Single layer centrifugation improves the quality of frozen-thawed sperm of giant panda (Ailuropoda melanoleuca).

The reproductive capacity of captive giant pandas is poor and sperm cryopreservation is necessary for the reproduction and conservation of this species. Cryopreservation, however, leads to a significant decrease in sperm quality, including sperm motility, acrosome integrity and DNA integrity. In the present study, a method was developed based on colloid single layer centrifugation that could significantly improve frozen-thawed sperm quality. Two colloids were compared for post-thaw giant panda sperm preparation; the sperm samples had greater total motility (Colloid 1: 44.5 ±â€¯16.0%, Colloid 2: 42.4 ±â€¯10.1% compared with Control: 25.4 ±â€¯8.4%, P < 0.05), linear velocity (Colloid 1: 17.2 ±â€¯8.3 µm/s; Colloid 2: 19.0 ±â€¯9.0 µm/s compared with Control: 6.6 ±â€¯1.7 µm/s, P < 0.05) and membrane integrity (Colloid: 46.9 ±â€¯13.2%; Colloid 2: 54.3 ±â€¯5.7% compared with Control: 36.0 ±â€¯9.1%; P < 0.05). This method could be a useful tool to enable the use of poor quality sperm samples and benefit this population by using available genetic material.

Alleviation of streptozotocin-induced diabetes in nude mice by stem cells derived from human first trimester umbilical cord.

Cells isolated from human first trimester umbilical cord perivascular layer (hFTM-PV) tissues display the pluripotent characteristics of stem cells. In this study, we examined whether hFTM-PV cells can differentiate into islet-like clusters (ILCs) in vitro, and whether transplantation of the hFTM-PV cells with and without differentiation in vitro can alleviate diabetes in nude mice. The hFTM-PV cells were differentiated into ILCs in vitro through a simple stepwise culture protocol. To examine the in vivo effects of the cells, the hFTM-PV cells with and without differentiation in vitro were transplanted into the abdominal cavity of nude mice with streptozotocin (STZ)-induced diabetes. Blood glucose levels, body weight, and the survival probability of the diabetic nude mice were then statistically analyzed. The hFTM-PV cells were successfully induced into ILCs that could release insulin in response to elevated concentrations of glucose in vitro. In transplantation experiments, we observed that mice transplanted with the undifferentiated hFTM-PV cells, embryonic body-like cell aggregations, or ILCs all demonstrated normalized hyperglycemia and showed improved survival rate compared with those without cell transplantation. The hFTM-PV cells have the ability to differentiate into ILCs in vitro and transplantations of undifferentiated and differentiated cells can alleviate STZ-induced diabetes in nude mice. This may offer a potential cell source for stem cell-based therapy for treating diabetes in the future.

Effect of You Gui Wan on mouse sperm fertilising ability in vivo and in vitro.

This study is to explore whether YGW has an impact on sperm fertilising ability in mice. Twenty male mice were randomly divided into two groups. In vivo experiments, one group of animals were orally administrated with YGW decoction and another group administered with saline for 14 days. Afterwards, the animals were mated with their female partners. Percentages of retrieved zygotes were then compared. In vitro experiments, in vitro fertilisation (IVF) assay, sperm acrosome reaction and acrosin activity were used to compare sperm fertilising ability between the two groups. The YGW-treated group had a significantly higher percentage of zygotes than the saline controls (P = 0.005). The IVF rates induced by spermatozoa from the herb-treated mice were also significantly higher than those from the control animals (P = 0.015). The sperm acrosin activity of the herb-treated group was significantly higher than that of the saline-treated group (P = 0.048), although there was no significant difference in testicular weight, sperm count and sperm motility. These data suggest that YGW decoction has a significant effect on normal sperm fertilising ability both in vivo and in vitro, which may be due to, at least in part, increments in the sperm acrosin activity.

Plasma soluble HLA-G is a potential biomarker for diagnosis of colorectal, gastric, esophageal and lung cancer.

Human leukocyte antigen-G (HLA-G) is a novel tumor marker and its soluble isoforms produce secretory proteins. Increased soluble HLA-G (sHLA-G) levels have been reported in patients with melanoma, neuroblastoma, lymphoproliferative disorders, breast, ovarian and colorectal carcinoma when compared to healthy controls or subjects with benign neoplasms. The aim of this study is to investigate whether or not plasma sHLA-G can be used as a potential biomarker for cancer diagnosis. We measured plasma sHLA-G levels in 166 patients with early stages of colorectal cancer (CRC, n = 37), gastric cancer (GC, n = 28), esophageal squamous cell carcinoma (ESCC, n = 58) and non-small cell lung cancer (NSCLC, n = 43), and compared them to healthy controls (n = 260) by using a specific HLA-G enzyme-linked immunosorbent assay (ELISA). We found that plasma sHLA-G levels were significantly higher in cancer patients than in healthy controls (all P < 0.0001). The areas under the receiver-operating characteristic (ROC) curves for sHLA-G were 0.97, 0.91, 0.98 and 0.80 for healthy controls vs CRC, GC, ESCC and NSCLC, respectively. At 100% specificity, the highest sensitivity achieved to detect CRC, GC, ESCC and NSCLC was 94% [95% confidence interval (CI), 89-99], 85% (95% CI, 76-94), 91% (95% CI, 88-94) and 51% (95% CI, 43-59) at a cutoff value of 49 U/ml, respectively. These findings suggest that plasma sHLA-G may be a useful molecule in the differential diagnosis of these malignancies against healthy controls.

Acrosin activity in pelleted frozen sperm does not correlate with in vitro fertilization of oocytes.

Spermatozoa pelleted after swim-up were frozen and then analysed in batches for acrosin activity, using a spectrophotometric method, and expressed as microIU micrograms DNA-1. A total of 259 sperm samples were analysed and the acrosin activity compared with fertilization in vitro. Of these, 224 samples fertilized at least one oocyte and 35 samples failed to fertilize any oocyte. Analysis by Student's t-test indicated that there was a statistically significant difference (P = 0.02) in acrosin activity between the two groups. However, when samples that failed to fertilize were matched for concentration, motility and normal morphology with samples that fertilized, this significance was lost (P = 0.77). It is concluded that total acrosin in pelleted sperm frozen after regular swim-up, does not correlate with fertilizing ability of spermatozoa used for insemination.

Polyploidy and failed fertilization in in-vitro fertilization are related to patient's age and gamete quality.

A review of 392 cycles of in-vitro fertilization (IVF) was carried out in order to identify whether any factors such as sperm concentration at insemination, sperm motility or morphology, oocyte grading, number of oocytes retrieved, patient age, follicular stimulation protocols or duration of follicular growth, could be associated with the incidence of polyploidy or completely failed fertilization. The majority of polypronuclear fertilizations occurred in mature oocytes and in patients < 37 years of age and the incidence of polyploidy was strongly associated with fertilization and pregnancy rates. Fertilization rates were highest with mature oocytes. A significant linear trend was observed with failed fertilization and sperm concentration, morphology and motility. High rates of failed fertilization were found in patients > 37 years of age and with one to five oocytes retrieved. No significant difference was seen in stimulation protocols and oocyte grading between oocytes that fertilized and those that did not. Cycles with good sperm morphology and motility, the presence of mature oocytes, the retrieval of a large number of oocytes and younger maternal age seem to provide the best chance for IVF success.

Effects of sex hormones on the pineal response to isoproterenol and on pineal beta-adrenergic receptors.

Several studies have shown sex hormone effects on pineal function. In order to clarify the role of adrenergic mechanisms in these effects, we investigated the pineal response to adrenergic stimulation and pineal beta-adrenergic receptors following castration and/or sex hormone treatment in 2-month-old male and female Sprague-Dawley rats. The urinary 6-sulphatoxymelatonin (aMT6s) response to isoproterenol (ISO) was compared in castrated and sham-operated animals. Ovariectomy caused an increase and orchiectomy a decrease in ISO-induced urinary aMT6s excretion. The melatonin response to ISO was examined in pineal glands and serum samples obtained from castrated, sex-hormone-treated castrated and sham-operated rats. Consistently, ovariectomy increased pineal and serum melatonin responses to ISO, while orchiectomy decreased the responses; oestradiol and testosterone treatments, respectively, reversed these effects. 3H-dihydroalprenolol binding was measured in single pineal glands from castrated, sex-hormone-treated castrated and sham-operated rats. Ovariectomy increased the density of beta-adrenoceptors, whereas orchiectomy decreased the density; oestradiol and testosterone replacement, respectively, blocked these effects. No significant effect on receptor Kd values was found. These data suggest that sex hormones regulate pineal melatonin production by modifying beta-adrenergic mechanisms.

Melatonin receptors on human granulosa cell membranes.

Putative melatonin binding sites were detected in the membrane fraction of gonadotropin-stimulated human granulosa cells using the melatonin analogue 2-[125I]-iodomelatonin (125I-IML). Saturation studies and Scatchard analysis revealed the presence of a major binding site with a Kd of 99 pM. Guanosine triphosphate shifted the receptor affinity to 380 pM. In competition studies, the rank order of potency of indoles for inhibition of 125I-IML binding at these sites was typical of melatonin receptors: 2-iodomelatonin > melatonin > N-acetylserotonin > 5-methoxytryptamine > serotonin. Culture of cells for 7 days in vitro increased receptor density but not the affinity. These findings strongly suggest that melatonin found in follicular fluid may have a physiological role.

Melatonin and steroids in human pre-ovulatory follicular fluid: seasonal variations and granulosa cell steroid production.

Follicular fluid samples were obtained from the largest pre-ovulatory follicle of 120 women undergoing in-vitro fertilization and were examined for melatonin by enzyme-linked immunosorbent assay and the steroids oestradiol and progesterone by radioimmunoassay. The concentrations (mean +/- SE) of melatonin (213.4 +/- 18.9 pmol/l) and progesterone (20.1 +/- 1.1 mumol/l) in follicular fluid during the autumn and winter (dark) months were significantly higher than during the spring and summer (light) months, melatonin (138.4 +/- 12.5 pmol/l) and progesterone (11.6 +/- 0.8 mumol/l). By contrast, oestradiol concentrations were significantly lower during the dark months than during the light months (264.7 +/- 44.1 and 661.8 +/- 55.1 nmol/l respectively). There was a positive correlation between follicular fluid melatonin and progesterone concentrations (r = 0.271, P < 0.05, n = 120) and a negative relationship between melatonin and oestradiol (r = -0.254, P < 0.05, n = 120). The effects of melatonin alone and in combination with human chorionic gonadotrophin (HCG) or follicle stimulating hormone (FSH) on steroidogenesis by human granulosa cell culture were also investigated. Melatonin had minimal effects on oestradiol or progesterone production by granulosa cells. Interestingly, the oestradiol response in culture appeared to be different according to the time of the year when harvested. During the light period oestradiol production was enhanced. Melatonin also synergized with HCG in increasing progesterone production on days 6 and 7 after treatment during both light and dark periods. FSH stimulated oestradiol production by the cells on day 2 of culture. Melatonin had no effect on FSH stimulation of oestradiol production. The results of this study suggest that melatonin may be involved in the regulation of steroidogenesis by the human ovaries.

Identification of a transforming growth factor alpha-like molecule in human seminal plasma.

OBJECTIVE: To characterize the putative seminal growth promoting factor serendipitously observed when human seminal plasma was analyzed for bioactive FSH. DESIGN: A pool of human seminal plasma was subjected to sequential Sephadex G-75 (superfine) chromatography and high-performance size exclusion liquid chromatography. The fractions were tested for mitogenic activity using a rat granulosa cell assay and normal rat kidney (NRK) cells. Properties of the factor were established and characterization by gel electrophoresis and neutralization with antibody were accomplished. SETTING: Reproductive Biology Research Laboratory at McMaster University Medical Centre. MAIN OUTCOME MEASURES: Ability of purified fractions of human seminal plasma to augment the uptake of tritiated thymidine into cell DNA. RESULTS: Mitogenic activity of human seminal plasma was augmented in the presence of FSH but not hCG, PRL, E2, T, P, or dihydrotestosterone. The putative growth factor synergized with insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), and transforming growth factor beta (TGF-beta) but not with TGF-alpha. Mitogenic activity was neutralized by a specific TGF-alpha antibody in a dose-dependent manner. The molecular weight of the factor as assessed by gel electrophoresis is 6 kd. CONCLUSIONS: Human seminal plasma contains a mitogen that is similar to TGF-alpha.

Competitive solid-phase enzyme immunoassay for melatonin in human and rat serum and rat pineal gland.

We describe a solid-phase competitive enzyme immunoassay for determination of melatonin in serum. The detection limit of the assay is 1.0 fmol/well. Low cross-reactivity of the antiserum with other indoles, parallel serum extract dilution and melatonin standard curves, good analytical recovery, and within- and between-assay CVs of 6.4-14.4% provide validation of the assay. Linear regression analysis of melatonin concentrations measured with this assay (y) and with a commercial 3H RIA (x) in 88 sera yielded the relation y = 0.62 x - 0.76, Sy/x = 0.03. Values for melatonin in serum samples from healthy subjects are lower during the day than during the night. Melatonin response in rat serum and pineal gland to isoproterenol injection is similar to published RIA data. The analytical procedure is also simple. Thus, the assay should have practical applications in investigation of pineal function in both clinical and basic studies.

Developmental patterns of bioactive and immunoreactive FSH in the female rabbit: effects of ovariectomy.

A longitudinal study was performed to determine the relationship between immunoreactive and biologically active FSH in the serum of sham-operated and ovariectomized female rabbits. Twenty-two-day-old female rabbits, 8 per group, were sham-operated or bilaterally ovariectomized on day 23. Blood was taken every 3-4 days from each rabbit until they achieved a weight of 3 kg or age 100 days. Sera were analysed by radioimmunoassay for LH and FSH or for bioactive FSH. In sham-operated animals, immuno-FSH levels showed a 10-fold increase from 0.36 +/- 0.04 ng/ml to greater than 35 ng/ml between days 45 and 80. By contrast, bio-FSH levels increased more gradually from a baseline of 5.4 +/- 0.4 ng/ml to about 8 ng/ml. Bilateral ovariectomy resulted in a significant increase in both bio-FSH, 5.5 +/- 0.4 ng/ml to 12.6 +/- 1.5 ng/ml and immuno-FSH levels from 0.4 ng/ml to 5.2 +/- 1.4 ng/ml 24 h later. These levels of FSH remained elevated throughout the sampling period in both groups of animals and then decreased after day 100. Peripheral LH levels showed much more variation but were 2-fold higher in ovariectomized rabbits, 0.4-1.4 ng/ml in sham-operated vs. 0.8-2.4 ng/ml in ovariectomized rabbits. These results emphasize the marked variations in FSH levels according to the method of analysis. They also suggest that extra-ovarian factors may play a role in inhibiting gonadotropin release especially LH.

Age-associated changes and sex differences in urinary 6-sulphatoxymelatonin circadian rhythm in the rat.

The circadian rhythm of 6-sulphatoxymelatonin (aMT6s) excretion has been determined in male and female rats at 3 weeks and at 2, 8, 14 and 20 months of age. All animals have a pronounced circadian pattern of aMT6s excretion under a 12 hour dark: 12 hour light cycle. A significant increase in aMT6s excretion is observed from 3 weeks to 14 months followed by a decrease at 20 months. There is a highly significant correlation between aMT6s excretion and body weight (r = 0.73 for female rats and r = 0.74 for male rats; p values are all less than 0.001). Thus, a decrease in aMT6s excretion associated with increasing age occurs when body weight is taken into consideration. aMT6s excretion is higher in males at 3 weeks and at 2 and 8 months of ages. Urinary testosterone in male rats and estradiol in female rats increase from 3 weeks to 8 months and decrease at older ages. These data suggest that increase of body weight from 3 weeks to 14 months is an important factor responsible for the age-related alteration. The sex differences in aMT6s excretion in younger rats may be associated with their sex hormonal milieu.

Melatonin and aromatase stimulating activity of human seminal plasma.

Melatonin concentrations and aromatase stimulating activity were determined in human seminal plasma and correlated with sperm density and motility. Aromatase stimulating activity was determined with an in vitro rat granulosa cell system and melatonin by radioimmunoassay. Compared to normal semen, aromatase stimulating activity was lower in azoospermic individuals, while melatonin was higher in oligospermic and azoospermic samples. Aromatase stimulating activity correlated positively with sperm concentrations and a negative correlation was found between melatonin and sperm progression. These findings suggest that low sperm production is associated with low aromatase stimulating bioactivity in seminal plasma; and melatonin may have an effect upon both sperm production and motility.

Urinary 6-sulphatoxymelatonin, an index of pineal function in the rat.

The objective of this study was to apply the radioimmunoassay for 6-sulphatoxymelatonin (aMT6s) to rat urine, and use it to study the source of aMT6s. The radioimmunoassay was found to have acceptable within- and between-assay variation, excellent specificity, and good parallelism between the standard and unknown. Because urine is highly contaminated we assessed whether preliminary purification was required and established that it was unnecessary. Using this assay a 24-hr rhythm in 6-sulphatoxymelatonin output was seen in pools of urine harvested at 3-hr intervals from Wistar rats on LD 12:12. The nocturnal rise in aMT6s was abolished by constant light. In contrast pinealectomy lowered aMT6s output significantly throughout both dark and light. This study confirms previous studies indicating that the pineal is the major source of 6-sulphatoxymelatonin. It is concluded that urinary 6-sulphatoxymelatonin as measured by radioimmunoassay is a valid measure of pineal gland activity in the rat.

Circadian variations of serum sex hormone binding globulin binding capacity in normal adult men and women.

Diurnal variations of serum sex hormone binding globulin (SHBG), testosterone (T) and estradiol (E2) in five normal adult men and five normal adult women were investigated. SHBG binding capacity was measured by both polyacrylamide gel electrophoresis and dextran-coated charcoal technique (DCC); T and E2 were assayed by RIA and free T and free E2 were determined by means of equilibrium dialysis. In male subjects the variations of SHBG binding capacity was associated with the changes of total T, free T and T/SHBG index, which had the highest concentrations in the morning and the lowest levels in the evening during the 24 h test period, but percentage free T remained unchanged. Serum protein concentrations did not change significantly during 24 h. No significant diurnal changes of SHBG binding capacity, total E2, free E2, percentage free E2 and percentage free T were found in female subjects in the mid-luteal phase of the menstrual cycle, although significant fluctuations of total T, free T and T/SHBG index were observed throughout the day. The results suggested that SHBG may play a buffer role in the presence of fluctuations of testosterone production during 24 h period, allowing stabilization of a bioactive fraction of the hormone both in normal adult male and female. However, the concentrations of T in normal adult women may be too low to drive any change of SHBG levels while there were no significant variations of E2 throughout a day in the mid-luteal phase of the menstrual cycle.