In Vitro Contraction of Isolated Cauda Epididymal Duct Smooth Muscle as a Complimentary Approach to Physiological, Pathological, Toxicological, and Pharmacological Studies on Epididymal Function.

Contraction of cauda epididymal duct (CE) smooth muscle is one of the very first events of the seminal emission phase of ejaculation. The contraction of CE smooth muscle is governed by a complex interaction of hormones, autacoids, and by the neurotransmitters released from the epididymal intramural nerve endings, and any impairment in the CE smooth muscle contraction has the potential to impair male fertility. Apart the obvious pathophysiological and toxicological importance of CE smooth muscle contraction, modulation of CE contraction has pharmaceutical interest offering a druggable target to development of drugs to improve/impair male fertility. The in vitro contraction experiments constitute a valuable approach to an in-depth evaluation of functional and molecular changes resulting from pathologies or drug exposure. Therefore, this chapter consists in a description of in vitro pharmacological reactivity contractility of the epididymal duct in a controlled medium, maintained at 30 °C of temperature and continuously bubbled with 95% O2 and 5% CO2 to obtain cumulative concentration-response curves that has been fundamental to some of our investigations on epididymal physiology, toxicology, and pharmacology.

Sperm parameters quality and reproductive effects of methanolic extract of Alchornea cordifolia leaves on senescent male rats.

The effect of the methanolic extract of Alchornea cordifolia leaves on the fertility of senescent male rats was assessed in this study. 40 rats received daily distilled water, testosterone, 200 and 400 mg/kg of extract of Alchornea cordifolia. The reproductive organs weight, the gonadotropins, testosterone and cholesterol level, the sperm parameters, histology of the testes and epididymis were assessed. The weight of testes and prostate (400 mg/kg) significantly increased (p < 0.05) as well as the level of FHS (p < 0.001), LH and testosterone (p < 0.01) at a dose of 400 mg/kg, respectively, while the cholesterol decreased at a dose of 200 mg/kg (p < 0.05) and 400 mg/kg (p < 0.01) respectively. The testes and epididymis were full of spermatozoa particularly at a dose of 400 mg/kg. The sperm count and morphology significantly increased at both doses of 200 mg/kg (p < 0.01; p < 0.001) and 400 mg/kg (p < 0.001; p < 0.01) respectively. The sperm motion (PROG, VAP, VSL, VCL) (p < 0.001), (ALH, BCF) (p < 0.05) increased at a dose of 200 mg/kg and decreased at a dose of 400 mg/ kg. The overall results provide the strong evidence of the fertility potential of the methanolic extract of Alchornea cordifolia leaves in senescent male rats.

DNA damaging effect of paclitaxel in the epididymal sperms as a chemotherapeutic agent and possible remedies to prevent this effect: A study on reproductive potential of male cancer patients of reproductive age.

Cancer is a major public health problem, young cancer patients therefore undergo chemotherapy, and most of them may lose their fertility. DNA damage level provides important clues about the quality and reproductive potential of spermatozoa. In this study, we evaluated the levels of both DNA fragmentation and abnormal DNA integrity in the epididymal sperms of New Zealand rabbit (Oryctolagus cuniculus) after cryopreservation using the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay and the toluidine blue (TB) staining methods and assessed the effects of paclitaxel, resveratrol, l-glutamine (LG), and basal medium eagle (BME) solution on DNA damage. Paclitaxel induced the levels of both DNA damages in the sperms, but resveratrol ameliorated this effect. LG and BME supplementation to the extender prevented the sperm samples from DNA fragmentation after cryopreservation. Chemotherapy drugs containing paclitaxel can cause the sperm DNA to be damaged, and hence adversely affect the fertility of male cancer patients of reproductive age. The administration of resveratrol together with paclitaxel may ameliorate the DNA damage inducing effect of paclitaxel. Sperm banking and cryopreservation with the appropriate cryoprotectants such as LG and BME prior to cancer treatment can also be suggested to all male cancer patients of reproductive age facing cancer treatment for fertility preservation.

Modification of sperm fatty acid composition during epididymal maturation in bats.

Biochemical properties of polyunsaturated fatty acids (PUFAs) are fundamental to sperm movements. Amongst all adjustments operated during epididymal maturation, sperm membrane lipid composition is remodelled. Specifically, the proportion of PUFAs usually increases from the caput towards the cauda epididymidis. In mammals, PUFAs are predominantly acquired through the diet, which can consequently impact male fertility. We aimed at analysing to what extent n-6 and n-3 PUFAs are incorporated into sperm in the Seba's short-tailed bat (Carollia perspicillata), and at demonstrating the effect of the sperm fatty acid composition on sperm mobility. We therefore provided food varying in fatty acid composition to males of C. perspicillata and measured the fatty acid composition and mobility traits in spermatozoa collected from the caput and cauda epididymides. We found that n-6 and n-3 PUFAs and saturated fatty acids were significantly related to sperm velocity but not to the proportion of progressive sperm (i.e. motility). Concomitant to an increase in sperm velocity, the level of fatty acid saturation increased from the caput to the cauda epididymidis, while the proportion of PUFAs remained similar along the epididymis. A reduction in n-6 PUFAs counterbalanced an increase in n-3 PUFAs. The food treatments did not affect the sperm fatty acid composition. Our results suggest that a precise endogenous control rather than dietary effects determines sperm fatty acid composition in C. perspicillata.

Cryopreservation of collared peccary (Pecari tajacu L., 1758) epididymal sperm using extenders based on Tris and powdered coconut water (ACP®-116c).

SummaryThe aim of this study was to establish a functional freezing-thawing protocol for epididymal sperm of collared peccaries (Pecari tajacu L., 1758) by comparing different extenders. The epididymal sperm from 12 sexually mature males was recovered by retrograde flushing using Tris-based or coconut water-based (ACP®-116c) extenders. After initial evaluation, samples were diluted and frozen with the same extenders to which 20% egg yolk and 6% glycerol were added. After 2 weeks, thawing was performed at 37°C/60 s and sperm motility, vigour, morphology, functional membrane integrity, sperm viability, sperm plasma membrane integrity, and a computer-assisted semen analysis (CASA) were assessed. In addition, to evaluate the survival of frozen-thawed sperm, a thermal resistance test (TRT) was executed. Samples preserved using Tris were in better condition compared with those preserved using ACP®, showing higher values for most assessments performed, including CASA and the TRT (P<0.05). After determining Tris to be the better of the two extenders, additional samples were thawed using different thawing rates (37°C/60 s, 55°C/7 s, 70°C/8 s). Sperm thawed at 37°C/60 s had the greatest preservation (P<0.05) of viability (54.1 ± 5.9%) and functional membrane integrity (43.2 ± 5.4%), and had higher values for various CASA parameters. In conclusion, we suggest the use of a Tris-based extender added to egg yolk and glycerol for the cryopreservation of epididymal sperm obtained from collared peccaries. In order to achieve better post-thawing sperm quality, we suggest that samples should be thawed at 37°C/60 s.

Resveratrol attenuates inflammation and oxidative stress in epididymal white adipose tissue: implications for its involvement in improving steroidogenesis in diet-induced obese mice.

Chronic, low-grade systemic inflammation has been shown to play an important role in the development of obesity-related complications. Epididymal white adipose tissue (WAT) can influence testicular function through its endocrine function. The purpose of this study was to assess the effects of resveratrol on the epididymal WAT inflammatory response and on testicular steroidogenesis in obese individuals. Seven-week-old male C57BL/6J mice were fed a high-calorie and high-cholesterol diet (HCD group) or HCD supplemented with resveratrol (HCD+Res group) for 18 weeks. As we previously showed that resveratrol protects against Leydig cell steroidogenesis in HCD-induced obese mice, this study assessed macrophage infiltration in fat depots by measuring crown-like structure (CLS) density. Histological analysis showed that adipocyte size was significantly smaller and CLSs were less numerous in the HCD+Res group than the HCD group (P < 0.01). Additionally, resveratrol supplementation decreased Nfkb1 expression (P < 0.01) and increased the IκB-α protein abundance (P < 0.01) in epididymal WAT. Consistent with this alteration in NF-κB signaling, the expression of two classic proinflammatory cytokines, TNF-α (Tnfa) and IL-1ß (Il1b), were significantly decreased in the HCD+Res group compared with the HCD group (P < 0.01). Significant differences were also found in the expression of sirtuin1 (Sirt1) (P < 0.01) and manganese superoxide dismutase (Sod2) (P < 0.01) between the HCD and HCD+Res groups. Our data suggest that resveratrol can attenuate obesity-induced inflammation and oxidative stress in epididymal WAT, which partly accounts for its beneficial effects in testicular steroidogenesis.

Reduced glutathione and Trolox (vitamin E) as extender supplements in cryopreservation of red deer epididymal spermatozoa.

The use of assisted reproductive techniques in cervids is increasing as the commercial use of these species increase. We have tested the suitability of the antioxidants Trolox and reduced glutathione (GSH) for freezing red deer epididymal spermatozoa, aiming at improving post-thawing quality. Samples from 19 stags were frozen in a TES-Tris-fructose extender (20% egg yolk, 8% glycerol), with 1 or 5 mM of antioxidant. Motility (CASA), lipoperoxidation (malondialdehyde -MDA- production), membrane status, mitochondrial activity, acrosomal status (flow cytometry) and chromatin status (SCSA: %DFI and %HDS; flow cytometry) were assessed after thawing and after 6 h at 39°C. There were few differences between treatments after thawing, with Trolox reducing MDA production in a dose-response manner. After the incubation, sperm quality decreased and %DFI increased moderately, with no change for MDA. GSH improved motility, kinematic parameters and mitochondrial status, with a slight increase in %HDS. GSH 5 mM also increased moderately MDA production and %DFI, possibly due to enhanced metabolic activity and reducing power. Trolox maintained MDA low, but was detrimental to sperm quality. Trolox might not be appropriate for the cryopreservation of red deer epididymal spermatozoa, at least at the millimolar range. GSH results are promising, especially regarding motility improvement after the post-thawing incubation, and should be selected for future fertility trials.

Alpha-mannosidase activity in stallion epididymal fluid and spermatozoa.

The expression of α-D-mannosidase activity was fluorometrically and electrophoretically assessed in spermatozoa, epididymal fluid and homogenates of stallion epididymal tissue. Enzyme activity had regional differences; it was higher (P<0.05) in samples from the cauda epididymal region than in samples from the proximal caput region (largely composed of efferent ducts). Based on enzyme activity, as a function of pH of the assay substrate, electrophoretic analysis in native and native/SDS-PAGE conditions, and the effect of inhibitors or activators, we inferred the presence of at least two catalytically active forms of α-D-mannosidase. The neutral form of the enzyme (α-mannosidase II) was activated by Co2+, whereas the acid form (optimum pH 3.5 to 4.0) was sensitive to swainsonine (an inhibitor of α-mannosidase I), stabilized or stimulated by Zn2+, and not activated by Co2+ (activator of the neutral form). The activity of the acid form of the enzyme was highest in the epididymal fluid, where it seemed to be mainly in a secretory form. This form of the enzyme may have a role in plasma membrane remodeling associated with sperm maturation. In contrast, the activity of α-mannosidase II was higher in mature spermatozoa. It has been postulated that α-mannosidase II may act as a receptor in the recognition and binding of the complementary carbohydrate moieties present on the zona pellucida. With non-denaturing electrophoresis, α-D-mannosidase had an electrophoretic mobility of 0.35 and 0.24. When resolved by 1D and 2D SDS-PAGE (under denaturing conditions) the enzyme had a major protein band of molecular weight 154 kDa in spermatozoa and epididymal samples. Based on its properties under native conditions, we inferred that this enzyme might interact with other proteins and form transitory aggregates.

Assessment of the contraceptive efficacy of the aqueous extract of Aegle marmelos Corr. leaves in male albino rats.

The present study was conducted to evaluate the contraceptive effect of an aqueous extract from the leaves of Aegle marmelos (AMLAq) on the reproductive organs of male rats with an emphasis on reversibility. Adult male rats were treated daily with different doses of AMLAq, i.e., 150, 300 and 600 mg/kg bw/day for 60 days. The data presented in this study demonstrate that the weight of the reproductive organs was reduced significantly in all the treatment groups. AMLAq induced a significant decrease in the sperm motility and sperm density of the Cauda epididymis and testes. The reduction in fertility was 50%, 85% and 100%, respectively, in the treatment groups. The testosterone level also significantly declined. Biochemical analysis of the reproductive tissues for sialic acid, protein, glycogen, fructose, ascorbic acid, acid and alkaline phosphatase indicated a significant decrease whereas testicular cholesterol level significantly increased indicating alterations in the biochemical milieu of the genital organs. Fertility and other effects gradually returned to control levels 120 days after cessation of treatment. No clinical signs of side effects on general metabolism were detected throughout the treatment, and after withdrawal, body weight gain was similar in all groups together with no alterations in the weight of vital organs', hematological and serological parameters.

Spermatotoxic effects of Cananga odorata (Lam): a comparison with gossypol.

OBJECTIVE: To compare the antifertility effects of 50% ethanolic extract of the root bark of Cananga odorata with gossypol. DESIGN: Controlled research laboratory study. SETTING: University research laboratory. ANIMAL(S): Male albino rats (Sprague Dawley, body weight 150 +/- 5 g) bred in university animal house. INTERVENTION(S): A 50% ethanolic extract of the root bark of Cananga odorata and gossypol was administered orally for 60 days. On day 61, one third of the animals in each group were killed for various analyses. One half of the remaining animals were used for the evaluation of fertility index, and other half was maintained for 15 days more on normal diet. MAIN OUTCOME MEASURE(S): The epididymal sperm motility, morphology, and count, the testicular activities of HMG CoA reductase, 3beta-hydroxy steroid dehydrogenase, glucose-6-phosphate dehydrogenase, and testicular cholesterol and serum testosterone were assessed. RESULT(S): Differences were not observed in the sperm count and fertility index of the gossypol group in comparison with the C odorata group. But statistically significant alterations were noted in the sperm morphology as well as in the activity of HMG CoA reductase, 3beta-hydroxy steroid dehydrogenase, glucose-6-phosphate dehydrogenase, cholesterol, and protein of the testis and in serum testosterone. On withdrawal of the drugs, sperms in C odorata group became completely motile but not in the gossypol group. The active component is a 52 kd protein. CONCLUSION(S): The ethanolic extract of Cananga odorata possesses antifertility effects.

Trans-11 vaccenic acid dietary supplementation induces hypolipidemic effects in JCR:LA-cp rats.

Trans-11 vaccenic acid [VA; 18:1(n-9)] is a positional and geometric isomer of oleic acid and is the precursor to conjugated linoleic acid (CLA) in humans. Despite VA being the predominant trans monoene in ruminant-derived lipids, very little is known about its nutritional bioactivity, particularly in conditions of chronic metabolic disorders, including obesity, insulin resistance, and/or dyslipidemia. The aim of this study was to assess the potential of VA to improve dyslipidemia, insulin sensitivity, or inflammatory status in obese and insulin-resistant JCR:LA-cp rats. The obese rats and age-matched lean littermates were fed a control diet or a control diet supplemented with 1.5% (wt:wt) VA for a period of 3 wk. The incorporation of VA and subsequent conversion to CLA in triglyceride was measured in adipose tissue. Glucose and insulin metabolism were assessed via a conscious adapted meal tolerance test procedure. Plasma lipids as well as serum inflammatory cytokine concentrations were measured by commercially available assays. VA supplementation did not result in any observable adverse health effects in either lean or obese JCR:LA-cp rats. After 3 wk of feeding, body weight, food intake, and glucose/insulin metabolism did not differ between VA-supplemented and control groups. The incorporation of VA and CLA into adipose triglycerides in obese rats fed VA increased by 1.5-fold and 6.5-fold, respectively, compared with obese rats fed the control diet. The most striking effect was a 40% decrease (P < 0.05) in fasting triglyceride concentrations in VA-treated obese rats relative to obese controls. Serum Il-10 concentration was decreased by VA, regardless of genotype (P < 0.05). In conclusion, short-term dietary supplementation of 1.5% VA did not result in any detrimental metabolic effects in JCR:LA-cp rats. In contrast, dietary VA had substantial hypo-triglyceridemic effects, suggesting a new bioactivity of this fatty acid that is typically found in ruminant-derived food products.

Effect of iodine supplementation on thyroid and testicular morphology and function in euthyroid rats.

Seventy-five male weaner euthyroid rats, randomly divided into three equal groups were used to evaluate the effect of iodine supplementation in the diet on growth and spermatogenesis. From the age of six weeks, the rat groups were fed normal diet containing 0.05 mg iodine/Kg diet (A); normal diet supplemented with 0.5 mg/Kg iodine (B) and normal diet supplemented with 3.0 mg/Kg iodine for a period of 90 days. Thereafter, all three groups were fed the normal diet for another 60 days. Body weight and feed consumption were determined; morphomeric studies of thyroid glands, testes and epididymes were carried out. Spermatogenesis was evaluated with epididymal (ESC) and testicular sperm counts (TSC). Increasing iodine intake significantly (p < 0.05) decreased mean body weight from day 30 of supplementation. Iodine supplementation influenced feed conversion ratio and efficiency in feed utilization in an inconsistent pattern. Supplementation did not significantly (p > 0.05) alter the size of thyroid glands, but increased the mean weights of the testes and epididymes to levels significantly (p < 0.05) higher than values for non-supplemented rats at specific stages of the study, especially at the highest (3 ppm) level of iodine supplementation. However, supplementation resulted generally in lower sperm counts, which was significant (p < 0.05) in the case of the epididymes. The results of the study show that iodine supplementation to weaner, non-iodine deficient euthyroid rats at 3ppm not only retard weight gain but could also reduce fertility by lowering epididymal sperm counts.

The inhibitory effects on adult male reproductive functions of crude garlic (Allium sativum) feeding.

AIM: to investigate the effects of crude garlic on adult male rat reproductive functions. METHODS: Thirty male rats were divided into five groups: group 1 (untreated) and groups 2, 3, 4 and 5 were fed for 30 days with 5%, 10%, 15% and 30% crude garlic, respectively. Testes and accessory organs were weighed and some markers were assessed. Light and electron microscopy observations were also performed. RESULTS: A significant decrease was observed in the body weight of groups 4 (14%; P < 0.01) and 5 (20%; P < 0.01); of the prostate weight in group 5 (29.1%; P < 0.05) and of seminal vesicle weight in groups 3 (14.4%; P < 0.01), 4 (18.3%; P < 0.01) and 5 (27.3%; P < 0.01). In contrast, testis and epididymis weights were unchanged. In epididymis tissue, the alpha glucosidase activity and the spermatozoa density were unchanged. The treatment resulted in a significant decrease in testosterone serum levels in groups 3 (77.3%; P < 0.01), 4 (77.3%; P < 0.01) and 5 (90.9%; P < 0.01), associated with a significant increase in LH serum levels (P < 0.01). Testicular histology showed a dose-dependent increase in the percentage of empty seminiferous tubules. Moreover, testicular function was affected; a significant decrease in phosphatase acid activity (P < 0.01) and testosterone (P < 0.05) contents were observed. CONCLUSION: Crude garlic consumption during 1 month reduced testosterone secretion and altered spermatogenesis at 10%, 15% and 30% doses.

[Comparison of the anti-fertility effects of four extracts from the roots of Rhynchosia volubilis Lour].

OBJECTIVE: To compare the anti-fertility effects of the four extracts from the roots of Rhynchosia volubilis Lour on male mice, that is, ethanolic extract, ethyl acetate extract, n-butanol extract and aqueous extract. METHODS: Four extracts from the roots of Rhynchosia volubilis Lour (1%, 0.1 ml/10 g), were administered orally for 11 weeks to adult male mice. The fertility and testicular function of the mice were assessed by mating tests and analyses of sperm motility in cauda epididymides and biochemical and histological indexes in the blood samples and reproductive organs. RESULTS: The four extracts, especially aqueous extract, gradually decreased the pregnancy rate of the experimental mice from the 77th day of the treatment, with an obvious reduction in the number of spermatozoa. Morphological observation of the reproductive organs by light microscopy showed that the numbers of the secondary spermatocytes and spermatids were decreased in varied degrees, and the seminiferous tubules were disarranged, while the numbers and shapes of and spermatids were decreased in varied degrees, and the seminiferous tubules were disarranged, while the numbers and shapes of spermatogonia, Sertoli cells and Leydig cells remained unchanged. CONCLUSION: The four extracts from the roots of Rhynchosia volubilis Lour all have anti-fertility effects on male mice, and that of the aqueous extract is more obvious.

Androgenic regulation of novel genes in the epididymis.

The epididymis is critically dependent on the presence of the testis. Although several hormones, such as retinoids and progestins, and factors secreted directly into the epididymal lumen, such as androgen binding protein and fibroblast growth factor, might play regulatory roles in epididymal function, testosterone (T) and its metabolites, dihydrotestosterone (DHT) and estradiol (E2), are accepted as the primary regulators of epididymal structure and functions, with the former playing the greater role. To ascertain the molecular action of androgens on the epididymis, three complementary approaches were pursued to monitor changes in gene expression in response to different hormonal milieux. The first was to establish changes in gene expression along the epididymis as androgenic support is withdrawn. The second was to determine the sequence of responses that occur in an androgen deprived tissue upon re-administration of the two metabolites of T, DHT and E2. The third was to study the effects of androgen withdrawal and re-administration on gene expression in immortalized murine caput epididymidal principal cells. Specific responses were observed under each of these conditions, with an expected major difference in the panoply of genes expressed upon hormone withdrawal and re-administration; however, some key common features were the common roles of genes in insulin like growth factor/epidermal growth factor and the relatively minor and specific effects of E2 as compared to DHT. Together, these results provide novel insights into the mechanisms of androgen regulation in epididymal principal cells.

Selenoprotein P is required for mouse sperm development.

Selenoprotein P (SEPP1), an extracellular glycoprotein of unknown function, is a unique member of the selenoprotein family that, depending on species, contains 10-17 selenocysteines in its primary structure; in contrast, all other family members contain a single selenocysteine residue. The SEPP1-null (Sepp1(-/-)) male but not the female mice are infertile, but the cellular basis of this male phenotype has not been defined. In this study, we demonstrate that mature spermatozoa of Sepp1(-/-) males display a specific set of flagellar structural defects that develop temporally during spermiogenesis and after testicular maturation in the epididymis. The flagellar defects include a development of a truncated mitochondrial sheath, an extrusion of a specific set of axonemal microtubules and outer dense fibers from the principal piece, and ultimately a hairpin-like bend formation at the midpiece-principal piece junction. The sperm defects found in Sepp1(-/-) males appear to be the same as those observed in wild-type (Sepp1(+/+)) males fed a low selenium diet. Supplementation of dietary selenium levels for Sepp1(-/-) males neither reverses the development of sperm defects nor restores fertility. These data demonstrate that SEPP1 is required for development of functional spermatozoa and indicate that it is an essential component of the selenium delivery pathway for developing germ cells.

The effects of long-term vitamin E treatment on gene expression and oxidative stress damage in the aging Brown Norway rat epididymis.

The male reproductive tract of the Brown Norway rat is profoundly affected by aging. In the epididymis, the site of sperm maturation and storage, aging results in histological and biochemical changes that are suggestive of oxidative stress. Vitamin E is a potent lipid-soluble antioxidant that ameliorates the oxidative stress load associated with some chronic disease conditions. To determine the effects of long-term (18-mo) vitamin E deficiency and supplementation on aging in the epididymis, we assessed gene expression changes using cDNA microarrays and lipid peroxidation using immunohistochemical detection of 4-hydroxynonenal (4-HNE) in 24-mo-old rats. Plasma vitamin E levels were significantly lower in vitamin E-deficient animals and higher in vitamin E-supplemented animals compared with age-matched controls. Vitamin E deficiency resulted in increased expression of oxidative stress-related transcripts along the epididymis. This effect was most marked in the corpus epididymidis, where expression of glutathione S-transferases pi, 8, and mu, as well as superoxide dismutase, increased by over 50%. The effect of vitamin E supplementation on the expression of oxidative stress-related transcripts was primarily decreased expression; however, the magnitude of the gene expression changes was smaller than that observed for vitamin E deficiency. 4-HNE immunostaining was present throughout the epididymis in control animals. Vitamin E deficiency both increased the intensity and altered the distribution of 4-HNE staining, while vitamin E supplementation had no observable effect. In summary, we found that long-term vitamin E treatment alters the expression of oxidative stress-related transcripts. Moreover, long-term vitamin E deficiency exacerbates the effects of age on the accumulation of oxidative stress damage in the epididymis.

Effect of olfactory stimulation with flavor of grapefruit oil and lemon oil on the activity of sympathetic branch in the white adipose tissue of the epididymis.

It has been mentioned in the field of aromatherapy that the fragrance of grapefruit has a refreshing and exciting effect, which suggests an activation of sympathetic nerve activity. It also can be assumed that the activation of sympathetic nerve innervating the white adipose tissue (WAT) facilitates lipolysis, then results in a suppression of body weight gain. This study addressed the effect of olfactory stimulation with the scent of grapefruit oil and lemon oil on the efferent nerve activity in the sympathetic branch of the WAT of the epididymis in the anesthetized rat. The results of the experiments demonstrated that the flavor of the citron group increased sympathetic nerve activity to WAT in anaesthetized rat, which suggests an increase in lipolysis and a suppression in body weight gain.

Nickel deficiency diminishes sperm quantity and movement in rats.

Early studies on nickel essentiality with rats and goats indicated that nickel deprivation impaired reproductive performance. Nickel also has been found to influence cyclic nucleotide gated channels (CNG); these types of channels are important in sperm physiology. Thus, two experiments were conducted to test the hypothesis that nickel deficiency affects sperm physiology in a manner consistent with nickel having an essential function related to CNG channel functions. The experiments were factorially arranged with four treatment groups of eight weanling rats in each. In experiment 1, the treatments were supplemental dietary nickel of 0 and 1 mg/kg and N(omega)-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor) added to the drinking water (50 mg/100 mL) the last 3 wk of an 8-wk experiment. In experiment 2, the treatments were supplemental dietary nickel at 0 and 1 mg/kg and supplemental dietary sodium chloride (NaCl) at 0 and 80 g/kg. The NaCl and L-NAME variables were included to act as stressors affecting CNG channel activity. The basal diet contained per kilogram about 27 microg of nickel and 1 g of sodium. After 8 wk in experiment 1 and 16 wk in experiment 2, urine while fasting and testes and epididymis in both experiments, and seminal vesicles and prostates in experiment 2 were harvested for analysis. Nickel deprivation significantly decreased spermatozoa motility and density in the epididymides, epididymal transit time of spermatozoa, and testes sperm production rate. Nickel deficiency also significantly decreased the weights of the seminal vesicles and prostate glands. Excessive NaCl had no effect on sperm physiology; however, it decreased prostate gland weights. The findings support the hypothesis that nickel has an essential function that possibly could affect reproductive performance in higher animals, perhaps through affecting a CNG channel function.

Ionic calcium levels in oviduct explant-conditioned media from an Australian marsupial, the brushtail possum (Trichosurus vulpecula) and its relevance to in vitro fertilization.

Gametes from the brushtail possum (Trichosurus vulpecula), an Australian marsupial, require exposure to oviductal cells and/or their secretions before sperm binding and penetration of the zona pellucida can occur. Sperm-egg fusion, the next critical step in fertilization has not previously been reported in vitro. Here we describe the refinement of an oviduct epithelial cell (OEC) explant culture system using two different media to obtain in vitro sperm-egg fusion in the brushtail possum for the first time. Conditioned media from OEC explant cultures were supplemented with either 1% fetal calf serum (FCS) or 1 mg/ml polyvinyl alcohol and used for co-culture of epididymal sperm and superovulated eggs. Under these conditions zona penetration rates varied from 0 to 46% and sperm-egg fusion from 0 to 20%. Analysis of explant conditioned media indicated that qualitative and quantitative differences between batches could account, at least partially, for the large variability in zona penetration rates. Conditioned media that contained approximately 1 mM of ionic calcium were most effective for achieving sperm capacitation, zona binding, and penetration and sperm-egg fusion. The reorientation of the sperm head to T-shape, an indicator of capacitation in the brushtail possum, was closely linked with the concentration of calcium present in vitro.