Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa.

BACKGROUND: Oxidative stress induced by reactive oxygen species (ROS) is associated with an impaired fertilization ability of spermatozoa. We investigated the effects of adding antioxidants to a sperm preparation medium on the functional parameters of the spermatozoa. METHODS: Spermatozoa were washed with Ham's F-10 media containing the antioxidants, ethylenediaminetetraacetic acid (EDTA) and catalase, at various concentrations, and then the ROS levels in sperm suspensions, and the forward motility, acrosome reaction, DNA integrity and lipid peroxidation of the spermatozoa were assessed. RESULTS: The ROS levels were significantly lower in sperm suspensions washed with the antioxidants (196 approximately 312 rlu; relative light units) than in control sperm (604 rlu, P < 0.05). The addition of 10 microM EDTA to the sperm preparation medium significantly improved the motility of the spermatozoa compared with the control group, the groups containing EDTA at other concentrations and the groups containing catalase. Catalase significantly increased the acrosome reaction rate of the spermatozoa. Both EDTA and catalase significantly decreased the DNA fragmentation rate of the spermatozoa. However, the antioxidants did not reduce lipid peroxidation. CONCLUSIONS: Supplementing sperm preparation medium with EDTA or catalase significantly improved the overall functional parameters of the spermatozoa by reducing the ROS levels.

Compaction in preimplantation mouse embryos is regulated by a cytoplasmic regulatory factor that alters between 1- and 2-cell stages in a concentration-dependent manner.

Present studies were performed to investigate what factors affect the morphogenesis of preimplantation mouse embryos, and to find the action mechanism of that factor by using cytoplasm removal and its reconstitution from a different developmental stage embryo. Half (HP group) or one-third of cytoplasm (TP group) was removed from 1-cell mouse embryos by micromanipulation, and their morphogenesis and genome expression were compared with sham-operated embryos (SP group). The compaction and blastocoel formation of embryos in both the HP and TP groups were accelerated in time and cell stage when compared with those of the SP group. However, the total activity and time of RNA synthesis, and gene expression of ZO-1alpha+ isoform were not different. To change the cytoplasm composition without altering the nucleus/cytoplasmic ratio, half a 1-cell embryo with both pronuclei was reconstituted with the half enucleated cytoplasm of 1-cell embryo (P + P group), 2-cell (P + 2 group) or 4-cell (P + 4 group) by electrofusion. Embryonic compaction, timing of RNA synthesis, and stage-specific gene expression of the ZO-1alpha(+) isoform in the P + 2 and P + 4 groups were accelerated in time and cell stage than that in the P + P group, but not different between the P + 2 and P + 4 groups. In addition, a blastomere of 2-cell embryo was reconstituted with the enucleated cytoplasm of 1-cell embryo (2 + P group) or 2-cell (2 + 2 group) in equal volume by electrofusion. Also, the karyoplast of 2-cell was fused with the enucleated 1-cell embryo (2 + PP group). Embryonic development, total activity of RNA synthesis, and gene expression of the ZO-1alpha(+) isoform of embryos in the 2 + P and 2 + PP groups were delayed when compared with those of the 2 + 2 group. Also, the phenomena of compaction and blastocoel formation were delayed in the development time and cell stage. From these results, the nucleus/cytoplasm ratio was found to have no direct effect on the regulation of embryonic morphogenesis, although it accelerated compaction and blastocoel formation. However, cytoplasmic factors that altered between 1- and 2-cell stages regulate embryonic morphogenesis, especially compaction, of preimplantation mouse embryos in concentration-dependent manner.

Ovarian expression of melatonin Mel(1a) receptor mRNA during mouse development.

Melatonin, secreted by the pineal gland, is involved in the regulation of many physiological functions of various species of animals. In the present study, the expression of gene for melatonin Mel(1a) receptor (MelR) was evaluated in the ovary, hypothalamus, and pituitary according to the developmental stages in female mice. Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) and in situ PCR techniques were applied. According to the developmental stages, gene for MelR was differently expressed on ovary, hypothalamus, and pituitary. MelR gene was first expressed on pituitary prior to the expression in hypothalamus and ovary. Ovarian MelR gene started to express at birth. Unlike hypothalamic expression of MelR gene which was identified after birth, in pituitary, it was expressed at 16 days post coitum. In the ovary, the expression signal of MelR gene was identified on granulosa cells. However, the signal was not detected in the theca cells. It was weak in the primordial and atretic follicles. Taken together, it can be considered that melatonin has a pivotal role in the folliculogenesis.

Expression of DAZ (deleted in azoospermia), DAZL1 (DAZ-like) and protamine-2 in testis and its application for diagnosis of spermatogenesis in non-obstructive azoospermia.

Spermatogenesis is regulated by hormones, local regulatory factors in the testes and specific gene expression of spermatogenic cells in humans. In this study, we have detected the expression of the deleted in azoospermia (DAZ), the DAZ-like autosome (DAZL1), and the protamine-2 genes in spermatogenic cells. Spermatogenesis in 38 male infertility patients was evaluated by the semen analysis and histological examination. Patients were diagnosed as Sertoli cell-only syndrome (n = 20), maturation arrest (n = 6), hypospermatogenesis (n = 6), and obstructive azoospermic patients with normal spermatogenesis (n = 6). After microscopic observation of the wet preparation of the testis tissues, seminiferous tubule contents were used for reverse transcription-polymerase chain reaction (RT-PCR) analysis of DAZ, DAZL1 and protamine-2. In cases with Sertoli-cell only syndrome, we found spermatogenic cells in 30% of patients (6/20) by the wet preparation method. There was no difference between the histology and the wet preparation results in maturation arrest and obstructive azoospermia; however, in one case of hypospermatogenesis, spermatozoa were not detectable by the wet preparation method. Using in-situ hybridization with DAZ and protamine-2 ribonuclear probes, we confirmed spermatogenic cell-specific expression of DAZ (spermatogonia/early spermatocyte) and protamine-2 (spermatid/spermatozoon). DAZ and protamine-2 expression can therefore be considered spermatogenic cell markers and could be useful in molecular diagnosis of spermatogenesis. In 13 patients with spermatozoa under the wet preparation, the expression of DAZ, DAZL1 and protamine-2 was detected in all the preparations. In one wet preparation showing only spermatogonia/spermatocyte, only DAZ and DAZL1 RNA were detected. In 14 wet preparations showing no spermatogenic cells, DAZ, DAZL1 and protamine-2 were not detected except in one preparation where DAZL1 expression was detected. In 10 wet preparations representing spermatogonia/spermatocyte to spermatids, but showing no spermaozoa, DAZ and DAZL1 were detected in eight and nine preparations respectively, and protamine-2 was detected in six preparations. These results of gene expression were similar to the wet preparation results. RT-PCR for DAZ, DAZL1 and protamine-2 was informative for the existence of germ cells, germ cell physiology and differentiation. From these results, we suggest that the analysis of DAZ, DAZL1 and protamine-2 expression by RT-PCR and wet preparation might offer a better method for finding the spermatogenic cells compared to the histological method.

Induction of acrosome reaction in human spermatozoa accelerates the time of pronucleus formation of hamster oocytes after intracytoplasmic sperm injection.

OBJECTIVE: To assess the relationship between the incidence of acrosome reaction (AR) and the timing of pronucleus (PN) formation after intracytoplasmic sperm injection (ICSI). DESIGN: Prospective study. SETTING: Infertility Research Center, Jeil Women's Hospital. MAIN OUTCOME MEASURE(S): Human semen obtained from fertile donors was prepared by one of the following methods: washing only (washed control); Percoll gradient; pentoxifylline; human follicular fluid (FF); pentoxifylline + FF; or platelet-activating factor (PAF) treatment. The AR of each group was assessed by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin or Arachis hypogea agglutinin. Spermatozoa of washed control, pentoxifylline + FF, and PAF treated groups, with significantly higher AR rate than others, were injected into mature hamster oocytes. Spermatozoon-injected oocytes were cultured for 6, 9, 12, or 15 hours. Then they were stained with Toluidine blue for PN formation examination under a light microscope. RESULT(S): Acrosome reaction rates of washed control, Percoll gradient, pentoxifylline, FF, pentoxifylline + FF, and PAF treated groups were 10.5% +/- 2.6%, 10.3% +/- 1.7%, 16.4% +/- 1.8%, 24.8% +/- 5.6%, 28.4% +/- 3.8%, and 33.3% +/- 5.2%, respectively. Pronuclear formation rate in washed control, pentoxifylline + FF, and PAF treated groups were 5.6% (3/54), 19.0% (11/58), and 18.9% (10/53) at 6 hours; 32.7% (18/55), 51.8% (29/56), and 57.4% (31/54) at 9 hours; 36.1% (22/61), 53.6% (30/56), and 50.0% (27/54) at 12 hours; and 47.2% (25/53), 64.8% (35/54), 53.6% (30/56) at 15 hours after ICSI. Pronuclear formation rate was significantly higher in pentoxifylline + FF, and PAF treated groups than that in the washed control group at 6 and 9 hours after ICSI. CONCLUSION(S): Pronuclear formation of oocytes takes place faster on those that were injected with acrosome-reacted spermatozoon than those injected with acrosome-intact spermatozoon. It could be concluded that induction of the AR of spermatozoa accelerates the time of PN formation and early development of the embryo in ICSI.

The supplementation of culture medium with protease improves the hatching rate of mouse embryos.

Mammalian embryos are known to exhibit delayed development and have lower hatching rates in vitro than in vivo because of inadequate culture condition. These discrepancies may be due to a deficiency of the paracrine factors and proteolytic enzymes which exist in the oviduct and uterus. In order to evaluate the effects of proteases on embryonic development and hatching, 2-cell mouse embryos were cultured for 72 h with or without proteases. The addition of 1.0 microg/ml pronase (PE) and/or 0.1 microg/ml proteinase K (PK) did not affect embryonic development up to the blastocyst stage (94.1% versus 88.2%; 92.2% versus 90.2%, respectively) but significantly increased the hatching rate (60.4% versus 39.2%, 71.8% versus 35.3%, respectively). However, the addition of alpha-chymotrypsin (Chymo) was detrimental to embryonic development and hatching. Changes in the structure of the zona pellucida (ZP) structure of embryos which had been cultured in human tubal fluid (HTF) medium with PE and PK were assessed by fluorescein isothiocyanate-conjugated (FITC)-casein. Embryos cultured in HTF-PE and PK were not stained with FITC-casein. When these embryos were cultured within oviducts, their perivitelline space (PVS) became strongly stained with FITC-casein which was easily removed by phosphate-buffered saline washing. This suggests that PE and PK altered the structure of the ZP. We suggest that the addition of PE and PK to culture media may accelerate the hatching of embryo, by structurally altering the ZP and PVS. This may provide a valuable and effective assisted hatching technique for human in-vitro fertilization-embryo transfer.

Intermittent GnRH antagonist plus progestin contraception conserving tonic ovarian estrogen secretion and reducing progestin exposure.

The present study was designed to evaluate the effectiveness of a once-weekly regimen of GnRH antagonist followed by a progestin as a potential new contraceptive method. On menstrual cycle days 2, 9, 16, and 23 (onset of menses = Day 1) monkeys were divided into two groups: 1) those injected sc with 0.1 mg/kg Nal-Glu GnRH antagonist in saline and those given only vehicle (control). On cycle days 15 to 26, each treated female was administered 25 micrograms norgestimate/day orally. This was continued for three treatment cycles (84 days). Weekly injections of Nal-Glu GnRH antagonist effectively blocked completion of folliculogenesis, ovulation, and corpus luteum function as judged by serum LH, E2, and P levels. Serum progesterone was undetectable (less than 0.1 ng/ml) during the treatment cycles. Importantly, serum estradiol levels during GnRH antagonist plus norgestimate treatments were maintained at 35 +/- 7 pg/ml. Upon the cessation of norgestimate treatment on day 26 in each cycle, menses uniformly began within 2 or 3 days. Regarding recovery, apparently normal and presumably ovulatory menstrual cycles, as judged by timely estradiol elevations, midcycle LH surges, and luteal phase progesterone patterns, were manifest immediately following termination of the final GnRH antagonist plus norgestimate treatment cycle. Endometrial biopsies removed on day 26 of control cycles, and on day 26 of the third treatment cycle revealed appropriate late secretory phase endometrium having tortuous endometrial glands and superficial stromal edema. Histological sections of ovaries removed at the end of the GnRH antagonist plus norgestimate treatment revealed multiple small and medium-sized developing and atretic follicles, having maintained serial ablation of the potentially maturing follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Probing studies on multiple dose effects of antide (Nal-Lys) GnRH antagonist in ovariectomized monkeys.

This study was designed to extend evaluation of the long-acting effects of a "third generation" Antide (Nal-Lys) GnRH antagonist on gonadotropin secretion in ovariectomized (OVX) monkeys, with special attention to recrudescence of pituitary gonadotropin secretion after multiple dose treatments, as well as pituitary secretory responsiveness to GnRH. The duration of FSH/LH inhibition by Antide was dose-dependent, as well as being much longer than for Nal-Glu GnRHant; however, full recrudescence of gonadotropin secretion, albeit gradual, did occur. The acute LH secretory response to serial iv boluses of GnRH, in the face of GnRHant-induced suppression of gonadotropin secretion, was transiently accelerated and biologically active. Thereafter, the state of FSH/LH inhibition was resumed chronically. Thus, treatment with Antide produced profound long-term inhibition of tonic gonadotropin levels, yet hyper-responsiveness to exogenous GnRH administration was maintained throughout.

Effects of progesterone and a progesterone antagonist (RU486) on germinal vesicle breakdown in the mouse.

The possibility that ovarian steroids may participate in the inhibition of meiosis has not been rigorously examined. Since progesterone levels are extremely high in follicular fluid prior to ovulation, we tested the possibility that this steroid may be involved in oocyte maturation. To this end, we collected follicular oocytes and cultured them in the presence of dibutyrl cAMP (Bt2), progesterone, and/or the progesterone antagonist RU486 and assessed maturation evidenced by germinal vesicle breakdown (GVBD). Denuded oocytes or cumulus masses collected in the presence of 1 mM Bt2 and subsequently cultured in 25 microM progesterone did not undergo GVBD. However, denuded oocytes and cumulus masses collected in the presence of progesterone and not Bt2 did undergo GVBD (93%). Concentrations of Bt2 (150 microM) that would not inhibit GVBD were inhibitory when used in the presence of progesterone (1-25 microM). Competition experiments using increasing concentrations of the progesterone antagonist RU486 (1-100 microM) did not block the ability of progesterone to enhance the activity of Bt2. We conclude that progesterone alone does not block GVBD; however, in the presence of low concentrations of cAMP it is extremely effective in blocking GVBD. The synergistic activity of progesterone does not appear to be mediated by the progesterone receptor. The data suggest that progesterone and cAMP may operate cooperatively to inhibit meiosis in the ovarian follicle.

Atypical luteinizing hormone rise and associated fertilization failure in non-male factor in vitro fertilization patients.

The purpose of this investigation was to analyze fertilization failure in non-male factor infertility in vitro fertilization (IVF) patients. Twenty-five nonfertilized IVF patients were compared with 57 successfully fertilized IVF patients. Patients were matched for age, ovulation-induction protocol, and cycle. The two groups were similar with respect to infertility etiology, peak estradiol, and total oocytes retrieved at laparoscopy. There was a greater incidence of primary infertility (64 versus 49%) and mean years of infertility (5.4 +/- 0.4 versus 4.6 +/- 0.3) in the nonfertilization group, although these differences were not statistically significant. Most important, the nonfertilization patients had a greater incidence of an atypical LH rise prior to hCG administration (60 versus 18%; P less than .001) and fewer mature oocytes (2.0 +/- 0.3 verus 3.4 +/- 0.3; P less than .01). Stepwise linear regression analysis yielded four primary factors for predicting fertilization failure: infertility duration, primary infertility, number of mature oocytes, and presence of LH rise. These findings help characterize several potential factors other than oligozoospermia that are associated with nonfertilization, and support LH monitoring in IVF and gamete intrafallopian tube transport patients.

The effects of progesterone antagonist RU 486 on mouse oocyte maturation, ovulation, fertilization, and cleavage.

The antiprogesterone RU 486 was utilized to evaluate the possible role of progesterone in ovum maturation, ovulation, fertilization, and embryo cleavage. After gonadotropin treatment, CD-1 mice received the following experimental agents: group 1, an oil vehicle; group 2, 1 mg progesterone; group 3, 1 mg antiprogesterone (RU 486); group 4, 1 mg RU 486 and 1 mg dexamethasone. Each group of animals was injected simultaneously for 2 days (concomitant with human chorionic gonadotropin and the day after coitus). Ova or embryos were obtained on day 1, 2, 3, or 4 after human chorionic gonadotropin by flushing uteri and tubes. No differences were apparent in number of oocytes ovulated, ovum maturation, or number of oocytes progressing to two-cell embryos. However, on day 3 a marked reduction in embryos retrieved from the oviduct and uterus was apparent in the RU 486-treated groups (group 1, 84; group 3.0; group 4, 17) (p less than 0.001). In addition, few cleavage stage embryos were recovered on day 4 in the RU 486-treated groups (group 1, 74; group 2, 70; group 3, 2; group 4, 0) (p less than 0.0001). Freshly ovulated cumulus masses were recovered from the oviducts on day 4 in groups 3 and 4 (coincident with resumption of the estrous cycle). In conclusion, periovulatory RU 486 injections had no effect on nuclear maturation, ovulation, fertilization, or first cleavage division. Progesterone may not have an obligatory role in these processes. However, RU 486 administration did result in a reduced number of embryos retrieved on days 3 and 4 because of either early expulsion or destruction of the embryos.

The effects of the progesterone antagonist RU-486 on mouse preimplantation development in vitro and in vivo.

We have evaluated the effects of RU-486 on preimplantation development in the mouse. Female mice received a subcutaneous injection of 1mg RU-486 with hCG on the morning post-coitus. Two-cell embryos were recovered from the oviducts and cultured in the presence or absence of RU-486. Three days later we found that 2-cell embryos exposed to RU-486 in vivo developed to the blastocyst stage at rates similar to control regardless of whether RU-486 was present in culture medium. Transfer experiments revealed that embryos retrieved from RU-486-treated females developed normally in the reproductive tracts of non-treated control foster mothers. However, when control 2-cell embryos were transferred to RU-486-treated foster mothers, development was not supported. We conclude that the contragestational properties of periovulatory treatment with RU-486 is not the result of a direct effect on the cleavage stage embryo but on the reproductive tract of the mother.

In vitro fertilization with concurrent pelvic reconstructive surgery.

The recent advent of ultrasound-guided follicular aspiration by various approaches now allows access to ovaries previously deemed inaccessible by laparoscopy; however, a small group of candidates for in vitro fertilization and embryo transfer (IVF-ET) require laparotomy for associated gynecologic disorders. Twenty-five IVF-ET cycles at the time of laparotomy were compared with 309 IVF-ET cycles in which oocytes were retrieved laparoscopically. Five pregnancies occurred in the IVF-ET cycle with laparotomy and one pregnancy occurred spontaneously following microsurgical tubal reconstruction. The pregnancy rate per embryo transfer was 25% in the laparotomy IVF-ET patients compared with 15.4% for the laparoscopy IVF-ET group. Obvious advantages of combining IVF-ET and pelvic reconstructive surgery include a single anesthesia exposure and economic benefits. Patients with a long history of infertility undergoing tubal reconstructive surgery may be offered combined IVF-ET. Extended anesthesia exposure with pelvic surgery demonstrated no adverse effects on the pregnancy rate.

In vitro fertilization and embryo transfer: treatment-dependent versus -independent pregnancies.

In order to determine the true incidence of treatment-dependent versus -independent pregnancy in an in vitro fertilization (IVF) program, 274 women who underwent 492 cycles of superovulation were studied. Overall, the treatment-dependent pregnancy rate was 15%. The treatment-independent pregnancy rate was 6.6%. When a subgroup of individuals with at least one patent fallopian tube was selected for analysis, the treatment-dependent and -independent pregnancy rates were 13.9% and 11.9%, respectively. While the mean observation interval following an attempt at IVF was 2 years, 83.3% of all treatment-independent pregnancies occurred within 6 months after a trial of IVF-ET (embryo transfer). Patient characteristics that predispose to treatment-independent pregnancy are discussed.

Local peritoneal factors: their role in infertility associated with endometriosis.

In order to detect peritoneal abnormalities that could account for infertility associated with endometriosis, 122 infertile individuals were studied at the time of laparoscopy for diagnostic purposes or for in vitro fertilization. Four groups were defined: group 1, laparoscopy without endometriosis; group 2, laparoscopy with endometriosis; group 3, in vitro fertilization without endometriosis; and group 4, in vitro fertilization with endometriosis. Mean peritoneal fluid volume was greater, although not significantly so, in group 4 (29.0 +/- 6.6 ml, mean +/- SEM) than in group 3 (18.2 +/- 2 ml). The concentration and total number of pelvic macrophages were similar for groups 1 and 2. The total number of pelvic macrophages was increased in group 4 (16.9 +/- 4.2 x 10(6)) versus group 3 (10.0 +/- 1.8 x 10(6)) (p = 0.08). The mean sperm phagocytosis in vitro did not differ among the four groups studied. Interleukin 1 activity within the peritoneal fluid and the in vitro interleukin 1 production rate did not differ between individuals with and without endometriosis. Peritoneal fluid and macrophage supernatants from individuals with endometriosis were not embryotoxic when studied in an in vitro mouse embryo system.