Showing papers in "Journal of Andrology in 1993"

Neonatal goitrogen treatment increases adult testis size and sperm production in the mouse

Abstract: Male rats made hypothyroid during neonatal life show unprecedented increases in adult testis size and daily sperm production (DSP). To determine if this effect was unique to the rat or could also be demonstrated in other species, we examined the effects of neonatal treatment with the reversible goitrogen 6-propyl-2-thiouracil (PTU) on adult testis size and function in the mouse. Male Swiss-Webster mice were untreated (control) or given PTU by adding 0.1% (w/v) to their mother's water from birth to day 25 postpartum. All pups were then weaned and given no further treatment. Sertoli cell proliferation was examined using tritiated thymidine autoradiography in some control and treated mice at 0, 5, 10, 15, 20, and 25 days, while the remainder were killed at 90 days to determine a variety of reproductive parameters. Neonatal PTU treatment decreased growth; body weight of treated mice at 4 weeks of age was 57% less than controls. Treated mice grew rapidly following cessation of PTU treatment, although their weights never equalled controls, remaining 17% smaller at 90 days of age. At 90 days of age, testis weight and DSP were increased by approximately 30% and 50%, respectively, in PTU-treated mice compared to controls. Despite the increased testis weight and function, serum testosterone concentrations were not different in control and treated mice. Testicular and epididymal histology in treated mice was similar to controls, while epididymal sperm in treated mice were motile and morphologically normal. Sertoli cell proliferation was altered in treated mice. The normal decrease in proliferative rate seen during early postnatal life was slowed, and by day 10 postnatal, the labeling index of treated Sertoli cells was about fourfold greater than that of controls. Furthermore, Sertoli cells in treated mice proliferated until day 25, whereas proliferation ceased in controls by day 15. In summary, neonatal PTU treatment increases testis weight, DSP, and the efficiency of sperm production (DSP/g testis) in the mouse, indicating that the PTU effect on testis development clearly occurs in other species. Furthermore, increased Sertoli cell proliferation appears to be the critical event for the development of this phenomenon in mice, as it is in rats. The existence of unique mutations that affect testicular development make the mouse an advantageous model for determining the mechanism of this effect. This technique may also be useful for increasing testicular size, sperm production, and fertility in various mutant mouse strains and transgenic mice in which these parameters are reduced.

Operational Standards for CASA Instruments

Abstract: Computer-aided sperm analysis (CASA) technology is 7 years old Over 120 papers have been written that verify the technology or apply it in basic and clinical studies Most of the technical problems with CASA, such as the dependence of velocity on video frame rate, inaccuracy of count and percent motility for low- and high-concentration specimens, parameter dependence on the number of frames analyzed, sensitivity of the subjective threshold setting, confusion over the presence of debris, and different implementations of algorithms across instruments, still persist A critical review of the literature reveals that no standard practices are followed within or across instruments Moreover, no standards have been embraced or recommended by professional societies Despite its potential to provide objective measurements of specimen and individual sperm parameters, and to automate the laboratory semen analysis, the promise of CASA has not been fulfilled Unless laboratory medicine defines instrument performance and laboratory standards and co-operates with industry to achieve these goals, CASA technology may remain a research curiosity This outcome is especially worrisome in the context of increasing requirements for laboratory accuracy, precision, standardization, and accreditation under the Clinical Laboratory Improvement Act of 1988

Evidence that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in cryopreserved human sperm: glycerol and other polyols as sole cryoprotectant.

Abstract: One effect of cryopreservation on human sperm is sublethal cryodamage, in which cell viability post-thaw is lost more rapidly at later times than in fresh cells. We hypothesized two modes of sublethal cryodamage: one is peroxidation-related involving plas- ma membrane damage due to lipid peroxidation; the other is mem- brane stress-related involving membrane embrittlement dunng phase transitions occurring during freeze-thaw. If the peroxidation-related mode contributed substantially to sublethal cryodamage, the hy- pothesis predicts that lipid peroxidation inhibitors should reduce this damage. To test this prediction, we examined the effect of the lipid peroxidation inhibitors, hypotaurine, bovine serum albumin (BSA), and a-tocopherol (Vit. E) on the time to loss of motility (TLM), taken as a measure of cell viability over time, for sperm samples cryopre- served in glycerol plus egg yolk medium. These agents had no effect on TLM of these samples, indicating that this mode contributes little to sublethal cryodamage. If the membrane stress-related mode con- tnbuted, the hypothesis predicts rapid recovery of motility in the presence of egg yolk plus glycerol, but slow recovery in the presence of glycerol alone. It also predicts that an appropriate polyol may be both necessary and sufficient for cryopreservation. In the presence of egg yolk plus glycerol, motility recovery was complete within 5 minutes, but the percent motile cells then decreased linearly with time. With glycerol alone in the range 3-12%, at 5 minutes post- thaw the percent motile cells was 5-1 0%, but by 40 minutes post- thaw had risen to 60-80%, approaching that in the fresh sample, and was maintained up to 4 hours. In the absence of glycerol, the percentage of motile cells post-thaw was nil and remained nil up to 4 hours. The polyols, erythntol, ribitol, and sorbitol had similar effects to that of glycerol, but the recovery of motility was not as complete. These results indicate that the membrane stress-related mode con- tributes substantially to sublethal cryodamage. They also indicate that glycerol and other polyols can function alone as cryoprotectants, but that recovery of motility is slow in these systems.

Analysis of the Responses of Human Spermatozoa to A23187 Employing a Novel Technique for Assessing the Acrosome Reaction

Abstract: Protocols for the use of A23187 in assessing the ability of human spermatozoa to acrosome-react and exhibit sperm-oocyte fusion have been developed and the results compared in two independent cohorts of infertile patients. Both bioassays were found to depend upon such factors as the dose and formulation of A23187, the duration of exposure, and the amount and type of protein used to supplement the medium. An optimal protocol for the hamster oocyte penetration test comprised a 3-hour exposure to 1.25-2.5 microM ionophore and gave penetration rates of 93.2 +/- 3.2% (11.26 +/- 1.27 sperm/egg) for a group of 33 fertile donors compared with 63.0 +/- 5.4% (4.73 +/- 0.81 sperm/egg) for a cohort of 56 patients consulting for infertility (P < 0.001). Higher doses (5.0-10.0 microM) of A23187 caused an inhibition of sperm-oocyte fusion in association with a loss of motility, although the integrity of sperm plasma membrane did not appear to be compromised and high rates (approximately 80%) of acrosome reaction were observed. A protocol for assessing the ability of viable human spermatozoa to acrosome-react in response to A23187 was developed, employing a fluorescein-conjugated lectin in concert with the hypoosmotic swelling test, which gave values of 20.1 +/- 2.6% and 13.6 +/- 1.6% for groups of fertile donors (n = 29) and infertile patients (n = 32) respectively (P < 0.05). Although only acrosome-reacted spermatozoa were capable of fusing with zona-free hamster oocytes, there was no significant correlation between the proportion of acrosome-reacted cells and the levels of sperm-oocyte fusion observed in two independent groups of patients, indicating that these bioassays are measuring different aspects of human sperm function. These results have implications for the way in which the responses of human spermatozoa to ionophore treatment are quantified and interpreted.

Motile sperm in human testis biopsy specimens

Abstract: We prospectively studied 62 consecutive infertile men who underwent 100 intraoperative wet prep cytological examinations of testis biopsy material obtained simultaneously with permanently fixed specimens. Wet preps were performed by placing a small sample of fresh testicular tissue on a slide, adding a drop of Ringer's lactate, and compressing the specimen under a glass coverslip. Among these 100 wet preps, complete sperm with tails were identified in 62 specimens, of which 44 contained nonmotile sperm and 18 contained motile sperm. Reproductive tract obstruction was documented in 65 testes (65%) on subsequent reconstructive surgery and/or inferred from histological evaluation, including mean mature spermatid counts on the permanent sections fixed in Bouin's solution. Obstruction was absent in the remaining testes (35%). All 18 testes with motile sperm found on wet prep were obstructed. These testes were also found to have complete spermatogenesis, a category selected to include normal spermatogenesis and slight hypospermatogenesis, determined by examination of the permanently fixed sections. The finding of motile vs. nonmotile sperm on a wet prep has positive predictive values of 100% vs. 81% for the presence of reproductive tract obstruction and 94% vs. 86% for complete spermatogenesis, respectively. The presence of motile sperm in human testis biopsy specimens is a novel finding. When any complete sperm with tail is found in a testis biopsy wet prep, obstruction is likely. When motile sperm are present, obstruction is almost certain, and immediate exploration and reconstructive surgery can be justified.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical Localization of the Yf Subunit of Glutathione S‐Transferase P Shows Regional Variation in the Staining of Epithelial Cells of the Testis, Efferent Ducts, and Epididymis of the Male Rat

Abstract: Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione (GSH), a tripeptide found in all mammalian cells; this function plays a protective role, as the addition of GSH to an electrophile generally forms a less toxic product The pi class of GSTs contains homodimers of the Yf subunit, also known as Yp or rat subunit 7; this subunit is found in high concentrations in the testis and epididymis The objective of the present study was to localize immunocytochemically the Yf subunit in the testis and in the various regions of the epididymis using light, electron, and confocal microscopy In the testis, immunoperoxidase staining was localized exclusively to Sertoli and Leydig cells The low cuboidal epithelial cells of the rete testis and the sparse ciliated cells of the ductuli efferents were also immunoreactive A distinct pattern of immunostaining for the Yf subunit was observed in the different regions of the epididymis The proximal area of the initial segment showed intense reactivity localized to epithelial basal cells Basal cells in the middle area of the initial segment were also reactive, as were a second unidentified population of cells located in the apical region of the epithelium The epithelium, including both principal and basal cells, in the distal initial segment, intermediate zone, and proximal caput epididymidis showed a weak, moderate, or strong degree of reactivity, respectively In the distal caput epididymidis, however, principal cells showed a checkerboard-like pattern of immunoreactivity, with some cells being intensely stained or faintly stained, whereas others were unreactive Strikingly, in the corpus and proximal cauda epididymidis, intense immunostaining was localized exclusively over the epithelial basal cells As viewed in the light and confocal microscope, the intensely stained basal cells showed extensive processes that covered most of the base of the epididymal tubule Upon quantitation of the immunogold labeling density (the number of gold particles/microns2) in principal and basal cells of the different regions of the epididymis, we observed a sharp decline in immunogold labeling of principal cells coupled with a dramatic increase in labeling of basal cells as we progressed along the tissue, particularly in the transition from the caput to the corpus epididymidis This study constitutes the first demonstration of a protein that is selectively expressed in epithelial basal cells of the corpus and proximal cauda epididymidis(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of Cryoprotective Additives and Cryopreservation Protocol on Sperm Membrane Lipid Peroxidation and Recovery of Motile Human Sperm

Abstract: Sperm membrane damage during cryopreservation reduces the recovery of motile sperm. The present study investigates changes in sperm motility and membrane lipid peroxidation (LPO) in response to two changes in the standard sperm cryopreservation/thawing methodology: 1) the addition of platelet-activating factor (PAF) and pentoxifylline (PTX) as cryoprotective additives, and 2) the alteration of sample thawing time. PAF (1 microM) and PTX (3 mM) were added to fresh sperm samples prior to cryopreservation. After 2 weeks the samples were thawed either quickly (5 minutes at 37 degrees C) or slowly (30 minutes at 4 degrees C) and evaluated for sperm motility and LPO. Thawing time influenced both post-thaw motility and LPO. Samples thawed quickly exhibited a 31% increase in motility recovery (35.2 +/- 4.3% in quick-thaw samples; 24.3 +/- 3.9% in slow-thaw samples) and a 23% lower LPO level (23.3 +/- 3.4% in quick-thaw samples; 30.09 +/- 4.4% in slow-thaw samples) compared to samples thawed slowly. Results also demonstrated that PAF (49 +/- 1.7%) or PTX (42.6 +/- 1.5%) enhance post-thaw motility in comparison to control (35.8 +/- 1.2%), whereas neither PAF nor PTX affect post-thaw LPO (19.1 +/- 2.2% in controls; 20.2 +/- 1.7% in PAF samples; 20.5 +/- 1.4% in PTX samples). These results support observations that there is a negative correlation between sperm motility and LPO in cryopreserved samples. The results also discount the hypothesis that LPO protection is a result of the cryoprotective action of PAF or PTX.

Testicular steroidogenesis in the aging brown Norway rat

Abstract: In seeking an animal model of age-associated changes in the male reproductive tract, we examined the effects of age on the health and testicular steroidogenic activity of the Brown Norway rat, with comparisons made to the Sprague-Dawley rat. When perfused in vitro under conditions of maximally stimulating luteinizing hormone significant age-associated reductions were seen in testosterone production by testes of Sprague-Dawley rats of 21-24 months of age and by testes of Brown Norway rats of 18-30 months of age. Decreases in the capacity of the testes to produce testosterone were reflected in age-associated decreases in both serum testosterone and in testosterone concentration within the seminiferous tubule fluid. In contrast to the Sprague-Dawley rat, changes in steroidogenic activity in the Brown Norway rat were not accompanied by the occurrence of pituitary adenomas, obesity, or testicular tumors. This along with its longevity, make the Brown Norway strain a highly promising model for testicular aging.

Validation of an acrosomal stain for equine sperm that differentiates between living and dead sperm.

Abstract: An acrosomal staining technique that can differentiate between living and dead sperm was developed for equine sperm. The fluoresceinated lectin Pisum sativum agglutinin (FITC-PSA) was used to identify the presence or absence of acrosomal contents, while the supravital nuclear dye Hoechst 33258 (H258) was used to assess viability. The accuracy of the FITC-PSA acrosomal stain was tested by comparing the percentage of sperm that had lost their acrosomal contents, detected by the staining method, with that detected by transmission electron microscopy (TEM). Following capacitation in vitro, the acrosomal status of sperm induced to acrosome react with A23187 and of control sperm were very similar with the staining technique and TEM, confirming the accuracy of the FITC-PSA acrosomal stain. We investigated the relationship between viability as measured by exclusion of H258 and motility as measured by three methods: one subjective and two objective. Although there was a good correlation between viability and motility as measured by all three methods (r = 0.88, 0.85, 0.75), there was always a proportion of viable sperm that were nonmotile. The physiology of the viable, nonmotile sperm was further investigated by comparing for individual sperm the viability as measured by exclusion of H258 with the mitochondrial function as measured by rhodamine 123. A good correlation (r = 0.99) was found to exist between viability and mitochondrial function, indicating that viable, nonmotile sperm possess functional mitochondria and confirming the ability of supravital staining to distinguish between living and dead sperm. We determined that 29-81% of the sperm in semen that had lost their acrosomal contents were in fact dead. Thus, this acrosomal staining technique can provide more relevant endpoints for future investigations of capacitation, the acrosome reaction, and sperm handling techniques in the horse.

Characterization of K Currents in Cultured Human Corporal Smooth Muscle Cells

Abstract: In order to gain more mechanistic insight into the regulation of corporal smooth muscle tone, we conducted electrophysiological studies on homogeneous explant cell cultures of human corpus cavernosum smooth muscle. Patch clamp analyses in the whole cell mode revealed a mean resting potential of -43 +/- 4.9 m V (n = 12 cells). Large whole cell outward K currents were very prominent in these cells, and ranged from 0.5 to 1.5 nA. In some cells, a transient, voltage-dependent A current accounted for a significant portion of the observed whole cell currents. Furthermore, stimulation with the calcium channel agonist BAY K 8644 or the K channel agonist pinacidil doubled the magnitude of the whole cell K current, as would be expected for maxi-K (KCa) and metabolically gated K channels (KATP), respectively. Single channel recordings in the detached patch mode consistently revealed the presence of at least two K channels: 1) a KCa channel, with a conductance of approximately 190 pS; and 2) a putative delayed rectifier channel with a conductance of approximately 50 pS. Furthermore, all channel types showed some degree of voltage and/or calcium sensitivity. In conclusion, the large magnitude of the whole cell K currents and the observed K channel heterogeneity indicate a potentially important role for these channels in modulating corporal smooth muscle tone.

The Effect of Aging on the Seminiferous Epithelium of the Brown Norway Rat

Abstract: Aging of the mammalian testis is often accompanied by loss of germ cells and, as a result, decreased daily sperm production. It is currently unknown whether cell loss is the result of aging-related changes in germ cells or whether there are also aging-related changes in the Sertoli cells that normally support germ development and differentiation. To begin to compare the effects of age on germ cells and on Sertoli cells, we examined brown Norway rats of 6, 12, 18, 21, and 24 months of age for the frequency of seminiferous tubule regression and total testis contents of transcripts for three Sertoli cell products: SGP-2, transferrin, and cyclic protein-2 (CP-2)/cathepsin L. Histological analysis revealed no changes in the seminiferous epithelium from 6 to 12 months of age. However, from 12 to 24 months of age, the percentage of normal tubules gradually decreased from 95% to 15% of the total while the percentage of fully regressed tubules (containing no germ cells per tubule cross section) increased from 0% to 78%. In our analysis of specific Sertoli cell transcripts, we noted no change in total testis content of SGP-2 mRNA from 6 to 24 months. However, total testis content of transferrin mRNA was unchanged from 6 to 18 months, but increased by 24 months to 368% of the content of a 6-month-old testis. In contrast, total testis content of CP-2/cathepsin L mRNA was unchanged from 6 to 12 months, but decreased by 24 months to 58% of the content of a 6-month-old testis.(ABSTRACT TRUNCATED AT 250 WORDS)

Germ cell-conditioned medium contains multiple factors that modulate the secretion of testins, clusterin, and transferrin by Sertoli cells

Abstract: In view of the evidence showing that germ cells regulate Sertoli cell (SC) function, the aim of this study was to examine if germ cell (GC)-conditioned media contained multiple biological factors that affect SC secretory functions. Total GC were isolated from adult rat testes. Pachytene spermatocytes (SPC) and early spermatids (SPT) were enriched to about 90% pure by centrifugal elutriation. GC, SPC, and SPT were cultured in serum-free medium for 20 hours with a viability greater than 95%. Conditioned media derived from these cells were fractionated by anion-exchange high-performance liquid chromatography (HPLC). An aliquot from each of these fractions was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were visualized by silver staining. The patterns of protein staining using media from GC, SPC, and SPT were similar. Bioassays of these column fractions on SC showed that the transferrin stimulatory, the testins inhibitory, and the clusterin inhibitory activities were eluted from the anion-exchange HPLC column in overlapping fractions. To determine whether these activities were confined to one or several molecules, further fractionations were performed. Eleven liters of GC-conditioned medium were fractionated by sequential HPLC using anion-exchange, gel permeation, and reversed-phase columns. Throughout the entire fractionation scheme, the HPLC fractions were bioassayed using primary SC-enriched cultures prepared from 20-day-old rats by incubating SC with aliquots of these fractions for 24 hours to monitor their effects on SC secretory function. The concentrations of transferrin, clusterin, and testins were quantified by specific radioimmunoassays. These studies showed that the transferrin stimulatory activity can be fractionated into four peaks (I, IIa, IIb, and IIc); clusterin inhibitory activity into three peaks (A, B, and C); and testins inhibitory activity into two peaks (1 and 2). Some of these bioactivities were eluted in overlapping fractions such as I and B, IIb and 1, and IIc and 2, whereas A, C, and IIa were not associated with any other assayed activities. In summary, additional GC modulators of SC function were identified for the first time, these include clusterin and testins inhibitors. The previously identified transferrin stimulatory activity was also resolved into multiple molecular forms.

Appearance of alpha-smooth muscle actin in peritubular cells of monkey testes is induced by androgens, modulated by follicle-stimulating hormone, and maintained after hormonal withdrawal.

Abstract: This study analyzed the hormonal requirements necessary for the development of primate testicular peritubular cells. Alpha-smooth muscle actin, as a specific differentiation marker for peritubular cells, was immunohistochemically detected in the testes of immature rhesus and adult rhesus and cynomolgus monkeys. Positive staining was localized in the wall of blood vessels and in peritubular myoid cells of adult animals. In the testis of vehicle-treated immature monkeys no positive staining could be detected in peritubular cells. Following follicle-stimulating hormone (FSH) or testosterone treatment, immunostaining for alpha-smooth muscle actin appeared in peritubular cells of immature animals. In comparison to FSH, testosterone was more effective in inducing the differentiation of peritubular cells. The most intense label for alpha-smooth muscle actin was, however, obtained after combined treatment with FSH and testosterone. A computer-assisted image analysis system was used to evaluate semiquantitative data for the alpha-smooth muscle actin positive area; this confirmed a significantly higher expression of alpha-smooth muscle actin in peritubular cells of immature monkeys after treatment with testosterone plus FSH in comparison to controls. These data present evidence that the differentiation of primate peritubular cells during pubertal sexual development is a hormone-dependent process, which may be predominantly regulated by androgens. FSH exerted additive effects on the androgen-induced differentiation of peritubular cells, suggesting that paracrine communication between peritubular cells and Sertoli cells existed. Maintenance of peritubular cell differentiation after withdrawal of hormonal stimuli in adult monkeys may be a possible explanation for different hormonal requirements during reinitiation of spermatogenesis after testicular involution.

Decreased Responsiveness to Progesterone of Spermatozoa in Oligozoospermic Patients

Abstract: Spermatozoa from oligozoospermic subjects are characterized by a reduced in vitro ability to penetrate hamster oocytes and by a decreased responsiveness to physiological stimuli that trigger the acrosome reaction. One of the first steps in the induction of the acrosome reaction is an increase of intracellular free calcium concentrations ([Ca2+]i). It has been recently shown that progesterone (P) is able to increase [Ca2+]i in capacitated human sperm at concentrations similar to those found in follicular fluid. We evaluated sperm [Ca2+]i increase in response to P (0.1 micrograms/ml) in 19 normo- and 17 oligozoospermic subjects. The average percentage of [Ca2+]i increase over the basal level was significantly lower in spermatozoa from oligozoospermic subjects when compared to normozoospermic subjects (138.7 +/- 8.22% increase in oligo- versus 263.3 +/- 39.7% increase in normozoospermic subjects; P < 0.001). Progesterone-stimulated [Ca2+]i increase was significantly correlated with sperm motility (r = 0.54), sperm concentration (r = 0.96), and sperm morphology (% of normal forms) (r = 0.49). In addition P induced a significant increase of acrosome-reacted spermatozoa in normospermic patients (n = 10), whereas no significant effect was observed in spermatozoa from oligozoospermic men (n = 7). Taken together, these results indicate that spermatozoa from oligozoospermic men have a reduced ability to initiate the cascade of events that lead to the acrosome reaction in response to a physiological stimulus, such as P, and might contribute to explaining the reduced fertilizing capacity of these patients.

Neuroendocrine Cells in the Human Prostate Gland

Abstract: O ne prostatic cell type, whose sole role is regulatory, which produces several different growth regulatory factors, which is abundantly present in all prostates, and which co-proliferates as a malignant epithelial component in most, if not all, prostatic adenocarcinomas has been almost totally ignored until very recently. This cell is the neuroendocrine (NE) cell (also known as the endocrineparacrine or APUD cell) (di Sant’Agnese, I 992a).

Antifertility effects of neem (Azadirachta indica) oil in male rats by single intra-vas administration : an alternate approach to vasectomy

Abstract: An alternate approach to vasectomy for long-term male contraception following a single intra-vas application of a traditional plant (Azadirachta indica) product having immunomodulatory properties is described. Male Wistar rats of proven fertility were given a single dose (50 microliters) of neem oil in the lumen of the vas deferens on each side; control animals received the same volume of peanut oil. Animals were put on continuous mating 4 weeks after the treatment, with females of proven fertility. While the control animals impregnated the female partners, all males treated with neem oil remained infertile throughout the 8 months of observation period. Epididymal and vas histology were normal without any inflammatory changes or obstruction. The intra-vas administration of neem oil resulted in a block of spermatogenesis without affecting testosterone production; the seminiferous tubules, although reduced in diameter, appeared normal and contained mostly early spermatogenic cells. No anti-sperm antibody could be detected in the serum. Unilateral administration of neem oil in the vas resulted in a significant reduction of testicular size and spermatogenic block only on the side of application; the draining lymph node cells of the treated side also showed enhanced proliferative response to in vitro mitogen challenge. These results indicate that the testicular effects following intra-vas application of neem oil may possibly be mediated by a local immune mechanism.

Relationship of semen quality, number of sperm inseminated, and fertility in rabbits.

Abstract: The relationship between the total number of sperm inseminated, semen quality, and fertility in rabbits was investigated, using fractionated or unfractionated semen and different diluting fluids. Semen was from Dutch-belted males collected twice weekly with an artificial vagina. All does were superovulated except in Experiment 3. In Experiment 1, sperm were fractionated on discontinuous 4% and 10% bovine serum albumin columns. Sperm from each portion of the gradient, along with unfractionated controls, were diluted to give 0.25 x 10(6), 0.5 x 10(6), 1.0 x 10(6), and 2.0 x 10(6) total sperm per insemination. In Experiment 2, sperm were diluted with Dulbecco's phosphate-buffered saline to provide 0.10 x 10(6), 0.50 x 10(6), and 1.0 x 10(6) total sperm per insemination, with minimal processing time. In Experiment 3, does were allowed to kindle after inseminating 0.1 x 10(6) or 1.0 x 10(6) sperm. In Experiment 4, sperm were diluted with TALP buffer: seminal plasma 1:1 to 0.025 x 10(6), 0.05 x 10(6), and 0.10 x 10(6) total sperm per insemination. Over 2,800 embryos or unfertilized oocytes were obtained either 24 or 48 hours after insemination to measure fertility. Sperm numbers required for normal fertility were 0.50 x 10(6) in Experiment 1 and only 0.05 x 10(6) in Experiment 4. This reduction presumably was due primarily to reduced processing time and diluent change. Litter size was normal with 0.1 x 10(6) sperm (Experiment 3). In Experiment 4, computer-assisted sperm analysis (HTM 2030 system; Beverly, Massachusetts) was adapted to successfully screen out some of the "interfering" granules in rabbit semen.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and localization of copper-zinc superoxide dismutase gene expression in rat testicular development.

Abstract: Reactive oxygen species are highly toxic agents that appear to have an important role in male infertility. In order to understand the potential for the testis to be protected from reactive oxygen, the mRNA levels of the natural reactive oxygen scavenger, copper-zinc superoxide dismutase (SOD), were determined in testes and other organs in rats using northern analysis and in situ hybridization. Northern analysis of total RNA from organs of 60-day-old rats demonstrated an SOD mRNA with a transcript length of 0.77 kb; its concentration was highest in the kidney, liver, testis, and epididymis. In testis, northern analysis of total RNA demonstrated two mRNA transcripts of 0.77 kb and 0.94 kb. The concentrations of the 0.77-kb transcript varied only slightly between 10 and 100 days of age. In contrast, the 0.94-kb transcript became detectable by northern analysis between 30 and 40 days of age, then its concentration rose progressively to peak at 60 days. In situ hybridization studies demonstrated a uniform distribution of SOD mRNA within seminiferous tubules of prepubertal rats at 10 days of age and a heterogeneous, stage-specific pattern in older animals. In mature rats, the highest level of SOD mRNA was detected in tubules just prior to spermiation (stages VI-VIII). In conclusion, two SOD mRNA transcripts were identified in the rat testes that followed significantly different patterns of expression during development. In situ hybridization studies revealed that accumulation of the SOD mRNA in the seminiferous tubule was stage specific. These data suggest that SOD may play an important role during testicular development and spermatogenesis in rats.

Endocrinological, Biophysical, and Biochemical Parameters of Semen Collected via Masturbation versus Sexual Intercourse

Abstract: In clinical programs of assisted reproduction involving infertile males, it is essential to obtain semen of maximum quality. To evaluate ways of achieving this objective, and to assess the fertilizing capacity of the sperm, six semen samples were collected from each of 38 infertile men via masturbation. Six more samples were then collected from each man at sexual intercourse using a semen collection device (SCD). We confirmed that the volume of seminal plasma, total sperm count, sperm motility, and percentage of morphologically normal spermatozoa were significantly higher in samples collected at intercourse than masturbation, as reported previously. In addition, the markers of the secretory function of the prostate and the outcome of sperm function tests (hypoosmotic swelling test, acrosin assay, and sperm penetration assay) were significantly higher for the samples collected at intercourse. There were no significant differences in markers of the secretory function of the seminal vesicles and epididymis between the samples. The improved spermatozoal parameters in the samples collected at intercourse may reflect a higher prostatic secretory function at that time. There were no significant differences in the serum concentrations of gonadotropins, or in the serum or seminal plasma concentrations of testosterone, before or after masturbation or sexual intercourse. Therefore, the differences in prostatic secretory function and semen parameters may not be attributed to differences in hormonal levels. Semen collection during intercourse using an SCD appears to be the method of choice for selecting semen samples for artificial insemination.

The acrosome reaction-inducing effect of human follicular and oviductal fluid.

Abstract: The human sperm acrosome reaction (AR) can be induced in vitro by a variety of naturally occurring and synthetic compounds. The present investigation determined the effect of human natural cycle periovulatory follicular (hFF) and oviductal fluids (hOF) on the human sperm AR in dose—response fashion using the synchronous AR assay. When hFF (30% v/v) or hOF (40% v/v) was added to non-capacitated spermatozoa, no significant (P > 0.05) increase in the % AR was detected in comparison to the non-treatment control. When either of these compounds was added (10%, 20%, and 30% v/v hFF; 20%, 30%, and 40% v/v hOF) to capacitated spermatozoa (3 hour incubation), a significant (P 0.05) increase in the % AR of capacitated spermatozoa in comparison to control. Inhibitors of protein kinases A (KT5720) and C (calphostin C) were tested to determine their effects on the hFF-induced AR. In comparison to hFF treatment alone, the kinase A inhibitor KT5720 (50 nM, 100 nM) prevented hFF (20%) stimulation of the AR when added at the end of the capacitation period and 5 minutes prior to the addition of inducer. Similarly, the kinase C inhibitor calphostin C (50 nM, 100 nM) prevented hFF (20%) stimulation of the AR. The present data demonstrate that periovulatory hFF and hOF stimulate the human sperm AR. hFF appears to stimulate the AR by activating multiple signal transduction pathways.

Protection by Gonadal Steroid Hormones Against Procarbazine-Induced Damage to Spermatogenic Function in LBNF1 Hybrid Rats

Abstract: Some anti-cancer drugs, such as procarbazine (PCZ), are associated with irreversible damage to spermatogenic function and cause sterility in men. In the present study, protection of spermatogenesis by gonadal steroid hormones during PCZ treatment was investigated in male rats. LBNF1 hybrid rats were chosen for these studies because the response of the testis to PCZ was more uniform than in a stock of Sprague-Dawley outbred rats. Mature male LBNF1 rats were subcutaneously implanted with cholesterol (C)-, testosterone (T)-, and/or estradiol-17 beta (E)-containing capsules, and 5-10 weeks later given one or four weekly intraperitoneal injections of PCZ at a dose of 200-250 mg/kg of body weight. Hormone capsules were removed 24 hours after the last PCZ injection and the animals were killed 10-12 weeks later. Testicular weights, sonication-resistant sperm head counts, and quantitative testicular histology revealed protection of the spermatogenic epithelium from PCZ toxicity by gonadal steroid hormones when compared with the C controls. Protection was observed against both single and multiple injections of PCZ. A combination of T and E capsules provided better protection than T alone. This model system is suitable for studies of the mechanism of protection of spermatogenesis from chemotherapy-induced damage.

Immunoreactive ubiquitin in human seminal plasma

Abstract: The polypeptide ubiquitin, up to now almost exclusively discovered in intracellular spaces, was measured immunologically in a total of 187 samples of human seminal plasma. The values were between 1.83 and 19.11 micrograms/ml. In spermatozoa ubiquitin was detected too; the values, however, were significantly lower than in the seminal plasma. The origin and function of ubiquitin in human seminal plasma is still unclear. The possible role of ubiquitin in reproduction is discussed.

Sampling Factors Influencing Accuracy of Sperm Kinematic Analysis

Abstract: Sampling conditions that influence the accuracy of experimental measurement of sperm head kinematics were studied by computer simulation methods. Several archetypal sperm trajectories were studied. First, mathematical models of typical flagellar beats were input to hydrodynamic equations of sperm motion. The instantaneous swimming velocities of such sperm were computed over sequences of flagellar beat cycles, from which the resulting trajectories were determined. In a second, idealized approach, direct mathematical models of trajectories were utilized, based upon similarities to the previous hydrodynamic constructs. In general, it was found that analyses of sampling factors produced similar results for the hydrodynamic and idealized trajectories. A number of experimental sampling factors were studied, including the number of sperm head positions measured per flagellar beat, and the time interval over which these measurements are taken. It was found that when one flagellar beat is sampled, values of amplitude of lateral head displacement (ALH) and linearity (LIN) approached their actual values when five or more sample points per beat were taken. Mean angular displacement (MAD) values, however, remained sensitive to sampling rate even when large sampling rates were used. Values of MAD were also much more sensitive to the initial starting point of the sampling procedure than were ALH or LIN. On the basis of these analyses of measurement accuracy for individual sperm, simulations were then performed of cumulative effects when studying entire populations of motile cells. It was found that substantial (double digit) errors occurred in the mean values of curvilinear velocity (VCL), LIN, and MAD under the conditions of 30 video frames per second and 0.5 seconds of analysis time. Increasing the analysis interval to 1 second did not appreciably improve the results. However, increasing the analysis rate to 60 frames per second significantly reduced the errors. These findings thus suggest that computer-aided sperm analysis (CASA) application at 60 frames per second will significantly improve the accuracy of kinematic analysis in most applications to human and other mammalian sperm.

Effects of Finasteride on the Rat Ventral Prostate

Abstract: The objectives of this study were to compare changes in the ventral prostate (VP) of young adult Sprague Dawley rats after 28 days of treatment with finasteride (F), a potent 5α-reductase inhibitor, with those caused by castration (Cx). VP concentrations of DHT were reduced to 20.2% and 6.6% of controls (1,947 ± 207 pg/VP, mean ± SE) by F (5 or 20 mg/kg/day) and to 2.6% of controls by Cx. VP weights were reduced 49% and 54% by F and 88% by Cx. DNA/VP fell 25% and 15% with F treatment and 72% after Cx, whereas RNA and protein/VP were reduced 37–51% by F and 91–93% by Cx. The RNA/DNA and the protein/DNA ratios fell to 30–36% of controls after F treatment and to 70% of controls after Cx. The mRNA concentrations of the C3subunit of prostatein 28S ribosomal RNA fell after treatment with F (5 mg/kg/day) and after Cx, whereas the mRNA for TRPM-2, an androgen-suppressed protein associated with apoptosis, was increased only after castration. To examine further the effects of F on the rate of DNA synthesis, 7-day regressed adult rats were treated for 3 days with testosterone propionate ± F, and incorporation of 3H-thymidine in minced ventral prostates was determined. F inhibited 3H-thymidine incorporation. We conclude that Cx causes a greater reduction in cell number/VP and a greater reduction in RNA and protein/cell than F and that the differences between F treatment and castration probably result from differences in prostatic concentrations of T. However, we cannot exclude the possibility that small differences in DHT contribute to these differences.

Effects of epidermal growth factor on human sperm cell function.

Abstract: The effects of human recombinant epidermal growth factor (EGF) on the fertilizing capacity of human sperm were investigated. At lower concentrations (0.1-10 nM) EGF did not significantly (P > 0.05) affect human sperm penetration of zona-free hamster oocytes (SPA). At higher concentrations (25-100 nM), EGF significantly (P = 0.01 to or = 25 nM), EGF also significantly inhibited the spontaneous as well as calcium ionophore-induced acrosome reaction and release of acrosin from human sperm. There was no effect of EGF on percent sperm motility, but at higher concentrations (> or = 25 nM) it significantly affected various sperm motility characteristics especially velocity and amplitude of lateral head displacement. EGF was detected by radioimmunoassay as well as radioreceptor assay in seminal plasma of fertile men. However, there were no statistical differences between the levels of EGF or EGF/transforming growth factor (TGF-alpha) in seminal plasma of fertile, infertile, and immunoinfertile men. Also, there was no significant correlation of the EGF or EGF/TGF-alpha levels with total sperm number, sperm motility characteristics, and penetration rates in SPA in these patients. These results indicate that EGF has either no effect or an inhibitory/negative effect on the human sperm cell capacitation and/or acrosome reaction, especially at higher concentrations (> 25 nM).

Studies on Sperm Chromatin Structure Alterations and Cytogenetic Damage of Mouse Sperm Following In Vitro Incubation. Studies on In Vitro-Incubated Mouse Sperm

Abstract: Mouse epididymal sperm incubated in Tyrode's T6 fertilization media were analyzed over time for chromosome damage by two methods. First, cytogenetic analysis was done on paternal pronuclei metaphase chromosomes. After 6 hours incubation 11% of the cells demonstrated chromosome structural abnormalities. Secondly, sperm nuclei were measured by the sperm chromatin structure assay, which is a measure of the susceptibility of sperm DNA to the nuclei demonstrated an increased susceptibility to DNA denaturation, reaching near 100% by 48 hours. Changes in chromatin structure at the molecular level may lead to chromosome breaks seen in pronuclear chromosomes.

Comparison of Motility and Flow Cytometric Assessments of Seminal Quality in Fresh, 24‐Hour Extended and Cryopreserved Human Spermatozoa

Abstract: Functional differences among fresh 24-hour extended and cryopreserved human spermatozoa were assessed using both computer-assisted semen analysis (CASA) and flow cytometry. The objective was to determine if there were interrelationships among various qualitative parameters of the fresh and treated samples when assessed by these two automated methods. Fertile donor specimens (n = 15) were split and examined for sperm motility and curvilinear velocity using CASA within 1 hour postejaculation, after 24 hours in TEST-yolk buffer at 5 degrees C and after cryopreservation in TEST-yolk-glycerol medium. Flow cytometric analyses were performed on 24-hour extended and cryopreserved (CP) samples after fluorescent staining with rhodamine 123 to quantify mitochondrial function and carboxydimethyl fluorescein diacetate and propidium iodide to assess plasma membrane integrity. The percentages of spermatozoa with functional mitochondria and intact membranes along with the proportion of dead cells were identified and quantified by flow cytometry. Quadrant analyses of these data were used to determine the relative red and green fluorescent intensities. The initial sperm motility was correlated to the motility observed for the 24-hour stored and the CP samples. The sperm velocity of both the initial and the 24-hour extended samples was correlated to the velocity of CP samples. As for the comparison of the two automated methods for assessing seminal quality, the only sperm motion parameter that was correlated with a sperm population identified by flow cytometry (quadrant 4) was the curvilinear velocity of the sperm after 24 hours storage (r = 0.69) and after cryopreservation (r = 0.74). The present findings indicate that additional research is needed to determine if prefreeze analyses of donor sperm could be useful in predicting the post-thaw integrity of CP samples and, thereby, be useful in screening potential semen donors.

Computer‐Assisted Semen Analysis (CASA) of Epididymal Sperm from the Domestic Cat

Abstract: Motion characteristics of epididymal sperm from domestic cats exhibiting a high (> 60%; normozoospermic; n = 21) or low (< 40%; teratozoospermic; n = 6) occurrence of structurally normal spermatozoa were correlated with morphology (MOR) using computer-assisted semen analysis (CASA). Mean values and standard errors for percent motility (MOT), curvilinear velocity (VCL), linearity (LIN), straight line velocity (VSL), and amplitude of lateral head displacement (ALH) were recorded for 3 hours. Average values for percent normal spermatozoa, MOT, VCL, VSL, and ALH were higher (P < 0.01) in samples from normozoospermic cats than from teratozoospermic cats at 0 hours, and there was no difference in motion parameters over the 3-hour incubation period in either group. Strong correlations (P < 0.01) existed between MOR and VCL, VSL, ALH, or MOT, but not LIN, upon regression analysis. We conclude that (1) motion parameters of domestic cat sperm are significantly correlated with morphology and (2) abnormal motion parameters associated with low fertility potential in other species are prevalent in samples from teratozoospermic cats. The correlation between morphology and altered sperm movement found in this study suggests that motion analysis of spermatozoa by CASA may be useful in evaluating fertilization potential in felids.

Comparison of Effects of 0.5 and 3.0 Gy X‐Irradiation on Lipid Peroxidation and Antioxidant Enzyme Function in Rat Testis and Liver

Abstract: The prooxidant effect of X-irradiation on rat testis and liver tissue was studied with doses of 0.5 and 3.0 Gy; the latter dose kills the proliferating spermatogonia and causes a maturation-depletion process in the germ cells. The level of lipid peroxidation, measured by the formation of diene conjugates and thiobarbituric acid-reactive substances (TBARS) and the activities of the antioxidant enzymes were determined 0.5 hours, 1 day, 7 days, and 31 days after the exposure. In the liver, increased levels of diene conjugation (+36%, P < 0.05) in the group of 3.0 Gy at 0.5 hours indicated increased lipid peroxidation. At the same time, TBARS were increased (+25%, P < 0.05) in the group of 0.5 Gy, but not in the 3.0-Gy group. In the testis, diene conjugation was not determined at 0.5 hours postirradiation, and at day 1 it was at the control level. The level of TBARS in the testis was below control (-11%, P < 0.01) in the 3.0-Gy group at day 1. At day 31 after 3.0 Gy in the testis, an increase in the amount of conjugated dienes (+24%, P < 0.01) was observed in parallel with a decreased level of TBARS (-15%, P < 0.01). The activity of superoxide dismutase (SOD) was decreased in the testis at 0.5 hours postirradiation (-28%, P < 0.05, and -29%, P < 0.05, in the groups of 0.5 and 3.0 Gy), whereafter it returned to normal by day 7. In the liver, such inactivation of SOD was not observed.(ABSTRACT TRUNCATED AT 250 WORDS)