scholarly journals Hypobaric hypoxia causes deleterious effects on spermatogenesis in rats

Reproduction ◽  
2010 ◽  
Vol 139 (6) ◽  
pp. 1031-1038 ◽  
Author(s):  
Weigong Liao ◽  
Mingchun Cai ◽  
Jian Chen ◽  
Jian Huang ◽  
Fuyu Liu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Germ Cells ◽  
Hypobaric Hypoxia ◽  
Diploid Cell ◽  
Seminiferous Tubules ◽  
Cell Populations ◽  
Control Group ◽  
Spermatogenic Cells ◽  
Tetraploid Cell ◽  
Normoxic Control

The study was conducted to explore the effects of hypobaric hypoxia on spermatogenesis in rats. Adult male Wistar rats were randomly divided into four groups: three hypoxia-exposed groups and one normoxic control group. Rats in the normoxic control group were raised at an altitude of 300 m, while rats in the 5-, 15-, and 30-day hypoxic groups were raised in a hypobaric chamber simulating a high altitude of 5000 m for 5, 15, and 30 days respectively. Flow cytometry was used to detect the DNA content of testicular spermatogenic cells in rats. The apoptosis of germ cells in testis was analyzed by using TUNEL assay. Spermatogenesis was also evaluated by morphology. Flow cytometry analysis revealed that 5–30 days of hypobaric hypoxia exposure significantly reduced the percentage of tetraploid cell population in rat testis. After rats were exposed to hypobaric hypoxia for 30 days, the ratio of haploid and diploid cell populations in testis reduced significantly. Seminiferous tubules with apoptotic germ cell increased after exposure to hypoxia. Most apoptotic germ cells were spermatogonia and spermatocytes. Hypoxia also caused decrease of cellularity of seminiferous epithelium, degeneration and sloughing of seminiferous epithelial cells occasionally. The data suggest that hypobaric hypoxia inhibits the spermatogenesis in rats. Decrease of tetraploid spermatogenic cells (primary spermatocytes) induced by hypoxia is an important approach to suppress spermatogenesis. The apoptosis of primary spermatocytes and spermatogonia may contribute to the loss of tetraploid cell populations.

286 EXPRESSION OF PLURIPOTENT STEM CELL MARKERS IN DOMESTIC CAT SPERMATOGONIAL CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 290 ◽  
Author(s):  
R. H. Powell ◽  
M. N. Biancardi ◽  
J. Galiguis ◽  
Q. Qin ◽  
C. E. Pope ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Basement Membrane ◽  
Germ Cell ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Cell Populations ◽  
Surface Markers ◽  
Cell Markers

Spermatogonial stem cells (SSC), progenitor cells capable of both self-renewal and producing daughter cells that will differentiate into sperm, can be manipulated for transplantation to propagate genetically important males. This application was demonstrated in felids by the successful xeno-transplantation of ocelot mixed germ cells into the testes of domestic cats, which resulted in the production of ocelot sperm (Silva et al. 2012 J. Androl. 33, 264–276). Spermatogonial stem cells are in low numbers in the testis, but have been identified and isolated in different mammalian species using SSC surface markers; however, their expression varies among species. Until recently, little was known about the expression of SSC surface markers in feline species. We previously demonstrated that many mixed germ cells collected from adult cat testes express the germ cell markers GFRα1, GPR125, and C-Kit, and a smaller population of cells expresses the pluripotent SSC-specific markers SSEA-1 and SSEA-4 (Powell et al. 2011 Reprod. Fertil. Dev. 24, 221–222). In the present study, our goal was to identify germ cell and SSC-specific markers in SSC from cat testes. Immunohistochemical (IHC) localization of germ cell markers GFRα1, GPR125, and C-Kit and pluripotent SSC-specific markers SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4 was detected in testis tissue from both sexually mature and prepubertal males. Testes were fixed with modified Davidson’s fixative for 24 h before processing, embedding, and sectioning. The EXPOSE Mouse and Rabbit Specific HRP/DAB detection IHC kit (Abcam®, Cambridge, MA, USA) was used for antibody detection. Staining for SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4 markers was expressed specifically at the basement membrane of the seminiferous tubules in both adult and prepubertal testes. The GFRα1 and GPR125 markers were detected at the basement membrane of the seminiferous tubules and across the seminiferous tubule section. However, C-Kit was not detected in any cell. Using flow cytometry from a pool of cells from seven adult testes, we detected 45% GFRα1, 50% GPR125, 59% C-Kit, 18% TRA-1-60, 16% TRA-1-81 positive cells, and a very small portion of SSEA-1 (7%) and SSEA-4 (3%) positive cells. Dual staining of germ cells pooled from 3 testes revealed 3 distinct cell populations that were positive for GFRα1 only (23%), positive for both GFRα1 and SSEA-4 (6%), and positive for SSEA-4 only (1%). Our IHC staining of cat testes indicated that cells along the basement membrane of seminiferous tubules were positive for SSC-specific markers, and flow cytometry analysis revealed that there were different cell populations expressing both germ cell and SSC-specific markers. Flow cytometry results show overlapping germ cell populations expressing SSEA-4 and GFRα1, and IHC results reveal that SSEA-4 positive cells are spermatogonia, whereas GFRα1 positive cells include other stages of germ cells, indicating that the small population of cells positive only for SSEA-4 is undifferentiated cat SSC.

201 TRANSPLANTATION OF SSEA-1+ AND SSEA-4+ SPERMATOGONIAL CELL SUBPOPULATIONS IN UNTREATED SEXUALLY IMMATURE DOMESTIC CATS

2014 ◽  
Vol 26 (1) ◽  
pp. 215
Author(s):  
R. H. Powell ◽  
J. L. Galiguis ◽  
Q. Qin ◽  
M. N. Biancardi ◽  
S. P. Leibo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Germ Cells ◽  
Cell Biology ◽  
Captive Breeding ◽  
Rete Testis ◽  
Domestic Cat ◽  
Seminiferous Tubules ◽  
Cell Populations ◽  
Specific Cell ◽  
Surface Markers

Captive breeding efforts in felids, including assisted reproduction techniques, have had varied success depending on species. Spermatogonial stem cells (SSC), comprising a small percentage of germ cells in the testis, are progenitor cells with the ability to both self-renew and differentiate into spermatozoa throughout the life of the male. Manipulation of SSC for transplantation (SSCT) may allow the propagation of genetically important males, as demonstrated by the production of ocelot sperm following transplantation of ocelot mixed germ cells to domestic cat testes (Silva et al. 2012 J. Androl. 33, 264–276). Using specific cell surface markers, SSC have been isolated from mixed germ cells in several other species for SSCT, culture, and studying germ cell biology; however, expression may differ with species. Using the domestic cat as a model for exotic felids, we recently began evaluating the expression of surface markers in feline SSC. Previously, we determined that pluripotent markers SSEA-1, SSEA-4, TRA-1–60, and TRA-1–81 were more specific to cat spermatogonia than SSC surface markers GFRα1 and GPR125 used in other species, with SSEA-1 and SSEA-4 expressed in the fewest cells (Powell et al. 2011 Reprod. Fertil. Dev. 24, 221–222; Powell et al. 2012 Reprod. Fertil. Dev. 25, 290–291). Our current goal was to 1) confirm the presence of SSC within SSEA-1+ and SSEA-4+ cell populations by the ability to colonize following SSCT; 2) compare the effectiveness of transplanting SSC purified by flow cytometry versus mixed germ cells; and 3) show that depletion of endogenous germ cells before SSCT, usually performed by irradiation or chemotherapy in other studies, is not necessary when using sexually immature recipients. Mixed germ cells from 8 to 12 adult testes were pooled, stained for SSEA-1 or SSEA-4, and sorted by flow cytometry. SSEA-1+, SSEA-4+, or mixed germ cells were then labelled with the membrane dye PKH26 (Sigma MINI26) and injected into the testes of six 5-month-old and six 6-month-old cats at the site of the external rete testis after carefully microdissecting the head of the epididymis away from the testis. Injections contained an average of 230 000 sorted or 10 × 106 mixed germ cells suspended in 80 μL of DMEM/F12 + 3 μL of Trypan Blue (T8154, Sigma, St. Louis, MO, USA). Testes were harvested 10 to 12 weeks post-SSCT and bisected, half snap-frozen for later cryosectioning and the other half enzymatically digested to loosen seminiferous tubules for immediate evaluation. Fluorescence was detected in the testes of both 6-month-old males that received injections of mixed germ cells, one 6-month-old male injected with SSEA-4+ cells, and two 5-month-old males, one injected with SSEA-4+ cells and one with SSEA-1+ cells. Results indicate that SSC are found in both SSEA-1+ and SSEA-4+ cell populations, but that purification of SSC is not necessary for successful SSCT. Additionally, SSC colonization in cats is possible without depletion of endogenous cells in sexually immature recipients.

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.

scholarly journals The Effect of Inhibin Immunization in Seminiferous Epithelium of Yangzhou Goose Ganders: A Histological Study

Animals ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 2801
Author(s):  
Muhammad Faheem Akhtar ◽  
Ejaz Ahmad ◽  
Ilyas Ali ◽  
Muhammad Shafiq ◽  
Zhe Chen
Keyword(s):  
Germ Cells ◽  
Marked Reduction ◽  
Histological Study ◽  
Seminiferous Epithelium ◽  
Histological Evaluation ◽  
Seminiferous Tubules ◽  
Control Group ◽  
Cell Numbers ◽  
Tubular Diameter ◽  
Reciprocal Expression

The current study investigated the effect of inhibin immunization on germ cell numbers (spermatogonia, spermatocytes, round, and elongated spermatids), seminiferous tubules (ST) diameter, Johnsen’s score, epithelial height (μm), luminal tubular diameter (μm), and number of ST per field (ST/field) of Yangzhou goose ganders. Histological evaluation showed apoptosis and regression of testes after inhibin (INH) immunization, with a concomitantly marked reduction in the round and elongated spermatids in the experiment (INH) group compared to the control group. The diameter of seminiferous tubules (ST) and epithelial height (EH) were positively correlated at 181, 200, and 227 days of age. In comparison, luminal tubular diameter (LD) was negatively correlated on day 227 to ST diameter and epithelial height. On day 227, many seminiferous tubules per field (ST/field) were negatively correlated to ST diameter, EH, and LD. INH immunization elevated ST diameter, EH, and LD, while Johnsen’s score and number of ST/field had reciprocal expression. In conclusion, the concomitant effect of INH immunization and seasonality in breeding regressed germ cells and damaged spermatogenesis in seminiferous epithelium Yangzhou ganders.

323. MEASURING CHANGES IN TESTICULAR CELL POPULATIONS USING FLOW CYTOMETRY

2010 ◽  
Vol 22 (9) ◽  
pp. 123
Author(s):  
G. Morin ◽  
K. Loveland
Keyword(s):  
Flow Cytometry ◽  
Germ Cells ◽  
Cell Biology ◽  
Leydig Cells ◽  
Cell Populations ◽  
Published Data ◽  
Wild Type ◽  
Proteomics Analysis ◽  
Kit Receptor ◽  
Different Levels

Spermatogenesis is first established during the first two weeks postpartum by the transition of undifferentiated (Kit–) into differentiated spermatogonia (Kit+). We recently showed that changes in the level of the growth factor activin alters the proportion of spermatogonial subtypes (1). However, detection of this transition by histology is unreliable. This project objective is to develop methods to efficiently measure changes in somatic and germ cell populations at the onset of spermatogenesis. Using surface (Kit receptor) and internal (mouse vasa homologue {MVH}) markers, we evaluated the proportion of differentiating germ cells in wild type Swiss mice by flow cytometry. Whole testes of mice at 7, 10, 14 days postpartum (dpp) were enzymatically dissociated and single cell suspensions were labelled with anti-Kit receptor antibody to detect Leydig cells and differentiating spermatogonia. These suspensions were then fixed and permeabilized in order to detect MVH, allowing spermatogonia to be distinguished from Leydig cells. Our present results show that combined Kit and MVH labelling is effective for evaluating the proportion of undifferentiating and differentiating germ cells. Our preliminary observations identified an elevation in the proportion of Kit+MVH+ cells between 7 and 10 days from 0.37 to 18%, indicating that spermatogonial differentiation advances dramatically between these ages. At day 14, the proportion of Kit+MVH+ cells decreased to 11%, as the emerging spermatocytes dilute spermatogonial numbers. These findings agree with published data (2). We have also used surface markers to discriminate between spermatogonia and Leydig cells without fixation or permeabilization, allowing us to isolate these cells for molecular and proteomics analysis. This will facilitate comparative profiling of germ cells with different levels of Kit, including those in mice with altered levels of growth factors (2) and hormones that govern the progression of testis development. (1) Mithraprabhu, 2010 Biology of Reproduction.(2) Bellve, 1977 Journal of Cell Biology.

Effects of subchronic exposure to carbendazim on spermatogenesis and fertility in male rats

2009 ◽  
Vol 25 (1) ◽  
pp. 41-47 ◽  
Author(s):  
G Yu ◽  
Q Guo ◽  
L Xie ◽  
Y Liu ◽  
X Wang
Keyword(s):  
Germ Cells ◽  
Flow Cytometric Analysis ◽  
Testicular Tissue ◽  
Male Rats ◽  
Seminiferous Tubules ◽  
Control Group ◽  
Testis Weight ◽  
Subchronic Exposure ◽  
Flow Cytometric ◽  
Sperm Counts

The aim of this study is to investigate the effects of subchronic exposure to carbendazim on spermatogenesis and fertility in male rats. Ninety-eight healthy male rats were divided into four groups: three exposure groups and a control group. Carbendazim was administered orally to male rats at 0, 20, 100 and 200 mg/kg for 80 days prior to mating. Each male was cohabited with an unexposed female for a maximum of 5 days. In 100 and 200 mg/kg groups, the mating index was relatively increased, the fertility index was decreased, and the testis weight, the sperm counts and motility were also decreased. The levels of luteinizing hormone (LH) showed a decreasing tendency and there was a statistical difference between the 200 mg/kg group and the control group. There were no obvious effects on the levels of follicle stimulating hormone (FSH) and testosterone (T). Histopathological evaluation showed atrophic seminiferous tubules, decreased germ cells, and increased sloughing of germ cells. Flow cytometric analysis of the testicular tissue revealed that carbendazim inhibited meiotic transformation and interfered with the spermatogenic process. These results suggest that carbendazim has adverse effects on spermatogenesis, resulting in reduced fertility in male rats.

Male reproductive toxicity and b-luteinizing hormone gene expression in sexually mature and immature rats exposed to 2-bromopropane

1999 ◽  
Vol 18 (11) ◽  
pp. 683-690 ◽  
Author(s):  
X Wu ◽  
A S Faqi ◽  
J Yang ◽  
X Ding ◽  
X Jiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dose Level ◽  
Germ Cells ◽  
Serum Testosterone Level ◽  
Seminiferous Tubules ◽  
Control Group ◽  
Dependent Manner ◽  
Immature Rats ◽  
The Absolute ◽  
Sexually Mature

1 The reproductive effects of 2-bromopropane (2-BP) in sexually mature and immature male Sprague-Dawley rats were investigated. The animals were randomly divided into three treatment groups and one control group each of which comprised six mature and six immature rats. The treated groups were injected s.c. 200, 600 and 1800 mg/kg of 2-BP on 5 days a week for 5-7 weeks and the control group received the vehicle. 2 The absolute and relative testis weights were significantly reduced in 600 and 1800 mg/kg b.w. dose groups in both mature and immature rats. The absolute epididymis, prostate, seminal vesicle, and pituitary weights and the relative epididymis weights, however, were significant only at the highest dose level used in both mature and immature rats. 3 The sperm concentration and sperm viability in epididymal duct decreased and the percentage of abnormal sperm increased in a dose-dependent manner in both mature and immature rats. Additionally, serum testosterone level was significantly decreased in all dose groups in mature rats, and was significantly reduced only in the group treated with the middle and highest dose in immature rats. 4 In both mature and immature rats treated with 200 and 600 mg/kg, the seminiferous tubules were atrophied and all types of germ cells were decreased in number. At the highest dose level, the effect was more marked showing severely atrophied seminiferous tubules and a complete loss of all types of germ cells. 5 The mating, pregnancy and fertility indices were significantly reduced in the 600 and 1800 mg/kg groups. Additionally, at the highest dose group the number of implantations and viable fetuses per litter were reduced and the resorption rate was increased significantly. 6 In the mature rats, the b-LH gene expression increased significantly in the 1800 mg/kg group when compared to the control group. 7 It can be concluded that 2-BP induces alterations in both neuro-endocrine axis and the reproductive tract under the present experimental conditions. The no observed adverse effect level (NOAEL) in this study could be estimated to be lower than 200 mg/kg/b.w. based on the alteration in testicular morphology as well as on sperm parameters observed at the dose level of 200 mg/kg.

scholarly journals Reproductive Hormones Imbalance, Germ Cell Apoptosis, Abnormal Sperm Morphophenotypes and Ultrastructural Changes in Testis of African Giant Rats (Cricetomys gambianus, Waterhouse, 1840) Exposed to Sodium Metavanadate Intoxication

2021 ◽  
Author(s):  
Ifukibot Levi Usende ◽  
Fatima Oyenike Oyelowo ◽  
Agbonu Oluwa Adikpe ◽  
Benjamin Obukowho Emikpe ◽  
Allam Abdel Hamid Mohamed Nafady ◽  
...  
Keyword(s):  
Germ Cells ◽  
Reproductive Toxicity ◽  
Treated Group ◽  
Reproductive Hormones ◽  
Ultrastructural Changes ◽  
Seminiferous Tubules ◽  
Control Group ◽  
Immunopositive Cell ◽  
Sodium Metavanadate ◽  
Cricetomys Gambianus

Abstract Environmental exposure to vanadium has been on the increase in recent time. This metal is a known toxicant. The current study was conducted to investigate the reproductive toxicity of sodium metavanadate (SMV) in male African giant rats. Administration of SMV was done intraperitoneally daily for 14 consecutive days at a dosage of 3mg/kg body weight. Sterile water was administered to the control group. We analyzed serum reproductive hormones, sperm reserve and quality as well as testicular ultrastructural changes following SMV treatment. Our results showed SMV exposed AGR group had statistically increased progesterone but decreased testosterone, FSH and LH concentrations. Also, SMV treated group had statistically decreased sperm motility and mass activity with increased percentage of abnormal morphophenotypes of spermatozoa and upregulation of P53 immunopositive cell. Ultrastructural study revealed vocuolation of germ and Sertoli cells, cytoplasmic and nucleus; and mitochondrial swelling and vacuolations were also observed. There was severe disintegration of the seminiferous tubules, atrophy and degeneration of myeloid cells and apoptosis of the Leydig, Sertoli and germ cells. In conclusion, intraperitoneal SMV exposure exerts severe adverse effects on some serum reproductive hormones, reduction of sperm reserve and quality, apoptosis and degenerative changes of the Leydig, Sertoli and germ cells which can lead to infertility.

scholarly journals Effect of Bleomycin, Etoposide, and Cisplatin Treatment on Spermatogonia Cell and Malondialdehyde Level in Male Rats

2020 ◽  
Vol 9 (3S) ◽  
pp. 293-297
Keyword(s):  
Germ Cells ◽  
Cell Number ◽  
Test Method ◽  
Male Rats ◽  
Seminiferous Tubules ◽  
Control Group ◽  
High Power Field ◽  
The Third ◽  
Spermatogonia Cells ◽  
Bep Chemotherapy

Co-administration of bleomycin, etoposide, and cisplatin (BEP) becomes standart chemotherapy for testicular cancer because it has brought a cure rate of more than 90%. Impact of the treatment to the outcome become a concern, particularly the adverse effect on a long-term reproductive health risk to cancer survivors. There is no evidence, when the damage to the testes began due to the administration of BEP chemotherapy, makes the indication of treatment still controversial. The aim of this study is to determine the effects of BEP on Spermatogonial cell and MDA levels outcome in an animal model. Male wistar rats (Rattus norvegicus) aged 13-15 weekswere treated daily with BEP for three cycles, 33 hours each. It was divided into one control group received 1cc of normal saline, and three groups received three cycles of 0.5 x dose-levels of BEP (Intraperitoneally; 0.75 mg/kg, 7.5 mg/kg, and 1.5 mg/kg). Cell number of Spermatogonia cells were calculated from HE-stained specimens and observed under light microscope (Olympus BX-51) using 400x magnification (high power field) Thiobarbituric acid (TBA) test method used to measure malondialdehyde (MDA) level by spectrophotometry. The result was a significant decrease in the average number of Spermatogonia cells (p = 0.003) between the control group and others. This is caused by excessive exposure to BEP chemotherapy, which cause atrophy of the seminiferous tubules and content of germ cells in the tubules has decreased, accompanied by the appearance of immature germ cells that enter the lumen. A significant increase in MDA levels (p = 0.001) occurred after the administration of the third cycle of BEP chemotherapy. In conclusion, BEP chemotherapy adversely affect the number of Spermatogonia cells and MDA level. The third cycle BEP chemotherapy significantly more destructive compared to the first and second cycle.

371 EVALUATION OF ENRICHMENT STRATEGIES FOR PORCINE SPERMATOGONIA BY EXPRESSION OF PROTEIN GENE PRODUCT 9.5, A SPERMATOGONIA-SPECIFIC MARKER IN THE PIG TESTIS

2006 ◽  
Vol 18 (2) ◽  
pp. 293
Author(s):  
J. Luo ◽  
S. Megee ◽  
R. Rathi ◽  
I. Dobrinski
Keyword(s):  
Gene Product ◽  
Germ Cells ◽  
Protein Gene ◽  
Velocity Sedimentation ◽  
Seminiferous Tubules ◽  
Cell Populations ◽  
Specific Marker ◽  
Pgp 9.5 ◽  
Protein Gene Product ◽  
Fraction I

Transplantation of genetically altered male germ cells is under investigation as a novel route to generate transgenic animal models. Identification and isolation of spermatogonial stem cells are a prerequisite for this strategy. The objectives of this study were to validate a marker for identification of undifferentiated porcine spermatogonia, and to use this marker to develop a practical enrichment strategy for spermatogonia from pig testis. We established that expression of protein gene product (PGP) 9.5 is a spermatogonia-specific marker in porcine testis through analysis of its expression pattern in testis cells, by comparison with the expression of the cell-type specific proteins GATA-4 (expressed in Sertoli cells) or PLZF (expressed in undifferentiated mouse spermatogonia) in seminiferous tubules at different ages, and by comparison of expression levels of PGP 9.5 and the germ cell-specific protein VASA in different cell fractions after differential plating. Using expression of PGP 9.5 as a marker, we characterized enrichment of porcine spermatogonia from two-week-old (2wo) and 10-week-old (10wo) pigs by immunofluorescence either after differential plating only or after velocity sedimentation at unit gravity followed by differential plating. After differential plating with overnight culture to deplete testicular somatic cells that firmly attach to culture dishes, spermatogonia (mean � SEM per 1000 cells) were 5-fold enriched (P < 0.05) in cells remaining in suspension (fraction I) (2wo: 54.0 � 9.1; 10wo: 162.7 � 30.5) and in populations slightly attached to the culture plate (fraction II) (2wo: 92.7 � 8; 10wo: 159.5 � 22.5) compared to the initial samples (2wo: 12.3 � 2.7; 10wo: 27.2 � 2.9). Slightly attached spermatogonia appear to be superior for future experiments due to higher viability (>90%) than spermatogonia remaining in suspension (<50%). Cell populations containing up to 70% spermatogonia with good viability (>80%) were achieved by velocity sedimentation isolation followed by differential plating. These results indicate that expression of PGP 9.5 is a useful marker for identification of undifferentiated porcine germ cells. Simple differential adhesion culture of testis cells harvested from pre-pubertal boars can supply cell populations enriched in spermatogonia for subsequent genetic manipulation and transplantation. This work was supported by 1 R01 RR17359-01.

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