Status Of Spermatogenesis Research Articles

Artificial intelligence: to a better predictive strategy for testicular sperm extraction outcome in azoospermia

Azoospermia, defined as the absence of sperm in the semen, is found in 10-15% of infertile patients. Two-thirds of these cases are caused by impaired spermatogenesis, known as non-obstructive azoospermia (NOA). In this context, surgical sperm extraction using testicular sperm extraction (TESE) is the best option and can be offered to patients as part of fertility preservation, or to benefit from in vitro fertilization. The aim of the preoperative assessment is to identify the cause of NOA and evaluate the status of spermatogenesis. Its capacity to predict TESE success remains limited. As a result, no objective and reliable criteria are currently available to guide professionals on the chances of success and enable them to correctly assess the benefit-risk balance of this procedure. Artificial intelligence (AI), a field of research that has been rapidly expanding in recent years, has the potential to revolutionize medicine by making it more predictive and personalized. The aim of this review is to introduce AI and its key concepts, and then to examine the current state of research into predicting the success of TESE.

Identification of a novel pyroptosis-related gene signature in human spermatogenic dysfunction.

To reveal the underlying roles that pyroptosis-related genes (PRGs) played in human spermatogenic dysfunction. One discovery set and three validation sets were employed to inspect the previously reported 33 PRGs in the human testis with different status of spermatogenesis. PRGs that differentially expressed in all sets were considered as key differentially expressed pyroptosis-related genes (PR-DEGs). The relationships between key PR-DEGs and samples' clinicopathological, therapeutic, and immune patterns were respectively studied. Single-cell RNA sequencing (scRNS-seq) analyses were conducted to show the expression changes and related mechanisms of key PR-DEGs at a single-cell resolution. CASP4 and GPX4 were identified as two key PR-DEGs. These two genes were significantly dysregulated in spermatogenic dysfunctional samples, but with opposite tendency. CASP4 was negatively correlated with Johnsen scores but positively correlated with follicle-stimulating hormone (FSH) levels (all p < 0.05), while GPX4 exhibited significant positive correlations with Johnsen scores and negative relevance with FSH. For treatments, both molecules showed a prospective value of being predictors for sperm retrieval surgeries. Moreover, CASP4 and GPX4 were potential immunoregulators in the testicular immune microenvironment and showed significant correlations to testicular macrophages and mast cell infiltration. In scRNA-seq analyses, GPX4 was highly expressed in germ cells, which therefore suffered a sharp reduction with the loss of germ cells in spermatogenic dysfunction. On the other hand, CASP4 were basically somatic cell-derived, and the proportion of CASP4-positive Leydig cells significantly increased in disease testes (p = 0.0001). In all, we revealed two key PRGs of human testes that might be functional in spermatogenic dysfunction.

Evaluation of cell-free seminal mRNA for the diagnosis of obstruction as the cause of azoospermia in infertile men: A prospective cohort study.

Differentiating obstructive (OA) from non-obstructive (NOA) azoospermia is clinically important in managing infertile men. Classically, the differentiation has been based on clinical, hormonal and histological analysis. Histological tests are invasive and may miss spermatogenic areas. Seminal fluid can serve as a medium to assess the status of spermatogenesis and presence or absence of certain markers can help diagnosing and differentiating azoospermia. We evaluated the role of cell-free seminal markers: DDX4, PRM1 and PRM2 in diagnosing and differentiating between OA and NOA and classifying their subtypes. We observed DDX4 was more sensitive for NOA compared with OA. Among various subtypes of NOA, DDX4 positivity was higher in patients with maturation arrest and hypospermatogenesis compared with Sertoli cell only syndrome. PRM1 and PRM2 had very low positivity rate for any meaningful comparison. Seminal cell-free markers can serve as non-invasive tests in diagnosing and differentiating etiologies of azoospermia but their validity needs to be proved in long-term trials with more refined molecular techniques.

Complete Androgen Insensitivity Syndrome: Diagnosis and Psychological Impact in Adolescence, A Case Report

This case report illustrates the case of complete androgen insensitivity syndrome (CAIS) which is a rare form of sexual development disorders (DSD). Complex critical thinking is needed for pathophysiology of primary amenorrhea causes and sex chromosomes differences of sexual development, such as primary ovarian failure, Mullerian Agenesis or disorders with abnormal androgen synthesis or response. This is a phenotypically female who presented with primary amenorrhea at the age of 19 years old. Normal levels of thyroid function test, serum prolactin and follicle stimulating hormone ruled out hypothyroidism, hyperprolactinemia, and primary ovarian failure. Magnetic resonance imaging showed absence of uterus, fallopian tubes, ovaries, but presence of proximal 1/3rd of the vagina. There is a single testis in the left inguinal region with unknown status of spermatogenesis. The chromosomal analysis revealed 46, XY karyotype conveying the patient is genotypically male. The testis-determining factor (TDF) test or sex-determining region Y (SRY) protein for male sex determination was not done. Similar presentation of primary amenorrhea diagnosis of CAIS was made to her 18-year-old sister. Women with CAIS are vulnerable to various psychological conditions caused by the appalling fact of being genotypically male when they have been raised female all their life. The gender confusion, reproductive issues and how others perceive them in the outside world require sensitive support. Hence, accentuate the need to address the emotional, psychological, and psychiatric vulnerabilities in issues pertaining to relationships, infertility and conception.International Journal of Human and Health Sciences Supplementary Issue-2: 2021 Page: S12

Stereological properties of seminiferous tubules in infertile men with chromosomal and genetic abnormalities.

Male infertility is caused by genetic anomalies in 15-30% of cases. This study aimed to determine stereological properties of seminiferous tubules in infertile men with genetic anomalies, including Klinefelter Syndrome (KS), Y chromosome microdeletions (MYC) and CFTR gene mutations (CFTR); and to compare them to seminiferous tubules of men with obstructive azoospermia of non-genetic origin (control group). The study was conducted on 28 human testis biopsy specimens obtained from 14 patients with MYC, 18 samples from 9 patients with KS, and 6 samples from 3 patients with CFTR. Whenever possible, a bilateral biopsy was included in the study. The control group had 33 samples from 18 patients (3 of them with a solitary testis). Qualitative and quantitative (stereological) analysis of seminiferous tubules (including the status of spermatogenesis, volume, surface area, length and number of tubules) were performed in all groups. Qualitative histological analysis revealed significant impairment of spermatogenesis in KS and MYC, whereas testicular parenchyma was fully maintained in CFTR and control groups. Spermatogenesis was most seriously impaired in KS. All stereological parameters were significantly lower in KS and MYC, compared to the CFTR and control groups. The total volume, surface and length of seminiferous tubules were significantly lower in KS compared with MYC. Stereological analysis is valuable in evaluating male infertility, whereas qualitative histological analysis can be helpful in assessing sperm presence in testicular tissue of patients with KS or MYK undergoing TESE.

F5-peptide enhances the efficacy of the non-hormonal male contraceptive adjudin

ObjectiveThe bioavailability of the non-hormonal male contraceptive adjudin is low in rats due to the blood-testis barrier (BTB). This study was designed to examine if F5-peptide, an endogenously produced reversible BTB modifier, could enhance the bioavailability of adjudin to affect spermatogenesis and provide a contraceptive effect in rats while reducing systemic toxicity. Study DesignWe overexpressed F5-peptide in adult male rats (n=10 rats; with 3 or 4 rats for each of the three different experiments noted in the three regimens) by intratesticular injection of a mammalian expression vector pCI-neo (pCI-neo/F5-peptide) vs. empty vector alone (pCI-neo/Ctrl) to be followed by treatment with adjudin by oral gavage at a dose of 10 or 20 mg/kg. The status of spermatogenesis was assessed by histological analysis and dual-labeled immunofluorescence analysis on Day 16. To assess fertility, we allowed treated males (n=3–4 rats) to mate with mature female rats (n=3–4) individually, and assessed the number of pups on Days 23, 36 and 82 to assess fertility and reversibility. ResultsAll 4 treated rats overexpressed with F5-peptide and low-dose adjudin were infertile by Day 36, and half of these rats were fertile by Day 82, illustrating reversibility. However, overexpression of F5-peptide alone (or low-dose adjudin alone) had no effects on fertility in n=3 rats. These findings were consistent with the histology data that illustrated the BTB modifier F5-peptide promoted the action of adjudin to induce germ cell exfoliation, mediated by changes in cytoskeletal organization of F-actin and microtubules across the epithelium, thereby reducing the systemic toxicity of adjudin. ConclusionIn this proof-of-concept study, it was shown that overexpression of the F5-peptide prior to administration of adjudin to rats at a low (and ineffective dose by itself) was found to induce reversible male infertility. ImplicationsOverexpression of F5-peptide, an endogenously produced biomolecule in the testis known to induce BTB remodeling, enhanced the contraceptive effect of adjudin in rats, supporting proof of concept studies of BTB disrupters in men.

DNA Flow cytometric analysis of the human testicular tissues to investigate the status of spermatogenesis in azoospermic patients

A single, rapid and reproducible diagnostic test to predict the type of azoospermia and outcome of sperm retrieval is not yet available. So the feasibility of employing DNA flow cytometry for rapid investigation of the status of spermatogenesis in the patients with azoospermia was investigated. Testicular biopsies of 44 patients with azoospermia undergoing sperm-retrieval surgery and 4 controls were analyzed by flow cytometry to ascertain their testicular germ-cell patterns. The observed germ-cell pattern was further confirmed by RT-PCR analysis of the cell-specific markers and histology for some patients. The patients with Obstructive Azoospermia (OA) exhibited normal spermatogenesis similar to the control fertile patients showing the presence of diploid, double-diploid and haploid cells. The non-obstructive azoospermia (NOA) patients exhibited disrupted spermatogenesis with arrest at the pre-meiotic (only diploid cells present) or meiotic (diploid and double-diploid cells present) stages. The germ-cell pattern, as ascertained by flow cytometry, provided a clear picture of the intra-testicular spermatogenesis and the presence of spermatozoa in the patients’ testes, which was prognostic of their sperm-retrieval. DNA flow cytometry test to ascertain the testicular germ-cell pattern is simple in execution, analysis and interpretation, requires small amount of tissue and provides quantitative data about the status of spermatogenesis in patients. This test would allow comparable analysis of the status of spermatogenesis in patients across clinics and may form the basis for deciding future treatment and intervention strategies.

Effects of Intratesticular Injection of 70% Glycerin on Stallions

The removal of endogenous germ cells of recipient stallions is a key step to produce donor germ cell-derived sperm using the germ cell transplantation technique. Six Thoroughbred stallions were divided into a treatment (n = 3) and a control group (n = 3), and 70% glycerin (1, 2, 3-trihydroxypropane, 40 mL per testis) or phosphate-buffered saline, respectively, was locally injected into testes. General semen evaluation, libido, and testicular volume were performed weekly from 3 weeks before to 10 weeks after treatment. The number of round germ cells in the ejaculate was counted using a hemocytometer. The hematoxylin and eosin staining was performed on the cross sections of testicular tissue obtained 11th week of treatment. Plasma testosterone levels in blood collected weekly were measured using a colorimetric competitive enzyme immunoassay kit. The sperm number was significantly lower than that of the control group at 5 and 10 weeks after glycerin injection. No differences in the status of spermatogenesis in the cross sections of seminiferous tubules and testicular volume were found between the two groups. The 70% glycerin-treated stallions had reduced total and progressively motile sperm and exhibited a significantly higher population of round germ cells in the ejaculate. Testosterone levels, testicular volumes, and libido of stallions were not significantly different between the groups. In conclusion, although intratesticular injection of 70% glycerin may have caused disassociation of some germ cells in the seminiferous tubules for several weeks, it did not significantly ablate germ cells in the tubules at 11 week in stallions.

MP70-18 SEMINAL LEVEL OF CLUSTERIN IN INFERTILE MEN AS A SIGNIFICANT BIOMARKER REFLECTING SPERMATOGENESIS

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology1 Apr 2016MP70-18 SEMINAL LEVEL OF CLUSTERIN IN INFERTILE MEN AS A SIGNIFICANT BIOMARKER REFLECTING SPERMATOGENESIS Teruo Fukuda, Hideaki Miyake, Noritoshi Enatsu, Kei Matsushita, and Masato Fujisawa Teruo FukudaTeruo Fukuda More articles by this author , Hideaki MiyakeHideaki Miyake More articles by this author , Noritoshi EnatsuNoritoshi Enatsu More articles by this author , Kei MatsushitaKei Matsushita More articles by this author , and Masato FujisawaMasato Fujisawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1443AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The objective of this study was to assess the impact of seminal clusterin level on spermatogenesis in infertile men. METHODS This study included 89 men who had visited our clinic due to infertility, consisting of 28, 33, and 28 diagnosed with normospermia, oligozoospermia and non-obstructive azoospermia (NOA), respectively. The seminal clusterin concentrations in these 89 men were measured by sandwich enzyme-linked immunosorbent assay. RESULTS The seminal clusterin level in men with NOA was significantly decreased compared with that of those with oligozoospermia or normospermia. Microdissection testicular sperm extraction (MD-TESE) was performed in the 28 NOA men, and sperm was successfully retrieved from 9. The expression level of clusterin protein in testes specimens from the 28 NOA men were evaluated by immunohistochemical staining, and a significant correlation was noted between testicular clusterin protein expression and seminal clusterin levels in these men. Furthermore, of the several parameters available prior to MD-TESE, the univariate analysis identified serum follicle stimulating hormone (FSH) level and seminal clusterin level as significant predictors of sperm retrieval, and of these only serum FSH level was shown to be independently associated with sperm retrieval in the multivariate analysis. CONCLUSIONS These findings suggest that the seminal clusterin level could be used as a biomarker reflecting the status of spermatogenesis in infertile men. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e913-e914 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Teruo Fukuda More articles by this author Hideaki Miyake More articles by this author Noritoshi Enatsu More articles by this author Kei Matsushita More articles by this author Masato Fujisawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

VASA (DDX4) is a Putative Marker for Spermatogonia, Spermatocytes and Round Spermatids in Stallions.

Expression of the protein DDX4/MVH, or VASA, has been reported in germ cells of several species. The main objectives of this study were to (i) investigate VASA expression patterns in testicular cells of stallions at two different reproductive stages (pre-pubertal and post-pubertal) and (ii) evaluate the use of VASA antibody as a molecular marker for single germ cells from stallions. Testicular tissues were obtained from stallions and categorized as pre-pubertal and post-pubertal based on the formation of lumen and status of spermatogenesis on the cross section of seminiferous tubules. The results of Western blot showed a VASA protein band located at 76 kDa, indicating that the rabbit antibody has a cross-reactivity with horse testicular tissues. The result of immunolabelling showed that VASA was expressed in the cytoplasm of spermatogonia at both reproductive stages and in spermatocytes and round spermatids at the post-pubertal stage. GATA4-positive Sertoli cells and Leydig cells located in the interstitial space were not immunolabelled with VASA. These results suggest that VASA can be utilized as a molecular marker for germ cells of stallions at pre-pubertal and post-pubertal stages. Interestingly, immunolabelling intensity was significantly higher in pachytene spermatocytes compared to spermatogonia and round spermatid. VASA antibody was also effective for staining of single germ cell preparations. In conclusion, VASA protein expression can be used as a marker for identification of spermatogonia, spermatocytes and round spermatids in testicular tissues of stallions.

Redefining abnormal follicle‐stimulating hormone in the male infertility population

Study Type - Diagnostic (validating cohort). Level of Evidence 2a. What's known on the subject? and What does the study add? FSH is a hormone released by the anterior pituitary gland via stimulation from gonadotrophin-releasing hormone and potentially other factors. FSH reflects the status of spermatogenesis (i.e. the ability to produce sperm) as a result of the feedback between the testis and hypothalamus/pituitary glands. An elevated FSH level is indicative of abnormal spermatogenesis and may indicate primary testicular failure. The range for 'normal' FSH varies somewhat between institutions but has been defined by the Strong Memorial Hospital (Rochester, NY, USA) clinical laboratory as 1.4-18.1 IU/L based on the ADVIA Centaur (Siemens Medical Solutions, Tarrytown, NY, USA) FSH assay. The findings obtained in the present study could be helpful for predicting male factor infertility in patients with a borderline high FSH level (≈4.5 IU/L) and a low testosterone level compared to someone with a borderline high FSH level and a normal testosterone level. Although the 'normal' range for FSH is qualified as a value in the range 1.4-18.1 IU/L, the present study shows that an FSH level >4.5 IU/L was associated with abnormal semen analysis in terms of morphology and sperm concentration in the present patient population. Therefore, these findings suggest that FSH values lower than those currently considered normal may be associated with abnormal semen analysis, and that the 'normal' range for FSH used in clinical settings may need to be reconsidered. • To examine the correlation between follicle-stimulating hormone (FSH) and testosterone/FSH levels with semen analysis parameters to evaluate whether the range for judging normal FSH levels should be reconsidered. • The present study included 610 male infertility patients from a single urology infertility clinic between 2004 and 2008. • Patients (n=153) were excluded for obstructive azoospermia, hypogonadotrophic hypogonadism, steroid use or failure to complete testing. • Abnormal semen analysis values were based on the WHO 1999 criteria. • We performed t-tests, anova, chi-squared tests and logistic regression to statistically examine the association between the FSH (or testosterone/FSH ratio) level and semen parameters. • The FSH level showed statistically significant associations, as well as evidence of a dose response, with abnormal sperm concentration and morphology but not with semen volume. • In men with FSH levels >7.5 IU/L, the risk of abnormal semen quality was five- to thirteen-fold higher than that of men with FSH levels <2.8 IU/L depending on the specific semen parameter. • Similarly, semen parameters were had a greater probability of being abnormal with decreasing testosterone/FSH ratios. • A significantly increased risk of abnormal semen analyses among men with FSH levels >4.5 IU/L and decreasing testosterone/FSH ratios suggests that redefining normal FSH in infertile men would be valuable.

Genotyping of Tunisian azoospermic men with Sertoli cell-only and maturation arrest.

Azoospermia factor (AZF) deletions were associated with severe oligospermia and azoospermia with testicular histologies varying from maturation arrest (MA) to Sertoli cell-only (SCO) phenotypes. Abnormal androgen receptor (AR) structure or function has also been implicated in male infertility. To assess the contribution of these genetic defects to azoospermic patients, 19 Tunisian men with SCO (n = 13) or MA (n = 6) were enrolled in this study. Using immunohistochemistry method, we evaluated the expression of AR in testicular biopsy for the two phenotypes. PCR with primers flanking the AR-(CAG)n region and direct sequencing were used to determine AR-(CAG)n length. And PCR amplification of 14 sequence-tagged sites (STSs) located at Yq was used to determine the rate and extent of Y microdeletions. We found a significant difference of the AR expression between SCO and MA cases. Hence, this expression in the testis depends on the status of spermatogenesis. However, we did not find any relationship between the (CAG) repeat and the testicular histology (neither for SCO nor MA). On the other hand, we found a high frequency of AZF deletions (46.2%) in SCOS and in MA (50%). The present results also suggest the contribution of Y chromosome microdeletions in SCO and MA pathogenesis.

Trials for development of once-a-month injectable, hormonal male contraceptive using dienogest plus testosterone undecanoate: dose standardization, efficacy and reversibility studies in rats

Background The study was conducted to test the potential of using dienogest (DNG) plus testosterone undecanoate (TU) in rats for development of a once-a-month injectable male hormonal contraceptive. Study Design Dose selection studies were initiated with administration of DNG in three different doses of 20, 30 and 40 mg/kg body weight (bw) per week plus TU 25 mg/kg bw once in every 6 weeks. Status of spermatogenesis and sperm count in epididymis was evaluated. The frequency of DNG intervention was later extended to every 2- and 4-week intervals. Mating studies, toxicity and reversibility of spermatogenesis following stoppage of treatment were carried out with DNG 40 mg/kg bw at 4-week intervals. Results Complete arrest of spermatogenesis was observed after 60 days of treatment at all doses of DNG (20, 30 and 40 mg/kg bw per week)+TU. However, weights of testis and accessory sex organs (epididymis, prostate and seminal vesicle) declined significantly 60 days post treatment compared to vehicle-treated controls. Epididymis in the treated animals was completely devoid of sperm. When the frequency of DNG injection (20 mg/kg bw) was extended to once every 15 days, a few immotile and decapitated sperm were observed in the epididymis. With TU treatment unchanged, animals receiving DNG (40 mg/kg bw) once either every 2- or 4-week intervals demonstrated good and uniform arrest of spermatogenesis. DNG 40 mg/kg per 4 weeks+TU also demonstrated a significant rise in germ cell apoptosis in the seminiferous epithelium. There was no significant increase in the serum high-density lipoprotein and low-density lipoprotein levels at the end of 120 days of treatment. Following withdrawal of treatment after 60 or 120 days, qualitative restoration of spermatogenesis was rapid in the former compared to the latter. Conclusion Dienogest plus TU has the potential for development as a monthly injectable showing reversible hormonal male contraception with good efficacy.

Adjudin Targeting Rabbit Germ Cell Adhesion as a Male Contraceptive: A Pharmacokinetics Study

Adjudin (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide; formerly called AF-2364) has been shown to inhibit spermatogenesis by disrupting anchoring junctions at the Sertoligerm cell interface. This, in turn, leads to germ cell loss from the seminiferous epithelium, and transient infertility. Adjudin's efficacyin inhibiting spermatogenesis, the recovery of spermatogenesis after cessation of the drug, and side effects were examined in adult male Japanese rabbits. The pharmacokinetics profiles of adjudin in rabbits after oral administration and after intravenous injection were compared. Rabbits received 25 mg/kg adjudin once weekly for 4 consecutive weeks either by intravenous injection or by gavage. Vehicle-treated rabbits were used as controls. At 1, 2, 3, 4, and 8 weeks after treatment, testes were removed for microscopic examination to assess the status of spermatogenesis. Four weeks after intravenous cessation of adjudin, the recovery of spermatogenesis also was monitored. Blood was withdrawn after first administration to measure plasma concentrations of adjudin by high-performance liquid chromatography. Four weeks after intravenous treatment, examination of testis sections showed rapid exfoliation of elongated/elongating spermatids and the presence of large multinucleated cells; more than 95% of germ cells were absent from the seminiferous epithelium. Intravenous treatment showed a more severe disturbance of spermatogenesis compared with gavage treatment, which was correlated with bioavailability of the drug. The areas under the curve for intravenous injection and gavage were 20.11 +/- 1.90 and 2.23 +/- 0.45 mg x h x L(-1), respectively. These results illustrate the potential of adjudin as a male contraceptive, and the efficacy is associated with the bioavailability of the drug.

Upregulation of AP1 by tertiary butyl hydroperoxide induced oxidative stress and subsequent effect on spermatogenesis in mice testis

Oxidative stress is detrimental to sperm function and a significant factor in the etiology of male infertility. Present study evaluates the effect of ter butyl hydroperoxide (TBHP)-induced oxidative stress on the spermatogenic process and cell number in the seminiferous tubules. Intraperitoneal injection of TBHP (84 mumol TBHP/100 g body weight) for 2 weeks to male Balb/c mice resulted in enhanced lipid peroxidation (P < 0.0001) decrease in reduced glutathione (P < 0.0001) and increase in the oxidized glutathione levels (P = 0.007) in the testis. Status of spermatogenesis after the treatment was assessed by the quantitative methods of germ cell evaluation in the seminiferous tubules. A significant decrease in the number of young spermatids (P = 0.0003) and pachytene cells (P = 0.022) was observed. A marked reduction was also seen in the mature spermatid number (P < 0.0001). An increase in testicular mRNA levels of redox-regulated cjun (P = 0.008) and cfos (P = 0.0006) subunits of activator protein 1 (AP1) was observed after TBHP treatment. Evaluation of AP1 regulated antioxidant enzymes in the testis revealed an increase in gamma-glutamyl cysteine synthetase (GCS) mRNA expression (P = 0.001). These results suggest a potential role of AP1 in oxidative stress-mediated meiotic and post meiotic changes in the spermatogenic process and regulation of cell number in male reproductive system.

Age‐dependent Expression and Localization of the Progesterone Receptor in the Boar Testis

Expression of the progesterone receptor (PR) was monitored in testes of groups of five boars aged 50, 100, 150, 200 and 250 days. The primary monoclonal antibody used for immunohistochemistry (IHC) was raised against a peptide mapping the amino acids 922-933 of the carboxy-terminus of the human PR, negative controls were set up using an irrelevant monoclonal isotype-specific antibody, porcine endometrium served as positive control tissue. In parallel, qualitative reverse transcription-polymerase chain reaction was applied. Based on the developmental status of spermatogenesis the 50- and 100-day-old boars were considered as immature, the boars aged 200 and 250 days as mature. Positive and negative controls confirmed specificity of IHC. In the 50-day-old boars 85.1% of the prespermatogonia that had reached the basal lamina and 18.2% of the centrally located prespermatogonia stained positive, while it was 92.1% respectively 2.1% in the 100-day-old boars. The effect of time and location was highly significant (p < 0.005 resp. 0.0001). In mature boars between 77 and 80% of the A and B spermatogonia stained positive, there was no effect of boar age and stage of spermatogenesis. In both groups also few peritubular myoid cells stained positive. It is hypothesized that Leydig cell-derived progesterone plays a functional role in spermatogoniogenesis in a synergistic manner with Leydig cell-derived oestrogens.

Laminin α 3 Forms a Complex with β3 and γ3 Chains That Serves as the Ligand for α 6β1-Integrin at the Apical Ectoplasmic Specialization in Adult Rat Testes

Apical ectoplasmic specialization (ES) is a testis-specific hybrid cell/cell actin-based adherens junction and cell/matrix focal contact anchoring junction type restricted to the interface between Sertoli cells and developing spermatids. Recent studies have shown that laminin gamma3, restricted to elongating spermatids, is a putative binding partner of alpha 6beta 1-integrin localized in Sertoli cells at the apical ES. However, the identity of the alpha and beta chains, which constitute a functional laminin ligand with the gamma3 chain at the apical ES, is not known. Using reverse transcription-PCR and immunoblotting to survey all laminin chains in cells of the seminiferous epithelium, it was noted that alpha 2, alpha 3, beta1, beta2, beta3, and gamma3 chains were found in germ cells, whereas alpha 1, alpha 2, alpha 4, alpha 5, beta1, beta2, gamma1, gamma2, and gamma3 chains were found in Sertoli cells, implying that alpha 3 and beta3 are the plausible laminin chains restricted to germ cells that may be the bona fide partners of gamma3. To verify this postulate, recombinant proteins based on domain G of alpha 3 and domain I of beta3 and gamma3 chains were produced and used to obtain the corresponding specific polyclonal antibodies. Additional studies have demonstrated that the laminin alpha 3, beta3, and gamma3 chains indeed are restricted to germ cells at the apical ES, co-localizing with each other and with beta1-integrin. Furthermore, co-immunoprecipitation studies have confirmed the interactions among laminin alpha 3, beta3, and gamma3, as well as beta1-integrin. When the functional laminin ligand at the apical ES was disrupted via blocking antibodies, such as using anti-laminin alpha 3 or gamma3 IgG, this treatment perturbed adhesion between Sertoli and germ cells (mostly spermatids), leading to germ cell loss from the epithelium. More important, a transient disruption of the blood-testis barrier was also detected.

Spermatogenesis following syngeneic testicular transplantation in Balb/c mice.

Transplantation of spermatogonial stem cells in cross-species has been widely used to study the function of Sertoli cells and the effect of phylogenetic distance between donor and recipient animals on the outcome of spermatogonial transplantation, whereas there have been only a few reports on the transplantation of testis tissue. The objective of the present study was to examine the development of grafted testes and the kinetics of spermatogenesis following syngeneic testicular transplantation in both male and female recipient Balb/c mice in an effort to establish an in vivo culture system and to compare the effects of host sex on spermatogenesis. The testes from 5-day-old Balb/c mice were transplanted under the dorsal skin of four-week-old mice. Twenty male and twenty female Balb/c mice were used as the hosts and each host received 4 grafts. The recipient mice were killed at 1, 2, 3, 5, 7, 9, 12 and 15 weeks after transplantation. The graft survival rate and graft size were measured. The status of spermatogenesis was assessed by histological analyses. The expression of the spermatid-specific Protamine-2 gene was examined by RT-PCR. Overall, 70.3% of the testicular grafts in male hosts and 67.2% in female hosts survived. All recovered grafts had increased in volume, some of them had increased by more than 30-fold. The architecture of the seminiferous tubules in female hosts appeared to be better than that in male hosts. The round spermatids were the most advanced germ cells until 15 weeks after transplantation, and no complete spermatozoon was observed in any of the grafts. The expression of protamine-2 was detected in grafts from 5 weeks posttransplantation in both male and female hosts, confirming that the spermatogenic cells differentiated into spermatids. In contrast to grafts, the testes of male hosts had a normal histological appearance. The results showed the schedule of spermatogenesis following syngeneic testicular transplantation in both male and female hosts. This model could be useful for further studies involving the endocrinology of the testis and the mechanisms of spermatogenesis.

Male factors determining the outcome of intracytoplasmic sperm injection with epididymal and testicular spermatozoa.

During a period of 8 years, 1,079 intracytoplasmic sperm injection (ICSI) procedures with aspirated epididymal or testicular spermatozoa were performed. Epididymal spermatozoa were used in 172 cycles and testicular spermatozoa or spermatids in 907 cycles. Multiple biopsies were obtained from at least two different locations in the testes. Retrieved spermatozoa were used after cryopreservation (frozen) or immediately after aspiration (fresh). Three hundred patients had obstructive azoospermia (OA) or ejaculation failure. In 414 cases, azoospermia was caused by impaired spermatogenesis resulting from maldescended testes, chemotherapy/radiotherapy, or by Sertoli-cell-only syndrome, genetic disorders or unknown aetiology. Transfer rates, pregnancy rates and birth rates per ICSI cycle showed no statistically significant differences between testicular and epididymal spermatozoa in men with OA (28% average birth rates in both cases). However, birth rates differed significantly with regard to the status of spermatogenesis. Treatment of men with nonobstructive azoospermia (NOA) resulted in a birth rate of 19% per cycle. In all patient groups, there was no difference in the birth rates achieved with fresh and cryopreserved spermatozoa. While testicular volume, follicle-stimulating hormone level and age of the male patient are no statistically significant prognostic factors, the underlying cause of azoospermia is the most important factor determining the outcome of ICSI with epididymal and testicular spermatozoa. The pregnancy rate is lower in NOA patients than in those with OA.

Mast cells in testicular biopsies of infertile men with 'mixed atrophy' of seminiferous tubules.

Mast cells in the bilateral testicular biopsies of 30 patients with a 'mixed atrophy' of seminiferous tubules were analysed. Seven biopsies from vasectomized patients served as controls. With regard to their characteristic location within testicular tissue, two groups of mast cells could be distinguished, in both control and infertile patients: 'interstitial' mast cells (located between Leydig and other interstitial cells as well as in the vicinity of blood vessels) and 'peritubular' mast cells (located in the close proximity of the tubular lamina propria or incorporated in the lamina propria itself). Morphometric data indicated a significant increase in the number and volume of mast cells in infertile patients when compared with controls. In the biopsies of infertile patients that were analysed both 'interstitial' and 'peritubular' mast cells showed a significant increase in their number and volume, although it appeared that 'peritubular' mast cells increased at a higher rate than 'interstitial' mast cells. A significant negative correlation was found between the following variables: volume and number of mast cells, testis volume and the status of spermatogenesis evaluated by Johnsen's scoring. It was concluded that the increased presence of mast cells is closely associated with an impairment of spermatogenesis.