Protective effect of astragaloside IV on cadmium-induced spermatogenesis microenvironment damage in rats.

The previous study using Sertoli cells cultured in vitro has shown that the protective effects of astragaloside IV (AsIV) on cadmium (Cd)-induced damage to Sertoli cells and its membrane proteins. Yet, it is not known if AsIV has an equivalent effect on Cd-induced damage to the spermatogenesis microenvironment in rats. Using an in vivo model, Cd-induced damage to the spermatogenesis microenvironment and the protective effects of AsIV were studied. Eighteen male Sprague Dawley (SD) rats were randomly divided into three groups (n = 6/group): Cd group, Cd&AsIV group, and control group. Cd was administered to the rats in the Cd group via i.p. at 1 mg/kg body weight once daily, Cd and AsIV was administered to the rats in the Cd&AsIV group via i.p. at 1 mg/kg body weight and 10 mg/kg body weight respectively once daily, and the same volume of saline was administered to the rats in control group via i.p. once daily. The rats in the three groups were injected continuously for 5 days. Vesicular formation in the seminiferous tubules was observed in the Cd treatment group. The average optical density of claudin-11, zonal occludin-1 (ZO-1), and connexin 43 (Cx43) decreased significantly in the Cd treatment group. The ultrastructural damage of the Sertoli cells and tight junctions were also observed by electron microscopy. AsIV treatment rescued the morphologic changes of the seminiferous tubules of the testis and the ultrastructural damage of the Sertoli cells and tight junctions. The average optical density of claudin-11, ZO-1, and Cx43 also increased significantly after AsIV treatment. Cd damages the spermatogenesis microenvironment in rats, which can be rescued by AsIV treatment. These results illustrate that AsIV may also have a protective effect on Cd-induced damage to the spermatogenesis microenvironment in rats.Abbreviations: AsIV: astragaloside IV; Cd: cadmium; SD: Sprague Dawley; ZO-1: zonal occludin-1; Cx43: connexin 43; BTB: blood-testis barrier; MAPKs: mitogen-activated protein kinases; OSP: oligodendrocyte-specific protein; Cxs: connexins; GJIC: gap junctional intercellular communication; ROS: reactive oxygen species; MDA: malondialdehyde; TGF: tumor growth factor; PBS: phosphate buffer saline; BSA: bovine serum albumin.

[Substitution of cordyceps cephalosporium mycelia for cordyceps sinensis in the prescription of Shengjing Capsules: Enhanced effect on spermatogenesis impairment].

OBJECTIVE: To screen out an effective substitute in the prescription of Shengjing Capsules (SJC), observe the effects of the redeveloped New SJC (NSJC) with cordyceps cephalosporium mycelia (CCM) substituted for the ingredient cordyceps sinensis in the treatment of spermatogenesis impairment (SI), and provide some experimental evidence for its application in the treatment of male infertility and sexual dysfunction. METHODS: We equally randomized 192 male mice into 16 groups: normal saline control, SI model, high-, medium- and low-dose fermented cordycepin powder (FCP, 1.60, 0.80 and 0.40 g/kg), high-, medium- and low-dose CCM (1.60, 0.80 and 0.40 g/kg), high-, medium- and low-dose cordyceps mortierella mycelia (CMM, 1.60, 0.80 and 0.40 g/kg), high-, medium- and low-dose fermented cordyceps sinensis (FCS, 1.60, 0.80 and 0.40 g/kg), SJC (0.80 g/kg), and vitamin E (VE, 0.25 g/kg), with the SI model established in all the mice and the normal controls injected intraperitoneally with cyclophosphamide at 60 mg/kg qd for 5 consecutive days. After intragastrical medication with respective drugs, we obtained the body mass index (BMI), sexual organ coefficient, sperm count, sperm motility, and percentage of morphologically abnormal sperm (MAS) of the mice. We also randomly divided 70 male rats into 7 groups of equal number: normal control, SI model, high-, medium- and low-dose NSJC (1.12, 0.56 and 0.28 g/kg), SJC (0.56 g/kg), and VE (0.18 g/kg), the SI model constructed in the latter 6 groups of rats by gavage of adenine at 200 mg/kg qd for 5 consecutive days. After intragastrical medication with respective drugs, we examined the BMI, coefficients of sexual and renal organs, levels of reproductive hormones, testicular morphology, and fertility of the animals. RESULTS: After medication, the mice in different groups showed different degrees of improvement in the cyclophosphamide-induced slow growth, significant increases in the testicular and epididymal coefficients, sperm count, motility and viability (P < 0.05 or P < 0.01), and a remarkable reduction in the percentage of MAS (P < 0.05 or P < 0.01). The effect was particularly significant in the CCM group and therefore CCM was chosen as the best substitute ingredient in the redeveloped NSJC. Compared with the rats in other groups, those treated with NSJC exhibited significant increases in the BMI, coefficients of sexual and renal organs and levels of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T) and estradiol (E2) (P < 0.05 or P < 0.01), improvement of the pathologically damaged testicular morphology, elevation of the pregnancy rate and litter size, and recovery from adenine-induced SI. CONCLUSIONS: The redeveloped New Shengjing Capsules with cordyceps cephalosporium mycelia substituted for the ingredient cordyceps sinensis can improve fertility and reverse spermatogenesis impairment in male rats. The new prescription may also be applied to the clinical treatment of male infertility and sexual dysfunction.

Cry1 deficiency leads to testicular dysfunction and altered expression of genes involved in cell communication, chromatin reorganization, spermatogenesis, and immune response in mouse testis.

Cryptochrome (Cry)1 is essential for generating circadian rhythm in central and many peripheral oscillators; however, its role in male reproduction remains unclear. We investigated this question using Cry1 knockout (KO) mice. We found that Cry1 is necessary for normal testicular function: Cry1 deficiency increased testicular germ cell apoptosis and decreased sperm count. A transcriptome analysis showed that the expression levels of 375 genes-including 12 encoding micro (mi)RNAs-were altered in the testis of Cry1 KO mice relative to wild-type controls. A bioinformatics analysis revealed that the differentially expressed genes were related to important biological processes including cell-cell communication, metabolism, chromatin reorganization, spermatogenesis, and the immune response. An integrative analysis of miRNA-mRNA networks suggested that the 12 dysregulated miRNAs may contribute to testis disorders through negative regulation of their target mRNAs expression in testis, and interestingly, all the 12 miRNAs are predicted to target core circadian clock components. These results provide the first evidence of a correlation between dysregulation of Cry1 and male reproductive defects in mice, indicating that Cry1 plays a critical role in maintaining normal testicular function.

Increased Sat2 expression is associated with busulfan-induced testicular Sertoli cell injury.

Busulfan is a chemotherapeutic agent used to treat chronic myelogenous leukemia and other myeloproliferative disorders. Increasing evidence has demonstrated that busulfan may induce testicular dysfunction by targeting genes that are expressed in the testis. Here, we showed that spermidine/spermine N1-acetyltransferase 2 (Sat2) was present in testicular Sertoli cells, and its expression was significantly increased by busulfan treatment. To investigate the implications of Sat2 upregulation for cell growth and function, a Sat2-overexpressing TM4 Sertoli cell model was established. Increased Sat2 expression led to inhibited cell proliferation and arrested cell cycle. Based on iTRAQ proteomics analysis, we revealed that Sat2 overexpression is detrimental to cell cycle progression and cell communication, and notably, Sat2 may disturb protein metabolic processes by altering translation regulation and protein complex subunit organization. In summary, the present study provides evidence that Sat2 upregulation induces alterations in the growth and function of Sertoli cells. In testis tissue subjected to busulfan, increased expression of Sat2 can cause cellular injury and subsequent organ damage, which could lead to male infertility. Therefore, Sat2 may be a novel molecular target for treating busulfan-induced testicular toxicity.

Overexpression of PRL7D1 in Leydig Cells Causes Male Reproductive Dysfunction in Mice.

Prolactin family 7, subfamily d, member 1 (PRL7D1) is found in mouse placenta. Our recent work showed that PRL7D1 is also present in mouse testis Leydig cells, and the expression of PRL7D1 in the testis exhibits an age-related increase. In the present study, we generated transgenic mice with Leydig cell-specific PRL7D1 overexpression to explore its function during male reproduction. Prl7d1 male mice exhibited subfertility as reflected by reduced sperm counts and litter sizes. The testes from Prl7d1 transgenic mice appeared histologically normal, but the frequency of apoptotic germ cells was increased. Prl7d1 transgenic mice also had lower testosterone concentrations than wild-type mice. Mechanistic studies revealed that Prl7d1 transgenic mice have defects in the testicular expression of steroidogenic acute regulatory protein (STAR) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase cluster (HSD3B). Further studies revealed that PRL7D1 overexpression affected the expression of transferrin (TF) in Sertoli cells. These results suggest that PRL7D1 overexpression could lead to increased germ cell apoptosis and exert an inhibitory effect on testosterone production in Leydig cells by reducing the expression of certain steroidogenic-related genes. In addition, PRL7D1 appears to have important roles in the function of Sertoli cells, which, in turn, affects male fertility. We conclude that the expression level of PRL7D1 is associated with the reproductive function of male mice.

Immunohistochemical study of a membrane skeletal molecule, protein 4.1G, in mouse seminiferous tubules.

Protein 4.1 families have recently been established as potential organizers of an adherens system. In the adult mouse testis, protein 4.1G (4.1G) localized as a line pattern in both basal and adluminal compartments of the seminiferous tubules, attaching regions of germ cells and Sertoli cells. By double staining for 4.1G and F-actin, their localizations were shown to be different, indicating that 4.1G was localized in a region other than the basal and apical ectoplasmic specializations, which formed the Sertoli-Sertoli cell junction and Sertoli-spermatid junction, respectively. By electron microscopy, immunoreactive products were seen exclusively on the cell membranes of Sertoli cells, attaching to the various differentiating germ cells. The immunolocalization of cadherin was identical to that of 4.1G, supporting the idea that 4.1G may be functionally interconnected with adhesion molecules. In an experimental mouse model of cadmium treatment, in which tight and adherens junctions of seminiferous tubules were disrupted, the 4.1G immunostaining in the seminiferous tubules was dramatically decreased. These results indicate that 4.1G may have a basic adhesive function between Sertoli cells and germ cells from the side of Sertoli cells.

Evidence that long-term hyperexcitability of the sensory neuron soma induced by nerve injury in Aplysia is adaptive.

Peripheral axotomy induces long-term hyperexcitability (LTH) of centrally located sensory neuron (SN) somata in diverse species. In mammals this LTH can promote spontaneous activity of pain-related SNs, and such activity may contribute to neuropathic pain and hyperalgesia. However, few axotomized SN somata begin to fire spontaneously in any species, and why so many SNs display soma LTH after axotomy remains a mystery. Is soma LTH a side effect of injury with pathological but no adaptive consequences, or was this response selected during evolution for particular functions? A hypothesis for one function of soma LTH in nociceptive SNs in Aplysia californica is proposed: after peripheral injury that produces partial axotomy of some SNs, compensation for sensory deficits and protective sensitization are achieved by facilitating afterdischarge near the soma, which amplifies sensory input from injured peripheral fields. Four predictions of this hypothesis were confirmed in SNs that innervate the tail. First, LTH of SN somata was induced by a relatively natural axotomizing event-a small cut across part of the tail in the absence of anesthesia. Second, soma LTH was selectively expressed in SNs having axons in cut or crushed nerves rather than nearby, uninjured nerves. Third, after several weeks soma LTH began to reverse when functional recovery of the interrupted afferent pathway was shown by reestablishment of a centrally mediated siphon reflex. Fourth, axotomized SNs developed central afterdischarge that amplified sensory discharge coming from the periphery, and the after-depolarization underlying this afterdischarge was enhanced by previous axotomy.

[Effects of captopril on myocardial energy metabolism in mice with viral myocarditis].

OBJECTIVE: To explore the changes of myocardial energy production in mice with viral myocarditis, and to observe the interventional effects of captopril. METHODS: The male Balb/c mice were randomly divided into three groups: coxsackie B3 virus (CVB3) infection group (infection group), CVB3 infection group with captopril treatment (treatment group) and control group. The morphology, membrane phospholipid, as well as activities of cytochrome oxidase (CCO) and succinate dehydrogenase (SDH) of myocardial mitochondria were studied by using transmission electron microscope, morphometry and enzyme cytochemical method respectively. Reversed phase high-performance liquid chromatography (RP-HPLC) was used to analyze contents of myocardial ATP, ADP and AMP. RESULTS: A large number of mitochondria were damaged in infection group, the apparent mitochondrial membrane phospholipid deletion and localization change were observed, the activities of CCO and SDH in mitochondria declined obviously, and the contents of ATP, ADP as well as AMP declined notably. All of these were improved significantly in treatment group. CONCLUSION: The myocardial mitochondrial structure destroys and its functions decline apparently in viral myocarditis. Captopril has effects against myocardial mitochondrial structure damage and function decline, ameliorate myocardial energy metabolism.

The use of elevated divalent cation solutions to isolate monosynaptic components of sensorimotor connections in Aplysia.

A commonly used method to remove polysynaptic components of test PSPs is to elevate action potential threshold of interneurons with high extracellular concentrations of divalent cations ('Hi-Di'). Extrapolation to normal conditions requires that Hi-Di have negligible effects on synaptic transmission. We examined effects of Hi-Di on EPSPs from sensory neurons (SNs) onto motor neurons (MNs) of Aplysia in the pleural-pedal and abdominal ganglia, and in dissociated cell culture. In ganglia, standard Hi-Di solutions eliminated spontaneous input from interneurons as well as polysynaptic components of PSPs evoked by single action potentials in single SNs, but failed to block polysynaptic PSPs evoked by nerve stimulation. Hi-Di solutions had no effect on activity-dependent synaptic depression or posttetanic potentiation, or facilitation by serotonin (5-HT). Unexpectedly, standard Hi-Di solutions substantially reduced sensorimotor EPSPs in all preparations, whereas a solution containing 2.2x[Ca(2+)] and 2x[Mg(2+)] blocked the polysynaptic component of EPSPs without obvious changes to the monosynaptic component. In contrast to previous observations in Aplysia, and to predictions of the (J. Physiol. 193 (1967) 419) model, tripling the normal extracellular concentrations of Ca(2+) and Mg(2+) failed to increase sensorimotor EPSPs. Depression of EPSPs by these Hi-Di solutions may result from reduced spike invasion into presynaptic terminals.