Journal of Reproduction & Infertility

Journal of Reproduction & Infertility

The effects of rFSH and Testosterone on in vitro maturation of mouse round spermatid in co-culture with Vero cells

Authors
Aliraza Ajeen 1
Mansoureh Movahedin 1
Mojtaba Rezazadeh Valojerdi 1
Anooshirvan Kazemnejad 2
1 Anatomy Department, Faculty of Medical Sciences, Tarbiyat Modares University, Tehran, Iran
2 Department of Biostatistics, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Abstract
Co-culture systems have important roles in maintenance of spermatogenic cells and process of spermatogenesis. These systems are used for in vitro maturation of germ cells and overcome on differentiation arrest of spermatogenic cells. FSH and Testosterone hormones are important in initiation and maintenance of spermatogenesis. Lack of these hormones causes spermatogenesis deficiency in vivo. In this study, the effects of both co-culture system with Vero cell and co-culture supplemented with rFSH and Testosterone were determined on maturation of round spermatids. Cell suspension was isolated from testis of NMRI male mice (8-12 weeks old) and divided into three parts. Suspensions in three groups include: control (culture on DMEM with 10% FBS), experimental 1 (culture on monolayer of Vero cell) and experimental 2 (culture on monolayer of Vero cell supplemented with rFSH and Testosterone), were cultured for 96-hours. The numbers of round, elongating and elongated spermatids, before and after of culture were recorded for 96-hr using light microscope. Survival rates of all kind of spermatids were evaluated using trypan blue test and the results of each group were compared statistically by repeated measure ANOVA test. The results of this study showed that in co-culture system on the first 24-hr, the number of round spermatid cells were reduced but elongating spermatid cells increased significantly(P < 0.0001). In co-culture system supplemented with rFSH and Testosterone, the numbers of elongated and elongating spermatid cells increased significantly after 24 and 48 hours respectively (P < 0.001) but after that the number of all kind of spermatid cells were reduced. Viability rates of all kinds of spermatid cells reduced during 96-hours culture and there were no significant differences. In conclusion, the results of this study confirms that round spermatid cells by using of co-culture system with and without hormones can progress into elongating over a short period (maximum 48 hours). Vero cell culture supplemented with rFSH and Testosterone can support in vitro maturation and viability of spermatogenic cells better than Vero cells without hormones.
Keywords
CO
culture
In vitro Maturation
Infertility
rFSH
Spermatid
Spermatogenesis arrest
testosterone
Barrat C.L.R., Grudzinsky J.G., Yovich, J.L. Spermatogenesis in gametes: the spermatozoon. Cambridge University Press: Cambridge.1995; 245-251.
Nomen M.N., Mousa M.M., Karasky A.O. Testicular biopsy in azoospermia and severe oligozoospermia. Arch Androl.1984;12:109-122.
Hosoi Y., Miyake M., Utsumu K., Iritani A. Development of rabbit oocytes after microinjection of spermatozoa. (Abstr.331). Proc11th Int. Cong Anim Reprod Artif. Insemin. ,1988.
Goto K., Kinoshita, A., Ogawa K. Fertilization of bovine oocytes by the injection of immobilized, killed spermatozoa. Vet Rec.1990;127:517-520.
Ahmadi A., Ng Sc., Liow, S.L. Intracytoplasmic sperm injection of mouse oocytes with 5mM Ca2+ at different intervals. Hum Reprod.1995;10:431-435.
Palermo G., Joris H., Devoroey, P., Van Steirtghem A.C. Pregnancies after intracytoplasmic injection single spermatozoon into an oocyte. Lancet.1992;340:17-18.
Balaban B., Urman B., Isiklar A. Progression to the blastocyst stage of embryos derived testicular round spermatids. Hum Reprod. 2000; 15:1377-1382.
Kimura Y., Yanagimachi R. Mouse oocytes injected with testicular spermatozoa or round spermatids can develop in to normal offspring. Development.1995;121:2397-2405.
Tesarik J. Sperm or spermatid conception? Fertil Steril.1997;68:214-216.
Bongso A., Fong C.Y., Ng, S.C., Ratnam, S. The search for improved in-vitro systems should not be ignored: Embryo co-culture may be one of them. Hum Reprod.1993;80:1155-1162.
Kervancioglu M.E., Djahanbakhch O., Aitken R.J. Epithelial cell co-culture and the induction of sperm capacitation. Fertil Steril.1994;61:1103-1108.
Mansour R.T., Aboulghar M.A., Serour G.I. The life span of sperm motility and pattern in co-culture. Fertil Steril.1995;63:660-662.
Cremades N., Bernabeu R., Barros A., Sousa M. In-vitro maturation of round spermatids using co-culture on vero cells. Hum Reorid. 1999; 14:1287-1293.
Cremades N., Sousa M., Bernabeu R., Barros, A. Developmental potential of elongating & elongated spermatids after in-vitro maturation of isolated round spermatids. Hum Reprod.2001; 16:1938-1944.
Tesarik J., Greco E. Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone. Hum Reprod. 1998; 13:2772-2781.
Tesarik J., Greco E. Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone. Hum Reprod. 1998;13:2772-2781.
Nematollahi N., Rezazadeh Valojerdi M. Effect of vero cell coculture on the development of frozen-thawed two-cell mouse embryos. J Assist Reprod Gen.1999;16:380-384.
Paria B.C., Dey S.K. Preimplantation embryo development in vitro cooperative interactions among embryos and role of growth factors. Proc Natl Acad Sci.1990;87:4756-4760.
Chen H.F. Peptides extracted from vero cell cultures overcome the blastocyst block of mouse embryos in a serum-free medium. J Assist Reprod Gen . 1994;11:165-171.
Menck M.C., Guyader- Joly C., Peyton N., Le Boyrhis, D., Lobo, R.B., Renard, J.P., Heyman, Y. Benefical effects of vero cells for developing IVF bovine eggs in two different co-culture systems. Reprod Nutr Dev.1997;37:141-150.
Maeda J., Kotsaji F., Negaimi A., Kamitami N., Tominagat T. In vitro development of bovine embryos in conditioned media for bovine granulose cells and vero cells cultured in exogenous protein and aminoacid free chemically defined human tubal fluid medium. Biol Reprold. 1996;54:930-936.
Tanaka A., Nagayoshi M., Awata S., Mawatari Y., Tanaka I., And Kusunoki H. Completion of meiosis in human primary spermatocytes through in vitro coculture with Vero cells. Fertil Steril.2003;79:795-801.
Jamal H.S., Amarin Z.O. Round spermatid separation and in vitro maturation. Saudi Med J.2000;21: 960-963.
Tesarik J., Guido M., Mendoza C. Humn spermatogenesis in vitro: Respecive effect of follicle-stimulatig hormone and testosterone on meiosis, spermiogenesis,and sertoli cell apoptpsis. J Clin Endocrinol Metab.1998;83:4467-4473.
Tesarik J., Nagy P., Abdelmassih R., Greco E., Mendoza C. Pharmacological concentrations of follicle-stimulating hormone and testosterone improve the efficacy of in vitro germ cell differentiation in men with maturation arrest. Fertil Steril.2002;77:245-251.
Tsutsumi O. and Takami, O.A. Physiological role of epidermal growth factor in male reproductive function. Science.1986;233:975-977.
Chen y., Dicou E., Diakiew D. Characterization of nerve growth factor precursor protein expression in rat round spermatids and the trophic effects of nerve growth factor in the maintenance of sertoli cell viability. Mol Cell Endocrinol.1997;127:129-136.
Sun, T., Wreford, N.G., Roberston, D.M. and De Krester, D.M. Quantitive cytological studies of spermatogenesis in intact and hypophysectomized rats: identification of androgen – dependent stages. Endocrinol.1990;127:1215-1223.
Tesarik, J. and Mendoza, C. In vitro differentiation of germ cells from frozen testicular biopsy specimens. Hum Reprod.2000;15:1713-1716.
Meachem SJ, Wreford NG, Robertson DM, McLachlan RI. Androgen action on the restoration of spermatogenesis in adult rats: effects of human chorionic gonadotrophin, testosterone and flutamide administration on germ cell number. Int J Androl. 1997;20(2):70-9.
Baarends W.M., Grootegoed J.A. Molecular biology of male gametogenesis. In Fauser, B.C., Rutherford, A.J., Strauss, J.F. and Van Steirteghem, A. (Edititors), Molecular biology in reproductive medicine. Parthenon New York, 1999; pp.271-295.
O´Donnel L., Mc Lachlan R.T., Wreford N.G. Testosterone withdrawal promotes stage specific detachment of round spermatids from the rat seminiferous epithelium. Biol Reprod.1996; 55:895-901.
Print C.G., Loveland K.L. Germ cell suicide: new insights into apoptosis during spermatogenesis. Bio Essay.2000;22:423-430.
Tesarik J., Mendoza C. and Greco E. In-vitro maturation of immature human male germ cells. Mol Cell Endocrinol.2000;166:45-50.
Journal of Reproduction & Infertility
Volume 4, Issue 4
Autumn 2003
Pages 295-305