L. M. Lewin

Carnitine and acylcarnitines in semen from azoospermic patients

Carnitine and its short-chain acyl esters were assayed in semen from normospermic and azoospermic men. Extremely low concentrations of free carnitine and acylcarnitine were found in semen from patients with obstructive azoospermia where the ejaculate was primarily of prostatis origin, and low values were also obtained in obstruction of the vas deferens, where the epididymal contents were not ejaculated. Semen from patients whose azoospermia was caused by testicular dysfunction had low acylcarnitine concentrations and normal levels of free carnitine in most cases, but a group of patients with severe testicular failure (including cases of Klinefelter syndrome and cryptorchidism) had low semen free carnitine concentrations. Whereas treatment with human chorionic gonadotropin increased serum testosterone levels in azoospermic patients, it did not increase the free carnitine concentration in semen, although it increased the proportion of carnitine found in acylcarnitines.

Loss of acid phosphatase from rat spermatozoa as a method for assessing the acrosome reaction.

Summary. A method is presented for evaluating the extent of the acrosome reaction by measuring the release of acrosomal acid phosphatase from rat spermatozoa during incubation under capacitating conditions. Treatment of spermatozoa with lysophosphatidylcholine or Triton X‐100 released the acid phosphatase from the sperm cell. Using this enzymatic method we could not detect an alteration in enzyme activity following 5 h incubation under capacitating conditions. The effect of in vitro capacitation for 5 h in the absence or presence of heparin or ionophore A23187 was studied. Incubation in the presence of heparin (10 μg ml−1) caused a 32% increase in enzyme activity. After exposure of the spermatozoa to ionophore A23187 (0.5 μM) 16% increase of enzyme activity could be detected.

Spermatogenesis in the golden hamster during the first spermatogenic wave: A flow cytometric analysis

In the present study propidium iodide was used as a fluorescent dye to stain DNA of cells of hamster testicular origin and fluorescent intensities were analyzed by flow cytometry. We used hamster testicular cells from the first spermatogenic wave to observe the consecutive appearance of the different types of cells during puberty. At 12 days postpartum (dpp) diploid cells (including spermatogonia) predominated and some tetraploid cells were also present. Tetraploid spermatocytes increased dramatically by 21 dpp. The first haploid cells appeared at 21 dpp but substantial numbers were first present at 23 dpp. Immature haploid cells predominated at 32 dpp. Elongating condensing spermatids appeared at 34 dpp and spermatozoa began to leave the testis to enter the epididymidis at 36–38 dpp marking the end of the first round of spermatogenesis. Using acridine orange staining flow cytometry, chromatin condensation was followed by measuring fluorescence decrease from early round spermatids to spermatozoa obtained from the initial segment and from the cauda epididymides. The major portion of sperm chromatin condensation (88–90%) in the hamster occurred in the testis and only 10–12% occurred during epididymal sperm maturation. Spermatozoa in the initial segment of the epididymidis of the hamster contained a small amount of RNA that was no longer present in sperm of the cauda epididymidis, indicating that RNA was lost during epididymal sperm maturation in this species. Mol. Reprod. Dev. 55:205–211, 2000.

Multiple Actions of a Hybrid PACAP Antagonist: Neuronal Cell Killing and Inhibition of Sperm Motilitya

Abstract: Pituitary stimulating adenylate cyclase (PACAP) is a major regulatory peptide with two active molecular forms: PACAP‐27 and PACAP‐38. Both molecular forms promote neuronal survival and protect against neurotoxicity. Based on our previous hybrid peptide strategy in designing vasoactive intestinal peptide (VIP) antagonists, novel PACAP analogues were synthesized (neurotensin6‐11 PACAP7‐27 and neurotensin6‐11 PACAP7‐38). In addition to the hybrid modification, the methionine in position 17 was replaced by norleucine (Nle). Treatment of rat cerebral cortical cultures for five days with the putative PACAP antagonists (1 nM) resulted in a 35‐45% reduction in neuronal cell counts as compared to controls. Neuronal cell death was already obtained at picomolar concentrations for the neurotensin6‐11PACAP7‐27 antagonist with 70% death at 10−8 M. Co‐administration of the PACAP hybrid analogue with picomolar amounts of PACAP‐27 or Nle17‐PACAP‐27 attenuated the reduction in neuronal cell counts. While the protective effects of both analogues exhibited a peak at 1 pM concentrations, the Nle‐containing agonist displayed a broader range of active concentrations (10−12 M‐10−9 M)

Alkaline phosphatase in human semen : an investigation using enzyme inhibitors and gel electrophoresis

Human seminal alkaline phosphatase was investigated with respect to its electrophoretic mobility, heat lability, and susceptibility to inhibition by phenylalanine, tartrate, and homoarginine. Total alkaline phosphatase activity in 30 samples of human semen was measured colorimetrically, using p-nitrophenylphosphate as substrate. Using linear regression analysis, no significant correlation was found between the enzyme activity and the sperm count, sperm motility, semen volume, and the concentrations of seminal inositol and fructose. The alkaline phosphatase activity was higher in the earlier portion of split ejaculate samples. Sodium DL-tartrate (42 mmol/l), which inhibits acid phosphatase, did not inhibit seminal alkaline phosphatase significantly. L-Homoarginine (10 mmol/l), an inhibitor of the liver and bone isoenzymes, inhibited the seminal enzyme (53%), whereas L-phenylalanine (12 mmol/l), a strong inhibitor of placental alkaline phosphate, decreased activity by about 10%. Electrophoresis of semen samples on agarose revealed a broad band which was not sharpened after treatment with neuraminidase. Semen total alkaline phosphatase was essentially totally inactivated by heating at 56 degrees C for 15 min or 10 min at 65 degrees C; similar behaviour has been reported for the liver and bone isoenzymes. Electrophoresis after heating did not reveal a residual band of heat-stable placental-like alkaline phosphatase. Semen alkaline phosphatase appears to contain more than one isoenzyme, but placental-like alkaline phosphatase cannot be more than a minor component.

Myo-inositol in the reproductive tract of the female rat

1. myo-Inositol concentrations in oviduct, ovary and uterus were many-fold those of blood serum during all four stages of the estrous cycle of the female rat. 2. Inositol concentration was higher in oviduct than in ovary or uterus and was lower in uterine fluid than in uterus. 3. Estrus uteri had higher inositol concentrations than uteri in other phases of the cycle. 4. In order to measure dynamic aspects of the distribution of inositol, the distribution of radioactivity among organs of the reproductive tract of mature female rats was measured 45 min after i.p. injection of [2-3H]myo-inositol. 5. These organs concentrated inositol from the blood, and the tissue radioactivity (expressed as dpm/mg wet wt of tissue) increased in the sequence: vagina less than cervix less than uterus less than ovary less than oviduct. 6. The uterus and ovary concentrated myo-inositol more strongly during proestrus than during metestrus, diestrus of estrus. 7. The contents of proestrus follicles were more highly radioactive than was the ovary itself, whereas proestrus uterine fluid was less radioactive than the uterine tissue.

Investigations on the Motility of Human Spermatozoa in a Defined Medium in the Presence of Metabolic Inhibitors and of Carnitine

Summary:  When washed human sperm were incubated in a modified Krebs‐Ringer buffer solution in the absence of exogenous metabolizable substrates at 30° C they maintained progressive motility for at least six hours. Under these conditions the spermatozoa apparently utilize endogenous substrates but addition of exogenous substrates (glucose, fructose, acetate, short‐chain fatty acids, branched chain amino acids) did not affect the % progressive motility or % total motility of the cells. The phospholipase inhibitors, quin‐acrine and Upjohn No. 1002, inhibited progressive motility when added to sperm utilizing endogenous substrate, and subsequent addition of oxidative or glycolytic substrates did not reverse the inhibition. In contrast, the inhibition by KCN of progressive motility based upon utilization of endogenous substrate was reversed upon addition of glycolyzable compounds (glucose or fructose).

Survey of carnitine content of human semen using a semiquantitative auxanographic method: decreased semen total carnitine concentration in patients with azoospermia or severe oligozoospermia.

A microbiological method, using the carnitine‐requiring yeast, Torulopsis bovina ATCC 26014, was developed to identify samples of human semen which contained low levels (< 250 μM) of total carnitine. Of 399 semen samples from a male infertility clinic which were tested, 30 (7.5%) were low in carnitine. Of these, 14 were azoospermic and 16 were severely oligozoospermic. Some azoospermic samples (19 = 58%) and severely oligozoospermic samples (51 = 79%) did not give evidence of low carnitine concentrations. These results indicate that decreased total carnitine concentration in semen occurs in certain classes of azoospermic and severely oligozoospermic patients.

Chromatin condensation during spermiogenesis in the golden hamster (Mesocricetus aureus): a flow cytometric study.

DNA‐staining of hamster testis cell suspensions followed by flow cytometry demonstrated appearance of the first haploid cells at 23 days post partum (dpp) and of condensed chromatin (in elongated spermatids and spermatozoa) at 33–34 dpp. Mature spermatozoa were first observed in the caput epididymis at 36–37 dpp, thus completing the first spermatogenic wave. Testicular cell suspensions from animals from 23 to 38 dpp were stained with acridine orange, and flow cytometer gating was adjusted to include only the haploid cells. Acridine orange intercalated into double‐stranded DNA to produce green fluorescence. The decrease in green fluorescence intensity from 23 until 37 dpp was caused by changes in the binding of DNA to basic proteins in such a fashion as to impede the access of the dye to the DNA double helix. When the green fluorescence values (of the most advanced spermatids) were plotted against the age of the hamsters (in dpp) or the corresponding steps of spermiogenesis, the decrease in fluorescence could be seen to occur in three phases. The inflection point between the first and second phases was observed at about spermiogenesis step 7, consistent with the hypothesis that this represents removal of histone from the chromatin. The second phase presumably represents the period in which transition proteins are bound to the DNA. At approximately steps 15 or 16 a further inflection point was seen where protamines replaced the transition proteins. The red fluorescence produced when acridine orange bound to RNA in spermatids, increased early in spermiogenesis and decreased dramatically at 34 dpp, consistent with the fact that elongating spermatids discard the bulk of their cytoplasm during the maturation process. Mol. Reprod. Dev. 56:105–112, 2000.

Androgen‐Binding Components of Human Semen

Summary:  Androgen‐binding components were detected in human semen, using the dextran‐coated charcoal method. Pre‐ and postvasectomy semen samples did not show significant differences in ability to bind dihydrotestosterone. Electrophoresis conducted in polyacrylamide gels in which tritiated dihydrotestosterone had been incorporated disclosed the presence of androgen‐binding protein with properties characteristic of the ABP of Sertoli cell origin in ram semen but ABP was not detectable in human semen, even after concentrating it two or four fold. The binding of dihydrotestosterone was slightly greater in the later portion of split‐ejaculate samples, but binding was found in the earlier fraction and in prostatic fluid, suggesting that androgen‐binding components enter human semen with secretions of the prostate and seminal vesicle glands. It was concluded that the extent of androgen‐binding by human seminal fluid is not a measure of Sertoli‐cell function.