Testicular histomorphometric patterns and spermatogenesis dynamics of Oecomys bicolor tomes, 1860 (Rodentia: Cricetidae).

Although the order Rodentia does not present a high risk of extinction compared to mammals as a whole, several families demonstrate high levels of threat and/or data deficiency, therefore highlighting the need for targeted research and the application of ecological and reproductive data to the development of conservation actions. The order Rodentia, the largest among mammals, includes 9 families, and the family Cricetidae is the most diverse of the Brazilian rodents. In Brazil, 12 of the 16 genera of Oecomys are found. Oecomys bicolor is known in Brazil as the 'arboreal rat' and is, found in dry, deciduous and tropical forests. The mean body weight of Oecomys bicolor was 35.8 g and the gonadal, tubular and epithelial somatic indexes were, 0.53%, 0.47% and 0.37%, respectively. Seminiferous tubules volume density was 89.72% and the mitotic and meiotic indexes corresponded to 8.59 and 2.45 cells, respectively, and the yield of spermatogenesis was 23.83 cells. The intertubular compartment represented 10.28% of the testis parenchyma and around 5% of the interstitial space was occupied by Leydig cells, whose number per gram of testis was 11.10 × 107 cells. By evaluating the biometric and histomorphometric characteristics of the testis, there is evidence that this species has a high investment in reproduction. Due to the high contribution of the seminiferous epithelium and the intertubular compartment in this species, compared to the others of the same family, it is possible to infer that the species Oecomys bicolor has a promiscuous reproductive behaviour.

Histological aspects of the renal sexual segment of Brazilian snakes of the Boidae family.

Snakes represent a wide and diverse group of species and have anatomical particularities, such as the renal sexual segment (RSS), a structure located in the kidneys and formed from the hypertrophy of the urinary ducts and nephrons. This study aims at describing the histological aspects of the RSS of Boa constrictor, Epicrates cenchria and Corallus hortulanus, all of which are Brazilian snake species from the Boidae family. The reproductive system and kidneys of five male specimens of E. cenchria, three male specimens of C. hortulanus and two male specimens of B. constrictor were obtained. Tissue samples were processed histologically and different stains used (Toluidine Blue, Alcian Blue and Periodic Acid Schiff). The histological evaluation of the RSS of E. cenchria, C. hortulanus and B. constrictor shows that the RSS in these species varies when comparing individuals in the reproductive period with those which are not. It also allows for the observation of the segment's secretory activity in animals in the reproductive stage (mature sperm in the lumen of the seminiferous tubules) as well as in those which are not. Finally, the histological evaluation also reveals the variation of the secretion product in individuals in the reproductive period, in those which are not, and also among individuals within the same reproductive stage.

Deep Learning-Based Spermatogenic Staging in Tissue Sections of Cynomolgus Macaque Testes.

The indirect assessment of adverse effects on fertility in cynomolgus monkeys requires that tissue sections of the testis be microscopically evaluated with awareness of the stage of spermatogenesis that a particular cross-section of a seminiferous tubule is in. This difficult and subjective task could very much benefit from automation. Using digital whole slide images (WSIs) from tissue sections of testis, we have developed a deep learning model that can annotate the stage of each tubule with high sensitivity, precision, and accuracy. The model was validated on six WSI using a six-stage spermatogenic classification system. Whole slide images contained an average number of 4938 seminiferous tubule cross-sections. On average, 78% of these tubules were staged with 29% in stage I-IV, 12% in stage V-VI, 4% in stage VII, 19% in stage VIII-IX, 18% in stage X-XI, and 17% in stage XII. The deep learning model supports pathologists in conducting a stage-aware evaluation of the testis. It also allows derivation of a stage-frequency map. The diagnostic value of this stage-frequency map is still unclear, as further data on its variability and relevance need to be generated for testes with spermatogenic disturbances.

Seasonal variations of testis anatomy and of G-coupled oestrogen receptor 1 expression in Gerbillus gerbillus.

The gerbil, Gerbillus gerbillus, a nocturnal desert rodent of northern Africa, exhibits a seasonal reproductive cycle with marked anatomical and behavioural shifts between breeding season and resting season. The aim of this study is to investigate key elements involved in these seasonal changes, specifically in males: the histology of the testis as well as the expression of the G-protein-coupled oestrogen receptor 1 (GPER1) in the testis. During the breeding season, the seminiferous tubules were full of spermatozoa, and their epithelium contained germinal cells embedded in Sertoli cells. Amidst tubules, well-developed Leydig cells were observed around blood vessels, with peritubular myoid cells providing structural and dynamic support to the tubules. GPER1 was largely expressed throughout the testis. Notably, Leydig cells, spermatogonia and spermatocytes showed strong immunohistochemical signals. Sertoli cells, spermatozoa and peritubular myoid cells were moderately stained. During the resting season, spermatogenesis was blocked at the spermatocyte stage, spermatids and spermatozoa were absent and the interstitial space was reduced. The weight of the testis decreased significantly. At this stage, GPER1 was found in Leydig cells, spermatocytes and peritubular myoid cells. Sertoli cells and spermatogonia were not marked. Overall, the testis of the gerbil, Gerbillus gerbillus, has undergone noticeable histological, cellular and weight changes between seasons. In addition, the seasonal expression pattern of GPER1, with pronounced differences between resting season and breeding season, indicates that this receptor is involved in the regulation of the reproductive cycle.

Deep SED-Net with interactive learning for multiple testicular cell types segmentation and cell composition analysis in mouse seminiferous tubules.

The development of mouse spermatozoa is a continuous process from spermatogonia, spermatocytes, spermatids to mature sperm. Those developing germ cells (spermatogonia, spermatocyte, and spermatids) together with supporting sertoli cells are all enclosed inside seminiferous tubules of the testis, their identification is key to testis histology and pathology analysis. Automated segmentation of all these cells is a challenging task because of their dynamical changes in different stages. The accurate segmentation of testicular cells is critical in developing computerized spermatogenesis staging. In this paper, we present a novel segmentation model, SED-Net, which incorporates a squeeze-and-excitation (SE) module and a dense unit. The SE module optimizes and obtains features from different channels, whereas the dense unit uses fewer parameters to enhance the use of features. A human-in-the-loop strategy, named deep interactive learning, is developed to achieve better segmentation performance while reducing the workload of manual annotation and time consumption. Across a cohort of 274 seminiferous tubules from stages VI to VIII, the SED-Net achieved a pixel accuracy of 0.930, a mean pixel accuracy of 0.866, a mean intersection over union of 0.710, and a frequency weighted intersection over union of 0.878, respectively, in terms of four types of testicular cell segmentation. There is no significant difference between manual annotated tubules and segmentation results by SED-Net in cell composition analysis for tubules from stages VI to VIII. In addition, we performed cell composition analysis on 2346 segmented seminiferous tubule images from 12 segmented testicular section results. The results provided quantitation of cells of various testicular cell types across 12 stages. The rule reflects the cell variation tendency across 12 stages during development of mouse spermatozoa. The method could enable us to not only analyze cell morphology and staging during the development of mouse spermatozoa but also potentially could be applied to the study of reproductive diseases such as infertility.

Expressions of cytochrome P450 aromatase and anti-Müllerian hormone in testes of fattening pigs by the timing of the first vaccination for immunocastration.

In practice, two injections of gonadotropin-releasing hormone (GnRH) vaccine are recommended for pig immunocastration for effective outcomes. The present study aimed to investigate the expressions of cytochrome P450 aromatase (P450arom ) and anti-Müllerian hormone (AMH) in testes, testicular length and testicular histomorphometry of the fattening pigs receiving the first injection of GnRH vaccine 6 weeks earlier than the standard protocol. Based on vaccination protocol, 24 pigs were equally divided into three groups: T1 was vaccinated at 15 and 19 weeks of age, T2 received vaccine at 9 and 19 weeks of age and C remained intact. P450arom and AMH expressions were analysed using immunohistochemistry and Western blot. The results revealed that testicular length was highest in C pigs, but not different between T1 and T2 groups (6.5 ± 0.2 versus 6.9 ± 0.3 cm, p = .538). Histomorphometry demonstrated that the height of spermatogenic epithelia, the diameter of seminiferous tubules and the number of seminiferous tubules between T1 and T2 groups were not different (p > .05). For P450arom , immunohistochemistry revealed that H-score of C group was significantly higher than that of both T1 and T2 groups. Western blot analysis showed that C group possessed the densest protein band. Moreover, H-score between T1 and T2 groups was not significantly different. Protein band intensity between both groups was not apparently different. As for AMH, C pigs had significantly lower H-score than both T1 and T2 pigs. Furthermore, T2 pigs possessed significantly higher H-score than T1 pigs. Western blot analysis showed that the most intense protein band was found in T2 group. In summary, GnRH vaccine affected testicular development and functions. The first injection could be performed either at 9 or 15 weeks of age since both protocols contributed to comparable results in aspect of testicular length, histomorphometry and expressions of P450arom and AMH.

Three-dimensional structure of seminiferous tubules in the Syrian hamster.

The hamster is useful for the study of male reproductive biology. However, unlike in the mouse and rat, the gross structure of seminiferous tubules in the hamster is largely unknown. The aim of the present study was to clarify the precise 3-dimensional (3D) structure of seminiferous tubules in hamsters. We reconstructed all seminiferous tubules in 3 and 1 testes from 0-day (P0) and 10-week (adult) Syrian hamsters, respectively, using serial paraffin sections and high-performance 3D reconstruction software. In P0 hamsters, the average numbers of seminiferous tubules, terminating points, branching points, and blind ends per testis were 9.0, 89.7, 93.0, and 0.7, respectively. There were two types of tubules: shorter and dominant ones. The dominant tubules, 2-4 in number per testis and accounting for 86% of the total tubule length, had many terminating and branching points and appeared to be derived from the anastomosis of many shorter tubules. In an adult hamster, there were 11 seminiferous tubules with a total length of 22 m, 98 terminating points, 88 branching points, and 2 blind ends per testis. Three of the 11 tubules were dominant ones, accounting for 83% of the total length, and occupied the testis from the surface over the circumference to the center, while the others were short and occupied only one side of the testis. The amplitude and direction of the curves of tubules were random, and there were no funnel-shaped networks of tubules present, in contrast to the mouse testis. The present study revealed the 3D structure of seminiferous tubules in developing and adult Syrian hamsters, which is different from that in mice and rats.

Effects of photoperiod on morphology and function in testis and epididymis of Cricetulus barabensis.

Photoperiod regulates the seasonal reproductive rhythms of mammals by influencing the development and function of sexual organs; however, the underlying mechanism remains unclear. We examined the morphology and functioning of the main sex organs of striped dwarf hamsters (Cricetulus barabensis) under different photoperiods (short daylight [SD], moderate daylight [MD], and long daylight [LD]) and further investigated the underlying molecular mechanisms. There was an inverse correlation between blood melatonin levels and photoperiod in the order SD > MD > LD. Decreases in body and tissue weights were observed under SD, whereas testis and epididymis weights between MD and LD were comparable. The diameters of the spermatogenic tubules, thickness of the spermatogenic epithelium, and the number of spermatogonia and Sertoli cells decreased under SD, whereas the serum-luteinizing hormone, follicle-stimulating hormone, and fecal testosterone concentrations decreased under LD. In SD, bax/bcl2 protein expression increased in the testes and decreased in the epididymides, whereas LC3II/LC3I remained unchanged in the testes and increased in the epididymides compared with the MD group. In LD, bax/bcl2 and LC3II/LC3I protein expression levels were unchanged in the testes but were decreased in the epididymides. In SD and LD, adenosine triphosphate synthase and citrate synthase protein expression levels were unchanged in the testes but were decreased in the epididymides. Drp1 and Mff protein expression increased in the testes and decreased in the epididymides. Overall, different regulatory mechanisms in the testis and epididymis led to degeneration under SD and maintenance under LD, preferentially protecting mitochondrial function in the testis by regulating mitochondrial fission.

Reproductive cycle and maturation of Swinhoe's tree lizard (Diploderma swinhonis (Günther, 1864)) in Hyuga City, Miyazaki Prefecture, Japan.

Swinhoe's tree lizard (Diploderma swinhonis) is an arboreal agamid that is native to Taiwan. The species has been introduced to some areas of Japan and is regarded as an invasive alien species. In 2016, a nonnative population of D. swinhonis was discovered in Hyuga City, Miyazaki Prefecture, Japan, but little information was available on the ecology of the population at the time. The main purpose of this study was therefore to investigate the reproductive cycle and maturation of this population. Field research was conducted from 2017 to 2019, and 764 lizards were collected. Euthanized lizards were dissected and the reproductive organs were examined to determine the reproductive period, clutch size, clutch frequency and size at sexual maturity. Females with oviductal eggs or vitellogenic ovarian follicles were observed from May to October. Clutch size ranged from 2 to 8, and clutch frequency was more than twice a year. In males, spermiogenesis started in early May and testicular regression was observed in September. Males with spermatozoa in the epididymides were found from May to November. Minimum snout-vent length at sexual maturity was 50.2 mm in females and 53.0 mm in males. Comparisons of the findings of this study and reports from Taiwan suggest that the nonnative population of D. swinhonis in Hyuga City has a higher fecundity than populations in Taiwan. It is therefore considered necessary to exterminate the population in Hyuga City before this species colonizes other areas.

Evaluation of reproductive parameters in male Neotropical bats during dry and rainy months in a specific area of the Cerrado biome.

The aim of this study was to analyse the reproductive aspects of male bats of three common species of the Phyllostomidae family: Artibeus lituratus, Platyrrhinus lineatus and Sturnira lilium, during dry and rainy months in a specific area of the Cerrado biome. Body weight was significantly higher during the dry months for S. lilium. The gonadosomatic index (GSI) and testicular weight were not significantly different between dry and rainy periods. The tubular parameters were significantly bigger in A. lituratus than in the other two species during both periods. No difference in the tubular/interstitial ratio was observed in any of the species during both periods. In both periods, all sperm cells and germ cell developmental stages were visible on seminiferous tubules whereas sperm cells were observed in epididymides of all sampled animals. The percentage of morphologically normal sperm was low (35%-60%), with no difference between periods. Spermatozoa from A. lituratus presented a leaf-shaped head, while the head was round-shaped in the other two species. In conclusion, our data suggest that males from the three studied species did not present reproductive latency during the most critical weather periods (dry and rainy months) in the metropolitan region of Brasilia, Brazil.

The effect of l-arginine and flaxseed on plasma testosterone concentration, semen quality and some testicular histology parameters in old broiler breeder roosters.

The objective of this study was to investigate the effect of dietary supplementation of l-arginine and flaxseed on testosterone and lipid parameters of serum and semen quality, as well as histological and morphometric parameters of testes in old broiler breeder rooster. Thirty six 52-week-old Ross 308 broiler breeder roosters with similar weights (4900 g ±â€¯210) were used for a 8-weeks trial period in a 3 × 3 factorial arrangement of dietary treatments that three levels of l-arginine (0.52, 0.68 and 0.83%) and flaxseed (0, 2 and 4%) with four replications in each. The ratio of left testicle weight/total testicular weight was significantly higher in A68F0 group. The maximum and minimum seminiferous tubule diameters were recorded for roosters fed no flaxseed and F2 flaxseed, respectively. The roosters fed A68 showed the highest serum testosterone. Seminal volume and sperm concentration, were not significantly affected by l-arginine × flaxseed interaction; however, sperm forward motility were significantly affected by arginine × flaxseed interaction (P < 0.05). A68F0 had significantly greatest tubular differentiation indices (TDI) in all of the treatments. Also, repopulation indices (RI) and spermiogenesis indices (SI) in A68F0 were significantly higher than other groups, but A52F0 and A52F4. However, testis index, testis density, serum lipids (cholesterol, LDL, HDL and HDL: LDL ratio), epithelium height of seminiferous tubules and number of Sertoli cells were not affected by the treatments. It is concluded that dietary supplementation of arginine (0.68%) has positive effects on blood testosterone, semen quality and spermatogenesis index of aged roosters.

Morphology of mouse seminiferous tubules.

We developed a technique to analyze the high-resolution three-dimensional (3D) structure of seminiferous tubules. It consists of segmentation of tubules in serial paraffin sections of the testis by marking the basement membrane with periodic acid-Schiff or a fluorescent anti-laminin antibody followed by 3D reconstruction of tubules with high-performance software. Using this method, we analyzed testes from mice at different ages and accurately elucidated the 3D structure of seminiferous tubules, including the number and length of tubules as well as the numbers of connections with the rete testis, branching points, and blind ends. We also developed a technique to identify the precise spermatogenic stage and cellular composition of the seminiferous epithelium. It consists of the combination of lectin histochemistry for acrosomes and immunohistochemistry for specific cell markers visualized with fluorescence. Using this method, we examined seminiferous tubules from normal mice and counted the number of each cell type at each stage, and thereby established a quantitative standard for the cellular composition of the seminiferous epithelium. We then investigated seminiferous epithelia from genetically modified infertile mice deficient in certain cell adhesion molecules and revealed characteristic abnormalities in the cellular composition. We also analyzed the distribution and direction of spermatogenic waves along the length of adult seminiferous tubules as well as the site of the first onset of spermatogenesis in postnatal seminiferous tubules. These methods will be useful for investigating the structure and function of seminiferous tubules in mice and humans under normal and pathological conditions.

The seminiferous tubule caliber pattern as evaluated at high magnification during microdissection testicular sperm extraction predicts sperm retrieval in patients with non-obstructive azoospermia.

BACKGROUND: microTESE proved to be the gold standard surgical approach for patients with non-obstructive azoospermia (NOA), but sperm retrieval rates (SRRs) vary considerably among centers. Some authors compared their SRRs with the pattern of seminiferous tubule caliber found at high magnification, but none provided diagnostic accuracy measures. OBJECTIVE: The present retrospective study sought to verify the diagnostic accuracy of the pattern of seminiferous tubule caliber in predicting the sperm retrieval in NOA patients. MATERIALS AND METHODS: Data from 143 infertile NOA men undergoing unilateral (64) or bilateral (79) microTESE (222 testes) were retrospectively evaluated. During microTESE, if present, dilated tubules (DTs) were retrieved, otherwise tubules with slightly larger caliber (SDT) (×24) than that of the surroundings were removed. When no DT or SDT were found, not dilated tubules (NDTs) were excised. RESULTS: Spermatozoa were retrieved in 95 of 222 testes (42.8%); sperm retrieval was successful in 90% of testes with DTs, in 47% of those with SDTs, and only in 7% of those with NDTs (p < 0.0001). Stepwise binary logistic regression revealed that the combination of seminiferous tubule pattern and testis histology was significantly predictive of SSR, being able to classify 86.8% of testes, with an excellent diagnostic accuracy (AUC 0.93). The median number of spermatozoa retrieved was significantly higher in DTs compared with SDTs and NDTs. DISCUSSION: The results of the present study provide reliable accuracy measures in support of the relationship between seminiferous tubule caliber pattern and SSR in patients with non-obstructive azoospermia. We are proposing for the first time that spermatozoa may be retrieved even from slightly dilated tubules in about half of cases. The pattern of tubules retrieved, together with histology, may represent an additional outcome measure of microTESE. CONCLUSION: The pattern of seminiferous tubules together with testis histology predicts sperm retrieval with an excellent diagnostic accuracy.

Stereological and morphological analysis of the effects of aging on spermatogenesis in rat testis 1.

PURPOSE: To quantify, through stereological and morphometric analysis, spermatogenesis in rats undergoing the natural aging process. METHODS: Seventy-two male Wistar rats were divided into 6 equal groups according to age at the time of killing: 3, 6, 9, 12, 18, and 24 months. All the rats were subjected orchiectomy and collection of testicular parenchymal fragments for histological and morphometric analysis. The numerical density of spermatids was calculated using a stereological study, and morphometric analysis was conducted to measure the height of the germinal epithelium and the area of the seminiferous tubules. RESULTS: We found that the 18 and 24 months groups showed a significant reduction in the number of round spermatids. However, the height of the germinal epithelium was not significantly different between the groups. The area of seminiferous tubules was also significantly reduced in the elderly rats compared to that in the young ones. CONCLUSION: Aging of rats showed a significant reduction in the number of round spermatids and the area of the seminiferous tubules, more pronounced in the rats at 18 and 24 months of life.

Stereological and morphological analysis of the effects of aging on spermatogenesis in rat testis

Abstract Purpose: To quantify, through stereological and morphometric analysis, spermatogenesis in rats undergoing the natural aging process. Methods: Seventy-two male Wistar rats were divided into 6 equal groups according to age at the time of killing: 3, 6, 9, 12, 18, and 24 months. All the rats were subjected orchiectomy and collection of testicular parenchymal fragments for histological and morphometric analysis. The numerical density of spermatids was calculated using a stereological study, and morphometric analysis was conducted to measure the height of the germinal epithelium and the area of the seminiferous tubules. Results: We found that the 18 and 24 months groups showed a significant reduction in the number of round spermatids. However, the height of the germinal epithelium was not significantly different between the groups. The area of seminiferous tubules was also significantly reduced in the elderly rats compared to that in the young ones. Conclusion: Aging of rats showed a significant reduction in the number of round spermatids and the area of the seminiferous tubules, more pronounced in the rats at 18 and 24 months of life.

[Lipoic acid protects spermatogenesis in male rats with ornidazole-induced oligoasthenozoospermia].

OBJECTIVE: To study the protective effect of lipoic acid (LA) on the spermatogenic function of the male rats with oligoasthenozoospermia induced by ornidazole (ORN). METHODS: Seventy male SD rats were equally randomized into groups A (solvent control: 1 ml 0.5% CMC-Na + 1 ml olive oil), B (low-dose ORN model: 400 mg/kg ORN suspension + 1 ml olive oil), C (low-dose ORN + low-dose LA treatment: 400 mg/kg ORN + 50 mg/kg LA), D (low-dose ORN + high-dose LA treatment: 400 mg/kg ORN + 100 mg/kg LA), E (high-dose ORN model: 800 mg/kg ORN suspension + 1 ml olive oil), F (high-dose ORN + low-dose LA treatment: 800 mg/kg ORN + 50 mg/kg LA), and G (high-dose ORN + high-dose LA treatment: 800 mg/kg ORN + 100 mg/kg LA), and treated respectively for 20 successive days. Then all the rats were sacrificed and the weights of the body, testis, epididymis and seminal vesicle obtained, followed by calculation of the organ index, determination of epididymal sperm concentration and motility, and observation of the histomorphological changes in the testis and epididymis by HE staining. RESULTS: Compared with group A, group E showed significantly decreased body weight (ï¼»117.67 ± 11.53ï¼½ vs ï¼»88.11 ± 12.65ï¼½ g, P < 0.01) and indexes of the testis (ï¼»1.06 ± 0.12ï¼½ vs ï¼»0.65 ± 0.13ï¼½ %, P < 0.01) and epididymis (ï¼»0.21 ± 0.03ï¼½ vs ï¼»0.17 ± 0.01ï¼½ %, P < 0.01). In comparison with group E, group F exhibited remarkable increases in the epididymal index (ï¼»0.17 ± 0.01ï¼½ vs ï¼»0.20 ± 0.02ï¼½ %, P < 0.01), and so did group G in the body weight (ï¼»88.11 ± 12.65ï¼½ vs ï¼»102.70 ± 16.10ï¼½ g, P < 0.05) and the indexes of the testis (ï¼»0.65 ± 0.13ï¼½ vs ï¼»0.95 ± 0.06ï¼½ %, P < 0.01) and epididymis (ï¼»0.17 ± 0.01ï¼½ vs ï¼»0.19 ± 0.02ï¼½ %, P < 0.05), but no obvious difference was observed in the index of seminal vesicle among different groups. Compared with group A, group B manifested significant decreases in sperm motility (ï¼»74.12 ± 8.73ï¼½ vs ï¼»40.25 ± 6.08ï¼½ %, P < 0.01), and so did group E in sperm count (ï¼»38.59 ± 6.40ï¼½ vs ï¼»18.67 ± 4.59ï¼½ ×105/100 mg, P < 0.01) and sperm motility (ï¼»74.12 ± 8.73ï¼½ vs ï¼»27.58 ± 8.43ï¼½ %, P < 0.01). Sperm motility was significantly lower in group B than in C and D (ï¼»40.25 ± 6.08ï¼½ vs ï¼»58.13 ± 7.62ï¼½ and ï¼»76.04 ± 8.44ï¼½%, P < 0.01), and so were sperm count and motility in group E than in F and G (ï¼»18.67 ± 4.59ï¼½ vs ï¼»25.63 ± 9.66ï¼½ and ï¼»29.92 ± 4.15ï¼½ ×105/100 mg, P < 0.05 and P < 0.01; ï¼»27.58 ± 8.43ï¼½ vs ï¼»36.56 ± 11.08ï¼½ and ï¼»45.05 ± 9.59ï¼½ %, P < 0.05 and P < 0.01). There were no obvious changes in the histomorphology of the testis and epididymis in groups A, B, C and D. Compared with group A, group E showed necrotic and exfoliated spermatogenic cells with unclear layers and disorderly arrangement in the seminiferous tubules and remarkably reduced sperm count with lots of noncellular components in the epididymal cavity, while groups F and G exhibited increased sperm count in the seminiferous tubules and epididymis lumen, also with exfoliation, unclear layers and disorderly arrangement of spermatogenic cells, but significantly better than in group E. CONCLUSIONS: LA can reduce ORN-induced damage to the spermatogenetic function of rats, improve sperm quality, and protect the reproductive system.

The balance between apoptosis and autophagy regulates testis regression and recrudescence in the seasonal-breeding South American plains vizcacha, Lagostomus maximus.

Mammalian testis undergoes deep changes in their architecture and function during photoregression conditions in seasonal breeders. Particularly, the testicular mechanisms that regulate the transition between the active (functional) and inactive (regression) stage vary between species. The aim of the present study was to analyze the incidence of proliferation, apoptosis and autophagy in the testicular seminiferous ephitelium of a seasonal breeder, Lagostomus maximus, during the annual reproductive cycle. We observed that proliferating spermatogonia increased from the active testis until reaching the maximum peak in the activating testis. During the annual reproductive cycle, the quantity of apoptotic-TUNEL positive spermatogonia and meiotic germ cells was constant and this might be regulated by the members of the BCL2 family. Only in the activating testis, apoptosis of germ cells was almost undetectable. The analysis of the autophagic-related proteins BECN1 and LC3 showed their localization in Leydig cells and the germ cells in the active and activating testis. In the inactive testis, BECN1 and LC3 ceased to be immunolocalized within the seminiferous tubules and the mRNA expression of both regulators decreased. Moreover, the expression of BECN1 and LC3 and also the apoptotic index were up regulated in testicular cultures subjected to nutritional stress. These results suggest a possible interaction between apoptosis and autophagy in the active and activating testis (characterized by high metabolic requirement and nutrient), where autophagy could promote survival over cell death. In the inactive testis, the absence of autophagic-related proteins might explain the massive loss of germ cells, suggesting that autophagy plays new and key role in the alterations of the seminiferous epithelium during photoregression.

Implications of body condition and seasonality on morphological and functional parameters of testes of Myotis nigricans (Chiroptera: Vespertilionidae).

The insectivorous bat Myotis nigricans is widely distributed throughout the Neotropics, including Brazil, and has a reproductive biology that is affected by climate and food availability. To evaluate the reproductive capacity of this species, morphofunctional parameters of the testes were correlated with environmental variables and the body condition of individuals captured. After bats had been killed, their testes were removed, fixed in Karnovsky's fluid for 24h and embedded in resin for evaluation by light microscopy. The mean annual tubulosomatic index (0.58%) and the percentage of seminiferous tubules in the testes (88.96%) were the highest ever recorded for the Order Chiroptera. The percentage of Leydig cells and volume of the cytoplasm of Leydig cells were higher in the rainy than dry season (80.62±3.19% and 573.57±166.95µm, respectively; mean±s.d.). Conversely, the percentage of nuclei of the Leydig cells in the dry season (26.17±3.70%; mean±s.d.) and the total number of Leydig cells (6.38±1.84×109; mean±s.d.) were higher in the dry season. The results of the present study could help in future conservation of these bats because they provide a better understanding of the bats' reproductive strategies and how the species can adapt to changes.

Roles of CD34+ cells and ALK5 signaling in the reconstruction of seminiferous tubule-like structures in 3-D re-aggregate culture of dissociated cells from neonatal mouse testes.

Tissue reconstruction in vitro can provide, if successful, a refined and simple system to analyze the underlying mechanisms that drive the morphogenesis and maintain the ordered structure. We have recently succeeded in reconstruction of seminiferous cord-like and tubule-like structures using 3-D re-aggregate culture of dissociated testicular cells. In testis formation, endothelial cells that migrated from mesonephroi to embryonic gonads have been shown to be critical for development of testis cords, but how endothelial cells contribute to testis cord formation remains unknown. To decipher the roles of endothelial and peritubular cells in the reconstruction of cord-like and tubule-like structures, we investigated the behavior of CD34+ endothelial and p75+ cells, and peritubular myoid cells (PTMCs) in 3-D re-aggregate cultures of testicular cells. The results showed that these 3 types of cells had the capacity of re-aggregation on their own and with each other, and of segregation into 3 layers in a re-aggregate, which were very similar to interstitial and peritubular tissues in vivo. Observation of behaviors of fluorescent Sertoli cells and other non-fluorescent types of cells using testes from Sox9-EGFP transgenic mice showed dynamic cell movement and segregation in re-aggregate cultures. Cultures of testicular cells deprived of interstitial and peritubular cells resulted in dysmorphic structures, but re-addition of them restored tubule-like structures. Purified CD34+ cells in culture differentiated into p75+ cells and PTMCs. These results indicate that CD34+ cells differentiate into p75+ cells, which then differentiate into PTMCs. TGFß signaling inhibitors, SB431542 and ALK5i, disturbed the reconstruction of cord-like and tubule-like structures, and the latter compromised re-construction of interstitial-like and peritubular-like structures, as well as the proliferation of CD34+, p75+, PTMCs, and Sertoli cells, and their movement and differentiation. These results indicate that CD34+ cells and signaling through ALK5 play pivotal roles in the morphogenesis of interstitial-like, peritubular-like and cord-like structures.