bluuebunny/crossref_metadata_2025 huggingface dataset:
Computer Tracking of Germ Cells in the Cycle of the Seminiferous Epithelium and Prediction of Changes in Cycle Duration in Animals Commonly Used in Reproductive Biology and Toxicology
Authors: HESS REX A. , CHEN PAUL
Published: October 2023

ABSTRACT: A computer program called Stages was written to aid the tracking of germ cells and stages forward and backward through time in the cycle of the seminiferous epithelium. The program incorporates the basic kinetics of spermatogenesis in the rat, mouse, hamster, guinea pig, dog, rabbit, bull, stallion, ram, boar, quail, monkey, and man. The program is flexible and permits the user to alter the cycle duration time and the frequency of each stage. Compiled for use on personal computers and available on floppy disks free of charge, Stages is menu‐driven and requires no knowledge of programming. The program was tested using published data of testicular toxicity and vitamin A synchronization of stages. In general, predicted cell types were similar to those observed; discrepancies between observed and predicted data are discussed. When cycle duration time was changed, predicted data for stage synchronization coincided with the observed data. This program will improve the speed and accuracy of studying factors that affect spermatogenesis. By using Stages, it is possible to predict the target cell types in a toxicity study and to track affected cells over long periods of time. Predicted cell types may also indicate which cells to examine biochemically as well as morphologically in subsequent experiments. The program will also be useful to beginning students learning the complex patterns of cellular associations and the progression of germ cells in the cycle of the seminiferous epithelium.
Effects of Caudal Elevation on Testicular Function in Rats Separation of Effects on Spermatogenesis and Steroidogenesis
Authors: DEAVER D. R. , AMANN R. P. , HAMMERSTEDT R. H. , BALL R. , VEERAMACHANENI D. N. R. , MUSACCHIA X. J.
Published: October 2023

ABSTRACT: A variety of biologic processes are perturbed when exposed to microgravity (space flight) for more than 7 days, including testicular function. Suspension of rats in a special harness (caudal elevation) to induce thoracic pooling of blood fluids and remove the support function of the hind limbs is used to mimic, on earth, the effects of microgravity encountered during space flight. Typically, this induces cryptorchidism in male rats. Three experiments were conducted to differentiate the effects of caudal elevation (30° angle) and anatomic location of testes on spermatogenesis and steroidogenesis. Rats were subjected to caudal elevation for 7 days using either a tail harness (experiments 1 and 2) or a whole‐body harness (experiment 3). Testes of rats fell into the abdominal cavity when a tail harness was used, but ligation of the inguinal canal prevented this repositioning. For rats with abdominal testes, testicular weight was reduced (P < 0.05) and histology of testes was abnormal; the number of spermatids per gram parenchyma was lower (P < 0.05) in tail‐suspended rats compared with control rats. In contrast, spermatogenesis was not affected by caudal elevation in most rats in which the inguinal canal was ligated or in rats elevated by whole‐body harness. Concentrations of testosterone in serum and testicular interstitial fluid were lower (P < 0.05) in suspended rats, regardless of the method used for caudal elevation or anatomic location of testes. Concentrations of luteinizing hormone in serum were elevated (P < 0.05) in rats with intra‐abdominal testes. The authors conclude that caudal elevation of rats using either a whole‐body harness or a tail harness after ligation of the inguinal canal results in altered steroidogenesis but normal spermatogenesis. Because this is similar to what occurs in the testes of male rats exposed to space flight, caudal elevation can be a useful model system to study the mechanisms responsible for reduced testosterone secretion during exposure to microgravity.
Evidence for Increased Lipid Peroxidative Damage and Loss of Superoxide Dismutase Activity as a Mode of Sublethal Cryodamage to Human Sperm During Cryopreservation
Authors: ALVAREZ JUAN G. , STOREY BAYARD T.
Published: October 2023

ABSTRACT: Cryopreservation of human sperm, now generally required in donor insemination programs, adversely affects the sperm in terms of standard sperm evaluation parameters and fertilizing ability. The freeze‐thaw process appears to produce sublethal damage that appears only after a delay. The authors hypothesized that cryopreservation enhanced peroxidation of sperm membrane lipids, based on previous studies of sperm lipid peroxidation, which showed that the effects of peroxidative damage became evident only after a delay, depending on the peroxidation rate. The effect of cryopreservation on the phospholipid content, the composition of the acyl moieties of the phospholipids, and the activities of the peroxidation protective enzymes, superoxide dimutase (SOD) and glutathione peroxidase plus reductase, in human sperm were examined to test the hypothesis. Parallel determinations were made of the percent motility, the average path velocity of the motile cells, and the time to loss of motility under specified aerobic incubation conditions, which gives a good estimate of the lipid peroxidation rate. The phospholipid content decreases after cryopreservation, with loss of phosphatidylcholine and phosphatidylethanolamine being the more pronounced. Polyunsaturated acyl moieties were also preferentially lost. This loss pattern is observed also from lipid peroxidation. The activities of glutathione peroxidase plus reductase remained unchanged. The sperm SOD activities varied widely between samples before cryopreservation. In all samples there was a decline in SOD activity after freeze—thaw, but the extent of the decline was also widely variable. The time to loss of motility declined in parallel with SOD activity, and a strong correlation (R2 > 0.9) between SOD activity and time to loss of motility was found for all samples, before and after freeze—thaw. The authors conclude that cryopreservation does enhance lipid peroxidation in human sperm, as hypothesized, and that this enhancement is mediated at least in part by the loss of SOD activity occurring during the process.
Cryopreservation of Human Semen: Comparison of Cryopreservatives, Sources of Variability, and Prediction of Post‐thaw Survival
Authors: CENTOLA G. M. , RAUBERTAS R. F. , MATTOX J. H.
Published: October 2023

ABSTRACT: Human semen was cryopreserved using Human Sperm Preservation Medium, TEST‐Yolk buffer, or glycerol alone. Sperm characteristics for each specimen were measured before and after freezing to determine which cryopreservative resulted in better cryosurvival and recovery of motile sperm. Sperm frozen in Human Sperm Preservation Medium had a significantly better recovery of all semen parameters (motility, velocity, and recovery) than either TEST‐Yolk or glycerol alone. Statistical analyses also were done to examine the variability between and within donor semen specimens. Differences between donors, between specimens, and measurements within donors all contributed to variability of sperm characteristics. Specimen‐to‐specimen variability for a given donor represented 12% to 47% of the total variability, whereas processing and measurement variability represented 12% to 41%. Donors also varied in the ability of their sperm to tolerate freezing. There was a relationship between motile count after dilution with cryopreservative and post‐thaw motile count. This relationship allows the prediction of poor‐thaw survival before freezing a specimen.
Reactive Oxygen Species and Human Spermatozoa
Authors: DE LAMIRANDE EVE , GAGNON CLAUDE
Published: October 2023

Abstract: Mammalian spermatozoa are sensitive to oxygen‐induced damages mediated by lipid peroxidation of the cell membrane. The aim of this study was to evaluate whether reactive oxygen species (ROS) could also induce axonemal damage. When Percoll‐separated spermatozoa were treated with hydrogen peroxide, or the combination xanthine and xanthine oxidase (X + XO), there was a progressive decrease, leading to a complete arrest, in sperm flagellar beat frequency. Once demembranated in a medium containing magnesium adenosine triphosphate (Mg.ATP), ROS‐immobilized spermatozoa still reactivated motility; however, the percentage and duration of motility obtained in these tests gradually decreased to zero in the next hour. In 50% of the cases, motility of intact spermatozoa spontaneously reinitiated after 6 to 24 hours of immobilization due to ROS treatment, although with percentages and beat frequencies lower than those of untreated spermatozoa. Studies using ROS scavengers (such as catalase, superoxide dismutase, and dimethylsulfoxide) indicated that hydrogen peroxide was the most toxic of the ROS involved, but that ·O2− and ·OH probably also played a role in immobilization of spermatozoa by ROS. The data suggest that ROS induce a chain of events leading to sperm immobilization, that axonemes are affected, and that limited endogenous repair mechanisms exist to reverse these damages.
Simultaneous Proliferation and Differentiation of Mast Cells and Leydig Cells in the Rat Testis
Authors: GAYTAN F. , ACEITERO J. , LUCENA C. , AGUILAR E. , PINILLA L. , GARNELO P. , BELLIDO C.
Published: October 2023

Abstract: The proliferation and differentiation of mast cells and Leydig cells were studied in adult sham operated or hypophysectomized rats after the administration of ethylene dimethane sulphonate (EDS) and in prepubertal rats after neonatal treatment with a gonadotropin‐releasing hormone (GnRH) antagonist (Organon 30276; Oss, The Netherlands). After treatment with EDS, two proliferative waves were found. On day 3, several interstitial cell types proliferated, whereas mitotic cells corresponded to differentiating Leydig cells and mast cells around day 20. Differentiating Leydig cells showed a higher mitotic index than that of differentiating mast cells. Hypophysectomized animals showed high mitotic activity 3 days after treatment, but 21 days after treatment differentiating Leydig cells were absent and proliferative activity was reduced. The number of mast cells increased from day 15 to day 30 in EDS‐treated rats and from day 15 to day 50 in hypophysectomized, EDS‐treated rats. GnRH antagonist‐treated rats showed poorly differentiated Leydig cells and abundant mitotic figures on day 23. Proliferation and differentiation of Leydig cells occurred concomitantly with the proliferation and differentiation of mast cells between 23 and 30 days of age. These results suggest that Leydig cells and mast cells in the rat testis share some common regulatory factors.
Atrial Natriuretic Factor Inhibits the Phosphorylation of Protein Kinase C in Plasma Membrane Preparations of Cultured Leydig Tumor Cells
Authors: PANDEY KAILASH N.
Published: October 2023

ABSTRACT: The peptide hormone atrial natriuretic factor (ANF) exerts its effect in a receptor‐mediated fashion and the membrane‐bound form of guanylate cyclase represents a biologically active ANF receptor; thus, cGMP has been considered a second messenger of ANF. To understand the mechanisms of ANF action, we have studied its effect on protein phosphorylation in the plasma membrane preparations of murine Leydig tumor (MA‐10) cells, which overexpress guanylate cyclase‐coupled ANF receptor molecules in high density. After pretreatment of the plasma membranes with ANF (100 nM), a marked decrease in phosphorylation of the 78‐kDa protein kinase C (PKC) and the 240‐kDa protein was observed. Phosphorylation of the 78‐kDa PKC was also inhibited by cGMP (0.1 mM); however, phosphorylation of the 240‐kDa protein was not affected by cGMP. The quantitative analyses, as determined by densitometric scanning, revealed that both ANF and cGMP inhibited phosphorylation of the 78‐kDa PKC by approximately 75% and 45%, respectively. The inhibitory effect of ANF on phosphorylation of the 240‐kDa protein was almost 90%, but cGMP did not show any discernible effect on its phosphorylation in plasma membranes of MA‐10 cells. Phosphorylation of the 78‐kDa PKC was stimulated by Ca2+ and phospholipids, and it immunologically cross‐reacted with antiserum against brain PKC. Furthermore, in these plasma membrane preparations, the 78‐kDa PKC was immunoprecipitated and its phosphorylation was inhibited by ANF. These data provide evidence for a new signal transduction mechanism of ANF that negatively regulates phosphorylation of the 78‐kDa PKC and the 240‐kDa protein in a cGMP‐dependent and ‐independent manner in Leydig cells.
Effects of Castration and Recombinant Human Inhibin Administration on Circulating Levels of Inhibin and Gonadotropins in Adult Male Monkeys
Authors: CHRISTENSEN RICHARD B. , FORAGE ROBERT G. , STEINER ROBERT A. , BREMNER WILLIAM J.
Published: October 2023

ABSTRACT: Inhibin has been suggested to play a role in gonadal feedback regulation of follicle‐stimulating hormone (FSH) secretion; however, neither the half‐life nor the time course of action of recombinant inhibin has been reported in any primate species. We sought to determine the disappearance half‐life of circulating endogenous inhibin following castration in adult male monkeys, Macaca fascicularis, and to determine the half‐life of administered recombinant human inhibin A and its effect on bioactive FSH and luteinizing hormone (LH) levels in castrate monkeys. Endogenous inhibin fell from 8,122 ± 2,077 U/L (mean ± SEM, n = 5) prior to castration to 383 ± 84 U/L at 24 hours and 269 ± 44 U/L at day 21 (P < 0.05 at 24 hours vs. day 21) (detection limit of assay 234 U/L). The early phase half‐life of endogenous inhibin was 34 minutes (between 8 and 60 minutes) and a later phase half‐life of 75 minutes was observed between 1 and 4 hours following castration. Recombinant inhibin exhibited a 14‐minute early phase half‐life between 8 and 60 minutes following the 5 μg intravenous (IV) recombinant inhibin dose, and a later phase half‐life of 70 minutes between 1 and 4 hours in castrate monkeys (n = 3). Serum inhibin levels were maintained within or above the precastration range for 15 minutes. Single dose recombinant inhibin, 100 μg subcutaneous (SC) or intramuscular (IM) administered to castrate monkeys (n = 3), achieved and maintained normal serum inhibin levels for 6 hours. When bioactive FSH and LH responses were assessed by either the posttreatment nadir value or the area under the curve of the posttreatment values relative to the pretreatment baseline, no significant effect was observed following the 0‐, 0.5‐, 5‐, or 50‐μg IV recombinant inhibin dosages over the 10‐hour sampling period. We conclude first that virtually all circulating inhibin is produced by the testis in male monkeys. Second, recombinant inhibin has a short early phase half‐life in monkeys, similar to that of endogenous inhibin. Third, inhibin's putative effect of suppressing FSH secretion may require more prolonged replacement in long‐term castrate monkeys. Fourth, IM or SC administration or recombinant inhibin appears to be a suitable means of achieving replacement serum inhibin levels in castrate monkeys.
Spermagglutination by Bacteria: Receptor‐Specific Interactions
Authors: MONGA MANOJ , ROBERTS JAMES A.
Published: October 2023

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 (α‐galp‐1–4‐β‐galp‐O‐methyl [gal‐gal], mannose) were evaluated. Bacterial concentrations of 104 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.
Zona Pellucida Piercing Enhances Zona Penetration by Spermatozoa from Normospermic and Teratospermic Domestic Cats
Authors: ROTH TERRI L. , HOWARD JOGAYLE , WILDT DAVID E.
Published: October 2023

ABSTRACT: Poor quality ejaculates appear associated with reduced zona penetration and poor fertilization in vitro in some felid species or populations. Even morphologically normal sperm from these teratospermic ejaculates are compromised in ability to penetrate the zona pellucida and fertilize oocytes. A domestic cat model and zona piercing were used to study the function of normal and malformed sperm. Male cats naturally producing different proportions of morphologically normal sperm (group I, >60%; group II, 45–55%; group III, <40%) were identified. Electroejaculates were subjected to sperm washing (WS) and half of these to swim‐up (SU) processing following WS, and all samples were assessed for a sperm motility index (SMI) over time. Zonae of salt‐stored (SS), domestic cat oocytes were mechanically pierced (ZnPd) three times each. Control (nonmanipulated) SS oocytes (n = 430) and ZnPd‐SS oocytes (n = 430) were coincubated with sperm in vitro. The proportion of morphologically normal sperm/ejaculate differed (P < 0.05) among groups averaging 68.8% for group I, 53.5% for group II, and 34.7% for group III. After SU processing, the percent normal sperm among groups was similar (P > 0.05; range, 70.8–80.2%). Average SMI for all groups was high (>70) at 0 hour and decreased over time, but the descent of slope was greater (P < 0.05) for group III than groups I and II. The proportion of oocytes penetrated was not influenced (P > 0.05) by sperm‐processing treatment. For all groups, inner zona penetration was greater (P < 0.05) in ZnPd‐SS than SS oocytes (group I, 52.3% vs. 34.9%; group II, 37.9% vs. 20.3%; group III, 32.0% vs. 17.2%). Similarly, zona piercing increased (P < 0.05) the proportion of oocytes with sperm in the perivitelline space (group I, 11.7% vs. 0.8%; group II, 5.6% vs. 0.0%; group III, 7.0% vs. 0.8%). Although the incidence of penetration by pleiomorphic sperm was low overall, more (P < 0.05) malformed sperm penetrated the inner zona of oocytes in groups II and III than in group I. Most importantly, zona piercing did not increase (P > 0.05) oocyte penetration by aberrant sperm. In summary, zona piercing increases the incidence of inner zona penetration by sperm from both normo‐and teratospermic cats without completely disrupting the ability of the zona to filter pleiomorphic sperm.
A Ten‐Year Safety Study of the Oral Androgen Testosterone Undecanoate
Authors: GOOREN LOUIS J. G.
Published: October 2023

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 5.4 ± 1.9 and 6.5 ± 1.9 nmol/L (normal range 8–24) and of 5α‐dihydrotestosterone between 3.2 ± 1.8 and 3.5 ± 1.7 nmol/L (normal range 0.8–2.5). 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.
Interleukin‐6 Enhances the Fertilizing Capacity of Human Sperm by Increasing Capacitation and Acrosome Reaction
Authors: NAZ RAJESH K. , KAPLAN PAUL
Published: October 2023

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 μl) 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 ionophoreinduced 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.
Suppression of Spermatogenesis by Low‐Level Glycerol Treatment
Authors: IGDOURA SULEIMAN A. , WIEBE JOHN P.
Published: October 2023

ABSTRACT: Previous studies have shown that a single intratesticular injection with 70% glycerol results in rapid and long‐term suppression of spermatogenesis. Because of the extensive testicular damage in the form of focal destruction and large numbers of acellular seminiferous tubules, it was difficult to examine the possible chemical mechanisms of antispermatogenic action. Our objective was to explore regimens of treatment that would result in significant suppression of spermatogenesis without significant testicular damage. Sexually mature Sprague‐Dawley rats were injected intratesticularly with various glycerol concentrations (0, 10, 20, 40, or 70%) and with volumes of either 200 or 350 μl per testis. At times between 1 and 36 weeks after the injection, the effect on weights of testes and accessory sex organs, testicular histology, sperm numbers, serum hormonal levels, and gonadotropin‐receptor binding was examined. An injection of 350 l/testis of a 10% glycerol solution led to a significant decrease in weights of testes and epididymides. The treatment resulted in an overall suppression of spermatogenesis, with about 90% fewer sperm in the epididymides than in control animals. Histologically, the treatment decreased the number of normal tubules from 97% (control) to 16% and resulted in testes in which about 75% of the tubules were aspermatogenic (containing only Sertoli cells and spermatogonia). The number of acellular tubules (tubules without cytological detail) was generally less than 5% of the total, and there was negligible focal destruction. Serum levels of gonadotropins and androgens were not altered significantly by the treatment, and Sertoli cell glutamyl transpeptidase activity appeared normal. An equi‐osmolar solution of glucose also resulted in significant suppression of spermatogenesis, but the effect of glycerol was significantly greater, suggesting a mechanism in addition to hyperosmolarity. This study, therefore, is further evidence for the specificity of glycerol actions and for its potential as an antispermatogenic agent.
Proacrosin Gene Expression in Rat Spermatogenic Cells
Authors: RAAB LINDA S. , HAMILTON DAVID W. , HANCOCK LARRY W.
Published: October 2023

ABSTRACT: Mammalian proacrosin gene expression was considered to be exclusively postmeiotic until recent studies detected the presence of proacrosin mRNA in mouse pachytene spermatocytes. To determine if rat proacrosin gene expression was initiated during meiosis, a 314‐bp proacrosin cDNA fragment was amplified from rat round spermatid RNA, using proacrosin‐specific primers, for use as a probe. Sequence analysis of the round spermatid 314‐bp cDNA fragment confirmed >99% identity with the rat proacrosin cDNA sequence. This 314‐bp fragment was subsequently used for Northern blot analysis of RNA isolated from testicular germ cells. A 1.6‐kb transcript was detected in pachytene spermatocytes, round spermatids, and a mixed population of condensing spermatids/residual bodies, with the highest level of expression in round spermatids. Northern blot analysis of testicular RNA during development revealed the earliest timepoint of expression to be at 24 days of age, further demonstrating the association of proacrosin mRNA with spermatocytes. These data demonstrate diploid expression of the rat proacrosin gene, in agreement with mouse proacrosin gene expression but in contrast to the apparent haploid expression of proacrosin described for the bull and the boar. These studies provide evidence that, in the rat, the process of acrosome biogenesis begins during meiosis.
Mechanism of Superoxide Dismutase Loss from Human Sperm Cells during Cryopreservation
Authors: LASSO JAIME L. , NOILES ESTHER E. , ALVAREZ JUAN G. , STOREY BAYARD T.
Published: October 2023

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 or 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 crypreserved 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.
An Orthotopic Model for the Study of Growth Factors in the Ventral Prostate of the Rat: Effects of Epidermal Growth Factor and Basic Fibroblast Growth Factor
Authors: MARENGO SUSAN RUTH , CHUNG LELAND W. K.
Published: October 2023

ABSTRACT: In order to determine if epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) are capable of stimulating prostatic growth in situ, we complexed EGF and bFGF to a Matrigel (MG) vehicle that was subsequently injected orthotopically into the ventral prostate of adult rats. Three weeks following a single injection of 0.1 ng of EGF or bFGF, the wet weight of the growth factor‐injected lobes of the ventral prostate was increased (P ≤ 0.025) 144 ± 14% and 138 ± 8%, respectively compared to contralateral lobes injected with MG only. Total DNA and protein per lobe (P ≤ 0.025) and the incorporation of bromodeoxyuridine (BrdUrd; P ≤ 0.01) were all significantly increased in lobes injected with EGF or bFGF compared to MG‐injected lobes. Thus, EGF and bFGF were stimulating true growth of the rat ventral prostate in situ. The induced prostatic growth was ligand specific, because co‐injection with neutralizing antibodies abolished EGF and bFGF stimulation of prostatic wet weight. The effect of a single injection of 0.1 ng of growth factor was long‐lasting and elevated prostatic wet weight for 4 (P ≤ 0.05; bFGF) to 6 (P ≤ 0.05; EGF) weeks. Histologic evaluation did not reveal any gross changes in the ratio of stroma to epithelium in either EGF‐ or bFGF‐injected lobes at the 0.1 ng/lobe dose. A slight hyperplasia of the prostatic epithelium was detected in lobes injected with 10 ng of EGF. Of note, the incorporation of BrdUrd was primarily localized in the luminal and basal epithelial cells, whereas incorporation into the prostatic stroma was scanty in lobes injected with either EGF or bFGF. In summary, we have developed an orthotopic model enabling the study of the roles of growth factors in prostatic physiology, function, and disease in situ. Additionally, we have demonstrated that when injected orthotopically into the rat ventral prostate, EGF and bFGF stimulate the growth of the prostatic epithelium to similar degrees.
Long‐term Alterations in the Permeability of the Blood‐Testis Barrier Following a Single Intratesticular Injection of Dilute Aqueous Glycerol
Authors: ENG F. , WIEBE JOHN P. , ALIMA LIMOR H.
Published: October 2023

ABSTRACT: The mechanism whereby glycerol exerts its antispermatogenic action is not known. The objective of this study was to determine, with the use of [3H]inulin and [125I]albumin by in vivo and in vitro methods, whether glycerol exerts its effect by altering the permeability of the blood‐testis barrier (BTB). Adult male rats received a single intratesticular injection of either glycerol (10% or 20%; treated) or saline (control), and 2, 4, 8, 26, and 56 weeks after treatment, either [3H]inulin or [125I]albumin was administered either by infusion or directly to the testicular tissues. Radioactivity was measured in testicular tissue, rete testis fluid, and seminiferous tubular fluid. Following in vivo administration, the uptake of [3H]inulin by seminiferous tubules, rete testis fluid, and seminiferous tubule fluid was significantly greater in the treated than in the control testes at all times after treatment. Radiolabeled inulin, injected into isolated testes or added to medium in which isolated tubules were incubated, accumulated at significantly higher levels in the seminiferous tubule compartment of treated than control tissues. Rete testis fluid from treated testes, collected by micropuncture following efferent duct ligation, contained about 5‐ to 13‐fold more radioactivity than fluid from controls. Following infusion of 50 μCi of [125I]albumin into the jugular vein, the accumulated radioactivity was significantly greater in testicular and epididymal tissues and in the seminiferous tubule fluid from treated than from control animals. In all experiments, the significant differences between treated and control were maintained during the period of 2–56 weeks following glycerol treatment. The results suggest that a single intratesticular injection of dilute glycerol solution alters the permeability of the BTB to macromolecules such as inulin and albumin, and the effect appears to be permanent.
Follicle‐Stimulating Hormone (FSH) Escape During Chronic Gonadotropin‐Releasing Hormone (GnRH) Agonist and Testosterone Treatment
Authors: BHASIN SHALENDER , BERMAN NANCY , SWERDLOFF RONALD S.
Published: October 2023

ABSTRACT: Observations that serum follicle‐stimulating hormone (FSH) levels begin to rise after Initial suppression during chronic gonadotropin‐releasing hormone (GnRH) agonist treatment of men with prostate cancer had led to speculation that FSH escape might in part account for the failure of GnRH agonist analogs to completely suppress spermatogenesis in normal eugonadal men. However, previous studies in healthy young men failed to report FSH escape during GnRH agonist treatment for up to 16 weeks. We considered the possibility that this may have been due to the insensitivity of the FSH assays. Accordingly, using highly sensitive and specific two‐site directed fluorometric assays and a sustained‐release GnRH agonist formulation, we reexamined the issue of whether serum FSH levels rise after initial suppression during chronic GnRH agonist treatment. Two groups of healthy normal men, 19–50 years of age, received 7.5 mg of a long‐acting GnRH agonist microcapsuie formulation (Lupron Depot; TAP Pharmaceutical Company, North Chicago, Illinois) on days 1 and 30. In addition, the subjects received eIther 4 or 8 mg/day testosterone replacement by means of a testosterone microcapsule injected intramuscularly on day 1. Serum luteinizing hormone (LH) and FSH levels were measured by sensitive and specific two‐site directed fluorometric assays on multiple occasions during the 3‐week control period and the 9‐week treatment period. Serum LH levels declined to a nadir between 2 and 4 weeks and stayed suppressed throughout the remainder of the treatment period in both the 4‐ and 8‐mg testosterone groups. In contrast, serum FSH levels, after reaching a nadir, began to rise towards pretreatment control levels in both treatment groups. These data provide unequivocal evidence that FSH levels escape from combined GnRH agonist‐ and testosterone‐induced suppression in healthy young men and add to a growing body of evidence that LH and FSH can be differentially regulated in vivo. Although the mechanisms of this phenomenon remain unclear, FSH escape may be clinically relevant for the therapeutic use of GnRH agonist analogs for male contraception.
Immunoreactive Parathyroid Hormone‐Related Protein Is Present in Human Seminal Plasma and Is of Prostate Origin
Authors: IWAMURA MASATSUGU , ABRAHAMSSON PER‐ANDERS , SCHOEN SUSAN , COCKETT ABRAHAM T. K. , DEFTOS LEONARD J.
Published: October 2023

ABSTRACT: Parathyroid hormone‐related protein (PTHrP) is a regulatory peptide that has been associated with normal fetal growth and differentiation as well as the regulation of fetal calcium. We recently demonstrated the expression of PTHrP in neuroendocrine cells of human prostatic glands. The present study was undertaken to determine if semen contained PTHrP and to determine if there were any differences in seminal PTHrP between normal and vasectomized men. Radioimmunoassay was used to examine whether immunoreactive PTHrP in seminal fluid is secreted by the prostate. Significant quantities of immunoreactive PTHrP were detected in all seminal samples from normal men of ages 24–34 years (mean ± SD, 12.94 ± 8.2 ng/ml). Vasectomy did not decrease the semen levels of PTHrP (25.53 ± 12.14 ng/ml). In addition, immunohisto‐chemical evaluation with a monoclonal antibody used in our previous study did not show PTHrP immunostaining in the seminal vesicles. These findings indicate that seminal PTHrP is predominantly of prostatic origin. Moreover, the significant correlations between PTHrP and calcium levels in human seminal plasma from both normal (P < 0.001) and vasectomized (P < 0.02) patients suggest that PTHrP may serve as a regulatory factor of calcium secretion in the prostate.
Immunocytochemical Localization of Glutathione S‐Transferase Yo Subunit in the Rat Testis and Epididymis
Authors: VERI J. P. , HERMO L. , ROBAIRE B.
Published: October 2023

ABSTRACT:The glutathione S‐transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione with electrophiles. These proteins exist as homo‐ or heterodimers and are separated into five classes (alpha, mu, pi, theta, and sigma). In the present study, the distribution of the GST Yo subunit, a member of the mu family, was examined immunocytochemically in the adult rat testis and epididymis using both light microscopy (LM) and electron microscopy (EM). In the testis, an intense immunoperoxidase reaction was observed over Leydig cells but not macrophages. Within the seminiferous epithelium, only weak reactivity was noted over Sertoli cells, spermatogonia, spermatocytes, and step 1–15 spermatids. There was, however, a progressive and dramatic increase in the intensity of staining in the cytoplasmic lobes of spermatids between steps 16 and 19. Residual bodies, representing the detached cytoplasmic lobes of the late step 19 spermatids, were also intensely stained. Initially seen near the lumen of the tubule, they eventually appeared at different levels of the tubule at stages IX‐XI; none were present at stage XII. Cytoplasmic droplets of step 19 spermatids were also intensely reactive. After spermiation, the cytoplasmic droplets of spermatozoa within the proximal region of the epididymis remained intensely stained. A noticeable decrease in staining was observed in the cauda epididymidis in those droplets that were still there. Quantitation of the labeling density (number of gold particles representing anti‐Yo antigenic sites/μm2) paralleled the LM results; for example, between step 15 and 19 spermatids, a greater than sevenfold increase in labeling density was noted. In the epididymis, a progressive increase in immunoreactivity was observed over epithelial principal cells from the initial segment to the cauda region of this tissue. There was little reactivity over basal, halo, or dear cells. In all reactive cells, gold particles were distributed randomly throughout the cytoplasmic matrix and nucleus. The present work thus demonstrates that, at the end of spermiogenesis, the GST Yo subunit is expressed at high levels in late spermatids. Furthermore, the presence of this protein in late spermatids and cytoplasmic droplets of spermatozoa suggests that this conjugating enzyme may play a role in protecting these cells from electrophilic attack. Also interesting is the correlation between the loss of reactivity in cytoplasmic droplets of spermatozoa of the distal region of the epididymis and the concomitant increase of reactivity in principal cells of this region.
Changes in the Endocrinological Milieu After Clomiphene Citrate Treatment for Oligozoospermia: The Clinical Significance of the Estradiol/Testosterone Ratio as a Prognostic Value
Authors: ITOH NAOKI , TSUKAMOTO TAIJI , NANBU AKIHITO , TACHIKI HITOSHI , NITTA TOSHIKAZU , AKAGASHI KEIGO , MARUTA HIROSHI , KUMAMOTO YOSHIAKI
Published: October 2023

ABSTRACT: We have already reported that the rate of increase in the estradiol/testosterone ratio (E2/T ratio) following human chorionic gonadotropin (hCG) injection has prognostic value in the treatment of oligozoospermia. It was found that the rate of increase in the E2/T ratio was statistically greater in the therapeutically ineffective cases compared to the therapeutically effective cases. The present study was planned in order to elucidate the relationship between the changes in the endocrinological milieu, especially the change in the E2/T ratio, and the therapeutic efficacy when clomiphene citrate was administered. Thirty‐eight oligozoospermic patients were administered 25 mg of clomiphene Citrate daily for 3 months. Sixteen of the 38 (42.1 %) cases showed improvement of more than 10 × 106/ml in sperm concentration. The rate of increase in the E2/T ratio of the effective cases (0.90 ± 0.43) during the administration of clomiphene citrate was statistically (P = 0.02) lower than that of the ineffective cases (1.44 ± 0.80). The rate of increase in the E2/T ratio during clomiphene citrate treatment showed a statistically positive correlation (r= 0.542) with the rate of increase following hCG injection performed before clomiphene citrate treatment. The rate of increase in follicle‐stimulating hormone (FSH) in the ineffective cases (2.16 ± 1.01) was statistically higher than in the effective cases (1.50 ± 0.43). The rate of increase in insulin‐like growth factor‐1 (IGF‐1) in the seminal plasma that was secreted by Sertoli cells was statistically higher in the effective cases (1.98 ± 1.23) than in the ineffective cases (1.19 ± 0.45). Based on these results, it is surmised that the relatively increased E2 level during clomiphene citrate treatment suppressed Sertoli cell function, and the serum FSH was elevated as a result. We concluded that the rate of increase in the E2/T ratio during clomiphene citrate treatment has prognostic value, and performing the hCG test before this treatment may be helpful in predicting the endocrinological milieu after it. If the rate of increase in the E2/T ratio following hCG injection is high, treatment should consist of a combination of clomiphene citrate and an aromatase inhibitor to decrease the E2/T ratio.
Effect of Bovine Oviductal Estrus‐Associated Protein on the Ability of Sperm to Capacitate and Fertilize Oocytes
Authors: King Robin S. , Anderson Sharon H. , Killian Gary J.
Published: October 2023

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.
Significant Role of 5α‐Reductase on Feedback Effects of Androgen in Rat Anterior Pituitary Cells Demonstrated With a Nonsteroidal 5α‐Reductase Inhibitor ONO‐3805
Authors: NAGAMOTO AKIHIRO , NOGUCHI KAZUMI , MURAI TETSUO , KINOSHITA YUZO
Published: October 2023

ABSTRACT: A 5α‐reductase inhibitor with nonsteroidal structure, ONO‐3805, has been used to study the role of 5α‐reductase on positive and negative feedback effects of androgen on gonadotropin secretion in the rat anterior pituitary gland. Initially, the potential of ONO‐3805 to inhibit the formation of 5α‐dihydrotestosterone (DHT) was evaluated. The activity of 5α‐reductase in rat anterior pituitary gland was approximately one‐fourth of that in rat prostate gland or epididymis, with a Michaelis' constant (Km) value for testosterone of 5–6 × 10−7 M. When homogenates of rat anterior pituitary glands were incubated with 14C‐testosterone (14C‐T) in the presence of ≥ 10−7 M ONO‐3805, the formation of labeled DHT and its metabolite 5α‐androstane‐3α, 17β‐diol was inhibited by > 90%. The inhibition pattern was non‐competitive, and the inhibition constant (Ki value) derived from Lineweaver‐Burk plots was 3.9 × 10−11 M. Dispersed pituitary cells (1–2 × 105/ml) were cultured for 48 hours and then further incubated with or without androgen and/or the inhibitor for 72 hours (basal secretion). After this incubation, the media were saved, and 10 nM of luteinizing hormone‐releasing hormone (LH‐RH) with the same concentration of androgen and/or the inhibitor as used in the 72‐hour incubation was added to the cultures for 6 hours (LH‐RH‐induced secretion). Secreted follicle‐stimulating hormone (FSH) and luteinizing hormone (LH) were assayed by radioimmunoassay (RIA). Both testosterone (T) and DHT stimulated 72‐hour basal FSH secretion from cultured pituitary cells in a dose‐dependent fashion. T was less effective than DHT. The addition of inhibitor at > 10−9 M concentration significantly reduced the stimulating effect of T on basal FSH secretion. Basal LH secretion was not affected by either T or DHT with or without the inhibitor. In the presence of 10 nM LH‐RH, 6‐hour secretion of both FSH and LH was significantly reduced by the addition of T or DHT at a concentration of ≥ 100 pg/ml. T (10 ng/ml)‐induced suppression of both FSH and LH was significantly but not completely returned by the addition of 10−7 M of ONO‐3805. These results strongly suggest a significant role of 5α‐reductase on both positive and negative feedback effects of androgen in the rat anterior pituitary gland.
Modulation of Androgen Receptor Transcriptional Activity by the Estrogen Receptor
Authors: KUMAR M. VIJAY , LEO MARK E. , TINDALL DONALD J.
Published: October 2023

ABSTRACT: Estrogen/androgen receptor interactions in naturally occurring physiological systems and the effect of their respective steroid hormones on transcriptional activity remain undefined. In an attempt to delineate further the nature of the interaction between these two steroid hormone receptors we have examined the effect of cotransfection of androgen (AR) and estrogen receptor (ER) cDNAs on the expression of the mouse mammary tumor virus long terminal repeat region (MMTV‐LTR) linked to the chloramphenicol‐acetyl‐transferase (CAT) reporter gene. In QT6 cells, which contain neither AR nor ER, cotransfection of AR cDNA with the MMTV‐LTR‐CAT reporter, resulted in transactivation only in the presence of dihydrotestosterone (DHT). Treatment with 10−8 M each of estradiol‐17β (E2), dexamethasone, or progesterone did not enhance CAT activity, whereas treatment with the androgens DHT and mibolerone resulted in 87% and 89% CAT activity. Transfection of increasing concentrations of ER cDNA in the presence of 100 ng of AR cDNA and 10−8 M each of DHT and E2 showed a dose‐dependent decrease in CAT activity as compared to the response with DHT alone. Cotransfection of AR and ER cDNA in the presence of 10−8 M DHT and increasing concentrations of E2 resulted in a dose‐dependent decrease in CAT activity. When cells were treated with increasing concentrations of DHT with 10−8 M E2 no significant increase in CAT activity was observed. In GC cells, which contain endogenous ER but no AR, cotransfection of AR cDNA and treatment with E2 and DHT, also reduced DHT‐induced CAT activity. Thus, we conclude that the E2 ER complex is capable of inhibiting transcriptional activity of the AR.