A 22-year-old woman with chronic, mild, intermittent knee pain.

The following case is presented to illustrate the roentgenographic and clinical findings of a condition of interest to orthopedic surgeons. The initial history, physical findings, and roentgenographic examination are found on the first page. The final clinical and roentgenographic diagnosis is presented on the following page.

A genetically encoded optical probe of membrane voltage.

Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is a fundamental problem for the study of neural development and information processing. To address this problem, we have constructed a novel, genetically encoded probe that can be used to measure transmembrane voltage in single cells. We fused a modified green fluorescent protein (GFP) into a voltage-sensitive K+ channel so that voltage-dependent rearrangements in the K+ channel would induce changes in the fluorescence of GFP. The probe has a maximal fractional fluorescence change of 5.1%, making it comparable to some of the best organic voltage-sensitive dyes. Moreover, the fluorescent signal is expanded in time in a way that makes the signal 30-fold easier to detect. A voltage sensor encoded into DNA has the advantage that it may be introduced into an organism noninvasively and targeted to specific developmental stages, brain regions, cell types, and subcellular compartments.

Genetic probes: new ways to watch cells in action.

Visualizing fast physiological changes in small living cells, such as synaptic transmission in a neural circuit, is a fundamental problem in biology. Progress towards this goal is being made with the development of new types of protein sensor that can be targeted within cells and organisms.

Effect of human sperm capacitation treatments on the penetration of freshly obtained and zona-free frozen hamster oocytes.

A study was conducted to evaluate the effect of sperm capacitation treatments on hamster egg penetration test (HEPT) values when using freshly obtained and zona-free frozen hamster oocytes. Semen was collected from 12 healthy donors with proven fertility and each sample processed by a sperm swim-up method, incubation in human follicular fluid (hFF), Percoll centrifugation, and refrigeration in test-yolk buffer (TYB). Capacitated sperm were then incubated with fresh and frozen-thawed eggs and the percent penetration scores and indices were calculated. Motility values of pre- and postprocessed sperm were also determined. A significant overall reduction in percent penetration scores but not penetration indices was observed when using frozen oocytes. However, within sperm treatments, differences between fresh and frozen oocytes were observed for the sperm swim-up, hFF incubation, and Percoll centrifugation groups, but not for TYB. Differences in sperm movement parameters according to sperm treatment did not appear to reflect specific penetration scores. In conclusion, despite the practical advantage of using frozen oocytes over freshly obtained oocytes for the HEPT, the specific sperm capacitation treatment must be considered a factor when the decision to use fresh or frozen oocytes is made. Furthermore, these findings reinforce the need to carefully reevaluate the predictive value of the HEPT any time modifications to the test are introduced.

The male fertility investigation and the role of the andrology laboratory.

The laboratory diagnosis and treatment of male infertility has been a topic of study for numerous decades. Despite significant advances in the understanding of male reproductive physiology, clinical tests with high predictive value related to fertility are still greatly needed in the assessment of semen fertility. Due to the variety of possible sperm and semen abnormalities, a comprehensive approach using several tests is generally employed to assess the integrity of semen specimens. Following assessment, the most common sperm treatment for couples with difficulties conceiving is the use of different sperm-processing procedures prior to intrauterine insemination. If this treatment is unsuccessful following several insemination cycles, the couple may then consider therapeutic donor insemination. The use of donor semen can result in pregnancy in many circumstances in which the couple's only other options might have been adoption or more costly assisted reproductive procedures. Donor programs using frozen sperm have become far more sophisticated in recent years, paying close attention to the efficacy and safety of specimens as well as the emotional needs of the recipient couple. This review is designed to present an overview of the methods of evaluation and treatment of male infertility as well as some of the controversies surrounding some of these approaches.

Influence of porcine follicular fluid on the fertilizing capacity of human spermatozoa.

The results of this study indicate that there are factors present in porcine FF that affect human sperm fertilizing potential. It appears that fluid from developmentally mature follicles stimulate sperm, whereas fluid from immature follicles can inhibit sperm. These findings also demonstrate that human sperm is influenced by factors present in nonhuman FF.

Effect of bromocriptine on sperm function in vitro and in vivo.

Vaginal bromocriptine is an effective method for the treatment of hyperprolactinemia, but it is unknown whether bromocriptine applied vaginally can interfere with sperm function. Thus, we sought to determine the effects in vitro and in vivo on sperm directly exposed to bromocriptine. Ten semen specimens from normal donors were diluted with Ham's F-10 medium and incubated with 0, 0.01, 0.1, and 1.0 mmol/L bromocriptine solution or diluent without bromocriptine. Computerized semen analysis revealed a 31% decrease in sperm motility, a 24% decrease in sperm average path velocity, and a 33% decrease in sperm average straight line velocity only using 1.0 mmol/L of bromocriptine (P less than .05). In addition, eight women with hyperprolactinemia and infertility who were receiving vaginal bromocriptine consented to a postcoital test. Five became pregnant and delivered normal infants. Four of the five women who had a postcoital test had six, eight, ten, and ten motile sperm per high-power field and one had one to two motile sperm per high-power field. Because sperm function was preserved enough to result in fertilization and term pregnancy, the clinical importance of the in vitro findings is probably minimal and it can be concluded that vaginal bromocriptine can be used in women with infertility due to hyperprolactinemia.

Influence of sperm processing on the fertilizing capacity and recovery of motile sperm from thawed human semen.

Artificial insemination programs now rely almost exclusively on frozen semen preparations as their source of sperm. Unfortunately, several reports indicate that conception rates using frozen-thawed semen are inferior to freshly ejaculated specimens. The present study was designed to investigate the fertilizing capacity of sperm and the recovery of motile sperm from thawed semen following different sperm processing methods. Washed resuspended pellets contained sperm with at least the same fertilizing potential as sperm from swim-up techniques. However, the recovery of motile sperm from the pellets was more than 3 times greater than from the swim-up techniques. Percentage motility, progressive velocity, and amplitude of lateral head displacement were generally higher in the sperm from swim-up techniques than in the sperm from the pellets, despite equivalent fertilizing potentials. It can be argued from these results that washed resuspended sperm preparations provide a significantly greater number of motile sperm without a loss in fertility when compared to sperm from swim-up techniques. Accordingly, this study raises questions about the use of the sperm swim-up as a procedure for processing thawed semen for use in intrauterine insemination in which maximal numbers of motile sperm are required. It also demonstrates the need to identify new methods for processing frozen-thawed specimens for assisted reproductive procedures.

Motile sperm recovery from fresh and frozen-thawed ejaculates using a swim-up procedure.

The recovery of motile sperm in swim-ups from fresh semen that was washed and centrifuged one time was 33% and after 2 washes and centrifugations was 22%. The recovery rates for frozen-thawed semen were only 7% and 5%, respectively. The straight line velocity of sperm was increased after sperm swim-up, whereas changes to the lateral sperm head movement were not as evident. However, the dramatic loss in motile sperm numbers especially after swim-up from thawed semen indicate that more efficient methods of isolating motile sperm from thawed semen may be required.

The influence of human follicular fluid on the acrosome reaction, fertilizing capacity and proteinase activity of human spermatozoa.

The present study was designed to assess physiological and enzymatic changes in human spermatozoa following incubation in human follicular fluid (HFF). Initially, it was determined that infertility patients (n = 29) scored dramatically higher on the hamster egg penetration test (HEPT) when spermatozoa were incubated with HFF (22.9 +/- 4.4%) rather than the standard swim-up alone (4.6 +/- 1.1%). To further evaluate this effect, in experiment I, spermatozoa were obtained from proven fertile individuals and evaluated following exposure to three different HFF samples as well as control treatments (medium, cul de sac fluid and heparin). There were no significant differences in HEPT scores following sperm incubation in the three different follicular fluids although incubation in all the fluids significantly (P less than 0.01) enhanced sperm penetration (%PEN) when compared to the standard swim-up and other control treatments. The absence of an effect of cul de sac fluid on spermatozoa indicates that not all body fluids contain factors which stimulate sperm fertilizing capacity. The effect of HFF was demonstrated in a infertile patient population as well as in donors of proven fertility. In experiment II, the effect of HFF on the acrosome reaction (%AR), sperm fertilizing capacity and changes in sperm proteolytic enzymes were determined. There was no significant difference in the %AR between freshly ejaculated (7.9 +/- 3.1) and medium incubated (9.4 +/- 1.6) spermatozoa; however, in both of these treatments the %AR was less (P less than 0.01) than for spermatozoa treated with HFF (45.6 +/- 4.7). The %PEN following incubation of spermatozoa in HFF (52.2 +/- 6.8) was greatly increased (P +/- 0.01) compared to the standard swim-up (19 +/- 3.9).

Absence of direct effect of beta-endorphin and calcitonin on human sperm motility.

Beta-endorphin and calcitonin are found in the male reproductive tract. To elucidate the role of these hormones in reproduction, we studied their effect on sperm motility in vitro. Eight semen specimens were obtained from healthy donors, washed, and incubated with different concentrations of human beta-endorphin and human calcitonin. After 30 min of incubation, percentage of motile sperm (% motility), mean progressive velocity (MPV), and lateral head displacement (LHD) were assessed by a computerized semen analyzer. There were no significant differences in any of the sperm motility parameters between control and treated sperm. There was also no correlation between the concentration of beta-endorphin or calcitonin and any sperm motility parameters. It would appear that beta-endorphin and calcitonin may not directly affect sperm motility parameters in vitro.

Biochemical and immunological comparisons between the human and boar proacrosin-acrosin proteinase systems.

Biochemical and immunochemical methods have been used to examine the proacrosin-acrosin system of human and boar spermatozoa. Marked biochemical similarities including the relative molecular weights of proacrosin (approx. 55,000), alpha-acrosin (45,000-49,000) and beta-acrosin (34,000-38,000) were observed for both species. In addition, the time course of proacrosin autoconversion between 0 to 60 min revealed that the purified proacrosin from both species autoconverted to alpha-acrosin and then to beta-acrosin at approximately the same time intervals. Despite these apparent biochemical similarities, distinct immunological differences between the human and boar proacrosin-acrosin systems were observed. The human proacrosin antibody immunoreacted with purified human proacrosin and alpha-acrosin but not with beta-acrosin. The antibodies to boar proacrosin cross-reacted with the purified boar proacrosin, alpha-acrosin and beta-acrosin. The antibodies to human proacrosin also cross-reacted with boar proacrosin and to a weak extent with boar alpha-acrosin but not with the boar beta-acrosin. While antibodies to boar proacrosin did not react with any of the components of the human proacrosin system. Additionally, in the non-purified sperm extracts the human proacrosin antibody preparation reacted with several proteins larger than proacrosin and one with a molecular weight of approximately 34,000. In the non-purified boar sperm extracts, the antibodies to boar proacrosin only cross-reacted with the known components of the proacrosin-acrosin system suggesting a high degree of specificity. Thus, immunochemical evidence is presented that indicates there are specific structural differences which occur in the proacrosin-acrosin system of mammalian sperm.

Comparison between proteinases of human seminal plasma and of sperm origin.

A comparison of the alkaline proteinase activity of human seminal plasma, the seminal non-gamete cellular material and spermatozoa was made by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) zymography. Several major (molecular weights = greater than 56,000) and minor (35,000 to 44,000) bands of proteinase activity were seen in the seminal plasma samples from nonvasectomized and vasectomized, healthy donors. Similar activity profiles were observed in the nongamete cellular material of vasectomized donor ejaculates. The major proteinase activity in sperm extracts was in the 47,000 to 55,000 (proacrosin-acrosin) and 34,000 to 37,000 (sperminogen-spermin) molecular weight ranges. These results suggest that the proacrosin-acrosin and sperminogen-spermin systems are of sperm origin and that there are considerable amounts of larger molecular weight trypsin-like enzymes in the soluble and nongamete cellular material of human seminal plasma.

Partial purification and characterization of human sperminogen.

A recently recognized non-proacrosin zymogen referred to as sperminogen has been purified from human spermatozoa, and several of its properties have been determined. The purification procedure included acid extraction of washed ejaculated sperm at pH 3.0, followed by gel filtration of the solubilized extract over a Sephadex G-75 superfine column. The sperminogen eluted from the column in a single band that was completely separated from the proacrosin band. This separation was confirmed by a gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) zymograph. This zymograph also demonstrated that the final sperminogen preparation contained four forms of zymogen, with molecular weights between 32,000 and 36,000. At neutral pH, the sperminogen was converted into spermin, its enzymatically active form, yielding a sigmoidal curve typical of zymogen autoactivation. The effects of several factors on the rate of this autoconversion indicate specific differences between sperminogen and proacrosin. Spermin hydrolyzed N-alpha-benzoyl-L-arginine ethyl ester (BzArgOEt), and was inhibited by lima bean trypsin inhibitor, pancreatic trypsin inhibitor, N-acetyl-L-leucyl-L-leucyl-L-argininal (leupeptin), and tosyl-L-lysine chloromethyl ketone, indicating that the enzyme has a trypsin-like specificity and probably belongs to the class of trypsin-like enzymes. Since acrosin is generally believed to be the only trypsin-like enzyme in mammalian sperm, the demonstration of human sperminogen and spermin necessitates further inquiry into the functions and the relationships between sperm proteinase systems.

The rapid purification and partial characterization of human sperm proacrosin using an automated fast protein liquid chromatography (FPLC) system.

A rapid and efficient procedure was developed for obtaining highly purified human proacrosin. Ejaculated spermatozoa were washed via centrifugation through 1 M sucrose containing 50 mM benzamidine and acid-extracted in the presence of benzamidine. The solubilized material was dialyzed then lyophilized. The sample was resuspended in 8 M guanidine hydrochloride in acetic acid (0.5 M) pH 2.5 and then subjected to gel permeation chromatography with an automated fast protein liquid chromatography system utilizing two Pharmacia Superose 12 columns set in tandem that were equilibrated in the same buffer. The proacrosin eluted as an individual peak that was well separated from another proteinase zymogen referred to as sperminogen. The proacrosin preparation was determined to be highly purified when observed on silver-stained SDS-polyacrylamide gels as well as on gelatin-SDS-polyacrylamide gels. The proacrosin appeared as a doublet (Mr = 55,000 and 53,000) on both of these systems. The autoconversion of proacrosin to acrosin at pH 8 resulted in a typical sigmoidal autoactivation curve. Following protein staining of SDS-polyacrylamide gels, it was shown that upon activation of purified proacrosin preparations the 55,000 and 53,000 molecular weight proteins were initially degraded to a 49,000 form and then to several lower molecular weight forms (Mr = 40,000-34,000). Similar findings with regard to proteolytic digestion were observed following gelatin-SDS-polyacrylamide zymography except that an increase with time in proteinase intensity between 58,000 and 53,000 was also observed. Cobalt and calcium were found to be potent inhibitors of the conversion of proacrosin into acrosin, while sodium resulted in much less inhibition of this process. Calcium was found to markedly enhance the proteolytic activity of human acrosin, while it had no observable influence on the acrosin hydrolysis of benzoylarginine ethyl ester. Thus, the described purification procedure resulted in a highly purified proacrosin preparation in sufficient yields to allow for its partial characterization.

Quantification and partial characterization of the hamster sperm proacrosin-acrosin system.

The proacrosin-acrosin proteinase system was measured and partially characterized in unpurified extracts of washed hamster epididymal sperm. Autoactivation experiments demonstrated that proacrosin accounted for greater than 98% of the acrosin activity in the sperm extracts from individual animals. Several bands of proteinase activity were observed on gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic (gelatin-SDS-PAGE) zymography. The major proteinase activities in the nonactivated extracts corresponded to relative molecular masses (Mr) of 51,000 to 56,000, while less distinct digestion occurred with relative molecular masses of 37,000 to 49,000. It was demonstrated that after a serial dilution of the sperm extract, the proteinase activity in as few as 6,000 sperm could readily be detected by the gelatin-SDS-PAGE methods. Time-course activation studies showed that the zymogen was completely converted to active proteinase in 45-60 min at pH 8.0 and 25 degrees C. This autoconversion process was markedly inhibited by calcium, sodium, and heparin. However, each of these compounds stimulated the proteolytic activity of acrosin. These studies demonstrate that the proacrosin-acrosin system can be investigated in extracts of nonpurified hamster epididymal sperm.

Comparative trial of ketoconazole and nystatin for prevention of fungal infection in neutropenic patients treated in a protective environment.

In a randomized trial we compared ketoconazole (400 mg once daily, 27 patients) and nystatin (3 X 10(6) units four times daily, 29 patients) for prevention of fungal infection in neutropenic patients undergoing marrow transplantation in a protective environment. Fewer weekly surveillance cultures contained Candida species in ketoconazole recipients than in nystatin recipients (70 [26%] of 274 vs. 151 [47%] of 322; P less than .001). When all fungi were considered, the difference in colonization was less but was still significant (117 [43%] of 274 vs. 173 [54%] of 322; P = .01), primarily due to increased colonization of the rectum with Torulopsis glabrata among ketoconazole recipients (P less than .001). No difference in the incidence of local mucosal infection was seen. Two disseminated fungal infections occurred, both in nystatin recipients. Compliance with ketoconazole was significantly better than was compliance with nystatin (96% vs. 68%; P less than .001), but similar effects on colonization were found in an analysis adjusting for compliance. Ketoconazole was better tolerated and more effective than nystatin in reducing colonization due to Candida species but was also associated with significantly increased rates of colonization with T. glabrata.

Evaluation of the human sperm proacrosin-acrosin system using gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophesis.

Proteolytic enzymes in extracts of human sperm have been identified and partially characterized using a technique which incorporates gelatin into a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) system. Initially, semen characteristics from four donors were evaluated. Following this, washed sperm were acid extracted and proacrosin and acrosin activities determined spectrophotometrically. Proteinase activity in unactivated sperm extracts was then extracts was then demonstrated using the gelatin-SDS-PAGE system. Three major (Mr approximately equal to 47,000-54,000) and four faint (Mr approximately equal to 34,000-38,000) bands of digestion were observed. Upon activation of sperm extracts it was observed that maximum esterase activity occurred within 7 min of activation while maximum proteinase activity required approximately 15 min. When gels were washed and incubated in the presence of 50 mM benzamidine, no digestion bands were observed. This indicates that all of the digestion bands were due to trypsin-like proteinases. Finally, upon serial dilution of sperm extracts it was found that this SDS-PAGE system is sensitive enough to detect proteinase activity from as few as 30,000 sperm.