Bloodmeal Identification in Field-Collected Sand Flies From Casa Branca, Brazil, Using the Cytochrome b PCR Method.

PCR-based identification of vertebrate host bloodmeals has been performed on several vectors species with success. In the present study, we used a previously published PCR protocol followed by DNA sequencing based on primers designed from multiple alignments of the mitochondrial cytochrome b gene used to identify avian and mammalian hosts of various hematophagous vectors. The amplification of a fragment encoding a 359 bp sequence of the Cyt b gene yielded recognized amplification products in 192 female sand flies (53%), from a total of 362 females analyzed. In the study area of Casa Branca, Brazil, blood-engorged female sand flies such as Lutzomyia longipalpis (Lutz & Neiva, 1912), Migonemyia migonei (França, 1924), and Nyssomyia whitmani (Antunes & Coutinho, 1939) were analyzed for bloodmeal sources. The PCR-based method identified human, dog, chicken, and domestic rat blood sources.

Ca(2+)-independent induction of acrosome reaction by protein kinase C in human sperm.

We report that activated protein kinase C (PKC) can induce acrosome reaction independently of elevated Ca2+. Addition of 12-O-tetradecanoyl phorbol-13-acetate or the membrane-permeable diacylglycerol analog 1-oleoyl-2-acetylglycerol to ejaculated human sperm resulted in stimulation of acrosomal reaction (2- to 3-fold), provided the sperm underwent capacitation. Induction of acrosome reaction by 12-O-tetradecanoyl phorbol-13-acetate was blocked by the PKC inhibitor staurosporine or by down-regulation of endogenous PKC, but not by removal of extracellular Ca2+. Acrosome reaction was also enhanced by the Ca2+ ionophore ionomycin in a Ca(2+)-dependent, PKC-independent fashion. Immunohistochemical analysis with type-specific PKC antibodies revealed the presence of PKC alpha and PKC beta II in the equatorial segment, whereas PKC beta I and PKC epsilon staining was found in the principal piece of the tail. Acrosome reaction, thus far believed to be induced only by elevated Ca2+, can therefore be triggered by activated PKC in a Ca(2+)-independent fashion. The PKC subtypes potentially involved in acrosome reaction are most likely alpha and beta II, whereas the beta I- and epsilon-subspecies might be involved in regulation of flagellar motility of human sperm.

Further studies on the involvement of protein kinase C in human sperm flagellar motility.

Addition of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or the membrane-permeable diacylglycerol analog, 1-oleoyl-2-acetylglycerol to human sperm resulted in increased motility. The biologically inactive 4 alpha-phorbol 12,13 didecanoate had no effect on flagellar motility. Basal motility was markedly reduced in the absence of Ca2+ in the incubation medium, but TPA-induced sperm motility persisted even in the absence of Ca2+. Sperm motility was also enhanced by the Ca2+ ionophore ionomycin in a Ca2(+)-dependent, protein kinase c (PKC)-independent fashion. Although all stimulants examined here reached maximal response at about 15 min of incubation, nevertheless whereas the effect of TPA and 1-oleoyl-2-acetylglycerol declined at 60 min of incubation, that of ionomycin still persisted. Human sperm PKC activity is extremely low and represents only about 20% and 25% of the specific activity recovered from PC-12 and rat pituitary cells, respectively. Immunohistochemical analysis using various type-specific PKC antibodies revealed staining only in the equatorial segment and the principal piece of the tail. Thus, PKC is present in human ejaculated sperm and is involved in flagellar motility.

Sperm penetration in vitro: correlations between parameters of sperm quality and the penetration capactiy.

A nonlinear regression analysis was used in order to fit a logistic model to 200 runs of human ejaculated spermatozoa penetrating cervical mucus of good quality. The data revealed excellent correlation between the number of sperm penetrating (sigma penetration, SP) and the motility (r = 0.776) and vitality (r = 0.534) of the spermatozoa. The percentage of abnormal spermatozoa found in the ejaculate was negatively correlated (r = -0.649), while sperm concentration showed a poor correlation with SP (r = 0.327). Furthermore, concentration was shown to have no effect in samples containing over 5 million sperm/ml. The conclusion of this study is that the two major parameters of sperm quality determining the capacity of spermatozoa to penetrate cervical mucus are motility and the presence of normal forms of spermatozoa.

Treatment of cervical ectropion by cryosurgery: effect on cervical mucus characteristics.

Eighteen women with cervical ectropion and 12 women with ectropion and vaginal discharge were treated by cryosurgery. Evaluation of the cervical mucus characteristics by cervical score and in vitro penetration test was performed before treatment and 2 months later. In the group with ectropion only (group A) the total cervical score was 5.7 +/- 0.4 and 11.9 +/- 0.06 (P less than 0.001) (mean +/- standard error) before treatment and 2 months later, respectively. In the group with ectropion and vaginal discharge (group B) the total cervical score before and after cryosurgery was 3.8 +/- 0.4 and 11.8 +/- 0.1 (P less than 0.001), respectively. In vitro penetration tests in group A before and after treatment were 0.72 +/- 0.1 and 2.9 +/- 0.08 (P less than 0.001), respectively. In group B, in vitro penetration tests before and after cryosurgery were 0.25 +/- 0.1 and 2.8 +/- 0.1 (P less than 0.001), respectively. It appears that cryosurgery improves the cervical mucus characteristics. It is recommended that infertile patients with hostile cervical mucus and ectropion will be treated by cryosurgery.

Penetration of human ejaculated spermatozoa into human and bovine cervical mucus. I. Correlation between penetration values.

Midcycle bovine cervical mucus (BCM), fresh (FBCM) or frozen at -20 degrees C (BCMF), and human cervical mucus (HCM) were collected and tested in in vitro penetration tests, using human ejaculated spermatozoa of good quality. Penetration tests were performed at 34 degrees C for 1 hour, and the penetration value (PV) was calculated. Duplicates of each run were in a narrow deviation. Correlations between PV rates of the same semen samples in the 3 sources were found to be significantly high (HCM versus FBCM, r = 0.958; HCM versus BCMF, r = 0.982, and FBCM versus BCMF, r = 0.985), suggesting that human ejaculated spermatozoa penetrate the cervical mucus of all three sources at the same rate.

Clomiphene citrate treatment in oligozoospermia: comparison between two regimens of low-dose treatment.

Clomiphene citrate (CC) is a well-known drug in fertility clinics that is used for increasing gonadotropin secretion. The present study was planned in order to evaluate the efficiency of a new regimen of treatment by 25 mg on alternate days (group A), compared to a daily dose of 25 mg (25 days on, 5 days off, group B), for 4 months. Semen quality was assessed in two matched groups, which consisted of 45 and 44 normogonadotropic oligoterato-asthenozoospermic (OTA) men, respectively. Nine men in group A and 22 in group B did not respond to therapy by improvement in semen quality. The statistical evaluation of the results revealed group A to yield the highest improvement in sperm concentration (P less than 0.0008) and total sperm count (P less than 0.004). Sperm motility was improved only in group A. No changes were recorded in the morphology of the sperm cells or in semen volume. Pregnancy rate after 6 months of follow-up was 26.7% and 20.5%, in couples of groups A and B, respectively. This study implicates the use of CC (25 mg on alternate days) in andrologic clinics as one of the recommended drugs for normogonadotropic OTA subfertile men in order to achieve a significant increase in sperm concentration and total sperm count.

Possible dopaminergic system involvement in LH and PRL responses to naltrexone treatment.

Naltrexone (Nalt) causes a rapid increase in luteinizing hormone (LH) level. This short term increase of LH concentration declines to baseline levels in less than 1 hour. Addition of pimozide (0.1 mg) caused a blunted response to Nalt challenge, with significantly reduced LH peak values compared with Nalt treatment alone. Pimozide alone caused a delayed decrease compared with baseline LH values. By following plasma prolactin (PRL) levels it was shown that pimozide administration increased PRL levels rapidly for more than 2 hours. Addition of Nalt to pimozide-treated rats significantly decreased plasma PRL values compared with pimozide alone. Nalt injected by itself attenuated PRL baseline levels. Thus, the mechanism by which pimozide caused PRL elevated level is via the dopaminergic as well as the opioid system. It is suggested that the opioid system controls plasma PRL and LH levels through other hypothalamic neurotransmitters in addition to dopamine.

Changes in LH and prolactin levels in diabetic male rats and the role of the opiate system in the control of their secretion.

Diabetic male rat has low serum levels of luteinizing hormone (LH) and testosterone (T), which are accompanied by atrophy of the testes and accessory glands. The present study investigated changes in the serum levels of LH, prolactin (PRL) and glucose, following diabetes induction by streptozotocin. In addition, involvement of the opiate system in the control of LH and PRL secretion was evaluated. There was no difference in PRL levels between diabetic and control animals, except at 8 hours after streptozotocin injection. In contrast, the diabetic animals had consistently lower levels of LH, starting on the second day of diabetes. Blockade of the opiate system by naltrexone caused a sharp increase of LH levels in normoglycemic rats, while only a gradual decrease was observed in hyperglycemic animals. PRL secretion was inhibited by naltrexone, both in diabetic and control groups. It is concluded that, unlike normoglycemic rats, inhibition of LH secretion in diabetes is not under the control of the opiate system, probably as a result of T deficiency. In contrast, PRL secretion in diabetic rats, as in the control group, is under the influence of endogenous opiates.

Opiate system involvement in the control of LH secretion in diabetic and normoglycemic male rats.

The effect of Naltrexone (Nalt), a specific opiate receptor blocker, on LH secretion was studied at frequent intervals during the first hour following treatment. Nalt was injected i.v. by one bolus (1 mg/rat) to diabetic and normoglycemic rats. Blood samples (0.8 ml) were withdrawn at short intervals after injection, through an indwelling cannula. The diabetic rats responded by secretion of LH, which was lower, but not significantly, than that of normal rats, (peak levels 0.74 +/- 0.17 and 0.97 +/- 0.21 ng/ml respectively). After 45 min., LH levels were in the same range as baseline level in the diabetic group; but were still significantly elevated in the control rats. Thus, it can be concluded that in normal rats, as well as in diabetics, LH secretion as a response to Nalt was episodic in spite of Nalt's long half life time. In order to explain the rapid fall in LH levels after Nalt administration, normal rats were injected with a second bolus of Nalt, 2 hours after the first. The second bolus caused only a blunted response of LH secretion. In another experiment, administration of morphine (1 mg/rat) 2 hours after pretreatment with Nalt did not stimulate the prolactin secretion which normally follows morphine treatment. These results indicate that the rapid decrease of LH levels after Nalt treatment in normal rats is not due to absence of the drug in the system. It is suggested that other neural mechanisms, such as the dopaminergic system, are activated during Nalt influence.

Developmental pattern of delta 5 3 beta-hydroxysteroid dehydrogenase activity in isolated rat Leydig cells.

A direct method for determination of delta 5 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity was employed in isolated Leydig cells (LC) derived from rats on fetal day 19 (Fig) and postnatal (N) days 1, 12, 24, 34 and 45 and adults. The activity of 3 beta-HSD in the adult LC was 1.15 +/- 0.02 (mumole/microgram DNA/hr, mean +/- SEM, n = 73). Activities in the other groups, expressed as a percentage of the respective adult control, were: Fig-38%; N1-39%; N12-8%; N24-89%; N34-166%; and N45-118%. A good correlation was found between histochemical staining for 3 beta-HSD and the quantitive method employed. Using (3H)-DHA as a substrate, LC isolated from F19, N1 and N12 produced testosterone in appreciable amounts (41%, 55% and 20% of the total products respectively) whereas at advanced stages of development (N24 to adulthood) the major product was androstenedione (93 +/- 1%). These findings may be explained by the observed decrease in 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity, due to an insufficient supply of NADPH, in the older vs. earlier stages of development. This study indicates the presence of steroidogenic enzymatic activity in LC throughout development in the rat. It also provides a relatively simple in vitro model for studies of testicular regulation during development.

Developmental pattern of testosterone production by the rat testis.

We studied the steroidogenic activity of isolated Leydig cells derived from rats on fetal day 19 (F19) and postnatal (N) days 1, 12, 24, 34, 45 and adults. Leydig cells, isolated at all ages by the collagenase method, increased in number throughout development with a doubling time of 8 days. Testicular content and serum concentrations of testosterone showed parallel changes during development. Moderate values were found at the early stages (F19 and N1), with a nadir on day 12, followed by a progressive increment to reach maximal values in adulthood. A reduction in steroidogenic activity of the testis during neonatal life was confirmed by in vitro studies with isolated Leydig cells. Maximal activity was found in group F19; testosterone production diminished after birth to reach a minimum in group N34 and rose thereafter to adulthood. Leydig cells were responsive to hCG stimulation at all ages in the following order: N1 greater than N34 greater than F19 greater than N24 greater than N45 greater than adult. The present study demonstrates the existence of an active and hCG-responsive population of Leydig cells in the rat testis from fetal life to adulthood.

Effect of the antifertility agent, gossypol acetic acid, on the metabolism and testosterone secretion of isolated rat interstitial cells in vitro.

Gossypol acetic acid is a polyphenolic compound present in the seed of cotton plants. Its antifertility activity by inhibition of spermatogenesis was proven in a large group of animals, including man. In the present study, the direct effect of gossypol acetic acid on collagenase isolated rat I-cells (interstitial cells) was investigated. It was shown that gossypol acetic acid depressed significantly the metabolic rate of the cells. Glucose utilization was abolished by a starting dose of 100 micrograms/ml. Oxygen consumption of I-cells was reduced even at a smaller dose of gossypol (50 micrograms/ml). At these doses, the vitality of the cells remained (proven by trypan blue exclusion test). 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) histochemical stain was slightly decreased. Increasing doses of gossypol caused a marked decrease in the vitality of I-cells and a dramatic drop in histochemical stain for 3 beta-HSD. The pH of the medium was not changed at any dose of treatment. In cultures of I-cells not stimulated by hCG, gossypol did not affect the tonic slow release of testosterone. Thus, gossypol acetic acid has a direct inhibitory effect on isolated rat I-cells, depressing cell metabolism. The failure of some of the other groups to show such an effect, especially in vivo, can be attributed to differences in the dose of treatment and strain of animals.

The possible use of phenoxybenzamine as a male contraceptive drug: studies on male rats.

Treatment of male rats with phenoxybenzamine hydrochloride (PBZ), 70 micrograms/100 g body weight for 5 weeks, caused infertility of the rats. This effect was fully reversible. Studies on the weights of the testes, epididymides, and male accessory sex glands, revealed increased glandular weights (significantly in the testes, epididymides and seminal vesicles). The number of spermatozoa found in the epididymis and vas deferens was significantly increased in the treated rats. Thus, PBZ caused a temporary cessation of sperm transport and paralysis of the muscles of the accessory glands, leading to the absence of ejaculation. In proestrous females mated with treated rats, this caused pseudopregnancy. No changes were found in the sexual behavior of the treated male rats, which was also supported by the absence of changes in the testosterone levels in serum and in testicular tissue. We recommend the use of PBZ in clinical trials, using this well-known drug as the active material for a future contraceptive pill.

Phenoxybenzamine--an effective male contraceptive pill.

Phenoxybenzamine (PBZ), administered in doses up to 20 mg/day, caused aspermia following male orgasm. This led to the development of a male contraceptive pill, PBZ being the active drug. It has been shown that small doses of the drug do not change the hormonal balance of the body, nor do they affect blood pressure. In 2 to 3 days, PBZ blocks ejaculation; this is fully reversed with the cessation of treatment. The drug does not affect semen quality (testicular function), even after a long period of medication. During treatment, the vas deferens, the ampulla and the ejaculatory ducts are probably paralyzed. Cessation of medication brought full recovery of these effects and the reappearance of normal ejaculation. Men complaining of premature ejaculation reported marked improvement in their sexual performance. The recommended regimen for administering PBZ as a male contraceptive is discussed.

Effects of long-term treatment with bromocriptine on pituitary prolactinoma in a male.

Poor surgical results are obtained when operating on large prolactinomas, especially when an extrasellar growth is demonstrated. Several groups have reported tumor regression following bromocriptine treatment. A case report is presented of a young hyperprolactinemic man with a large pituitary adenoma with suprasellar extension, normal visual fields, decreased libido, impotence, hypogonadism and azoospermia. This man was treated with high doses of bromocriptine for a period of 5 months. A significant improvement of potency libido and sperm concentration associated with radiological evidence of a marked reduction of tumor size was noted following suppressive treatment with this ergot alkaloid.

A direct effect of alpha-chlorohydrin on rat epididymal spermatozoa.

The effect of alpha-chlorohydrin (alpha-CH) on rat epididymal spermatozoa was studied in vivo and in vitro. Alpha-CH was injected sc in doses of 5 and 20 mg daily for 16 days. The 20 mg dose resulted in diminished epididymal spermatozoal content (8 +/- 4 vs. 44 +/- 5 million, m +/- SE, n = 5) and motility (13 +/- 7 vs. 74 +/- 4%) as compared to saline injected-controls. Fertility rates were significantly reduced; control-100% (5/5), 5 mg - 25% (1/4), 20 mg - 0% (0/4). Alpha-CH was added to suspensions of spermatozoa in vitro and a level of 132 micrograms/ml depressed motility by 90% (P less than 0.0001) and O2 consumption by 40% (P less than 0.05). Intrauterine insemination of in vitro treated spermatozoa was performed in 61 pro-oestrous rats. Alpha-CH treated spermatozoa (from 5.3 to 26.400 micrograms/ml) were found to be completely infertile compared to untreated spermatozoa which showed a 63% fertility rate. There was almost complete absence of oocytes in the flushed ampullas of recipients of alpha-CH treated sperm, in the lowest dose which did not affect sperm motility. Thus, alpha-CH has direct effect upon spermatozoal function and also has a possible effect on the female reproductive tract.