bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Sperm Lipid Peroxidation on Fertilization
Authors: KODAMA HIDEYA , KURIBAYASHI YASUSHI , GAGNON CLAUDE
Published: October 2023

ABSTRACT: This study was aimed at determining, with homologous mouse gametes, whether a level of membrane lipid peroxidation insufficient to affect sperm motility parameters can alter sperm fertilizing potential. The addition of ferrous ions and ascorbic acid (Fe2+/ Asc) to mouse sperm suspensions increases the formation of thiobarbituric acid‐reactive substances (TBARS), an indicator of lipid peroxide breakdown products, without significantly affecting the level of sulfhydryl groups. In the presence of Fe2+/Asc concentrations above >0.4/2.0 mM, spermatozoa become immotile. However, at concentrations ≥0.4/2.0 mM of Fe2+/Asc, i.e., conditions in which the TBARS formation is increased by ≥4.6‐fold over that of controls, motility remains unaffected for up to 3 hours. In the presence of 0.4/2.0 mM Fe2+/Asc, treated spermatozoa increase their fertilizing potential by 50%, as measured by the formation of two‐cell embryos.

This increase is not caused by improvements in sperm motility parameters (percentage, linearity, velocity, hyperactivation) or sperm capacitation. On the other hand, there is a significant increase in the capacity of mouse spermatozoa to bind to homologous zona pellucida. In conclusion, mild peroxidative conditions, that increase lipid peroxide formation 4.6‐fold without significantly modifying free sulfhydryl groups and sperm motility parameters, improve the fertilizing potential of spermatozoa by increasing their binding capacity to zona pellucida.
Cytokine Levels in the Seminal Plasma of Infertile Males
Authors: Gruschwitz Matthias S. , Brezinschek Ruth , Brezinschek Hans‐Peter
Published: October 2023

ABSTRACT: Cytokines released by various ceil subsets in the male urogenital tract are capable of markedly influencing sperm function and fertility. We determined the cytokine content in the seminal plasma of patients with unexplained infertility and correlated the results with urogenital infections and sperm parameters. Routine sperm parameters, bacterial culture of seminal plasma and blood follicle‐stimulating hormone (FSH), luteinizing hormone (LH), and testosterone were obtained from 14 infertile males and 8 healthy control subjects. Interleukin 1β (IL‐1β), interleukin 6 (IL‐6), and tumor necrosis factor alpha (TNFα) levels in the seminal plasma were measured by enzyme‐linked immunosorbent assay (ELISA). IL‐1β, IL‐6, and TNFα levels in the seminal plasma were negatively correlated with the number of progressively motile sperm, but there was no correlation with total sperm counts, viability, pH, morphological alterations, type of abnormality, and hormone parameters. Cytokine levels were significantly elevated in seminal plasma exhibiting bacterial or mycoplasmal infections of the urogenital tract. Urogenital infections lead to an release of inflammatory cytokines, most probably by immunocompetent cells of the lymphocyte/macrophage origin. Cytokines such as IL‐1, IL‐6, and/or TNFα might influence sperm motility via direct or indirect effects, resulting in reduced mucosa penetration properties. Therefore, our data suggest that cytokines may be involved in reduced male fertility.
Simple, Rapid Staining Method for Immediate Intraoperative Examination of Testicular Biopsies
Authors: BELKER ARNOLD M. , SHERINS RICHARD J. , DENNISON‐LAGOS LISA
Published: October 2023

ABSTRACT: Before patients with obstructive azoospermia undergo surgical reconstruction, testicular biopsy is required to establish that spermatogenesis is normal. To perform the corrective surgical procedure at the same time as the testicular biopsy, a touch imprint method of processing the biopsy specimen has replaced frozen section processing, which distorts the germinal cells. We report a rapid, simple method of staining a touch imprint specimen in the operating room. It requires less than 5 minutes and can be performed by the andrology laboratory technologist, operating room staff, or the surgeon, thereby avoiding the need to transport the biopsy specimen to the pathology laboratory.
Ultrastructural Observations of Spermatogenesis in Mice Resulting From Transplantation of Mouse Spermatogonia
Authors: RUSSELL LONNIE D. , FRANÇA LUIZ R. , BRINSTER RALPH L.
Published: October 2023

ABSTRACT: The objective of the present study was to provide a morphological characterization of spermatogenesis following germ cell transplantation into the seminiferous tubular lumen of another mouse. The recipient mice (W‐locus) were sterile because of a defect in spermatogenesis resulting from the failure of virtually all germ cell precursors to migrate to the genital ridge during embryonic development. Recipient mice containing intratubular injections of testis cell suspensions from C57 mice were allowed to develop for over 1 year, whereupon animals were sacrificed and testis tissue examined by light and electron microscopy. Donor mouse cells formed normal cell associations (stages) as viewed in cross‐sectioned tubules. Spermatogonia were found exclusively in the basal compartment, indicating that they were translocated from the tubule lumen through the Sertoli cell junctions, eventually to reside on the basal lamina. Some tubules looked entirely normal from both a quantitative and qualitative standpoint. Others showed qualitative and quantitative impairment. In some tubules a generation of cells was missing from a cell association. A variety of degenerating cells and structural abnormalities were responsible for this impairment, however, the most common abnormalities were seen during the elongation phase of spermatogenesis. Elongation abnormalities and the subsequent degeneration of these cells led to the presence of fewer‐than‐expected elongate spermatids. There were regions of the testis where no spermatogenesis was noted and only Sertoli cells were present. These regions were generally typical of the testis histology seen in animals not exposed to injected germ cells. However, Sertoli cells in these regions phagocytosed sperm produced in spermatogenically active regions of the tubules. Because transplantation of germ cells, either from fresh or from frozen cells, had wide‐ranging implications in biology and medicine, characterization of spermatogonial transplants is an important step in improving this procedure.
Polyamine Levels in Testes and Seminal Vesicles From Adult Golden Hamsters During Gonadal Regression‐Recrudescence
Authors: FRUNGIERI MÓNICA B. , GONZALEZ‐CALVAR SILVIA I. , CALANDRA RICARDO S.
Published: October 2023

ABSTRACT: The exposure of golden hamsters to short days results in early regression of the reproductive organs and subsequent spontaneous recrudescence characterized by active cellular regeneration and differentiation. Thus, adult male hamsters were subjected to short photoperiod (SP, 6L:18D) for 9, 12, 14, 16, 18, and 22 weeks or maintained under long photoperiod (LP, 14L:10D) for 22 weeks, to assess photoperiodic‐related changes in testicular and seminal vesicle (SV) levels of polyamines (PA) that are involved in cell growth and differentiation. During the regression phase, the weights of the organs and the circulating levels of luteinizing hormone (LH), follicle‐stimulating hormone (FSH), prolactin, testosterone, dihydrotestosterone, and 5α‐androstane‐3α, 17β‐diol were significantly diminished and, thereafter, during the recrudescence phase, they recovered total or partially their control values. In both tissues, the exposure to SP for 14–16 weeks resulted in an increase of PA concentrations, followed by a return to control levels in the recrudescence period. At the time of maximal tissue involution, the ornithine decarboxylase (ODC) activity (key regulatory enzyme of PA biosynthesis) showed a significant increase in testis, preceding the sharp peak of PA concentration. However, a marked decrease in ODC activity was detected in SV. The concentration of N‐acetyl PA in SV showed an increment at 16 weeks of SP, while no modifications were detected in testicular concentration. When PA, N‐acetyl PA, and ODC activity were expressed per testis and per SV, values fell significantly during the involution period, but in the recrudescence phase levels were recovered concomitantly with the restoration of the organ weight and function. In conclusion, the photoperiodic‐related changes in PA and their N‐acetyl derivatives might play a crucial role in regrowth and differentiation of the male sexual organs during the spontaneous recrudescence phase. Additionally, organ‐specific regulation of the PA biosynthesis pathway could also take place.
The Relation Between Reactive Oxygen Species and Cytokines in Andrological Patients With or Without Male Accessory Gland Infection
Authors: DEPUYDT CHRISTOPHE E. , BOSMANS EUGÈNE , ZALATA ADEL , SCHOONJANS FRANK , COMHAIRE FRANK H.
Published: October 2023

ABSTRACT: The presence of various cytokines, namely hepatocyte growth factor (HGF), interleukin‐1 receptor antagonist (IL‐1 RA), and interleukins (IL‐1α, IL‐6, and IL‐8), as well as the production of reactive oxygen species (ROS) was investigated in seminal plasma of fertile and infertile patients in order to evaluate the possible value of measuring these substances for the diagnosis of male accessory gland infection, and to assess the possible relationship between oxidative stress and cytokines during leucocytospermia and male accessory gland infection (MAGI). Our findings indicate that all of the measured cytokines seem to be produced locally as well as by white blood cells (WBC) and that, due to the presence of higher numbers of WBC, accessory gland infection may exert a deleterious effect on sperm quality through the production of ROS and/or of particular cytokines such as IL‐1α, IL‐1 RA, and IL‐8. The most specific marker for a sensitivity of 95% in discriminating between cases with or without MAGI is the measurement of IL‐6 in seminal plasma. In the absence of WBC several cytokines are constitutively produced and correlate with sperm concentration (HGF, IL‐8), α‐glucosidase (IL‐6), and γ‐glutamyltransferase activity (HGF). The measurement of these cytokines in semen may provide clinically useful information for the diagnosis of male accessory gland infection, as well as in the absence of WBC where it can provide information about certain mechanisms of male reproductive function and dysfunction.
Prevention of Seminiferous Tubular Atrophy in a Naturally Cryptorchid Rat Model by Early Surgical Intervention
Authors: LUGG JAMES A. , PENSON DAVID F. , SADEGHI FARSHID , PETRIE BEVERLY , FREEDMAN ANDREW L. , GONZALEZ‐CADAVID NESTOR F. , HIKIM AMIYA SINHA , RAJFER JACOB
Published: October 2023

ABSTRACT: In an attempt to determine whether the seminiferous tubular atrophy of the cryptorchid testis is preventable by early surgical correction of the cryptorchid state, aberrantly developed gubemacula destined to result in a cryptorchid testis in the Long‐Evans cryptorchid (LE/ORL) rat were surgically reimplanted to the bottom of the scrotum on day 10 to 12 of age. Testis descent was monitored and the changes in testicular histology and in the volumes of the seminiferous tubules and Leydig cells were examined at day 60. As expected, normal testis descent occurred on or about day 25. Compared to untreated undescended testes at day 60, relative seminiferous tubular volumes (volume: % ± SEM) were significantly increased by early surgical reimplantation of the gubemacula (89 ± 1 vs. 66 ± 3; P < 0.01). Absolute seminiferous tubular volumes (μ1 ± SEM) were also significantly increased by early surgical intervention when compared to undescended nontreated testes (893 ± 27 vs. 170 ± 12; P < 0.01). The testes of the surgically corrected cryptorchid animals were similar in all respects to those found in the descended testes of the sham‐operated controls. Relative Leydig cell volume (% ± SEM) was increased in the untreated cryptorchid testes compared to the surgically corrected testes (5.2 ± 0.6 vs. 1.2 ± 1.0; P < 0.05). Relative Leydig cell volumes in the surgically corrected testes were not significantly different from those found in the sham‐operated descended controls. A modest but significant (P < 0.05) increase in absolute Leydig cell volume was also noted in the cryptorchid testes when compared both to normal controls or surgically corrected cryptorchid testes. From these observations, we conclude that early gubemaculopexy reverses the histologic changes normally seen in the cryptorchid rat testis to a relatively normal histologic architecture. These data provide experimental evidence to support the value of orchiopexy in the treatment of cryptorchidism.
Reactivity of Human Deferential Artery to Constrictor and Dilator Substances
Authors: MEDINA PASCUAL , CHUAN PASCUAL , NOGUERA ROSA , VILA JOSé M. , ALDASORO MARTíN , LLUCH SALVADOR
Published: October 2023

ABSTRACT: The present study was designed to investigate general morphology and the response of human deferential artery to constrictor and dilator substances with special emphasis on endothelium‐dependent responses. Human deferential artery segments were obtained from patients undergoing radical cystectomy (n = 7), suprapubic prostatectomy (n = 6), or radical prostatectomy (n = 6). Light microscopy revealed that human deferential artery is of muscular type, and fluorescence microscopy showed a dense adrenergic innervation. Paired rings, one normal and the other de‐endothelialized by gentle rubbing, were mounted for isometric recording of tension in organ baths. Vasopressin, endothelin, serotonin, and potassium chloride induced endothelium‐independent contractions, whereas norepinephrine and electrical field stimulation caused frequency‐dependent contractions that were of greater magnitude in arteries denuded of endothelium. In precontracted arterial rings, acetylcholine and substance P induced endothelium‐dependent relaxations. In contrast, papaverine and sodium nitroprusside caused concentration‐dependent relaxations that were similar in the presence and in the absence of endothelium. NG‐nitro‐L‐arginine methyl ester(10−4 M), an inhibitor of nitric oxide synthase, potentiated the responses to norepinephrine in artery rings with endothelium, nearly abolished the acetylcholine‐induced relaxation, and attenuated the relaxation induced by substance P. Incubation with methylene blue (10‐5 M), an inhibitor of guanylate cyclase, completely prevented the relaxation induced by acetylcholine in arteries with endothelium. The results of this study indicate that the human deferential artery has a dense adrenergic innervation and marked ability to contract or relax in response to different agonists. Some of these responses are in part endothelium dependent and mediated through release of nitric oxide. These morphological and pharmacological observations could play an important role in regulating flow or pressure of blood that arrives to the vas deferens.
Hyaluronic Acid Enhances the Zona Pellucida—Induced Acrosome Reaction of Macaque Sperm
Authors: VANDEVOORT CATHERINE A. , CHERR GARY N. , OVERSTREET JAMES W.
Published: October 2023

ABSTRACT: Hyaluronic acid (HA) not only surrounds the zona pellucida as part of the cumulus matrix but also is present throughout the zona pellucida and the perivitelline space of many mammalian oocytes. However, most in vitro techniques to study sperm—oocyte interaction eliminate HA from the oocyte through enzymatic digestion and/or do not expose sperm to HA prior to zona pellucida binding. This study explores the effect of preincubation of sperm or oocytes with HA on sperm—zona pellucida binding and subsequent acrosome reaction of bound sperm. Cynomolgus macaque semen was washed, incubated, chemically capacitated with dibutryl cyclic adenosine monophosphate (dbcAMP) and caffeine, and sperm—zona pellucida binding assays were performed. In one experiment, sperm were pretreated with HA (100 μg/ml) during the last 10 minutes of the 30‐minute period for chemical capacitation. In another experiment, only the oocytes were preincubated in the media containing HA. In the third experiment, gametes were exposed to HA for 10 minutes after sperm had been allowed to bind to zonae pellucida. The preincubation of either sperm or zonae pellucida with HA enhanced the percentage of bound sperm that were acrosome reacted. However, HA did not affect the number of sperm bound to zonae pellucida. When sperm already bound to the zona pellucida were exposed to HA, there was no increase in the percentage of bound acrosome‐reacted sperm. The HA enhancement of the acrosome reaction of sperm bound to the zona pellucida shown in this study required less than 1 minute of sperm exposure to HA‐treated zonae during the zona binding process. However, this enhancement was observed only if the HA exposure preceded sperm—zona binding. This result suggests that HA is interacting with the sperm surface, possibly via a receptor, at the time of initiation of the acrosome reaction. It is unlikely that the effects noted in the current experiments were the result of motility retention or improvement because the full enhancement of the acrosome reaction was observed when only the oocytes were pretreated with HA.
Androgenic Regulation of NO Availability in Rat Penile Erection
Authors: REILLY CHRISTOPHER M. , ZAMORANO PEDRO , STOPPER VIVIENNE S. , MILLS THOMAS M.
Published: October 2023

ABSTRACT: Prior studies from this laboratory, using untreated castrated (CASTRATE) rats and testosterone‐treated castrated (TES‐TO) rats, have shown that the magnitude of the intracavernosal pressure increase during erection is androgen dependent. Studies from this and other laboratories have also presented evidence suggesting that penile erection is mediated principally by nitric oxide (NO). The present report was designed to confirm that androgens maintain the availability of cavernosal NO and to determine if this androgenic action is exerted at the genomic level modulating the expression of the neuronal form of the nitric oxide synthase gene (nNOS). The results showed that administration of supplemental L‐arginine failed to augment the erectile response in either group, suggesting that substrate availability is not a cause of the reduced response in CASTRATE animals. Inhibition of NO synthesis with a nitro‐arginine competitive inhibitor of nitric oxide synthase enzyme protein (NOS) resulted in strong inhibition of erection in both TESTO and CASTRATE rats. When given in conjunction with ganglionic stimulation to induce erection, the NO releasing drug, sodium nitroprusside (SNP), increased intracavernosal pressure in CASTRATE rats but not in TESTO rats, suggesting a deficiency of the available NO in CASTRATE animals. Finally, reverse transcription‐polymerase chain reaction (RT‐PCR) demonstrated that mRNA levels for the enzyme nNOS in the penis were greater in TESTO animals than in CASTRATE rats. These results support the hypothesis that androgens mediate the erectile response in the rat penis by stimulating the expression of the neuronal isoform of nitric oxide synthase, thus maintaining an adequate supply of NO.
Partial Sympathetic Denervation of the Rat Epididymis Permits Fertilization But Inhibits Embryo Development
Authors: RICKER DEBORAH D. , CRONE JULIE K. , CHAMNESS SHELLY L. , KLINEFELTER GARY R. , CHANG THOMAS S. K.
Published: October 2023

ABSTRACT: The rat cauda epididymidis receives sympathetic innervation from the inferior mesenteric ganglion (IMG). We have previously demonstrated that surgical removal of the IMG and proximal hypogastric nerves (IMG denervation) results in significant and cauda‐specific changes in epididymal sperm transport, sperm motility, luminal fluid protein composition, and tissue histology. In the present study we used natural mating trials and intrauterine insemination (IUI) techniques to determine whether or not IMG denervation affects male fertility and reproductive capacity. For the initial studies, adult male Sprague Dawley rats were mated with estrous females 1 and 4 weeks following IMG denervation. Nine days after mating, uterine implantation sites and corpora lutea (CL) were counted. In females mated with sham‐operated control males, 85.8% of ovulated oocytes were fertilized and subsequently implanted. In contrast, females mated with IMG‐denervated males 1 or 4 weeks following surgery had 0% and 3.5%, respectively, of ovulated oocytes fertilized and implanted. For rats maintained 21 days after mating, an average of 13 ± 1 pups were delivered by each of nine females mated with sham‐operated control male rats; whereas, only seven morphologically normal pups were delivered by one of 14 females mated with IMG‐denervated male rats. Additional experiments demonstrated that the decrement in offspring was, in part, due to a significant decrease in the number of spermatozoa in the female uterus following mating with IMG‐denervated males. To determine whether IMG denervation exerted an additional effect directly on the fertilizing ability of spermatozoa, IUI experiments were performed. Six million cauda epididymal spermatozoa from 1‐ or 4‐week IMG‐denervated males were inseminated into the uterine horns of luteinizing hormone‐releasing hormone (LHRH)‐synchronized females and 9 days later implantation sites and CL were counted. Implantations were observed for 78%, 28%, and 25% of ovulated oocytes following IUI with spermatozoa from sham‐operated controls and from 1 ‐ and 4‐week IMG‐denervated rats, respectively. To determine whether the reduction in implantation sites following IUI with spermatozoa from IMG‐denervated rats resulted from impaired oocyte fertilization, studies were performed in which oocytes were retrieved and stained 24 hours after IUI. Comparable fertilization rates of 76.5% and 89.0% were observed using cauda epididymal spermatozoa from IMG‐denervated and sham‐operated control males, respectively, indicating that oocyte fertilization was not affected by the loss of innervation. These studies establish the importance of innervation from the IMG for ejaculatory competence and sperm reproductive capacity in the male rat. These data further suggest that sympathetic innervation in the epididymis critically influences paternal factors associated with embryonic development.
The Effects of Spinal Cord Injury on the Status of Messenger Ribonucleic Acid for TRPM 2 and Androgen Receptor in the Prostate of the Rat
Authors: HUANG HOSEA F. S. , LI MING‐TANG , LINSENMEYER TODD A. , OTTENWELLER JOHN E. , POGACH LEONARD M. , IRWIN ROBERT J.
Published: October 2023

ABSTRACT: The prostate is one of the male accessory sex glands that produce fluid components of the seminal plasma. In addition to androgen, a normal innervation of the prostate is believed to be important for maintaining normal function of the prostate. Previously we noted that, in the rat, the weight of the prostate decreased following surgically induced spinal cord injury (SCI). This observation suggests that growth, and possibly function, of the prostate may be compromised after SCI. To explore this possibility, we examined the effects of SCI on the androgen‐related biochemical properties and morphology of the prostate in the rat at various times after surgically induced SCI. SCI resulted in an acute decrease in prostate weight and an increase in steady state level of mRNA for testosterone‐repressed prostate message 2 (TRPM 2) during the first 2 weeks postinjury. These changes perhaps relate to an increase in cell death or a decrease in secretory activity due to an acute suppression of serum testosterone after the injury. Concomitantly, there was a transient, but significant, decrease in the steady state level of androgen receptor (AR) mRNA in the prostate during the first 2 weeks after SCI, an indication of an altered autoregulation of AR by its own ligand. Despite the fact that growth of the prostate, as indicated by weight increase, in SCI rats resumed 2 weeks postinjury, prostate weights were persistently lower in SCI rats than sham‐operated controls for at least 3 months. Furthermore, prostate TRPM 2 mRNA levels remained elevated throughout the recovery period even after a normal prostate weight had been restored. In addition, a decrease in the height of ventral prostate epithelial cells was noted in SCI rats 28 and 90 days postinjury. These results demonstrate a prolonged effect of SCI on prostate function. These findings and our unreported observation of persistently smaller seminal vesicles in the same groups of SCI rats suggest that functions of male accessory sex glands may also be compromised after SCI. These changes may affect biochemical properties of the secretory products of these glands and may provide some explanation for the reported changes in the composition of the seminal plasma and abnormal sperm motility seen in the semen of SCI men.
Sertoli Cells in Culture and mRNA Differential Display Provide a Sensitive Early Warning Assay System to Detect Changes Induced by Xenobiotics
Authors: SYED VIQAR , GU WEI , HECHT NORMAN B.
Published: October 2023

ABSTRACT: We have used cultured rat Sertoli cells as an “early warning system” to monitor for morphological and biochemical changes induced by two different xenobiotics—cadmium acetate and polychlorinated biphenyls (PCBs). Sertoli cells begin to round, become vacuolized, and detach from their substrate within 24 hours of culture in the presence of cadmium at concentrations of 0.5–1.0 μM. Similar results were obtained with a lower dose of cadmium (0.01 μM) after 72 hours. When Sertoli cells are cultured for 24 hours in the presence of a mixture of PCBs (3,3‘,4,4’‐tetrachlorobiphenyl, 2,2,4,6,6‘‐pentachlorophenyl, and 2,2’,3,3‘,4,5,5’,6,6‘‐nonachloro‐biphenyl) at concentrations of 1.0–2.0 μM, they enlarge. After 72 hours, a lower dose of PCBs (0.01 μM) produces similar cellular enlargement. Despite their changes in morphology, no reduction in Sertoli cell viability was seen at any of the concentrations or time points studied for either toxicant. Using mRNA differential display, a number of novel cDNAs were detected when cells were cultured with either cadmium or the PCBs, demonstrating that changes in gene expression accompany the changes in Sertoli cell structure. We propose that Sertoli cells in culture and mRNA differential display provide a sensitive morphological and biochemical assay system to detect early direct effects of low concentrations of toxicants on mammalian Sertoli cells.
Effects of Heat Stress on Mouse Testicular Cells and Sperm Chromatin Structure
Authors: SAILER BRIAN L. , SARKAR LINDA J. , BJORDAHL JANET A. , JOST LORNA K. , EVENSON DONALD P.
Published: October 2023

ABSTRACT: Scrotal regions of mice were exposed to a 38.0, 40.0, or 42.0°C (±0.1) H2O bath for 60 minutes to determine the effects of elevated temperatures on testicular cells and sperm chromatin structure. Mice were killed on various days after exposure, and ratios of acridine orange‐stained testicular cell populations were determined by flow cytometry. Testicular weights of mice exposed to 42.0°C decreased significantly day 1 (P < 0.01) through 35 (P < 0.001). Also, a significant relative decrease in testicular haploid cells was seen on days 3–35 (P < 0.001) with a corresponding increase in the diploid population (P < 0.001). Testicular analyses of mice exposed to 38.0°C were not significantly different from control values. Testis weights of mice exposed to 40.0°C were not affected, but a relative decrease in percent haploid cells occurred on days 11 and 14 (P < 0.001). The sperm chromatin structure assay (SCSA) was used to measure the susceptibility of cauda epididymal sperm DNA to in situ denaturation at low pH. Caudal epididymides of mice exposed to 42.0°C had no sperm. Caudal epididymal sperm from mice exposed to 40.0°C were most susceptible to acid‐induced DNA denaturation on days 3 (P < 0.05), 7, 11, and 14 (all P < 0.001). The 38.0°C exposed mice showed some minor sperm chromatin abnormalities at later time points (days 11–35). When compared to sperm head morphology measurements, SCSA parameters were more sensitive indicators of heat‐induced sperm abnormalities. These results show that mouse spermatogenesis is disrupted by scrotal exposure to environmental temperatures several degrees over normal physiological temperature and, of more biological interest, that some thermal ranges above normal allowed production of sperm with compromised nuclear chromatin structure.
Copulatory Behavior and Fertility in Transgenic Male Mice Expressing Human Placental Growth Hormone Gene
Authors: MELISKA C. J. , BARTKE A.
Published: October 2023

ABSTRACT: Male transgenic (TG) mice overexpressing the human placental growth hormone (GH) variant gene (hGH‐V) exhibit reproductive deficits in spite of normal testosterone levels and normal sperm counts. To evaluate the relationship of copulatory behaviors to fertility, we first measured mount, intromission, and ejaculation indices in 2–5‐month‐old mice (10 TG and 10 normal litter mate controls) during 1 hour tests with ovariectomized, estrogen‐, and progesterone‐primed females. After eight tests, each male was housed with three intact females for 27 consecutive days. Females were checked daily for vaginal plugs and sacrificed 14 days after insemination to determine the numbers of corpora lutea and live and dead fetuses. Relative to their normal siblings, TG mice mounted less often and intromitted sooner after the initial mount, made marginally more intromissions (with and without ejaculation), and were slower to ejaculate. In subsequent fertility tests, TG males inseminated fewer females and sired fewer live fetuses per insemination than non‐TG controls. Across TG and normal males, the length of interval between initial mount and initial intromission was inversely correlated with the number of live offspring sired. This suggests that reduced fertility in hGH‐V transgenic male mice may be related to altered copulatory behavior, including a rapid progression from first mount to first intromission.
Effect of Semen Dilution on Bovine Sperm Viability as Determined by Dual‐DNA Staining and Flow Cytometry
Authors: GARNER DUANE L. , THOMAS CHERYL A. , ALLEN CHARLES H.
Published: October 2023

ABSTRACT: Living and dead spermatozoa were examined for the effects of sperm concentration level on sperm viability. Semen was collected from two different bulls on each of four collection dates. A ninth bull was collected on all four collection dates as a control for effects of collection date. The ejaculates from these nine bulls were diluted to 30 × 106 spermatozoa/0.5 ml and then serially diluted to 20, 10, 5, or 1 × 106 spermatozoa/0.5 ml French straw. One‐half of the straws for each dilution series was stored 24 hours at 5° C, while the other half was cryopreserved. Spermatozoa were stained with SYBR‐14 and propidium iodide (PI) to assess viability. Flow cytometry yielded dot plots showing three distinct sperm populations: dead red‐stained spermatozoa (PI), viable green‐stained spermatozoa (SYBR‐14), and moribund spermatozoa that stained both red and green (doubly‐stained). Populations were expressed and analyzed in terms of mean percentage of viable spermatozoa and by actual numbers of viable spermatozoa per insemination dose. The mean percentage of living spermatozoa decreased linearly with decreasing sperm concentration; whereas the decrease was parabolic when those same samples were expressed as the mean number of living spermatozoa per insemination dose. The percentage of SYBR‐14‐stained spermatozoa differed among concentration levels and among bulls (P < 0.01). There were no differences among straws from the same ejaculate. The total volume of ejaculated semen and the concentration of spermatozoa in that ejaculate were both significantly positively correlated with the percentage of SYBR‐14‐stained spermatozoa in that semen when it was cryopreserved and diluted to <10 × 106 spermatozoa/0.5 ml. In contrast, there were no significant correlations between the initial ejaculate characteristics and the proportion of SYBR‐14‐stained spermatozoa in the 24‐hour‐stored samples at any concentration. In conclusion, the percentage of viable spermatozoa in an ejaculate significantly decreased with increasing dilution. Further, in cryopreserved samples, the percentage of living spermatozoa <10 × 106 spermatozoa/0.5 ml depended on the original volume and the sperm concentration of that particular ejaculate.
Successful Treatment of Severe Oligozoospermia With Sperm Washing and Intrauterine Insemination
Authors: CENTOLA GRACE M.
Published: October 2023

ABSTRACT:During the period January 1, 1991 through December 31, 1995, 258 patients, in whom motile sperm counts for insemination (postwash, processed) were 10.0 million motile sperm or less were seen in the andrology unit for sperm washing and intrauterine insemination (IUI). No significant female factors were noted on history; all female partners had patent Fallopian tubes and were ovulatory spontaneously or were treated by the referring gynecologist with clomiphene citrate, human menopausal gonadotropin (hMG), or follicle‐stimulating hormone (FSH) ovulation induction in both anovulatory or ovulatory women. Of the total of 258 patients, 15 achieved a pregnancy in 284 cycles of IUI in which the inseminating motile count was <1.0 million motile sperm, resulting in a monthly fecundity (f) of 5.3%. The mean (±SD) motile count for IUI in this group was 0.61 (±0.29) million sperm, with a range of 0.19–0.95 million motile sperm. The initial motile count was 2.97 (3.2) million sperm, with a range of 0.2–12.81 million sperm. With inseminating motile counts of 1.0–10.0 million motile sperm, there were 83 pregnancies after 467 cycles of IUI, resulting in a monthly f of 17.8%. The mean (±SD) motile count for IUI in this group was 4.9 (±2.7) million motile sperm with a range of 1.0–9.9 million motile sperm. The initial sperm count in this group was 10.9 million (±7.1), with a range of 1.1–23.7 million motile sperm. These data suggest that acceptable pregnancy rates can be achieved with IUI, even in severely Oligozoospermie specimens. Intrauterine insemination is less invasive and less costly than other assisted reproductive techniques. These data are supportive of IUI prior to attempting other more invasive and potentially costly reproductive technologies.
The Impact of Reactive Oxygen Species on Bovine Sperm Fertilizing Ability and Oocyte Maturation
Authors: BLONDIN PATRICK , COENEN KARINE , SIRARD MARC‐ANDRé
Published: October 2023

ABSTRACT: The objective of this study was to examine the effects of reactive oxygen species (ROS) on bovine sperm function and on the developmental competence of in vitro‐matured bovine oocytes. In a first series of experiments, spermatozoa were exposed to ROS generated through the use of the hypoxanthine‐xanthine oxidase system ± catalase prior to the conduct of in vitro fertilization (IVF). Reactive oxygen species exposure reduced significantly (P < 0.001) the rates of oocyte penetration (control: 56% ± 4 SEM; ROS: 16 ± 2–23% ± 7 SEM), and this effect was reversed by adding catalase (ROS + catalase: 67% ± 0.3 SEM). During IVF, addition of superoxide dismutase (SOD: 1, 10, or 100 U/ml) had no effect on penetration rates. However, increasing concentrations of catalase (0.1 or 1 mg/ml) reduced these rates significantly (control: 70% ± 3 SEM; treated: 45% ± 5 and 1% ± 1 SEM; P < 0.001). In a second series of experiments, when oocytes were matured in vitro in the presence of exogenous antioxidants (SOD: 10,100, or 1000 U/ml; β‐mercaptoethanol: 0.01, 0.1, or 0.5 mM; ascorbic acid: 0.05 mg/ml), the developmental competence of the oocytes after IVF was not significantly improved. On the other hand, presumed production of ROS using the hypoxanthine‐xanthine system at the beginning of the in vitro maturation period did improve subsequent developmental competence of the oocytes under some conditions and when catalase was present (control: 14% ± 4 SEM and treated: 23% ± 9 and 27% ± 8 SEM; P < 0.05). These observations demonstrate that ROS may be beneficial to gamete function under specific conditions.
Premature Capacitation of Bovine Spermatozoa Is Initiated by Cryopreservation
Authors: CORMIER NATHALY , SIRARD MARC‐ANDRé , BAILEY JANICE L.
Published: October 2023

ABSTRACT: Poor motility and abnormal acrosomal morphology only partially explain the reduced fertility of cryopreserved bovine spermatozoa. To test the hypothesis that cryopreservation procedures (dilution, cooling, freeze‐thaw) induce capacitation in bovine spermatozoa, two experiments were conducted using semen diluted in egg yolk—Tris—glycerol extender (EYTG) (Tris, tris(hydroxymethyl)aminomethane). Capacitation was determined prior to and following incubation with various concentrations of heparin using the chlortetracycline (CTC) fluorescence assay or after preexposure to EYTG using in vitro fertilization (IVF) of bovine cumulus—oocyte complexes (COC) in the absence of heparin. Fresh ejaculates were divided into four treatments and the first was diluted with noncapacitating medium, NCM (+ 0.3% polyvinyl alcohol (PVA); control), then maintained at 23°C for 4 hours. The remaining semen was diluted with EYTG; the second treatment was held at 4°C (EYTG‐4), and the third treatment was held at 23°C (EYTG‐23) for 4 hours. The fourth treatment was cooled to 4°C over 4 hours, as per the normal industry protocol, cryopreserved, and thawed (frozen‐thawed). After the 4‐hour maintenance periods or thawing, all treatments were resuspended either in capacitating medium (CM; + 0.6% BSA) for the CTC experiment (n = 3) or in NCM for the IVF experiment (n = 9–11). Prior to incubation in conditions that support capacitation, the percentage of cells exhibiting pattern B (capacitated according to the CTC assay) was similar for all treatments with fresh‐extended spermatozoa. Immediately following the addition of heparin (0, 2, or 10 μg/ml), three times more frozen‐thawed than fresh‐extended spermatozoa exhibited pattern B (P < 0.05). After 3 or 6 hours of incubation, however, the percentages of cells displaying pattern B did not differ among treatments. In the absence of heparin, spermatozoa preexposed to EYTG‐4 fertilized 2.6x more COC than did control cells (P < 0.001) and 9.2 x more than spermatozoa preexposed to EYTG but held at 23°C (EYTG‐23; P < 0.0001). No differences were observed among fertilization rates for fresh‐extended (EYTG‐4) and frozen‐thawed spermatozoa. This study provides evidence that premature capacitation occurs in partially (extended and cooled) and fully cryopreserved bovine spermatozoa.
Prevalent Decrease of the EGF Content in the Periurethral Zone of BPH Tissue Induced by Treatment With Finasteride or Flutamide
Authors: MONTI SALVATORE , SCIARRA FRANCESCO , ADAMO M. VITTORIA , TOSCANO VINCENZO , TROTTA M. CRISTINA , MARTINI CHIARA , LANZARA STEFANIA , SILVERIO FRANCO D.
Published: October 2023

ABSTRACT: The aim of the present investigation is to verify whether treatment with Finasteride or Flutamide influences the regional distribution of testosterone (T), dihydrotestosterone (DHT), and epidermal growth factor (EGF) in benign prostatic hyperplasia (BPH) tissue. Thirty seven BPH patients were studied: 15 untreated, 9 treated with Flutamide (750 mg/day for 2 months), and 13 treated with Finasteride (5 mg/day for 3 months). Testosterone and DHT were evaluated by radioimmunoassay (RIA) after purification of the extracts on celite columns, and EGF was evaluated by RIA after purification on Sep‐pak C18 cartridges in total tissue and in periurethral, subcapsular, and intermediate zone. In the untreated group, T, DHT, and EGF of the periurethral region are higher than those of the subcapsular zone (P < 0.01 for T and P < 0.001 for DHT and EGF). In the Flutamide group, DHT is not modified, T is increased (P = 0.045), and EGF is decreased in total tissue (P < 0.02) and in the periurethral zone (P < 0.01). In the Finasteride group, T is increased (P < 0.001), and DHT and EGF are decreased (P < 0.001), particularly in the periurethral zone. A positive linear correlation between DHT and EGF is observed in the Finasteride and in the untreated groups. In conclusion, in BPH the production of EGF is a DHT‐dependent receptor‐mediated function. The reduction of this growth factor during both treatments, associated with a fall of DHT in only the Finasteride group, is particularly evident in the periurethral zone. Since Finasteride reduces prostatic volume, mainly of the periurethral zone, we can speculate that DHT is responsible, either directly or indirectly through growth factors such as EGF for the enlargement of this region and thus responsible for urinary obstruction.
Stability and Transcriptional Regulation of Gamma‐Glutamyl Transpeptidase mRNA Expression in the Initial Segment of the Rat Epididymis
Authors: RUDOLPH DANIEL B. , HINTON BARRY T.
Published: October 2023

ABSTRACT: Gamma‐glutamyl transpeptidase (GGT) is an enzyme believed to play a role in the protection of maturing spermatozoa in the epididymis. Our previous studies have shown that four GGT mRNAs (I—IV) transcribed from the single‐copy rat GGT gene are differentially expressed and regulated in the rat epididymis. In particular, the normal expression of GGT mRNAiv in the epididymal initial segment is dependent upon the presence of testicular factors. The objective of this study was to test the hypothesis that the decreased expression of GGT mRNAiv in the initial segment following the in vivo removal of testicular factors by efferent duct ligation (EDL) is due to a decrease in stability and/or transcription rate. The stability of the GGT mRNAs was evaluated by measuring the rate of mRNA decay. These stability studies showed that GGT mRNAiv exhibited a rapid initial decay that slowed at later times to a decay rate similar to that of GGT m‐RNAsu.m. The decay rates were not different following sham‐operation or EDL, and thus the stability of GGT mRNAs were not influenced by the in vivo loss of testicular factors. Results of transcription analysis revealed that the transcription rate of GGT mRNAiv in the initial segment fell by approximately 68% following a 12‐hour EDL. Additionally, secondary‐structure models indicate two families of folding patterns for GGT mRNAiv, which could be the reason for the two decay regimes detected in the stability study. Thus, the decreased expression level of GGT mRNAiv in the initial segment following the in vivo loss of testicular factors is a function of a decreased transcription rate and intricate decay kinetics.
The Cellular Mechanisms of Corticotropin‐Releasing Hormone (CRH)‐Stimulated Steroidogenesis in Mouse Leydig Cells Are Similar to Those for LH
Authors: HUANG BU‐MIIN , STOCCO DOUGLAS M. , NORMAN REID L.
Published: October 2023

ABSTRACT: Previous reports have demonstrated that corticotropin‐releasing hormone (CRH) treatment of primary cultures of mouse Leydig cells and MA‐10 mouse Leydig tumor cells results in a dose‐dependent stimulation of steroidogenesis, probably by acting through the cAMP/protein kinase A second messenger pathway. Based on this observation, the mechanism of CRH‐stimulated steroidogenesis is now further investigated and compared to trophic hormone stimulation. Both cell types were treated with human chorionic gonadotropin (hCG) or CRH in the absence and presence of the following agents: the translation inhibitor cycloheximide, the transcription inhibitor actinomycin D, the protonophore carbonyl cyanide m‐chlorophenylhydrozone (mCCCP), which disrupts the mitochondrial electrochemical gradient or the phorbol ester, phorbol‐12‐myristate 13‐acetate (PMA), a stimulator of protein kinase C. Cortico‐releasing hormone‐stimulated steroidogenesis was completely blocked by cycloheximide in both cell types, indicating that CRH‐stimulated steroidogenesis in mouse Leydig cells requires ongoing protein synthesis. Actinomycin D had profound inhibitory effects on CRH‐stimulated steroidogenesis in MA‐10 cells, and this inhibition was greater than that seen in mouse primary Leydig cells. mCCCP severely inhibited CRH‐stimulated steroid production in both cell types, indicating that an electrochemical gradient across the inner mitochondrial membrane is required for CRH‐stimulated steroidogenesis. In addition, PMA inhibited hCG‐ and CRH‐stimulated steroidogenesis in MA‐10 cells and CRH‐stimulated steroidogenesis in primary Leydig cells, suggesting that activation of the protein kinase C pathway can influence protein kinase A stimulated steroidogenesis. Results of these studies suggest that the mouse Leydig cell steroidogenic response to CRH shares many similarities to that of the LH response.
Mild Hypothermia Induces Apoptosis in Rat Testis at Specific Stages of the Seminiferous Epithelium
Authors: BLANCO‐RODRíGUEZ JOSEFA , MARTíNEZ‐GARCíA CARMEN
Published: October 2023

ABSTRACT: This study was designed to determine the effects of short episodes of mild hypothermia on cell apoptosis in the testis of the adult rat. Apoptosis was assessed by in situ DNA 3′‐end labeling in the testes from rats killed 2 hours after cooling (10°C for 30 minutes), quantitated, and compared with spontaneous apoptosis found in control animals. A significantly increased number of dying germ cells appeared after cold treatment at specific stages (stage XIV, P < 0.0001; stage XII, P < 0.001) of the seminiferous epithelium, mainly due to dying primary metaphasic spermatocytes (P < 0.0001), followed by secondary interphase spermatocytes (P < 0.001), and A2 spermatogonia (P < 0.05). The highly specific effect of mild hypothermia on germ cell apoptosis suggests that the process is tightly regulated.
Regional Distribution of 5α‐Reductase Type 1 and Type 2 mRNA Along the Nonhuman Primate (Macaca fascicularis) Epididymis
Authors: MAHONY MARY C. , HEIKINHEIMO OSKARI , GORDON KEITH , HODGEN GARY D.
Published: October 2023

ABSTRACT: The 5α‐reduced metabolites of testosterone, including dihydrotestosterone, are considered the primary regulators of epididymal function. Two genes encode two 5α‐reductase isozymes. We examined 5α‐reductase type 1 and type 2 mRNA tissue distribution and relative abundance in cynomolgus monkey (Macaca fascicularis) testicular and epididymal tissues using semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR). mRNA extracted from monkey tissues including the testis (T) and the proximal caput (PCp), the caput (Cp), the midcorpus (Co), and the distal cauda (Cd) epididymis was reverse transcribed to produce cDNAs. 5α‐reductase type 1 and 2 cDNAs were subsequently coamplified with the housekeeping gene, cyclophilin, in a PCR spiked with MP‐dCTP. Relative abundance was reported as the cpm ratios of type 1 or type 2/cyclophilin mRNA. Semiquantitative RT‐PCR results indicated that type 1 mRNA was most abundant in the testis (0.48 ± 0.06) and significantly decreased distally along the monkey epididymis (PCp: 0.29 ± 0.04; Cp: 0.29 ± 0.04; Co: 0.21 ± 0.03; Cd: 0.07 ± 0.01) (P < 0.001). Type 2 mRNA was undetectable in the testis but was present throughout the epididymis at uniform levels (PCp: 1.6 ± 0.2; Cp: 1.4 ± 0.3; Co: 1.6 ± 0.2; Cd: 1.5 ± 0.2). These data demonstrate that 5α‐reductase type 1 mRNA is differentially expressed but of low abundance along the nonhuman primate epididymis, whereas 5α‐reductase type 2 gene expression is uniform.