An integrase facilitates long-lasting foreign gene expression in vivo in mouse spermatogenic cells

The objective of the present study was to attain long-lasting foreign gene expression in vivo in spermatogenic cells in the mouse testis for establishing spermatogenic-cell mediated gene transformation. Prior to in vivo gene transfer, surgical cryptorchidism was performed by retaining the testis into the abdominal cavity for 1 month to remove differentiated spermatogenic cells. Subsequently, in vivo gene transfer was conducted by electroporation with a lacZ reporter gene in combination with a retroviral integrase gene, and the testis was descended immediately to the scrotum to recover from the cryptorchidism, and restart spermatogenesis. At 1 month post-transfection in vivo, lacZ gene expression was detected in some spermatocyte-like cells in seminiferous tubules of the mouse testis. However, the recovery period of 1 month appeared to be too short, since no elongated and fully differentiated spermatids were found. At 2 months post-transfection, fully differentiated spermatogenic cells expressing the lacZ gene, albeit at low frequency, were detected when the integrase gene was co-transfected, while virtually no lacZ-positive cells were found in the absence of the integrase gene. It was concluded, therefore, that stable transformation of spermatogenic cells in vivo would be facilitated by integrase gene co-transfection.     

Elucidating the identity and behavior of spermatogenic stem cells in the mouse testis

Spermatogenesis in mice and other mammalians is supported by a robust stem cell system. Stem cells maintain themselves and continue to produce progeny that will differentiate into sperm over a long period. The pioneering studies conducted from the 1950s to the 1970s, which were based largely on extensive morphological analyses, have established the fundamentals of mammalian spermatogenesis and its stem cells. The prevailing so-called A(single) (A(s)) model, which was originally established in 1971, proposes that singly isolated A(s) spermatogonia are in fact the stem cells. In 1994, the first functional stem cell assay was established based on the formation of repopulating colonies after transplantation in germ cell-depleted host testes, which substantially accelerated the understanding of spermatogenic stem cells. However, because testicular tissues are dissociated into single-cell suspension before transplantation, it was impossible to evaluate the A(s) and other classical models solely by this technique. From 2007 onwards, functional assessment of stem cells without destroying the tissue architecture has become feasible by means of pulse-labeling and live-imaging strategies. Results obtained from these experiments have been challenging the classical thought of stem cells, in which stem cells are a limited number of specialized cells undergoing asymmetric division to produce one self-renewing and one differentiating daughter cells. In contrast, the emerging data suggest that an extended and heterogeneous population of cells exhibiting different degrees of self-renewing and differentiating probabilities forms a reversible, flexible, and stochastic stem cell system as a population. These features may lead to establishment of a more universal principle on stem cells that is shared by other systems.     

Effects of calpain and Rho-kinase inhibitors on the acrosome reaction and motility of fowl spermatozoa in vitro

At the avian body temperature of 40 degrees C, intact fowl spermatozoa require Ca(2+) for the initiation of motility and a combination of both Ca(2+) and homogenized inner perivitelline layer (IPVL) together to induce the acrosome reaction. Within the range of 1-100 micromol/l, neither PD 150606 (a Ca(2+)-dependent calpain inhibitor) nor Y-27632 (an inhibitor of Ca(2+)-dependent Rho-kinase) were able to inhibit the acrosome reaction induced by the presence of Ca(2+) and IPVL. However, PD 150606, although not Y-27632, was able to inhibit sperm motility initiated by Ca(2+), as well as motility initiated by calyculin A -- a specific inhibitor of protein phosphatases, which also initiates sperm motility at 40 degrees C. The addition of PD 150606 did not reduce the ATP concentrations of intact spermatozoa, nor the motility of demembranated spermatozoa. Immunoblot analysis of sperm extract using a polyclonal antibody against calpain 12 revealed a cross-reacting protein of approximately 80 kDa. These results suggest that Rho-kinase is not involved in the regulation of the acrosome reaction or of motility in fowl spermatozoa. In contrast, calpain appears to be involved in the regulation of flagellar movement, but not izn that of the acrosome reaction. Furthermore, it seems that endogenous calpain is present in the cytoplasmic matrix and/or the plasma membrane, but not retained in the axoneme and/or accessory cytoskeletal components.     

Temperature dependence of intracellular Ca2+ homeostasis in rat meiotic and postmeiotic spermatogenic cells

The hypothesis that intracellular [Ca2+] is a cell parameter responsive to extreme temperatures in rat meiotic and postmeiotic spermatogenic cells was tested using intracellular fluorescent probes for Ca2+ and pH. In agreement with this hypothesis, extreme temperatures induced a rapid increase of cytosolic [Ca2+] in rat pachytene spermatocytes and round spermatids. Oscillatory changes in temperature can induce oscillations in cytosolic [Ca2+] in these cells. Intracellular [Ca2+] homeostasis in round spermatids was more sensitive to high temperatures compared with pachytene spermatocytes. The calculated activation energies for SERCA ATPase-mediated fluxes in pachytene spermatocytes and round spermatids were 62 and 75 kJ mol(-1), respectively. The activation energies for leak fluxes from intracellular Ca2+ stores were 55 and 68 kJ mol(-1) for pachytene spermatocytes and round spermatids, respectively. Together with changes in cytosolic [Ca2+], round spermatids undergo a decrease in pH(i) at high temperatures. This temperature-induced decrease in pH(i) appears to be partially responsible for the increase in cytosolic [Ca2+] of round spermatids induced by high temperatures. This characteristic of rat meiotic and postmeiotic spermatogenic cells to undergo an increment in cytosolic Ca2+ at temperatures > 33 degrees C can be related to the induction of programmed cell death by high temperatures in these cells.     

Protective effect of rutin against ultraviolet b-induced cyclooxygenase-2 expression in mouse epidermal cells

Rice bran was heated at 120°C for 0 to 30 min to extend the oxidative stability. Also, effects of visible light irradiation on the crude rice bran oil (RBO) from rice bran heated at 120°C for 20 min were studied. As heat treatment time increased from 0 to 30 min, rice bran had significantly high oxidative stability at 40°C for 12 days (p<0.05). Headspace oxygen content in rice bran without heat treatment decreased significantly (p<0.05) whereas those in heat treated rice bran did not change significantly (p>0.05). Results of acid value and conjugated dienoic acid (CDA) confirmed the higher oxidative stability of heat treated rice bran. γ-Oryzanol content was not significantly different among crude RBO during heat treatment and storage (p>0.05). Visible light irradiation caused similar degree of lipid oxidation in crude RBOs from rice bran irrespective of heat stabilization for 48 h, which may be due to the presence of photosensitizers in crude RBO like chlorophylls. This study showed that heat treatment was not effective to enhance the oxidative stability of RBO under visible light irradiation and products containing rice bran should be stored in the dark conditions.     

Long-term storage of mouse spermatozoa after evaporative drying

To determine if mouse spermatozoa could be preserved long-term without using liquid nitrogen, mouse spermatozoa in trehalose-EGTA solution were partially evaporatively dried under nitrogen gas (5 min at flow rate10 l/min) and stored for 1 week and 5 months at 4, -20, and -80 degrees C before intracytoplasmic sperm injection. Fertilization rates were neither different with spermatozoa stored at 4, -20, or -80 degrees C for 1 week or 1, 3, and 5 months respectively, nor blastocyst formation rates with spermatozoa stored for 1 week and 1 month. However, spermatozoa stored at 4 and -20 degrees C for 3 months resulted in fewer blastocysts (35.1 and 54.3% respectively) when compared with spermatozoa stored at -80 degrees C (74.4%). Blastocyst formation rates using spermatozoa stored for 5 months at -20 degrees C (57.4%) or -80 degrees C (74.5%) were not significantly different from those stored for 3 months at the same temperatures respectively, but were significantly better than those stored for 5 months at 4 degrees C (10.2%). Blastocysts derived from spermatozoa stored for 3 and 5 months at -20 and -80 degrees C respectively, were then transferred to pseudopregnant mothers to develop into healthy liveborn offspring. No significant differences were found in embryo transfer rates (number of pups born/number of embryos transferred), weaning rates, or sex ratios of resultant pups, which were healthy and reproductively sound. These results demonstrate for the first time that partially evaporatively dried mouse spermatozoa in trehalose-EGTA solution can be preserved for long term at -20 and -80 degrees C. The possibility that the storage temperature must be less than the glass transition temperature is discussed.     

Expression of Col1a1, Col1a2 and procollagen I in germ cells of immature and adult mouse testis

The objective of this study was to compare the expression of Col1a1, Col1a2, and procollagen I in the seminiferous tubules of immature and adult mice and to characterize the cellular expression pattern of procollagen I in germ cells during spermatogenesis in order to provide necessary groundwork for further functional studies in the process of spermatogenesis. Microarray analysis demonstrated that Col1a1 and Col1a2 were abundantly expressed in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice, and the expression levels of Col1a1 and Col1a2 mRNA were validated using a semi-quantitative RT-PCR assay. Western blot analysis further confirmed that procollagen I was expressed at a higher level in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice. Immunohistochemical analysis revealed that type A spermatogonia were positive for procollagen I in the testis of 6-day-old mice, whereas Sertoli cells were negative for this protein. The in vivo procollagen I staining in type A spermatogonia was corroborated in spermatogonia exhibiting a high potential for proliferation and the ability to form germ cell colonies in in vitro culture. Moreover, procollagen I was also detected in type A spermatogonia, intermediate spermatogonia, type B spermatogonia, and preleptotene spermatocytes in the adult mouse testes, but positive staining disappeared in more differentiated germ cell lineages detaching from the basement membrane, including leptotene spermatocytes, pachytene spermatocytes, round spermatids and elongated spermatids. These data suggest that Col1a1, Col1a2 and procollagen I are associated with type A spermatogonia and play a potential role in mediating the detachment and migration of germ cells during spermatogenesis.     

Effects of ACTH and expression of the melanocortin-2 receptor in the neonatal mouse testis

ACTH has been shown to stimulate androgen production by the fetal/neonatal mouse testis through the melanocortin type 2 receptor (MC2R). This study was designed to localize the expression of MC2R in the neonatal mouse testis and characterize the effects of ACTH on testicular androgen production. Using immunohistochemistry, MC2R was localized to the fetal-type Leydig cell population of the neonatal testis. ACTH caused a time-dependent increase in cyclic AMP (cAMP) and testosterone production by isolated cells with an increase in cAMP apparent in < 3 min. There was no additive effect of maximally stimulating doses of ACTH and human chorionic gonadotropin (hCG). Androgen production in response to ACTH and hCG was reduced by UO126 and dexamethasone, which are the inhibitors of ERK1/2 and phospholipase A2 respectively. Expression of mRNA encoding StAR was increased fourfold by both ACTH and hCG, although expression of mRNA encoding for steroidogenic enzymes was not markedly affected. The potency of N-terminal fragments of ACTH to stimulate androgen production was similar to that seen previously in the adrenal. Data indicate that both LH and ACTH, acting through their respective receptors, stimulate steroidogenesis by fetal-type Leydig cells via arachidonic acid, protein kinase A, and ERK1/2 activation of StAR.     

海参精囊水提物对模型小鼠睾丸生精细胞凋亡及 Bcl-2和Bax表达的影响

目的 探讨海参精囊水提物对模型小鼠睾丸生精细胞凋亡的影响及相关蛋白的表达情况。方法 昆明种小白鼠72只随机分6组,除正常空白组用生理盐水外其他各组均腹腔注射环磷酰胺(CP,28mg/kg),每天1次,连续5天,复制睾丸生精障碍小鼠模型;同时治疗组每天灌胃不同剂量的药物1次,连续4周。用TUNEL法检测各组小鼠睾丸生精细胞的凋亡情况;用免疫组化的方法检测Bcl-2和Bax的表达,并与模型组进行比较。结果 海参精囊水提物能够抑制小鼠睾丸生精细胞的凋亡,使Bcl-2的表达增加,Bax的表达降低,与模型组比较有统计学意义(P<0.05、0.01)。结论 海参精囊水提物对环磷酰胺所致生精障碍模型小鼠的生精细胞凋亡有一定的抑制作用,其作用可能与调控Bcl-2和Bax的表达有关。     

Angiotensin II stimulates cAMP production and protein tyrosine phosphorylation in mouse spermatozoa

Angiotensin II (AII), found in seminal plasma, has been shown to stimulate capacitation in uncapacitated mammalian spermatozoa. The present study investigated the location of AII receptors on spermatozoa and AII's mechanism of action. AT1 type receptors for AII are present on the acrosomal cap region and along the whole of the flagellum of both mouse and human spermatozoa. Because combinations of low concentrations of AII and either calcitonin or fertilization-promoting peptide (FPP), both known to regulate the adenylyl cyclase (AC)/cAMP signal transduction pathway, elicited a significant response, this study investigated the hypothesis that these peptides act on the same pathway. AII was shown to significantly stimulate cAMP production in both uncapacitated and capacitated mouse spermatozoa and this was associated with increases in protein tyrosine phosphorylation. Using an anti-phosphotyrosine antibody to visualize the location of tyrosine phosphoproteins within individual cells, AII significantly stimulated phosphorylation within 20 min in both the head, especially in the acrosomal cap region, and the flagellum, especially in the principal piece, of uncapacitated mouse spermatozoa; combined AII + FPP was stimulatory within 5 min. In addition, Western blotting revealed that AII stimulation increased phosphorylation in a number of tyrosine phosphoproteins in both uncapacitated and capacitated mouse spermatozoa, with some being altered only in the latter category of cells. These results support the hypothesis that AII stimulates AC/cAMP in mammalian spermatozoa.     

The conceptus increases secreted phosphoprotein 1 gene expression in the mouse uterus during the progression of decidualization mainly due to its effects on uterine natural killer cells

Within the mouse endometrium, secreted phosphoprotein 1 (SPP1) gene expression is mainly expressed in the luminal epithelium and some macrophages around the onset of implantation. However, during the progression of decidualization, it is expressed mainly in the mesometrial decidua. To date, the precise cell types responsible for the expression in the mesometrial decidua has not been absolutely identified. The goal of the present study was to assess the expression of SPP1 in uteri of pregnant mice (decidua) during the progression of decidualization and compared it with those undergoing artificially induced decidualization (deciduoma). Significantly (P<0.05) greater steady-state levels of SPP1 mRNA were seen in the decidua when compared with deciduoma. Further, in the decidua, the majority of the SPP1 protein was localized within a subpopulation of granulated uterine natural killer (uNK) cells but not co-localized to their granules. However, in addition to being localized to uNK cells, SPP1 protein was also detected in another cell type(s) that were not epidermal growth factor-like containing mucin-like hormone receptor-like sequence 1 protein-positive immune cells that are known to be present in the uterus at this time. Finally, decidual SPP1 expression dramatically decreased in uteri of interleukin-15-deficient mice that lack uNK cells. In conclusion, SPP1 expression is greater in the mouse decidua when compared with the deciduoma after the onset of implantation during the progression of decidualization. Finally, uNK cells were found to be the major source of SPP1 in the pregnant uterus during decidualization. SPP1 might play a key role in uNK killer cell functions in the uterus during decidualization.     

Effects of flavonoids on CYP1 expression in RL95-2 endometrial carcinoma cells

RL95-2 endometrial cancer cells were used to study cytochrome P450-mediated chemopreventative mechanisms of four flavonoids found in foods. To investigate enzymatic CYP1 inhibition, intact cells were induced with benzo(a)pyrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Quercetin, kaempferol and myricetin inhibited CYP1 activity dose-dependently with IC₅₀s ranging from 2.2 to 4 μM; while amentoflavone was inactive. Further experiments were designed to determine if flavonoids also interacted with the AhR or caused a decrease in CYP1 protein or mRNA expression. CYP1A1 protein expression was inhibited in cells co-treated with TCDD and quercetin, kaempferol or myricetin compared with TCDD alone, but amentoflavone was ineffective. Relatively higher (∼7-fold) basal levels of CYP1B1 protein were not significantly affected by flavonoid treatments. In general, at the message level significant inhibition of induced CYP1A1 or CYP1B1 was not detected following flavonoid cotreatment. Despite the common inhibitory effects of quercetin, kaempferol, and myricetin on induced CYP1A1-dependent activity and protein expression, the mechanisms of CYP1 inhibition in this cell line are complex and dependent on the CYP gene, AhR inducer and the flavonoid.     

Vitamin D3 regulation of body fat,cytokines, and calpain gene expression

BACKGROUND: We conducted an in vivo experiment to determine whether vitamin D₃ acts as a fat synthesizer and/or meat tenderizer in mice. At 6 weeks of age, 20 male C57BL/6 wild‐type mice were randomly divided into two groups (10 mice per group) and fed a modified AIN93G diet with (vitamin D₃ diet) or without (basal diet) 10 IU 25‐OH‐cholecalciferol kg^?3 for 3 weeks. RESULTS: When vitamin D₃ was fed to mice for 3 weeks, body fat was significantly increased compared to mice fed a basal diet. There was, however, no difference in body weight between the two groups. Vitamin D₃ increased the gene expressions of pro‐inflammatory cytokines and peroxisome proliferator‐activated receptor gamma, but decreased interleukin‐15 in adipose tissue through nuclear vitamin D receptor and uncoupling protein‐2 signals. The muscle inducible nitrate oxide synthase content of mice fed vitamin D₃ was higher than those fed a basal diet, while muscle arginase l showed a reverse phenomenon. longissimuss dorsi muscle of vitamin D₃‐fed mice showed more severe fat deposition than those fed a basal diet. Vitamin D₃ amplified muscle u‐ and m‐calpain protein content and suppressed muscle calpastatin protein content. CONCLUSION: These findings suggest that vitamin D₃ can be used as a fat synthesizer and meat tenderizer in meat‐producing animals. Copyright © 2011 Society of Chemical Industry     

Gene expression in the mouse preimplantation embryo

Mouse preimplantation development represents a tightly controlled programme of gene expression and cell division, which starts with the fertilized egg and ends with implantation of the blastocyst approximately 4.5 days later. Spatial and temporal differences in gene expression underpin establishment of axes at the two-cell stage and development of the trophectoderm and inner cell mass after embryo compaction at the eight-cell stage. Approximately 15 700 mouse genes expressed during preimplantation development have been identified from cDNA sequences deposited in the UniGene database of the National Institutes of Health. This inventory of preimplantation genes is the starting point for identifying signalling modules that function in preimplantation development.     

Investigation on CAST, CAPN1 and CAPN3 porcine gene polymorphisms and expression in relation to post-mortem calpain activity in muscle and meat quality

This study aimed to detect variability in CAST, CAPN1 and CAPN3 porcine genes and to investigate the effect of CAST and CAPN1 polymorphisms on the activity of native and autolyzed μ-calpain and m-calpain, measured from 1 to 72 h post-mortem in Longissimus dorsi (LD) muscle of 30 pigs. Effects of polymorphisms on meat quality parameter such as pH, color and drip loss were also evaluated. Samples carrying CAST EU137105:g.76,872AA genotype showed higher autolyzed μ-calpain activity 24 and 72 h post-mortem, as well as lower drip loss values. Expression of CAST, CAPN1 and CAPN3 was assessed in LD muscles divergent for shear force. Higher CAST and CAPN3 expression was found in LD with high shear force (P<0.2), confirming a direct role for calpastatin but not for calpain 3 in meat tenderization. In conclusion, CAST gene affected post-mortem activation time of calpain and drip loss.     

钙蛋白酶的活性调节及其与肉嫩度的关系

钙蛋白酶(calpain)是与肉嫩度相关的重要内源性蛋白酶之一,广泛存在于细胞质中,且需要一定量的Ca2 激活。研究表明,钙蛋白酶在Ca2 的激活下能降解肌原纤维蛋白,从而改善肉品嫩度。但是,由于钙蛋白酶抑制蛋白(calpastatin)等因素对钙蛋白酶活性的抑制作用,钙蛋白酶的活性调节成为一系列高度可调的复杂过程。在综述钙蛋白酶结构与功能关系的基础上,进一步阐述了钙蛋白酶的活性调节如何影响肉品嫩度。