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6 TRANSGENESIS IN SPERMATOGONIAL STEM CELLS
Innovations in Assisted Reproductive Technologies V. Most recently revolutionary data were presented, whereby mouse ovaries or even bone marrow may possess mitotically active germ cells that continuously replenish the pool of immature follicles. These Germline Stem Cells (GSC) may exist in the mouse ovary and/or bone marrow and regenerate the primordial follicle pool. These observations contradict the basic doctrine of reproductive biology whereby most mammalian females lose the capacity for germ-cell renewal during fetal life, such that a fixed reserve of germ cells (oocytes) enclosed within follicles is endowed at birth. One may speculate that the GnRH-a protective effect may possibly be through protection of the undifferentiated GSC, who ultimately generates de-novo primordial follicles. Indeed, the observation of temporary, high, reversible FSH concentrations in a third of our patients, several months after the chemotherapy and GnRH-a co-treatment, even in those who spontaneously conceived later on, may point towards reversible gonadotoxicity. The possible de-novo formation of follicles by the surviving germline stem-cells brings about a decrease in FSH concentration and return of regular cycles, ovulation, and even gestations. Multicenter, prospective, randomized studies are awaited to substantiate the in-vivo effect of GnRH-a as an unequivocal means for minimizing follicular apoptosis. Most recently, the resulta of the Italian, five year, multicenter, prospective, randomized studiy has validated the protective role of GnRH-a in preservation of ovarian function despite chemotherapy in young women with breast cancer. 5 THE MECHANISMS OF ENDOMETRIOSIS AND THEIR IMPACT ON FERTILITY K. Buehler. Centre for Gynaecological Endocrinology & Reproductive Medicine, Langenhagen, Hanover & Wolfsburg, Germany Endometriosis is a prevalent disease usually associated with subfertility. A supposed link between presence of endometriosis and subfertility is supported by the facts that endometriosis is seen more frequently in infertile women than in fertile controls and the cumulative conception rate is lower in women with endometriosis. Own clinical studies on fallopian tube flow and perfusion pressure were reported [1]. When diagnosis of endometriosis is performed by laparoscopy and the fallopian tubes permeability is proven, treatment options like cycle monitoring or cycle optimisation with Clomiphen or Gonadotropine with or without intrauterine insemination are recommended normally. But there is a lot of evidence that in women with endometriosis tubal function is significantly reduced even when the blue dye test is positive. Another factor is that oocyte maturation in endometriotic milieu seems to be disturbed and oocyte quality is reduced. So, if in women with endometriosis and associated subfertility treatment option by the natural ways is attempted it should not exceed a restricted number of cycles. Especially, taking into consideration that prolonged ovarian stimulation could induce adverse effects on endometriosis extracorporeal fertilisation in the so-called ultra-long GnRH agonist protocol should be a first-line approach in the management of infertility and endometriosis [2]. Reference(s) [1] B¨ uhler K. Diagnostik der Endometriose. Aus der Sicht des niedergelassenen Gyn¨ akologen. Endometriose 1998;Kongreßheft 98:3 10 [2] Dmowski WP, Pry M, Ding J, Rana N. Cycle-specific and cumulative fecundity in patients with endometriosis who are undergoing controlled ovarian hyperstimulation-intrauterine
S3 insemination or in vitro fertilisation-embryo transfer. Fertil Steril 2002;78:750 6 6 TRANSGENESIS IN SPERMATOGONIAL STEM CELLS W.G. Cao1 , H.W. Dou1 , M.X. Cui2 , Y.H. Ma1 , H.Y. Hu3 , Y.X. Li3 . 1 IAS-CAAS, Beijing, China, 2 CAAS, Beijing, China, 3 Beijing Ovine Breeding Center, Beijing China Gene therapy has potential applications in reproductive medicine and clinical genetics. However, most currently used methods are inefficient. Here we introduce a more efficient technique in transgenesis, i.e. transgenesis though spermatogonia using an ovine animal model. Spermatogonia were enzymatically isolated from 3-month-old ram testes, purified by discontinuous Percoll gradient centrifugation, and cultured on the monolayer of testis somatic cells in DMEM/F-12 supplemented with 10 ng/ml of EGF and 0.01% 2-mercaptoethanol, with or without 2.5% FBS in a humidified atmosphere of 95% air 5% CO2 at 37ºC. Medium was changed every 2 3 days and the monolayer changed monthly. Cells and their morphological changes in culture were studied and photographed with a camera installed on a Nikon inverted microscope (TS-100). The cells were also stained with PGP 9.5, BrdU and C-kit to help identify the types, mitosis and meiosis of the germ cells. Spermatogonia were collected after culture and cryopreserved in liquid nitrogen. Transgenesis in spermatogonia was done in vitro by e-trans in 6-well plate. The best results of thransgensis was found at 250 volts for 5 ms and repeat twice in serum free medium with more than 75% cells express report genes (GFP and RFP) and oIGF-1. In the depletion experiments, four 3-month-old Han rams (multiparous) were used. The animals were treated with buslfan (2.5 mg/kg body weight) every three weeks for three times. After three weeks from the last treatment, the GFP trnsfected spermatogonia were transplanted into the testes of the recipient rams through rete testis guided by ultra sound. The samples of testes were collected after two weeks and embedded in OCT and frozen at 86C to protect the expressed proteins. Cryosections were performed using a Leica Cryotome 195, observed and photographied with an AMG all-inone microscope. Results: (1) A number of clones/colonies of spermatogonia were formed in a couple of weeks of culture in the culture conditions, and the spermatogonia in the culture maintained over an experimental period of 3 months (2) Treatment with buslfan resulted in significant reduction in the size of testes and complete depletion of germ cells in rams; (3) the transplanted GFP expression spermatogonia in the foster testes continue to express GFP vividly in most somniferous tubules. oIGF-1 expression was also detected by RT-PCR, Southern blot and Western blot. 7 PROTEOMICS ANALYSIS OF MALE REPRODUCTIVE PHYSIOLOGY BY TOONA SINENSIS ROEM S.-J. Chang1 , W.-J. Yu2 , C.-C. Chang3 , Y.-H. Chen1 . 1 Dept. of Life Sciences, National Cheng Kung University, Taiwan, 2 Dept. of Biotechnology, Huangkung University, Taiwan, 3 Dept. of Living Science, Tainan University of Technology, Taiwan Oxidative stress has been implicated in the male infertility. Our previous studies demonstrated that several extracts from Toona sinensis Roem leaves (TSL) exhibited anti- or pro-oxidant effects on the sperm functions. However, the mechanism of these TS extracts on the sperm functions is not clear. In this study, we employed differential proteomic analysis using 2D-gel electrophoresis combined with RP-nano-HPLC-ESI-MS/MS to analyze the protein expression in testes of rats under oxidative stress induced by injecting H2 O2 and fed with TS extracts
S3 insemination or in vitro fertilisation-embryo transfer. Fertil Steril 2002;78:750 6 6 TRANSGENESIS IN SPERMATOGONIAL STEM CELLS W.G. Cao1 , H.W. Dou1 , M.X. Cui2 , Y.H. Ma1 , H.Y. Hu3 , Y.X. Li3 . 1 IAS-CAAS, Beijing, China, 2 CAAS, Beijing, China, 3 Beijing Ovine Breeding Center, Beijing China Gene therapy has potential applications in reproductive medicine and clinical genetics. However, most currently used methods are inefficient. Here we introduce a more efficient technique in transgenesis, i.e. transgenesis though spermatogonia using an ovine animal model. Spermatogonia were enzymatically isolated from 3-month-old ram testes, purified by discontinuous Percoll gradient centrifugation, and cultured on the monolayer of testis somatic cells in DMEM/F-12 supplemented with 10 ng/ml of EGF and 0.01% 2-mercaptoethanol, with or without 2.5% FBS in a humidified atmosphere of 95% air 5% CO2 at 37ºC. Medium was changed every 2 3 days and the monolayer changed monthly. Cells and their morphological changes in culture were studied and photographed with a camera installed on a Nikon inverted microscope (TS-100). The cells were also stained with PGP 9.5, BrdU and C-kit to help identify the types, mitosis and meiosis of the germ cells. Spermatogonia were collected after culture and cryopreserved in liquid nitrogen. Transgenesis in spermatogonia was done in vitro by e-trans in 6-well plate. The best results of thransgensis was found at 250 volts for 5 ms and repeat twice in serum free medium with more than 75% cells express report genes (GFP and RFP) and oIGF-1. In the depletion experiments, four 3-month-old Han rams (multiparous) were used. The animals were treated with buslfan (2.5 mg/kg body weight) every three weeks for three times. After three weeks from the last treatment, the GFP trnsfected spermatogonia were transplanted into the testes of the recipient rams through rete testis guided by ultra sound. The samples of testes were collected after two weeks and embedded in OCT and frozen at 86C to protect the expressed proteins. Cryosections were performed using a Leica Cryotome 195, observed and photographied with an AMG all-inone microscope. Results: (1) A number of clones/colonies of spermatogonia were formed in a couple of weeks of culture in the culture conditions, and the spermatogonia in the culture maintained over an experimental period of 3 months (2) Treatment with buslfan resulted in significant reduction in the size of testes and complete depletion of germ cells in rams; (3) the transplanted GFP expression spermatogonia in the foster testes continue to express GFP vividly in most somniferous tubules. oIGF-1 expression was also detected by RT-PCR, Southern blot and Western blot. 7 PROTEOMICS ANALYSIS OF MALE REPRODUCTIVE PHYSIOLOGY BY TOONA SINENSIS ROEM S.-J. Chang1 , W.-J. Yu2 , C.-C. Chang3 , Y.-H. Chen1 . 1 Dept. of Life Sciences, National Cheng Kung University, Taiwan, 2 Dept. of Biotechnology, Huangkung University, Taiwan, 3 Dept. of Living Science, Tainan University of Technology, Taiwan Oxidative stress has been implicated in the male infertility. Our previous studies demonstrated that several extracts from Toona sinensis Roem leaves (TSL) exhibited anti- or pro-oxidant effects on the sperm functions. However, the mechanism of these TS extracts on the sperm functions is not clear. In this study, we employed differential proteomic analysis using 2D-gel electrophoresis combined with RP-nano-HPLC-ESI-MS/MS to analyze the protein expression in testes of rats under oxidative stress induced by injecting H2 O2 and fed with TS extracts