Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility. In the present study, we de...Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility. In the present study, we developed in vitro culture conditions using three-dimensional agar culture system （SACS）, which has the capacity to induce testicular germ cells to reach the final stages of spermatogenesis, including spermatozoa generation. Seminiferous tubules from testes of 7-day-old mice were enzymatically dissociated, and intratubular cells were cultured in the upper layer of the SACS in RPMI medium supplemented with fetal calf serum （FCS）. The lower layer of the SACS contained only RPMI medium supplemented with FCS. Colonies in the upper layer were isolated after 14 and 28 days of culture and were classified according to their size. Immunofluorescence and real-time PCR were used to analyse specific markers expressed in undifferentiated and differentiated spermatogonia （Vasa, Dazl, OCT-4, C-Kit, GFR- a-l, CD9 and a-6-integrin）, meiotic cells （LDH, Crem-1 and Boule） and post-meiotic cells （Protamine-1, Acrosin and SP-IO）. Our results reveal that it is possible to induce mouse testicular pre-meiotic germ cell expansion and induce their differentiation to spermatozoa in SACS. The spermatozoa showed normal morphology and contained acrosomes. Thus, our results demonstrate that SACS could be used as a novel in vitro system for the maturation of pre-meiotic mouse germ cells to post-meiotic stages and morphologically-normal spermatozoa.展开更多
Spermatogenesis is a complex process regulated by endocrine and testicular paracrine/autocrine factors. Gonadotropins are involved in the regulation of several testicular paracrine factors, mainly of the IL-1 family a...Spermatogenesis is a complex process regulated by endocrine and testicular paracrine/autocrine factors. Gonadotropins are involved in the regulation of several testicular paracrine factors, mainly of the IL-1 family and testicular hormones. Testicular cytokines and growth factors (such as IL-1, IL-6, TNF, IFN-γ, LIF and SCF) were shown to affect both the germ cell proliferation and the Leydig and Sertoli cells functions and secretion. Cytokines and growth factors are produced by immune cells and in the interstitial and seminiferous tubular compartments by various testicular cells, including Sertoli, Leydig, peritubular cells, spermatogonia, differentiated spermatogonia and even spermatozoa. Corresponding cytokine and growth factor receptors were demonstrated on some of the testicular cells. These cytokines also control the secretion of the gonadotropins and testosterone in the testis. Under pathological conditions the levels of pro-inflammatory cytokines are increased and negatively affected spermatogenesis. Thus, the expression levels and the mechanisms involved in the regulation of testicular paracrine/autocrine factors should be considered in future therapeutic strategies for male infertility.展开更多
Poor ovarian response (POR)—retrieval of 3 or fewer eggs, is a challenging issue in IVF. A retrospective study included POR women who underwent 386 IVF cycles. The data were classified in four groups according to wo...Poor ovarian response (POR)—retrieval of 3 or fewer eggs, is a challenging issue in IVF. A retrospective study included POR women who underwent 386 IVF cycles. The data were classified in four groups according to women’s age (years) during the treatment cycle: 1) 20 - 34 (n = 133), 2) 35 - 39 (n = 133), 3) 40 - 42 (n = 78), 4) 43 - 47 y (n = 42), and correlated with the characteristics of the population. The clinical pregnancy rates for groups 1, 2, 3, and 4 were: 23.3%, 12%, 2.6%, 4.8%, respectively. It was found to be significantly higher (p < 0.001) comparing group 1 with group 3 patients. The “take home baby” rate was much lower (p < 0.001) in group 3;there were no deliveries in group 4. Delivery rates for groups 1, 2, 3, and 4 were: 19.5%, 10.5%, 1.3% and 0%, respectively. Intraabdominal adhesions were more common (p = 0.005) as the cause of infertility in group 3 women compared to groups 2 and 1: 24.4% compared with 9% and 9.8%, respectively. According to multivariate regression analysis, the parameters that negatively reflect on the pregnancy rate in POR women are intraabdominal adhesions, POR in the past, and increased age. We suggest encouraging young POR patients to pursue IVF treatments since the “take home baby” rates are reasonably good.展开更多
文摘Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility. In the present study, we developed in vitro culture conditions using three-dimensional agar culture system （SACS）, which has the capacity to induce testicular germ cells to reach the final stages of spermatogenesis, including spermatozoa generation. Seminiferous tubules from testes of 7-day-old mice were enzymatically dissociated, and intratubular cells were cultured in the upper layer of the SACS in RPMI medium supplemented with fetal calf serum （FCS）. The lower layer of the SACS contained only RPMI medium supplemented with FCS. Colonies in the upper layer were isolated after 14 and 28 days of culture and were classified according to their size. Immunofluorescence and real-time PCR were used to analyse specific markers expressed in undifferentiated and differentiated spermatogonia （Vasa, Dazl, OCT-4, C-Kit, GFR- a-l, CD9 and a-6-integrin）, meiotic cells （LDH, Crem-1 and Boule） and post-meiotic cells （Protamine-1, Acrosin and SP-IO）. Our results reveal that it is possible to induce mouse testicular pre-meiotic germ cell expansion and induce their differentiation to spermatozoa in SACS. The spermatozoa showed normal morphology and contained acrosomes. Thus, our results demonstrate that SACS could be used as a novel in vitro system for the maturation of pre-meiotic mouse germ cells to post-meiotic stages and morphologically-normal spermatozoa.
文摘Spermatogenesis is a complex process regulated by endocrine and testicular paracrine/autocrine factors. Gonadotropins are involved in the regulation of several testicular paracrine factors, mainly of the IL-1 family and testicular hormones. Testicular cytokines and growth factors (such as IL-1, IL-6, TNF, IFN-γ, LIF and SCF) were shown to affect both the germ cell proliferation and the Leydig and Sertoli cells functions and secretion. Cytokines and growth factors are produced by immune cells and in the interstitial and seminiferous tubular compartments by various testicular cells, including Sertoli, Leydig, peritubular cells, spermatogonia, differentiated spermatogonia and even spermatozoa. Corresponding cytokine and growth factor receptors were demonstrated on some of the testicular cells. These cytokines also control the secretion of the gonadotropins and testosterone in the testis. Under pathological conditions the levels of pro-inflammatory cytokines are increased and negatively affected spermatogenesis. Thus, the expression levels and the mechanisms involved in the regulation of testicular paracrine/autocrine factors should be considered in future therapeutic strategies for male infertility.
文摘Poor ovarian response (POR)—retrieval of 3 or fewer eggs, is a challenging issue in IVF. A retrospective study included POR women who underwent 386 IVF cycles. The data were classified in four groups according to women’s age (years) during the treatment cycle: 1) 20 - 34 (n = 133), 2) 35 - 39 (n = 133), 3) 40 - 42 (n = 78), 4) 43 - 47 y (n = 42), and correlated with the characteristics of the population. The clinical pregnancy rates for groups 1, 2, 3, and 4 were: 23.3%, 12%, 2.6%, 4.8%, respectively. It was found to be significantly higher (p < 0.001) comparing group 1 with group 3 patients. The “take home baby” rate was much lower (p < 0.001) in group 3;there were no deliveries in group 4. Delivery rates for groups 1, 2, 3, and 4 were: 19.5%, 10.5%, 1.3% and 0%, respectively. Intraabdominal adhesions were more common (p = 0.005) as the cause of infertility in group 3 women compared to groups 2 and 1: 24.4% compared with 9% and 9.8%, respectively. According to multivariate regression analysis, the parameters that negatively reflect on the pregnancy rate in POR women are intraabdominal adhesions, POR in the past, and increased age. We suggest encouraging young POR patients to pursue IVF treatments since the “take home baby” rates are reasonably good.
