bluuebunny/crossref_metadata_2025 huggingface dataset:
Cognitive Changes Associated With Supplementation of Testosterone or Dihydrotestosterone in Mildly Hypogonadal Men: A Preliminary Report
Authors: Cherrier Monique M. , Craft Suzanne , Matsumoto Alvin H.
Published: December 2015

ABSTRACT: This study prospectively examined changes in cognition in hypogonadal men given testosterone (T) or older hypogonadal men given dihydrotestosterone (DHT) gel. A battery of cognitive tests assessing verbal and spatial memory, language, and attention was administered at baseline (prior to medication) and again at days 90 and 180 of treatment for men receiving T gel and at baseline and days 30 and 90 of treatment for men receiving DHT gel. For men receiving T gel, circulating total T and estradiol (E2) were significantly raised compared with baseline, and a significant improvement in verbal memory was observed. For men receiving DHT gel, serum DHT levels increased and T levels decreased significantly compared with baseline, and a significant improvement in spatial memory was observed. The results suggest that beneficial changes in cognition can occur in hypogonadal men using T replacement levels and DHT treatment, and these changes in cognition can be reliably measured during a relative steady‐state dose level. Further, our results suggest that aromatization of T to E2 may regulate verbal memory in men, whereas nonaromatizable androgens may regulate spatial memory.
Effects of Ethanol on Embryonic and Neonatal Rat Testes in Organ Cultures
Authors: Li Hui , Kim Kwan Hee
Published: December 2015

ABSTRACT: Ethanol exposure in adult animals and humans has shown to elicit significant inhibitory effects on the function of male reproduction, but consequences of ethanol exposure on the embryonic and early postnatal testis development are not known. The current study investigated the effect of ethanol on embryonic and neonatal testis development using an organ culture technique. In embryonic day 13 (E13) testis organ cultures, ethanol had no effect on the testicular cord formation, the expression of Müllerian‐inhibiting substance (MIS) in Sertoli cells or the number of gonocytes. Similarly, in the ethanol‐treated embryonic day 18 (E18) testes, both the number of gonocytes and the expression of GATA‐4 and MIS were similar to those from the control testes. In contrast, in postnatal day 3 (P3) testes, ethanol at concentrations of 150 and 200 mM significantly decreased the number of gonocytes without affecting the expression of GATA‐4 and MIS in Sertoli cells. This effect was shown to be resulting from the enhanced apoptosis of gonocytes. In addition, ethanol abnormally activated retinoic acid receptor alpha (RARα), as indicated by increased nuclear localization of RARα with increasing doses of ethanol treatment. These observations suggest that the effect of ethanol on testis varies at different stages during embryonic and neonatal testis development. Furthermore, germ cells may be the main target for the action of ethanol on the early postnatal testis.
Enhanced Chemiluminescence Assay vs Colorimetric Assay for Measurement of the Total Antioxidant Capacity of Human Seminal Plasma
Authors: Said Tamer M. , Kattal Namita , Sharma Rakesh K. , Sikka Suresh C. , Thomas Anthony J. , Mascha Edward , Agarwal Ashok
Published: December 2015

ABSTRACT: Although the enhanced chemiluminescence assay is commonly used to measure the nonenzymatic total antioxidant capacity (TAC) of the human seminal plasma, it is cumbersome, expensive, and time‐consuming. We describe herein an alternate method to measure TAC that is based on the ability of antioxidants in seminal plasma to interfere with a reaction between 2,2′‐azino‐di‐[3‐ethylbenzthiazoline sulphonate] and metmyoglobin with H2O2. This reaction produces a relatively stable blue‐green color with absorbance maxima at 600 nm. We compared this colorimetric assay with our established chemiluminescence method and assessed quality control parameters (ie, intra‐assay and interassay variabilities) in addition to intraobserver and interobserver differences. Our results show that the colorimetric assay was fairly predictive of antioxidant capacity similar to the chemiluminescence assay (P < .001). Furthermore, there was a high level of agreement between the duplicate measures by the same observer (intraobserver) and intra‐assay variability, with a concordance correlation coefficient of 0.99. The interassay coefficient of variation was 4.7% (overall). The mean ± SD of the difference between the 2 observers was 2.98% ± 4.1%. In conclusion, we found that the colorimetric assay is a reliable and accurate method to evaluate seminal TAC, and it could be used as a simpler, rapid, and cheaper alternative to the chemiluminescence assay.
Androgen‐Induced Prostate‐Specific Antigen Gene Expression Is Mediated via Dihydrotestosterone in LNCaP Cells
Authors: Zhu Yuan‐Shan , Cai Li‐Qun , You Xueke , Cordero Juan J. , Huang Ying , Imperato‐McGinley Julianne
Published: December 2015

ABSTRACT: Prostate cancer is a leading cause of cancer death in American males. Androgens play an essential role in prostate development, growth and pathogenesis of benign prostate hyperplasia, and prostate cancer. Although testosterone is the main androgen secreted from the testes, dihydrotestosterone (DHT), a more potent androgen converted from testosterone by 5α‐reductase isozymes, type 1 and 2, is the major androgen in the prostate cells. Thus, 5α‐reductase(s) are critical in determining androgen activity in the prostate. However, it is unclear in prostate tumor cells whether 1 or 2 5α‐reductase isozymes are expressed and whether they are functionally important. In the present report, we studied the importance of 5α‐reductase isozymes in the androgen induction of prostate‐specific antigen (PSA) gene expression in LNCaP prostatic tumor cells. Treatment with either testosterone or DHT in LNCaP cells produced dose‐ and time‐dependent increases in PSA levels in the cell media and in PSA messenger RNA (mRNA) levels in the cells. However, testosterone‐induced but not DHT‐induced PSA gene expression was significantly inhibited by finasteride, a 5α‐reductase inhibitor, in a dose‐dependent manner. Furthermore, we demonstrated for the first time that both 5α‐reductase‐1 and 5α‐reductase‐2 mRNAs were expressed in LNCaP cells using reverse transcriptase‐polymerase chain reaction (RT‐PCR) and RT‐PCR Southern blot analysis. These results suggest that both 5α‐reductase isozymes are present and functionally important in prostatic tumor LNCaP cells and that DHT is a major mediator of androgen induction of PSA gene expression in these cells.
A Simple Self‐Report Diary for Assessing Psychosexual Function in Hypogonadal Men
Authors: Lee Ka Kui , Berman Nancy , Alexander Gerianne M. , Hull Laura , Swerdloff Ronald S. , Wang Christina
Published: December 2015

ABSTRACT: To examine the performance of a self‐report diary to assess psychosexual function in hypogonadal men, 2 groups of eugonadal men and 2 groups of hypogonadal men were asked to record and score parameters for sexual desire, sexual enjoyment, sexual performance, sexual activity, and positive and negative moods daily for 7 days before a clinic visit (data set 1 and 2). The hypogonadal men were also assessed after testosterone replacement and some of the eugonadal men were studied while they were on placebo treatment. In this retrospective analysis, sexual function parameters (sexual desire, performance, and activity score) in the diary discriminated between the hypogonadal and eugonadal men with all measures significantly lower in hypogonadal men (all parameters P < .0001). Significant improvements in sexual desire and performance as well as sexual activity scores (P < .0001 for all parameters) in hypogonadal men after testosterone treatment were readily detected within 30 days. Mood and functional parameters did not show any change over time in eugonadal men on placebo treatment. The mood parameters assessed by the diary showed an excellent correlation with those assessed by the Profile of Mood States. Mood parameters were not clearly different between eugonadal and hypogonadal men at baseline. With testosterone treatment positive mood parameters were significantly increased (P < .0028 and .0001 set in data 1 and 2, respectively), and negative mood parameters improved in hypogonadal men (P < .0003 in data set 2). We conclude that this simple self‐report diary is useful in assessing the sexual function and mood profile of hypogonadal subjects in clinical research.
Male Genital Tract Antioxidant Enzymes: Their Source, Function in the Female, and Ability to Preserve Sperm DNA Integrity in the Golden Hamster
Authors: Chen Hong , Chow Pak Ham , Cheng So Kwan , Cheung Annie L. M. , Cheng Lydia Y. L. , O Wai‐Sum
Published: December 2015

ABSTRACT:Recently, we reported that male accessory sex gland (ASG) secretions protect sperm genomic integrity by demonstrating that DNA damage was more extensive in sperm not exposed to the secretions. The present study was conducted to find out if ASGs secrete the main antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx or GSH‐Px), and catalase (CAT) and if the most abundant one, SOD, can protect those sperm that were not exposed to ASG secretions against NADPH‐induced oxidative stress. Four experimental groups of male golden hamsters were used: intact animals with proven fertility, animals with all major ASGs removed (TX), animals that were bilaterally vasectomized, and sham‐operated controls. SOD, CAT, and GPx activities were measured in secretions from all 5 ASGs and sperm‐free uterine flushing from virgin females and those mated with the experimental males. The alkaline comet assay was used to analyze DNA integrity of the TX group sperm after incubation in a medium containing 50 U/mL of SOD along with 0 to 20 mmol/L NADPH. The main antioxidant enzyme in ASGs was SOD from coagulating glands (P< .05) and GPx together with CAT from ampullary glands (P< .05). Uterine flushing of ejaculates that contained ASG secretions had more SOD and CAT activities than those with epididymal secretions alone (P< .05 andP< .001, respectively), whereas activity of GPx was the same (P> .05). Addition of SOD in vitro dose dependently decreased the incidence of single‐strand DNA damage in sperm not exposed to ASG secretions incubated in the presence of 0 to 20 mmol/L NADPH (P< .001). These results indicated that, in terms of abundance, SOD was the main antioxidant enzyme secreted by male ASGs, whereas CAT was the second one. The GPx activity came from both epididymis and ASGs. We conclude that ASG secretions play a significant role in protecting sperm against oxidative stress.
Diagnostic Value of Differential Quantification of Spermatids in Obstructive Azoospermia
Authors: Nistal Manuel , González‐Peramato Pilar , Paniagua Ricardo
Published: December 2015

ABSTRACT: Testicular biopsies from 80 azoospermic young men were revised and the average numbers per cross‐sectioned tubule of each germ cell type were calculated and compared with those of control normal testes. In 53 patients, azoospermia had an obstructive cause, and in 22 of those 53 patients more adult spermatids were found by testicular biopsy than young spermatids (over 100% in some testes), in one or both testes. However, in normal testes fewer mature spermatids than young spermatids (23.3%) were found. In the 22 patients, the causes of azoospermia were: vasectomy (7 patients), bilateral agenesis of the vas deferens (3 patients), Young syndrome (3 patients), bilateral cysts in the caput epididymidis (1 patient), bilateral inguinal herniorrhaphy (1 patient), left varicocele (1 patient), and unknown causes (6 patients). Biopsies were bilateral except for 3 cases (a vasectomized patient, a patient with Young syndrome, and a patient with obstruction due to an unknown cause). Hormonal levels were normal in the 22 patients. In addition, testicular biopsies of 3 twisted testes from 3 young adult men showing a number of adult spermatids higher than that of young spermatids were also included in the study. All testicular biopsies—including those of the twisted testes—showed an obstructive histologic pattern, consisting of a mosaic distribution of testicular lesions: mainly tubular ectasis and germ cell sloughing into the adluminal compartment of seminiferous tubules. The increase in the number of adult spermatids was bilateral in 1 of the 6 vasectomized men who underwent bilateral biopsy, and in 7 of the 11 bilaterally biopsied patients with obstructive azoospermia due to other causes. The most probable explanation for the increased number of adult spermatids is stagnation of testicular fluid, caused by sperm excretory duct obstruction. The unilateral increase in the number of adult spermatids in vasectomized men might be related to the occurrence of a spermatic granuloma (a frequent finding in vasectomy) in the proximal end of the sectioned ductus deferens ipsilateral to the testis with nonincreased adult spermatid numbers, and the absence of spermatic granuloma in the ductus deferens ipsilateral to the testis with increased adult spermatid numbers. This granuloma would produce, in addition to spermatozoon destruction, reabsorption of the testicular and epididymal fluids. The higher rate of bilateral increase, in the number of young spermatids observed in the patients with congenital lesions of the ductus deferens or the ductus epididymidis, might be related to the absence of spermatic granulomas in congenital obstructions.
Effects of Thawing Procedure on Postthawed In Vitro Viability and In Vivo Fertility of Red Deer Epididymal Spermatozoa Cryopreserved at −196°C
Authors: Soler Ana J. , García Andrés J. , Fernández‐Santos María R. , Esteso Milagros C. , Garde José J.
Published: December 2015

ABSTRACT: In this study, we have determined the effects of individual factor and thawing procedure on in vitro viability and in vivo fertility of frozen‐thawed red deer epididymal spermatozoa. The spermatozoa that were collected from 13 Iberian deer stags were diluted at room temperature in a Triladyl®‐20% egg yolk medium and frozen in nitrogen vapors. In the first experimental series, sperm samples were collected from 10 mature stags. For thawing, the frozen straws were subjected to 3 different procedures: I (37°C for 20 seconds), II (60°C for 8 seconds) and III (70°C for 5 seconds). Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SM) and of plasma membrane and acrosome (NAR) integrities. Statistically significant variations were found (P < .05) between stags for most of the seminal parameters evaluated. The thawing procedure did not have an effect on the seminal characteristics evaluated after this process, except for SM (P < .05), with the best overall recovery rates after freezing and thawing found with the use of protocol I. Our results also show a differential resistance to return to isosmotic conditions of spermatozoa thawed using the different thawing protocols. In the second experimental series (insemination artificial trial), with spermatozoa from 3 stags, results of fertility were statistically higher (69.7% vs 42.4%, P = .014) when spermatozoa were thawed at 37°C for 20 seconds than were warmed at 60°C for 8 seconds. Therefore, thawing protocol I, which provides slow thawing rates, was the most beneficial for epididymal spermatozoa thawing of the cervid subspecies analyzed in this study. In summary, high in vitro survival and in vivo fertility of frozen‐thawed deer epididymal spermatozoa were dependent on warming rates, but each stag exhibited its own sensitivity to cryopreservation.
Characterization of Membrane Rafts Isolated From Rat Sertoli Cell Cultures: Caveolin and Flotillin‐1 Content
Authors: Evans William E. , Coyer Rachel L. , Sandusky Matthew F. , Van Fleet Melinda J. , Moore Joseph G. , Nyquist Sally E.
Published: December 2015

ABSTRACT: Membrane rafts from Sertoli cell cultures were isolated as detergent‐insoluble glycosphingolipid‐enriched (DIG) fractions on the basis of their enriched content of glycosphingolipids and cholesterol and the resulting insolubility in 1% Triton X‐100 and their low buoyant density. Because lipid rafts have been implicated in numerous cell functions, including cell signaling and sites for actin/membrane attachment, studies were initiated to characterize Sertoli cell rafts. This study reports the distribution of the raft structural proteins, caveolin and flotillin‐1, implicated in raft microdomain organization. Methods employed included the immunoblotting of cell lysates and detergent‐insoluble glycosphingolipid‐enriched (DIG) fractions, the immunofluorescent microscopy of peritubular myoid cell (PMC) cultures and cryostat‐sectioned testis, and the immunohistochemical staining of paraffin‐embedded sections following microwave antigen retrieval techniques. Sertoli cells and Sertoli DIG fractions were found to lack the common raft‐associated protein, caveolin, a marker protein for caveolae, but they are enriched in the 48‐kd protein, flotillin‐1, a protein also implicated in raft formation, cell signaling, and cell motility. Since the primary cell contaminant of Sertoli cell cultures is the PMC, these cells, along with spermatogenic cell fraction (SPGC), were also examined for caveolin and flotillin‐1 content. The PMCs contained significant concentrations of both caveolin and flotillin‐1. PMCs in culture exhibited a punctate caveolin staining pattern at the cell surface characteristic of a caveolar location. These data support the idea that the pinocytotic vesicles observed in PMCs are caveolae. PMCs also show a perinuclear location for caveolin characteristic of a Golgi location. Cryostat sections of rat testis showed a marked concentration of caveolin in the PMCs. The PMC location of caveolin was also confirmed by the immunohistochemical staining of sections from paraffin‐embedded rat testis following microwave antigen retrieval techniques. Similar experiments showed a more ubiquitous, stage‐specific distribution of flotillin‐1 among testicular cell types.
Quantification of Seminal Plasma Motility Inhibitor/Semenogelin in Human Seminal Plasma
Authors: Yoshida Kaoru , Yamasaki Tadashi , Yoshiike Miki , Takano Shoichi , Sato Itaru , Iwamoto Teruaki
Published: December 2015

ABSTRACT: Semenogelin I and II (Sg I and II) are the major components of human semen coagulum. The protein is rapidly cleaved after ejaculation by the chymotrypsin‐like protease prostate‐specific antigen (PSA), which results in the liquefaction of the semen coagulum and the progressive release of motile spermatozoa. One of the cleavage products of the protein, a 14‐kDa protein, is a sperm motility inhibitor (seminal plasma motility inhibitor [SPMI]). We developed a monoclonal antibody (mAb) that is specific to the fragment of Sgs, SPMI, and a sandwich enzyme‐linked immunosorbent assay (ELISA) system for the quantification of Sgs using this mAb. Then, we measured SPMI/Sg levels in human seminal plasma from healthy male volunteers (n = 100, aged 18–24 years). The mean level of SPMI/Sg in seminal plasma was 19 ± 13 mg/mL (range, 4–68 mg/mL). Log‐transformed SPMI/Sg levels were negatively correlated with the sperm motility (r = −0.229, P = .0220) and positively correlated with the total protein concentration (r = 0.793, P < .0001). This result supports that SPMI, one of the fragments of Sg, has its inhibitory effect on ejaculated spermatozoa in liquefied semen under physiological conditions.
Effect of Simulated Microgravity on Testosterone and Sperm Motility in Mice
Authors: Kamiya Hiroyuki , Sasaki Shoichi , Ikeuchi Takahito , Umemoto Yukihiro , Tatsura Hiroyuki , Hayashi Yutaro , Kaneko Shigeo , Kohri Kenjiro
Published: December 2015

ABSTRACT:We examined changes in the serum testosterone level and in sperm in the testis and epididymis by using tail‐suspended mice, which are a simulation model of the body fluid shift in space, to evaluate the possibility of spermatogenesis failure in space environment. We also studied pathological disorders of the testis in the tail‐suspended mice. Tail suspension was imposed with a tail harness to a degree at which the hindlegs of mice did not touch the floor of the housing unit. In control mice, the tail was similarly fixed with a tail harness to impose the same stress, except that a hindleg remained on the floor. Body weight was not significantly different between the 2 groups during 7 days, and testicular weight was significantly different. The testosterone level was significantly lower in the tail‐suspended group (0.71 ± 1.24 ng/mL) than in the control group (2.38 ± 3.50 ng/mL;P< .05). Microscopy with hematoxylin and eosin (HE) and periodic acid‐Schiff (PAS) staining showed a small proportion of seminiferous tubules with impairment of spermatogenic function in the tail‐suspended group, and multinucleated giant cells were occasionally noted. Terminal deoxynucleotidyl tranferase‐mediated nick end‐labeling staining revealed positive cells even in animals in which impairment was considered to be mild based on HE and PAS staining. Many cells showed intense p53 immunostaining compared to the control group, with more intense staining of the nucleus in the tail‐suspended group. The proportion of motile sperm was slightly but not significantly reduced in the tail‐suspended group. However, the mean movement velocity of the motile spermatozoa was significantly decreased.
Lack of Voltage‐Dependent Calcium Channel Opening During the Calcium Influx Induced by Progesterone in Human Sperm. Effect of Calcium Channel Deactivation and Inactivation
Authors: Guzmán‐Grenfell Alberto Martín , González‐Martínez Marco T.
Published: December 2015

ABSTRACT: Progesterone induces calcium influx and acrosomal exocytosis in human sperm. Pharmacologic evidence suggests that voltage‐dependent calcium channels (VDCCs) are involved. In this study, membrane potential (Vm) and intracellular calcium concentration ([Ca2+]i) were monitored simultaneously to assess the effect of VDCC gating on the calcium influx triggered by progesterone. Holding the Vm to values that maintained VDCCs in a deactivated (‐71 mV) closed state inhibited the calcium influx induced by progesterone by approximately 40%. At this Vm, the acrosomal reaction induced by progesterone, but not by A23187, was inhibited. However, when the Vm was held at −15 mV (which maintains VDCCs in an inactivated closed state), the progesterone‐induced calcium influx was stimulated. Furthermore, the progesterone and voltage‐dependent calcium influxes were additive. These findings indicate that progesterone does not produce VDCC gating in human sperm.
Identification of Glutamate Receptors and Transporters in Mouse and Human Sperm
Authors: Hu Jia Hua , Yang Na , Ma Ying Hua , Jiang Jie , Zhang Jin Fu , Fei Jian , Guo Li He
Published: December 2015

ABSTRACT: γ‐Aminobutyric acid (GABA) and glutamate (Glu) are considered as the predominant inhibitory and excitatory neurotransmitters in mammalian central nervous systems (CNS), respectively. The presence of the GABA system and metabotropic glutamate receptors in sperm prompted us to explore the existence of ionotropic glutamate receptors and glutamate transporters in sperm. Immunofluorescent analysis was used to investigate the existence and location of glutamate, glutamate receptor (NR2B), and glutamate transporter (GLT1) in mouse and human sperm. Our present results showed that NR2B was located in the midpiece of sperm, whereas GLT1 mainly existed in the head. Moreover, glutamate uptake activity was detected in mouse sperm and it could be blocked by dihydrokainic acid (DHK, GLT1‐selective inhibitor) and DL‐threo‐beta‐hydroxyaspartic acid (THA, nonselective inhibitor). In addition, reverse transcription‐polymerase chain reaction technique and sequencing analysis revealed that glutamate transporters (GLT1 and EAAC1) and ionotropic glutamate receptors (NR1, NR2B, GluR6, and KA2) existed in mouse sperm as well as in human sperm. The present findings are the first direct evidence for the existence of ionotropic glutamate receptors and glutamate transporters in sperm. It also indicates that, in sperm, glutamate receptors and transporters might have functions other than neurotransmission.
Spermiogenic Germ Cell Phase—Specific DNA Damage Following Cyclophosphamide Exposure
Authors: Codrington Alexis M. , Hales Barbara F. , Robaire Bernard
Published: December 2015

ABSTRACT: The production of genetically competent spermatozoa is essential for normal embryo development. The chemotherapeutic drug cyclophosphamide creates cross‐links and DNA strand breaks in many cell types, including germ cells. This study assessed the phase specificity of the susceptibility of spermiogenic germ cells to genetic damage induced by cyclophosphamide. Adult male rats were given cyclophosphamide using one of four schedules: 1) high dose/acute— day 1, 100 mg/kg; 2) low dose/subchronic, 4 days—days 1–4, 6.0 mg/kg/d; 3) high dose/subchronic, 4 days—day 1, 100 mg/kg, and days 2–4, 50 mg/kg/d; and 4) low dose/chronic—daily, 6.0 mg/kg/d for 14–28 days. To capture cauda epididymal spermatozoa exposed to cyclophosphamide during late, mid‐, and early spermiogenesis, animals were sacrificed on days 14, 21, and 28, respectively. Spermatozoa were analyzed for DNA strand breaks using the comet assay. No dramatic increases in damage were seen after high‐dose/acute exposure to cyclophosphamide. Subchronic exposure showed a dose‐related increase in DNA damage; maximal damage, as demonstrated by comet tail parameters, was seen after 21 days, reflecting an increased susceptibility of step 9–14 spermatids. Low‐dose chronic exposure to cyclophosphamide induced DNA damage, which reached a plateau by day 21. The magnitude of damage at all time points after low‐dose chronic exposure was much greater than that following low‐dose exposure for 4 days, indicating an accumulation of damage over time. Thus, the DNA damage induced by cyclophosphamide is germ cell phase—specific. The most damaging effects of cyclophosphamide occurred during a key point of sperm chromatin remodeling (histone hyperacetylation and transition protein deposition). We speculate that strand breaks disrupt chromatin remodeling, hence affecting chromatin structure and embryo development.
Compound Heterozygous Mutations in the SRD5A2 Gene Exon 4 in a Male Pseudohermaphrodite Patient of Chinese Origin
Authors: Fernández‐Cancio Mónica , Nistal Manuel , Gracia Ricardo , Molina M. Antonia , Tovar Juan Antonio , Esteban Cristina , Carrascosa Antonio , Audí Laura
Published: December 2015

ABSTRACT: The goal of this study was to perform 5‐α‐reductase type 2 gene (SRD5A2) analysis in a male pseudohermaphrodite (MPH) patient with normal testosterone (T) production and normal androgen receptor (AR) gene coding sequences. A patient of Chinese origin with ambiguous genitalia at 14 months, a 46,XY karyotype, and normal T secretion under human chorionic gonadotropin (hCG) stimulation underwent a gonadectomy at 20 months. Exons 1–8 of the AR gene and exons 1–5 of the SRD5A2 gene were sequenced from peripheral blood DNA. AR gene coding sequences were normal. SRD5A2 gene analysis revealed 2 consecutive mutations in exon 4, each located in a different allele: 1) a T nucleotide deletion, which predicts a frameshift mutation from codon 219, and 2) a missense mutation at codon 227, where the substitution of guanine (CGA) by adenine (CAA) predicts a glutamine replacement of arginine (R227Q). Testes located in the inguinal canal showed a normal morphology for age. The patient was a compound heterozygote for SRD5A2 mutations, carrying 2 mutations in exon 4. The patient showed an R227Q mutation that has been described in an Asian population and MPH patients, along with a novel frameshift mutation, Tdel219. Testis morphology showed that, during early infancy, the 5‐α‐reductase enzyme deficiency may not have affected interstitial or tubular development.
The Contractile Wall of the Caput Epididymidis in Men Affected by Congenital or Postinflammatory Obstructive Azoospermia
Authors: Pelliccione Fiore , Cordeschi Giuliana , Giuliani Valeria , D'Abrizio Piera , Necozione Stefano , Negri Luciano , Mancini Mario , Sagone Patrizia , Francavilla Felice , Colpi Giovanni Maria , Francavilla Sandro
Published: December 2015

ABSTRACT: The transport and storage of spermatozoa in the epididymis depend on the contractile activity of its tubular wall. It is not known what differences exist in the contractile wall of the human epididymis in cases of obstructive azoospermia. The contractile wall in the tubules of the caput epididymidis was analyzed by light microscopy and transmission electron microscopy in 10 azoospermic men, 5 with a bilateral congenital absence of vas deferens (CBAVD) and 5 with a bilateral postinflammatory congestive obstruction of the epididymis. Five specimens from the same region of the caput epididymidis, obtained from fertile men who had undergone an orchidectomy because of testicular cancer, served as controls. No differences were observed between congenital and congestive obstructions. The contractile wall in caput tubules proximal to the obstructed level was strongly thickened when compared with controls (62.98 ± 5.84 μ; 80.82 ± 7.72 μ vs 19.59 ± 2.23 μ, respectively, for congestive and congenital obstructions vs controls; P < .0001 vs controls), and the spindle‐shaped myoid cells, which formed the contractile wall in normal cases, were replaced by large smooth muscle cells (SMCs) that showed features of coexisting contractile and secretory functions. The former included crowded cytoplasmic bundles of thin myofilaments (5–6 nm in diameter) converging to a large number of dense bodies, numerous micropinocytotic vesicles of the plasma membrane, and a continuous cell basement membrane. The presence of a developed rough endoplasmic reticulum and a Golgi complex, associated with the accumulation of thick layers of pericellular basement membrane—like material and ground substance, was indicative of a secretory phenotype of SMCs. The increased mechanical forces on the epididymal wall upstream from the obstruction might eventually activate the differentiation of myoid cells into SMCs, leading to an altered physiology of the contractile wall that could have possible clinical relevance in the case of microsurgical epididymovasostomy.
Gene Expression in the Spermatogenically Inactive “Dark” and Maturing “Light” Testicular Tissues of the Prepubertal Colt
Authors: Ing Nancy H. , Laughlin Andy M. , Varner Dickson D. , Welsh Thomas H. , Forrest David W. , Blanchard Terry L. , Johnson Larry
Published: December 2015

ABSTRACT:In the testis of the 1.5‐year‐old horse, spermatogenesis initiates locally in grossly light, central areas that contrast with grossly dark, peripheral areas that are as yet inactive in spermatogenesis. Gene expression was compared between “light” and “dark” tissues of 1.5‐year‐old horse testes to identify mechanisms important to the initiation of spermatogenesis. Microarrays containing human cDNAs were used to assess expression levels of 9132 genes simultaneously in matched pairs of dark and light testis tissues from 3 prepubertal colts. In all 3 analyses, dysferlin (DYS), down‐regulated in ovarian cancer 1 (DOC1), and Golgi apparatus protein 1 (GLG1) genes were preferentially expressed in dark tissues, while outer dense fiber of sperm tails (ODF2) and phosphodiesterase 3B (PDE3B) genes were more highly expressed in light testis tissue (>1.7 balanced difference value, Incyte GEM tools software). Expression levels of 88 additional genes appeared to be different between dark and light tissues in 2 of the 3 microarray analyses. The preferential expression of DYS, DOC1, ODF2, and PDE3B genes in dark or light testis tissues was confirmed on Northern blots and localized to cell types by in situ hybridization. Future studies to determine the role of genes regulated during the initiation of spermatogenesis may aid in elucidating molecular mechanisms during this critical time as well as in identifying new therapies for enhancing male fertility.
Sperm Morphology and Preparation Method Affect Bovine Embryonic Development
Authors: Walters Anneke H. , Eyestone Willard E. , Saacke Richard G. , Pearson Ronald E. , Gwazdauskas Francis C.
Published: December 2015

ABSTRACT: This study was conducted to evaluate the effect of sperm separation methods of semen samples collected from bulls subjected to scrotal insulation on embryonic development after in vitro fertilization (IVF) and to determine whether IVF results would be affected by various heparin concentrations. Morphologically abnormal semen samples were obtained and cryopreserved from Holstein bulls following scrotal insulation for 48 hours. Standard protocols using the Percoll gradient (90%/45%) method and the swim‐up method were used to separate spermatozoa fractions in experiment I. The pellet (Ap) and the 45% layer (Bp) were isolated from the Percoll separation, while for the swim‐up separation, the supernatant (As) and the interphase (Bs) were isolated. The overall blastocyst rate for our laboratory control semen was 23.1 ± 2.1% for Percoll separations (Ap and Bp) and 18.2 ± 2.0% for swim‐up (As and Bs) separations. This rate was higher (P < .01) than the rate observed for the semen from the bull that had the greatest response to scrotal insult 5 days prior to the insult, when it was 9.2 ± 2.1% for the Percoll separation and 20.7 ± 2.3% for the swim‐up separation, while semen from 27 days after scrotal insulation (D +27) resulted in no blastocyst formation for the Percoll separation and a 4.2 ± 2.1% rate for the swim‐up separation. In experiment II, semen was sampled from the bulls that responded in the greatest and least degrees to scrotal insult 5 days before scrotal insulation (D −5) and on days 23 (D +23) and 34 (D +34) after scrotal insulation. These samples were exposed to IVF mediums with 3 different heparin concentrations (0.1, 1.0, and 10 μg/mL). There was a significant difference (P < .05) in developmental scores between the D −5 (1.08 ± 0.08), D +23 (0.9 ± 0.08), and D +34 (0.8 ± 0.08) samples, but no differences were observed in blastocyst formation based on the number of cleaved embryos. Increasing the heparin concentration resulted in higher (P < .01) embryonic developmental scores. In conclusion, when semen samples with high percentages of abnormal spermatozoa are used for IVF, semen separation preparation methods affect results. Our results show that the separation methods used under these conditions were inadequate in their ability to provide potentially competent sperm for IVF. However, selecting appropriate sperm separation procedures could improve in the IVF embryonic development of semen from bulls used in artificial insemination. Also, an increase in the heparin concentration was able to partially overcome deficiencies, which suggests that morphologically abnormal spermatozoa undergo capacitation despite possible structural changes to the plasma membrane.
Standardized Methods for Semen Evaluation in a Multicenter Research Study
Authors: Brazil Charlene , Swan Shanna H. , Drobnis Erma Z. , Liu Fan , Wang Christina , Redmon J. Bruce , Overstreet James W. ,   Study for Future Families Research Group
Published: December 2015

ABSTRACT: Semen evaluation methodology is complex and difficult to standardize. Rigorously standardized laboratory protocols and strict quality control (QC) are essential for meaningful comparison of data from multiple sites. We describe the methods used for determination of semen volume, sperm concentration, and percent sperm motility in the Study for Future Families, a multicenter study of semen quality in the United States. Each of these 3 semen parameters was assessed using 2 techniques, which provided the opportunity to compare precision and assess suitability for multicenter studies. Detailed protocols were used, and technicians were centrally trained. A total of 509 semen evaluations were performed. Semen volume measured by weight was greater (P < .0001) than that determined by pipetting (3.7 ± 1.6 mL vs 3.2 ± 1.6 mL). Sperm concentration determined using hemacytometer chambers was consistently higher (P < .001) than that using disposable MicroCell chambers (81.0 × 106/mL vs 65.9 × 106/mL). Precision was slightly greater for the MicroCell chamber. The percentage of motile sperm was assessed by a simple counting technique as well as by the World Health Organization categorical method that assigns individual motile sperm to “a,” “b,” and “c” categories on the basis of progression. When these 3 categories were collapsed, the methods provided values that were not statistically different (P > .05), although the collapsed values tended to be higher (58.1% vs 51.6%) and less precise (CV 7.7% vs 4.1%) for the categorical method than for motility determined using the simple method. The data obtained in this study demonstrate the critical need for rigorous standardization of protocols and techniques for multicenter studies.
Quality Control of Laboratory Methods for Semen Evaluation in a Multicenter Research Study
Authors: Brazil Charlene , Swan Shanna H. , Tollner Charlene R. , Treece Cathy , Drobnis Erma Z. , Wang Christina , Redmon J. Bruce , Overstreet James W. ,   Study for Future Families Research Group
Published: December 2015

ABSTRACT:Rigorously standardized laboratory protocols and strict quality control (QC) are essential for meaningful comparisons between semen quality data from multiple sites. We describe our experience with the Study for Future Families (SFF), a multicenter study of semen quality in the United States. Detailed protocols were developed, and technicians from each study site attended a training session at the central laboratory. Technicians received blinded replicates from diluted semen specimens for counting by MicroCell and hemacytometer. Sperm motility was assessed using videotaped recordings for simple percent motility and categorical assessment of individual sperm progression as recommended by the World Health Organization (WHO). The mean intertechnician coefficient of variation for individual specimens was 12.6% for MicroCell counts, 15.2% for hemacytometer counts, and 10.5% for percent motility. Intratechnician coefficients of variation averaged 10.3% for MicroCell counts, 12.5% for hemacytometer counts, and 5.2% for percent motility. The average percent differences between the technicians' values and the central standard for individual specimens were 13.5%, 16.6%, and 11.9% for MicroCell counts, hemacytometer counts, and simple percent motility, respectively. We achieved our goal of maintaining mean intratechnician coefficients of variation and mean percent differences from the standard values of 15% or less for measurements of simple percent motility and sperm concentration by MicroCell. Standardization using the Improved Neubauer hemacytometer chamber proved more difficult. We were not successful in standardizing a method for categorical assessment of individual sperm progression.
Preservation of Testicular Arteries During Subinguinal Microsurgical Varicocelectomy: Clinical Considerations
Authors: Grober Ethan D. , O'brien Jeanne , Jarvi Keith A. , Zini Armand
Published: December 2015

ABSTRACT: Microsurgical varicocelectomy with intentional preservation of the testicular artery(ies) is regarded as the gold standard approach to varicocele repair. We sought to determine whether the number of testicular arteries preserved at the time of micro‐surgical varicocelectomy predicts improvement in postoperative semen parameters. We analyzed the records of 334 infertile men who underwent varicocelectomy performed by a single surgeon using a subinguinal microsurgical technique between July 1996 and January 2003. We examined the association between the number of testicular arteries preserved at the time of varicocelectomy and serum follicle‐stimulating hormone (FSH), luteinizing hormone (LH), varicocele grade, testicular volume, and postoperative improvement in semen parameters. Unilateral, left‐sided varicocelectomy was performed in 194 men, while bilateral varicocelectomy was performed in 140 men. Mean (±SE) sperm concentration (20.1 ± 1.5 × 106/mL to 26.7 ± 1.9 × 106/mL, P = .001), percent motility (24.7 ± 1.0% to 30.9 ± 1.2%, P = .001), and percent normal morphology (35.8 ± 1.4% to 37.7 ± 1.5%, P = .046) improved significantly following varicocelectomy. The mean number of preserved testicular arteries was 1.5 on the left (range, 1–4) and 1.5 on the right (range, 1–4). The number of testicular arteries preserved at the time of varicocelectomy did not correlate significantly with preoperative assessment of serum FSH, LH, varicocele grade, and testicular volume or with postoperative improvement in semen parameters. Our data indicate that preoperative parameters are not predictive of the number of testicular arteries identified at the time of microsurgery. These data also suggest that the number of arteries identified and preserved with meticulous spermatic cord dissection does not correlate with improvement in semen parameters.
Males With Subnormal Hypo‐Osmotic Swelling Test Scores Have Lower Pregnancy Rates Than Those With Normal Scores When Ovulation Induction and Timed Intercourse Is Used as a Treatment for Mild Problems With Sperm Count, Motility, or Morphology
Authors: Tartagni Massimo , Cicinelli Ettore , Schonauer Matteo Maria , Causio Franco , Petruzzelli Filomena , Loverro Giuseppe
Published: December 2015

ABSTRACT:This study was designed to evaluate the effectiveness and clinical usefulness of the hypo‐osmotic swelling (HOS) test in predicting successful conception in couples in which men with mild male‐factor infertility criteria were undergoing a timed vaginal inter‐course protocol. One hundred couples, in which mild male infertility was the only abnormality, were included in the study. Semen was analyzed according to standard World Health Organization (WHO) criteria and subjected to the HOS test. Patients were divided into 2 groups: group 1 (n = 39) with normal HOS test and group 2 (n = 61) with abnormal HOS test. All women underwent three consecutive cycles of follicular growth ultrasound monitoring and timed intercourse. Ten couples were exclude from the study. Ten clinical pregnancies were achieved in group 1 with a pregnancy rate per patient and per cycle of 28.5% and 9.5%, respectively. In group 2, 6 pregnancies were achieved, with a pregnancy rate per patient and per cycle of 10.9% and 3.6%, respectively. Both pregnancy rates per patients and per cycle was significantly higher (P < .05) in group 1 than in group 2. The HOS test may be considered an easy and reliable test in identifying among subfertile men those who have a greater possibility of causing pregnancy.
Comparison of Six Density Gradient Media for Selection of Cryopreserved Donor Spermatozoa
Authors: Mousset‐Siméon Nathalie , Rives Nathalie , Masse Lydie , Chevallier Florence , Mace Bertrand
Published: December 2015

ABSTRACT: The aim of our study was to evaluate the efficiency of 4 density gradient media for motile cryopreserved spermatozoa selection to Percoll (Kabi Pharmacia, Uppsala, Sweden) and to Puresperm (J.C.D. International Laboratory, L'Aigle, France). Puresperm was the new medium chosen in our laboratory in 1996 as the substitute for Percoll. The solutions tested were 3 colloidal silane‐coated silica particle media (Isolate, SpermGrad‐100, Sil‐Select Plus) and iodixanol (Optiprep). Semen parameters analyzed after selection were concentration, motility, and morphology. Semen parameters after Puresperm gradient had similar values compared to Percoll. Optiprep was less efficient with a poor concentration. Isolate had a comparatively better concentration, but the capacity of selection was not satisfactory. SpermGrad‐100 and Sil‐Select Plus were less effective than Puresperm. In conclusion, Puresperm could be considered a better alternative to Percoll for cryopreserved spermatozoa migration.
Molecular Cloning of Rat Spergen‐3, a Spermatogenic Cell‐Specific Gene‐3, Encoding a Novel 75‐kDa Protein Bearing EF‐Hand Motifs
Authors: Iida Hiroshi , Yamashita Haruhiro , Doiguchi Masamichi , Kaneko Takane
Published: December 2015

ABSTRACT: By use of differential display in combination with cDNA cloning approach, we isolated a novel rat gene designated as Spergen‐3, which has an open reading frame of 2055‐length nucleotides encoding a protein of 685 amino acids. Spergen‐3 gene is composed of 15 exons and mapped on chromosome 5q36, and its mouse counterpart, which shares 85.5% identity to rat Spergen‐3 at the amino acid level, is mapped on chromosome 4E1. Spergen‐3 encodes a 75‐kDa soluble protein bearing putative 2 EF‐hand motifs, proline‐repeat, and a putative nuclear localization signal. Of the 2 EF‐hand motifs in Spergen‐3, the second one seems to match the consensus sequence. Reverse transcription‐polymerase chain reaction analysis showed that the expression of Spergen‐3 is developmentally up‐regulated and that it is exclusively expressed in testis. In situ hybridization revealed that Spergen‐3 mRNA was exclusively expressed in haploid spermatids, but its signal was weak or undetectable in spermatogonia, spermatocytes, and Sertoli cells as well as in interstitial cells. We interpreted these data as a potential role of Spergen‐3, a new member of EF‐hand family, in differentiation of haploid spermatids in testis.
Intratesticular Testosterone Concentrations Comparable With Serum Levels Are Not Sufficient to Maintain Normal Sperm Production in Men Receiving a Hormonal Contraceptive Regimen
Authors: Coviello Andrea D. , Bremner William J. , Matsumoto Alvin M. , Herbst Karen L. , Amory John K. , Anawalt Bradley D. , Yan Xiaohua , Brown Terry R. , Wright William W. , Zirkin Barry R. , Jarow Jonathan P.
Published: December 2015

ABSTRACT: Intratesticular testosterone (ITT) is thought to play a key role in the control of spermatogenesis in man but is rarely measured. The purposes of this study were 1) to examine the relationship between intratesticular fluid and serum testosterone (T) at baseline and during treatment with a contraceptive regimen known to suppress spermatogenesis and 2) to measure intratesticular fluid androgenic bioactivity. Seven men received 6 months of T enanthate (TE) 100 mg weekly intramuscularly plus levonorgestrel (LNG) 62.5 or 31.25 μg orally daily. Testicular fluid was obtained by percutaneous aspiration at baseline and during month 6. Mean luteinizing hormone (LH) was suppressed 98% from 3.79 ± 0.80 IU/L at baseline to 0.08 ± 0.03 IU/L. Mean follicle stimulating hormone (FSH) was suppressed 97%, from 3.29 ± 0.67 IU/L to 0.10 ± 0.03 IU/L. Mean serum T levels were similar before (22.8 ± 1.9 nmol/L) and during treatment (28.7 ± 2.0 nmol/L) (P = .12). ITT (822 ± 136 nmol/L) was ∼40× higher than serum T (P < .001) at baseline. ITT was suppressed 98% during treatment to 13.1 ± 4.5 nmol/L, a level similar to baseline serum T (P = .08) but significantly lower than on‐treatment serum T (P = .01). At baseline, intratesticular fluid androgenic bioactivity (583 ± 145 nmol/L) was 70% of the ITT concentration measured by radioimmunoassay. Intratesticular androgenic bioactivity was suppressed 93% to 40 ± 22 nmol/L (P < .01) during treatment, but was 3× higher than ITT (13.1 ± 4.5 nmol/L). Sperm counts declined from 65 ± 15 million/mL to 1.3 ± 1.3 million/mL. In summary, TE plus LNG dramatically suppressed ITT (98%) and intratesticular androgenic bioactivity (93%) to levels approximating those in serum. ITT levels comparable with serum T were insufficient to support normal spermatogenesis. Intratesticular androgenic bioactivity was higher than ITT during treatment, suggesting that other androgens may be prevalent in the low‐ITT environment.