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).

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.

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.

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).

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.

scholarly journals Testicular and seminal evaluation of goats fed hay Cenostigma pyramidale

2020 ◽  
Vol 40 (12) ◽  
pp. 963-969
Author(s):  
Maria V.B. Santos ◽  
Ana K.S. Cavalcante ◽  
Juliana T.S.A. Macêdo ◽  
Marilúcia C. Santos ◽  
Laiara F. Rocha ◽  
...  
Keyword(s):  
Gonadosomatic Index ◽  
Live Weight ◽  
Seminiferous Tubules ◽  
Control Group ◽  
Blood Collection ◽  
Panicum Maximum ◽  
Alternative Source ◽  
Parametric Data ◽  
Testicular Weight ◽  
Student’S T

ABSTRACT: This study aimed to assess the possible occurrence of reproductive changes in male goats associated with ingestion of Cenostigma pyramidale hay. Sixteen animals divided into two experimental groups, G1 and G2 (control group) were used. Animals in G1 received 2% of forage, based on live weight (LW), composed of 100% of C. pyramidale, and animals in G2 received 2% of Panicum maximum “Massai’ grass hay, based on LW. Both groups received 1% of concentrated feed supplementation based on LW, along with mineralized salt and water ad libitum. The goats were subjected to weighing, testicular biometry, and semen and blood collection every 30 days. After 120 days, the animals were castrated and their testes were collected. Testicular measurements were performed and fragments were collected for histological processing to determine the gonadosomatic index (GSI), diameter of the seminiferous tubules, height of the germinal epithelium (HGE), volumetric proportion and volume of the testicular parenchyma components, total length of the seminiferous tubules, length of the seminiferous tubules per gram of testis, and leydigosomatic and tubulosomatic indexes. The data were evaluated for normality using the Student’s t-test. Data with normal distribution were assessed using analysis of variance (ANOVA) and the non-parametric data were evaluated using the Kruskal-Wallis test, both at 5% probability. Statistically significant differences (p<0.05) were observed for GSI (G1=0.48 ±0.08 and G2=0.34 ±0.09) and HGE (G1=52.95 ±2.99 and G2=.47.63 ±2.67) between treatments. Consumption of C. pyramidale hay increased LW and, consequently, testicular weight, contributing to high GSI. In conclusion, ingestion of C. pyramidale has no toxic effect on the testicular, seminal and histological parameters of goat testis. Due to its nutritional characteristics, consumption of this plant improves animal body development. Because C. pyramidale is adapted to semi-arid regions, it can be an alternative source of feed for goats during periods of shortage.

HORMONAL REGULATION OF ANDROGEN-BINDING PROTEIN IN LAMB TESTES

1980 ◽  
Vol 85 (3) ◽  
pp. 443-448 ◽  
Author(s):  
S. CARREAU ◽  
M. A. DROSDOWSKY ◽  
C. PISSELET ◽  
M. COUROT
Keyword(s):  
Sertoli Cells ◽  
Hormonal Regulation ◽  
Positive Response ◽  
Binding Protein ◽  
Seminiferous Tubules ◽  
Control Group ◽  
Supporting Cells ◽  
Testosterone Treatment ◽  
Androgen Binding Protein ◽  
Androgen Binding

Androgen-binding protein (ABP) was measured in the testes of 50-day-old lambs. The animals were hypophysectomized and treatment lasting for 5 days was begun 15 days after surgery. In hypophysectomized but otherwise untreated lambs (control group), no 5α-dihydrotestosterone binding was detectable in testicular cytosol. One out of four lambs gave a positive response with FSH treatment (25 fmol ABP/mg protein), whereas a restoration of the synthesis of ABP was noted in all LH-treated animals (19 ± 9 (s.e.m.) fmol ABP/mg, n = 4). No synergism between the two gonadotrophins was observed in lambs treated simultaneously with FSH and LH (19 ± 4 fmol ABP/mg, n = 5). Testosterone treatment elicited a greater response (37 ± 9 fmol ABP/mg, n = 5) than FSH or LH alone and the response was not increased by the simultaneous addition of FSH (38 ± 10 fmol ABP/mg, n = 5). Whatever the treatment, no influence was observed either on the number of supporting cells (undifferentiated Sertoli cells) or the length of the seminiferous tubules (P > 0·05); the diameter of tubules was significantly increased in the group treated with FSH and LH. It is postulated that testosterone may have a direct effect on the production of ABP by the supporting cells of the impuberal lamb.

scholarly journals Curcumin Effectivity on Hepar and Reproductive Organ Recovery Male Mice (Mus musculus L) after Methoxychlor Exposure

Biota ◽  
2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Mahriani Mahriani ◽  
Susantin Fajariyah ◽  
Eva Tyas Utami
Keyword(s):  
Mus Musculus ◽  
Sertoli Cells ◽  
Leydig Cells ◽  
Reproductive Organ ◽  
Seminiferous Tubules ◽  
Control Group ◽  
Disruptive Effect ◽  
Curcumin Treatment ◽  
Cell Counts ◽  
Male Reproductive Organ

Methoxychlor (MXC) is an insecticide (DDT derivates) that has the potential for bioaccumulation in mammal and causes a disruptive effect on the hepar and reproductive system. This study was done to find out the benefits of curcumin as a natural ingredient to overcome the negative impact of Methoxychlor (MXC) on hepar and male reproductive organ of Balb’C mice (Mus musculus L). The study was carried out in a Completely Randomized Design (CRD) Posttest Only Control Group Design used four treatments and six replications. The curcumin treatment after administration of MXC was carried out by gavage with curcumin doses: 0.05; 0,1; and 0.2 mg/g body weight, every day for two weeks, respectively. Histological observations of the liver, and testis was performed using the paraffin method and Hematoxylin Eosin stained. The results showed that MXC exposure caused liver disruption by increasing the number of pycnotic necrotic hepatocytes and hydrophic degeneration hepatocytes. On the male reproductive organ, MXC caused testis impairment by reducing the number of Sertoli cells and Leydig cells, spermatogenic cell counts, and the diameter of seminiferous tubules. The administration of curcumin at doses of 0.1 mg/g bw in mice exposed to methoxychlor can reduce the number of hydrophic degeneration hepatocytes and tend to reduce the number of pycnotic hepatocytes; and also increase the number of Sertoli cells, the number of spermatogenic cells, and the diameter of the seminiferous tubules, and tend to reduce the amount of Leydig cells. Curcumin treatment tends to recover hepar dan testis disruption of mice that were exposed by MXC.

scholarly journals 315 AN ATTEMPT AT INDUCING DIFFERENTIATION INTO ROUND SPERMATIDS OF RAT SPERMATOGONIA BY CO-CULTURING WITH SERTOLI CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 308 ◽  
Author(s):  
Y. Iwanami ◽  
T. Kobayashi ◽  
M. Kato ◽  
M. Hirabayashi ◽  
S. Hochi
Keyword(s):  
Cell Population ◽  
Sertoli Cells ◽  
Cultured Cells ◽  
Uv Light ◽  
Close Association ◽  
Type A ◽  
Round Spermatid ◽  
Full Term ◽  
Male Rats ◽  
Seminiferous Tubules

Mammalian spermatogenesis is a complex process of germ cell development at the seminiferous tubules whereby diploid spermatogonia proliferate and differentiate into haploid spermatozoa via round and elongating spermatids in close association with somatic Sertoli cells. In the present study, the potential of rat spermatogonia to undergo meiosis during co-culture with Sertoli cells was assessed. The type-A spermatogonia and Sertoli cells were prepared from Day 7 heterozygous transgenic male rats carrying EGFP DNA, and co-cultured on the dishes (coated; BD Falcon™ 35-3801, or non-coated: BD Falcon™ 35-1008, 4 × 106 cells/4-mL dish) at 37°C for 3 days and at 34°C for a subsequent 7 days in 5% CO2 in air. The culture medium was DMEM medium supplemented with 10% fetal bovine serum, growth factors (10 ng/mL EGF and 10 ng/mL IGF) and various hormones (500 ng/mL FSH, 133 μIU/mL hGH, 5 μg/mL insulin, 0.1 μM testosterone and 0.1 μM dihydrotestosterone). During culture, appearance of round spermatid-like cells (ca. 15 μm in cellular diameter and 7–8 μm in nuclear diameter) was traced. The ploidy of the cells was also analyzed by flow cytometry (FCM). At the end of culture, the proportion of EGFP DNA-bearing cells in the total cultured cell population was examined under UV light at 365 nm. Thereafter, continuation of the spermatid-like cells to full-term development was examined by ooplasmic microinjection (Kato et al. 2004 Contemp. Top. Lab. Anim. Sci. 43/2, 13). Briefly, oocytes from the Sprague-Dawley rats were denuded, activated with two direct-current pulses at 100 V/mm for 99 μs and held in 2 mM 6-dimethylaminopurine for 20 min. The nuclei of spermatid-like cells were microinjected into the oocytes by using a piezo impact driving unit, and the injected oocytes after 24 h culture were transferred into recipients. Round spermatid-like cells were first observed at the 5th day of culture on both dishes, but the proportion of spermatid-like cells on the coated dish was higher than that on the non-coated dish. The FCM analysis showed that a single peak of haploid cells was detected in the cell population cultured on the coated dish at the 5th day of culture, while no haploid peak was detected on the non-coated dish. The cultured cells exhibited two distinct patterns of EGFP fluorescence, with a proportion of EGFP-positive cells at 53.5% (total 1,000 counts). The microinsemination into 263 oocytes resulted in the production of 27 oocytes with two pronuclei (10.3%) and 15 cleaved oocytes (5.7%). However, the oviductal transfer of 143 microinseminated oocytes resulted in only 8 implantation sites without viable offspring (5.6%). These results indicated that rat type-A spermatogonial cells seemed to undergo meiosis, but the potential of the cultured spermatid-like cells to participate into full-term development was questionable.

scholarly journals Granulomatous Epididymitis Related to Rhodotorula glutinis Infection in a Dog

1995 ◽  
Vol 32 (6) ◽  
pp. 716-718 ◽  
Author(s):  
K. Kadota ◽  
K. Uchida ◽  
T. Nagatomo ◽  
Y. Goto ◽  
T. Shinjo ◽  
...  
Keyword(s):  
Connective Tissue ◽  
Sertoli Cells ◽  
Periodic Acid ◽  
Rhodotorula Glutinis ◽  
Seminiferous Tubules ◽  
Spermatogenic Cells ◽  
Cell Debris ◽  
Periodic Acid Schiff ◽  
Microbiological Characteristics ◽  
Cut Surface

A 4-year-old, male Great Dane dog developed severe swelling of the scrotum on 9 December 1991, and the testes and epididymides were removed surgically on 12 December 1992. The cut surface of the epididymides consisted of hard connective tissue and several small abcesses with slight hemorrhage. Histopathologically, the seminiferous tubules in the testes had only a few spermatogenic cells, but Sertoli cells were well preserved. Both epididymides consisted entirely of a proliferation of fibrous connective tissue, and only a few ducts deferens containing cell debris, neutrophils, and macrophages in the lumina were present. In all lesions of the epididymides, the macrophages contained periodic acid–Schiff– and Grocott's silver–positive round granules, 5-8 μm in diameter. Microbiologically, smooth salmon-pink colonies consisting of ovoidal yeast, about 10 μm in diameter, were isolated from the samples of epididymides but not from those of the testes. The isolated yeast had microbiological characteristics of Rhodotorula glutinis. From these observations, we diagnosed the present case as granulomatous epididymitis due to Rhodotorula infection.

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