25 Cryopreservation of horse testicular tissue as a model for rhinoceros

2019 ◽  
Vol 31 (1) ◽  
pp. 138 ◽  
Author(s):  
M. C. Gómez ◽  
A. Alrashed ◽  
C.-Y. Su ◽  
B. Durrant
Keyword(s):  
Basement Membrane ◽  
Dimethyl Sulfoxide ◽  
Structural Integrity ◽  
Membrane Damage ◽  
Genetic Material ◽  
Testicular Tissue ◽  
Live Cells ◽  
Seminiferous Tubules ◽  
Positive Interaction ◽  
Cryoprotectant Solution

Cryopreservation of testicular tissue (TT) allows retention of valuable genetic material that can be used for conservation of endangered species, such as the northern white rhinoceros (NWR; Ceratotherium simum cottoni). Previously, we found that cryopreservation of NWR TT with a slow controlled cooling rate (CR) method induced morphological alterations in the seminiferous tubules (ST). However, the relative influence of CR, type of medium, and condition of TT from the aged NWR male on TT integrity was not clear. Due to the limited availability of rhinoceros TT, we used the horse as a model for optimization of TT cryopreservation. We evaluated the effect of (1) cryoprotectant solution [PBS (PBS +1.5M dimethyl sulfoxide) v. DMEM (DMEM/F12+10.0% fetal bovine serum+0.05M sucrose+1.5M dimethyl sulfoxide)] and (2) CR [CR1 (−2.0°C min−1 from 0°C to −4.0°C, −15°C min−1 to −12°C, and −0.3°C min−1 to −40°C in a programmable freezer) v. CR2 (same as CR1 but cooled to −8°C and held for 5min before cooling to −40°C) v. CR3 (−1.0°C min−1 from 0°C to −80°C in a CoolCell® freezing device; Corning, Corning, NY, USA)] on the structural integrity of ST from a 2-year-old horse (n=20 ST), cell viability, and expression of spermatogonial stem cells (SSC; GFRα1, and GRP125) and pluripotent markers (SSEA-4, SSEA-1, and OCT-4) in spermatogonial cells isolated from TT frozen with the above treatments (n=3). We found a positive interaction between CR and cryoprotectant solution on structural integrity of fixed and stained TT after freezing in PBS and CR2 that resulted in lower detachment of epithelium cells from the basement membrane (score±standard error of the mean; 0.50±0.1) than that of TT frozen in PBS and CR1 and CR3 (1.00±0.1 and 1.80±0.1, respectively; P<0.001) or in DMEM and CR1 (1.25±0.1), CR2 (1.35±0.1), and CR3 (1.40±0.1; P<0.01) and in lower incidence of basement membrane damage (0.75±0.1) than that of TT frozen in PBS and CR1 (1.17±0.07) and CR3 (1.16±0.07) or in DMEM and CR1 (1.10±0.1), CR2 (1.15±0.1), or CR3 (1.45±0.1; P<0.01). A lower rate of pyknosis was observed in TT frozen with PBS (1.15±0.06) than in TT frozen in DMEM (1.43±0.06; P<0.001). Overall, integrity of ST was improved when TT was frozen in PBS at CR2 having similar percentages of ST with intact epithelium (60%) and basement membrane (35%) as that of refrigerated TT (45 and 50%, respectively) but different from that of TT frozen with PBS at CR1 (10 and 15%, respectively; P<0.05). Flow cytometry analysis of spermatogonial cells revealed that the percentages of live cells from TT frozen in PBS (CR1: 61.5±7.4%; CR2: 59.7±4.8%; CR3: 51.5±4.1%) or DMEM (CR1: 66.2±6.0%; CR2: 59.8±6.0%; CR3: 58.9±6.9%), and expression of SSC and pluripotent markers was similar among all freezing treatments. However, the percentages of live cells from frozen-thawed TT were lower than those of cells isolated from refrigerated TT (80.6±2.2%; P<0.001). Overall, our results showed that (1) structural integrity of horse ST was better maintained when TT was frozen in PBS at CR2 and (2) SSC can be isolated from frozen-thawed TT with a similar relative frequency to that of refrigerated TT.

scholarly journals Formalin Versus Bouin Solution for Testis Biopsies: Which Is the Better Fixative?

2020 ◽  
Vol 13 ◽  
pp. 2632010X1989726 ◽  
Author(s):  
James L Ellenburg ◽  
Peter Kolettis ◽  
Joseph C Drwiega ◽  
Anna M Posey ◽  
Matthew Goldberg ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Nuclear Membrane ◽  
Cytoplasmic Membrane ◽  
Testicular Tissue ◽  
Seminiferous Tubules ◽  
High Quality ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Histologic Evaluation

Purpose: Some resources recommended Bouin solution for the fixation of testis biopsy specimens. We compared the histologic quality of rat testicular tissue using buffered formalin and Bouin solution as fixatives. Methods: We prospectively compared the histologic quality of rat testicular tissue fixed in Bouin solution versus formalin. Testicular tissue was harvested post-mortem from six rats. Each testis was removed and sectioned in half; one half was fixed in formalin and one half in Bouin solution. Testicular tissue histology (nuclear membrane detail, nuclear granularity, cytoplasmic granularity, cytoplasmic membrane detail, and basement membrane detail) was graded as high quality (2) or low quality (1). Sloughing of cells into the lumens of the seminiferous tubules was graded on a 0-3 scale (0=none, 1=mild, 2=moderate, 3=extensive). Results: All slides regardless of fixative were of appropriate quality for the histologic evaluation of spermatogenesis. The average sloughing score for formalin cases was 1.4 and for Bouin cases 1.6. Formalin fixed tissue was found to have high quality nuclear membrane detail (2), nuclear granularity (1.9), and basement membrane detail (2). Cytoplasmic granularity was of lesser but adequate quality (1.4). Cytoplasmic membrane detail was poor, (1). Tissue fixed with Bouin solution had high quality basement membrane detail (2) and adequate cytoplasmic granularity (1.5), nuclear membrane detail (1.3) and nuclear granularity (1.4). Cytoplasmic membrane detail was poor (1). Conclusion: Compared to Bouin solution, formalin fixation of rat testicular tissue produced adequate histology for the evaluation of spermatogenesis and may be superior to Bouin solution for certain cytologic features.

187 Morphological Appearance and Expression of Spermatogonial Stem Cell Markers in White Rhinoceros Testicular Tissue

2018 ◽  
Vol 30 (1) ◽  
pp. 233
Author(s):  
M. C. Gomez ◽  
Y. Cates ◽  
D. B. Stansfield ◽  
C. Young ◽  
R. Klee ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Mammalian Species ◽  
Testicular Tissue ◽  
Seminiferous Tubules ◽  
Stem Cell Markers ◽  
Tissue Preparation ◽  
Fresh Tissue ◽  
Morphological Alterations ◽  
White Rhinoceros

Spermatogonial stem cells (SSC) have been isolated from testicular tissue (TT) of several mammalian species and differentiated into mature spermatozoa following transplantation or in vitro culture. The northern white rhinoceros (NWR; Ceratotherium simum cottoni) is critically endangered. Thus, frozen NWR TT, cryopreserved and stored at the San Diego Zoo’s Frozen Zoo®, potentially contain SSC that could be a source of spermatozoa. The method used for cryopreserving TT may affect the integrity and number of SSC. Therefore, identifying alterations in the seminiferous tubules (ST) of frozen-ndash;thawed-NWR TT will provide insight into the condition of the SSC. Therefore, our aims were to (1) determine the effect of freezing rhinoceros TT on the structure of epithelium, and (2) identify SSC (GFRα1, GPR125) and pluripotent (SSEA-4 and Oct-4) markers. Testicular tissue of an adult NWR and a stillborn southern white rhinoceros (SWR) were frozen by equilibration of TT for 30 min at 4.0°C in PBS and 1.5 M dimethyl sulfoxide (DMSO), cooled at 2.0°C/min to −4.0°C, 0.3°C/min to −40°C, and plunged into liquid nitrogen. Tissues were thawed at 37°C in a water bath and DMSO removed in a 4-step dilution. Tissue was then fixed, dehydrated, and paraffin embedded. For morphological evaluations, frozen-ndash;thawed tissue was sectioned and stained with hematoxylin and eosin (H&E). The TT from both rhinoceros collected immediately after death (fresh) and stained with H&E were used as a control for cryopreservation. Localization of SSC and pluripotent markers in ST of frozen-ndash;thawed TT was detected by immunohistochemistry. Morphologically, fresh-NWR TT was severely altered, displaying large epithelium gaps and partial (62.2%) or total detachment (37.7%) from, and slight damage (35.5%) to, the basement membrane. The number of pyknotic nuclei per ST was moderate (15.6 ± 7.2%). Many of these changes could have resulted from autolysis and handling before tissue preparation. In contrast, histological appearance of fresh-SWR was good, with 98.3% of the tubules intact, and a small proportion of pyknotic cells (0.8 ± 1.5%). Seminferous tubule (n = 30/male) length and width (μm; ± SEM) differed between NWR (635.2 ± 34.4 × 214.6 ± 10.8) and SWR (277.7 ± 13.8 × 73.2 ± 2.4; P < 0.05). Damages after cryopreservation compared with fresh tissue comprised (1) epithelium detachment, NWR = 100% (P < 0.0001), and SWR = 43.3% (P < 0.001); (2) basement membrane alteration, only in NWR (93.0%; P < 0.001); and (3) decreased length and width in the ST, NWR = 409.4 ± 18.1 × 173.4 ± 8.2 (P < 0.05), and SWR = 195.2 ± 8.3 × 61.6 ± 2.8 (P < 0.05), with loss of lumen in both males. Immunohistochemistry revealed that NWR expressed GFRα1 and GPR125 at various stages of spermatogenqaesis, whereas Oct-4 was detected in few cells. In contrast to NWR, Oct-4 expression in SWR was located at the basement membrane; SSEA-4 was not detected in either male. In conclusion, freezing-induced morphological alterations in rhinoceros ST and positive expression of markers for SSC and pluripotency suggest the presence of SSC. Further studies are required to evaluate the viability of rhinoceros SSC.

scholarly journals The Expression Levels and Cellular Localization of Pigment Epithelium Derived Factor (PEDF) in Mouse Testis: Its Possible Involvement in the Differentiation of Spermatogonial Cells

2021 ◽  
Vol 22 (3) ◽  
pp. 1147
Author(s):  
Noy Bagdadi ◽  
Alaa Sawaied ◽  
Ali AbuMadighem ◽  
Eitan Lunenfeld ◽  
Mahmoud Huleihel
Keyword(s):  
Cellular Localization ◽  
Pigment Epithelium ◽  
Testicular Tissue ◽  
Seminiferous Tubules ◽  
Target Cells ◽  
Isolated Cells ◽  
Expression Levels ◽  
Cellular Origin ◽  
Pigment Epithelium Derived Factor

Pigment epithelium derived factor (PEDF) is a multifunctional secretory soluble glycoprotein that belongs to the serine protease inhibitor (serpin) family. It was reported to have neurotrophic, anti-angiogenic and anti-tumorigenic activity. Recently, PEDF was found in testicular peritubular cells and it was assumed to be involved in the avascular nature of seminiferous tubules. The aim of this study was to determine the cellular origin, expression levels and target cells of PEDF in testicular tissue of immature and adult mice under physiological conditions, and to explore its possible role in the process of spermatogenesis in vitro. Using immunofluorescence staining, we showed that PEDF was localized in spermatogenic cells at different stages of development as well as in the somatic cells of the testis. Its protein levels in testicular homogenates and Sertoli cells supernatant showed a significant decrease with age. PEDF receptor (PEDF-R) was localized within the seminiferous tubule cells and in the interstitial cells compartment. Its RNA expression levels showed an increase with age until 8 weeks followed by a decrease. RNA levels of PEDF-R showed the opposite trend of the protein. Addition of PEDF to cultures of isolated cells from the seminiferous tubules did not changed their proliferation rate, however, a significant increase was observed in number of meiotic/post meiotic cells at 1000 ng/mL of PEDF; indicating an in vitro differentiation effect. This study may suggest a role for PEDF in the process of spermatogenesis.

An electron microscope study of kidney basement membrane changes in the mouse by lipoteichoic acid from Streptococcus pyogenes

1987 ◽  
Vol 33 (8) ◽  
pp. 709-717 ◽  
Author(s):  
Ofra Leon ◽  
Charles Panos
Keyword(s):  
Basement Membrane ◽  
Streptococcus Pyogenes ◽  
Membrane Damage ◽  
Lipoteichoic Acid ◽  
Poststreptococcal Glomerulonephritis ◽  
Membrane Morphology ◽  
Proximal Tubular ◽  
Weight Analysis ◽  
Membrane Changes ◽  
Almost All

Mice injected repeatedly, intraperitoneally or intravenously, for approximately 1 month with a total of 1.04 mg lipoteichoic acid from a nephritogenic strain of Streptococcus pyogenes lost weight. Analysis by electron microscopy revealed that they also exhibited extensive kidney changes in basement membrane morphology which resembled, in part, those observed in human poststreptococcal glomerulonephritis. For example, the glomerular basement membrane became electron dense and exhibited at least a twofold increase in width sporadically within the same preparation after exposure to lipoteichoic acid. Also, whereas appreciable loss or reduction in epithelial foot processes as a result of fusion was clearly evident, epithelial slits and slit membranes or diaphragms between normal foot processes were not selectively affected. In addition, another mostly thickened, highly coiled or serpentinelike basement membrane with amorphous nodules appeared in these preparations. This type membrane was not observed surrounding the capillary lumina and was the most pronounced abnormality apparent in almost all preparations from mice exposed to lipoteichoic acid. Likewise, the proximal tubular basement membrane became variable in width and increased in electron density in mice given lipoteichoic acid as compared with controls. In addition, this membrane was often punctuated with various morphological protrusions originating from only its thickened areas and which extended away from, and not into, the capillary space. This change was only associated with the basement membrane of the proximal tubular capillaries. All membrane changes persisted but gradually subsided, with normal kidney membrane morphology reappearing on the 4th day following the last injection of lipoteichoic acid. The use of mice and minute amounts of lipoteichoic acid to study membrane damage or change in the fine structure of the glomerular or proximal tubular areas at the immediate presymptomatic, and very early symptomatic, phase of streptococcal glomerulonephritis is noted.

Consequences of Microwave oven Exposed Diet on The Basal Lamina of Seminiferous Tubules of Mice

2021 ◽  
Vol 15 (8) ◽  
pp. 2141-2144
Author(s):  
Kishwar Naheed ◽  
Muhammad Saad Abdullah ◽  
Maria Yousaf ◽  
Humaira Ali ◽  
Fareeha Mushtaq ◽  
...  
Keyword(s):  
Basal Lamina ◽  
Treated Group ◽  
Testicular Tissue ◽  
Microwave Oven ◽  
Mentha Piperita ◽  
Seminiferous Tubules ◽  
Control Group ◽  
Protective Effects ◽  
Trial Methodology ◽  
Experimental Group

Usage of electronic gadgets like microwave oven is increasing day by day that heats the food by exposing it to electromagnetic radiations which has many hazardous effects on human health including fertility. Aim: To find the effects of microwave oven exposed diet on basal lamina of seminiferous tubules of mice alongwith protective effects of Mentha piperita and melatonin on the same tissue. Study Design: Randomized control trial. Methodology: Adult male mice (n=32) were divided into four groups. Control group (G1) received standard pellets prepared for mice. Second group (G2) was given mice pellets exposed to microwave oven. Third group (G3) received Mentha Piperita leaf extract along with mice pellets exposed to microwave oven and the fourth group (G4) received oral melatonin along with pellets exposed to microwave oven. Later their testicular tissue was removed for histological examination while basal lamina disruption was assessed by scoring. Data analyzed by SPSS 22.0v. Results: In group G2, there was slight disruption in the basal lamina in 75% of the cases while in experimental group G3, there was slight disruption of basal lamina only in 12.5% of the cases. However, in group G4, only 25% specimen had slight disruption of basal lamina Conclusion: It was concluded that microwave oven exposed diet produced severe disruption of basal lamina in group G2 that decreased in Mentha piperita and melatonin treated groups. However, Mentha piperita treated group produced better results than melatonin treated group. Keywords: Mice, Testis, Basal Lamina, Mentha piperita and Melatonin

Anionin sites of the basement membrane of rat seminiferous tubules during ontogenesis

1988 ◽  
Vol 63 (1) ◽  
pp. 83-87
Author(s):  
Bruno P. Leheup ◽  
Jean-Louis Gelly ◽  
Jean-Luc Delongeas ◽  
Georges Grignon
Keyword(s):  
Basement Membrane ◽  
Seminiferous Tubules

scholarly journals The Effects of Vasectomy on Testicular Tissue of Mice: Histological Changes and DNA Fragmentation Study

2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Salman MO ◽  
Al-Wasiti EA ◽  
Thamir KA ◽  
Al-Ani IM ◽  
Al-Salihi AR
Keyword(s):  
Single Cell ◽  
Dna Fragmentation ◽  
Gel Electrophoresis ◽  
Testicular Tissue ◽  
Seminiferous Tubules ◽  
Control Group ◽  
Sham Operation ◽  
Male Mice ◽  
Histological Changes ◽  
Single Cell Gel Electrophoresis

Introduction: We aim to investigate the effect of vasectomy on the histology of the testis as well as to evaluate DNA fragmentation in testicular tissue of male mice. Methods: Bilateral vasectomy was performed on 20 mature male mice; 10 control mice underwent sham-operation. After 6 weeks, the testes were evaluated for histological changes and DNA fragmentation by single cell gel electrophoresis (comet assay). Results: Marked alterations were observed in the testes of vasectomized mice, including degeneration of spermatids, thickened basement membrane, dilatation of the seminiferous tubules, exfoliation of germ cells, reduction in the seminiferous cell population, vacuolated appearance of the epithelium in the tubules and marked interstitial fibrosis. Single cell gel electrophoresis showed a highly significant (P<0.0001) increase in DNA damage among vasectomized mice (46.02%) compared with control group (%27.17) after six weeks of operation. Conclusion: Vasectomy induced deterioration in the seminiferous tubules associated with increased testicular cell’s DNA fragmentation.

scholarly journals Meiosis, spermatogenesis and ultrastructure of the basement membrane of seminiferous tubules in patients with azoospermia

2019 ◽  
Vol 20 (1) ◽  
pp. 43-54
Author(s):  
E. E. Bragina ◽  
I. I. Vityazeva ◽  
M. A. Lelekova ◽  
A. A. Kashintsova ◽  
S. V. Bogolyubov ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Seminiferous Tubules
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