Studies suggest a relationship between semen quality and the concentration of trace elements in serum or seminal plasma. However, trace elements may be linked to DNA and capable of altering the gene expression patterns. Thus, trace element interactions with DNA may contribute to the mechanisms for a trans-generational reproductive effect. We developed an analytical method to determine the amount of trace elements bound to the sperm DNA, and to estimate their affinity for the sperm DNA by the ratio: R = Log [metal concentration in the sperm DNA/metal concentration in seminal plasma]. We then analyzed the concentrations of 15 trace elements (Al, Cd, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Ti, V, Zn, As, Sb, and Se) in the seminal plasma and the sperm DNA in 64 normal and 30 abnormal semen specimens with Inductively Coupled Plasma/Mass Spectrometry (ICP-MS). This study showed all trace elements were detected in the seminal plasma and only metals were detected in the sperm DNA. There was no correlation between the metals’ concentrations in the seminal plasma and the sperm DNA. Al had the highest affinity for DNA followed by Pb and Cd. This strong affinity is consistent with the known mutagenic effects of these metals. The lowest affinity was observed for Zn and Ti. We observed a significant increase of Al linked to the sperm DNA of patients with oligozoospermia and teratozoospermia. Al’s reproductive toxicity might be due to Al linked to DNA, by altering spermatogenesis and expression patterns of genes involved in the function of reproduction.

During spermiogenesis the spermatid nucleus is elongated, and dramatically reduced in size with protamines replacing histones to produce a highly compacted chromatin. After fertilisation, this process is reversed in the oocyte to form the male pronucleus. Emerging evidence, including the coordinated loss of the nuclear lamina (NL) and the histones, supports the involvement of the NL in spermatid nuclear remodelling, but how the NL links to the chromatin is not known. In somatic cells, interactions between the NL and the chromatin have been demonstrated: LEM-domain proteins and LBR interact with the NL and respectively, the chromatin proteins BAF and HP1. We therefore sought to characterise the lamina-chromatin interface during spermiogenesis, by investigating the localisation of six LEM-domain proteins, two BAF proteins and LBR, in human spermatids and spermatozoa. Using RT-PCR, IF and western blotting, we show that six of the proteins tested are present in spermatids: LEMD1, LEMD2 (a short isoform), ANKLE2, LAP2β, BAF and BAF-L, and three absent: Emerin, LBR and LEMD3. The full-length LEMD2 isoform, required for nuclear integrity in somatic cells, is absent. In spermatids, no protein localised to the nuclear periphery, but five were nucleoplasmic, receding towards the posterior nuclear pole as spermatids matured. Our study therefore establishes that the lamina-chromatin interface in human spermatids is radically distinct from that defined in somatic cells. In ejaculated spermatozoa, we detected only BAF and BAF-L, suggesting that they might contribute to the shaping of the spermatozoon nucleus and, after fertilisation, its transition to the male pronucleus.

Due to their catalytic and oxidative properties, cerium dioxide nanoparticles (CeO(2)NPs) are widely used as diesel additive or as promising therapy in cancerology; yet, scarce data are available on their toxicity, and none on their reproductive toxicity. We showed a significant decrease of fertilization rate, assessed on 1272 oocytes, during in vitro fertilization (IVF) carried out in culture medium containing CeO2NP at very low concentration (0.01mg.l(-1)). We also showed significant DNA damage induced in vitro by CeO2NP on mouse spermatozoa and oocytes at 0.01mg.l(-1) using Comet assay. Transmission Electron Microscopy did not detect any nanoparticles in the IVF samples at 0.01mg.l(-1), but showed, at high concentration (100mg.l(-1)), their endocytosis by the cumulus cells surrounding oocytes and their accumulation along spermatozoa plasma membranes and oocytes zona pellucida. We did not observe any nanoparticles in the cytoplasm of spermatozoa, oocytes or embryos. This study demonstrates for the first time the impact of CeO2NP on in vitro fertilization, as well as their genotoxicity on mouse spermatozoa and oocytes, at low nanoparticle concentration exposure. Decreased fertilization rates may result from: (1) CeO2NP's genotoxicity on gametes; (2) a mechanical effect, disrupting gamete interaction and (3) oxidative stress induced by CeO2NP. These results add new and important insights with regard to the reproductive toxicity of nanomaterials requesting urgent evaluation, and support several publications on metal nanoparticles reprotoxicity. Our data highlight the need for in vivo studies after low-dose exposure.

Alteration of sperm parameters related to occupational exposures is the subject of several studies, often on a case-control approach. The study populations usually comprise men consulting in infertility clinics for couple infertility. The objective of this review is to identify, from these case-control studies, the main occupational factors that may be associated with altered sperm parameters. We selected 13 articles in the PubMed database. Participation in these studies varied from 61 to 2619 subjects, with great methodological heterogeneity, particularly in the characterization of exposure. The main occupations that appear significantly associated with a risk of altered sperm parameters are workmen, painters, farmers, welders, plumbers and technicians. When analysis focuses on occupational exposures, a significant result is reported for solvents, heavy metals, heat, vibrations and non-ionizing radiation. None of the selected studies has found a link with exposure to pesticides.

BACKGROUND:Alterations in the nutritional perinatal environment, such as intrauterine growth retardation with subsequent postnatal catch-up growth, program cardiovascular disease in adulthood, possibly through alterations in matrix metalloproteinase (MMP)-2 and -9. However, experimental evidences demonstrating that changes in the nutritional perinatal environment can program MMP-2 and -9 with subsequent alterations of vessel wall are lacking.AIM:The current study evaluated whether immediate postnatal overfeeding is able to alter vascular morphological indexes and circulating and/or vascular MMP2-2 and -9 status.METHODS:Aortic morphology (wall thickness and percentage of incomplete elastin lamellae) and circulating and aortic MMP-2 and -9 activity (measured by gelatin zymography) and aortic MMP-2 and -9 mRNA (measured by reverse transcription polymerase chain reaction (RT-PCR)) were studied in adult male rats overfed (OF) or normofed (NF) during the immediate postnatal period.RESULTS:Postnatal overfeeding induced early onset obesity. Adult OF rats presented with increased blood pressure and circulating MMP-2 and -9 activity. In the thoracic aorta, postnatal overfeeding increased wall thickness and decreased elastin integrity (as demonstrated by an increased percentage of incomplete elastin lamellae). OF rats showed enhanced aortic MMP-2 activity and MMP-9 mRNA levels. Circulating and aortic MMP-2 activity correlated positively with the percentage of incomplete elastin lamellae and aortic wall thickness, respectively.CONCLUSION:Our data demonstrate for the first time that immediate postnatal nutritional programming induces increases in circulating and aortic MMP-2 activity with parallel aortic wall alterations, such as decreased elastin integrity and enhanced thickening, showing that this experimental model is suitable for the study of perinatal nutritional programming of vascular functions.

Spinal cord injury (SCI) most often affects young men of reproductive age: several millions of men between 16 and 45 years of age face quadriplegia or paraplegia worldwide. Acute SCI modifies hormonal and neural regulation of Sertoli and Leydig cells, which may affect the endocrine and/or paracrine microenvironment within the testis and impair the Sertoli support for normal spermatogenesis. A study on SCI dogs showed a significant impairment of sperm motility and spermatogenesis 3 weeks after SCI, compared with non-SCI controls. In animal models, during the chronic phase of SCI, auto-immunity and oxidative stress impair semen parameters. In chronic SCI patients, increased scrotal temperature is caused by the continuous sitting position and by a dysregulation of scrotal thermoregulation. Chronic SCI patients show high levels of reactive oxygen species in seminal fluid, which are inversely correlated to sperm motility. The management of SCI patients' fertility involves a multidisciplinary staff and should be patient centered.

The nuclear lamina (NL) is a filamentous protein meshwork, composed essentially of lamins, situated between the inner nuclear membrane and the chromatin. There is mounting evidence that the NL plays a role in spermatid differentiation during spermiogenesis. The mouse spermatid NL is composed of the ubiquitous lamin B1 and the spermatid-specific lamin B3, an N-terminally truncated isoform of lamin B2. However, nothing is known about the NL in human spermatids. We therefore investigated the expression pattern and localization of A-type lamins (A, C and C2) and B-type lamins (B1, B2 and B3) during human spermiogenesis. Here, we show that a lamin B3 transcript is present in human spermatids and that B-type lamins are the only lamins detectable in human spermatids. We determine that, as shown for their mouse counterparts, human lamin B3, but not lamin B2, induces strong nuclear deformation, when ectopically expressed in HeLa cells. Co-immunofluorescence revealed that, in human spermatids, B-type lamins are present at the nuclear periphery, except in the region covered by the acrosome, and that as the spermatid matures the B-type lamins recede towards the posterior pole. Only lamin B1 remains detectable on 33-47% of ejaculated spermatozoa. On spermatozoa selected for normal head density, however, this fell to <6%, suggesting that loss of the NL signal may be linked to complete sperm nucleus compaction. The similarities revealed between lamin expression during human and rodent spermiogenesis, strengthen evidence that the NL and lamin B3 have conserved functions during the intense remodelling of the mammalian spermatid nucleus.

Objectives. - To evaluate our elective single embryo transfer policy performed at 48/72 h and define predictive factors of pregnancy after frozen/thawed embryo transfer.Methods. - Analysis of 289 elective single embryo transfers (eSET) performed in a selected population in the ART center of Marseille University Hospital from January 2005 to December 2011, as well as the 325 following frozen/thawed embryo transfers performed in this population between May 2005 and December 2012.Results. - Cumulative pregnancy rate/oocyte retrieval was of 62.6%; 45% of the couples obtained the birth of at least one child. During this studied period, cumulative pregnancy and delivery rates in the whole population remained stable while multiple delivery rate/delivery clearly decreased. Elective single embryo after frozen/thawed transfer gave satisfying results (24.6% pregnancy/transfer) only in the lack of lysis or in case of mild lysis (1-25%) of the transferred embryo.Conclusion. - The implementation of an eSET policy gives satisfying results, depending largely on embryo quality. By proposing eSET to a well-targeted population, chosen both on clinical and biological criteria, a clear reduction of cumulative multiple delivery rate/delivery was obtained in our center over this period, without any global decrease of cumulative pregnancy rate/attempt. Embryo quality is a major factor of success, especially in frozen/thawed cycles. The elective single embryo frozen/thawed transfer should be carried out only if embryo lysis after thawing does not exceed 25%. (C) 2015 Elsevier Masson SAS. All rights reserved.

Évaluer notre politique de transfert électif d’un embryon à 48/72 h et rechercher des facteurs prédictifs de grossesse après transfert d’embryons congelés (TEC). Étude des 289 transferts électifs d’un embryon (eSET) effectués chez une population sélectionnée au centre d’AMP du CHU de la Conception à Marseille de janvier 2005 à décembre 2011, ainsi que des 325 TEC réalisés dans cette population entre mai 2005 et décembre 2012. Les taux cumulés de grossesses débutantes/ponction (après transferts frais et TEC) ont été de 62,6 % ; 45 % des couples ont obtenu la naissance d’au moins un enfant. Pendant cette période, les taux cumulés de grossesses et d’accouchements/tentative dans la population générale sont restés stables alors que les taux d’accouchement multiple/accouchement ont nettement chuté. Le TEC électif d’un embryon a donné des résultats satisfaisants (24,6 % grossesse/TEC) seulement en l’absence de lyse ou en cas de lyse modéré (de 1 à 25 %) de l’embryon transféré. Notre politique d’eSET donne des résultats satisfaisants, dépendant en grande partie de la qualité embryonnaire. En proposant le transfert électif à une population bien ciblée, choisie à la fois sur des critères cliniques et biologiques, une réduction nette des taux cumulés d’accouchements multiples dans notre centre a été obtenue sur cette période, sans diminution globale des taux cumulés de grossesses/tentative. La qualité embryonnaire est un facteur essentiel de succès, surtout lors de la congélation/décongélation. Le TEC électif d’un embryon devrait être envisagé uniquement si la lyse embryonnaire après décongélation n’excède pas 25 %. To evaluate our elective single embryo transfer policy performed at 48/72 h and define predictive factors of pregnancy after frozen/thawed embryo transfer. Analysis of 289 elective single embryo transfers (eSET) performed in a selected population in the ART center of Marseille University Hospital from January 2005 to December 2011, as well as the 325 following frozen/thawed embryo transfers performed in this population between May 2005 and December 2012. Cumulative pregnancy rate/oocyte retrieval was of 62.6%; 45% of the couples obtained the birth of at least one child. During this studied period, cumulative pregnancy and delivery rates in the whole population remained stable while multiple delivery rate/delivery clearly decreased. Elective single embryo after frozen/thawed transfer gave satisfying results (24.6% pregnancy/transfer) only in the lack of lysis or in case of mild lysis (1–25%) of the transferred embryo. The implementation of an eSET policy gives satisfying results, depending largely on embryo quality. By proposing eSET to a well-targeted population, chosen both on clinical and biological criteria, a clear reduction of cumulative multiple delivery rate/delivery was obtained in our center over this period, without any global decrease of cumulative pregnancy rate/attempt. Embryo quality is a major factor of success, especially in frozen/thawed cycles. The elective single embryo frozen/thawed transfer should be carried out only if embryo lysis after thawing does not exceed 25%.

Coxiella burnetii, the agent of Q fever, is known to persist in humans and rodents but its cellular reservoir in hosts remains undetermined. We hypothesized that adipose tissue serves as a C. burnetii reservoir during bacterial latency. BALB/c and C57BL/6 mice were infected with C. burnetii by the intraperitoneal route or the intracheal route. Adipose tissue was tested for the presence of C. burnetii several months after infection. C. burnetii was detected in abdominal, inguinal and dorsal adipose tissue 4 months post-infection, when no bacteria were detected in blood, liver, lungs and spleen, regardless of the inoculation route and independently of mouse strain. The transfer of abdominal adipose tissue from convalescent BALB/c mice to naïve immunodeficient mice resulted in the infection of the recipient animals. It is likely that C. burnetii infects adipocytes in vivo because bacteria were found in adipocytes within adipose tissue and replicated within in vitro-differentiated adipocytes. In addition, C. burnetii induced a specific transcriptional program in in-vivo and in vitro-differentiated adipocytes, which was enriched in categories associated with inflammatory response, hormone response and cytoskeleton. These changes may account for bacterial replication in in-vitro and chronic infection in-vivo. Adipose tissue may be the reservoir in which C. burnetii persists for prolonged periods after apparent clinical cure. The mouse model of C. burnetii infection may be used to understand the relapses of Q fever and provide new perspectives to the follow-up of patients.

Objective: To identify the prognostic factors for pregnancy after intrauterine insemination with the husband's sperm (IUI-H).Design: Retrospective study.Setting: A single university medical center.Patient(s): 851 couples, for 2,019 IUI-H cycles.Intervention(s): After controlled ovarian stimulation, IUI-H performed 36 hours after ovulation triggering or 24 hours after a spontaneous luteinizing hormone (LH) surge.Main Outcome Measure(s): Clinical pregnancy rate per cycle (PR) and delivery rate per cycle (DR).Result(s): The overall PR was 14.8% and DR 10.8%. Higher PR and DR were observed for patients presenting with ovulation disorders (particularly polycystic ovary syndrome) or with male infertility. Secondary infertility in the woman appeared to be a positive prognostic factor as did a basal follicle-stimulating hormone (FSH) level <= 7 IU/L and ovulation triggering over spontaneous LH rise. The other parameters influencing the results were the women's age, the number of mature follicles obtained (>= 2), the endometrial thickness (10-11 mm), and the number of progressive motile spermatozoa inseminated (> 1 million).Conclusion(s): In women aged <= 38 years, IUI-H should be considered as an option, particularly in cases of female infertility from ovulation disorders, in cases of a normal ovarian reserve, in cases of secondary infertility, or when >= 1 million progressive sperm are inseminated. Bifollicular stimulation is required. In other cases, in vitro fertilization should be discussed as the first-line treatment. (c) 2014 by American Society for Reproductive Medicine.

Background: Epicardial adipose tissue (EAT) is in close contact with coronary vessels and therefore could alter coronary homeostasis. Glucocorticoids are pathophysiological mediators of visceral fat deposition and its associated atherogenic complications. Aim: We investigated in EAT the expression of the glucocorticoid receptor (GR) and its various (A, B, C) promoters. Materials and methods: Paired subcutaneous adipose tissue (SAT) and EAT biopsies were obtained from 15 patients with coronary artery disease (CAD) and 12 patients without CAD (NCAD). GR and 11 β-hydroxysteroid dehydrogenase type 1 protein (11 β-HSD-1, the enzyme which converts inactive cortisone into active cortisol) were studied by immunohistochemistry and GR and its various promoters were studied by mRNA quantitative RT-PCR. Results: GR and 11 β-HSD-1 protein were expressed in adipocytes, stromal areas, isolated stromal cells close to adipocytes, and blood vessels. Total GR mRNA levels did not differ in SAT obtained from NCAD or CAD patients and were decreased in EAT, irrespectively of the coronary status, with parallel changes in promoter B- and C-, but not promoter A-associated transcripts. Total GR mRNA and adipocyte surface in EAT obtained from CAD patients were correlated negatively (p<0.035, r=0.39). Conclusions: Our findings demonstrate that in EAT, GR gene promoters could play a role in tissue-specific GR expression levels. EAT may be less sensitive to glucocorticoids than SAT, preventing the EAT mass development in CAD patients and suggesting a protective role on coronary homeostasis.

Using a validated model of culture of rat seminiferous tubules, we assessed the effects of 0.1, 1 and 10 μg/L cadmium (Cd) on spermatogenic cells over a 2-week culture period. With concentrations of 1 and 10 μg/L in the culture medium, the Cd concentration in the cells, determined by ICP-MS, increased with concentration in the medium and the day of culture. Flow cytometric analysis enabled us to evaluate changes in the number of Sertoli cells and germ cells during the culture period. The number of Sertoli cells did not appear to be affected by Cd. By contrast, spermatogonia and meiotic cells were decreased by 1 and 10 μg/L Cd in a time and dose dependent manner. Stage distribution of the meiotic prophase I and qualitative study of the synaptonemal complexes (SC) at the pachytene stage were performed by immunocytochemistry with an anti SCP3 antibody. Cd caused a time-and-dose-dependent increase of total abnormalities, of fragmented SC and of asynapsis from concentration of 0.1 μg/L. Additionally, we observed a new SC abnormality, the "motheaten" SC. This abnormality is frequently associated with asynapsis and SC widening which increased with both the Cd concentration and the duration of exposure. This abnormality suggests that Cd disrupts the structure and function of proteins involved in pairing and/or meiotic recombination. These results show that Cd induces dose-and-time-dependent alterations of the meiotic process of spermatogenesis ex-vivo, and that the lowest metal concentration, which induces an adverse effect, may vary with the cell parameter studied.

Semen from 10 932 male partners of infertile couples was analysed and sperm parameter trends were evaluated at the Reproduction Biology Laboratory of the University Hospital of Marseille (France) between 1988 and 2007. After 3-6 days of abstinence, semen samples were collected. Measurements of seminal fluid volume, pH, sperm concentration, total sperm count, motility and detailed morphology of spermatozoa were performed. Sperm parameters were analysed on the entire population and in men with normal total numeration (≥40 million per ejaculate). The whole population demonstrated declining trends in sperm concentration (1.5% per year), total sperm count (1.6% per year), total motility (0.4% per year), rapid motility (5.5% per year) and normal morphology (2.2% per year). In the group of selected samples with total normal sperm count, the same trends of sperm quality deterioration with time were observed. Our results clearly indicate that the quality of semen decreased in this population over the study period.

visceral obesity is a risk factor for cardiovascular diseases. Perivascular adipose tissue (PVAT) is a visceral fat depot close to the vessel wall which therefore could directly influence vascular reactivity. Diet-induced alterations in endothelium-dependent vascular reactivity have been well investigated. Diet-induced changes in endothelium-independent vascular reactivity have received less attention. we studied in 5-month-old rats fed low-fat (LFD) or high-fat diet (HFD) from weaning vascular reactivity using phenylephrine (PE)-stimulated isolated endothelium-removed aortic rings without or with their PVAT (PVAT - or PVAT +, respectively), HFD induced an increase in mesenteric and PVAT fat mass, and enhanced systemic and mesenteric AT Tbars while it decreased aortic and PVAT Tbars. When PVAT - rings were incubated with increasing doses of PE, maximal contraction and EC50 were not different between groups. The presence of PVAT decreased the maximal contraction to the same extent in rings obtained from LFD- or HFD-fed rats. Incubation with catalase suppressed the anticontractile properties of PVAT, indicating that this effect was mediated through H2O2. Transfer experiments of PVAT conditioned medium demonstrated that PVAT was the site of H2O2 synthesis. In LFD PVAT- rings, oxidative stress-associated procontractile activity was generated by xanthine oxidase and cytochrome c oxidase. In HFD PVAT- rings oxidative stress-associated procontractile activity increased and was generated, in addition to the above-mentioned enzymes, by NADPH oxidase. Catalase-induced H2O2 dismutation had prorelaxing properties, comparable in rings obtained from LFD or HFD fed rats. Superoxide dismutase (SOD) was devoid of effect in LFD rings, whereas it had a prorelaxing activity in HFD rings. These observations suggest that, in addition to H2O2, O2.- has a procontractile activity in the aortic wall of HFD rats. In aorta HFD increased the mRNAs coding for NADPH oxidase (p47 phox, p67phox, and NOX4), xanthine oxidase, SOD and catalase, suggesting an increased dismutation activity. PVAT modified the effect of the various drugs tested. The increased anticontractile activity of PVAT found after reactive oxygen species scavenging was reduced in HFD fed rats compared with LFD fed animals. Inhibition of O2.- dismutation resulting from DETC-induced SOD blocking led to a procontractile effect of PVAT in both groups, this effect being reduced in HFD compared to LFD rats. NADPH blockade did not affect the anticontractile properties of PVAT in both groups. Xanthine oxidase blockade amplified the anticontractile effect of PVAT in LFD animals while it reversed this effect in HFD rats. In rings obtained from LFD animals cytochrome c oxidase blockade reversed the anticontractile effect of PVAT in LFD animals whereas it was devoid of effect in HFD rats. In PVAT, HFD increased the mRNAs coding for cytochrome c oxidase, glutathione peroxidase and UCP-1 and -3. our data show that HFD-induced obesity was associated with NADPH-dependent increased oxidative stress-associated procontractile activity in the aortic wall. Such phenomenon was counteracted by enhanced dismutation activity in the aortic wall and decreased procontractile activity provided by the PVAT. As a consequence, these observations predict that any defect in the above-mentioned counterregulatory mechanisms can have deleterious functional consequences.

A low-protein diet applied during pregnancy in the rat results in intrauterine growth restricted (IUGR) fetuses. In humans, IUGR is associated with increased perinatal morbidity, higher incidence of neuro-developmental defects and increased risk of adult metabolic anomalies, such as diabetes and cardiovascular disease. Development and function of many organs are affected by environmental conditions such as those inducing fetal and early postnatal growth restriction. This phenomenon, termed "fetal programming" has been studied unconnectedly in some organs, but very few studies (if any) have investigated at the same time several organs, on a more comparative basis. However, it is quite probable that IUGR affects differentially most organ systems, with possible persistent changes in gene expression. In this study we address transcriptional alterations induced by IUGR in a multi-organ perspective, by systematic analysis of 20-days rat fetuses. We show that (1) expressional alterations are apparently stronger in organs functioning late in foetal or postnatal life than in organs that are functioning early (2) hierarchical classification of the deregulations put together kidney and placenta in one cluster, liver, lungs and heart in another; (3) the epigenetic machinery is set up especially in the placenta, while its alterations are rather mild in other organs; (4) the genes appear deregulated in chromosome clusters; (5) the altered expression cascades varies from organ to organ, with noticeably a very significant modification of the complement and coagulation cascades in the kidney; (6) we found a significant increase in TF binding site for HNF4 proteins specifically for liver genes that are down-regulated in IUGR, suggesting that this decrease is achieved through the action of HNF transcription factors, that are themselves transcriptionnally induced in the liver by IUGR (x 1.84 fold). Altogether, our study suggests that a combination of tissue-specific mechanisms contributes to bring about tissue-driven modifications of gene cascades. The question of these cascades being activated to adapt the organ to harsh environmental condition, or as an endpoint consequence is still raised.

Testicular sperm extraction is widely used in the treatment of male infertility in cases of non-obstructive azoospermia.Identifying spermatogenetic foci within the testes is critical for testicular sperm extraction.Two-photon laser scanning microscopy (TPLSM) is an autofluorescence-based microscopy technique that allows observation at a cellular level in the depth of fresh living tissues and does not require any histological processing (fixation or staining).The wavelengths previously used have shown no phototoxicity on sperm.We used TPLSM to detect spermatogenetic foci in fresh mouse testicular parenchyma without disrupting the tunica albuginea.Fresh surgically retrieved testes were observed using TPLSM within 1 h after extraction.Contralateral testes for each animal were observed using standard histology.Using TPLSM we were able to observe and measure the diameter of seminiferous tubules through the tunica albuginea, similar to the histological control.Structures within epithelial tubules were also observed, although their nature has yet to be identified.TPLSM is a real-time microscopy technique that could detect spermatogenetic foci.