Effects Of Wuzi Yanzong Pills On Ki67, AR Expression And Sex Hormones In Testis Of Young Male Rats

Background and Objective:In recent years, the proportion of male infertility increases year by year.And among infertile couples who accounts for about 15% of the couples of child-bearing age in the world, male infertility accountsfor about 50%. A variety of reasons can lead to male infertility, of which no sperm, less sperm, weak sperm or deformed sperm are the most common causes of spermatogenesis dysfunction. The essence of traditional Chinese medicine (TCM) theory is that kidney stores sperm and regulate reproduction, which means that the amount of kidney essence decides the reproductive ability. Generations of physicians have attaches great importance to reinforcing kidney to replenish essence method in treating the male reproductive system diseasesby recognizing kidney essence deficiency is the leading cause of weakened male reproductive ability. Wu zi yan zong Pill(WP), consists of medlar, raspberry, plantain seed, dodder and fructus schisandrae, has the effect of reinforcing kidney to replenish essence and promoting reproduction, and thus is a classic formula in TCM for the treatment of male infertility.In current clinical and animal experimental studies, patients with infertility or a variety of established experimental animal models are used as the research subjects to observe the therapeutic effect of WP on the reproductive dysfunction and pathological state. However, it is not clear whether WP can promote young rat reproduction maturity under the physiological state. In addition, previous studies were confined only to general sperm function, such as sperm number and activity, but seldom explored specifically the juvenile rat testicle reproductive function based on the observation of the functional changesof sustentacular cells and spermatogenic cells in seminiferous tubule.The aim of the study is to investigate whether the WP can promote reproductive functions of young rats through observing the change of FSH, LH, T, Ki67, AR, CK-18,leydig cells and sperm cells in young male rats and to provide themodern scientific basis for kidney governs reproductively theory in traditional Chinese medicine.Research methods:Experiment Ⅰ:(1) 40 male 20-day-old Wistar rats were randomly divided into five groups:20 days group,30 days group,40 daysgroup,50 days group and 60 days group, with 8 rats in each group. Blood samples were collected and bilateral testes were excised by operation after 0 day, 10 days,20 days,30 days and 40 days respectively since grouping.(2) Rat serum hormone levels were tested by radioactive immunization:follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T).(3) Ki67、AR、CK-18 gene expression were tested by RT-PCR.Experiment II:(1) 16 male Wistar rats, only aged 20 days, were randomly divided into normal group and medicine group, with 8 rats in each group. Animals in medicine group weregiven WP by intragastric administration once a day,10 days in a row, and animalsin normal group were given physiological saline by intragastric administration once a day,10 days in a row. After 10% chloral hydrate anesthesia, blood samples were collected by abdominal aortic method, and bilateral testes were excised by operation.(2)Rat serum hormone levels were tested by radioactive immunization:FSH. LH、T.(3) Seminiferous tubule wasobserved for morphometric studies by HE staining.(4) The expression of Ki67 was measured by immunohistochemical method.(5) CK-18, AR, Ki67 gene expression were tested by RT-PCR.(6) CK-18, AR protein expression was measured by Western Blot technique.Results:Experiment I:(1) Serum hormone results:compared with 20 days group, T level of 30 days group significantly decreased (P<0.05), and there were no significant changein other groups (P>0.05); And compared with 20 days group, FSH and LH in all other groups had no significant change (P>0.05).(2) RT-PCR results:compared with 20 days group, Ki67 mRNA expression of 30 days group have no significant change (P>0.05), while Ki67 mRNA expression of other groups decreased significantly (P＜0.05); compared with 20 days group, AR mRNA and CK-18 mRNA expression decreased significantly in the rest groups (P＜0.05).Experiment II:(1) Serum hormone results:compared with normal group, medicine group T level increased significantly (P＜0.05); serum FSH and LH levels of medicine group hada rising trend (P>0.05).(2) HE staining results:no deformed sperm cells and sperm were detected in rats aged 30 days, but theirseminiferous tubule was in two distinct forms:type I that only contained SSCS and spermatocyteand type Ⅱ that also contained round sperm cells. Compared with normal group, medicine groupshowed the following changes:the proportion of type I seminiferous tubule number to all seminiferous tubule number decreased significantly (P＜0.05); the proportion of type Ⅱ seminiferous tubule number to all seminiferous tubule number increased significantly (P<0.05); average number of type Ⅰ and type Ⅱ seminiferous tubule cells increased significantly (P<0.05); and average number of mesenchymal cells increased significantly (P<0.05).(3) Immunohistochemical results:in type I seminiferous tubule, Ki67 was mainly expressed in SSCS and primary spermatocyte; while in type Ⅱ seminiferous tubule, Ki67 was mainly expressed in SSCS. And compared with normal group, Ki67 expression levels were significantly increased in type Ⅰ and type Ⅱ seminiferous tubule of the medicine group (P＜0.05).(4) RT-PCR results:compared with normal group, Ki67 mRNA and AR mRNA expression in medicine group were significantly higher (P<0.05), and there was no significant change (P>0.05) in the expression of CK-18.(5) Western blot results:compared with normal group, AR protein level increased significantly (P＜0.05) in medicine group; and there was no significant change in CK-18 protein levels (P>0.05).Conclusions:(1) During the developmental process of immature male rats, Sertoli cells matured and Spermatogenic cells proliferated due to the changes of T and AR, thus the improvement of reproductive function was promoted. Furthermore, the 30th day of male rats is a critical period of adjustment.(2) The target of WP is the testis, probably by promoting immature leydig cells proliferation, thereby enabling increased testosterone synthesis.(3) In physiological conditions, WP can promote the juvenile male rats support cell growth,and promote early SSCS and primary spermatocyte proliferation by raising T and AR level, thus promoting male reproductive function.