Involvement of the calcium-specific protease, calpain, in the fertilizing capacity of human spermatozoa.

We recently reported the novel finding that human spermatozoa contain the calcium (Ca2+)-dependent protease, calpain. In somatic cells this protease mediates several cellular activities regulated by Ca2+ including membrane fusibility during cell-to-cell interactions. In this paper we examined the participation of sperm calpain in sperm-oocyte penetration, a process that is dependent on Ca2+ and involves membrane fusion between the two cells.

Changes in expression of adenyl cyclase activity in human endometrium during hormone replacement therapy and ovarian stimulation.

We have investigated membrane fractions prepared from human endometrium for activity of the signalling adenyl cyclase (AC). We characterized the AC guanine nucleotide-binding proteins (G proteins) and examined the changes in AC activity during evaluation cycles of oestrogen and progesterone replacement therapy as well during ovarian stimulation cycles. AC activity was determined by the conversion of substrate ATP into cyclic AMP under basal conditions and in the presence of guanine nucleotide or forskolin.

Calpain-calpastatin: a novel, complete calcium-dependent protease system in human spermatozoa.

Calpain, a calcium (Ca2+)-activated cysteine protease presents in several somatic mammalian cells, has been demonstrated to mediate specific Ca2+-dependent reactions including cell fusion. Because spermatozoa cells have an absolute Ca2+ requirement for penetration of oocytes, we have postulated that calpain would also be found in mammalian spermatozoa. Here we show that whole sperm homogenate and cell fractions prepared from ejaculated human spermatozoa contain calpain activity.

Enzymatic amplification of specific deoxyribonucleic acid sequences from single cells: evaluation of a simplified and rapid method for use in preimplantation genetic diagnosis.

Objective: To develop a simplified polymerase chain reaction (PCR) protocol on single cells for the purpose of preimplantation genetic diagnosis. Also to evaluate a new thermal cycler, RoboCycler 40 (Stratagene, La Jolla, CA), for reducing the time to complete PCR amplification.

Design: PCR amplification without DNA purification or reamplification of a 149 base pair (bp) segment of the human Y chromosome was used as a model.

Status of hCG/LH receptor and G proteins in human endometrium during artificial cycles of hormone replacement therapy.

Objectives: We examined the existence of hCG/LH receptors and associated GTP-binding (G) proteins in membrane fractions of nonpregnant human endometrium and investigated whether their expression is affected, in vivo, by estrogen and progesterone replacement therapy.

Methods: A pool of normal endometrial biopsy specimens (n = 5) was initially used to characterize receptors and G proteins. Subsequently, biopsy specimens (n = 22) were obtained from 11 patients undergoing evaluation cycles of hormone replacement therapy (HRT).

Evidence for a novel adenylyl cyclase in human epididymal sperm.

Since cAMP is considered to play a major role in the acquisition of maturation and fertilizing capacity of mammalian sperm, we investigated the expression of cAMP-synthesizing adenylyl cyclase (AC) in sperm retrieved directly from the human epididymis. Particulate fractions were prepared from purified epididymal sperm samples and AC was monitored by the direct conversion of ATP into cAMP. We report that in great contrast to human ejaculated sperm and other mammalian sperm cells, the human epididymal sperm do not express a Mn(2+)-sensitive AC.

Relationship of epididymal sperm antibodies to their in vitro fertilization capacity in men with congenital absence of the vas deferens.

Objectives: To test, using the immunobead binding technique, for the presence of antisperm antibodies on epididymal sperm, in epididymal fluid, and in serum of men with congenital absence of the vas deferens. To evaluate the in vitro fertilization (IVF) capacity of human epididymal sperm in the presence of antisperm antibodies.

Design: Prospective.

Regulation of cyclic adenosine monophosphate synthesis in human ejaculated spermatozoa. II. The role of calcium and bicarbonate ions on the activation of adenylyl cyclase.

In this study, we have applied our previous data describing the experimental conditions necessary for expression of cyclic adenosine monophosphate (cAMP)-synthesizing adenylyl cyclase in human ejaculated spermatozoa, to investigate the direct effects of calcium (Ca2+) and bicarbonate (HCO3-) upon activation of the enzyme in vitro. We report that the effects of Ca2+ and HCO3- were significantly dependent on the status of the enzyme activity. Thus, at a near saturating (10 mM) concentration of MnCl2 giving high enzyme activity, addition of less than 10 mM HCO3- did not affect adenylyl cyclase activity and higher concentrations inhibited the enzyme, with 50 mM HCO3- reducing the activity by 33%.

The role of the adenylyl cyclase system in the regulation of corpus luteum function in the human and in nonhuman primates.

We have reviewed the properties of luteinizing hormone/human chorionic gonadotropic (LH/hCG)-sensitive adenylyl cyclase (AC) of human corpus luteum (CL) and its regulation by several hormones and nonhormonal activators. We have also described the changes in enzyme activity in membrane preparations of human and cynomolgus monkey CL obtained at various stages of the menstrual cycle and pregnancy. The data have been analyzed with respect to the functional status of the luteal tissue and to the species differences among primate CL.

Insulin-like growth factors and thecal-granulosa-cell function.

Insulin-like growth factors (IGFs) belong to a family of low mol. wt, single chain polypeptides inducing growth promotion and insulin-like metabolism effects and regulating both cell replication and differentiation. Recent studies in laboratory animals suggest that IGFs play an important role as intraovarian regulators in several mammalian species.

Intrauterine inseminations with washed human spermatozoa does not induce formation of antisperm antibodies.

In this study we investigated the possible development of serum antisperm antibodies in women receiving repeated IUI. Patients acted as its own control and were evaluated before and after various (1 to 15) IUI cycles using three different assays for antisperm antibodies. It was found that only 2 out of 41 women developed antisperm antibodies.

Low incidence of sperm antibodies in men with congenital absence of the vas deferens.

The incidence of antisperm antibodies in serum and seminal fluid of 27 azoospermic men with congenital absence of the vas deferens is evaluated. The presence of antisperm antibodies was assessed using the immunobead test, the agglutination test, and immobilization test. Five patients with vasovasostomy or vasoepididymostomy attempts were included in the study and tested for the presence of antisperm antibodies.

Expression of insulin-like growth factor-II (IGF-II) messenger ribonucleic acid (mRNA), but not IGF-I mRNA, in human preovulatory granulosa cells.

Increasing evidence suggests that insulin-like growth factors (IGFs) play an important role as intra-ovarian regulators in several mammalian species. Recently, we and others have reported the presence of both IGF-I and IGF-II in human follicular fluid. The source of these follicular IGFs, however, has not been determined.

Delayed occurrence of an LH surge after HCG administration during ovarian stimulation with gonadotrophins: effect of LHRH treatment.

Previous studies have shown the appearance of a spontaneous luteinizing hormone (LH) surge after human chorionic gonadotrophin (HCG) administration in human menopausal gonadotrophin (HMG)/HCG-stimulated menstrual cycles. In this report we investigated the effect of leuprolide acetate, a long-acting luteinizing hormone releasing hormone (LHRH) agonist, on the occurrence of these post-HCG rises in serum LH. Two groups of patients were included.

Reproducibility of the indirect immunobead assay for detecting sperm antibodies in serum.

Although an immunobead assay (IBA) for the detection of antisperm antibodies was developed several years ago and has been used for the study of immunologic infertility, no data regarding its variability and reproducibility are yet available. We evaluated the intraassay reproducibility of the indirect IBA by testing aliquots of antisperm-antibody-positive sera from two patients against the same donor sperm sample. The interassay reproducibility was evaluated by testing a positive serum sample first with different sperm samples from the same donor and second with sperm samples from different donors.

Changes in adenylyl cyclase activity of the human and nonhuman primate corpus luteum during the menstrual cycle and pregnancy.

Adenylyl cyclase (AC) activity in membrane particles of corpora lutea (CL) from humans and cynomolgus monkeys was examined at various stages of the menstrual cycle and pregnancy. AC activity was monitored by the conversion of [alpha-32P]ATP into [32P]cAMP under basal conditions and in the presence of several activators: NaF (10 mmol/L) plus forskolin (100 mumol/L); hCG (10 micrograms/mL); guanyl 5'-yl-imidodiphosphate [GMP-P(NH)P; 100 mumol/L]; and hCG (10 micrograms/ml) plus GMP-P(NH)P (100 mumol/L). The groups of human CL were midluteal (n = 10), late luteal (n = 4), following cycle (old CL; n = 5), and early pregnancy (6-11 weeks; n = 10).

Regulation of gonadotropin-stimulable adenylyl cyclase of the primate corpus luteum.

In an effort to understand the molecular mechanisms that control luteal function in the human and nonhuman primates, we have investigated the experimental conditions for expression of gonadotropin-induced adenylyl cyclase (AC) in membrane particles from primate corpus luteum (CL) and some of the factors modulating the enzyme activity. We also examined the usefulness of the cell-free model for studying the role of AC in the regulation of CL functions in human and nonhuman primates. Enzyme activity was dependent on guanine nucleotide and Mg ion.

Immunoreactive insulin-like growth factor I in human follicular fluid.

Recent studies in laboratory animals suggest that insulin-like growth factor I (IGF-I) plays an important role in the regulation of granulosa cell function. The purpose of the present study was to investigate the presence of immunoreactive IGF-I in human follicular fluid (FF) and compare the levels of follicular IGF-I (64 follicles) with those detectable in serum (n = 19) in hyperstimulated cycles from 25 infertile patients. Also, the FF IGF-I levels were correlated to corresponding follicular volume (n = 62) and oocyte maturation (n = 37).

A cDNA for the estradiol-regulated 24K protein: control of mRNA levels in MCF-7 cells.

We have previously demonstrated an estradiol-regulated 24 kDa (24K) protein in human breast cancer tissue culture cells and human tumor biopsies. The presence of 24K correlates well with the presence of steroid hormone receptors. In order to further study the hormonal regulation of the 24K protein and gene, we have isolated cDNA clones corresponding to the 24K mRNA.

Assessment of serum luteinizing hormone during ovarian stimulation with gonadotrophins.

An immunoradiometric assay (IRMA), using monoclonal antibodies with high affinity for human luteinizing hormone (HLH), was evaluated for quantitative measurement of serum LH after human chorionic gonadotrophin (HCG) administration in patients undergoing stimulation of multiple follicular development. Compared to a radioimmunoassay (RIA) commonly used to monitor serum LH, LH IRMA was more effective by several orders of magnitude in discriminating between HLH and HCG and showed no cross-reactivity at HCG concentrations normally found in serum after hormone treatment. Assays of serum samples obtained from 10 patients receiving HCG as part of an HMG/HCG protocol to induce ovulation for IVF/GIFT also demonstrated that RIA values were greatly affected by exogenous HCG.

P-glycoprotein expression in human breast cancer cells.

Multidrug resistance in Chinese hamster ovary cells is associated with the Mr 170,000 surface glycoprotein. Using our monoclonal antibody to this protein, we have isolated a complementary DNA clone from an expression vector library. This complementary DNA recognizes a 4.

Serotonergic and adrenergic regulation of skeletal muscle metabolism in the rat. II. The use of [125I]iodolysergic acid diethylamide and [125I]iodopindolol as probes of sarcolemmal receptor function and specificity.

To evaluate serotonin receptor kinetics in skeletal muscle, we synthesized and developed 2-[125I]iodolysergic acid diethylamide [( 125I]iodoLSD) as a high affinity, high specific activity probe of serotonergic receptor function. The kinetics of binding of this probe and the profile of agonist and antagonist displacement have been compared to results obtained using [125I] iodopindolol as a probe for beta-adrenergic receptor binding. [125I]IodoLSD was prepared by chloramine-T iodination and purified by high pressure liquid chromatography.