Experimental Study On Separation And Culture Of The Spermatogonia In Mice And Its Influencing Factors

According to the report, male infertility has been increasing in recent years and men with aspermia,oligospermia and weak-spermia have increased too. as a result, the study on the spermatogenesis and its influencing factors changes into a researching hot spot . Spermatogenesis is refering to the courses from spermatogonia through spermatocyte and spermatid reaching to the mature spermatozoa , during which experiencing polymitosis and two miosis , regulated by nerve, endocrine, paracrine and many cytokines.The study of the complicated event and its influencing factors is a foundment of prevention and therapy male infertility and develop male contraception method . At present, the study relating to this is mainly confining to the in vivo study , the in vitro study is rarely less . The results of the in vivo study show that the agent leading to the decrease of the quality and quantity of sperm mainly concluding genetic defect , chronical affection , excess exertion and mind burden , environmental pollution , infaust diet and living habit etc . Because the in vivo study is affected by complicated agent such as nerve and humor, the in vitro study becomes the inexorable trend.The most initial spermatogonia assuming as type A spermatogonia are the spermatogenic stem cells,some of which differentiate into type B spermatogonia by dissymmetric division , the other part of which still maintain as spermatogenic cells . When studying the spermatogenesis and its influencing factors in vitro,the first problem met with is how to extract and purification culture of spermatogeniccells and the factors that influence the cyto-bionomics such as cell survivaK cell proliferation and the apoptosis and so on . This study developed a preferred way of the extraction > purification culture, detection the factors contribute to the cell survivaK cell proliferation and the apoptosis of the spermatogenic cells such as the co-culture with sertoli celK tempreature> EGF^ recombinant follicle stimulating hormone (rFSH) -. hyperoxia etc . 1. The isolation and purification culture of spermatogoniaThe most initial type A spermatogonia are spermatogenic stem cells ,some of which can differentiate into type B spermatogonia by dissymmetric division ,the other carried out to autoduplicate . At present , there is no way to extract and purify the spermatogonial stem cell and researchers have to be fored to extract and purify the whole spermatogonia including the spermatogenic stem cells . This experiment screeninged a reasonable method of separation > culture and the in vitro amplification of spermatogonia, providing an important technological support for the study on the spermatogenesis and its influencing factors .The amount of spermatogonia is very less in testis.There are only 1~2 spermatogonia per 104 testis cells,consequently,it's rather difficulty to obtain sufficient spertogonia in the testis . First of all,this experiment carried out the crude separation of spermatogonia with density gradient centrifugation , then differential adherent culture and multiple medium changing to removal of the fibroblast and sertoli cells which is easier to adherence,the purity of the spermatogonia obtained reached to 75.2% . The spermatogonia was cultured in DMEM/F12 medium containing 10% fetal bovine serum(FBS) in 5% CC>2> saturated humidity ^ 37°C incubator . The spermatogonia can adherence to the somatic cells and proliferate into spermatogonia clones and chains , the conspicuous cell bridge can be seen between the catenation spermatogonia under the inverted phase contrast microscope;At the same time,spermatogonia apoptosis can be seen under laser confocal scanning microscope , too . 2.Separation and culture of sertoli cell and its co-culture with spermatogoniaThere are two kinds of cell components : spermatogenic cells and sertoli cells in seminiferous epithelium. The survival, proliferation, differentiation and the maturation of spermatogonia depends on sertoli cell in vivo . During the in vitro culture of spermatogonia, can sertoli cells contribute to the survival and proliferation of spermatogonia ? Sertoli cells were separated and co-cultured with spermatogonia as the feeder cells layer.There are more sertoli cells than spermatogonia in testis of neonatal mice.Sertoli cells can be isolated and crudely purified from testis cell suspension by centrifuge gradient centrifugation combined with differential adherent culture in the 5% CC>2, saturated humidity 37°C incubator . 2~3 hours later,the cell suspension which hadn't adherenced were discarded,the other were preserved and cultured . The sertoli cells present polygon shape,divide and proliferate rapidly.About 7 days later,a monolayer of sertoli cells can be seen,the purifity of sertoli cell can reach to 95% above after being identified by Sudan IV staining and observed under the inverted phase contrast microscope . When sertoli cells reach to about 90% confluens,they can be subcultured.During the sertoli cells subculture,we passaged for four generations,the sertoli cells maintains its property. The sertoli monolayer serve as feeder layer after being treated with lOug/ml mitomycin C . Spermatogonia isolated and enriched were seeded on it to be the experimental group;At the same time the control was established without the feeder layer. Spermatogonia of both groups were cultured in DMEM/F12 medium containing 10% fetal bovine serum in the 37°C incubator with 5% CO2 and saturated humidity . MTT colorimetric method was used to detect the cell viability and Annexin V-FITC/PI staining was used to detect apoptosis under the laser confocal scanning microscope on the 3rd day during the cell culture . The results show that adherence rate, proliferation pace and cell viability of spermatogonia in the experimental group were higher than that in the control group;Apoptosis rate of spermatogonia in experimental group was lower than that in control group.When spermatogonia clone or chains were subcultured ,they canadherence to the sertoli feeder layer to survive and proliferate . The results tells us that sertoli cells can support the survival and growth of spermatogonia in vitro culture , and support the passage cell to survive and proliferate. 3. Influence of temperature on spermatogonia culture in vitroTestis is located in scrotum which protrude out of the body and the pampiniform in testicular cord make temperature in testis much lower than body temperature, which maintain under 35°C . The experimental results in vivo show that spermatogenic cells were very sensitive to temperature, if the temperature of testis keep at 43 °C for 30min, Apoptosis occurred in spermatogonia and primary spermatocyte at leptotene stage . The disability of spermatogenesis in cryptorchidism patient is the best example of the influence of the temperature on the process of spermatogenesis. Hypothermy can also disturb the process of spermatogenesis . If the male rat was put to the environment at -10°C for 30min or -5°C for 15min,the spermatogenesis was obviously hindered, apoptosis occurred also in spermatogenic cells heavily . Up to now, the influence of temperature on survival and proliferation of spermatogenic cells , especially monofactorial experiment under limited condition in vitro has not been reported.This article studied influence of temperature on proliferation and differentiation of spermatogonia.The isolated and enriched spermatogonia were cultured in DMEM/F12 medium containing 10% FBS, in 5% CO2, saturated humidity, respectively 32°C\ 34°C\ 37°C and 39°C incubator, the adherence and survival of spermatogonia ^ the number and the size of the spermatogonia clone were observed and recorded;MTT colorimetric method was used to detect the viability of the spermatogonia, Annexin V-FITC/PI staining was used to detect the apoptosis of the spermatogonia cultured in vitro. The results show that under 34 °C condition,spermatogonia adherent rate and the survival period is much higher and longer than the other groups,the number and size of spermatogonia clones displayed that cell multiplication was productive,the viability of the spermatogonia was much higher than the other groups , apoptosis of spermatogonia was ratherless than that of the other groups;under 32°C condition, although cell adherent rate > survival period and the cell proliferation were lower than that under 34°C condition, on the 3r day of culture, viability of the spermatogonia was also lower than that under 34°C condition, but higher than that in the other groups, the apopsosis of the spermatogonia rate is higher than that under 34°C condition, but lower than that in the oter groups;under 37°C condition, the spermatogonia proliferate pace is very quick at the beginning, but on the 3rd day of the culture, the viability of the spermatogonia was the lowest and the apopsosis rate is the highest among the experimental groups.under 39°C condition , spermatogonia scarcely proliferate and apoptosis was observed quickly;on the 3rd day of the culture,the viability of the spermatogonia can not be detected, apoptosis were seen in nearly all spermatogonia. The above results show that the the tolerance of spermatogonia in hyperthermal was rather poor , even under normal body temperature 37°C, apoptosis of spermatogonia took place,under the condition higher than normal temperature , spermatogenic cells scarecely divided and proliferate,moreover, apoptosis took place quickly. 4.1nfluence of EGF on spermatogonia culture in vitroEGF in mice is a peptide whose molecular weight is low , cytology and biochemistry study show that EGF can promote cell proliferation as well as cell differentiation in many kinds of cells to quicken the protein DNA and RNA synthesis.Experiment results proved that EGF can influence the function of rat reproductive system, moerover there is obvious sex differences . EGF content in male rat is ten times higher than that in female rat;EGF can also promote division > proliferation and synthesis of steroid hormone in interstitial cells in testis, at the same time, EGF can enhance the sertoli cell activity;experiments in recent years show that expression level of EGF has close coorelation to spermatogenesis.Add 4 different concentration of EGF(l(h 10(h 200 %tt 300 ng/ml) into the culture medium and take the group without EGF as the control. Cyto-morphologychanges and cell survival and proliferation were observed under inverted phase contrast microscope;on the 3r day of the culture,MTT colorimetric method was used to detect the viability of the spermatogonia, Annexin V-FITC/PI staining was used to detect the cell apoptosis under laser confocal scanning microscope.The results showed that the growth state of spermatogonia in each experimental group was obviously better than that in control;on the 3rd day of the culture,the viability of the spermatogonia in each experimental group was higher than that in control;apoptosis rate of spermatogonia in each experimental group was lower than that in control . Moreover , in the range of 10~200 ng/ml,the effect of EGF intensified gradually with the EGF concentration stepping up,but when the concentration reaches to 200 ng/ml,the effect of EGF does not intensify with the EGF concentration stepping up.The results showed that EGF can promote the division growth of the spermatogonia culture in vitro,when the concentration reaches to 200 ng/ml,the effect was the strongest, the effect of EGF does not intensify with the EGF concentration increasing any more.5.1nfluence of follicle stimulating hormone(FSH) on spermatogonia culture in vitroFSH is a kind of glycoprotein hormone secreted by lobus anterior hypophyseos in animal.Experiment show that, for women, it can facilitate the development and the maturation of ovauian follicle;for men,it can facilitate sertoli cell to synthesize and secret androgen binding protein(ABP) consequently elevate and maitain the androgenic hormone concentration in seminiferous tubule and enhance the spermatogenesis. Can FSH play an important role on the spermatogonia culture in vitro;Except the indirect effect via sertoli cell , can it play a direct role on spermatogenesis ? This experiment focus the emphasis on the effect of the FSH on spermatogonia culture in vitro.Add different concentration of rFSH into the spermatogonia culture system as the experimental group and the control without FSH . Cytomorphology changes and the survival and proliferation were observed under inverted phase contrastmicroscope;on the 3rd day of culture, MTT colorimetric method was used to detect the viability of the spermatogonia, Annexin V-FITC/PI staining was used to detect the apoptosis of spermatogonia under laser confocal scanning microscope.The results showed that the growth state of spermatogonia in each experimental group was better than that in control;on the 3rd day of culture,the viability of spermatogonia in each experimental group was obviously higher than that in control and the apoptosis of spermatogonia in each experimental group was less than that in control . Moreover,in the range of 0.075 ~ 0.75 IU/ml FSH,the effect intensified with the FSH concentration stepping up.All results illustrated that FSH can play a direct role on spermatogonia culture in vitro and promote the division of spermatogonia and inhibit the apoptosis of spermatogonia , but the mechanism remains an enigma. 6. Influence of hyperxia on spermatogonia culture in vitroThe normal oxygen content in tha air is very important for the maintenance of human life and the living and work ability,but too much oxygen content in the environment will have the adverse effect for the human body . In recent years,the relationship between the oxygen content and the spermatogenesis attract the attention with the increasing male infertility ^ oligospermia and aspermia . Some regard that elevation of oxygen content can cure male inferlity,some propose the contradictory opinion . Which is right and which is wrong ? All wait for the verification of the scientific experiment . This experiment undertook the primary exploration.The isolated and enriched spermatogonia were seeded in DMEM/F12 medium containing 10% FBS and cultured in 34 °C incubator containing 5% CC^ 40%C>2 and saturated humidity . Take spermatogonia cultured under ordinary culture condition as the control . The adherent rate and survival of spermatogonia were observed under the inverted phase contrast microscope;on the 3rd day of culture,MTT colorimetric method was used to detect the viability of the spermatogonia, Annexin V-FITC/PI staining was used to detect the apoptosis ofthe spermatogonia under the laser confocal scanning microscope . The results showed that the cell growth state of spermatogonia in the first 2 days in the experimental group has no obvious difference from that in control group,but with the culture period prolonged , the amount of spermatogonia in experimental group decreased sharply;On the 3r day of culture , spermatogonia viability in experimental group was obviously lower than that in control , spermatogonia apoptosis in experimental group was much more than that in control . The above results show that hyperxia environment can inhibit the survival and division growth of the spermatogonia culture in vitro.