Showing papers in "Journal of Andrology in 1994"

Dual DNA staining assessment of bovine sperm viability using SYBR-14 and propidium iodide.

Abstract: A new membrane-permeant DNA stain, SYBR-14, was used in combination with propidium iodide (PI) to estimate the proportion of living sperm in bovine semen. The SYBR-14 stained living sperm while PI only stained degenerate cells that had lost their membrane integrity. Staining with SYBR-14 resulted in the nuclei of living sperm fluorescing bright green. Aliquots containing nearly all living bovine sperm were prepared using glass wool/Sephadex filtration to remove dead and damaged cells. A portion of this filtered sample was killed by unprotected freeze-thawing and used to provide mixed aliquots containing known ratios of living and dead sperm. Flow cytometry was used to assess the green and red fluorescence of these mixtures. The percentages of living sperm, as determined by the log of green fluorescence, were 85.1, 68.8, 39.8, 20.7, and 1.4 for ratios of 100:0, 75:25, 50:50, 25:75, and 0:100 of the filtered, killed mixtures. Also, bovine semen was diluted 1:60 in HEPES-0.1% bovine serum albumin and incubated for 0, 3, 6, and 24 hours at 36 degrees C to assess changes in cell viability. As cell death occurred during this incubation period, a relatively rapid transition of staining from green to red occurred as sperm died. Three replicates of cryopreserved sperm from six bulls were also examined using SYBR-14 and PI to assess the proportion of living and dead cells. Flow cytometric analyses of these samples, which had been processed and stored in homogenized milk, indicated that this stain combination was useful in assessing the quality of cryopreserved sperm. The combination of SYBR-14 and PI was determined to be an effective tool for assessing the viability of fresh or cryopreserved sperm.

Leukocytic infiltration into the human ejaculate and its association with semen quality, oxidative stress, and sperm function

Abstract: Immunocytochemical techniques have been used to monitor the size and composition of the leukocyte population in unfractionated human semen samples and sperm populations generated by Percoll gradient centrifugation. The characteristics of the leukocyte population have then been related to the quality of the semen profile, the production of reactive oxygen species, and the functional competence of the spermatozoa. A majority (97%) of the ejaculates examined contained leukocytes, and in 82.4% the major cell type was the granulocyte. Small numbers of T cells, B cells, and monocytes/macrophages could also be found in 62%, 43%, and 21% of samples, respectively, and patients were occasionally identified in whom one of these cell types became the predominant leukocyte species. Although a subpopulation of patients was identified in whom the infiltration of multiple leukocyte species was positively correlated with the concentrations of spermatozoa and precursor germ cells in semen, in general, the presence of leukocytes, to the point of leukocytospermia, did not significantly influence any component of the semen profile. Similarly, the fertilizing potential of the washed spermatozoa, as assessed by in vitro tests of the acrosome reaction and sperm-oocyte fusion, was not correlated with the concentration of seminal leukocytes. In contrast, the carryover of leukocytes into the washed sperm preparations profoundly influenced the fertilizing potential of the spermatozoa via mechanisms that were associated with the production of reactive oxygen species. These results have implications for the diagnostic significance of leukocyte contamination in the context of male infertility and assisted conception.

A ten-year safety study of the oral androgen testosterone undecanoate

Abstract: Testosterone undecanoate (Andriol) is an oral androgen that provides the hypogonadal patient with the unmodified testosterone molecule It was introduced in the mid-1970s This is a report on the safety of this oral androgen Of 35 men originally included in the study, 33 could be followed up for a minimum of 10 years In them no alteration in the biochemical parameters of liver function could be detected Upon annual measurements (7-9 hours after ingestion of testosterone undecanoate), serum levels of testosterone ranged between 54 +/- 19 and 65 +/- 19 nmol/L (normal range 8-24) and of 5 alpha-dihydrotestosterone between 32 +/- 18 and 35 +/- 17 nmol/L (normal range 08-25) These levels remained constant during the study period, indicating that there is no increased hepatic enzymatic breakdown of the androgen over time Eight men were older than 50 years at the start of the study Over the 10-year period in two of them a mild reduction in urine flow was measured, whereas in the other six this could not be demonstrated Digital examination of the prostate did not reveal signs of prostate tumors Testosterone undecanoate appears to be a safe oral androgen A yearly checkup of the patient on therapy with this androgen seems adequate

The Genetic Basis of Congenital Bilateral Absence of the Vas Deferens and Cystic Fibrosis

Abstract: Congenital bilateral absence of the vas deferens (CBAVD) has long been thought to be a rare and distinct clinical and genetic entity. However, it occurs in 1-2% of the infertile male population (Jequier et al, 1985). Recently, this disorder has been shown to represent a mild (reproductive) form of cystic fibrosis (CF) (Anguiano et al, 1992). This finding mandates that proper genetic counseling take place for patient, spouse, and appropriate family members. With the advent of techniques such as microscopic epididymal sperm aspiration (MESA) or Moni’s Window, pregnancies have been achieved in couples where the male partner is afflicted with CBAVD. What was once a brief and frustrating conversation between patient and urologist has now been markedly expanded to include a discussion on the hopeful possibilities of fatherhood but also on the myriad of other concerns that the diagnosis of potential CF raises. Complete bilateral absence of the scrotal vasa is the hallmark of CBAVD. For the purposes of this discussion, CBAVD will be defined in a strict sense because some of the partial forms of vasal agenesis, such as unilateral vasal agenesis, may not have the same genetic or pathophysiological basis as does classic CBAVD in otherwise healthy men. Therefore, CBAVD will be defined as occurring only when both scrotal vasa are deficient to palpation and/or exploration, either partially or totally. If either vasa is palpable from its origin to the external ring, CBAVD is not present. By adherence to this definition, we can segregate various types of vasal agenesis into different clinical Minireview

The direct pituitary effect of testosterone to inhibit gonadotropin secretion in men is partially mediated by aromatization to estradiol.

Abstract: In men, administration of exogenous testosterone (T) exerts direct negative feedback effects at the pituitary as well as at the hypothalamic level. This study was undertaken to determine whether T itself causes the inhibitory effects on the pituitary, or whether conversion to estradiol (E2) or dihydrotestosterone (DHT) is required. We assessed the biological activity of serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as immunoactivity. Blood samples were drawn before, during, and after a continuous, 72-hour i.v. infusion of T (15 mg/day), E2 (90 micrograms/day), or DHT (500 micrograms/day). Each of these doses is twice the daily production rate of the steroid. Each man received each of the three steroid infusions. We studied four men, ages 23-35, with idiopathic hypothalamic hypogonadism (IHH), who were treated with pulsatile gonadotropin releasing hormone (GnRH) until their gonadotropins reached the normal range. Serum levels of T, E2, DHT, and levels of immunologically active and biologically active LH and FSH were measured. We found that administration of each steroid increased serum levels of the infused steroid to the upper physiologic range. Administration of T or E2 resulted in decreased mean levels of biologically and immunologically active LH and FSH; administration of DHT did not alter gonadotropin secretion. These data suggest that some of the direct effect of T at the pituitary level in men is mediated by E2, whereas peripherally formed DHT may not play an important role in this process.

Age-Related Decreased Leydig Cell Testosterone Production in the Brown Norway Rat

Abstract: Previous studies have demonstrated that Leydig cell testosterone production diminishes with age in Brown Norway rats. The objective of the studies presented herein was to test the following possible explanations for age-related decline in steroidogenesis: (1) decline in Leydig cell number; (2) understimulation by luteinizing hormone (LH); (3) reduced ability of individual Leydig cells to produce testosterone; and (4) influence of loss of germ cells. Leydig cells isolated from the testes of young and aged rats by centrifugal elutriation and Percoll density gradient centrifugation were examined for their ability to produce testosterone when stimulated maximally with LH or with dibutyryl cyclic AMP (dbcAMP). Leydig cell number and volume were examined in situ using stereological methods. Serum LH levels were measured using a highly sensitive immunofluorometric assay. Average Leydig cell volume decreased with age, and consistent with this observation, individual Leydig cells isolated from aging rats produced significantly less testosterone than those from young rats whether the cells were cultured in vitro with maximally stimulating LH or with dbcAMP. The age-associated diminished testosterone production could not be explained by changes in Leydig cell number, serum LH levels, Leydig cell responsiveness to LH, or testicular germ cell content. These results, taken together, suggest that the reduced testosterone production seen in aged rats is related to defects in the steroidogenic pathway beyond the LH receptor-cAMP cascade. The nature of the initial age-related changes that cause reduced steroidogenesis is not known, and therefore it is not known whether such changes are intrinsic or extrinsic to the Leydig cells.

Effect of low-dose testicular irradiation on sperm count and fertility in patients with testicular seminoma.

Abstract: The treatment of seminoma with radiation therapy risks transient infertility. We have prospectively followed eight patients with stage I seminoma of the testicle. All patients underwent radical orchiectomy of the affected testis. The mean age of the patients was 32.9 years (range 24-40). Each patient was treated with megavol- tage radiation with a 10- or 18-MV linear accelerator. The remaining testicle was shielded using a standard lead enclosure, and the mean testicular dose was 44 cGy (range 20.8-78.2). Semen specimens were delivered to the lab within 30 minutes of ejaculation. All spec- imens were analyzed using a computer-assisted sperm analyzer. Pretreatment parameters were within normal limits for all but one patient; one patient presented with a borderline normal sperm count at 18 and 22 x 10#{176}/mI. Following treatment, there was a decrease in sperm count, detected at 3 months, to <10 x 10#{176}/mi (range 4.4- 8.6 x 106) in all patients except one, who presented with an initial pretreatment count of 189 x 10#{176}/mI, which decreased to 58 x 10#{176}/ ml at 3 months, 32 x 10#{176}/mI at 6 months, and rose to 325 x 106/ ml by 12 months following treatment. Although the sperm count for this patient (D.L.) was within the normal range, the post-radiation sperm count was less than 20% of the pretreatment count. There was no difference in the motility at 3 months, the mean of which was 51.3%. One patient's (F.C.) wife conceived at 9 months following treatment, one at 12 months (J.R.), and one (J.S.) at 14 months, and all have delivered normal infants. Of the remaining patients, four patients recovered sperm counts within the normal range by 1 year (range 38-325 x 10#{176}/mI), one by 18 months, and one by 30 months. Of the two remaining patients, one did not return for sperm analysis after the couple had a child using banked sperm, and the second was able to conceive a child at 14 months and believed that a sperm count was superfluous. The earliest recovery was seen at 12 months. In the present study, recovery was seen at doses of �65 cGy, although the one patient receiving a dose of 90 cGy conceived nat- urally and did not return for follow-up sperm analysis after a sperm

Correlation between the rate of lipid peroxidation and cellular maturity as measured by creatine kinase activity in human spermatozoa.

Abstract: We have demonstrated previously that creatine kinase (CK) activity is a measure of cellular maturity and fertilizing potential in human spermatozoa. In the present work we have examined whether there is a relationship between sperm CK activity and the rate of lipid peroxidation (LP) as measured by malondialdehyde (MDA) formation. Both MDA production and CK activity were higher in oligospermic than in normospermic specimens (P < 0.001, N = 41 and 101, respectively), and there was a close correlation (R = 0.43, P < 0.001) between these two biochemical parameters. As demonstrated previously with the CK measurements, there was a heterogeneity among the groups: About 40% of the oligospermic men had MDA and CK activity values similar to that of the normospermic group, and 12% of the normospermic men had MDA and CK activity values similar to that of the oligospermic group. We have also examined in three experimental paradigms the question of sperm-to-sperm propagation of increased LP and the possible increase in LP following centrifugation as used in sperm preparation for assisted reproduction: The MDA differences among Percoll sperm fractions originating within the same specimens, the lack of change in MDA production after co-centrifugation and co-incubation of samples with high and low sperm LP rates, and the repeated centrifugation of the same specimens without an increase in MDA production all indicated the lack of sperm-to-sperm propagation of LP or increase in LP due to mechanical stress. However, the iron content of Ham's F-10 medium or iron added to human tubal fluid medium has significantly increased the rate of LP in various sperm samples. Exposure to iron during sperm preparation procedures in assisted reproduction is likely to affect sperm integrity and fertilizing potential. We can con-clude that there are highly significant differences in the rate of LP among men and among sperm within the same specimen. We suggest, based on the relationship between LP and CK activity, that the increased rate of LP along with the increased CK content are due to incomplete cytoplasmic extrusion during terminal spermatogenesis. The retained excess cytoplasm in immature sperm fuels LP. Thus, the increased rate of spontaneous LP is an “inborn” rather than an “acquired” property of spermatozoa.

Mechanism of superoxide dismutase loss from human sperm cells during cryopreservation

Abstract: Earlier studies on human sperm cryodamage have shown that plasma membrane stress is the primary process and that phospholipid peroxidation in cryopreserved samples is not inhibited by addition of antioxidants. One consistent effect of cryopreservation is loss of enzymatic activity of the peroxidation defense enzyme, superoxide dismutase (SOD). To clarify this aspect of the freeze-thaw process and to develop a more complete resolution of the reactions leading to cryodamage, we sought to identify which of the two most probable mechanisms, loss of enzyme protein from the cells of denaturation of the protein, operates. If the first operates, cellular enzymatic activity and enzyme protein as identified by immunocytochemistry should give a linear correlation. If the second operates, there should be no correlation. In this study, five individual samples were analyzed before and after cryopreservation for immunoreactive Cu/Zn SOD and cell intactness by flow cytometry, for SOD enzymatic activity by a highly sensitive fluorimetric method, and for motility characteristics by Hamilton-Thorn motility analyzer. Fresh samples were obtained by the "swim-up" method and had > 95% intact cells with > 78% motile cells. After freeze-thaw, about half the cells were intact. SOD enzymatic activity was determined on Triton X-100 cell extracts, a method that removes all enzymatic activity from the cell structure, and compared with immunoreactive SOD in the cells as determined by indirect immunofluorescence mean intensities. Residual immunofluorescence was observed in the cells after Triton X-100 treatment; if this was taken into account, a close linear correlation between SOD enzyme activity and SOD immunoreactivity was obtained (r = 0.90; P = 0.00014). There was no correlation between SOD enzyme activity ratios for cryopreserved and fresh cells and fraction of intact cells after freeze-thaw. We conclude that loss of SOD protein from the subset of cells undergoing acute membrane damage is the most probable primary mechanism of SOD enzymatic activity loss from the sample and that resistance to cryodamage and SOD activity in any given cell are quite independent of one another.

Acute Immobilization Stress Disrupts Testicular Steroidogenesis in Adult Male Rats by Inhibiting the Activities of 17α‐Hydroxylase and 17,20‐Lyase Without Affecting the Binding of LH/hCG Receptors

Abstract: We have investigated the effect of acute immobilization (3 hours) stress on testicular steroidogenesis in the adult rat. Immobilization did not alter plasma luteizing hormone (LH) levels, but plasma testosterone (T) levels were reduced by 82%. Plasma levels of corticosterone in stressed rats were elevated more than ninefold over control levels. After 3 hours of stress, testicular levels of progesterone were elevated 33%, and levels of 17 alpha-hydroxyprogesterone and T were reduced 47% and 37%, respectively, compared to controls. Immobilization for 3 hours had no effect on the association or dissociation rate constants of LH/human chorionic gonadotropin (hCG) receptors of testicular interstitial cells and did not alter specific hCG binding. The effect of 3 hours of immobilization on testicular 17 alpha-hydroxylase and 17,20-lyase was assessed by incubating testicular microsomes from stressed and control animals in the presence of 21[14C]progesterone and [3H]17 alpha-hydroxyprogesterone. Immobilization of rats reduced the Vmax values of 17 alpha-hydroxylase and 17,20-lyase by 47% and 48%, respectively, but had no effect on the Km values. These results support the hypothesis that stress for 3 hours disrupts rat testicular steroidogenesis via a mechanism that is independent of changes in circulating levels of LH and the binding characteristics of LH/hCG receptors. The effects of immobilization on the content of testicular steroids and on the activities of 17 alpha-hydroxylase and 17,20-lyase suggest that stress inhibits the activities of both 17 alpha-hydroxylase and 17,20-lyase.

Effect of bovine oviductal estrus-associated protein on the ability of sperm to capacitate and fertilize oocytes.

Abstract: At estrus, an oviduct-specific, estrus-associated glycoprotein (EAP) of molecular weight 85–95,000, is secreted by the oviductal epithelium and found in cannula-derived bovine oviductal fluid (ODF). The otyectives of these studies were to determine if bovine sperm were capacitated by EAP in vitro, whether this effect differed for EAP derived from ODF versus conditioned medium from oviduct ampullar explants, and to determine if sperm capacitated in vitro with EAP-fertilized bovine eggs. Sperm were incubated for up to 6 hours with partially purified EAP derived from ODF and assessed for capadtation by their ability to undergo the acrosome reaction following exposure to lysophosphatidyichotine. At 4 hours of incubation, the number of capacitated sperm in treatments containing 50% ODF or EAP plus bovine serum albumin (BSA) was similar, and it was significantly greater than the number of capacitated sperm in treatments containing antibodies to EAP. Using purified EAP derived from ampullar explant-conditioned medium, twice the number of sperm were capacitated after 4 hours with EAP from conditioned medium or with ODF than with treatments containing anti-EAP. The fertilizing ability of sperm incubated with EAP was significantly greater than that for sperm incubated without EAP or with anti-EAP. We conclude that bovine EAP, derived from both ODF and in vitro cultures, promotes sperm fertilizing ability.

The Testicular Iron Shuttle: A “Nurse” Function of the Sertoli Cells

Abstract: The techniques of cell culture and molecular biology first revealed that Sertoli cells synthesize transferrin. Accumulated biological information led to a plausible model for the role of testicular transferrin in an iron shuttle system designed to transport ferric ions around the cellular tight junctions to the germ cells inside the blood-testis barrier. Experiments done in culture and in vivo have supported many aspects of this model. A mutant mouse model that lacks the ability to synthesize transferrin is defective in spermatogenesis and may help to delineate the nature of the iron requirement by germ cells. The levels of seminal transferrin, possibly of Sertoli cell origin, are proportional to sperm production in humans and cattle and may be an effective indicator of Sertoli cell function. The testicular iron shuttle thus represents an important "nurse cell" function of the Sertoli cells.

Regulation of Sperm Motility: Emerging Evidence for a Major Role for Protein Phosphatases

Abstract: Lesions in the normal expression of sperm motility are associated with certain types of male infertility. Therefore, an understanding of the signal transduction pathways regulating sperm motility will be an important part of our understanding of the mechanisms underlying these types of male infertility. In this connection, the regulation of sperm fiagellar motility by second messengers is a rapidly expanding area of research in cell and reproductive biology. While abundant information can be found concerning protein kinases and phosphoproteins in relation to sperm motility, relatively little attention has been paid to the potential role for protein phosphatases in this process. This review will focus attention on protein phosphatases, in the context of current literature and more recent studies, as a growing area of interest in relation to the role that protein phosphorylation plays in the regulation of motility of sperm and other axoneme-containing cells.

Lipid Peroxidation and Antioxidant Enzyme Activities in the Rat Testis after Cigarette Smoke Inhalation or Administration of Polychlorinated Biphenyls or Polychlorinated Naphthalenes

Abstract: Lipid peroxidation products and antioxidant enzyme activities were studied in the rat testis following exposures to cigarette smoke, polychlorinated biphenyls (PCBs), or polychlorinated naphthalenes (PCNs). Three hours after a single 1-hour period of smoke inhalation, the levels of fluorescent chromolipids and thiobarbituric acid-reactive species (TBARS) were markedly increased in the testis (+49%, P < 0.01, and +43%, P < 0.05, respectively). Twelve hours after daily smoking for 1 hour, for 1, 5, or 10 days, such an increase was not found. Activities of the antioxidant enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), glutathione transferase (GSH-Tr), or hexose monophosphate shunt (HMS) were not affected immediately, 3 hours, or 12 hours after a single smoking session. Twelve hours after smoking for 5 days, the activity of catalase was decreased (-16%, P < 0.05). Smoking exposures had no consistent effects on serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), or testosterone concentrations. Single i.p. injections of PCB or PCN mixtures resulted in decreases in testicular SOD activity 1 day after the exposures (-14%, P < 0.05, and −51%, P < 0.01, respectively). Catalase activity also decreased after both exposures (−30 to −42%, P < 0.05, at days 1–7 after PCB exposure, and −37 to −43%, P < 0.05, at days 3–7 after PCN exposure). Ninety days after the PCN exposure, activities of GSH-Px and GSH-Tr were decreased in the testis (−20%, P < 0.05, and −26%, P < 0.05, respectively). The only statistically significant change in lipid peroxidation measurements in the testes of PCB- or PCN-treated rats was a decrease in TBARS by 13% (P < 0.01) 1 day after PCN exposure. The main findings of this study were the increase in lipid peroxidation in the rat testis after cigarette smoke inhalation and the impairment of the function of the enzymatic antioxidant defense after exposures to PCBs or PCNs. These results suggest that free radical-dependent mechanisms may play an important role in the testicular toxicity of environmental chemicals.

Spermagglutination by Bacteria: Receptor‐Specific Interactions

Abstract: The influence of genital infection on infertility has yet to be elucidated. We examined receptor-ligand interactions between sperm and Escherichia coli from patients with prostatitis. Two E. coli surface adhesins (P-fimbriae, type 1 fimbriae) and their specific receptor saccharides (alpha-galp-1-4-beta-galp-O-methyl [gal-gal], mannose) were evaluated. Bacterial concentrations of 10(4) caused spermagglutination. P-fimbriae caused tail-tail spermagglutination that was inhibited by gal-gal. D-mannose concentrations are highest in the acrosomal region and type 1 fimbriae caused head-head agglutination that was inhibited by mannose. Strains with both fimbriae caused head-head and tail-tail agglutination that was inhibited by a mannose/gal-gal combination. E. coli agglutinated 40-75% of motile sperm. Seminal fluid provided 50-100% protection, with lower effectiveness against type 1 fimbriae. Understanding bacteria-spermatozoa interactions at the receptor-ligand level holds potential for treatment of infertility and development of spermagglutinating contraceptives.

Is FSH required for adult spermatogenesis

Abstract: in the adult testis and for the restoration of spermatogenesis (and fertility) in testes induced experimentally to become azoospermic. In contrast, despite years of intensive study, the role of follicle-stimulating hormone (FSH) in the regulation of spermatogenesis in the adult mammal remains far from settled. In particular, whether or not FSH is required to maintain spermatogenesis once it is established, and whether or not it is required to restore spermatogenesis after azoospermia is reached, are still debated in the literature and remain matters of controversy.

Human Androgen Receptor Binding to the Androgen Response Element of Prostate Specific Antigen

Abstract: This study examined the in vitro interaction of the human androgen receptor with a putative androgen response ele- ment (ARE) in the promoter region of the prostate specific antigen (PSA) gene. To characterize the androgen receptor's interactions with its DNA response elements we expressed the full length human androgen receptor protein in a baculovirus expression system. The receptor was shown to be 110 kDa by sodium dodecyl sulfate poly- acrylamide gel electrophoresis (SOS-PAGE) and was purified using ion-exchange column chromatography. Binding of the synthetic an- drogen Ri 881 to the unpurified recombinant receptor exhibited a k. of 7.6 nM by Scatchard analysis. In DNA gel electromobility shift assays the promoter region from PSA (a 313-bp fragment) was bound by the unpurified recombinant androgen receptor in a se- quence-specific manner. An ARE-containing sequence from the pro- moter region of the PSA gene was synthesized as a 30-bp oligo- nucleotide and was shown to bind specifically to the human androgen receptor in gel electromobility shift assays by DNA competition and by antibody supershifts of the receptor-ARE complex. The specific binding of the insect cell expressed androgen receptor to its ARE was shown to occur even in the absence of androgen. Androgen receptors purified by ion-exchange chromatography were unable to bind to ARE, suggesting the presence of other factors required for DNA binding.

Increased Levels of Interleukin‐6 in Seminal Plasma of Infertile Men

Abstract: The presence of various cytokines, namely the tumor necrosis factor (TNF-alpha), interferon (IFN-gamma), and interleukins (IL-1 beta and IL-6), was investigated in seminal plasma of fertile, infertile, and immunoinfertile men using specific immunoradiometric assays. TNF-alpha and IL-1 beta were not detected. IFN-gamma was detected, but the differences between the levels of fertile and infertile/immunoinfertile were not significant (P > 0.05). IL-6 was detected in seminal plasma with significantly higher levels in infertile/immunoinfertile men compared to those of fertile men. IL-6 was also present in sera, and interestingly, the levels in sera were lower than those in seminal plasma. IL-6 levels in seminal plasma correlated significantly with some sperm parameters and penetration rates in the human sperm penetration assay (SPA). These findings suggest that IL-6 is associated with infertility and may be of importance in specific diagnosis and treatment of male infertility.

DNA Organization in Human Spermatozoa

Abstract: Previous studies from this laboratory on hamster spermatozoa have demonstrated that rodent sperm DNA is packaged into the sperm nucleus in a specific manner by nuclear structures The entire genome is organized into DNA loop domains attached at their bases to a sperm nuclear matrix, the skeletal structure of the nucleus When nuclei are completely decondensed, the nuclear matrix dissipates, and the entire genome remains anchored to a single structure located at the base of the tail, termed the nuclear annulus Here, we have extended these studies to human sperm nuclei, which were found to be similar to hamster Human sperm DNA was found to be organized into loop domains attached at their bases to a nuclear matrix The average size of the human sperm halo of DNA surrounding the extracted sperm nucleus (made up of DNA loop domains) was about 50% smaller than those that have been reported for somatic cells (this corresponds to an approximate loop domain size of 268 ± 21 kb) Human sperm DNA also remained anchored to the base of the tail when completely decondensed, indicating the existence of a nuclear annulus-like structure in human spermatozoa; but, unlike the hamster nuclear annulus, the human annulus could not be isolated because of its structural instability when separated from the tail Using human centromere repeats as a probe for in situ hybridization, we examined the packaging of individual DNA sequences within the sperm nucleus These studies demonstrate that human sperm DNA is highly organized by nuclear structures

Human Osteoblast-Like Cells Contain Specific, Saturable, High-Affinity Glucocorticoid, Androgen, Estrogen, and 1α,—Dihydroxycholecalciferol Receptors

Abstract: Glucocorticoids, androgens, estrogens and 1 alpha,25-dihydroxycholecalciferol (1 alpha,25-(OH)2D3) exert a variety of effects on bone homeostasis We have measured the receptors for these hormones in cultured human osteoblast-like cells Specific binding was found with each of the four steroids tested, namely maximum binding capacities, Bmax, of 2,581 (1,368-4,223), 146 (101-237), 176 (20-552), and 387 (290-620) fmolmg DNA-1 (mean, range) for [3H]dexamethasone, [3H]5 alpha-dihydrotestosterone, [3H]17 beta-estradiol ([3H]E2), and [3H]1 alpha,25-(OH)2D3, respectively The corresponding apparent dissociation constants were 133 (92-222), 043 (023-094), 045 (017-088), and 0020 (0008-0030) nM, respectively Both high-affinity, low-capacity and low-affinity, high-capacity specific [3H]E2 binding were demonstrable In conclusion, we could demonstrate that human osteoblast-like cells contain specific glucocorticoid receptors In addition specific androgen, estrogen, and 1 alpha,25-(OH)2D3 binding was demonstrated These cells contain several times more binding sites for dexamethasone than for dihydrotestosterone, estradiol, and 1 alpha,25-(OH)2D3, a feature that might contribute to the marked sensitivity of human bone to glucocorticoids

Comparative study of experimentally induced benign and atypical hyperplasia in the ventral prostate of different rat strains.

Abstract: The induction of prostatic lesions in the rat by hormonal manipulation and/or chemical carcinogens in different strains is well documented in literature. However, the selection of a strain for such studies was merely based on the laboratory husbandry facilities rather than on the animal genotype. It is well accepted that the morphology and function of the prostate exhibit a marked species-specific difference that makes extrapolation to other animals and man difficult. Our group has developed an experimental model for the study of rat prostatic hyperplasia and/or dysplasia induced by citral—a flavor constituent. Previous observations have revealed that the Wistar rat ventral prostate is reactive to citral, especially during its adolescent growth period as well as during the redifferentiation stage among postcastrated rats treated with exogenous testosterone. The present study presents data on the ventral prostate susceptibility of three additional strains selected by their interrelated endocrine morbidity. In order to facilitate an objective comparative analysis of the prostatic lesions a histopathological score chart was elaborated. The present findings showed that the Wistar and Sprague-Dawley strains were the most susceptible to developing benign and atypical prostatic hyperplasia both in intact and postcastrated rats. The F344 and ACI/Ztm rat strains remained refractory toward all of these treatments. Our data provide evidence that the strain genotype and, more precisely, their endocrine background, play a determinative role in the prostatic responsiveness towards citral. It is suggested that additional consideration be given to the selection of an adequate animal strain when studying experimental neoplastic transformation, especially when hormonal interactions might be involved.

Effects of macrophage depletion at different times after treatment with ethylene dimethane sulfonate (EDS) on the regeneration of Leydig cells in the adult rat

Abstract: Testicular macrophages were selectively depleted in the right testes of adult rats by an intratesticular injection of dichloromethylene diphosphonate-containing liposomes (Cl2MDP-lp), whereas the left testes were injected with 0.9% NaCl and served as control. Before or after Leydig cell destruction with ethylene dimethane sulfonate (EDS), treatment with Cl2MDP-lp/NaCl was given at different times to study the requirements of macrophages in the different stages of Leydig cell regeneration. On day 30 after EDS treatment, new Leydig cells were abundant in the left, macrophage-containing testes. However, in the right, macrophage-depleted testes, the number of Leydig cells was related to the time elapsed between EDS treatment and macrophage depletion. When macrophages were depleted on day 10 before or on days 4 or 10 after EDS treatment, new Leydig cells were nearly absent at 30 days. However, when macrophages were depleted on days 16 or 22 after EDS treatment, Leydig cells were found at 30 days, but their numbers were equivalent to the number of Leydig cells that were already present in EDS-treated animals at the time the macrophages were depleted. These results indicate that macrophages are needed for the differentiation of Leydig cells from mesenchymal precursors, as well as for the proliferative activity of the newly formed Leydig cells, possibly through the secretion of essential growth factors.

Interleukin‐6 Enhances the Fertilizing Capacity of Human Sperm by Increasing Capacitation and Acrosome Reaction

Abstract: The effects of human recombinant interleukin-6 (IL-6) on human sperm penetration of zona-free hamster ova (SPA) and human sperm cell capacitation and acrosome reaction were investigated. IL-6 at higher concentrations (60-600 pg/100 microliters) significantly (P < 0.001) increased rather than decreased the human sperm penetration rates in SPA. The effects were specific because immunoadsorption with the specific neutralizing antibody completely abolished the enhancing effects of IL-6. At the same concentrations, IL-6 also significantly enhanced spontaneous as well as calcium ionophore-induced acrosome reaction and release of acrosin from the human sperm cells. There was no effect of IL-6 on percent sperm motility, although it significantly affected various motility characteristics such as velocity, linearity, amplitude of lateral head displacement (ALH), and beat frequency of sperm cells--the motility parameters involved in hyperactivation phenomenon of sperm cells. These results demonstrate for the first time that IL-6 provides a positive signal in enhancing fertilizing capacity of human sperm by increasing capacitation leading to acrosome reaction, and thus it may have clinical applications in the treatment of male infertility in humans.

Coenzyme Q10 concentrations in normal and pathological human seminal fluid.

Abstract: Coenzyme Q10 (CoQ10) levels were assayed in total seminal fluid or both in seminal fluid and seminal plasma in 77 subjects with normal or pathological findings at standard semen analysis CoQ10 levels showed a significant correlation with sperm count and with sperm motility An interesting exception was constituted by patients with varicocele, in whom the correlation with sperm concentration was preserved, whereas the correlation with sperm motility was lacking Moreover, they showed an increased ratio of plasma CoQ to total seminal CoQ10 in comparison with the other subjects These data suggest a pathophysiological meaning of CoQ10 in human seminal fluid and a possible molecular defect in varicocele patients CoQ10 measurement could represent an important examination in infertile patients; moreover, from these results a rationale might arise for a possible treatment with exogenous CoQ10 in dyspermic patients

Atrial natriuretic peptide (ANP) as a stimulus of the human acrosome reaction and a component of ovarian follicular fluid : correlation of follicular ANP content with in vitro fertilization outcome

Abstract: Atrial natriuretic peptides (ANPs) from several species induced the human acrosome reaction. The maximal response was highest for human ANP (18.6% above unstimulated or baseline values) and decreased progressively for peptides derived from animals lower on the phylogenetic scale. ANP concentrations required for a half-maximal effect in noncapacitated spermatozoa ranged from 0.07 to 0.38 nM. ANP induced the acrosome reaction in capacitated spermatozoa, but the concentration required was higher than in noncapacitated cells. The response in noncapacitated spermatozoa was independent of added extracellular Ca2+ and was completely inhibited by 1 microM LY83583 (inhibits particulate guanylate cyclase). However, 10 microM N omega-nitro-L-arginine (inhibits soluble guanylate cyclase) had no effect. ANP (80 pM) and 3 microM 1,2-dihexanoyl-sn-glycerol each induced a nearly half-maximal acrosome reaction. Added in combination, they produced no increased response, suggesting antagonism. Follicular fluid had variable levels of immunoreactive ANP. Average ANP content was nearly zero in samples that contained no oocyte at the time of aspiration but was higher (6.9 pM; 90% confidence limits = 1.67-28.72 pM) in follicular fluid containing oocytes that did not fertilize in vitro. Highest concentrations of ANP were present in follicular fluid containing oocytes that fertilized in vitro (72.8 pM; 90% confidence limits = 38.1-139.1 pM). These data suggest that noncapacitated spermatozoa can acrosome react without added extracellular Ca2+ in response to an extracellular ligand. Also, human spermatozoa appear to contain receptors for ANP similar to those found in other cell types. The ANP content of follicular fluid might partly explain the ability of follicular fluid to induce the acrosome reaction.

Scrotal and Oral Temperatures are not Related to Semen Quality or Serum Gonadotropin Levels in Spinal Cord-Injured Men

Abstract: Scrotal temperature, oral temperature, and the differ- ence between oral and scrotal temperature were measured in spinal cord-injured subjects (SCl) and non-injured subjects as controls. We statistically correlated these measures to semen quality and serum gonadotropin levels in both groups. No difference was found between SCI and control subjects on any temperature measurement. Mean sperm motility, mean sperm morphology, and mean serum gonad- otropin levels were significantly lower in SCI compared to control subjects, but these measures were not correlated to scrotal tem- perature, oral temperature, or the difference between oral and scrotal temperature in SCI or control subjects. These data indicate that: 1) there is not a generalized scrotal thermoregulatory dysfunction in SCI men; 2) scrotal temperature does not appear to contribute to poor semen quality in SCI men; and 3) elevated gonadotropin levels are not related to elevated scrotal temperatures in SCI men, as has been reported in non-injured, infertile men.

Effect of Follicular or Oviductal Fluids on Movement Characteristics of Bovine Sperm during Capacitation In Vitro

Abstract: Mammalian sperm exhibit characteristic motility changes associated with capacitation. Movement characteristics of bovine sperm incubated in noncapacitating (control, medium alone), capacitating (oviduct fluid, nonluteal, and luteal), or capacitating, acrosome reaction inducing (follicular fluid) conditions were investigated using a computer-assisted automated semen analysis system. Sperm were incubated up to 4 hours in a modified Tyrode's medium (control), 20 and 60% nonluteal (NL) or luteal (L) oviduct fluid (ODF), or 20 and 60% follicular fluid (FF). Relative to sperm incubated in control medium, motility of sperm treated with ODF or FF had increased linearity and vigorous motility. Sperm incubated in 60% ODF or FF showed a small decrease in mean trajectory/path straightness and velocity over time compared to 20% fluid treatments and control. Frequency distribution graphs were symmetric for 20% NL- and L-ODF treated sperm. However, 20% FF and 60% ODF and FF treatments had distributions skewed to the left, indicating smaller values for lateral head displacement (ALH) and curvilinear velocity (VOL). Me dian values for ALH and VCL were determined for control-treated sperm, and subtracted from individual sperm values for all treatments to estimate deviation from control, designated ALHc and VCLc. Three-dimensional plots of ALHc, VCLc and corresponding frequency indicated shifts in peak patterns for fluid-treated sperm compared to control sperm. Incubation in 20% ODF and FF resulted in peak shift for ALH and VCL values; yet, little change in peak position was observed in sperm incubated in 60% ODF and FF. Although mean values decreased for ALH and VCL during the 4-hour incubation, three-dimensional plots suggested an increase in the frequency of individual sperm with these parameters. This study provides additional information on bovine sperm motion during in vitro capacitation and indicates that the presence of ODF and FF influences sperm motion characteristics.

Consistency of Sperm Morphology Classification Methods

Abstract: The World Health Organization (WHO) recently suggested new morphometric dimensions for normal sperm heads and new sperm head classification rules. These specifications differ from previous WHO methods and from the Kruger method for strict morphology assessment. We analyzed the WHO and Kruger sperm classification rules to determine their appropriateness as predictive linear models. We reviewed the theoretical requirements for allometric modeling, a more traditional approach for specifying the size relationships between phenotypic characters, and developed valid allometric models based on the statistical properties of over 3,700 measurements of individual sperm head dimensions. We implemented the WHO, Kruger, and Allometric methods on the same set of digitized sperm heads to study the empirical consequences of different metric requirements and classification methods. Results show that the WHO and Kruger methods are internally consistent, but specify nonconstant variance. Hence, they are inappropriate linear models. The allometric models were internally consistent and specified constant variance. Hence, they are appropriate linear models. Significant differences were found in the percentage of normal sperm between the methods. On average, the Kruger method produced the lowest value and the Allometric methods produced significantly higher values than the WHO methods, but no average difference was found between WHO methods or between Allometric methods. Nevertheless, the percentage of normal sperm did change significantly for individual specimens. These results indicate that small differences in metric requirements and classification rules can dramatically change the percentage of normal sperm. Until more rigorous definitions of sperm features can be developed, and valid statistical models can be used to describe the population characteristics of fertile sperm, the prognostic value of sperm morphology is limited.

The ethane dimethanesulfonate-induced decrease in the fertilizing ability of cauda epididymal sperm is independent of the testis

Abstract: Several decades ago it was reported that when adult male rats were exposed to a single injection of 50 mg/kg body weight ethane dimethanesulfonate (EDS) and mated with untreated females, average litter size was significantly reduced as early as 2 weeks later. Recently, we demonstrated that EDS exerts multiple effects in the epididymis of adult rats. Some of these effects were independent of reduced serum testosterone (T) levels. Later we found that EDS has direct effects on epididymal epithelial cells in vitro. Herein, we sought to determine whether EDS perturbs the fertilizing ability of cauda epididymal sperm. Four days after exposure to 50 mg/kg EDS, sperm from the proximal cauda epididymidis were inseminated into adult receptive females in utero; on the next day the percentage of fertilized eggs was determined. Exogenous T administration and castration were used to determine what role, if any, androgen deprivation and the testis had on the fertilizing ability of proximal cauda epididymal sperm. Sperm motion parameters, serum T, T in the caput/corpus epididymidis, and detergent-extracted sperm protein were evaluated and correlated with fertilizing ability. We found that both castration and EDS exposure significantly compromised the fertilizing ability of sperm in proximal cauda epididymidis 4 days after exposure. Exogenous T, sufficient to maintain serum T, completely restored the fertilizing ability of sperm following castration, but not after EDS exposure. Moreover, exogenous T failed to restore fertilizing ability when castrated animals were exposed to EDS. Thus, the effects that EDS exerts on sperm maturation in vivo are independent of the testis. Finally, the only endpoint that was well correlated with fertilizing ability was the relative amount of an acidic 18-kDa sperm protein.

Stimulation of cryopreserved epididymal spermatozoa of the domestic cat using the motility stimulants caffeine, pentoxifylline, and 2'-deoxyadenosine.

Abstract: We have investigated the effects of caffeine, pentoxifylline, and 2'-deoxyadenosine on the motion characteristics and longevity of domestic cat spermatozoa. Freshly collected or cryopreserved domestic cat epididymal sperm were incubated with 0.01-20 mM caffeine, pentoxifylline, or 2'-deoxyadenosine for 15 minutes at 23 degrees C. The percent motility (MOT), curvilinear velocity (VCL), linearity (LIN), straight line velocity (VSL), and amplitude of lateral head displacement (ALH) were determined for each group using computer-assisted semen analysis. Freshly collected domestic cat sperm exhibited a strong forward progressive movement, and treatment with caffeine, pentoxifylline, or 2'-deoxyadenosine did not consistently alter sperm motion. Following cryopreservation, spermatozoa exhibited decreased (P < 0.05) MOT, VCL, VSL, and ALH. Caffeine and pentoxifylline increased (P < 0.05) the MOT, VSL, VCL, and ALH of cryopreserved sperm at 0.01-20 mM, in a dose-dependent manner. 2'-Deoxyadenosine also increased (P < 0.05) both VSL and VCL at 1.0 mM, and MOT, VSL, VCL, and ALH at 10 mM. All treatments shifted the percentage of nonhyperactive sperm to either a transitional or hyperactivated state. The motility indices of cryopreserved samples were examined during a 6-hour incubation to assess the effects of caffeine, pentoxifylline, and 2'-deoxyadenosine on sperm longevity. Compared to untreated control samples, the longevity of stimulated cryopreserved sperm was not reduced. These results indicate that motility stimulants may prove useful for enhancing the fertility of cryopreserved cat sperm by increasing their motility and producing hyperactivated motion.