Evaluating testis function non-invasively: how epidemiologist-andrologist teams might better approach this task.

Opinions herein focus on epidemiology-based publications using semen to study testis function, but several have broader applicability. 'Opinion 1': authors often fail to write out an explicit question(s) or hypothesis, and to stipulate how measured outcomes will be used to refute or support the hypothesis. Might critical thinking be lax? 'Opinion 2': authors often fail to consider the biology underlying a question or hypothesis, and/or which analytical methods really provide meaningful information or should be rejected. 'Opinion 3': spermatogenesis cannot be evaluated in a meaningful manner via conventional semen attributes. Quantitative evaluation of spermatogenesis requires a 'rate attribute', not provided by number of sperm per milliliter of semen or total number per ejaculate (TSperm). Influence of abstinence interval is under-appreciated. The rate attribute, TSperm per hour of abstinence (TSperm/h), meaningfully estimates sperm production if the abstinence interval is 42-60 h. Most attributes of individual sperm do not reflect quality at spermiation. 'Opinion 4': reliance on a single semen sample per subject might hamper detection of the association sought, because an imprecise value might not establish if a subject's testes were dysfunctional or not. 'Opinion 5': curve-fitting, to adjust quantitative data, for a sample provided after an abstinence interval falling within a broad range, to a standardized abstinence interval, distorts outcomes for many samples provided after approximately 60 h abstinence. TSperm values for individuals with good daily sperm production are artifactually low and those for individuals with poor daily sperm production are artifactually high. Hence, it is important to explain the importance of abstinence interval to participants and censor samples outside an acceptable 37-64 h abstinence range.

Sequelae in male rabbits following developmental exposure to p,p'-DDT or a mixture of p,p'-DDT and vinclozolin: cryptorchidism, germ cell atypia, and sexual dysfunction.

Rabbit does (7-9 per group) were treated daily per orum from gestation day 15 through post-natal week 4 to provide per kg body wt 25 micaromol (low) or 250 micromol (high) p,p'-DDT or a mixture of DDT and vinclozolin (12.5 and 125 micromol each). Developmental as well as post-pubertal reproductive sequelae of male progeny were studied. Testicular descent in some pups was impaired by DDT. Serum LH or testosterone was not affected. FSH was lower in mixture- but not in DDT-exposed rabbits. Lack of sexual interest, penile erection and ejaculation were observed in some mixture rabbits. Sperm counts were unaffected, but morphologically normal spermatozoa were fewer; nuclear and acrosomal morphogenesis was disrupted. Atypical germ cells resembling carcinoma in situ were found. Also considering data for vinclozolin [Veeramachaneni DNR, Palmer JS, Amann RP, Kane CM, Higuchi TT, Pau K-YF. Disruption of sexual function, FSH secretion, and spermiogenesis in rabbits following developmental exposure to vinclozolin, a fungicide. Reproduction 2006;131:805-16], we concluded that DDT causes cryptorchidism and germ cell atypia, vinclozolin permanently disrupts FSH secretion and sexual function, and the mixture causes the full spectrum of dysgenesis.

Steroids in fluids and sperm entering and leaving the bovine epididymis, epididymal tissue, and accessory sex gland secretions.

Ten steroids which may have a role in the process of sperm maturation within the epididymis were quantified by competitive protein binding or radioimmunoassay. Rete testis fluid (RTF) carrying testicular sperm into the epididymis was rich in dehydroepiandrosterone and testosterone (21 +/- 2 and 33 +/- 3 ng/ml) while cauda eipididymal plasma (CEP) around sperm which have completed maturation had high levels of progesterone, dihydrotestosterone, 3beta-androstanediol, dehydroepiandrosterone and testosterone (7.4 +/- 0.8, 20.3 +/- 1.1, 6.5 +/- 0.4, 8.0 +/- 0.7 and 11.5 +/- 0.7 ng/ml). About 4 mug of steroids enter the epidymis daily in RTF, but less than 1% was found in CEP; the balance presumably was absorbed by the epithelium in the proximal caput epididymidis. Nevertheless, tissue levels of total 17beta-OH androgens were lower in the proximal caput than in the distal caput or corpus epididymidis. In all zones of the epididymis, dihydrotestosterone accounted fro about 70% of the total 17beta-OH androgens found in the nuclear fraction. In the cytoplasmic fraction, however, dihydrotestosterone predominated only in the distal caput and corpus epididymidis. In the cauda epididymidis, CEP and sperm probably accounted for less than 35% of the total 17beta-OH androgens and less than 25% of the dihydrotesterone. The progesterone concentration of the cauda than in the caput epidymidis. Twice washed testicular sperm contained more testosterone than cauda epididymal or ejaculated sperm (16.6 +/- 1.9, 1.6 +/- 0.2 and 1.5 +/- 0.3 ng/10(9) sperm, respectively), but less progesterone (0.5 +/- 0.1, 1.3 +/- 0.2 and 1.0 +/- 0.4 ng/10(9) sperm, respectively). As a consequence of mixture with estrogen-rich prostatic fluid (150 +/- 9 pg/ml), ejaculated sperm contained a relatively high amount of estrogens (112 +/- 15 pg/10(9) sperm). These studies revealed marked differences in steroid profiles of fluids entering and leaving the epididymis and of infertile testicular and fertile cauda epididymal sperm.

Oxytocin in the ovine ductuli efferentes and caput epididymidis: immunolocalization and endocytosis from the luminal fluid.

The presence, possible biosynthesis, and uptake of oxytocin from luminal fluid in the ductuli efferentes and caput epididymidis of the ram were studied. Specific immunostaining for oxytocin, but not neurophysin, was observed in the ductuli efferentes as well as caput epididymidis. This indicates the presence, but not production, of oxytocin in epithelial cells of these ducts. Staining was predominantly present in the epithelium, especially in the middle lobules of the ductuli efferentes and initial segment of the epididymis. Endocytosis of oxytocin was studied by electron microscopy after intraluminal microinjections of oxytocin conjugated to colloidal gold (8-10 nm), a 20-fold excess of oxytocin followed by oxytocin-gold, or plain colloidal gold into the ductuli efferentes and four successive regions of the caput epididymidis. Specific uptake by a receptor-mediated process was evidenced by the presence of more gold particles within epithelial cells after oxytocin-gold injections than after control injections. The quantity of oxytocin-gold endocytosed was 3.7-fold greater in the ductuli efferentes than in the initial segment of the epididymis. Within the caput epididymidis, more oxytocin-gold was endocytosed in the initial segment and proximal caput epididymidis than in two distal regions. We conclude that localization of oxytocin in epithelia of the excurrent ducts is a consequence of endocytosis (predominantly receptor mediated) of luminal oxytocin entering in rete testis fluid; however, uptake of blood-borne oxytocin cannot be excluded. Although oxytocin may have a role in sperm transport via action on smooth muscle in the ductal wall, the regional pattern of endocytosis of oxytocin is suggestive of a role for oxytocin in epithelial function in the ductuli efferentes and proximal portions of the caput epididymidis.

Detection of alterations in testicular and epididymal function in laboratory animals.

The potential impact of an agent altering male reproductive function is greater for humans than for animals. Consequently, it is essential that sensitive criteria be used to look for effects on a multiplicity of target sites when an agent is evaluated using an animal model. No animal model has reproductive characteristics similar to those of humans, but this does not negate the validity of using animal models. Classic methodologies for reproductive toxicology are limited by the approaches used for subjective evaluation of testicular histology and use of natural mating for fertility tests. After dosing for an interval at least equal to six times the duration of one cycle of the seminiferous epithelium, sperm from ejaculated semen or the cauda epididymidis can be evaluated for normalcy of morphology or function and should be used for artificial insemination of females to critically evaluate fertility. Normal males of animal models ejaculate a great excess of sperm. A 50 or 90% reduction in the number of fertile sperm deposited during mating probably will not markedly reduce fertility. Artificial insemination of a critical number of sperm, selected to result in slightly less than maximal fertility for control animals, will maximize the probability of detecting a decrease in fertility if the same critical number of sperm is inseminated for treated animals as for control animals. Testicular function should be evaluated by objective, rather than subjective, criteria. For each male, a piece of testicular tissue should be appropriately fixed and an aliquot of parenchyma should be homogenized to allow enumeration of homogenization-resistant spermatids. Among the more sensitive criteria of testicular function are the minor diameter of essentially round seminiferous tubules, the ratio of leptotene spermatocytes to Sertoli cells, the corrected numbers of germ cells per seminiferous tubule cross section, and the number of homogenization-resistant spermatids per testis.

Effects of microgravity or simulated launch on testicular function in rats.

Testes from flight rats on COSMOS 2044 and simulated-launch, vivarium, or caudal-elevation control rats (5/group) were analyzed by subjective and quantitative methods. On the basis of observations of fixed tissue, it was evident that some rats had testicular abnormalities unassociated with treatment and probably existing when they were assigned randomly to the four treatment groups. Considering rats without preexisting abnormalities, diameter of seminiferous tubules and numbers of germ cells per tubule cross section were lower (P less than 0.05) in flight than in simulated-launch or vivarium rats. However, ratios of germ cells to each other or to Sertoli cells and number of homogenization-resistant spermatids did not differ from values for simulated-launch or vivarium controls. Expression of testis-specific gene products was not greatly altered by flight. Furthermore, there was no evidence for production of stress-inducible transcripts of the hsp70 or hsp90 genes. Concentration of receptors for rat luteinizing hormone in testicular tissue and surface density of smooth endoplasmic reticulum in Leydig cells were similar in flight and simulated-launch rats. However, concentrations of testosterone in testicular tissue or peripheral blood plasma were reduced (P less than 0.05) in flight rats to less than 20% of values for simulated-launch or vivarium controls. Thus spermatogenesis was essentially normal in flight rats, but production of testosterone was severely depressed. Exposure to microgravity for greater than 2 wk might result in additional changes. Sequelae of reduced androgen production associated with microgravity on turnover of muscle and bone should be considered.

Scanning electrons and light microscopy of the equine seminiferous tubule.

Changes within the equine seminiferous tubules during the cycle of the seminiferous epithelium were studied light and scanning electron microscopy (SEM). Once observed with SEM, tubules were sectioned and staged using light microscopy. As viewed by SEM, the weblike, spongy cytoplasm of germ cells or Sertoli cells in stages I and II extended over the entire height of the germinal epithelium. The cytoplasm of the basal portion of the germinal epithelium in stages III to VIII was similar to that in stages I and II. However, the cytoplasm which occupied the luminal third of the epithelium in stages III to VII was smooth appearance. The smooth-surfaced, periluminal cytoplasm diminished in stages VIII. Principal pieces of flagella from spermatids extended into the tubular lumina in all stages whereas the middle pieces extended into the lumen only in stage VIII. Later in stage VIII, the middle pieces, which were thickened with cytoplasm, were connected to the germinal epithelium by stalks. After spermiation, the diameter of the middle pieces was similar to that of ejaculated spermatozoa. Thus, the cytoplasm within the thickened middle pieces contributed to the formation of the cytoplasmic droplets.

Can the fertility potential of a seminal sample be predicted accurately?

This paper highlights the most critical aspects of the problem of predicting fertility. To determine if a laboratory test(s) is highly correlated with fertility it is essential to have: a) specific, precise and accurate laboratory tests, and b) precise and accurate fertility data. Acquisition of precise and accurate data for laboratory tests and fertility of spermatozoa in the same sample is not easy. Data derived from in vitro fertilization are not tests of fertility, because only a subset of the attributes important for fertilization in vivo are tested. Because of deficiencies in fertility data, there probably is no valid report for human spermatozoa correlating results of laboratory tests and fertility, and very few valid studies for laboratory or domesticated animals. There is little doubt that objective measures of sperm motion, acrosomal status, or other characteristics are significantly correlated with fertility. However, establishment of the correlations between a group of attributes and fertility is not the question of interest. The goal is prediction of fertility. There has been no recent effort to develop a prediction of fertility or fecundity based on sperm characteristics, and achievement of this goal may be elusive.

Endocytosis of androgen-binding protein, clusterin, and transferrin in the efferent ducts and epididymis of the ram.

The amount of androgen-binding protein (ABP) in luminal fluid from the central caput epididymidis of the ram, on a per sperm basis, remains the same as that in rete testis fluid (RTF) entering the ductuli efferentes, although greater than 85% of the testicular protein is absorbed in proximal sites. To determine if ABP is spared from endocytosis in proximal sites and if proteins are differentially and selectively absorbed at specific sites in the excurrent ducts, we studied the endocytosis of ABP, clusterin, transferrin, and a 26/35-kd dimer isolated from ovine RTF. Each protein was labeled with colloidal gold and microinjected into the lumen of a ductulus efferens and five specific sites in the ductus epididymidis; uptake was quantified by electron microscopy. Endocytosis of each protein, including ABP, was substantially greater in the ductuli efferentes than in any site in the ductus epididymidis. More ABP was endocytosed in proximal regions of the epididymis than any other protein studied. Endocytosis of the 26/35-kd dimer, like ABP, was greater in proximal sites of the epididymis, whereas endocytosis of clusterin and transferrin was greater in distal sites. Thus, there was a differential absorption, since proteins were endocytosed in one or another specific region of the epididymis, depending on the protein. ABP was endocytosed in the ductuli efferentes and caput epididymidis in amounts similar to or greater than other major testicular proteins, and was not spared from endocytosis in the proximal excurrent ducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationships between computerized measurements of motion of frozen-thawed bull spermatozoa and fertility.

A computerized system (CellSoft, CRYO Resources, Ltd.) was validated using video tapes of frozen-thawed bull spermatozoa diluted in filtered (0.2 micron) egg yolk-citrate extender (8 X 10(6) spermatozoa/ml) and analyzed at 30 frames/sec for the percentage of motile spermatozoa (greater than or equal to 20 microns/sec) and linear velocity of motile spermatozoa. Virtually all motile spermatozoa were detected and debris rarely were classified as immotile spermatozoa if the extender had been filtered. Variation about the mean for percent motile cells was similar when only 12 rather than 20 or 30 frames/field were analyzed. Use of 20 frames/field was adequate to determine the percentage of motile bull spermatozoa. Five mixtures of live and killed spermatozoa were analyzed (four bulls) to evaluate accuracy. Percent motile spermatozoa was correlated (r = 0.97) with the ratio of live:killed spermatozoa. Mean linear velocity of motile spermatozoa was similar for each mixture (P greater than 0.05). To further evaluate accuracy, percent motile spermatozoa was determined by computer and by "track motility" (20 samples; 0 to 63% motile spermatozoa); values were correlated (r = 0.95). The system was precise (CV of 6% based on triplicate analyses of the same samples) and reasonably accurate for evaluating bull sperm motility if the extender had been filtered and 20 to 25 fields (greater than or equal to 200 spermatozoa) were evaluated. Correlations between measurements of sperm motion and fertility were studied using cryopreserved semen from two fertility trials. For the first, 75-day nonreturn rate data for 20 samples of bull semen (10 bulls) were not significantly correlated with evaluations made by CellSoft. For the second fertility trial, the competitive fertility index (a measure of relative fertility) for nine bulls was correlated (r greater than or equal to 0.68; P less than 0.05) with percent motile spermatozoa, linear velocity and straight-line velocity. Multiple correlations based on six characteristics evaluated by CellSoft, at 0 or 1.5 hours, and the competitive fertility index were greater than or equal to 0.94. Based on the latter data, the system may facilitate prediction of the relative fertility of bull spermatozoa.

Sperm-bound amidase activity as a marker for acrosomal integrity in bull spermatozoa.

An assay for sperm-bound amidase activity was validated using bovine spermatozoa and N-benzoyl-DL-arginine p-nitroanilide as substrate. The assay had intra- and interassay coefficients of variations of 5 and 12%, respectively. It is an inexpensive, simple and rapid assay since 100 samples can be evaluated in 2 hours and it requires only 4 X 10(6) spermatozoa per sample. Sperm-bound amidase activity was proportional (r = 0.95) to the percentage of spermatozoa with an intact acrosome, as determined by differential interference-contrast microscopy. A change of five percentage units in the incidence of damaged spermatozoa was detectable. Using this procedure, assessment of sperm-bound amidase activity is therefore a sensitive and efficient means of evaluating acrosomal integrity.

In vitro sperm-binding assay to distinguish differences in populations of human sperm or damage to sperm resulting from cryopreservation.

Annually, >1.3 million men are members of couples seeking help because of infertility. Semen from many of these men contains reasonable numbers of motile and normal sperm, but for a subset of individuals, many sperm are deficient in ability to bind to the zona pellucida during in vitro fertilization. Diagnosis of this defect has been hampered by lack of a low-cost test. Molecular similarity exists between the perivitelline membrane of a hen's egg and the mammalian zona pellucida. These facts and some preliminary data led to evaluation of binding of human sperm during incubation for 60 minutes at 37 degrees C to an extract of chicken perivitelline membrane coated in microwell assay plates. The sperm-binding assay had inter- and intraassay plate variations of 21 and 12%, respectively, using washed fresh sperm. All seminal samples were normal, except a few that had 36 to 50% motile sperm with a low rate of sperm movement (if there is a low rate of movement, World Health Organization [WHO] criterion for normalcy is >50% motile). Nevertheless, this sperm-binding assay detected differences among individuals in percentage of sperm bound. Based on data for two to four ejaculates from each of eight occasional sperm donors, the coefficient of variation for ejaculates within donor averaged 31%, and means for the donors differed (P < 0.02). Percentage of sperm bound ranged from <1 to 38% for fresh semen from 57 men and from <1 to 13% for frozen-thawed semen from 34 men. Percentage of motile sperm accounted for <30% of the variation in percentage of sperm bound. In a direct comparison based on 17 ejaculates, aliquots evaluated fresh averaged 13% sperm bound, versus 2% for frozen-thawed aliquots. We concluded that the egg membrane substrate used in these microwell assay plates might serve as the basis for a diagnostic assay. However, it remains to be established whether samples of human semen with a low percentage of sperm binding indeed have relatively low fertilizing potential.

Increased in vitro binding of fresh and frozen-thawed human sperm exposed to a synthetic peptide.

Prosaposin is a well-characterized, approximately 68-kDa protein found in many tissues and as a normal component of human semen. A fragment of prosaposin apparently is involved in primary sperm-egg binding. We hypothesized that binding of sperm from some men to egg investments would be increased by in vitro exposure of their sperm to a synthetic fragment of human prosaposin (FertPlus peptide). Hence, we evaluated samples of washed fresh or frozen-thawed human sperm after a 10-minute exposure to synthetic FertPlus peptide at 0 (control), 80, 160, 320, 640, or 1280 pM, followed by 1:50 dilution for evaluation of binding. The criterion of response was mean percentage of sperm bound to a substrate prepared from chicken egg membranes after sperm were incubated for 60 minutes at 37 degrees C in substrate-coated wells of a sperm-binding assay plate. For each seminal sample, data were normalized against the percentage of sperm bound for control aliquots, providing values for relative binding. With fresh sperm, relative binding was increased (P < 0.01) by exposure of sperm to peptide, and the effect was especially obvious at 1280 pM. Higher doses were not tested. Collectively at three study sites, exposure of fresh sperm to 1280 pM peptide substantially increased (above 99% confidence interval; on the basis of duplicate control samples) percentage of sperm bound for 25 of 74 (34%) samples. For frozen-thawed sperm, exposure to 1280 pM peptide increased binding for 29 of 65 (45%) samples. We concluded that for >30% of men, exposure of their sperm to this synthetic fragment of prosaposin at 1280 pM increased binding of sperm to an egg membrane substrate similar to that offered by the zona pellucida.

Steroidogenesis and testosterone metabolism in cultured principal cells from the ram epididymis.

To determine if ram principal cells can synthesize or metabolize testosterone, or metabolize other steroids present in rete testis fluid, principal cells from the initial segment, central caput, and proximal corpus epididymidis were isolated and cultured in a floating collagen matrix with medium containing 20% dialyzed rete testis fluid. In the first experiment, each matrix was washed twice in testosterone-free medium on day 2.8, transferred into culture medium containing 100 nM of a tritiated steroid and incubated for 4 hours at 34 C. The tritiated steroids were pregnenolone, 5-androstene-3 beta,17 beta-diol, progesterone, 4-androstene-3,17-dione, testosterone, and dihydrotestosterone. Since testosterone was not formed from 5-androstene-3 beta,17 beta-diol or 4-androstene-3,17-dione, testosterone synthesis by ram principal cells is unlikely Pregnenolone and 5-androstene-3 beta,17 beta-diol were not metabolized and only slight metabolism of dihydrotestosterone occurred. Progesterone, 4-androstene-3,17-dione, and testosterone were metabolized to 5 alpha-reduced products tentatively identified as 5 alpha-pregnane-3,20-dione and 5 alpha-pregnan-3 beta-ol-20-one and/or 5 alpha-pregnan-20 alpha-ol-3-one; 5 alpha-androstane-3,17-dione and 5 alpha-androstan-3 alpha-ol-17-one, and dihydrotestosterone, respectively. The second experiment evaluated testosterone metabolism by both cultured principal cells and minced epididymal tissue. On day 1 of culture, during 12 hours the accumulation of dihydrotestosterone in medium from cells of the central caput was 48 X and 1.1 X that in medium from cells of the initial segment and proximal corpus epididymidis.(ABSTRACT TRUNCATED AT 250 WORDS)

Exposure of thawed frozen bull sperm to a synthetic peptide before artificial insemination increases fertility.

We evaluated the effect on fertility of in vitro exposure of thawed frozen bull sperm to synthetic FertPlus peptide prior to artificial insemination (AI). The peptide represented a 60-amino acid sequence within rat prosaposin. Commercial cryopreserved semen was from three Holstein bulls. Onset of estrus in groups of Holstein nulliparous heifers was synchronized via injection of prostaglandin F2-alpha, and heifers were scheduled for AI 8-24 hours after estrus was detected. Semen was thawed, diluted to 2.4 x 10(6) sperm/ml with buffer, and split to provide control and exposed aliquots (0 or 30 microM peptide) that were incubated at 37 degrees C for 10 minutes and then were held at 32 degrees C. The two aliquots of semen then were used on an alternate basis 2-65 minutes later to inseminate females. Each AI (one per female) involved the deposit of approximately 250,000 sperm into each uterine horn. This procedure for AI was used to reduce the pregnancy rate with control semen to below the maximum value for a given bull and to facilitate detection of any beneficial effect of the peptide. For each bull, approximately 32 heifers were inseminated with control semen, and approximately 32 heifers were inseminated with peptide-exposed semen. Pregnancy was evaluated ultrasonically approximately 60 days after AI. After excluding one group of heifers with unusually low fertility, averaged across all animals, a 29% increase in pregnancy rate resulted from exposure of sperm to peptide (P < 0.04; one-tailed chi-square test; means were 48 vs. 62%). Pregnancy rates for the three bulls for control and peptide-exposed semen, respectively, were 42 and 62%, 44 and 64%, and 56 and 61%; means in the first two pairs of values tended to differ (P approximately equal to 0.10). These observations should be confirmed with sperm from other bulls used in a more conventional manner. However, with insemination of a limiting number of cryopreserved sperm, brief exposure of the thawed bull sperm to FertPlus peptide appeared to improve fertility dramatically.

Exposure of human, boar, or bull sperm to a synthetic peptide increases binding to an egg-membrane substrate.

We evaluated the effects of in vitro exposure of sperm to synthetic FertPlus peptide, which represents a 60-amino acid sequence within rat prosaposin, using a microwell sperm-binding assay (SBA), in which an extract of hen's egg served as the binding substrate. Sperm suspensions were incubated with FertPlus peptide (six to eight concentrations; 0 and 20-1,280 pM) at 37 degrees C for 10 minutes, diluted > or = 20 times, and placed onto SBA plates. After 60 minutes at 37 degrees C, unbound sperm were washed away and the DNA of bound sperm was quantified. Percentage of sperm bound was independent of the percentage of motile sperm, but immotile sperm did not bind. For fresh human sperm (25 ejaculates), the percentage of sperm bound was increased by exposure to 640 pM peptide (P < 0.01). For 11 of 25 samples, the percentage of sperm bound for the aliquot exposed to 640 pM peptide was > or = 1.4 times the value for a 0 pM control aliquot. With frozen-thawed human sperm, for six of seven samples, binding was > or = 1.4 times greater after exposure to 640 pM peptide. For boar sperm held for approximately 24 hours at approximately 18 degrees C before use (28 ejaculates), there was a higher percentage of sperm bound for aliquots previously exposed to 1,280 pM peptide than there was for control aliquots (P < 0.01). For 16 of 28 samples, exposure to peptide increased the percentage of sperm bound by > or = 1.4 times. For frozen-thawed bull sperm, percentage of sperm bound was > or = 1.4 times greater for 4 of 10 samples that were briefly exposed to 160 pM peptide. Clearly, human, boar, and bull sperm were beneficially modified by brief in vitro exposure to FertPlus peptide, so that for many samples a greater percentage of sperm was bound in vitro. As presented in an accompanying paper, fertility of bull sperm was increased by brief exposure to FertPlus peptide.

Culture of principal cells from the ram epididymis. A comparison of the morphology of principal cells in culture and in situ.

A procedure was developed to isolate and culture principal cells from the initial segment, central caput, distal caput, and proximal corpus epididymidis. The morphology of cultured cells and of cells in situ was compared. Over four to ten days, principal cells cultured in a floating collagen matrix with serum-free medium formed clusters that developed into either large sheets of columnar cells or tubular structures of cuboidal cells. Structural polarity was evident and junctional complexes reformed. The distribution and relative abundance of organelles in principal cells in situ differed depending on the region examined, and most of these regional characteristics were retained by principal cells in culture. Microvilli and membrane-bound vesicles were less conspicuous in cultured cells. Cultured principal cells were shorter than principal cells in situ, but were of similar volume. The high purity (90%) and viability (70%) of principal cells after seven to ten days in culture, their retention of morphologic characteristics unique to the region of origin, and the formation of function units are evidence that such cultures should be valuable for studying regional differences in the function of principal cells.

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.

Effects of caudal elevation on testicular function in rats. Separation of effects on spermatogenesis and steroidogenesis.

A variety of biologic processes are perturbed when exposed to microgravity (space flight) for more than 7 days, including testicular function. Suspension of rats in a special harness (caudal elevation) to induce thoracic pooling of blood fluids and remove the support function of the hind limbs is used to mimic, on earth, the effects of microgravity encountered during space flight. Typically, this induces cryptorchidism in male rats. Three experiments were conducted to differentiate the effects of caudal elevation (30 degrees angle) and anatomic location of testes on spermatogenesis and steroidogenesis. Rats were subjected to caudal elevation for 7 days using either a tail harness (experiments 1 and 2) or a whole-body harness (experiment 3). Testes of rats fell into the abdominal cavity when a tail harness was used, but ligation of the inguinal canal prevented this repositioning. For rats with abdominal testes, testicular weight was reduced (P less than 0.05) and histology of testes was abnormal; the number of spermatids per gram parenchyma was lower (P less than 0.05) in tail-suspended rats compared with control rats. In contrast, spermatogenesis was not affected by caudal elevation in most rats in which the inguinal canal was ligated or in rats elevated by whole-body harness. Concentrations of testosterone in serum and testicular interstitial fluid were lower (P less than 0.05) in suspended rats, regardless of the method used for caudal elevation or anatomic location of testes. Concentrations of luteinizing hormone in serum were elevated (P less than 0.05) in rats with intra-abdominal testes.(ABSTRACT TRUNCATED AT 250 WORDS)

A fragment of prosaposin (SGP-1) from rooster sperm promotes sperm-egg binding and improves fertility in chickens.

A protein isolated from the supernatant of cryopreserved rooster sperm was found to increase the capability of cryopreserved rooster sperm to bind in vitro to the perivitelline membrane of a chicken egg and substantially raise fertility after artificial insemination (AI). That activity was partially purified and termed universal primary sperm-egg binding protein (UPSEBP). Insufficient protein remained from 6 x 10(11) sperm, despite retention of bioactivity, to allow sequencing. We deduced that the protein may be related to prosaposin (also termed SGP-1, for sulfated glycoprotein-1), and we used published amino acid sequences of prosaposin as a guide for synthesis of peptides. Certain peptides were found to increase in vitro sperm-egg binding and increase fertility of frozen-thawed or fresh rooster sperm, in a manner similar to semipurified UPSEBP. Active epitopes were in a 60 amino acid sequence, reflecting the intervening sequence between saposins A and B, plus short extensions into saposins A and B. Highest activity was found when this synthetic peptide was oxidized to form a disulfide bond between terminal cysteines. Antibody against a synthetic peptide consisting of 58 of these 60 amino acids bound to a 7-9 kilodalton protein in UPSEBP. Collectively, the data support the conclusion that UPSEBP is a fragment of prosaposin. Because prosaposin is in semen in humans and animals, these observations have broad implications for possible cause and therapy of one type of subfertility.