137 Recovery of quail spermatogenesis by donor spermatogonia transplantation

2020 ◽  
Vol 32 (2) ◽  
pp. 195
Author(s):  
A. N. Vetokh ◽  
E. K. Tomgorova ◽  
L. A. Volkova ◽  
N. A. Volkova ◽  
N. A. Zinovieva
Keyword(s):  
Germ Cells ◽  
Recombinant Dna ◽  
Seminiferous Tubules ◽  
Spermatogenic Cells ◽  
Optimal Parameters ◽  
Histological Studies ◽  
Sperm Dna ◽  
Donor Cells ◽  
Reported Study ◽  
Series Of Experiments

Spermatogonia, the stem cell precursors of male germ cells, are used as convenient biological material for the preservation of genetic resources (cryobanks) and the introduction of recombinant DNA (transgenesis). Donor spermatogonia subsequently differentiate into mature germ cells (spermatozoa), which are used to produce offspring. Our laboratory is investigating methods to improve the efficiency of spermatogonial germ cell transplantation in quail. The objectives of this study were to (1) determine the optimal age for spermatogonia isolation from the testes of donor male quail and (2) identify the most appropriate concentration of busulfan for treatment of recipient quail testes. Statistical analysis was performed using SPSS ver. 15.0 (IBM Corp.; analysis of variance test). In order to determine the optimal age for spermatogonia isolation, testes from male quail at 1-4 weeks of age were isolated and histological studies were performed on a population of spermatogenic cells in the seminiferous tubules. Histological studies of quail testes isolated at different ages showed that the optimal age for obtaining a culture of spermatogonia is a period from 1-2 weeks of age. During this period, spermatogenic cells were represented mainly by spermatogonia (P<0.01). Therefore, testes of 1-week-old quails were used to obtain a culture of spermatogonia. The resulting cell culture consisted mainly of spermatogonia (85%) with a small number of Sertoli cells. Next, a series of experiments introducing busulfan into quail testes was carried out using concentrations from 10-150mg kg−1 of liveweight. Experiments showed that an effective dose to remove the recipient male's own spermatogenic cells was a concentration of 100mg kg−1 of liveweight (P<0.05). Finally, using the optimal parameters described above, spermatogonia cultures were obtained and introduced into the testes of quail recipients (n=6), following administration of busulfan for 2-3 weeks before donor spermatogonia were introduced. The effectiveness of spermatogenesis recovery was assessed based on the analysis of sperm from quail recipients at 3 months after the injection of donor cells. The presence of donor germ cells in the testes of quail recipient drakes was confirmed by microsatellite analysis of DNA isolated from the blood and sperm of recipients as well as the donor cells (spermatogonia). The microsatellite profiles of the blood and sperm DNA in quail recipient males were different, which confirms the restoration of spermatogenesis in the studied recipients due to the development of donor germ cells. The reported study was funded by RFBR, project number 18-29-07079.

201 Spermatogonia Transplantation in the Chicken

2018 ◽  
Vol 30 (1) ◽  
pp. 241
Author(s):  
A. N. Vetokh ◽  
N. A. Volkova ◽  
T. O. Kotova ◽  
E. N. Antonova ◽  
A. V. Dotsev ◽  
...  
Keyword(s):  
Cell Population ◽  
Sertoli Cells ◽  
Recombinant Dna ◽  
Genetic Material ◽  
Live Weight ◽  
Seminiferous Tubules ◽  
Control Group ◽  
Spermatogenic Cells ◽  
Donor Cells ◽  
Male Chicken

Spermatogonia are the precursors of male germ cells. They are a valuable genetic material for the production of transgenic poultry. This technology includes isolation of the spermatogonia from male donor’s testes, transformation, and transplantation of donor cells into the sterilized recipient’s testes. The transplanted spermatogonia subsequently differentiate into male sex cells (sperm). The aim of this study was to optimize the individual stages of donor spermatogonia transplantation into the recipient’s testes to increase the effectiveness of spermatogenesis recovery. In the first stage, the spermatogenesis in male chicken was examined to determine the optimal age for isolation of spermatogonia from testes. Histological examinations of male chicken testes (n = 80 birds) were done for 8 age categories, from 1 week to 3 months. It was found that under the age of 4 weeks, the cell population in the seminiferous tubules of male chickens was represented mainly by Sertoli cells and spermatogonia. Maximum percentage of spermatogonia was 69 ± 3% at 4 weeks. At the next stage, a culture of spermatogonia was obtained. Testes of 3-week-old male chickens were used. Separation of the spermatogonia from other types of cells was based on a differential adhesive capacity. The maximum homogeneity of the cell population was established by transfer (3 times) of the supernatant containing unattached cells after 24 h of cultivation into a new culture dish for further cultivation. The cell population is represented mainly by the spermatogonia (89 ± 3%). The lentiviral transduction (pHAGE vector, ZsGreen under CMV promotor) was used to transform the resulting culture of the spermatogonia. The efficiency of spermatogonia infection with lentiviral particles (TU/mL = 2.5 × 108) was 65 ± 2%. After transformation, spermatogonia were introduced into the testes of busulfan-sterilized recipients. The optimal concentration of busulfan treatment after series of experiments from 40 to 100 mg/kg was determined. The effective dose for the removal of own spermatogenic cells was revealed at a concentration of 80 mg/kg of live weight. With complete elimination of other types of spermatogenic cells, the number of Sertoli cells and spermatogonia in the testicle tubules decreased by 39 ± 2% and 98 ± 1%, respectively, compared with the control group. The efficiency of spermatogenesis recovery was assessed based on sperm analysis that was obtained from male recipients (n = 5 birds) 4 months after the introduction of donor cells using PCR. The presence of recombinant DNA (ZsGreen) in recipients’ sperm was shown. Thus, our results indicate the prospect of using spermatogonia as a genetic material for the production of transgenic poultry. Study was supported by the Russian Science Foundation (Project no.16-16-10059).

250 ALL REGIONS OF THE MOUSE EPIDIDYMIS ARE ABLE TO PHAGOCYTIZE IMMATURE SPERMATOGENIC CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 272
Author(s):  
P. Ramos-Ibeas ◽  
E. Pericuesta ◽  
R. Fernandez-Gonzalez ◽  
M. A. Ramirez ◽  
A. Gutierrez-Adan
Keyword(s):  
Quality Control ◽  
Green Fluorescent Protein ◽  
Germ Cells ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Sperm Quality ◽  
Seminiferous Tubules ◽  
Spermatogenic Cells ◽  
Green Fluorescent ◽  
Immature Cells

Successful mammalian fertilization requires gametes with an intact structure and functionality. Although it is well known that epididymal functions are sperm maturation, sustenance, transport, and storage, there is controversial information about its role in sperm quality control, and it has been suggested that some regions of the rat epididymis are able to phagocytize germ cells. Our objective was to analyse whether different segments of the mouse epididymal epithelium act as a selection barrier for abnormal spermatogenic cells by removing immature cells from the lumen by phagocytosis. To detect the presence of immature germ cells along the epididymis, transgenic mice expressing enhanced green fluorescent protein under a Deleted in Azoospermia-Like (mDazl) promoter were generated. The transgenic animals express specifically enhanced green fluorescent protein in spermatogonias, spermatocytes, and spermatids; thus, immature spermatogenic cells can be easily identified by fluorescence microscopy. Colchicine, a microtubule disruptor that leads to severe alterations in the architecture of the seminiferous tubules, was administered in the rete testis to induce the release of immature germ cells into the epididymis. Mice were killed daily, from Day 1 to 8 post-administration, and epididymides were collected and observed under a fluorescence stereoscope to determine the transit of immature germ cells along the epididymis. Epididymides from control mice without colchicine administration were also collected. Fluorescent immature germ cells were present in the caput epididymis 24 h after colchicine administration, and they progressed through the corpus and cauda, leaving the epididymis 7 days after colchicine administration. After fluorescence observation, epididymides were fixed, sectioned, and stained with hematoxylin solution. Immature germ cells and phagosomes were not observed in control epididymides. By contrast, the presence of phagosomes in the principal cells of the epididymal epithelium containing immature germ cells in different degrees of degradation was observed by light microscopy in mice injected with colchicine. Phagocytosis was observed along the epididymis following the main wave of fluorescent immature cells. Thus, when immature cells had reached the corpus epididymis, phagocytosis was detected in several segments of the caput epididymis. Later, once the immature cells had arrived to the cauda epididymis or had abandoned the epididymis, phagocytosis was observed in the corpus and cauda epididymis. The presence of phagosomes was observed in all epididymal tubules within a phagocytosis area. In conclusion, we demonstrated that the epididymal epithelium is engaged in sperm quality control by clearing immature germ cells after a massive shedding into the epididymal lumen, and that this phenomenon is not restricted to a specific segment of the epididymis.

142 Isolation of the Quail Spermatogonia

2018 ◽  
Vol 30 (1) ◽  
pp. 211
Author(s):  
E. R. Mennibaeva ◽  
N. A. Volkova ◽  
E. K. Tomgorova ◽  
L. A. Volkova ◽  
V. A. Bagirov ◽  
...  
Keyword(s):  
Growth Factor ◽  
Sertoli Cells ◽  
Recombinant Dna ◽  
Initial Study ◽  
Seminiferous Tubules ◽  
Target Cells ◽  
Spermatogenic Cells ◽  
Russian Science ◽  
Epidermal Growth ◽  
The Individual

Spermatogonia are testicular stem cells, the precursors of male sex cells. They are target cells for introduction of recombinant DNA and suitable for creation of cryobanks to preserve biological materials. The aim of our research was to optimize the individual stages culturing quail spermatogonia. In an initial study, dynamics of change in the composition of spermatogenic cells in the seminiferous tubules were assessed histologically, at weekly intervals from 1 week to 1.5 months of age. Thereafter, spermatogonia were isolated from quail testes. Disaggregation of the testis tissue was carried out by consecutive enzymatic treatment in 0.25% trypsin and 0.1% collagenase solution. Purification of spermatogonia from other types of spermatogenic cells was conducted by separation of the cells by adhesion. The duration and conditions of cultivation of spermatogenic cells were selected experimentally. Cultivation of spermatogonia was performed on feeder layers, including quail primary Sertoli cells, STO cell line, and transplanted porcine Sertoli cells. Growth medium for culturing spermatogonia was DMEM HG medium supplemented with 5% FCS, 2 mM α-glutamine, MEM (10 μL mL−1), antibiotic (100×), insulin-transferrin-selenium (ITS, 10 μL mL−1), 2-mercaptoethanol (5 × 10−5 M), albumin (5 mg mL−1), epidermal growth factor (EGF, 20 ng mL−1), basic fibroblast growth factor (bFGF, 10 ng mL−1), and leukemia inhibitory factor (LIF, 2 ng mL−1). For identification of spermatogonia colonies, SSEA-1 antibodies were used. The maximum number of spermatogonia in seminiferous tubules of quail occurred at 3 weeks of age; there were mainly spermatogonia and Sertoli cells at this time. The percentage of spermatogonia from the total number of spermatogenic cells in the seminiferous tubule reached 76 ± 2%. In view of this, spermatogonia were isolated from the testes of 2-week-old quail. Spermatogenic cells were cultured for 24 h, after which the supernatant with unattached cells, mainly spermatogonia, was transferred to a new dish and cultured. Maximum homogeneity of the cell population was detected by dividing the cells by 3-fold transfer of the cell supernatant at an interval of 24 h; the proportion of spermatogonia in the suspension reached 88%. Quail Sertoli cells were the optimal feeder layer for cultivation of quail spermatogonia. Formation of spermatogonia colonies was observed on Day 5 to 7 of cultures, and their identity confirmed by immunohistochemical staining for SSEA-1. The study was supported by the Russian Science Foundation within Project no.16-16-04104.

143 The Dynamics of Spermatogenesis in Guinea Fowls

2018 ◽  
Vol 30 (1) ◽  
pp. 211
Author(s):  
N. A. Volkova ◽  
A. N. Vetokh ◽  
I. P. Novgorodova ◽  
A. V. Dotsev ◽  
N. A. Zinovieva
Keyword(s):  
Germ Cells ◽  
Sertoli Cells ◽  
Genetic Material ◽  
Guinea Fowl ◽  
Seminiferous Tubules ◽  
Spermatogenic Cells ◽  
Russian Science ◽  
Effective Selection ◽  
Generative Cells ◽  
Selection Of

Male gonads are valuable genetic material for creation of biomaterial cryobanks to preserve the genes of various animals, including poultry. Spermatogonia, which are stem cells of the testes, are of greatest interest. For effective selection of spermatogenic cells, including spermatogonia, it is necessary to know the specific features of spermatogenesis of the species of interest. In this regard, the aim of this study was to investigate the dynamics of spermatogenesis in guinea fowl. Histological examinations of guinea fowl testes (n = 90 birds) were done for 9 age categories, from 2 wk to 6 months. For each individual, at least 30 seminiferous tubules were examined. Seminiferous tubule diameters and numbers and types of spermatogenic cells (based on morphology) were determined. Overall, the histologic structure of guinea fowl testes was similar to that of mammals. Cell populations of the seminiferous tubules included Sertoli cells and generative cells, including spermatogonia, spermatocytes, spermatids, and sperm, at various stages of differentiation. Diameter of seminiferous tubules was (mean ± SEM) 36 ± 1, 58 ± 1, 64 ± 1, 65 ± 1, 110 ± 3, 178 ± 4, 233 ± 4, 274 ± 6, and 295 ± 5 µm at 2 wk, 1, 1.5, 2, 2.5, 3, 4, 5, and 6 months, respectively. Furthermore, at those ages, the number of spermatogenic cells per tubule was 18 ± 1, 20 ± 1, 29 ± 2, 30 ± 2, 68 ± 5, 114 ± 8, 186 ± 10, 400 ± 20, and 447 ± 24. Maximum percentage of spermatogonia was 72 ± 2% at 6 wk. Primary and secondary spermatocytes were first observed at 10 and 12 wk of age, respectively, whereas spermatids were first apparent at 4 months. Sperm were first identified at 5 months, with more present at 6 months. We concluded that the optimal age for retrieving testicular germ cells in guinea fowl was no later than 8 wk, as that represented the age when seminiferous tubules were dominated by spermatogonia. The study was supported by the Russian Science Foundation (Project no.16-16-04104).

scholarly journals PSII-36 The application of busulfan to inhibit the spermatogenesis in quail testis

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 373-373
Author(s):  
Tatyana Kotova ◽  
Anastasia N Vetokh ◽  
Ludmila A Volkova ◽  
Natalia Volkova ◽  
Natalia A Zinovieva
Keyword(s):  
Stem Cells ◽  
Body Weight ◽  
Germ Cells ◽  
Genetic Modification ◽  
Sertoli Cells ◽  
Seminiferous Tubules ◽  
Multiple Injection ◽  
Spermatogenic Cells ◽  
Methodological Approaches ◽  
Spermatogonia Cells

Abstract The use of testicular stem cells (spermatogonia) is of most interest for obtaining individuals with predetermined traits and genome genetic modification and for conservation of poultry gene pool. A significant population of mature donor germ cells (sperm) is formed upon successful spermatogonia cells transplantation into the testes of male recipients. Obtained sperm can be used to produce offspring with the desired traits. A key step in this technology is the removal of own spermatogenic cells (inhibition of spermatogenesis) in male recipients. The aim of research was to develop and optimize methodological approaches to inhibit the spermatogenesis in quail using busulfan. This drug was injected directly into the testes parenchyma of mature males by multiple injection at the concentration from 20 to 100 mg per 1kg of body weight (n = 25). Histological preparations of testes from the experimental quails were obtained to study composition of spermatogenic cells in the seminiferous tubules after busulfan administration. The male peers who were not injected with busulfan were used as a control. Experimental quails showed a decrease in the number of spermatogenic cells in the seminiferous tubules 32, 75, 111, 119 and 118 times compared with the control when using busulfan in concentrations 20, 40, 60, 80 and 100 mg/kg of weight, respectively (P < 0.001). The cells composition in the seminiferous tubules from experimental quails was represented mainly by Sertoli cells and spermatogonia. After busulfan introduction at the concentrations 20, 40, 60, 80 and 100 mg/kg, the percentage of spermatogonia was 55±5 %, 24±4 %, 6±2 %, 5±2 % and 4±1 %, respectively. The use of busulfan at the concentration of 80–100 mg/kg led to high mortality of quails. Thus, it was found that the optimal busulfan concentration for elimination of quail spermatogenic cells was 60 mg/kg. Supported by RFBR within Project №18-29-07079.

scholarly journals PSII-35 The age-related characteristics of spermatogenesis in gander

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 372-373
Author(s):  
Anastasia N Vetokh ◽  
Ludmila A Volkova ◽  
Baylar S Iolchiev ◽  
Natalia Volkova
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Seminiferous Tubule ◽  
Seminiferous Tubules ◽  
Spermatogenic Cells ◽  
Age Related ◽  
Effective Selection ◽  
The Creation ◽  
Spermatogonia Cells

Abstract Cryopreservation of testicular stem cells - spermatogonia is of interest along with the creation of semen cryobanks. During transplantation into recipients’ testes, spermatogenic cells can create a significant population of germ cells in the process of differentiation. The knowledge about spermatogenesis course in males is necessary for the effective selection spermatogenic cells. The research aim was to study the age-related characteristics of spermatogenesis in geese. The histostructure of gander testes (n = 35) at the age of 1 to 7 months was studied. The diameter of seminiferous tubules, and the types and number of spermatogenic cells in them were evaluated. From each gander at least 30 seminiferous tubules were examined. At the age of 1 month, the diameter of the seminiferous tubules was 51±1 μm. In subsequent age periods, this indicator increased and amounted to 63±2, 65±3, 66±2, 79±3, 98±6 and 170±5 μm at the age of 2, 3, 4, 5, 6 and 7 months, respectively. Diameter increase with the age was associated with an increase of spermatogenic cells number inside tubules. At the 1 months age, the number of spermatogenic cells in one seminiferous tubule did not exceed 22±1. At the age of 2, 3, 4, 5, 6, and 7 months, this indicator increased by 2.1, 2.7, 3.1, 6.4, 8.5 and 21.2 times. At the age from 1 to 3 months, the main cells types were Sertoli and spermatogonia cells. Primary and secondary spermatocytes from 4 months of age and spermatids from 5 months of age were visualized in the seminiferous tubules. Sperm were detected in the seminiferous tubules at 6 months old, the number of which increased towards the age of 7 months. The study was supported by RSF within Project №16–16–04104.

scholarly journals The efficiency of use lentiviral vectors for the transformation of rooster spermatogenic cells in vivo

2019 ◽  
Vol 14 (2) ◽  
pp. 162-169
Author(s):  
Natalya Alexandrovna Volkova ◽  
Anastasia Nikolaevna Vetokh ◽  
Lyudmila Aleksandrovna Volkova ◽  
Anatolievna Zinovyeva Nataliya
Keyword(s):  
Genetic Transformation ◽  
Reporter Gene ◽  
Germ Cells ◽  
Lentiviral Vectors ◽  
Seminiferous Tubules ◽  
Target Cells ◽  
Spermatogenic Cells ◽  
Viral Preparation

Male gonad cells are considered as promising target cells for the introduction of recombinant DNA within obtaining genetically modified individuals with given characteristics. The use of testicular spermatogonial stem cells is of the greatest interest. In the process of differentiation, this type of cell gives rise to a significant population of mature male germ cells. In the case of their genetic transformation, differentiated cells can be used to inseminate females in order to produce transgenic progeny. The aim of the research was to study the efficiency of using lentiviral vectors for the local transformation of roosters’ testicular spermatogenic cells. We used a lentiviral vector containing the ZsGreen reporter gene under the control of the CMV promoter. In vitro transformation of rooster spermatogenic cells was carried out by infection with a viral preparation, in vivo through multiple injections of the viral preparation into the testicular parenchyma of roosters ( n = 5). The efficiency of transformation was assessed by expression of the reporter ZsGreen gene in transfected spermatogenic cells. The success of using lentiviral vectors for the genetic transformation of rooster spermatogenic cells was shown in experiments in vitro and in vivo . The transformation efficiency of this cells types in an in vitro culture varied from 45 to 57% and averaged 48 ± 4%. The expression of the ZsGreen reporter gene in the cells of the spermatogenic epithelium of the testes was established in almost all experimental roosters in the in vivo experiments. The number of seminiferous tubules with transformed spermatogenic cells varied in the studied experimental roosters from 10 to 22%. The effectiveness of genetic transformation of the testes spermatogenic cells was 1.8 ± 0.2%. The obtained results indicate to the success of using lentiviral vectors for the genetic transformation of spermatogenic cells of rooster testes in vivo in order to create individuals with genetically transformed germ cells for the further production of transgenic offspring with given characteristics.

147 Dynamics of drake spermatogenesis

2020 ◽  
Vol 32 (2) ◽  
pp. 200
Author(s):  
L. A. Volkova ◽  
A. N. Vetokh ◽  
E. K. Tomgorova ◽  
N. A. Volkova ◽  
H. V. Ashraf ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genetic Material ◽  
Cell Types ◽  
Seminiferous Tubules ◽  
Age Group ◽  
Spermatogenic Cells ◽  
Russian Science ◽  
Donor Cells ◽  
Development And Differentiation ◽  
Species Specific

The examination of age dynamics for the development and differentiation of spermatogenic cells is of great importance to the study of spermatogenesis in poultry. Testicular stem cells, represented by spermatogonia, are a valuable genetic material for creating cryobanks of biomaterial. This is especially important when preserving and maintaining the gene pool of valuable breeds of poultry. In the process of differentiation, these cells give rise to a significant population of germ cells, so they can be used as donor cells for transplantation into the testes of male recipients. Thus, understanding species-specific characteristics of spermatogenesis in males is an important step for obtaining the spermatogenic cell population of interest. The aim of this research was to study the dynamics of spermatogenesis in drake. For the study, 10 groups of males were formed depending on age: 2 weeks and 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 7 months. There were eight males in each age group from 2 weeks to 6 months and six males in the 7-month age group. The testes were isolated postmortem, fixed in Bouin's fixative solution, and embedded in paraffin, and histological sections (5µm) were cut. The following indicators were evaluated: diameter of the seminiferous tubules, types of spermatogenic cells in the seminiferous tubules, and the amount of these cells within seminiferous tubules. Statistical analysis was performed using a t-test in SPSS ver. 15.0 (IBM Corp.). The types of spermatogenic cells were identified by morphology, and no fewer than 30 seminiferous tubules were examined from each individual. The diameter of the seminiferous tubules in the drake testes increased with age. At the ages of 2 weeks and 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 7 months, these indicators were 38±2, 55±2, 60±2, 61±3, 62±2, 62±4, 65±2, 76±3, 94±5, and 163±7µm, respectively. This was due to an increase in the number of spermatogenic cells within the seminiferous tubules to 23±1, 27±1, 38±1, 44±2, 47±3, 57±2, 68±2, 140±5, 187±7, and 466±13 at the ages of 2 weeks and 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 7 months, respectively. The presence, number, and ratio of the cell populations varied depending on age. At the ages of 1-12 weeks, the main cell types in the seminiferous tubules were Sertoli cells and spermatogonia. After the age of 4 months, primary spermatocytes began to appear in the seminiferous tubules. Secondary spermatocytes were visualised at 5 months of age, whereas spermatids could be detected at 6 months of age. Mature sperm cells were detected in the seminiferous tubules of drakes at the age of 7 months. Based on the data obtained, the following conclusion can be made: from 1-12 weeks of age, the generative cells of the seminiferous tubules in drakes are represented mainly by spermatogonia (P<0.05). Therefore, this period can be considered optimal for obtaining testicular stem cells and carrying out manipulations with them. This study was supported by the Russian Science Foundation within project no.16-16-04104.

scholarly journals PSXIII-5 The age dynamics of the spermatogenic cells development in the roosters’ testes

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 373-373
Author(s):  
Anastasia N Vetokh ◽  
Natalia A Volkova ◽  
Evgeniya K Tomgorova ◽  
Ludmila A Volkova ◽  
Natalia A Zinovieva
Keyword(s):  
Germ Cells ◽  
Sertoli Cells ◽  
Genetic Material ◽  
Cell Types ◽  
Seminiferous Tubules ◽  
Sperm Cells ◽  
Spermatogenic Cells ◽  
Different Cell Types ◽  
Hematoxylin Eosin ◽  
Testis Tissue

Abstract The cells of the male gonads are considered as a valuable genetic material for the conservation of the gene pool of breeds and lines of agricultural birds, as well as the directed modification of the poultry genome. Mature germ cells – spermatozoa and their predecessors – spermatogonia, spermatocytes and spermatids can be used for these purposes. To obtain these types of cells, it is necessary to know the characteristics of their development (spermatogenesis). The dynamics of the development of certain spermatogenic cell types in the testicular tubules of different-aged roosters has been studied. Histological studies were performed on testes of roosters aged from 1 week to 6 months with an interval of 2 weeks. Samples of testis tissue were fixed in Bouin’s solution. Histological sections were stained with hematoxylin-eosin. Identification of different cell types (Sertoli, spermatogonia, spermatocytes, spermatids, sperm cells) was carried out according to their morphology. At the age of 1–6 weeks in the seminiferous tubule of roosters, the mainly presence of two cell types was noted: Sertoli cells and spermatogonia. From 7 weeks of age, spermatocytes were detected in the seminiferous tubules, in the 4 months - spermatids, in the 5.5 months - sperm cells. The number of Sertoli cells remained almost unchanged with age and was 21 ± 2. The percentage of these cells decreased with age from 71 ± 3 % to 5 ± 1 %. The percentage of spermatogonia also decreased with age from 75 ± 2 % to 7 ± 1 %. The number of spermatids and spermatozoa, on the contrary, increased to puberty (6 months) and reached 54 %. The study was supported by the RFBR within Project no.18-29-07079.

Xenogeneic transplantation of human spermatogonia

Zygote ◽  
2000 ◽  
Vol 8 (2) ◽  
pp. 97-105 ◽  
Author(s):  
Marcos M. Reis ◽  
Ming C. Tsai ◽  
Peter N. Schlegel ◽  
Miriam Feliciano ◽  
Ricciarda Raffaelli ◽  
...  
Keyword(s):  
Germ Cells ◽  
Cell Types ◽  
Testicular Biopsy ◽  
Testicular Tissue ◽  
Seminiferous Tubules ◽  
Obstructive Azoospermia ◽  
Spermatogenic Cells ◽  
Immunodeficient Mice ◽  
Xenogeneic Transplantation ◽  
Immunological Rejection

In the last 3 years, several studies have shown that xenogeneic transplantation of rodent spermatogonia is feasible. The treatment of infertile patients with spermatogenic arrest using the injection of immature germ cells has yielded only poor results. We attempted to establish a complete spermatogenetic line in the testes of mutant aspermatogenic (W/Wv) and severe combined immunodeficient mice (SCID) transplanted with germ cells from azoospermic men. Spermatogenic cells were obtained from testicular biopsy specimens of men (average age of 34.3 ± 9 years) undergoing infertility treatment because of obstructive and non-obstructive azoospermia. Testicular tissue was digested with collagenase to promote separation of individual spermatogenic cells. The germ cells were injected into mouse testicular seminiferous tubules using a microneedle (40 μm inner diameter) on a 10 ml syringe. To assess the penetration of the cell suspension into the tubules, trypan blue was used as an indicator. Mice were maintained for 50 to 150 days to allow time for germ cell colonisation and development prior to them being killed. Testes were then fixed for histological examination and approximately 100 cross-sectioned tubules were examined for human spermatogenic cells. A total of 26 testicular cell samples, 16 frozen and 10 fresh, were obtained from 24 men. The origin of the azoospermia was obstructive (OA) in 16 patients and non-obstructive (NOA) in 8 patients. The concentration of spermatogenic cells in the OA group was 6.6 × 106 cells/ml, and 1.3 ? 106 cells/ml in the NOA group (p < 0.01). The different spermatogenic cell types were distributed equally in the OA samples, ranging from spermatogenia to fully developed spermatozoa, but in the NOA group the majority of cells were spermatogonia and spermatocytes. A total of 23 testes from 14 W/Wv mice and 24 testes from 12 SCID mice were injected successfully, as judged by the presence of spermatogenic cells in histological sections of testes removed immediately after the injection. However, sections from the remaining testes examined up to 150 days after injection showed tubules lined with Sertoli cells and xenogeneic germ cells were not found. The reason why the two strains of mouse used as recipients did not allow the implantation of human germ cells is probably due to interspecies specificity involving non-compatible cell adhesion molecules and/or immunological rejection.

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