Inn Soo Kang

Cocaine Disrupts Estrous Cyclicity and Alters the Reproductive Neuroendocrine Axis in the Rat

Although a common drug of abuse, cocaines effects on cyclic reproductive functions and the neuroendocrine systems regulating these functions have not been studied. Here, we report the effects of cocaine on (1) estrous cyclicity and ovulation rates and (2) the stimulated in vitro release of hypothalamic GnRH and aminergic neurotransmitters directly involved in regulating or modulating GnRH release. Within 7 days of treatment with 10 mg kg-1 day-1 of cocaine HCl subcutaneously, rats demonstrated significant estrous cycle irregularity including repetitive days of estrus and prolonged periods of diestrus. After 6 weeks of treatment, cocaine-treated rats exhibited a 44.3% decrease in ovulation rates. For the in vitro studies, bilaterally ovariectomized rats were injected with cocaine (10 mg kg-1 day-1) or with saline for 2 weeks. Each rat received estradiol benzoate (50 mg kg-1 day-1 s.c.) for 2 days before sacrifice. Hypothalamic slices were prepared, placed in 0.1 ml microchambers and perfused with modified Krebs buffer (pH 7.4) using a programmable perfusion system. Basal release of norepinephrine (NE) and serotonin (5HT) was significantly increased in the cocaine-treated group versus controls. Ten-minute pulses of 10(-7)M progesterone (P4) increase NE and 5HT, but not dopamine (DA), release in the saline-treated group. In contrast, pulses of P4 increased NE, but not 5HT or DA, in the cocaine-treated rats. Ten-minute pulses of 0.1 microM NE increased GnRH release in both saline- and cocaine-treated rats. However, the response to pulsed NE was significantly attenuated in the cocaine-treated group.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of treatment with gonadotropin-releasing hormone analogues on pregnancy outcome in the baboon * †

The effect of a gonadotropin-releasing hormone (GnRH) agonist, Zoladex (Imperial Chemical Industries, PLC, London, England), and an antagonist, Organon 30276 (Organon, Oss, Holland), on the outcome of pregnancies when administered just after implantation was examined. The antagonist, Organon 30276, was administered continuously from days 14 through 21, and the agonist, Zoladex, was injected as long-acting pellets on day 14 after conception to pregnant baboons. Maternal baboon chorionic gonadotropin and progesterone levels and sonographic measurement were made before, during, and after treatment and throughout pregnancy of control and treated animals. Two pregnant animals were treated with 3.6 mg of Zoladex; one aborted and one had a stillbirth. Another four pregnant baboons were treated with 7.2 mg of Zoladex; two aborted, one had a premature with a low-birthweight infant, and one had a normal liveborn. Organon 30276, at 50 mg, was administered to three pregnant baboons and resulted in one stillbirth, one neonatal death, and one normal liveborn. The two pregnant baboons treated with 100 mg Organon 30276 both aborted. Therefore, treatment with these GnRH analogues in very early baboon pregnancy could adversely affect the outcome of pregnancy. Thus attention should be paid to the possible presence of early pregnancy at the time of GnRH analogue therapy, which might adversely affect the outcome of the pregnancy.

Expression pattern of germ cell-specific genes in the testis of patients with nonobstructive azoospermia : usefulness as a molecular marker to predict the presence of testicular sperm

OBJECTIVE To analyze the expression pattern of testis-specific genes of patients with various spermatogenic defects and their usefulness as a molecular marker to predict the presence of testicular spermatozoa in patients with nonobstructive azoospermia undergoing IVF. DESIGN Prospective, controlled study. SETTING Hospital-based infertility research laboratory. PATIENT(S) Fifty-eight men with azoospermia or severe oligozoospermia. INTERVENTION(S) Testicular biopsy was done in the patients with obstructive or nonobstructive azoospermia, including Sertoli cell-only syndrome, maturation arrest, severe hypospermatogenesis, and normal spermatogenesis. MAIN OUTCOME MEASURE(S) Reverse transcriptase polymerase chain reaction was performed using 1 microgram of total RNA extracted from testicular tissues. Three pairs of primers were used for amplification of male germ cell-specific genes (DAZ, transcribed in male germ cells; PGK2, in late spermatocytes and spermatids; protamine-2, in spermatids) as molecular markers. Testicular sperm was obtained by multiple testicular sperm extraction. RESULT(S) The DAZ, PGK2, and protamine-2 genes were expressed in 38, 30, and 21 of the 43 patients with nonobstructive azoospermia, respectively. Testicular spermatozoa were successfully extracted in 4 of 43 patients with nonobstructive azoospermia with the use of multiple testicular sperm extraction. Detection of protamine-2 transcripts predicted the presence or absence of spermatozoa in the testicular tissue in 39 of 43 patients (91%). CONCLUSION(S) Expression of the protamine-2 gene may be a useful molecular marker to predict the presence of testicular sperm in patients with nonobstructive azoospermia.

Ubiquitin-specific protease activity of USP9Y, a male infertility gene on the Y chromosome.

Deletions of USP9Y have been observed among infertile males with defective spermatogenesis. Therefore, the gene has been designated as a male infertility gene on the Y chromosome. However, it remains to be determined how male infertility results from deletions of this gene. In order to initiate an investigation into the cellular functions of USP9Y in male germ cell development, in the present study we characterized the enzymatic specificity of USP9Y. Our results show that both USP9Y and Fam, the mouse infertility protein Usp9x, possess a protease activity specific to ubiquitin. These results suggest that, through de-ubiquitination, USP9Y may stabilize a specific target protein that is important for male germ cell development.

Characterization and purification of a placental protein that inactivates GnRH, TRH and angiotensin II☆

A protein that inactivates the immunoreactivity of GnRH, TRH and angiotensin II has been isolated from human term placentae. Only in the presence of DTT, a sulphydryl agent, are OXY and SRIF also inactivated by this protein. However, it is without effect on CRF, hCS, or hCG. It also inhibits the biological activity of GnRH, i.e. its ability to stimulate pituitary LH and FSH. The ability of this protein to inactivate GnRH, TRH or angiotensin II can be inhibited by various peptidase inhibitors. Thus, we have postulated that it is a chorionic peptidase, specific for these peptides, and herein called chorionic peptidase-1 (C-ase-1). Isolation of this protein, C-ase-1, has been effected using permeation, ion exchange and affinity chromatography. As estimated by SDS-PAGE and HPLC analyses, C-ase-1 has an apparent molecular weight of 58,000. It is proposed that C-ase-1 may be an important chorionic regulator of GnRH, TRH and angiotensin II levels during pregnancy.

Effect of exogenous arachidonic acid and enzyme inhibitors on placental prostanoid production

Prostanoids play an important role throughout pregnancy and during the initiation and progress of labor. The human placenta, at term, produces large quantities of prostanoids, yet little is known of the rate-limiting steps regulating their biosynthesis. In these studies, the effect of exogenous arachidonic acid and of enzyme inhibitors on the release of placental prostanoids was investigated. Basal prostaglandin E (PGE), prostaglandin F (PGF), thromboxane (TxB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) increased from the fifth hour in culture, yet the increased PGF release did not result in further 13,14-dihydro-15-keto-PGF2 alpha (PGFM) production. Addition of varying doses of arachidonic acid (0.2-10 micrograms/ml) had no significant effect on PGFM, TxB2 or 6-keto-PGF1 alpha, although the endogenous arachidonate was similar to or much less than the doses studied. Only at the 10 micrograms/ml dose was a delayed increase of PGE and PGF observed. Incubation with indomethacin resulted in an immediate inhibition of PGE, PGF, TxB2 and 6-keto-PGF1 alpha, with a delayed inhibition of PGFM. The phospholipase A2 inhibitor, quinacrine (10 microM), had no significant effect on PGE, PGFM, TxB2 or 6-keto-PGF1 alpha. PGF was inhibited within the first hour of quinacrine exposure, but no significant inhibition was observed thereafter. Ca2+ chelator, EDTA, effected an inhibition of only 6-keto-PGF1 alpha. Nordihydroguaiaretic acid, a leukotriene inhibitor, reduced PGE release as well as 6-keto-PGF1 alpha. These data demonstrate the high biosynthetic competence of the human term placenta to produce prostanoids. This capacity does not appear to be rate-limited by arachidonic acid availability. However, the metabolism of PGF to PGFM appears to be saturated. In addition, the production of placenta prostacyclin may be affected by Ca2+ levels, as chelating agent inhibited the release of 6-keto-PGF1 alpha.

Dose-related action of gonadotropin-releasing hormone on basal prostanoid production from the human term placenta.

The dose-related effect of gonadotropin-releasing hormone on placental prostanoids was studied with a perifusion system. Villous tissues were perifused with medium 199 (1 ml/hr) and at the beginning of the fifth hour, either 0, 10(-10), 10(-9), 10(-8), 10(-7), or 10(-6) mol/L gonadotropin-releasing hormone was added to the medium of triplicate chambers. The concentration of prostaglandin E, prostaglandin F, 13,14-dihydro-15-keto-prostaglandin F2 alpha, 6-keto-prostaglandin F1 alpha, and thromboxane B2 in the effluent medium, collected every hour, was determined by specific radioimmunoassay. The cumulative release after gonadotropin-releasing hormone treatment for each chamber was calculated, and replicate chambers were averaged. Linear regression analysis of the average for each dose from three different placentas was used to determine the dose-response relationship. Gonadotropin-releasing hormone significantly inhibited the release of placental prostaglandin E, prostaglandin F, and thromboxane B2 in a dose-dependent fashion. Gonadotropin-releasing hormone had no significant effect on 13,14-dihydro-15-keto-prostaglandin F2 alpha and 6-keto-prostaglandin F1 alpha, although there was an apparent increase in 13,14-dihydro-15-keto-prostaglandin F2 alpha. These data support the hypothesis that chorionic gonadotropin-releasing hormone inhibits prostanoid production from the placenta, which in turn may regulate various functions of prostanoids during pregnancy.

Chorionic peptidase inactivates GnRH as a post-proline peptidase

Recently, we have described a chorionic peptidase (C-ase-1) which inactivates gonadotropin releasing hormone (GnRH), oxytocin, angiotensin II and thyrotropin releasing hormone. Since all these hormones contain a proline residue, we proposed that C-ase-1 may act as a post-proline peptidase. Using HPLC and amino acid analyses, we have defined the products which resulted from enzymatic inactivation of GnRH by C-ase-1. The N-terminal nonapeptide of GnRH was isolated by HPLC and confirmed by amino acid composition analyses. Thus, it was demonstrated that C-ase-1 acts as a post-proline peptidase when inactivating GnRH, yielding the nonapeptide, i.e., des-Gly10-NH2-GnRH, and Gly-NH2. The levels of intrauterine GnRH, angiotensin II, oxytocin and thyrotropin releasing hormone may be affected and integrated by this enzyme. Thus, C-ase-1 may play an important role in the regulation of the paracrine and endocrine function during pregnancy.

Alternatively spliced variants of the follicle-stimulating hormone receptor gene in the testis of infertile men

OBJECTIVE To investigate whether or not alternatively spliced variants of the FSH receptor gene occur in human testis and whether the presence of the splicing variants is associated with spermatogenic defects and serum FSH concentration in infertile men. DESIGN A prospective case control study. SETTING An IVF clinic and infertility laboratory at a university hospital. PATIENT(S) Forty-three infertile patients undergoing testicular biopsy. INTERVENTION(S) Total RNA was extracted from the testicular tissues and used for reverse transcriptase-polymerase chain reaction (RT-PCR). MAIN OUTCOME MEASURE(S) Expression pattern was analyzed by nested RT-PCR using primers designed to amplify a fragment of FSH receptor gene. PCR products of splicing variants were cloned and sequenced. RESULT(S) The PCR products showed three kinds of additional bands corresponding to alternatively spliced isoforms of the FSH receptor gene. Exon 9 deleted variant was detected in all patients and inclusion variant of small extra exon was detected in 64% (9/14) of the patients with normal spermatogenesis and 34% (10/29) of the patients with spermatogenic defects. The presence of inclusion variant was not significantly associated with spermatogenic defects but was associated with a low level of serum FSH. On the other hand, exon 6 deleted variant was detected in only one patient having a high level of FSH concentration (30 IU/L) and Sertoli cell only syndrome. CONCLUSION(S) We identified three different types of alternatively spliced variants of the human FSH receptor. However, it is not clear whether or not there is an association between three variants and spermatogenic defects.

Comparison of the efficacy and safety of a new recombinant human follicle-stimulating hormone (DA-3801) with follitropin-α (Gonal-F®) in women undergoing controlled ovarian hyperstimulation for assisted reproductive technology

Aim:  To compare the efficacy and safety of a new recombinant human follicle‐stimulating hormone (FSH; DA‐3801) with follitropin‐α (Gonal‐F®) in women undergoing controlled ovarian hyperstimulation (COH) for assisted reproductive technology (ART).