Rapid detection of mouse spermatogenic defects by testicular cellular composition analysis via enhanced deep learning model.

BACKGROUND: Histological analysis of the testicular sections is paramount in infertility research but tedious and often requires months of training and practice. OBJECTIVES: Establish an expeditious histopathological analysis of mutant mice testicular sections stained with commonly available hematoxylin and eosin (H&E) via enhanced deep learning model MATERIALS AND METHODS: Automated segmentation and cellular composition analysis on the testes of six mouse reproductive mutants of key reproductive gene family, DAZ and PUMILIO gene family via H&E-stained mouse testicular sections. RESULTS: We improved the deep learning model with human interaction to achieve better pixel accuracy and reduced annotation time for histologists; revealed distinctive cell composition features consistent with previously published phenotypes for four mutants and novel spermatogenic defects in two newly generated mutants; established a fast spermatogenic defect detection protocol for quantitative and qualitative assessment of testicular defects within 2.5-3 h, requiring as few as 8 H&E-stained testis sections; uncovered novel defects in AcDKO and a meiotic arrest defect in HDBKO, supporting the synergistic interaction of Sertoli Pum1 and Pum2 as well as redundant meiotic function of Dazl and Boule. DISCUSSION: Our testicular compositional analysis not only could reveal spermatogenic defects from staged seminiferous tubules but also from unstaged seminiferous tubule sections. CONCLUSION: Our SCSD-Net model offers a rapid protocol for detecting reproductive defects from H&E-stained testicular sections in as few as 3 h, providing both quantitative and qualitative assessments of spermatogenic defects. Our analysis uncovered evidence supporting the synergistic interaction of Sertoli PUM1 and PUM2 in maintaining average testis size, and redundant roles of DAZ family proteins DAZL and BOULE in meiosis.

Ultrastructural changes in the spermatogenic cells of domestic chicken (Gallus gallus domesticus) observed at different reproductive stages.

Spermatogenesis is a complex process. It is the modification of progenitor spermatogonia into mature spermatozoa. The stages are similar in all-male vertebrates, as well as avian species. However, studies on spermatogenesis in birds are fewer compared to mammals. The current study investigated the ultrastructural changes in the spermatogenic cells of domestic chickens in different reproductive stages. Thirty (30) male birds, ten (10) in each of the three reproductive stages: pre-pubertal, pubertal, and adult were used in the study. Testicular tissues from all age groups were processed for transmission electron microscopy (TEM). TEM results showed spermatogonia and primary spermatocytes in the pre-pubertal testis, and the seminiferous tubule lumen was wide and empty. Also, the nuclei of spermatogonia at this stage did not contain condensed chromatin material at the center nor scattered at the periphery of the nuclear membrane. There were slight differences between the spermatogenic cells in the pubertal and adult age groups. The spermatogonia, primary and secondary spermatocytes, and round spermatids with scanty chromatin material were observed in both age groups. In the adult age group, round and elongated spermatids with condensed chromatin materials were observed besides the other spermatogenic cells. Also, the seminiferous tubule lumen was filled with sperm cells and cellular debris, unlike in the pre-pubertal and pubertal age groups where they were wide and empty. The presence of numerous oval mitochondria were observed in all age groups. This signifies the active process of spermatogenesis in pre-pubertal, pubertal, and adult male domestic chickens.

Pathological and vascular changes in the rat testiсle after experimental trauma.

Objective: Mechanical trauma to the testicles poses a potential risk of tissue destruction, disruption of local blood supply, and impairment of spermatogenesis, which can ultimately lead to infertility. Therefore, investigating this topic is crucial. The study aimed to identify cytological and morphological changes in the testicular tissue of laboratory rats following mechanical trauma to the organ. Methods: Observations were recorded on days 7, 14, 30, and 90 post-trauma. The experiment involved two groups of animals: a control group of healthy animals and an experimental group that sustained blunt mechanical trauma. Tissue samples were collected, fixed, dehydrated, and embedded in paraffin; subsequently, sections were prepared and stained. Structural changes in tissues and cells were documented using light and transmission electron microscopy. Results: In the experimental sample, notable changes included a decrease in organ weight, thickening of the protein shell and tubule walls, sclerotisation of the tubule membrane, narrowing of tubule diameter, reduced spermatozoa and spermatids titre, diminished capillary network and spermatogenic epithelium, uneven blood vessel lumen expansion, and decreased volume of Leydig cell nuclei. Additionally, in cells under different functional loads, the cytoplasm was vacuolated, mitochondrial cristae and the Golgi apparatus were diminished, cytoplasm volume decreased, karyopyknosis was observed, and uncharacteristic protrusions appeared on the surface of the cytoplasmic membrane. The severity of destruction at the cellular and tissue levels showed a positive correlation with time. Conclusion: The data obtained from these model sites can be predictive for clinical trials.

The possible effects of chronic administration of amiodarone hydrochloride on the seminiferous tubules of adult male albino rats: histological and biochemical study.

Amiodarone hydrochloride is an antiarrhythmic agent that is widely prescribed. However, it has serious side effects that approximately affect the whole body organs. In our study, we aimed to assess the possible effects of chronic administration of two different doses of amiodarone hydrochloride on the oxidative and inflammatory parameters as well as the histological morphology and ultrastructure of the seminiferous tubules of adult male albino rats. Forty rats were divided into four groups; Control group 1: each rat did not receive any drugs at all. Control group 2: each rat received 3 ml of 0.16% methylcellulose, orally and daily for 4 weeks. Low dose amiodarone group: each rat received 3 ml of 0.16% methylcellulose contained 3.6 mg amiodarone, orally and daily for 4 weeks. High dose amiodarone group: each rat received 3 ml of 0.16% methylcellulose contained 7.2 mg amiodarone, orally and daily for 4 weeks. Blood samples were collected for measuring serum levels of malondialdehyde, superoxide dismutase, interleukin-6 and tumor necrosis factor-alpha. Testes specimens were examined to assess the morphological changes and the level of expression of caspase-3 apoptotic marker. The results indicated that; amiodarone hydrochloride could induce a dose-dependent toxicity, causing oxidative stress, inflammation, cellular degeneration, deposition of collagen and enhanced apoptosis in the seminiferous tubules.

Evaluation of the Potential Protectivity of Both Allium sativum and Zingiber officinale on the Cadmium-Induced Testicular Damage in Rats.

Background: Cadmium (Cd) is a widely spread environmental pollutant, listed among the unsafe metals due to known toxic effects on multiple organs, including the testes. In this study, we aim to evaluate the potential protectivity of garlic and ginger extracts on Cd-induced damage of the testis in rats. Materials and Methods: Fifty-six adult male albino rats were alienated into seven groups; control group, garlic-treated group, and ginger-treated group were given garlic and ginger extracts at doses of 250 mg and 120 mg/kg b.wt/day, Cd-treated group received 8.8 mg/Kg b.wt/day of Cd chloride, and the protected groups were given Cd and co-treated with garlic, ginger, or both extracts. The testes were subjected to different procedures to assess the oxidative status and histopathological changes. Results: Cd-treated rats showed a significant reduction in the testis weight and morphometric measurements of the seminiferous tubules compared to the control group. Cd administration resulted in a marked drop in the testosterone level and activities of antioxidative enzymes. Moreover, Cd induced histopathological changes in the seminiferous tubules. Co-administration of garlic and ginger extracts with the Cd showed partial improvement in the investigated parameters toward the control figures and improvement in the morphological changes. Co-treating both extracts together and the Cd resulted in complete normalization of these adverse effects of Cd. Conclusion: These findings indicated that garlic and ginger extracts could ameliorate the harmful effects of Cd on the testis. This effect was more prominent when garlic and ginger extracts were co-administered together with Cd.

Cyto- and Histopographic Assessment of CPA3-Positive Testicular Mast Cells in Obstructive and Non-Obstructive Azoospermia.

Infertility is an important personal and society disease, of which the male factor represents half of all causes. One of the aspects less studied in male infertility is the immunological testicular microenvironment. Mast cells (MCs), having high potential for regulating spermatogenesis due to fine-tuning the state of the integrative buffer metabolic environment, are one of the most crucial cellular subpopulations of the testicular interstitium. One important component of the MC secretome is proteases that can act as proinflammatory agents and in extracellular matrix (ECM) remodeling. In the testis, MCs are an important cell component of the testicular interstitial tissue (TIT). However, there are still no studies addressing the analysis of a specific MC protease-carboxypeptidase A3 (CPA3)-in cases with altered spermatogenesis. The cytological and histotopographic features of testicular CPA3+ MCs were examined in a study involving 34 men with azoospermia. As revealed, in cases with non-obstructive azoospermia, a higher content of CPA3+ MCs in the TIT and migration to the microvasculature and peritubular tissue of seminiferous tubules were observed when compared with cases with obstructive azoospermia. Additionally, a high frequency of CPA3+ MCs colocalization with fibroblasts, Leydig cells, and elastic fibers was detected in cases with NOA. Thus, CPA3 seems to be of crucial pathogenetic significance in the formation of a profibrogenic background of the tissue microenvironment, which may have direct and indirect effects on spermatogenesis.

Single-Cell Transcriptome Sequencing Reveals Molecular Expression Differences and Marker Genes in Testes during the Sexual Maturation of Mongolian Horses.

This study aimed to investigate differences in testicular tissue morphology, gene expression, and marker genes between sexually immature (1-year-old) and sexually mature (10-year-old) Mongolian horses. The purposes of our research were to provide insights into the reproductive physiology of male Mongolian horses and to identify potential markers for sexual maturity. The methods we applied included the transcriptomic profiling of testicular cells using single-cell sequencing techniques. Our results revealed significant differences in tissue morphology and gene expression patterns between the two age groups. Specifically, 25 cell clusters and 10 cell types were identified, including spermatogonial and somatic cells. Differential gene expression analysis highlighted distinct patterns related to cellular infrastructure in sexually immature horses and spermatogenesis in sexually mature horses. Marker genes specific to each stage were also identified, including APOA1, AMH, TAC3, INHA, SPARC, and SOX9 for the sexually immature stage, and PRM1, PRM2, LOC100051500, PRSS37, HMGB4, and H1-9 for the sexually mature stage. These findings contribute to a deeper understanding of testicular development and spermatogenesis in Mongolian horses and have potential applications in equine reproductive biology and breeding programs. In conclusion, this study provides valuable insights into the molecular mechanisms underlying sexual maturity in Mongolian horses.

Feasibility of high-frequency ultrasound for seminiferous tubule assessment and correlation of B-mode imaging with pathological findings in the testis in azoospermia.

PURPOSE: To determine the feasibility of high-frequency ultrasound (HFUS) for assessing seminiferous tubules and to understand high-resolution B-mode images of the testes in cases of azoospermia. METHODS: We verified how the histopathological images of testicular biopsy specimens can be observed using HFUS images and measurement analysis of seminiferous tubules was performed to 28 testes of 14 cases with azoospermia who underwent preoperative ultrasound and microdissection testicular sperm extraction (micro-TESE). The population consisted of obstructive azoospermia (OA) and non-obstructive azoospermia (NOA), including Sertoli cell-only syndrome (SCOS), and the other pathologies. Statistical verification of differences in seminiferous tubule diameters among preoperative ultrasound examination, ultrasound examination of pathological specimens, and histopathological specimens. We also examined the imagingpathology correlation via a case series presentation, aiming to identify imaging markers of testicular pathology and determine the possibility of predicting each condition. RESULTS: A comparison between HFUS images and histopathology from the same biopsy specimens suggested that ultrasonography could be seen as stereoscopic images due to its significantly greater slice thickness. The diameters of tubules were generally larger in pathological tissues as compared to ultrasonographic findings in OA and SCOS, but not in the other conditions. Comparisons provided insights into the predictability of SCOS and revealed imaging findings such as gaps between tubules and decreased diameter reflective of testicular damage. CONCLUSION: Seminiferous tubules can be observed however the diameter of seminiferous tubules varies in imaging and histopathology depending on the pathology. Imaging findings that reflect testicular damage and the predictability of SCOS were revealed in this study, but further verification is required.

Identification of spermatogenesis in individual seminiferous tubules and testicular tissue of adult normal and busulfan-treated mice employing Raman spectroscopy and principal component analysis.

The present study aims to identify spermatogenesis in testicular seminiferous tubules (ST) and testicular tissue of adult normal and busulfan-treated mice utilizing PCA and Raman spectroscopy. Raman measurements were conducted on single tubules and testes samples from adult and immature mice, comparing them with those from busulfan-treated adult mice, with validation through histological examination. The analysis revealed a higher signal variability (30 %-40 % at the peaks), prompting scrutiny of individual Raman spectra as a means of spermatogenesis measurement. However, principal component analysis (PCA) demonstrated significant cluster separation between the ST of mature and immature mice. Similar investigations were performed to compare ST from normal mature mice and those from busulfan-treated (BS-treated) mature mice, revealing substantial separation along PC1 and PC2 for all comparison sets. Additionally, comparing testicular samples from mature and immature mice revealed distinct separation in PCA. The study concludes that the combined approach of PCA and Raman spectroscopy proves to be a noninvasive and potentially valuable method for identifying spermatogenesis in seminiferous tubules and testicular samples.

Protective role of hesperidin in finasteride-induced testicular toxicity in adult male Wistar rats: Insights into oxidative stress, apoptosis, and ultrastructure of seminiferous tubules.

A negative impact of finasteride on fertility has been reported, in which over production of reactive oxygen species and apoptosis were implicated. Hesperidin, a plant-derived bioflavonoid with antioxidant and anti-apoptotic effects, may mitigate these adverse effects. In order to investigate the possible protective role of hesperidin against finasteride-induced seminiferous tubules toxicity in adult male Wistar rats, 60 rats were randomized into five groups (I-V) receiving distilled water, 0.5% sodium carboxymethylcellulose solution, hesperidin, finasteride, and combined hesperidin and finasteride respectively. Testicular weight, sperm count and motility were determined. Testicular tissue homogenates were prepared to measure the level of malondialdehyde (MDA), total antioxidant capacity (TAC), reduced glutathione (GSH) and the gene expression of caspase-3 and B-cell lymphoma 2 (Bcl2). Testes were processed for light and electron microscopic evaluation. Johnsen score was calculated. Administration of finasteride resulted in significantly decreased testicular weights, sperm count and motility, Johnsen score, tissue levels of TAC and GSH together with significant increase in tissue MDA. Gene expression revealed significantly increased caspase-3 and decreased Bcl2. Furthermore, finasteride disrupted the seminiferous tubules, causing degenerative changes affecting Sertoli cells and spermatogenic cells. Co-administration of hesperidin with finasteride resulted in improvement in testicular weights, TAC, GSH, Bcl2, Johnsen score, sperm count and motility as well as preservation of the structure of the seminiferous tubules. To conclude, hesperidin was found to have a protective potential on finasteride-induced oxidative stress, apoptosis and testicular structural damage.

Three-dimensional morphometry of the testis in dog using design-unbiased stereology.

Testis is considered the main organ of the male reproductive system. Dogs are used as a suitable experimental model of testicular diseases in humans. From the veterinary aspect, several disorders have been reported to affect the testis in dogs. Thus, the objective of the present study was to investigate the morphometrical features of the dog testis using design-based stereology. The testes of six male dogs were used. Isotropic, uniform random sections were obtained and processed for light microscopy. Testicular total volume and the fractional volume of the seminiferous tubules, interstitial tissue and germinal epithelium were measured using the Cavalieri's estimator and the point counting system. Germinal epithelial surface area was estimated using test lines, and total length of seminiferous tubules was analysed using the counting frames. The total volume of testis was calculated 13.64 ± 1.94 cm3 . The relative volume fractions of the seminiferous tubules, interstitial tissue and germinal layer expressed as a percentage of total testicular volume were found to be 75.87 ± 6.11%, 23.68 ± 5.15% and 64.15 ± 4.82%, respectively. The surface area of the germinal layer was 915.25 ± 150.48 cm2 . The thickness of germinal layer was estimated to be 96.18 ± 10.72 µm. The total length of seminiferous tubules measured 290.8 ± 35.86 m. No statistical difference in investigated parameters was found between the left and right testes (p > 0.05). Our data might contribute to the male reproductive knowledge, help develop experimental studies in this field and possibly lead to advancement in the diagnosis and treatment of testicular diseases in the dog.

A Prediction Model of Sperm Retrieval in Males with Idiopathic Non-obstructive Azoospermia for Microdissection Testicular Sperm Extraction.

Patients with Idiopathic non-obstructive azoospermia (iNOA) can achieve fertility by extracting testicular sperm through microdissection testicular sperm extraction (mTESE). But more than half of iNOA patients still cannot benefit from mTESE. In recent years, some studies had reported that serum hormones may be related to the outcome of sperm retrieval, but few had been verified. We hope to obtain a predictive method that is convenient for clinical application and can help judge the outcome of sperm extraction before implementing mTESE. We performed a retrospective analysis of NOA patients who underwent mTESE in the same andrology center from June 2020 to November 2022. A total of 261 patients with complete data were collected, logistic regression analysis was performed and a predictive model was constructed. Then, from December 2022 to May 2023, one prospective cohort of 48 NOA patients who met the inclusion criteria from the same center was recruited to validate the risk prediction model. We successfully constructed a logistic regression model to predict the outcome of iNOA patients undergoing mTESE and found that higher serum anti-Müllerian hormone (AMH) levels were associated with failure sperm retrieval, resulting in an AMH cut-off of 2.60 ng/ml. The area under the receiver operating curve was 0.811, the sensitivity was 0.870, and the specificity was 0.705. Decision curve analysis demonstrated that the threshold probability was above 4%, and unnecessary mTESE could be reduced using this model. In a prospective cohort at the same center, 85.42% (41/48) of iNOA patients correctly identified the mTESE outcome using this model. A logistic regression model with AMH as an independent predictor can predict mTESE outcomes in iNOA patients. Preoperative selection of mTESE in patients with iNOA using this model had clinical benefit in reducing unnecessary surgery. The model demonstrated good accuracy in a small prospective cohort validation.

Intratesticular transplantation of allogenic mesenchymal stem cells mitigates testicular destruction after induced heat stress in Miniature-horse stallions.

Testicular degeneration (TD) is the most frequent cause of sub or infertility in stallions. Currently, mesenchymal stem cells (MSC) have been studied as a therapeutic option for several diseases including induced-TD in laboratory animals. Therefore, this study aimed to evaluate the effect of intratesticular MSC therapy on the testicular histology of stallions submitted to scrotal heat stress. Ten healthy Miniature-horse stallions were submitted to testicular heat stress induced by a heating wrap device (42-45°C). Afterward, the stallions were divided into two groups and treated seven days later. MSCs-treated stallions were treated with an intratesticular injection of 10 × 106 of MSCs diluted in 5 mL of PBS, whereas placebo-treated stallions had 5 mL of PBS intratesticular injected. All stallions had testicular biopsies collected seven days before and one- and 14-days post-heat stress and were castrated 30 days after testicular insult. Tissue sections were stained with H&E and evaluated for the tubular and luminal diameter, epithelial thickness, seminiferous tubules (STs) integrity, the number of spermatozoa in the STs, and the percent of abnormal STs. Significance was set at P≤0.05. In both groups, testicular heat stress damaged the STs (P<0.05). However, STs' parameters were improved in MSCs-treated stallions compared to placebo-treated stallions 30 days after the testicular insult (P<0.05). In conclusion, the results of the present study suggest that intratesticular MSC therapy provided a therapeutic advantage in rescuing acute TD in stallions. However, further studies are essential to evaluate the benefits of this therapy on semen parameters and stallions with idiopathic TD.

The Role of Chlorella vulgaris in Attenuating Infertility Induced by Cadmium Chloride via Suppressing Oxidative Stress and Modulating Spermatogenesis and Steroidogenesis in Male Rats.

Cadmium (Cd) is an environmental pollutant known as endocrine disruptor . Cd has been reported to induce perturbations of the testicular functions and the subsequent decline of the male fertility of both animals and humans. Chlorella vulgaris (ChV) a species of green microalga has been reported to have multiple beneficial activities such as anti-inflammatory, antioxidant, and antiapoptotic effects. Thus, this work was conducted to declare the benefits of Chlorella vulgaris (ChV) (500 mg/kg doses) against cadmium chloride CdCl2 (2 mg/kg doses) toxicity on the main and accessory reproductive organs' weight, structure, and function of male rats. Briefly, 40 adult male rats in 4 groups (n = 10) were used as follows; control, ChV, CdCl2, and CdCl2+ChV. (i) The 1st group was kept as control fed on pellet chow and water ad libitum. (ii) The second group is Chlorella vulgaris (ChV) group fed with C. vulgaris alga for 10 days (500 mg/kg BW). (iii) The third group was administrated CdCl2 (2mg/kg BW) via subcutaneous injection (S/C) daily for 10 days. (iv) The fourth group administered both CdCl2 and ChV with the abovementioned doses daily for successive 10 days. Our observations declared that cadmium exhibited an adverse influence on the testes and prostate gland architecture indicated by seminiferous tubule destruction, testicular edema, degeneration of Leydig cells, and prostate acini damage. All together affect the epididymal semen quality and quantity including sperm viability, motility, and count. Interestingly, ChV could restore the testicular architecture and spermatozoa regeneration accompanied by semen quality improvement and increased reproductive hormones including testosterone. On the other side, ChV suppresses reactive oxygen species (ROS) formation via enhancement the antioxidant-related genes in the testicular tissue including SOD, CAT, GSH, and MDA and maintaining spermatocyte survival via suppression of apoptotic related genes including caspase3 and activating steroidogenic related genes including StAR and HSD17ß3 in the cadmium-treated testes. In this study, ChV could enhance male fertility under normal or stressful conditions and ameliorate the adverse effects of hazardous heavy metals that are widely distributed in our environment.

Characterization of sperm motility and testosterone secretion in the taiep myelin mutant, a model of demyelination.

Presently, demyelinating diseases have been reported to affect the reproductive life of patients who suffer from them, but the progression of the alterations is unknown, especially in men. To better understand these effects, it is necessary to perform studies in animal models, such as the male taiep rat, which exhibits progressive demyelination of the central nervous system, altered kisspeptin expression at the hypothalamic level, and decreased luteinizing hormone, which could alter sperm quality and testicular diameter. Thus, the objective of the present study was to analyze the diameter of the seminiferous tubules, the sperm motility, and the testosterone levels of 90-day-old male taiep rats. The obtained results indicate that male taiep rats show an increase in testicular size accompanied by an increase in the diameter of the seminiferous tubules of the left testicle. There was also a decrease in progressive motility in sperm samples from the left epididymis of male taiep rats compared to the control group, with no changes in serum testosterone concentration. Therefore, we conclude that male taiep rats with central demyelination show altered testicular diameter and decreased motility in sperm from the left side. This type of studies serves as a basis for proposing possible reproductive strategies to improve the fertility and testicular function of men with demyelinating diseases of the central nervous system.

Sox2 Localization During Spermatogenesis and Its Association with other Spermatogenesis Markers Using Protein-Protein Network Analysis.

Background: Sox2 (SRY box2) is an essential transcription factor that plays a vital role in spermatogenesis and regulates the genes in this process. Sox2 is important for pluripotency, self-renewal, and even spermatogonial stem cell differentiation. This gene is found in pluripotent and specialized cells, and it is involved in their biological activities. Methods: Protein-protein interaction (PPI) network analysis was performed during spermatogenesis using NCBI, STRING, and Cytoscape databases. Then, after isolating spermatogonial stem cells from 6 C57BL/6 mice, mouse embryonic stem cells and ES-like cells were prepared. In the following, Sox2 expression was examined in differentiated and undifferentiated spermatogonia by immunohistochemistry (IMH), immunocytochemistry (ICC), and Fluidigm PCR (polymerase chain reaction). Finally, the results were compared using the Kruskal-Wallis and Dunn tests at the significance level of p<0.05. Results: The results of this experiment showed that contrary to expectations, Sox2 has cytoplasmic expression in undifferentiated cells and nuclear expression in differentiated cells in in vitro conditions. In addition, the expression of Sox2 increased during differentiation. Fluidigm PCR showed a significantly higher expression of Sox2 (p<0.05) in differentiated compared to undifferentiated spermatogonia. Sox2 has an interaction with other genes during spermatogenesis such as Oct4, Nanog, Klf4, Stra8, Smad1, Tcf3, and Osm. Conclusion: Sox2, which is known as a pluripotency marker, has a vital role in spermatogenesis and could be a differential marker. Sox2 has strong connections with other genes such as Oct4, Nanog, Klf4, Tcf3, Osm, Stra8, Lim2, Smad1, Gdnf, and Kit.

Deep learning-based method for segmenting epithelial layer of tubules in histopathological images of testicular tissue.

Purpose: There is growing concern that male reproduction is affected by environmental chemicals. One way to determine the adverse effect of environmental pollutants is to use wild animals as monitors and evaluate testicular toxicity using histopathology. We propose an automated method to process histology images of testicular tissue. Approach: Testicular tissue consists of seminiferous tubules. Segmenting the epithelial layer of the seminiferous tubule is a prerequisite for developing automated methods to detect abnormalities in tissue. We suggest an encoder-decoder fully connected convolutional neural network model to segment the epithelial layer of the seminiferous tubules in histological images. The ResNet-34 is used in the feature encoder module, and the squeeze and excitation attention block is integrated into the encoding module improving the segmentation and localization of epithelium. Results: We applied the proposed method for the two-class problem, where the epithelial layer of the tubule is the target class. The F-score and Intersection over Union of the proposed method are 0.85 and 0.92. Although the proposed method is trained on a limited training set, it performs well on an independent dataset and outperforms other state-of-the-art methods. Conclusion: The pretrained ResNet-34 in the encoder and attention block suggested in the decoder result in better segmentation and generalization. The proposed method can be applied to testicular tissue images from any mammalian species and can be used as the first part of a fully automated testicular tissue processing pipeline. The dataset and codes are publicly available on GitHub.

Pfaffia glomerata polyploid accession compromises male fertility and fetal development.

ETHNOPHARMACOLOGICAL RELEVANCE: Pfaffia glomerata (Spreng.) Pedersen has traditionally been used as a tonic and a stimulant by the Brazilian population. It shows higher biomass accumulation and production of secondary compounds, such as the phytosterol 20-hydroxyecdysone. AIMS: The present study aimed to evaluate the effects of the hydroalcoholic extract of the root of tetraploid P. glomerata (BGEt) on testicular parenchyma, and its implications on fertility. MATERIAL AND METHODS: Adult Swiss mice were divided as: control (water) and sildenafil citrate (7 mg/kg), BGEt at 100, 200, and 400 mg/kg, and BGEtD 200 mg/kg (treated with BGE every three days). Males (n = 4/group) were mated with normal untreated adult females to assess fertility rates, while other animals (n = 6/group) were euthanized for testis, epididymis, and oxidative stress analyses. RESULTS: Increase in tubule diameter and epithelium height in the discontinuous group, in addition to an increase in the proportion of tubules with moderate pathologies was observed. The pre-implantation loss was lower in all treated groups. The post-implantation loss was significantly increased in all treated groups, except for the lowest BGEt dose. BGEt intake caused a decrease in daily sperm production, along with the number and quality of sperm in the epididymis. Changes were observed in protein carbonylation and hydrogen peroxide and nitric oxide levels, characterizing oxidative stress. CONCLUSIONS: The hydroalcoholic extract of P. glomerata tetraploid altered sperm and testicular parameters, compromising embryonic development after implantation.

Deep learning-based method for segmenting epithelial layer of tubules in histopathological images of testicular tissue.

Purpose: There is growing concern that male reproduction is affected by environmental chemicals. One way to determine the adverse effect of environmental pollutants is to use wild animals as monitors and evaluate testicular toxicity using histopathology. We propose an automated method to process histology images of testicular tissue. Approach: Testicular tissue consists of seminiferous tubules. Segmenting the epithelial layer of the seminiferous tubule is a prerequisite for developing automated methods to detect abnormalities in tissue. We suggest an encoder-decoder fully connected convolutional neural network model to segment the epithelial layer of the seminiferous tubules in histological images. The ResNet-34 is used in the feature encoder module, and the squeeze and excitation attention block is integrated into the encoding module improving the segmentation and localization of epithelium. Results: We applied the proposed method for the two-class problem, where the epithelial layer of the tubule is the target class. The F -score and Intersection over Union of the proposed method are 0.85 and 0.92. Although the proposed method is trained on a limited training set, it performs well on an independent dataset and outperforms other state-of-the-art methods. Conclusion: The pretrained ResNet-34 in the encoder and attention block suggested in the decoder result in better segmentation and generalization. The proposed method can be applied to testicular tissue images from any mammalian species and can be used as the first part of a fully automated testicular tissue processing pipeline. The dataset and codes are publicly available on GitHub.

LHX2 in germ cells control tubular organization in the developing mouse testis.

In the gonads of mammalian XY embryos, the organization of cords is the hallmark of testis development. This organization is thought to be controlled by interactions of the Sertoli cells, endothelial and interstitial cells with little or no role of germ cells. Challenging this notion, herein we show that the germ cells play an active role in the organization of the testicular tubules. We observed that the LIM-homeobox gene, Lhx2 is expressed in the germ cells of the developing testis between E12.5-E15.5. In Lhx2 knockout-fetal testis there was altered expression of several genes not just in germ cells but also in the supporting (Sertoli) cells, endothelial cells, and interstitial cells. Further, loss of Lhx2 led to disrupted endothelial cell migration and expansion of interstitial cells in the XY gonads. The cords in the developing testis of Lhx2 knockout embryos are disorganized with a disrupted basement membrane. Together, our results show an important role of Lhx2 in testicular development and imply the involvement of germ cells in the tubular organization of the differentiating testis. The preprint version of this manuscript is available at https://doi.org/10.1101/2022.12.29.522214.