[Linggui Fang protects the reproductive system of asthenospermia rats: An experimental study based on the L-carnitine pathway].

OBJECTIVE: To explore the protective effect of the Chinese medicinal prescription Linggui Fang (LGF) on the reproductive system of the ornidazole-induced asthenospermia (AS) rat and its possible action mechanisms. METHODS: Forty male SD rats weighing 200－230 g were equally randomized into four groups, blank control, AS model control, LGF treatment and L-carnitine (LC) intervention. The AS models were made in the latter three groups by intragastrical administration of ornidazole at 400 mg/kg. Meanwhile, the rats in the LGF group were treated intragastrically with LGF at 17.5 g/kg, those in the LC group with LC at 100 mg/kg, and the control animals with 0.5% sodium carboxymethylcellulose (CMC-Na), all once a day for 4 successive weeks. Then, all the rats were sacrificed for examination of the semen parameters, determination of the LC content and OCTN2 mRNA expression in the epididymis and observation of the histopathological changes in the testis. RESULTS: Compared with the AS model controls, the rats in the other groups showed significantly higher percentages of progressively motile sperm and total motile sperm (P < 0.01) as well as a higher LC content in the epididymis (P < 0.01), but no statistically significant difference in sperm concentration (P > 0.05). The expression of OCTN2 mRNA was remarkably upregulated in the LGF and LC groups in comparison with that in the AS model control (P < 0.05). Compared with the rats in the blank control group, the AS model controls exhibited markedly increased morphologically abnormal seminiferous tubules, irregularly arranged, with narrowed lumens and reduced numbers of sperm and sperm cells, as well as significantly increased hollow seminiferous tubules with deficient and disorderly arranged spermatogenic cells and partial epithelial degeneration and vacuolization. Those in the LGF and LC groups, however, manifested almost normal testicular histomorphology, with basically regular arrangement of different layers of seminiferous tubules. CONCLUSIONS: ①Ornidazole induces AS in rats by reducing the LC content in the epididymis, while LGF can improve the sperm motility and testicular morphology of the rats and upregulate the expression of OCTN2 mRNA in the epididymis by increasing the LC concentration.

Zinc: A Necessary Ion for Mammalian Sperm Fertilization Competency.

The importance of zinc for male fertility only emerged recently, being propelled in part by consumer interest in nutritional supplements containing ionic trace minerals. Here, we review the properties, biological roles and cellular mechanisms that are relevant to zinc function in the male reproductive system, survey available peer-reviewed data on nutritional zinc supplementation for fertility improvement in livestock animals and infertility therapy in men, and discuss the recently discovered signaling pathways involving zinc in sperm maturation and fertilization. Emphasis is on the zinc-interacting sperm proteome and its involvement in the regulation of sperm structure and function, from spermatogenesis and epididymal sperm maturation to sperm interactions with the female reproductive tract, capacitation, fertilization, and embryo development. Merits of dietary zinc supplementation and zinc inclusion into semen processing media are considered with livestock artificial insemination (AI) and human assisted reproductive therapy (ART) in mind. Collectively, the currently available data underline the importance of zinc ions for male fertility, which could be harnessed to improve human reproductive health and reproductive efficiency in agriculturally important livestock species. Further research will advance the field of sperm and fertilization biology, provide new research tools, and ultimately optimize semen processing procedures for human infertility therapy and livestock AI.

[Tongjingling improves sperm DNA integrity and reduces oxidative stress in the testis of experimental varicocele rats].

OBJECTIVE: To explore the protective effect of Tongjingling (TJL) against sperm DNA damage and oxidative stress in the rat model of experimental varicocele (EVC). METHODS: We randomly divided 75 Wistar male rats into five groups of equal number: sham operation, EVC model, high-dose TJL, mid-dose TJL, and low-dose TJL. The EVC model was established in the rats by partial ligation of the left renal vein, followed by 8 weeks of medication from the 4th week after modeling. Then we observed the general status of the rats, detected the sperm DNA fragmentation index (DFI) in the epididymis by sperm chromatin structure assay (SCSA), and measured the content of hydroperoxide (H2O2) and the activities of catalase (CAT) and superoxide dismutase (SOD) in the testis by colorimetry. RESULTS: Compared with the sham operation group, the EVC models showed significantly increased sperm DFI in the epididymis (P <0.01) and elevated level of H2O2 and activities of CAT and SOD in the testis (P <0.01). In comparison with the EVC models, the rats of the TJL groups exhibited remarkably reduced sperm DFI and H2O2 content, but increased activities of SOD and CAT. CONCLUSIONS: TJL can improve sperm DNA integrity by increasing the activities of SOD and CAT and reducing the H2O2 level and hence oxidative stress in the testis tissue.

Effects of Anethum graveolens L. on fertility in male rats.

OBJECTIVES: The effects of Anethum graveolens seed extract on fertility of male rats were investigated. METHODS: Male Wistar rats were divided into five groups according to the treatment they received during 42 days: control, low dose (0.5 g/kg) and high dose (5 g/kg) of aqueous extracts, and low dose (0.045 g/kg) and high dose (0.45 g/kg) of ethanol extracts of Anethum graveolens seed. Sperm count and motility and testosterone concentration were measured. Sections of the testes, epididymis, and seminal vesicles were stained with peroxidase-conjugated lectins of Ulex europaeus agglutinin, peanut agglutinin, Dolichos biflorus agglutinin, soy bean agglutinin and concanavalin A. The treated male rats were mated with females and the crown-rump lengths and weights of their newborn pups were measured. RESULTS: No significant differences in sperm count, sperm motility or testosterone concentration were observed in the experimental groups. However, female rats did not become pregnant after mating with rats given the high dose of the ethanol extract. The distribution of terminal sugars on the epithelial surface of the reproductive structures decreased in the experimental groups. CONCLUSION: Anethum graveolens extract decreased fertility rate by modifying some terminal sugars on the cell surface of male reproductive organs involved in sperm maturation, capacitation and oocyte recognition.

Effects of conjugated linoleic acid supplementation on fatty acid composition in the plasma, liver, and epididymal fat pads of male-Sprague Dawley rats.

The purpose of this study was to investigate the effects of conjugated linoleic acid (CLA) on the fatty acid composition of plasma, liver, and epididymal fat pads in rats. Seventy-two male Sprague-Dawley rats were divided into four groups and received beef tallow (BT), fish oil (FO), beef tallow with CLA (BTC), and fish oil with CLA (FOC). All the rats were fed an experimental diet containing 12% (wt/wt) total fat, including CLA at 1%, for 30 weeks. The fatty acid analyses of the plasma, liver, and epididymal fat pads were performed by the one-step methylation method, followed by gas chromatography. Monounsaturated fatty acid (MUFA) levels in the plasma were significantly reduced in the BTC group as compared to the BT group. The levels of C18:1 and C20:4 in the liver were significantly lower in the BTC group than in the BT group (P < .05). CLA was detected in the epididymal fat pads of the rats that did not receive supplemental CLA (the BT and FO groups), indicating de novo synthesis. CLA caused significant decreases in C18:1 fatty acid levels in both the BTC and FOC groups in epididymal fat pads. C22:6 was significantly higher in the liver microsomes of the BTC group, as compared to the BT group. MUFA was stored at higher levels in the epididymal fat pads and was significantly lower in level in the CLA-supplemented (BTC and FOC) groups as compared to the BT and FO groups (P < .05). In conclusion, dietary CLA supplementation in rats altered fatty acid composition in a tissue-specific manner. CLA was stored mainly in the epididymal fat pad. The results also suggested that CLA may inhibit the conversions of C18:0 to C18:1 and C18:2 to C20:4 fatty acid, resulting in significantly lower levels of C18:1 and C20:4 in the livers of the BTC group as compared to the BT group.

Chronic consumption of a low-fat diet leads to increased hypothalamic agouti-related protein and reduced leptin.

OBJECTIVE: This study examined the hypothesis that dietary fat under ad libitum feeding conditions influences expression levels (mRNA) of the mouse agouti-related protein (AgRP), leptin, leptin receptor (OBRb), and neuropeptide Y (NPY) at early stages of development. METHODS: C57Bl/6J male mice were placed on a high-fat diet (HFD) or a low-fat diet (LFD) shortly after weaning. Groups of mice were euthanized at various ages and real-time one-step reverse transcriptase polymerase chain reaction was used to analyze gene expression in the hypothalamus (AgRP, NPY, OBRb), the adrenal gland (AgRP), the testis (AgRP), and epididymal fat (leptin). RESULTS: Leptin expression increased linearly with age but only under the HFD despite body weight gain under both diets. This pattern of expression coincided with reduced expression of hypothalamic AgRP under an HFD, whereas OBRb and NPY did not fluctuate in response to diet. By contrast, consumption of an LFD (i.e., high carbohydrate) increased hypothalamic AgRP and suppressed adipose leptin, which is consistent with the notion that leptin could regulate AgRP centrally. In contrast, AgRP expression in the adrenal gland initially decreased and then increased with age under both diets. CONCLUSIONS: Dietary fat can have a tissue-dependent effect on AgRP that may be unfettered by leptin under an HFD.

Marginal zinc deficiency in rats decreases leptin expression independently of food intake and corticotrophin-releasing hormone in relation to food intake.

Zn deficiency reduces food intake and growth rate in rodents. To determine the relationship between Zn deficiency and the regulation of food intake, we evaluated leptin gene expression in epididymal white adipose tissue (eWAT), and hypothalamic corticotropin-releasing hormone (hCRH) and hypothalamic neuropeptide Y (hNPY) of rats Zn-deficient only to show reduced food intake and growth rate but not food intake cycling. Growing male Sprague-Dawley rats (240 g) were randomly assigned to one of four dietary groups: Zn-adequate (ZA; 30 mg/kg diet), Zn-deficient (ZD; 3 mg/kg diet), pair-fed with ZD (PF; 30 mg/kg diet) and Zn-sufficient (ZS; 50 mg/kg diet) (n 8), and were fed for 3 weeks. Food intake and body weight were measured, as were blood mononuclear cells and pancreas Zn levels. eWAT leptin, hCRH and hNPY mRNA levels were determined. Food intake was decreased by about 10 % in ZD and PF rats compared to ZA and ZS rats. Growth and eWAT leptin mRNA levels were unaffected in PF rats but were significantly (P < 0.05) decreased in ZD rats. However, hNPY showed a tendency to increase, and hCRH significantly (P < 0.05) decreased, in both ZD and PF rats. These results suggest that while leptin gene expression may be directly affected by Zn, hNPY and hCRH are likely responding to reduced food intake caused by Zn deficiency.

Tissue expression of the nuclear progesterone receptor in male non-human primates and men.

In females, progesterone is associated with reproductive functions. In males, its role and the expression of its genomic receptor are not very well understood. In attempts to achieve a hormonal male contraceptive method, gestagens are used to downregulate gonadotropin and sperm production. It is therefore essential to understand the mechanism of action of progesterone at the molecular level in males, especially in primates. This investigation was undertaken: (a) to determine whether the genomic progesterone receptor is expressed in males; and (b) to locate it in various organs that are potential targets of gestagens. Human tissues were obtained at surgery for benign prostatic hyperplasia or prostate cancer and at autopsy. Non-human primate tissues were obtained at autopsy. This study was performed by analyzing the genomic progesterone receptor by immunohistochemistry, Western blot and RT-PCR. The nuclear progesterone receptor was expressed in pituitary and hypothalamus of both monkeys and men. In the testis progesterone receptor expression was found in a few peritubular and interstitial cells, but not in germ cells. In addition, expression was detected in the epididymis, prostate and male mammary gland. Reverse transcriptase (RT)-PCR experiments indicated that progesterone receptor A and B are expressed in all tissues analyzed. These data exclude direct genomic effects of gestagens at the spermatogenic level but indicate that a male contraceptive based on gestagens might have some effects on other tissues, such as the epididymis, prostate and mammary gland.

Antispermatogenic and hormonal effects of Crotalaria juncea Linn. seed extracts in male mice.

AIM: To evaluate the antifertility activity of various extracts of Crotalaria juncea seeds in male mice. METHODS: Adult male mice were gavaged the petroleum ether, benzene and ethanol extracts of C. juncea seeds, 25 mg x (100g)(-1) x day(-1) for 30 days. On day 31 the animals were sacrificed by cervical dislocation and the testes, epididymis, vas deferens, seminal vesicles, prostate gland, bulbourethral gland and levator ani were dissected out and weighed. The organs were processed for biochemical and histological examination. RESULTS: In petroleum ether, benzene and ethanol extracts treated rats, there was a decrease in the weights of testis and accessory reproductive organs. The diameters of the testis and seminiferous tubules were decreased. Spermatogonia, spermatocytes and spermatids in the testis and the sperm count in cauda epididymis were also decreased. There was a significant reduction in the protein and glycogen contents and an increase in the cholesterol content in the testis, epididymis and vas deferens. Of the 3 extracts, the ethanol extract appeared to be the most potent in antispermatogenic activity. When the ethanol extract was tested in immature male mice, there was an antiandrogenic effect as the weights of accessory organs were reduced. CONCLUSION: The various extracts of C. juncea seeds arrest spermatogenesis and are likely to have an antiandrogenic activity.

Age-induced apoptosis in the male genital tract of the mouse.

We have examined the effects of ageing on the increase in apoptotic cells numbers in the male genital tract of the house mouse (Mus musculus). We have found that not all organs have the same response. There is an induction of apoptosis in both the epididymis and ventral prostate. However, seminal vesicles and other prostatic lobes remain unaffected. Apoptosis was assessed by several methods: TUNEL, detection of the active fragment of caspase-3 and the pattern of DNA fragmentation on agarose gels. This increase in apoptosis is related to the fall in testosterone levels, although there is only a partial decrease in androgen receptor (AR). AR is still present in all tissues and only moderately reduced in the epididymis and ventral prostate. A more intense increase of lipofuscin granules, which may be indicative of oxidative stress, occurred in these tissues. Finally, testosterone supplementation reverses the changes (both in apoptosis and lipofuscin content in the tissue), suggesting a role of androgens in these processes.

Expression and structure-function analysis of de, a sperm cysteine-rich secretory protein that mediates gamete fusion.

Rat sperm epididymal glycoprotein DE belongs to the cysteine-rich secretory protein (CRISP) family and participates in sperm-egg fusion through its binding to complementary sites on the egg surface. To investigate the molecular mechanisms underlying the role of DE in gamete fusion, in the present work we expressed DE in a prokaryotic system, and examined the relevance of carbohydrates and disulfide bonds for the biological activity of the protein. Immunofluorescence and sperm-egg fusion assays carried out in the presence of recombinant DE (recDE) revealed that this protein exhibits the ability to bind to the DE-egg binding sites and to inhibit gamete fusion, as does native DE (nDE). Comparison of the proteins indicated, however, that the inhibitory ability of recDE was significantly lower than that of nDE. This difference would not be due to the lack of carbohydrates in the bacterially expressed protein because enzymatically deglycosylated nDE was as able as the untreated protein to inhibit gamete fusion. To examine whether disulfide bridges are involved in DE activity, the presence of sulfhydryls in nDE and recDE was evaluated by the biotin-maleimide technique. Results indicated that, unlike nDE, in which all cysteines are involved in disulfide bonds, recDE contains free thiol groups. Subsequent experiments showed that reduction of nDE with dithiothreitol significantly decreased the ability of the protein to inhibit gamete fusion. Together, these results indicate that whereas carbohydrates do not have a role in DE-mediated gamete fusion, disulfide bridges are required for full biological activity of the protein. To our knowledge, this is the first study reporting the relevance of structural components for the function of a CRISP member.

Age-related changes in plasma testosterone concentrations and genital organs content of bulk and trace elements in the male dromedary camel.

There is a paucity of information regarding the influence of plasma testosterone concentrations and inorganic cations secreted in the different seminal fractions on the spermatozoon activity throughout the reproductive life of the one-humped camels. To demonstrate these relationships, the genital organs of 12 prepubertal (<3 years), 9 peripubertal (3-<5 years), 16 mature (5-<15 years) and 15 aged (>/=15 years) camels were collected from the Buraidah slaughter house (Al-Qassim Province, Saudi Arabia) during two consecutive breeding seasons (November-April) over 2 years. Plasma testosterone concentrations (mean+/-S.E.) did not exceed 1.4 ng/ml in prepubertal animals with a 3-4 fold increase in peripubertal (3.2+/-0.4 ng/ml) and mature (4.8+/-0.6 ng/ml) camels followed by about 50% decrease (2.6+/-0.3 ng/ml) in aged ones. These hormonal changes were correlated significantly with concentrations of certain elements in the testes (highest Na, Ca and Cu contents), epididymides (highest P and Fe contents), prostate (highest Zn content), and bulbo-urethral glands (highest K and Mg contents). The significance of some interrelationships among the different cations and their biological effects on sperm production and metabolic activity were discussed.

In vivo regulation of rat epididymal proteins by retinoids: analysis by two-dimensional electrophoresis.

The role of retinoids in the regulation of epididymal fluid protein expression was investigated. We compared the patterns of two-dimensional electrophoretic gels of proteins from luminal fluids, cytosols and spermatozoa (from control rats only) of control, retinoid-depleted, retinoid-depleted retinoic acid-complemented and retinoid-depleted testosterone-supplemented rats. This study compared the luminal fluid patterns from the 4 diets and observed 13 proteins whose expression was dependent on nutritional status. Eight were either absent or very weakly expressed in retinoid-depleted animals only, while their presence was obvious in control rats and in the retinoid-deficient retinoic acid- and testosterone-complemented groups. The expression of 8 proteins was greatly enhanced in retinoid-depleted testosterone-supplemented fluids as compared to control fluids. Five of the regulated proteins seemed to be captured by spermatozoa as they were observed in sperm protein patterns of control rats. These results clearly show that the synthesis of several epididymal proteins is influenced by retinoids. Since testosterone-supplemented animals on retinoid-free diet elicited the same response as retinol and retinoic acid ones, testosterone is likely to be the mediator of retinoid action on epididymal protein synthesis. Nevertheless, the observation of one protein whose expression is stimulated by retinoic acid only and is totally independent of testosterone also favors the direct influence of this retinoid.

5c,11c,14c-eicosatrienoic acid and 5c,11c,14c,17c-eicosatetraenoic acid of Biota orientalis seed oil affect lipid metabolism in the rat.

The effects of 5c,11c,14c-eicosatrienoic acid (20:3BSO) and 5c,11c,14c,17c-eicosatetraenoic acid (20:4BSO), polyunsaturated fatty acids (PUFA) contained in Biota orientalis seed oil (BSO), on lipid metabolism in rats were compared to the effects of fats rich in linoleic acid (LA) or alpha-linolenic acid (ALA) under similar conditions. The potential effect of ethyl 20:4BSO as an essential fatty acid also was examined in comparison with the ethyl esters of LA, ALA and gamma-linolenic acid (GLA). BSO- and ALA-rich fat decreased the concentration of plasma total cholesterol, high density lipoprotein cholesterol, triglyceride and phospholipid as compared to LA-rich fat. BSO was more effective in reducing plasma cholesterol concentrations than was the ALA-rich fat. Dietary BSO markedly decreased the hepatic triglyceride concentration as compared to the LA-rich or ALA-rich fats. Aortic production of prostaglandin I2 tended to decrease in rats fed BSO or ALA-rich fat compared to those fed the LA-rich fat. Adenosine diphosphate-induced platelet aggregation was similar in the three groups. The proportion of arachidonic acid (AA) in liver phosphatidylcholine (PC) of rats fed BSO was lowest compared to that of rats fed ALA-rich or LA-rich fats. Administration of 20:4BSO, ALA or GLA to essential fatty acid-deficient rats decreased the ratio of 20:3n-9 to AA in liver PC to the same extent; administration of LA was more effective. The results indicate that the effects of specific PUFA contained in BSO on lipid metabolism are different from those of LA and ALA. It is also suggested that 20:4BSO may exhibit some essential fatty acid effects.