Role of the hypothalamus in ghrelin effects on reproduction: sperm function and sexual behavior in male mice.

In brief: Ghrelin signals to the hypothalamus inhibit reproduction during times of food scarcity. In this study, we demonstrate that ghrelin impairs sperm quality in male mice. Abstract: Ghrelin (GHRL) is an orexigenic peptide that has been investigated as one of the signals responsible for the reproductive performance of mammals under fluctuating metabolic conditions. Central GHRL administration impairs spermatogenesis in mice by regulating the hypothalamic-pituitary-gonadal axis function. In the present study, the hypothalamus role as a mediator of GHRL effects on sperm fertilizing capacity and male sexual behavior was evaluated. After 42 days of hypothalamic GHRL infusion or artificial cerebrospinal fluid, in vitro and in vivo sperm fertilizing capacity, testicular α-tubulin, speriolin gene expression and spermatic α-tubulin protein were evaluated. Hypothalamic expression of genes Kiss1, Gpr54 and Gnrh was also studied. The second group of animals was infused with one time only GHRL or artificial cerebrospinal fluid into the hypothalamus to evaluate the effects on sexual behavior. Results demonstrated that chronic GHRL administration to male mice significantly increased the percentages of pre-implantation embryo loss and the number of post-implantation embryo loss. In relation to the gene expression, our results show a relative decrease of Kiss1, Gpr54 and Spatc1. Although no significant differences were observed in the quantitative expression of α-tubulin protein, qualitative changes in its expression pattern were observed. In addition, a dual effect on sexual behavior was observed: 40% of the treated animals showed a significant reduction in the number of mounts and intromissions, while a 60% showed a significant decrease in ejaculation latency vs control animals. In conclusion, our results provide evidence that central GHRL administration possibly induces failure in embryo development and/or implantation in the females mated with treated males, possibly because of a negative effect in the α-tubulin pattern.

Effects of dietary omega-3 PUFAs on growth and development: Somatic, neurobiological and reproductive functions in a murine model.

Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) are relevant to fetal and infant growth and development. Objective: to assess whether long-term exposure to dietary ω-3 PUFA imbalance alters pre- and/or postnatal pups' development and reproductive function later in life. Mice dams were fed with ω-3 PUFA Control (soybean oil, 7%), Deficient (sunflower oil, 7%) or Excess (blend oil; 4.2% cod-liver+2.8% soybean) diet before conception and throughout gestation-lactation and later on, their pups received the same diet from weaning to adulthood. Offspring somatic, neurobiological and reproductive parameters were evaluated. Excess pups were lighter during the preweaning period and shorter in length from postnatal day (PND) 7 to 49, compared to Control pups (P<.05). On PND14, the percentage of pups with eye opening in Excess group was lower than those from Control and Deficient groups (P<.05). In Excess female offspring, puberty onset (vaginal opening and first estrus) occurred significantly later and the percentage of parthenogenetic oocytes on PND63 was higher than Control and Deficient ones (P<.05). Deficient pups were shorter in length (males: on PND14, 21, 35 and 49; females: on PND14, 21 and 42) compared with Control pups (P<.05). Deficient offspring exhibited higher percentage of bending spermatozoa compared to Control and Excess offspring (P<.05). These results show that either an excessively high or insufficient ω-3 PUFA consumption prior to conception until adulthood seems inadvisable because of the potential risks of short-term adverse effects on growth and development of the progeny or long-lasting effects on their reproductive maturation and function.

Reproductive performance of male mice after hypothalamic ghrelin administration.

It has been demonstrated that food intake and reproductive physiology are both simultaneously modulated to optimize reproductive success under fluctuating metabolic conditions. Ghrelin (GHRL) is an orexigenic peptide identified as the endogenous ligand of the growth hormone secretagogue receptor that is being investigated for its potential role on reproduction. Considering that data available so far are still limited and characterization of GHRL action mechanism on the reproductive system has not been fully elucidated, we studied the participation of hypothalamus in GHRL effects on sperm functional activity, plasma levels of gonadotropins and histological morphology in mice testes after hypothalamic infusion of 0.3 or 3.0 nmol/day GHRL or artificial cerebrospinal fluid (ACSF) at different treatment periods. We found that GHRL 3.0 nmol/day administration for 42 days significantly reduced sperm concentration (GHRL 3.0 nmol/day = 14.05 ± 2.44 × 106/mL vs ACSF = 20.33 ± 1.35 × 106/mL, P < 0.05) and motility (GHRL 3.0 nmol/day = 59.40 ± 4.20% vs ACSF = 75.80 ± 1.40%, P < 0.05). In addition, histological studies showed a significant decrease percentage of spermatogonia (GHRL 3.0 nmol/day = 6.76 ± 0.68% vs ACSF = 9.56 ± 0.41%, P < 0.05) and sperm (GHRL 3.0 nmol/day = 24.24 ± 1.92% vs ACSF = 31.20 ± 3.06%, P < 0.05). These results were associated with a significant reduction in luteinizing hormone and testosterone plasma levels (P < 0.05). As GHRL is an orexigenic peptide, body weight and food intake were measured. Results showed that GHRL increases both parameters; however, the effect did not last beyond the first week of treatment. Results presented in this work confirm that central GHRL administration impairs spermatogenesis and suggest that this effect is mediated by inhibition of hypothalamic-pituitary-gonadal axis.

Role of ghrelin in fertilization, early embryo development, and implantation periods.

In order to clarify the physiological role of ghrelin in gestation, we evaluated the effects of administration of exogenous ghrelin (2 or 4 nmol/animal per day) or its antagonist (6 nmol/animal per day of (d-Lys3)GHRP6) on fertilization, early embryo development, and implantation periods in mice. Three experiments were performed, treating female mice with ghrelin or its antagonist: i) starting from 1 week before copulation to 12 h after copulation, mice were killed at day 18 of gestation; ii) since ovulation induction until 80 h later, when we retrieved the embryos from oviducts/uterus, and iii) starting from days 3 to 7 of gestation (peri-implantation), mice were killed at day 18. In experiments 1 and 3, the antagonist and/or the highest dose of ghrelin significantly increased the percentage of atrophied fetuses and that of females exhibiting this finding or a higher amount of corpora lutea compared with fetuses (nCL/nF) (experiment 3: higher nCL/nF-atrophied fetuses: ghrelin 4, 71.4-71.4% and antagonist, 75.0-62.5% vs ghrelin 2, 46.2-15.4% and control, 10-0.0%; n=7-13 females/group; P<0.01). In experiment 2, the antagonist diminished the fertilization rate, and both, ghrelin and the antagonist, delayed embryo development (blastocysts: ghrelin 2, 62.5%; ghrelin 4, 50.6%; and antagonist, 61.0% vs control 78.4%; n=82-102 embryos/treatment; P<0.0001). In experiment 3, additionally, ghrelin (4 nmol/day) and the antagonist significantly diminished the weight gain of fetuses and dams during pregnancy. Our results indicate that not only hyperghrelinemia but also the inhibition of the endogenous ghrelin effects exerts negative effects on the fertilization, implantation, and embryo/fetal development periods, supporting the hypothesis that ghrelin (in 'adequate' concentrations) has a physiological role in early gestational events.

Developmental and neurobehavioral effects of perinatal exposure to diets with different omega-6:omega-3 ratios in mice.

OBJECTIVE: To investigate in mice the effect of diets enriched with soy or sunflower oil with different omega-6:omega-3 ratios on gestation, reproductive success, physical maturation, and the neurobiological development of the pups. METHODS: Dams were assigned, throughout gestation and lactation, to different groups: a commercial diet (CD), a soy oil-enriched diet (SOD), or a sunflower oil-enriched diet (SFOD). Measurements during gestation were dams' body weights and daily food intakes. Measurements in the offspring were physical parameters (body weight, body length, body mass index, fur appearance, pinna detachment, incisor eruption, eye opening, and puberty onset) and behavioral preweaning tests (surface righting reflex, negative geotaxis, and cliff avoidance). RESULTS: The SOD and SFOD dams became significantly heavier than the CD dams from gestational days 14 and 19, respectively, to parturition. There were no significant differences in gestational length or food consumption during pregnancy or lactation or in maternal weight during lactation. Diets did not modify litter size, sex ratio, survival index at weaning, or body weight. The SFOD and SOD offspring were significantly shorter than the CD offspring at weaning. The mean offspring physical scores of SOD and SFOD offspring were higher than CD offspring and simple reflexes were earlier in the SOD and SFOD groups. In SFOD offspring, puberty onset was significantly delayed, at postnatal days 26 and 27 in male and female offspring, respectively. CONCLUSION: This study suggests that the maintenance of an adequate omega-6:omega-3 ratio is necessary for the optimal growth and development of murine offspring. In populations that do not have sufficient provision of polyunsaturated fatty acids in the diet, their consumption would be advisable during gestation and lactation because these improve most neurodevelopmental outcomes included in this study.

Chronic administration of nonsteroidal-antiinflammatory drugs (NSAIDS): effects upon mouse reproductive functions.

Although nonsteroidal-antiinflamatory drugs (NSAIDs) are widely employed, reproductive side effects of prostaglandins long-term inhibition remain unknown. The objective of the present study was to evaluate the effects of chronic low/moderate NSAIDs doses upon mice reproductive functions. Male or female mice were injected (i.p. for 60 or 35 days respectively) with: ibuprofen doses A, B or C (0.56, 1.12 or 1.68 mg/100 g/day respectively) or piroxicam doses A, B or C (0.028, 0.056 or 0.084 mg/100 g/day respectively). Parameters evaluated were: a) in females, spontaneous and induced ovulation, oocyte maturity and spermatozoa migration through genital tract, b) in males, epididymal spermatozoa concentration, motility, viability, resistance to hypoosmotic shock, acrosomal status and membrane maturity and c) in both genders, in vitro and in vivo fertilization, reproductive hormones plasma levels and cyclooxigenase inhibition in reproductive tissues. In females ibuprofen (dose A) elicited a significant reduction in spontaneous and induced ovulation rates and piroxicam (dose A) diminished the concentration of spermatozoa found in the uterus after mating. Males treated with ibuprofen (dose B) showed a reduction in the in vitro fertilization ability. Our data reveal that chronic administration of ibuprofen or piroxicam can exert detrimental effects upon reproductive physiology, which depends on the doses and/or the drug employed.

Neutral alpha-glucosidase activity in mouse: a marker of epididymal function?

Neutral alpha-glucosidase (NAG) activity is considered a functional epididymal marker in several species. Unlike the rat, no NAG activity has been detected in mice. The aims of the present study were to evaluate NAG secretory activity (the supernatant of the incubated tissue) in mouse epididymis and to determine whether it could be used as a functional epididymal marker. Epididymides (whole or in parts) were incubated in the presence or absence of testosterone (10(-5) m) and secretory NAG activity was compared with known positive controls. Furthermore, we compared enzyme activity in epididymides from well-fed and undernourished mice (50% food restriction for 21 days), a model that alters the epididymal maturation processes. Spectrophotometric analysis revealed NAG activity in mouse epididymis (22.6 +/- 3.7 mU g(-1) tissue; n = 4), being higher in the caput. NAG activity was statistically higher in the caput than in the corpus and in the cauda. No significant differences existed between the caput NAG activity and complete epididymis NAG activity. In undernourished mice, we confirmed changes in epididymal maturation observed previously (i.e. increased number of immature spermatozoa and diminution of the sperm concentration). Concordantly, the epididymides of undernourished mice exhibited decreased enzyme secretory activity, which increased to values similar to those seen in controls following incubation in the presence of testosterone (22.5 +/- 2.6, 12.5 +/- 1.0 and 22.4 +/- 3.7 mU g(-1) tissue, n = 9 in control (n = 7), undernourished (n = 9) and undernourished + testosterone groups (n = 9), respectively). In conclusion, NAG activity was detected in mouse epididymis. Although the present study supports the possibility of using NAG as an epididymal marker, more studies are necessary to effectively prove that NAG activity can be used as an epididymal marker.

Effects of PDC-109 on bovine sperm functional activity in presence or absence of heparin.

PDC-109, a heparin-binding protein (from the seminal vesicles) that binds to sperm surface phospholipids at ejaculation, may modulate several aspects of sperm activity. The objectives of the present study were to determine: (1) in the presence or absence of heparin, the effects of exogenous PDC-109 on sperm motility (Makler chamber), viability (Hoechst 33258) and membrane functional integrity (hypoosmotic swelling test) of bovine spermatozoa; (2) the role of PDC-109 as a capacitation-inducing factor; and (3) its ability to induce the acrosome reaction (fluorescein staining). After 4-h capacitation in the presence of heparin, the addition of PDC-109 (0.5, 1.5 or 3.0mg/ml) significantly decreased the percentages of motile, progressive, and viable cells; these effects were also detected in the absence of heparin. However, PDC-109 elicited a twofold increase (from 14 to 28%) in the proportion of acrosome-reacted spermatozoa, but only in the presence of heparin. Progesterone (10 microM) or angiotensin II (100 or 1000 nM) stimulated the acrosome reaction after capacitation in the presence of PDC-109 without heparin (from 10 to 17, 23 and 22%, respectively). In conclusion, PDC-109 appears to modulate sperm functional activity, with some effects manifest in the absence of heparin.

The effect of alcohol, tobacco, and aspirin consumption on seminal quality among healthy young men.

In this study, the authors examined the effects of alcohol, tobacco, and drug use on plasma testosterone and seminal parameters (in accordance with the World Health Organization's standards) in healthy Argentine medical students (n = 34). Some alterations in seminal parameters were detected in 19 (56%) subjects. Alcohol and tobacco use were correlated significantly, p = 0.005; subjects who used these substances exhibited a nonsignificant reduction in sperm concentration, motility, viability, and normal morphology. There was a significant decrease in sperm motility among students who used moderate amounts of aspirin (i.e., > or = 500 mg/wk). The authors concluded that alcohol, tobacco, and aspirin use could have had detrimental effects on seminal parameters and that men who wish to procreate should be warned of such effects. Doses, exposure time, and interactions with other variables deserve additional study.

Estudio comparativo de la prueba de resistencia osmótica en semen de pacientes normospérmicos o dispérmicos / Comparative study of the test of osmotic resistence in semen of normospermic or dispermic patients

La alta incidencia de factor masculino en los casos de esterilidad conyugal justifican los intentos de un estudio más completo del semen. La prueba de resistencia osmótica (PRO), ha sido propuesta como un detector precoz de fallas en la integridad funcional de la membrana espermática. En el presente trabajo estudiamos las posibles correlaciones entre PRO y los parámetros convencionales del espermograma (de acuerdo a las técnicas recomendadas por la OMS) y comparamos los resultados de la PRO en especímenes normospérmicos y oligo, terato y/o astenospérmicos. En 75 casos se cuantificó: concentración espermática (en hemocitómetro), motilidad, velocidad de traslación (rápidos, lentos y no progresivos), morfología (coloración de Papanicolaou) y PRO (incubación del semen en solución de citrato de Na y fructosa, 150 mOsm/l, 37§C, 60 min). En todos los casos, la PRO se correlacionó significativamente con el porcentaje de formas móviles, rápidas o de morfología normal (r = 0.39; r = 0.39 y r = 0.35 respectivamente). A su vez, disminuye significativamente al incrementarse el número de alteraciones en las determinaciones del espermograma de rutina. En las muestras asteno, terato y oligospérmicas, los resultados de la PRO son significativamente inferiores a las con motilidad, morfología o concentración normal, respectivamente (p < 0.005 en todos los casos). Consideramos que la PRO representa una técnica accesible y de relativa sencillez que constituye un aporte relevante en el diagnóstico de la esterilidad masculina

Estudio comparativo de la prueba de resistencia osmótica en semen de pacientes normospérmicos o dispérmicos / Comparative study of the test of osmotic resistence in semen of normospermic or dispermic patients

La alta incidencia de factor masculino en los casos de esterilidad conyugal justifican los intentos de un estudio más completo del semen. La prueba de resistencia osmótica (PRO), ha sido propuesta como un detector precoz de fallas en la integridad funcional de la membrana espermática. En el presente trabajo estudiamos las posibles correlaciones entre PRO y los parámetros convencionales del espermograma (de acuerdo a las técnicas recomendadas por la OMS) y comparamos los resultados de la PRO en especímenes normospérmicos y oligo, terato y/o astenospérmicos. En 75 casos se cuantificó: concentración espermática (en hemocitómetro), motilidad, velocidad de traslación (rápidos, lentos y no progresivos), morfología (coloración de Papanicolaou) y PRO (incubación del semen en solución de citrato de Na y fructosa, 150 mOsm/l, 37ºC, 60 min). En todos los casos, la PRO se correlacionó significativamente con el porcentaje de formas móviles, rápidas o de morfología normal (r = 0.39; r = 0.39 y r = 0.35 respectivamente). A su vez, disminuye significativamente al incrementarse el número de alteraciones en las determinaciones del espermograma de rutina. En las muestras asteno, terato y oligospérmicas, los resultados de la PRO son significativamente inferiores a las con motilidad, morfología o concentración normal, respectivamente (p < 0.005 en todos los casos). Consideramos que la PRO representa una técnica accesible y de relativa sencillez que constituye un aporte relevante en el diagnóstico de la esterilidad masculina (AU)

Correlación entre parámetros convencionales del espermograma y la preuba de resistencia osmotica en pacientes integrantes de parejas fertiles o infertiles / The correlation between the conventional spermogram parameters and the osmotic resistance tests in patients from among fertile and infertile couples

It is well accepted that routine semen analysis is a useful tool for valuation of male potential fertility. In our environment, HOST has been recently applied as an additional approach which early detects structural and/or functional alterations of the sperm membranes. We investigated the possible relationship between some semen parameters and HOST in males from A) fertile couples (23-32 years old; n = 11) and B) sterile couples (25-35 years old; n = 171. Sperm concentration, motility, morphology and HOST (semen incubation in hypoosmotic saline solution-sodium citrate and fructose, 150 m0sm/1--, 37 degrees C during 60 min) were evaluated. In HOST determinations, results higher than 60 of swollen cells are considered within the normal range. In our study, sperm from males of B group showed a significantly lower percentage of hyperhydrated cells than those from A group (55.7 + 2.2 and 70.4 + 2.3 respectively; p < .001). In addition, a significant statistical correlation between HOST vs motility or normal morphology was found. On the contrary, we detected no correlation between HOST vs sperm concentration or volume. We suggest that development and application of HOST as a routine, can play an important role in the evaluation and prognosis of an infertile couple.

Correlación entre parámetros convencionales del espermograma y la preuba de resistencia osmotica en pacientes integrantes de parejas fertiles o infertiles / The correlation between the conventional spermogram parameters and the osmotic resistance tests in patients from among fertile and infertile couples

It is well accepted that routine semen analysis is a useful tool for valuation of male potential fertility. In our environment, HOST has been recently applied as an additional approach which early detects structural and/or functional alterations of the sperm membranes. We investigated the possible relationship between some semen parameters and HOST in males from A) fertile couples (23-32 years old; n = 11) and B) sterile couples (25-35 years old; n = 171. Sperm concentration, motility, morphology and HOST (semen incubation in hypoosmotic saline solution-sodium citrate and fructose, 150 m0sm/1--, 37 degrees C during 60 min) were evaluated. In HOST determinations, results higher than 60 of swollen cells are considered within the normal range. In our study, sperm from males of B group showed a significantly lower percentage of hyperhydrated cells than those from A group (55.7 + 2.2 and 70.4 + 2.3 respectively; p < .001). In addition, a significant statistical correlation between HOST vs motility or normal morphology was found. On the contrary, we detected no correlation between HOST vs sperm concentration or volume. We suggest that development and application of HOST as a routine, can play an important role in the evaluation and prognosis of an infertile couple.(Au)