Seminiferous Tubule Cross-sections Research Articles

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.

Impact of Hydroxyurea to Treat Haematological Disorders on Male Fertility: Two Case Reports and a Systematic Review.

Hydroxyurea (HU) is a cytoreductive agent used as standard treatment option for sickle cell anaemia/disease (SCD), essential thrombocythemia (ET), and polycythaemia vera (PV). Despite its overall good safety profile, its use also in relatively young patients raises an interest on its potential impact on spermatogenesis. To perform a systematic review of all published articles investigating fertility in male patients affected by SCD, ET, and PV and treated with HU. Two paradigmatic case reports of patients affected by PV and ET, respectively, have been also reported. PubMed, EMBASE, and Cochrane databases were queried for all the published studies indexed up to November 15th, 2022. A combination of the following keywords was used: "hydroxyurea," "fertility," "male," "sperm," "sickle cell anaemia," "sickle cell disease," "essential thrombocythemia," "polycythaemia vera." Of 48 articles identified, 8 studies, involving 161 patients, were eligible for inclusion. Overall, the number of spermatogonia per round cross section of seminiferous tubule were decreased in patients with SCD compared to healthy males. HU treatment was always associated with a worsening of semen parameters, even up to azoospermia. Notably, treatment discontinuation was associated with an improvement of semen parameters and a trend toward normalization in the case of PV and ET, with a less clear amelioration in men with SCD. In both our patients with either PV or ET, HU discontinuation was associated with a significant improvement of spermatogenesis with successful spontaneous pregnancies. Published evidence do not consistently report normalization of spermatogenesis after HU discontinuation in SCD cases. Conversely, the literature almost consistently reported an improvement of semen parameters at the discontinuation of HU therapy in PV and ET cases. Our real-life two cases confirmed those findings. The willing of fatherhood and the need for effective fertility treatment warrant further research to improve work-up management in men with hematological disorders.

RNA sequencing profiles reveals progressively reduced spermatogenesis with progression in adult cryptorchidism.

The fertility of cryptorchidism patients who didn't perform corrective surgery will decrease with age. Herein, we elucidate the histological alterations and underlying molecular mechanism in patients with an increase in the disease duration from 20 to 40 years. Testicular tissues were obtained from three patients with cryptorchidism, ranging in age from 22 to 44 years. Three benign paracancerous testicular samples of matched ages were used as controls. The normal and undescended testicular tissues were stained with hematoxylin and eosin (HE) and immunofluorescence and all six testicular samples were subjected to RNA sequencing. RNA sequencing data were subjected to gene set enrichment analysis (GSEA), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI) network analysis, and Gene Ontology (GO) searches. Real-time reverse transcriptase polymerase chain reaction was used to confirm the DEGs. The seminiferous tubules' basement membrane thickens with age in healthy testes. As the period of cryptorchidism in the cryptorchid testis extended, the seminiferous tubules significantly atrophy, the number of spermatogenic cells declines, and the amount of interstitial fibrous tissue increases in comparison to normal tissues. The number of germ cells per cross-section of seminiferous tubules was significantly lower in cryptorchidism than in normal testicular tissues, according to immunofluorescence staining, but the number of Sertoli cells remained stable. RNA sequencing analysis identified 1150 differentially expressed genes (DEGs) between cryptorchidism and normal testicular tissues (fold change >2 and p<0.05), of which 61 genes were noticeably upregulated and 1089 were significantly downregulated. These genes were predominantly linked to sperm development and differentiation, and fertilization, according to GO analysis. Meiosis pathways were significantly downregulated in cryptorchidism, according to KEGG pathway analysis and GSEA (P<0.001). PPI analysis was used to identify the top seven downregulated hub genes (PLCZ1, AKAP4, IZUMO1, SPAG6, CAPZA3, and ROPN1L), which were then further verified by qPCR. By describing the histological changes and differential gene expression patterns in adult cryptorchid patients of different age groups, we discovered the progression mechanisms of undescended testes in adults with aging and identified seven significantly downregulated hub genes (PLCZ1, AKAP4, IZUMO1, SPAG6, CAPZA3, and ROPN1L) in cryptorchid testis compared to normal testicular tissues. These genes played a role in the process of spermgenesis and are directly linked to the steady decline in fertility caused by cryptorchidism. Our study provided a better understanding of the molecular mechanisms underlying the loss of spermatogenesis in adult cryptorchidism, and give support for the development of adult cryptorchidism treatments.

Impact of low- or moderate-risk gonadotoxic chemotherapy prior to testicular tissue freezing on spermatogonia quantity in human (pre)pubertal testicular tissue.

What is the impact of low- or moderate-risk gonadotoxic chemotherapy received prior to testicular tissue freezing (TTF), and of the cancer itself, on spermatogonia quantity in testicular tissue from (pre)pubertal boys? Vincristine, when associated with alkylating agents, has an additional adverse effect on spermatogonia quantity, while carboplatin has no individual contribution to spermatogonia quantity, in testicular tissue of (pre)pubertal boys, when compared to patients who have received non-alkylating chemotherapy. The improved survival rates after cancer treatment necessitate the inclusion of fertility preservation procedures as part of the comprehensive care for patients, taking into consideration their age. Sperm cryopreservation is an established procedure in post-pubertal males while the TTF proposed for (pre)pubertal boys remains experimental. Several studies exploring testicular tissue of (pre)pubertal boys after TTF have examined the tubular fertility index (TFI, percentage of seminiferous tubule cross-sections containing spermatogonia) and the number of spermatogonia per seminiferous tubule cross-section (S/T). All studies have demonstrated that TFI and S/T always decrease after the introduction of chemotherapeutic agents, especially those which carry high gonadotoxic risks such as alkylating agents. Testicular tissue samples from 79 (pre)pubertal boys diagnosed with cancer (from 6 months to 16 years of age) were cryopreserved between May 2009 and June 2014. Their medical diagnoses and previous chemotherapy exposures were recorded. We examined histological sections of (pre)pubertal testicular tissue to elucidate whether the chemotherapy or the primary diagnosis affects mainly TFI and S/T. (Pre)pubertal boys with cancer diagnosis who had been offered TTF prior to conditioning treatment for hematopoietic stem cell transplantation were included in the study. All the patients had previously received chemotherapy with low- or moderate-risk for future fertility. We have selected patients for whom the information on the chemotherapy received was complete. The quantity of spermatogonia and quality of testicular tissue were assessed by both morphological and immunohistochemical analyses. A significant reduction in the number of spermatogonia was observed in boys treated with alkylating agents. The mean S/T values in boys exposed to alkylating agents were significantly lower compared to boys exposed to non-alkylating agents (P = 0.018). In contrast, no difference was observed for patients treated with carboplatin as the sole administered alkylating agent compared to the group of patients exposed to non-alkylating agents. We observed an increase of S/T with age in the group of patients who did not receive any alkylating agent and a decrease of S/T with age when patients received alkylating agents included in the cyclophosphamide equivalent dose (CED) formula (r = 0.6166, P = 0.0434; r = -0.3759, P = 0.0036, respectively). The TFI and S/T decreased further in the group of patients who received vincristine in combination with alkylating agents (decrease of 22.4%, P = 0.0049 and P < 0.0001, respectively), but in this group the CED was also increased significantly (P < 0.0001). Multivariate analysis, after CED adjustment, showed the persistence of a decrease in TFI correlated with vincristine administration (P = 0.02). This is a descriptive study of testicular tissues obtained from (pre)pubertal boys who were at risk of infertility. The study population is quite heterogeneous, with a small number of patients in each sub-group. Our results are based on comparisons between patients receiving alkylating agents compared to patients receiving non-alkylating agents rather than chemotherapy-naive patients. The French national guidelines for fertility preservation in cancer patients recommend TTF before highly gonadotoxic treatment. Therefore, all the patients had received low- or moderate-risk gonadotoxic chemotherapy before TTF. Access to testicular tissue samples from chemotherapy-naive patients with comparable histological types of cancer was not possible. The functionality of spermatogonia and somatic cells could not be tested by transplantation or in vitro maturation due to limited sample sizes. This study summarizes the spermatogonial quantity of (pre)pubertal boys prior to TTF. We confirmed a negative correlation between the cumulative exposure to alkylating agents and spermatogonial quantity. In addition, the synergistic use of vincristine in combination with alkylating agents showed a cumulative deleterious effect on the TFI. For patients for whom fertility preservation is indicated, TTF should be proposed for chemotherapy with a predicted CED above 4000 mg/m2. However, the data obtained from vincristine and carboplatin use should be confirmed in a subsequent study including more patients. This study had financial support from a French national research grant PHRC No. 2008/071/HP obtained by the French Institute of Cancer and the French Healthcare Organization. The sponsors played no role in the study. The authors declare no conflicts of interest. N/A.

Immunohistochemical analysis of the vimentin filaments in Sertoli cells is a powerful tool for the prediction of spermatogenic dysfunction

The close interaction between male germ cells and Sertoli cells, a type of somatic cell found in the seminiferous tubules of mammalian testis, is essential for the normal progression of spermatogenesis in mammals. Vimentin is an intermediate filament protein that primarily provides mechanical support, preserves cell shape, and maintains the nuclear position, and it is often used as a marker to identify Sertoli cells. Vimentin is known to be involved in many diseases and aging processes; however, how vimentin is related to spermatogenic dysfunction and the associated functional changes is still unclear. In a previous study, we reported that vitamin E deficiency affected the testes, epididymis, and spermatozoa of mice, accelerating the progression of senescence. In this study, we focused on the Sertoli cell marker vimentin and explored the relationship between the cytoskeletal system of Sertoli cells and spermatogenic dysfunction using testis tissue sections that caused male reproductive dysfunction with vitamin E deficiency. The immunohistochemical analysis showed that the proportion of the vimentin-positive area in seminiferous tubule cross-sections was significantly increased in testis tissue sections of the vitamin E-deficient group compared with the proportion in the control group. The histological analysis of testis tissue sections from the vitamin E-deficient group showed that vimentin-positive Sertoli cells were greatly extended from the basement membrane, along with an increased abundance of vimentin. These findings suggest that vimentin may be a potential indicator for detecting spermatogenic dysfunction.

MP55-04 LOW SERUM INHIBIN B/FOLLICLE-STIMULATING HORMONE AND ANTI-MULLERIAN HORMONE/FOLLICLE-STIMULATING HORMONE RATIOS AS MARKERS OF DECREASED GERM CELLS IN INFANTS WITH BILATERAL CRYPTORCHIDISM

You have accessJournal of UrologyPediatric Urology V (MP55)1 Sep 2021MP55-04 LOW SERUM INHIBIN B/FOLLICLE-STIMULATING HORMONE AND ANTI-MULLERIAN HORMONE/FOLLICLE-STIMULATING HORMONE RATIOS AS MARKERS OF DECREASED GERM CELLS IN INFANTS WITH BILATERAL CRYPTORCHIDISM Taiki Kato, Kentaro Mizuno, Hidenori Nishio, Akihiro Nakane, Satoshi Kurokawa, Hideyuki Kamisawa, Tetsuji Maruyama, Shoichiro Iwatsuki, Yukihiro Umemoto, Takahiro Yasui, and Yutaro Hayashi Taiki KatoTaiki Kato More articles by this author , Kentaro MizunoKentaro Mizuno More articles by this author , Hidenori NishioHidenori Nishio More articles by this author , Akihiro NakaneAkihiro Nakane More articles by this author , Satoshi KurokawaSatoshi Kurokawa More articles by this author , Hideyuki KamisawaHideyuki Kamisawa More articles by this author , Tetsuji MaruyamaTetsuji Maruyama More articles by this author , Shoichiro IwatsukiShoichiro Iwatsuki More articles by this author , Yukihiro UmemotoYukihiro Umemoto More articles by this author , Takahiro YasuiTakahiro Yasui More articles by this author , and Yutaro HayashiYutaro Hayashi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002085.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: In cryptorchid boys, germ cell development failure occurs during infancy. Thus, the establishment of serum markers for predicting infertility is warranted. Serum inhibin B may reflect the state of the germinative epithelium. In this retrospective single-center observational study, we assessed the correlations between testicular histopathology and serum hormone levels according to testicular position in cryptorchid boys. METHODS: A total of 145 prepubertal boys underwent orchidopexy between December 2014 and May 2019 at our university hospital. Of them, 122 had unilateral cryptorchidism and 23 had bilateral cryptorchidism. Testicular biopsy was performed at orchidopexy. We examined the serum levels of inhibin B, anti-Mullerian hormone (AMH), gonadotropins, and testosterone; histological parameters; and the mean number of germ cells per seminiferous tubule cross-section (G/T). The area under the receiver operating characteristic (ROC) curve (AUC) showed the efficiency of each marker in predicting impaired germ cell numbers (below the lower limit of 0.4 G/T for boys without cryptorchidism). RESULTS: The mean G/T and serum inhibin B levels were significantly lower in the bilateral than in the unilateral cryptorchidism group (0.20 vs. 0.65, p<0.001; 96 [range 46–197] vs. 125 [range 21–354] pg/mL, p=0.026) (Table 1). Seven testes (4.1%) showed no germ cells (five boys with unilateral cryptorchidism and two boys with bilateral cryptorchidism). Inhibin B/follicle-stimulating hormone (FSH) and AMH/FSH ratios were correlated with the mean G/T in bilateral cryptorchid boys who were ≤24 months old at orchidopexy (n=12; p=0.008 and p=0.019, respectively). ROC curves showed significant correlations between impaired G/T and inhibin B/FSH (p=0.019, AUC 0.847) and AMH/FSH (p=0.004, AUC 0.932) ratios. CONCLUSIONS: In bilateral cryptorchid boys with a maximal age of 24 months, low inhibin B/FSH and AMH/FSH ratios may predict high risks for later infertility. Additional treatment and follow-up may be needed for such cases. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e959-e959 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Taiki Kato More articles by this author Kentaro Mizuno More articles by this author Hidenori Nishio More articles by this author Akihiro Nakane More articles by this author Satoshi Kurokawa More articles by this author Hideyuki Kamisawa More articles by this author Tetsuji Maruyama More articles by this author Shoichiro Iwatsuki More articles by this author Yukihiro Umemoto More articles by this author Takahiro Yasui More articles by this author Yutaro Hayashi More articles by this author Expand All Advertisement Loading ...

Reproduction and reproductive tract morphology of male and female pigs whose mothers were heat stressed during the second month of gestation.

The primary objective was to assess the development of fetal gonads and measure the subsequent reproductive capacity of boars and gilts whose mother was either subjected to gestational heat stress (GHS) or thermoneutral (GTN; control) conditions during pregnancy. Gilts were subjected to either GHS (28 to 38 °C; 65% to 88% relative humidity [RH]; n = 30) or GTN (17 to 22 °C; 56% to 65% RH; n = 29) for the second month of gestation (a period that coincides with a critical window of gonadal development). A subset of GHS (n = 12) and GTN (n = 11) gilts was sacrificed immediately following treatment for the collection of pregnancy data. The remaining gilts (n = 18 GHS and n = 18 GTN) were allowed to farrow. Female offspring from the farrowed gilts were studied through puberty, first insemination, and early pregnancy when fetal tissues were again collected. During the treatment period, GHS gilts had greater (P < 0.001) rectal temperature and respiration rate at both measurement time points (morning and afternoon) compared with GTN gilts. When assessed at the end of the second month of gestation, the total number of viable fetuses did not differ (P > 0.10) for GHS vs. GTN. Likewise, the weight of the fetus, placenta, fetal testes, and fetal ovaries were similar (P > 0.10) for GHS and GTN pregnancies. There was a tendency for an effect of treatment (63.3 ± 2.3 vs. 70.1 ± 2.6; GHS vs. GTN; P < 0.073) on the number of oogonia per histological section in the fetal ovaries. There was no effect of treatment on the number of prespermatogonia per histological section in the fetal testis. For gilts farrowing after treatment, litter size, piglet birth weight, and weaning weight were similar (P > 0.10) for the GHS and GTN gilts. Testes collected from castrated GHS boars had fewer prespermatogonia per seminiferous tubule cross section (P < 0.049). Female offspring from the GHS (n = 30) or GTN (n = 37) sows reached puberty at a similar age, and their pregnancies (ninth week of gestation) had fewer corpora lutea (15.6 ± 0.5 vs. 17.1 ± 0.4; GHS vs. GTN; P < 0.038) but the number of fetuses was similar for GHS and GTN. In summary, compared with GTN, GHS during a critical window of gonadal development tended to reduce the number of oogonia in the fetal ovary, reduced the number of prespermatogonia in the neonatal testes, and reduced ovulation rate at first pregnancy in gilts.

Computerized spermatogenesis staging (CSS) of mouse testis sections via quantitative histomorphological analysis.

Spermatogenesis in mammals is a cyclic process of spermatogenic cell development in the seminiferous epithelium that can be subdivided into 12 subsequent stages. Histological staging analysis of testis sections, specifically of seminiferous tubule cross-sections, is the only effective method to evaluate the quality of the spermatogenic process and to determine developmental defects leading to infertility. Such staging analysis, however, is tedious and time-consuming, and it may take a long time to become proficient. We now have developed a Computerized Staging system of Spermatogenesis (CSS) for mouse testis sections through learning of an expert with decades of experience in mouse testis staging. The development of the CSS system comprised three major parts: 1) Developing computational image analysis models for mouse testis sections; 2) Automated classification of each seminiferous tubule cross-section into three stage groups: Early Stages (ES: stages I-V), Middle Stages (MS: stages VI-VIII), and Late Stages (LS: stages IV-XII); 3) Automated classification of MS into distinct stages VI, VII-mVIII, and late VIII based on newly developed histomorphological features. A cohort of 40 H&E stained normal mouse testis sections was built according to three modules where 28 cross-sections were leveraged for developing tubule region segmentation, spermatogenic cells types and multi-concentric-layers segmentation models. The rest of 12 testis cross-sections, approximately 2314 tubules whose stages were manually annotated by two expert testis histologists, served as the basis for developing the CSS system. The CSS system's accuracy of mean and standard deviation (MSD) in identifying ES, MS, and LS were 0.93±0.03, 0.94±0.11, and 0.89±0.05 and 0.85±0.12, 0.88±0.07, and 0.96±0.04 for one with 5 years of experience, respectively. The CSS system's accuracy of MSD in identifying stages VI, VII-mVIII, and late VIII are 0.74±0.03, 0.85±0.04, and 0.78±0.06 and 0.34±0.18, 0.78±0.16, and 0.44±0.25 for one with 5 years of experience, respectively. In terms of time it takes to collect these data, it takes on average 3 hours for a histologist and 1.87 hours for the CSS system to finish evaluating an entire testis section (computed with a PC (I7-6800k 4.0 GHzwith 32GB of RAM & 256G SSD) and a Titan 1080Ti GPU). Therefore, the CSS system is more accurate and faster compared to a human histologist in staging, and further optimization and development will not only lead to a complete staging of all 12 stages of mouse spermatogenesis but also could aid in the future diagnosis of human infertility. Moreover, the top-ranking histomorphological features identified by the CSS classifier are consistent with the primary features used by histologists in discriminating stages VI, VII-mVIII, and late VIII.

Effect of GnRH agonist deslorelin implant on spermatogenesis and testosterone concentration in Guinea pigs (Cavia aperea porcellus)

Guinea pigs are social animals that are often kept in groups regardless of their gender. Due to reproduction control and male aggressiveness prevention, surgical castration is commonly required. In the present study, we evaluated the effect of GnRH agonist implant (4.7 mg deslorelinum) on the serum testosterone concentration (T) and spermatogenesis in male guinea pigs. Twenty-four animals were divided into two groups. All animals in the first group were neutered (Group 1), animals in the second group (Group 2) were administered the implant subcutaneously and then neutered in one-month intervals. A histological examination was performed when cross sections of seminiferous tubules were assessed. Subsequently, these tubules were divided based on the most developed germ cell observed: spermatogonia, spermatocytes, round spermatids, elongating spermatids and elongated spermatids. The anticipated decrease in testosterone concentration and cessation of spermatogenesis was not achieved. Thus, the results obtained proved the inefficacy of the deslorelin implant in male guinea pigs so the alternative methods of contraception remain the methods of choice.

Low protein diet and methyl-donor supplements modify testicular physiology in mice.

The link between male diet and sperm quality has received significant investigation. However, the impact diet and dietary supplements have on the testicular environment has been examined to a lesser extent. Here, we establish the impact of a sub-optimal low protein diet (LPD) on testicular morphology, apoptosis and serum fatty acid profiles. Furthermore, we define whether supplementing a LPD with specific methyl donors abrogates any detrimental effects of the LPD. Male C57BL6 mice were fed either a control normal protein diet (NPD; 18% protein; n = 8), an isocaloric LPD (LPD; 9% protein; n = 8) or an LPD supplemented with methyl donors (MD-LPD; choline chloride, betaine, methionine, folic acid, vitamin B12; n = 8) for a minimum of 7 weeks. Analysis of male serum fatty acid profiles by gas chromatography revealed elevated levels of saturated fatty acids and lower levels of mono- and polyunsaturated fatty acids in MD-LPD males when compared to NPD and/or LPD males. Testes of LPD males displayed larger seminiferous tubule cross section area when compared to NPD and MD-LPD males, while MD-LPD tubules displayed a larger luminal area. Furthermore, TUNNEL staining revealed LPD males possessed a reduced number of tubules positive for apoptosis, while gene expression analysis showed MD-LPD testes displayed decreased expression of the pro-apoptotic genes Bax, Csap1 and Fas when compared to NPD males. Finally, testes from MD-LPD males displayed a reduced telomere length but increased telomerase activity. These data reveal the significance of sub-optimal nutrition for paternal metabolic and reproductive physiology.

Embryo production by intracytoplasmic injection of sperm retrieved from neonatal testicular tissue of Agu pigs after cryopreservation and grafting into nude mice.

The Agu is the only indigenous pig breed in Japan but its population is very small. In order to estimate the efficacy of testicular xenografting for the conservation of Agu pigs, we investigated whether neonatal testicular fragments would acquire the capacity to produce sperm after they had been cryopreserved and grafted into nude mice. Although on day 180 (day 0=xenografting), grafts showed a low proportion of seminiferous tubule cross-sections containing sperm (0.1±0.1%, mean±SEM for four mice), the proportion reached 36.9±16.7% (n=4 mice) by day 240. When single sperm obtained on day 240 was injected into individual porcine oocytes, 28.2% of the oocytes were found to contain one male and one female pronuclei with the second polar body. Moreover, the blastocyst formation rate after injection of the xenogeneic sperm was 28.4%, whereas that in the absence of sperm injection (attributable to parthenogenesis) was 13.3%. These findings suggest that more than half of the blastocysts resulted from fertilization. Thus, testicular xenografting could assist the conservation of Agu pigs by salvaging germ cells present in neonatal testes even after cryopreservation.

PSII-12 Comparison of fine needle aspiration and tissue sections to determine Sertoli and germ cell counts in prepubertal beef bulls

Abstract Daily sperm production in bulls is determined by the size of the testicular Sertoli cell (SC) population established before puberty. Fine needle aspiration (FNA) is currently used as a diagnostic tool, but its value to determine SC and germ cell (GC) counts remains unexplored. The objectives were to compare three sampling techniques [(smears produced via 22G fine needle aspiration (SMR) vs. tissue sections collected via 14G aspiration (FNS) vs. conventional tissue sections (HIS)] and two stains [immunohistochemistry with GATA4 (IHC) and HE] to determine SC density in prepubertal bulls. Fourteen age-matched Angus bulls were surgically castrated. Testicular parenchyma samples and smears were stained using anti-GATA4 as a specific SC marker and HE. For stain comparison, mirrored tissue cuts were produced allowing cell counts of complementary seminiferous tubule (ST) cross sections. All data were analyzed using Pearson’s product moment correlations and linear regression. Testicular weights were correlated with IHC SC density (P = 0.018). High correlations existed for SC and GC counts done on ST cross sections using IHC (P &amp;lt; .0001) and HE (P &amp;lt; .0001). Within HIS and FNS, high correlations (r = 0.955; P ≤ 0.0001) existed between SC and automated GATA4+ cell densities. However, when comparing IHC SC density across techniques, no significant correlations (P ≤ 0.587) existed. The high correlation observed between SC counts done on mirrored ST cross sections for HE and IHC underlines the value of GATA4 as a specific SC marker in prepubertal bulls. Furthermore, the high correlation established between SC and GATA4+ cell densities within IHC HIS and FNS collection methods infers SC density determination can be automated. However, lack of correlation for SC density among techniques indicates that aspiration and conventional methods lead to different results. Research supported by North Dakota EPSCoR Doctoral Dissertation Assistantship (DDA) program.

Fertility Potential is Compromised in 20% to 25% of Boys with Nonsyndromic Cryptorchidism despite Orchiopexy within the First Year of Life

One of the concerns surrounding cryptorchidism is the risk of impaired fertility. Current guidelines recommend orchiopexy at age 6 to 12 months to optimize fertility outcome. We evaluated the fertility potential of boys with nonsyndromic cryptorchidism who underwent orchiopexy within the recommended age range to clarify the need for eventual supplemental treatment modalities. We retrospectively evaluated mini-puberty hormones (follicle-stimulating hormone, luteinizing hormone and inhibin B) and testicular biopsies from boys with cryptorchidism who underwent orchiopexy within the first year of life between 2010 and 2019. We histologically analyzed germ cell number and type A dark spermatogonia number per seminiferous tubule cross-section in relation to normal values. Of the 333 boys with nonsyndromic cryptorchidism 83 (25%, 21% with bilateral cryptorchidism) had a reduced number of germ cells. A total of 70 boys (21%) had low serum inhibin B, of whom 32 (46%) had a decreased number of germ cells and 23 (33%) had a decreased number of type A dark spermatogonia (p <0.01). Overall, 75 boys (23%) had no type A dark spermatogonia present. Despite early and successful orchiopexy, 20% to 25% of boys with cryptorchidism may be at risk for infertility based on hormonal and histological data. Blood test and testicular biopsy are mandatory to identify boys at high risk for infertility, in whom additional treatment modalities and followup may be needed.

KIF11 as a Potential Marker of Spermatogenesis Within Mouse Seminiferous Tubule Cross-sections.

The arrangement of immature germ cells changes regularly and periodically along the axis of the seminiferous tubule, and is used to describe the progression of spermatogenesis. This description is based primarily on the changes in the acrosome and the nuclear morphology of haploid spermatids. However, such criteria cannot be applied under pathological conditions with arrested spermatid differentiation. In such settings, the changes associated with the differentiation of premeiotic germ cells must be analyzed. Here, we found that the unique bipolar motor protein, KIF11 (kinesin-5/Eg5), which functions in spindle formation during mitosis and meiosis in oocytes and early embryos, is expressed in premeiotic germ cells (spermatogonia and spermatocytes). Thus, we aimed to investigate whether KIF11 could be used to describe the progression of incomplete spermatogenesis. Interestingly, KIF11 expression was barely observed in haploid spermatids and Sertoli cells. The KIF11 staining allowed us to evaluate the progression of meiotic processes, by providing the time axis of spindle formation in both normal and spermatogenesis-arrested mutant mice. Accordingly, KIF11 has the potential to serve as an excellent marker to describe spermatogenesis, even in the absence of spermatid development.

Postnatal germ cell development in cryptorchid boys.

Cryptorchidism is associated with infertility in adulthood. Early orchiopexy is suggested to reduce the risk. Information is lacking on the potential link between infant germ cell maturation and the risk of future infertility. The objective of the study was to evaluate age-related germ cell development in cryptorchidism. Immunostaining for markers of germ cell development (octamer-binding transcription factor 3/4 [OCT3/4], placental alkaline phosphatase [PLAP], KIT proto-oncogene [C-KIT], podoplanin [D2-40], Lin-28 homolog A [LIN28], and G antigen 7 [GAGE-7]) was performed in testicular biopsies from 40 cryptorchid boys aged 4–35 months. Germ cell numbers and distributions were evaluated in cross sections of seminiferous tubules, with and without immunostaining. OCT3/4, D2-40, and LIN28 were generally expressed in the early stages of germ cell development, as shown by positive expression in germ cells in the central region of seminiferous tubules. In contrast, PLAP and GAGE-7 were expressed in both central and peripheral parts of the tubules in the early stages of development and expressed mainly in a peripheral position with advancing age. Germ cell maturation was delayed in this study population as compared with that observed in our previous study on germ cell markers in a healthy population. The number of GAGE-7-positive germ cells per tubular cross section obtained by immunostaining was significantly higher than that obtained by standard hematoxylin and eosin staining. Double immunostaining revealed heterogeneity in germ cell development in cryptorchid testes. These results shed light on the pathophysiology of germ cell development in boys with cryptorchidism.

Effect of Erythropoietin Administration on Spermatogonium Amount, Sertoli Cell and Leydig on Rats Testis (Wistar Strain) After Vas Deferens Ligation Released

The study aimed to determine the effect of Erythropoietin on spermatogonia number, Sertoli cells and Leydig cells in white rat Wistar strain testis after ligation vas deferens release. Twenty-four Wistar strain eats were grouped into four group. The control group only performed an orchiectomy for testicular examination, ligation group vas deferens, group performed release ligation of vas deferens and group performed release ligation of vas deferens and given EPO injection with dose of 1000 iu/kg BW intraperitoneally for a week (three times per week). The observation of spermatogonium, Sertoli cells and Leydig cells by determined amount on 5 cross sections of seminiferous tubules used 400x light magnificent microscope with Hematoxylin Eosin staining. The result showed ligation of vas deferens had significant decreased spermatogonia number and Sertoli cells (p 0.05). Meanwhile, release ligation of vas deferens had no significant different in spermatogonia, Sertoli cells and Leydig cells with ligation of vas deferens group. Besides, treatment ligation of vas deferens release and an EPO injection for a week also no significant different in number compared ligation release group of vas deferens.

Royal jelly protection on flunixin meglumine-induced spermiotoxicity and testicular degeneration in mice

Current study was designed to investigate the protective effects of royal jelly on Flunixin me- glumine (FM)-induced spermiotoxicity related to sperm concentration, abnormal spermatozoa count and histopathological changes in mice testis. The subjects were divided into five groups according to FM and/or royal jelly intake: Control group; group 1, FM alone (25 mg/kg, im); group 2, combination of FM (25 mg/kg, im) and royal jelly (200 mg/kg, oral); group 3, FM alone (50 mg/kg, im); and group 4, combination of FM (50 mg/kg, im) and royal jelly (200 mg/kg, oral). The animals were fed once daily for 15 days and they were sacrificed last day. Epididymal sperm concentration and abnormal spermatozoa count were noted. Testicular histological findings were evaluated. On purpose, organization of each animal was graded according to Johnsen's scoring to assess the spermatogenesis relying on seminiferous tubule cross-section scores. Comparing to controls, FM administration caused a decrease in sperm concentration (p⟨0.05), an increase in total abnormal spermatozoa rates (p⟨0.05) and more degenerative changes in testes in mice. Royal jelly supplementation ameliorated both sperm concentration and abnormal spermato- zoa (p⟨0.05) comparing to the control group. In conclusion, we suggested that royal jelly might have protective effects in the FM-induced reductions in epididymal sperm concentration and in- crease in abnormal spermatozoa rate.

Transient Hypothyroidism: Dual Effect on Adult-Type Leydig Cell and Sertoli Cell Development.

Transient neonatal 6-propyl-2-thiouracil (PTU) induced hypothyroidism affects Leydig and Sertoli cell numbers in the developing testis, resulting in increased adult testis size. The hypothyroid condition was thought to be responsible, an assumption questioned by studies showing that uninterrupted fetal/postnatal hypothyroidism did not affect adult testis size. Here, we investigated effects of transient hypothyroidism on Leydig and Sertoli cell development, employing a perinatal iodide-deficient diet in combination with sodium perchlorate. This hypothyroidism inducing diet was continued until days 1, 7, 14, or 28 postpartum (pp) respectively, when the rats were switched to a euthyroid diet and followed up to adulthood. Continuous euthyroid and hypothyroid, and neonatal PTU-treated rats switched to the euthyroid diet at 28 days pp, were included for comparison. No effects on formation of the adult-type Leydig cell population or on Sertoli cell proliferation and differentiation were observed when the diet switched at/or before day 14 pp. However, when the diet was discontinued at day 28 pp, Leydig cell development was delayed similarly to what was observed in chronic hypothyroid rats. Surprisingly, Sertoli cell proliferation was 6- to 8-fold increased 2 days after the diet switch and remained elevated the next days. In adulthood, Sertoli cell number per seminiferous tubule cross-section and consequently testis weight was increased in this group. These observations implicate that increased adult testis size in transiently hypothyroid rats is not caused by the hypothyroid condition per se, but originates from augmented Sertoli cell proliferation as a consequence of rapid normalization of thyroid hormone concentrations.

Inhibitory effect of &lt;i&gt;Taraxacum officinale&lt;/i&gt; L (Compositae) aqueous root extract on spermatogenesis

Purpose: To investigate if T. officinale root aqueous extract has anti-spermatogenic activity similar to that of the whole plant which was shown previously to inhibit spermatogenesis. Methods: T. officinale aqueous extract was prepared by soaking 100 g of dried materials in 1 L distilled water for two days at 45 oC. Fifty adult male rats were divided into five groups and treated for 60 days. Four groups were gavaged with the whole plant or root aqueous extract in low or high doses. The male rat rats were allowed to mate with female rats. The control group received distilled water. Sperm count, motility and morphology as well as chromatin integrity were evaluated. Results: Serum testosterone level, sperm parameters, pregnancy rate and average number of fetuses per pregnant females decreased significantly in the treated groups compared to control and in the rootreceiving rats compared to the whole plant-receiving rats. Female rats which were mated with high dose root-receiving males did not deliver fetuses. Cross sections of seminiferous tubules of T. officinale treated rats showed lesions and disorganized germinal epithelium. Late spermatogenesis maturation arrest (spermatid stage) was observed in all of the treated groups except the high dose root-receiving group which showed early maturation arrest (spermatocyte stage). In addition, the mRNA level of two spermatogonial stem cell markers responsible for self-renewal and proliferation of spermatogonia increased in high dose-receiving rats. Conclusion: T. officinale root aqueous extract has inhibitory effects on spermatogenesis. Further studies are required to identify specific ingredient(s) in T. officinale that may be useful as male contraceptive(s). Keywords: Taraxacum officinale , Dandelion, GDNF family receptor alpha 1, Macrophage Colony- Stimulating Factor, Promyelocytic Leukaemia Zinc-Finger, Testosterone, Sperm count

МОНГОЛ ҮҮЛДРИЙН АЗАРГАНЫ ТӨМСӨГНИЙ МОРФО-ГИСТОЛОГИЙН СУДАЛГААНЫ ЗАРИМ ҮР ДҮН

The present study aimed to investigate correlation between testicle and body weights, cross section of seminiferous tubule and relationship between the Sertoli cells and matured sperms in stallion of Mongolian breed. It was found that there is significant difference (p&lt;0.001) in testicle weights depending on ages of stallions (121.1 g at 36 months old age and 169.2 g at 48 months old). Changes in the body and testicle weights of stallions to be used for breeding are significantly different at two age classes. There were moderate (r=0.59) and strong (r=0.72) correlations at the ages of 36 and 48 months respectively. Both the testicle weight and diameter of seminiferous tubule increases in mutually related ways and tubule diameter was 72.6 µm and 94.8 µm at 36 and 48 months of stallions age respectively. Sertoli cells play key role in production of sperms from primordial germ cells in the testicle. Interrelationship between Sertol cells and mature sperms in seminiferous tubule is moderate (R2=0.316) and satisfactory (R²=0.445) correlations at 36 and 48 months of ages and it demonstrates differences between the ages are stable.