Normal Testicular Histology Research Articles

Therapeutic Effects of Mesenchymal Stem Cell Conditioned Medium in Rat Varicocele Model.

This study aimed to examine the therapeutic effects of injection of conditioned medium of adipose-derived mesenchymal stem cells (ADMSC-CM) in a surgically created varicocele model in comparison with varicocelectomy. Twenty-eight male Wistar Albino rats were randomly divided into four groups: sham group, varicocele group, varicocelectomy group, and ADMSC-CM injection group. Sperm parameters were analyzed in samples taken from the epididymis after treatment. Malondialdehyde and superoxide dismutase (SOD) levels in blood samples were examined by biochemical analysis. The testicular tissues were stained with hematoxylin-eosin for histological examination (Johnsen's Score). Additionally, Western Blot analyzes were performed to detect Claudin-11 levels, the functional protein of the blood-testis barrier, in testicular tissues. Varicocelectomy and ADMSC-CM treatments significantly improved mean sperm parameters (concentration, progressive motility, motility, normal sperm morphology) (p≤0.05 for all). Both treatment groups had increased SOD levels along with a decrease in malondialdehyde levels (p≤0.05 for all). No significant difference was observed between the ADMSC-CM group and the varicocelectomy group in preserving normal testicular histology according to Johnsen's Score (p=0.114). Levels of Claudin-11 were significantly higher in the varicocelectomy and ADMSC-CM groups compared to the varicocele group (p≤0.05 for all). The therapeutic effects of ADMSC-CM in varicocele model may involve secretion of anti-inflammatory and regenerative factors from ADMSC. ADMSC-CM injection appears to be a promising new strategy in the treatment of varicocele.

Pharmacological inhibition of ER stress mitigates testicular pathology in hind‐limb unloaded mice

Reduced physical activity and cephalic fluid shift are common occurrences in prolonged bed rest and space flight, and can lead to testicular dysfunction. Experimental mouse model of hind‐limbs‐unloading (HU) mimics spaceflight and bed rest and recapitulates several features of testicular anomalies in such conditions including disruption of tubular architecture and associated histological changes that together contribute to male infertility. However, an effective pharmacological intervention to these changes remain elusive, partly because molecular mechanisms are poorly understood. Disruption of protein folding by endoplasmic reticulum (ER) or ER stress is a common etiology in several diseases but its contribution to testicular pathology is not known. We hypothesize that HU is associated with activation of ER stress and subsequent disruption of normal testicular histology in a time‐dependent manner, while treatment with 4‐Phenylburtyrate (4‐PBA; an ER‐stress inhibitor) will reverse these changes to normal levels. To test this hypothesis, 4‐months‐old male c57B16/j mice were subjected to 14 and 28‐days of HU. A significant reduction in thickness and organization of seminiferous tubular epithelium and discontinuation of germ cell layers was seen after 14 days of HU. We also observed a significant increase in the tubular and luminal diameter along with a reduction in the density of spermatozoa at 14 days. These changes were further intensified at 28 days of HU and were associated with signature alterations in the Raman spectroscopic analysis of the HU testis. Importantly, treatment with 4‐PBA for 3‐4 weeks (100mg/kg BW/d via intraperitoneal injections) partially reversed most of the pathological changes in mouse testis.Collectively, our findings reveal the pivotal role of ER stress in triggering testicular pathology in conditions mimicking spaceflight and present 4‐PBA as a potential therapeutic agent to prevent and/or reduce testicular disruption in such cases.Currently, we are characterizing the associated molecular phenotype in testicular disruption by mapping the global transcriptome and detailed Raman spectroscopic alterations in mouse testes under HU with and without 4‐PBA treatment.

Testicular thrombotic microangiopathy: An unrecognized complication

Thrombotic microangiopathy (TMA) causes global endothelial damage and multiorgan injury. No study has described reproductive organ involvement in TMA. Our study aimed to characterize testicular involvement in TMA. We reviewed autopsies from four patients who underwent hematopoietic cell transplant (HCT) complicated by TMA. Three patients had striking histologic evidence of TMA, while the fourth had normal testicular histology. This suggests that TMA injures the testicles and may adversely affect fertility. There is now an urgent need for a larger analysis of reproductive organ involvement in TMA.

DILTIAZEM PREVENTS MONOSODIUM GLUTAMATE TOXICITY IN THE RAT TESTES

Increased exposure to food additives may exhibit harmful effect on the male reproductive system. Neonatal application of high doses of popular taste enhancer monosodium glutamate (MSG) to different kinds of animals cause lesions of the hypothalamic nuclei and the retina. Later in adulthood, animals exhibit a series of neuroendocrine disorders (stunted growth, obesity and decreased fertility). The mechanism of MSG action is not completely explained yet. We hypothesized that high concentration of MSG could alter permeability of neural membrane for calcium. The objective of our study was to find out whether the pretreatment with diltiazem, a calcium channel blocker, could prevent harmful effect of MSG in the rat testes. Male rat pups were treated with: 0.9% sodium chloride (C group), 4 mg/g BW of MSG (M group), 5 mg/g BW of diltiazem (D group) and diltiazem 5 mg/g BW with MSG (DM group) on 2nd , 4th , 6th , 8th, 10th postnatal day. Animals were sacrificed at the age of six months. MSG treatment resulted in: stunted growth (decreased naso-anal length and tail length; p < 0.05), decreased relative testis weight (p < 0.05), and increased adipose tissue mass (Lee index; p < 0.05), testicular atrophy and decreased histomorphometric parameters (tubular area, tubular perimeter, Feret diameter, tubular diameter, epithelial height; p < 0.001). The rats of C, D and DM groups had normal testicular histology and morphometric parameters. Pretreatment with diltiazem has prevented the development of morphological disorders of testes. Our results suggest that calcium overloading may play an important role among mechanisms of MSG testicular toxicity.

Abnormal histology in testis from prepubertal boys with monorchidism

BackgroundLittle is known about the histology of contralateral descended testes in boys with unilaterally absent testis. We investigated whether absence of one testis is associated with abnormal tissue architecture of the solitary contralaterally descended testis.Design, setting, and patientsFor this retrospective study, we examined the results of biopsies of the contralateral descended testis in 43 boys with monorchidism. Data from 26 control testes from boys of matching ages were selected from results published in 1977 and 2009. During surgery, any nubbins were removed. In each case, the scrotal testis was biopsied, and the testis fixed by subdartos pouch or suture.ResultsOf the 43 affected boys, 23 had normal testicular histology in the contralateral descended testis, whereas 20 (46%) had abnormal histology. Eight of the abnormal biopsies matched the criteria for high infertility risk. Samples from three boys in this latter group revealed a Sertoli-cell-only phenotype. Immunohistochemical assays were positive for steroidogenic acute regulatory (STAR) protein in Leydig cells and spermatogonia. STAR expression was stronger in the monorchid group with normal testicular histology.ConclusionsAlmost half of the patients with unilateral absent testis were at risk for subfertility or infertility. Our results emphasize the need for testicular biopsy of the solitary testis in boys with monorchidism to appropriately assess infertility risk.

Mageb4 , a Testis-Specific Gene, is Dispensable for Mouse Spermatogenesis

Objective: It has recently been shown that the melanoma antigen gene (MAGE) family is expressed in various tumor cell lines but silent in normal tissues, except germ cell lines. Mageb4, a member of the MAGE family, is highly expressed in the testis and homologous in humans and mice. Whole-exome sequencing studies have identified Mageb4 as a possible X-linked cause of inherited male infertility. However, the function of Mageb4 protein remains largely unknown. Methods: Using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 technology, we generated a Mageb4 knockout mouse model (Mageb4−/Y) to explore the role of this gene in spermatogenesis. Results: First, immunostaining of testicular cells showed that Mageb4 is localized in the cytoplasm of spermatogonia. Second, Mageb4−/Y male mice displayed significant increases in apoptosis. However, Mageb4−/Y male mice showed normal fertility, including normal sperm concentration, sperm motility, and testicular and epididymal histology. Conclusions: These findings suggest that, despite testis-exclusive expression, Mageb4 is dispensable for mouse spermatogenesis. Future research should focus on the role of this gene in apoptosis, aiming to provide clinical guidance regarding male infertility.

Prolonged Oral Administration of a Pan-Retinoic Acid Receptor Antagonist Inhibits Spermatogenesis in Mice With a Rapid Recovery and Changes in the Expression of Influx and Efflux Transporters.

We have previously shown that oral administration of a pan-retinoic acid receptor antagonist in mice daily at 2.5 mg/kg for 4 weeks reversibly inhibited spermatogenesis, with no detectable side effects. To elucidate the lowest dose and the longest dosing regimen that inhibits spermatogenesis but results in complete restoration of fertility upon cessation of administration of the drug, we examined the effects of daily doses as low as 1.0 mg/kg with dosing periods of 4, 8, and 16 weeks. We observed 100% sterility in all regimens, with restoration of fertility upon cessation of the drug treatment even for as long as 16 weeks. There was no change in testosterone levels in these males and the progeny examined from 2 of the recovered males were healthy and fertile, with normal testicular weight and testicular histology. Strikingly, a more rapid recovery, as assessed by mating studies, was observed at the lower dose and longer dosing periods. Insight into possible mechanisms underlying this rapid recovery was obtained at 2 levels. First, histological examination revealed that spermatogenesis was not as severely disrupted at the lower dose and with the longer treatment regimens. Second, gene expression analysis revealed that the more rapid recovery may involve the interplay of ATP-binding cassette efflux and solute carrier influx transporters in the testes.

A testis‐specific gene, Ubqlnl, is dispensable for mouse embryonic development and spermatogenesis

Spermatogenesis is a complex process through which male germ-line stem cells proliferate and differentiate, eventually becoming spermatozoa. Successful spermatogenesis requires precise regulation of gene expression at transcriptional, post-transcriptional, translational and post-translational levels. Ubiquitination is a post-translational modification known to control protein turnover and, thus, plays a critical role in numerous biological processes, including spermatogenesis (Hou et al., 2012). The ubiquilin family consists of five ubiquitin-like proteins (Ubqln1-4 and Ubqlnl), all of which contain an N-terminal UBL domain and a C-terminal ubiquitin-associated (UBA) domain (Hou et al., 2012). We previously demonstrated that Ubqln3 is exclusively expressed in the testis, yet ablation of Ubqln3 causes no discernable phenotype, suggesting it has a dispensable role in mouse spermatogenesis (Yuan S, 2015). We subsequently discovered that another member of the ubiquilin gene family, Ubqlnl, is only ~5 kb apart from Ubqln3 on mouse chromosome 7. Of 10 different mouse organs assessed using quantitative reverse-transcriptase PCR (qPCR), we found that, similar to Ubqln3, Ubqlnl was exclusively detected in the testis (Fig. 1A). Ubqlnl transcript was first detected in the testes by postnatal day 28 (P28) and plateaued in adults (P56) (Fig. 1B). The timing of Ubqlnl expression onset at ~P28 suggests that it is mainly expressed in elongating/elongated spermatids, which is a pattern similar to that of Ubqln3 (Yuan S, 2015). Figure 1 Ubqlnl is a testis-specific gene dispensable for spermatogenesis. A: qPCR analyses of expression levels of five ubiquilin genes (Ubqln1-4 and Ubqlnl) in 10 organs of adult mice. B: Expression levels of five ubiquilin genes in developing testes, based ... To study the physiological role of Ubqlnl, we generated Ubqlnl global knockout (KO) mice using cryopreserved sperm carrying a null Ubqlnl allele (allele information: Ubqlnl_A06, C57Bl/6N-Ubqlnltm1(KOMP)Vlcg) that is available from the Knockout Mouse Project (KOMP) repository. The Ubqlnl-null allele was generated using the “gene trap” strategy, in which a gene-trap cassette (ZEN-UB1) was inserted into the Ubqlnl locus (KOMP project ID: VG11289). Genotyping analyses demonstrated that Ubqlnl KO mice were homozygous for Ubqlnl-null alleles (Fig. 1C), and qPCR analyses confirmed the absence of Ubqlnl expression in the testes of KO males (Fig. 1D). Both female and male Ubqlnl-KO mice were viable, and did not exhibit discernable differences in either growth or behavior compared to their wild-type (WT) or heterozygous littermates. A 5-month-long fecundity test using Ubqlnl-KO males bred with WT females of proven fertility revealed no significant difference in either litter size (7.7 ± 1.2 for WT and 7.3 ± 1.1 for KO, n=6, P > 0.05; t-test) or litter interval (22.8 ± 2.8 for WT and 23.3 ± 2.2 for KO, n=6, P > 0.05; t-test) compared to WT breeding pairs, suggesting that Ubqlnl KO males are fertile. Consistent with their normal fertility, testis size and weight of adult Ubqlnl KO males were similar to those of WT males (Fig. 1E), and Ubqlnl KO males displayed normal testicular histology with robust spermatogenesis (Fig. 1F) and normal sperm morphology (Fig.1G). Taken together, our data suggest that, despite its testis-exclusive expression, Ubqlnl is not required for spermatogenesis or fertility in male mice. We also analyzed transcripts levels of the other four ubiquilin genes (Marin, 2014), in WT and Ubqlnl-KO testes using qPCR. Interestingly, levels of Ubqln1 and Ubqln4 mRNAs were significantly increased in Ubqlnl-KO compared to WT testes (Fig. 1D). This suggests that other ubiquilin family members may have compensated for the loss of Ubqlnl, thus maintaining a normal phenotype in Ubqlnl-KO males. Together, these findings demonstrate that Ubqlnl is dispensable for both embryonic and postnatal development and for spermatogenesis in mice.

The management of the persistent Müllerian duct syndrome

ObjectivesTo report the findings and management of patients with persistent Müllerian duct syndrome (PMDS). Patients and methodsNineteen phenotypically male patients (aged 8months to 27years) presented with testicular maldescent. All of them had normal male external genitalia. Two of them had had a previous diagnosis of persistent Müllerian structures. All patients were karyotyped, and had a hormonal profile, diagnostic laparoscopy, retrograde urethrocystogram, gonadal biopsies, and surgical management according to the findings. The follow-up was based on a clinical examination, abdominal ultrasonography (US) and scrotal colour-Doppler US at 3 and 6months after surgery, and every 6months thereafter. ResultsDiagnostic laparoscopy showed the presence of persistent Müllerian structures in all 19 patients. All patients had a normal male karyotype (46XY). Ten patients had a laparoscopic excision of their Müllerian structures while the remaining nine patients had their Müllerian structures left in place. No malignant changes were found in the excised Müllerian tissues. Of the 37 gonadal biopsies taken, 31 (84%) indicated normal testes. ConclusionsThe incidence and prevalence of PMDS are not well estimated. Müllerian structures should be removed whenever possible to avoid the risk of malignant transformation. The early diagnosis of PMDS makes possible the excision of Müllerian structures and a primary orchidopexy. A long-term follow-up is needed for patients with intact Müllerian structures and magnetic resonance imaging might be a better method than US for that purpose. Most of the patients had normal testicular histology, which might allow fertility.

Regional differences in maturation of germ cells of cryptorchid testes: role of environment

To investigate differences in maturation of germ cells in cryptorchid testes in three different regions. A total of 103 consecutive patients were operated for unilateral undescended testis in Vojvodina, from March 2006 until September 2007, and had a testicular biopsy performed. Germ cells were counted, and the presence of Ad spermatogonia was noted. Biopsies were compared to biopsies of similar patients from two different regions: Philadelphia, USA (130), and Liestal, Switzerland (55 patients). In Vojvodina, 84.5% of patients had Sertoli cells only, or some spermatogonia, but no Ad spermatogonia, and 15.5% had Ad spermatogonia. In Philadelphia, 59.3% of patients had poor testicular histology, and 40.7% had Ad spermatogonia. In Liestal, 61.8% of patients had no, or some, spermatogonia, but no Ad spermatogonia, and 38.2% had Ad spermatogonia. There was a difference (p = 0.000025) between the patients with normal testicular histology from Philadelphia and those from Vojvodina, as well as between the patients from Vojvodina and Liestal (p = 0.0027). The reduction in the number of germ cells in patients with cryptorchidism from Vojvodina is more pronounced than patients from either Switzerland or USA. This is a unique observation, since such a study has not been published yet.

Genetic evidence against a role for W-linked histidine triad nucleotide binding protein (HINTW) in avian sex determination

Birds have a ZZ/ZW sex chromosome system, but the mechanism of sex determination remains unknown. The heterogametic sex is female (ZW) and one hypothesis holds that the W chromosome carries a dominant-acting ovary-determining gene. The strongest candidate ovary-determinant on the W chromosome is HINTW, which encodes an aberrant nucleotide hydrolase enzyme. HINTW is conserved amongst all carinate (flying) birds and it is strongly expressed in the gonads and other tissues of female chicken embryos. This and other lines of circumstantial evidence support the proposal that HINTW is the female-determining gene in birds. However, in vivo gain-of-function or loss-of-function studies have not hitherto been reported to test this hypothesis. We tested the potential role of HINTW by mis-expressing it in genetically male (ZZ) embryos, using the RCASBP avian retroviral vector. Strong, widespread expression was delivered throughout the embryo, including the urogenital system, as assessed by whole mount in situ hybridisation. This expression pattern mimicked that seen in normal ZW females, in which HINTW is widely expressed. Strong mis-expression was observed throughout the gonads of genetic male (ZZ) embryos. However, despite strong HINTW expression, ZZ gonads developed normally as bilateral testes. In tissue sections of ZZ urogenital systems transgenic for HINTW, normal testicular histology was observed. Female (ZW) gonads over-expressing HINTW also developed normally, with normal ovarian structure and left/right asymmetry. These results provide genetic evidence against a dominant role for HINTW in avian sex determination.

Testicular Histology in Boys With Prader-Willi Syndrome: Fertile or Infertile?

Prader-Willi syndrome is associated with hypogonadism. Cryptorchidism is found in 93% of cases and considered a phenotypic criterion. Men with Prader-Willi syndrome are thought to be infertile. To study the fertility probability in boys with Prader-Willi syndrome we analyzed testicular histology in 8 prepubertal boys and 1 man. Eight boys 16 months to 14 years old with a proven molecular diagnosis of Prader-Willi syndrome, including 6 with a deletion on chromosome 15 and 2 with uniparental maternal disomy of chromosome 15, underwent orchiopexy and the man underwent unilateral orchiectomy. Prepubertal testes were classified into 4 Nistal categories according to mean tubular diameter, the tubular fertility index (average percent of tubules containing spermatogonia) and the Sertoli's cell index. Two of 8 prepubertal boys showed a favorable Nistal score of I, 1 showed a Nistal score of II and 5 showed a Nistal score of III. The testis in the man showed diffuse tubular atrophy with tubular hyalinization, a Sertoli's cell nodule, vacuolized Leydig cells, peritubular hyalinization and small tubuli. Prader-Willi syndrome appears to be a heterogenic disorder with respect to testicular histology. Although most boys showed absent spermatogonia, 2 of 8 had normal testicular histology. Therefore, it is suggested that it is uncertain what the fertility outcome is in boys with Prader-Willi syndrome.

Extensive nonhomologous meiotic synapsis between normal chromosome axes of an rcp(3;6)(p14;q21) translocation in a hairless Mexican boar

Due to its low fertility, expressed as small litter size, a Mexican hairless boar was subjected to cytogenetic investigation. Analysis of G-banded mitotic chromosomes revealed a reciprocal chromosome translocation, rcp(3;6) (p14;q21). Synaptonemal complex analysis showed a regular pairing behavior of the translocation chromosome axes, always resulting in a quadrivalent configuration. However, due to extensive nonhomologous pairing between the axes of nonderivative chromosomes 3 and 6, the quadrivalent mostly had an asymmetrical cross-shaped morphology. The nonhomologous pairing occurred not only at mid and late pachytene, but also at the earliest stage of pachytene. It seems that early pachytene heterosynapsis is a common phenomenon in the pairing behavior of pig reciprocal translocations. Therefore, heterosynapsis may reduce apoptosis of germ cells due to partial absence of homologous synapsis during the pairing phase of meiosis. The frequency of spermatocytes showing quadrivalent configurations with unpaired axial segments apparently did not affect germ cell progression in the boar, since fairly normal testicular histology was noticed.

Status of the executioner step of apoptosis in human with normal spermatogenesis and azoospermia

To localize the different proteins involved in the executioner step of apoptosis in the human testicular tissue: effector caspases (3, 6, and 7), caspase inhibitors called inhibitor of apoptosis proteins (IAPs, such as XIAP), and IAP inhibitors such as Smac/DIABLO and to investigate XIAP and Smac expression and activation of caspase-3 in azoospermia. Retrospective study. The Biology of Reproduction Center of Poissy and Inserm U407, France. Twenty-five patients diagnosed as azoospermic and 4 patients with normal testicular histology. Testicular biopsies for histopathological assessment. Localization of proteins by immunohistochemistry. Effector caspase-7 seemed to be absent from normal human testes, whereas procaspase-3 and procaspase-6 were detected in both somatic and germ cells. XIAP was mainly expressed in Sertoli cells, whereas its inhibitor Smac was detected in pachytene spermatocytes. On the other hand, although few apoptotic germ cells were detected in biopsies from patients with obstructive azoospermia, increased levels of apoptotic germ cells were detected in spermatogenetic arrest. This increase in apoptotic germ cells was associated with increased levels of active caspase-3 in patients with spermatogenetic arrest, whereas the expression of XIAP and Smac/DIABLO was at similar levels in all groups. Active caspase-3 might be important in the apoptotic process observed in spermatogenetic arrest.

Relationship between adult dark spermatogonia and secretory capacity of Leydig cells in cryptorchidism

To examine whether hormonal therapy before orchidopexy affects the histology of the testis and to assess the responsiveness of the Leydig cells, as it has been shown that although basal plasma testosterone levels are within the 'normal' range in cryptorchid boys there is an insufficient increase of testosterone after a human chorionic gonadotrophin (hCG) stimulation in approximately 30% of cryptorchid boys. In all, 55 boys (aged 1-7 years) with a unilateral undescended testis were included in the study and divided into two groups. Group I (32 boys) received hormonal therapy before orchidopexy; 17 boys received a long-acting LHRH analogue (buserelin) administered as a nasal spray in doses of 20 microg/day for 28 days, followed by 1500 IU hCG intramuscularly (i.m.) once a week for 3 weeks, and the remaining 15 received 1500 IU hCG i.m. once a week for 3 weeks. Group II (33 boys) had orchidopexy alone. During orchidopexy biopsies were taken from the undescended and contralateral descended testes of the boys in both groups for histological analyses. Variations in the number of adult dark (Ad) spermatogonia per tubule (Ad/T) were assessed and testosterone levels were measured during the course of the hormonal therapy (before treatment, 14 days after initiation of buserelin administration, 24 h after each hCG injection, and 3 months after cessation of therapy). In group I, 17 boys (53%) had a 'normal' Ad/T after hormonal treatment vs only six (18%) in group II after orchidopexy alone (P = 0.019). In the hormonally treated boys (group I) we compared the testosterone values 24 h after the second injection of hCG (when the response was most pronounced). Those with a normal Ad/T had a mean (sd) testosterone level of 199.5 (97.6) ng/dL vs 99.6 (85) ng/dL in those with an inadequate Ad/T response to hormonal therapy (P < 0.003). We have confirmed that there are two subgroups of cryptorchid boys. Patients with a sufficient Leydig cell secretory capacity will have normal testicular histology and Ad spermatogonia count after hormonal treatment. While those with a suboptimal Leydig cell capacity will have a low Ad spermatogonia count and consequently poor prognosis for future fertility, despite successful surgery. As to whether different types and durations of the hormonal therapy in patients with impaired Leydig cell response could lead to improved testicular histology and consequently improved prognosis for future fertility, remains to be answered.

Can inhibin-B predict the outcome of microsurgical epididymal sperm aspiration in patients with suspected primary obstructive azoospermia?

To evaluate whether inhibin-B can predict the outcome of a microsurgical epidymal sperm aspiration (MESA) procedure in patients with suspected primary obstructive azoospermia (OA) and if inhibin-B can replace testicular biopsy in the diagnostic work-up of these patients. Inhibin-B levels and testicular biopsy scores were related to the outcome of MESA in 43 patients with suspected primary OA. MESA was considered to be successful when epididymal sperm could be identified during the procedure. Spermatozoa were present in the epididymal aspirate in 28 out of the 43 patients (65%). Inhibin-B values were not significantly different in patients with successful or unsuccessful MESA. The modified Johnsen score, however, was significantly lower in patients with unsuccessful MESA (P = 0.003). A rete testis obstruction or epididymal malfunctioning was found in 15% of patients with suspected primary OA, reflected by unsuccessful MESA despite normal inhibin-B levels and normal testicular histology. Inhibin-B cannot replace testicular biopsy as a diagnostic tool in the work-up of patients with suspected primary OA. Testicular biopsy is useful in identifying patients with spermatogenic arrest, who might have normal inhibin-B values.

Synaptonemal complex analysis in a boar with tertiary, trisomy, product of a rcp(7;17)(q26;q11) translocation.

Synaptonemal complex analysis by electron microscopy showed a trivalent formed by pairing of chromosome 17(7) with chromosome pair 7 in two zygotene cells. In 89 pachytene cells (75.4%) the chromosome 17(7) occurred as a univalent with the axis being unpaired, in the shape of a ring (60.6%), a rod (38.2%) or self-paired (1 cell). Thirty-four (38.2%) of the univalents were paired or associated with the sex bivalent. Twenty-five (21.2%) of the 118 pachytene cells analysed demonstrated different types of trivalent pairing: the 17(7) chromosome paired with chromosome pair 7 in 24 cells and with pair 17 in one cell. Moreover, in 4 cells, chromosome 17(7) was paired with both pair 7 and pair 17, forming a pentavalent. Trivalent association with XY was observed in 4 cells. Nineteen bivalents plus a univalent (8 cells), and eighteen bivalents plus a trivalent (11 cells), were found during conventional meiotic investigation of diakinesis-metaphase I. Though the boar demonstrated a normal testicular histology, a qualitatively deviant semen picture was evident.

Inherited adrenal hypoplasia: not just for kids!

Inherited adrenal hypoplasia: not just for kids!

Chromatin condensation and DNA damage of human epididymal spermatozoa in obstructive azoospermia

Obstructive azoospermia (OA) includes patients with different aetiologies of obstruction such as congenital bilateral absence of vas deferens (CBAVD), post-vasectomy/failed vaso–vasostomy, or unknown (idiopathic). The frequency of DNA-damaged spermatozoa and abnormal chromatin condensation in epididymal spermatozoa of OA patients was investigated for the different aetiology subgroups. DNA damage in caput epididymis spermatozoa was assessed with the TdT-UTP nick-end labelling (TUNEL) assay and chromatin condensation status was measured with chromomycin A3 (CMA3) stain ( n = 60 patients). All epididymal sperm samples showed high levels of TUNEL positivity (mean and SD, 43 ± 17%). CMA3 rates showed large variation within the samples (mean and SD, 53 ± 21%, range 7–97%); however, a significant difference in chromatin condensation was found between the different aetiologies of obstruction. The highest percentage of CMA3 positivity was found in the CBAVD and idiopathic groups (60 ± 17 and 65 ± 18% respectively) compared with the post-vasectomy samples (37 ± 17%) or ejaculated spermatozoa from normozoospermic males (29 ± 12%). In conclusion, despite the normal testicular histology in OA men, chromatin condensation differed between OA patients. The physiological heterogeneity found in chromatin condensation between patient subgroups is probably influenced by the aetiology of the obstruction.

A simple and reliable method for meiotic studies on testicular samples used for intracytoplasmic sperm injection

Objective: To develop a reliable and simple method allowing meiotic studies to be performed on testicular samples used for ICSI. Design: Evaluation of meiotic abnormalities in patients with severe spermatogenic impairment. Setting: Centre de Médecine de la Reproduction, Marseille. Patient(s): Two azoospermic men undergoing testicular biopsy for ICSI and one control individual with normal testicular histology. Intervention(s): The immature germ cells from the patients came from testicular biopsy used for ICSI, after dispersal into a thin cell suspension. Cells were cytocentrifuged to obtain well-spread spermatocytes and then immunocytochemical techniques were performed. We used rabbit polyclonal antibodies against the specific meiotic proteins Cor1 and Syn1 and a human CREST anti-kinetochore antibody. Main Outcome Measure(s): Synapsis abnormalities in patients with severe spermatogenesis impairment. Result(s): Pachytene spermatocytes are easily analyzed with this technique, without damage of the axial core and synaptonemal complex. The loss of germ cells is limited. Conclusion(s): The cytocentrifugation method is the most suitable technique for meiotic studies in patients with severe spermatogenic failure, because it can be used on the testicular cell suspension remaining after ICSI with testicular spermatozoa.