that might contribute to reduced fertility.

Human and rat pineal melatonin secretion decline with aging, whereas visceral fat and plasma insulin levels increase. Melatonin modulates fat metabolism in some mammalian species, so these aging-associated melatonin, fat and insulin changes could be functionally related. Accordingly, we investigated the effects of daily melatonin supplementation to male Sprague-Dawley rats, starting at middle age (10 months) and continuing into old age (22 months). Melatonin was added to the drinking water (92% of which was consumed at night) at a dosage (4 microg/ml) previously reported to attenuate the aging-associated decrease in survival rate in male rats, as well as at a 10-fold lower dosage. The higher dosage produced nocturnal plasma melatonin levels in middle-aged rats which were 15-fold higher than in young (4 months) rats; nocturnal plasma melatonin levels in middle-aged rats receiving the lower dosage were not significantly different from young or middle-aged controls. Relative (% of body wt) retroperitoneal and epididymal fat, as well as plasma insulin and leptin levels, were all significantly increased at middle age when compared to young rats. All were restored within 10 weeks to youthful (4 month) levels in response to both dosages of melatonin. Continued treatment until old age maintained suppression of visceral (retroperitoneal + epididymal) fat levels. Plasma corticosterone and total thyroxine (T4) levels were not significantly altered by aging or melatonin treatment. Plasma testosterone, insulin-like growth factor I (IGF-I) and total triiodothyronine (T3) decreased by middle age; these aging-associated decreases were not significantly altered by melatonin treatment. Thus, visceral fat, insulin and leptin responses to melatonin administration may be independent of marked changes in gonadal, thyroid, adrenal or somatotropin regulation. Since increased visceral fat is associated with increased insulin resistance, diabetes, and cardiovascular disease, these results suggest that appropriate melatonin supplementation may potentially provide prophylaxis or therapy for some prominent pathologies associated with aging.