Developmental potential of human spermatogenic cells co-cultured with Sertoli cells

Sousa, Mário; Cremades, Nieves; Alves, Cláudia; Silva, Joaquina; Barros, Alberto

doi:10.1093/humrep/17.1.161

Cited by 126 publications

(112 citation statements)

References 55 publications

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“…Wikstrom et al (2004) found that only 50% of the boys with KS had germ cells in their testes, indicating a severely impaired fertility potential even in the peripubertal period. Therefore, a potential strategy for infertility in patients with KS is cryopreservation of ejaculated spermatozoa or testicular tissue early in the patients' adolescence (Sousa et al, 2002;Ichioka et al, 2006). Our present study partially supported that age may affect sperm recovery from KS patients.…”

Section: Discussionsupporting

confidence: 78%

Sperm retrieval from patients with nonmosaic Klinefelter’s syndrome by semen cytology examination

Jiang¹,

Dong²,

Yu³

et al. 2014

Genet. Mol. Res.

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ABSTRACT. Successful sperm retrieval from ejaculates of nonmosaic Klinefelter's syndrome (KS) patients by using semen cytology examination was described in this report. The clinical parameters of KS patients with sperm compared to patients without sperm were described. One hundred and fifty-one patients were proven to suffer from KS by chromosomal analysis using G-banding. Spermatozoa were obtained from 10 patients (10/151, 6.6%) using semen analysis. After semen cytology examination, 32 patients (32/151, 21.2%) were found to have sperm or germ cell in their ejaculate. The patients with successful sperm retrieval were significantly younger (27.1 ± 3.7 years) than the patients for whom sperm retrieval failed (28.9 ± 4.2 years). The mean serum testosterone level and the mean T/LH ratio of KS patients with successful sperm retrieval were significantly higher in men with sperm than in men without sperm (testosterone: 3.2 ± 2.1 ng/mL vs 2.7 ± 1.5 ng/mL; T/LH ratio: 0.2 ± 0.3 vs 0.1 ± 0.1). In conclusion, semen Sperm retrieval from patients with Klinefelter's syndrome cytology examination should be performed to identify sperm and germ cells in the ejaculate of KS patients if no sperm can be detected by traditional semen analysis. The serum testosterone level and T/LH ratio revealed an association between impaired Leydig cell function and impaired spermatogenesis in KS males. KS patients should receive earlier diagnosis and treatment.

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Section: Discussionsupporting

confidence: 78%

Sperm retrieval from patients with nonmosaic Klinefelter’s syndrome by semen cytology examination

Jiang¹,

Dong²,

Yu³

et al. 2014

Genet. Mol. Res.

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“…The observation of a high percentage of spermatozoa with an apparently normal chromosomal content (85.1%), almost as high as in the general population, 18 is of very good prognosis for this patient, since the selection of these cells for ICSI does not represent an increased risk for aneuploidies. However, selection of female embryos by PGD should be considered at genetic counselling.…”

Section: Discussionmentioning

confidence: 70%

“…21,22 In the present study, an abnormal segregation of the (Y;13) was found during meiosis I, as only 13.6% of the secondary spermatocytes showed a normal chromosome complement. In this situation, most of the abnormal secondary spermatocytes might become arrested and degenerated, 18 which explains why the large majority of the round spermatids were normal (70%), as were in consequence most of the sperm (85.1%). Results also evidenced unpaired chromosome centromeric signals in most (73.5%) of the diploid germ cells, thus suggesting that the majority of the cells might not overcome meiosis I, blocking and degenerating at the primary spermatocyte stage.…”

Section: Discussionmentioning

confidence: 99%

“…Classification of germ cells was made using inverted Hoffman microscopy and after fluorescent in situ hybridization was mainly based on the size and density of the cell nucleus. 18 PCR amplification for SRY and AZF regions Peripheral blood samples (8 -10 ml) were pelleted and stored at 7208C until DNA extraction. High molecular weight DNA was isolated using a salting-out method.…”

Section: Meiotic Studiesmentioning

confidence: 99%

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Unique (Y;13) translocation in a male with oligozoospermia: cytogenetic and molecular studies

et al. 2002

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The incidence of Y/autosome translocations is low. Whereas involvement of non-acrocentric chromosomes often leads to infertility, cases related with acrocentric chromosomes are usually familial with no or minimal effect on fertility. A de novo (Yp/13p) translocation was found in a 32-year-old male referred for severe oligozoospermia. Conventional cytogenetic procedures (GTG, CBG and NOR banding) and molecular cytogenetic techniques (Fluorescence In Situ Hybridization, FISH) were performed on high-resolution chromosomes obtained after peripheral blood lymphocyte culture as also on interphase nuclei of spermatogenic cells from semen samples. Screening of AZF microdeletions in the Yq11.2 region known to be involved with spermatogenesis defects was also performed. GTG banding showed a (Yp/13p) translocation in all scored metaphases. CBG and NOR staining of the derivative chromosome revealed the maintenance of Yq heterochromatin and of the 13p NOR region. FISH with centromeric Y and 13/21 probes, SRY specific probe and X/Y (p and q arms) sub-telomeric probes gave the expected number/location of fluorescent signals. Hybridisation with a pan-telomeric repeat (TTAGGG) probe showed an absence of the telomeric sequences at the fusion point of the rearranged chromosome. FISH analysis with probes to chromosomes X, Y, 13 and 18 showed an abnormal segregation of the translocated chromosome during meiosis I, which explains that only 13.6% of the secondary spermatocytes were normal. Most of these became arrested, as after meiosis II the large majority of the round spermatids were normal (70%), as were in consequence most of the sperm (85.1%). Multiplex-PCR confirmed the intactness of the SRY region and showed absence of AZF microdeletions. We report a novel de novo (Yp;13p) translocation characterised by loss of the 13p and Yp telomeres. Meiotic studies using FISH demonstrated meiosis I chromosome unpairing and mal segregation that justifies the severe oligozoospermia. Although most sperm have a normal chromosomal constitution, preimplantation genetic diagnosis should be considered an option for this patient.

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“…The requirement of glial cell line-derived neurotrophic factor (GDNF) was also reported for long-term in vitro culture of self-renewing SSCs [25,31]. Subsequently, coculturing of SSC on feeder layers (viz., Sertoli cells, STO cells and embryonic fibroblast cells) was exploited as an alternative [46][47][48]. Among the co-culturing system, Sertoli cells have been emerged as the most commonly used feeder layers [48].…”

Section: Discussionmentioning

confidence: 99%

In vitro culture and characterization of spermatogonial stem cells on Sertoli cell feeder layer in goat (Capra hircus)

Pramod

Mitra

2014

J Assist Reprod Genet

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Purpose To develop an efficient protocol for isolation, purification and long-term culture of spermatogonial stem cell (SSC) in goat. Methods The isolation of SSC was performed by testicular disaggregation by enzymatic digestion using collagenase IV, trypsin and DNase I. Further SSCs were enriched using Percoll density gradient centrifugation. The purity of SSCs was assessed by immunocytochemistry (ICC) using α6 integrin. The SSCs were co-cultured on Sertoli cell feeder layer. The SSC colonies were characterized by studying the expression of SSC specific markers (viz., α6 integrin and PLZF) using ICC. The abundance of mRNAs encoding the markers of SSC (viz., β1 integrin and Oct-4) and Sertoli cells (viz., vimentin) was also assayed using quantitative real-time PCR (qPCR). Results The viability of isolated testicular cells was>90 % and the Percoll density gradient method resulted in 3.65 folds enrichment with a purity of 82.5 %. Co-culturing of SSCs with Sertoli cell feeder layer allowed the maintenance of stable SSC colonies even after one and half months of culture. The results of ICC analysis showed the expression of α6 integrin and PLZF in almost all the SSC colonies. qPCR analysis revealed a differential expression of mRNAs encoding β1 integrin, Oct-4 and vimentin markers. Conclusion Results of this study demonstrate a simple enzymatic digestion and Percoll density gradient method for isolation and enrichment of SSCs, and suitability of Sertoli cell feeder layer for long term in vitro culture of SSC in goats.Results also suggest the possible application of non-caprine antibodies against SSC specific markers for the identification and subsequent assessment of SSCs in goats.

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Developmental potential of human spermatogenic cells co-cultured with Sertoli cells

Cited by 126 publications

References 55 publications

Sperm retrieval from patients with nonmosaic Klinefelter’s syndrome by semen cytology examination

Sperm retrieval from patients with nonmosaic Klinefelter’s syndrome by semen cytology examination

Unique (Y;13) translocation in a male with oligozoospermia: cytogenetic and molecular studies

In vitro culture and characterization of spermatogonial stem cells on Sertoli cell feeder layer in goat (Capra hircus)

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