Ovarian carcinoid with severe constipation due to peptide YY production.

We report a patient with primary trabecular carcinoid of the ovary with severe constipation probably due to peptide YY production by the tumor. A 43-year-old female had complained of severe constipation for several months and was found to have a left ovarian tumor. The surgically resected tumor was diagnosed as trabecular carcinoid by light microscopic examination. The carcinoid tumor cells were intensely and uniformly stained by the Grimelius technique. Immunohistochemically, the tumor cells were strongly positive for peptide YY, which has a strong inhibitory action on intestinal motility. The patient has been free from constipation since the removal of the tumor. The present case supports previously reported findings that not typical carcinoid syndrome but rather severe constipation accompanies primary trabecular carcinoid of the ovary, and that peptide YY is presumably the cause of the constipation.

Is treatment of long-term and consecutive use of clomiphene citrate effective in anovulatory patients? Results of multi-centric retrospective studies.

The efficacy of long term consecutive treatment of clomiphene citrate (CC) was investigated in 608 infertile women associated with anovulatory disorders (classified as WHO group II amenorrhea). This study was performed in multi-centric facilities of obstetrics and gynecology by a retrospective randomized assessment. Infertile women associated with anovulation were treated by 50-150 mg of CC for five consecutive days in each treatment cycle. In this study, 200 pregnancies were observed and the total pregnancy rate was 32.9%. The number of abortions were 33 out of the 200 pregnancies (16.5%). The cumulative pregnancy rate within the pregnant subjects reached 90% in initial 10 treatment cycles and there was no difference between 50 mg and 100 mg of CC treatment. There were remarkable differences among the pregnancy rates achieved during consecutive treatments at the primary (clinics and hospital), secondary (regional center hospitals) and advanced facilities. Consecutive treatment by CC for infertile women associated with anovulation may be effective during initial 10 cycles. Although ovulation can be confirmed during the following cycles, the stimulation protocol should be reconsidered after 12 consecutive cycles therapy with CC, because cycle fecundity decreases. Hence, to improve pregnancy rates, it is important to check the previous administration of CC which reflects adversely on the subsequent conception rate especially in advanced facilities where these patients are referred to.

Localization of fibronectin on the surface of human spermatozoa and relation to the sperm-egg interaction.

OBJECTIVES: The mechanism of human sperm-oolemma adhesion and penetration as well as localization of fibronectin on the sperm head and its relation to fertilization were investigated. DESIGN: Sperm-oolemma interaction was examined with an in vitro assay of the human sperm-zona-free hamster egg interaction. Localization of fibronectin on the surface of human spermatozoa was observed by the back scattered electron imaging mode of the scanning electron microscopy (SEM). RESULTS: It was confirmed by observations under SEM that the anterior tip of the sperm head is the first to come into contact with the egg plasma membrane but that the equatorial segment of the sperm head is the first to be trapped by microvilli of the plasma membrane and that the postacrosomal region is first incorporated into ooplasm. Localization of fibronectin on the equatorial segment of the human sperm head was detected by SEM. Antifibronectin antibodies inhibited human sperm-oolemma adhesion significantly. CONCLUSIONS: Important involvement of fibronectin in the gamete interaction was made clear by the fact that fibronectin is localized in the region where a spermatozoon is fused first with the egg plasma membrane during fertilization and that the sperm adhering to the egg is inhibited by antifibronectin antibodies.

The role of inhibin on pituitary FSH secretion and maturation of pituitary-gonadal axis in immature female rats.

The possible physiological significance of endogenous inhibin was evaluated in prepubertal female rats during sexual maturation. Ovarian/serum inhibin, serum FSH levels were measured from 4th day to 35th day of life by RIA. Serum inhibin levels were first detected on 4th day of life, thereafter, progressively increasing up to 27th day. Serum FSH levels gradually increased up to the 14th day with a sharp fall in the FSH levels on day 15 which afterwards was maintained at a lower level. Pregnant mare serum gonadotropin (PMSG) treatment on day 14 and day 22 rats showed different patterns of serum FSH and inhibin changes. In 14 day group, there was no difference between control and PMSG treatment, but in 22 day group, not only significant difference between control and PMSG treatment but also inverted relations between FSH and inhibin after PMSG treatment were found. It can be postulated from these findings that secretion of inhibin from the granulosa cells is initiated by FSH by its direct action on the ovary whereas the level of FSH itself is controlled by inhibin after FSH reaches peak values. Also it can be inferred that the feedback control mechanism of inhibin seems to be established around 3rd week of life.

Tenascin expression in normal and abnormal human endometrium.

Tenascin has been recently characterized as an extracellular matrix glycoprotein involved in tissue interactions during fetal development and oncogenesis. In order to study the possible involvement of tenascin in epithelial growth of the human endometrium, we evaluated the expression of tenascin in 84 cases of normal, hyperplastic, or neoplastic human edometrium. The specimens were obtained by curettage and/or biopsy and analyzed by immunohistochemistry utilizing a newly developed monoclonal antibody against human tenascin. Weak periglandular immunoreactivity was observed in 50% of proliferative phase, but not in secretory endometrium. Approximately 60% of endometrial hyperplasia specimens had weak periglandular tenascin immunoreactivity, but its distribution was irregular and not necessarily correlated with degree of cell atypia. Invasive endometrial carcinomas displayed intense and diffuse staining around the carcinoma cells, in addition to thin periglandular immunoreactivity similar to that seen in hyperplasia. The intensity of tenascin staining in endometrial carcinoma was not related to the degree of tumor differentiation. These results suggest that tenascin appears as a result of interactions between neoplastic epithelium and stroma in tumor development and that diffuse and intense staining could be a stromal marker for the invading capacity of human endometrial malignancies.

Fibronectin in reproduction.

The fibronectin (FN) levels in human follicular fluids have been shown to correlate well with follicular size and oocyte maturity, suggesting a role of FN in oocyte maturation. When added to the culture medium, the synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS), which specifically inhibits the cell-binding of FN, has been shown to inhibit both spontaneous resumption of meiosis and gonadotropin-releasing hormone-induced meiosis of the oocytes. In another set of experiments, GRGDS has been found to inhibit the in vitro cleavage of mouse embryos by a still unknown mechanism.

Artificially induced menstrual cycle with natural estradiol and progesterone.

In order to study the feasibility and efficacy of using natural 17 beta-estradiol (E2) and progesterone (P) to induce endometrial changes, a group of patients with history of premature ovarian failure and bilateral oophorectomy, also interested in the embryo transfer program with donor ovum, were given transdermal E2 (Estraderm, Ciba Pharmaceutical Co., Summit, NJ), and vaginal progesterone suppositories. Serial serum E2, P, follicle-stimulating hormone, and luteinizing hormone were determined by radioimmunoassays. The dosages of E2 and P were adjusted according to the levels of E2 and P so that their changes could follow the same pattern as that of a normal spontaneous menstrual cycle. Serial ultrasonic evaluation of the endometrium and endometrial biopsy during the late luteal phase also was performed. Preliminary data indicated that transdermal E2 patches and vaginal P suppositories, while being as effective in inducing endometrial development for the embryo transfer procedure with donor ovum as synthetic steroids, can also provide a more physiologic approach that may conveniently and safely be extended into the second trimester of pregnancy.

Effects of fibronectin and its peptide fragment on preimplantation mouse embryo.

To investigate the involvement of fibronectin in the early development of mouse embryo, embryos at the two-cell stage were cultured in serum-free Ham F-10 medium with or without fibronectin-related substances. When antibody raised against fibronectin was added at concentrations of 0, 10(-3), 10(-2), and 2 X 10(-2) (vol:vol), the percentages of cells in the blastocyst stage at 72 hours were 88.5%, 67.2%, 48.1%, and 0%, respectively. No inhibitory effects were observed with nonspecific antibody. A low concentration of exogenous fibronectin (up to 0.2 mumol/L) slightly enhanced mouse embryo development after 48 hours. However, a higher concentration (greater than 0.4 mumol/L) of fibronectin inhibited mouse embryo development. A competitive blocker of cell membrane fibronectin receptors, oligopeptide (Gly-Arg-Gly-Asp-Ser), inhibited mouse embryo development in a dose-dependent manner. Meanwhile, control peptide (Gly-Arg-Gly-Glu-Ser-Pro), which does not contain the crucial cell binding sequence, did not show any inhibitory effects. These findings suggest the necessity of endogenous fibronectin-blastomere interaction in early mouse embryo development.

Fibronectin and glycosaminoglycans in human preovulatory follicular fluid and their correlation to follicular maturation.

The purpose of this study was to investigate the involvement of two extracellular matrix constituents--fibronectin (FN) and glycosaminoglycans (GAG)--in the development of human ovarian follicles. One-hundred-and-one samples of human follicular fluid (HFF) aspirated from patients participating in an IVF-ET programme were assayed for FN, GAG, protein, progesterone (P) and oestradiol (E2). FN/protein and FN/GAG ratios increased significantly with volume of HFF and with follicular P levels. In contrast, GAG/protein ratios decreased significantly with HFF volume, follicular P and E2. Ratios of FN/protein correlated positively with maturity of oocytes; 3.1 +/- 0.5 with dysmature, 4.2 +/- 0.5 with immature, 8.5 +/- 1.0 with intermediate and 7.8 +/- 0.5 with mature oocytes. Ratios of GAG/protein were inversely correlated with maturity of oocytes; 11.0 +/- 1.0 with dysmature, 12.5 +/- 1.4 with immature, 8.9 +/- 0.9 with intermediate and 9.2 +/- 0.5 with mature oocytes. Ratios of FN/GAG correlated with maturity of oocytes; 0.3 +/- 0.02 with dysmature, 0.4 +/- 0.1 with immature, 1.2 +/- 0.2 with intermediate and 1.1 +/- 0.1 with mature oocytes. Furthermore, follicles leading to oocytes which fertilized showed significantly higher FN/protein and FN/GAG ratios than those yielding oocytes which remained unfertilized. In contrast, GAG/protein ratios were significantly lower in follicles with which oocytes fertilized than in those with oocytes which did not fertilize. These results suggest that FN and GAG can be useful markers for follicular development and the potential of the oocyte to be fertilized.

Effects of androgen on 17 beta-estradiol production by cultured human granulosa cells.

In order to determine whether androgen acts solely as a substrate for aromatization or whether it also influences on the activity of aromatase enzyme, human granulosa cells were incubated in vitro with or without androgen. Although basal production of 17 beta-estradiol (E2) in cultured granulosa cells obtained from follicles about 26 hr after the initiation of the LH surge was restricted in small quantities, a marked increase in E2 production occurred in the presence of testosterone (T) (10(-6) M) as aromatizable substrate. The non-aromatizable androgen, 5 alpha-dihydrotestosterone (DHT) (10(-7) M, 10(-6) M), slightly enhanced E2 production and it did not inhibit T aromatization in these cells. By contrast, DHT did not increase E2 production in granulosa cells obtained from follicles 2-5 days before the LH surge. The results indicate that androgen can enhance the estrogen biosynthesis of granulosa cells obtained from follicles during the LH surge not only by acting as a substrate for aromatization but also by participating in some process of the estrogen synthesis. This effect of androgen was not clearly seen in granulosa cells obtained before the LH surge, suggesting that the response of granulosa cells to exogenous androgen varies with the stage of the cell differentiation.

[Effects of androgen on 17 beta-estradiol and progesterone secretion by cultured human granulosa cells].

Human granulosa-luteal cells obtained from dominant preovulatory follicles at the time of oocyte retrieval for in vitro fertilization (about 10 h after the peak of the endogenous LH surge) produced large quantities of progesterone (P) (8.7 pg/cell/first 2 days) in a monolayer culture. Although basal production of estradiol (E2) in these granulosa-luteal cells was restricted to small quantities, a marked increase in E2 production occurred in the presence of testosterone (T) (10(-6) M) as aromatizable substrate. The non-aromatizable androgen, 5 alpha-dihydrotestosterone (DHT) (10(-7) M, 10(-6) M) slightly enhanced E2 production in these granulosa-luteal cells and it did not inhibit T aromatization. In contrast, DHT did not increase E2 production in cultured granulosa cells obtained from follicles 2 approximately 5 days before the LH surge. Thus, the response of granulosa cells to exogenous androgen seems to vary at different stages of cell differentiation. Neither T nor DHT elicited significant changes in the production of P by granulosa-luteal cells obtained from preovulatory follicles during the LH surge. However, T (10(-6) M) suppressed hCG-stimulated P production in these cells. The results suggest that androgen enhances the estrogen biosynthesis of granulosa-luteal cells obtained from preovulatory follicles during the LH surge not only by acting as substrate for aromatization but also by participating in some process of estrogen synthesis. Further, it is suggested that androgen has a negative effect on P biosynthesis in these cells.

Steroid profiles of follicular fluids from a patient with the empty follicle syndrome.

Steroid profiles of follicular fluid obtained from a patient during a cycle in which no oocytes were recovered in 12 follicular aspirates were compared with those of fluid obtained both from cycles of the same patient and from cycles of other patients when oocytes were recovered. Follicles aspirated in the cycle when no oocytes were recovered were shown to be neither atretic follicles, follicular cysts, nor prematurely luteinized follicles. The steroid profile of follicular fluid from the index cycle was characterized by a markedly increased estradiol-to-progesterone ratio and an increased androstenedione level. This, together with a comparison to the steroid profiles of fluid from follicles containing either fertilizable or nonfertilizable oocytes, suggests that the empty follicle syndrome may reflect a dysfunctional ovulation induction.

[Ultrastructural study of the intercellular junction and lanthanum penetration in the mouse blastocyst].

Cell junctions in mouse blastocyst were ultrastructurally investigated with or without lanthanum tracer. Tight junctions, gap junctions and desmosomes were observed in the trophectoderm. The tight junction was located near the zona pellucida in all trophoblast interspaces, whereas the gap junction and the desmosome, which were infrequently observed, were localized far from the zona pellucida. However, the desmosomes in the trophectoderm of the expanded blastocyst after culture increased in number and came to be located near the zona pellucida. The trophoblast layer excluded lanthanum whose invasion was interrupted by the tight junction. There were a few intermediate junctions in the interspace between the trophoblast and the inner cell mass cell as well as between the inner cell mass cells. These findings indicate that a substances whose molecular weight exceeds that of lanthanum (138.9) may not flow into the blastocyst through the intercellular space. Moreover, it is speculated that the intercellular connection is strengthened during the expansion of the blastocyst.

Sperm-egg interactions observed by scanning electron microscopy.

Successful in vitro fertilization requires mature oocytes in which the first polar body has been extruded and capacitated sperm capable of penetrating the zona pellucida. In this study we made a time sequential observations on human sperm-egg interactions by SEM in two experimental systems. Human sperm-human zona pellucida interaction: Cytoplasmic processes of corona cell extend around sperm head. Spermatozoa took different angles in attaching or penetrating to the zona pellucida. The head of some spermatozoa bound to the zona were vesiculated, suggesting the progression of the acrosomal reaction. Initially, the anterior part of the sperm head penetrates from the pore of the zona pellucida. Human sperm-zona-free hamster egg interaction: Most spermatozoa lie flat on the vitellus surface covered with numerous microvilli, but a few are oriented perpendicular to the vitellus surface. Most bound sperm had lost their acrosomal caps, because a ridge exists at the leading edge of the equatorial segment. Initially most microvilli appeared to grasp and immobilize the anterior tip of the sperm head. But as gamete interaction proceeded, microvilli were overlying the postacrosomal region and were observed adjacent to the plasma membrane of the postacrosomal region. The postacrosomal region is first incorporated into the ooplasma, the anterior tip of sperm head being the last portion to be incorporated. The microvilli of the oolemmal surface where sperm penetrated did not show major changes in size or in appearance, and the so-called incorporation cone was not observed.

[The evaluation of serum estradiol, progesterone, testosterone concentrations and urinary estrogen excretion level for the monitoring indices of follicular maturation].

We have evaluated serum estradiol, progesterone, testosterone, and urinary estrogen excretion in 24 hour urine samples to monitor indices of follicular maturation. The serum steroid levels were determined with the direct radioimmunoassay kit. The urinary estrogen level was measured with the estrogen micrometering kit using hemagglutination inhibition reaction. Moreover, relationships between these steroid levels and the follicular size measured with ultrasound were analyzed. The serum estradiol concentration and the estrogen excretion in 24 hour urine samples mostly showed a continuous increase during the late follicular phase, and had a positive correlation to the maximum follicular diameter of the leading follicle (MxFD) and to the total of the maximum diameter of the follicles (TFD). The serum progesterone concentration showed a remarkable increase especially on the day of the LH surge onset in many cases, and had a significant (p less than 0.01) correlation to MxFD but not to TFD. The serum testosterone concentration, however, showed neither a specific tendency on its daily change nor a correlation with the follicular size. These results indicated that the serum estradiol and progesterone, and the urinary estrogen excretion can be utilized as indices of follicular maturation.

Relationship between follicular fluid steroid concentrations and in vitro fertilization.

Steroid concentrations were measured in follicular fluid obtained from patients fertilized in vitro. Progesterone and estradiol-17 beta concentrations showed positive correlations with the growth of follicles, whereas the testosterone concentration had negative correlations not only with follicle growth but also with progesterone and estradiol-17 beta concentrations. The testosterone concentration was significantly lower in follicles with cleaved oocytes than in those with uncleaved oocytes or those with cleaved but degenerated oocytes. After the luteinizing hormone (LH) surge, the fluid contained significantly higher progesterone and lower testosterone concentrations than after human chorionic gonadotropin (hCG) injection. These results indicate that steroid concentrations vary with the growth of follicles, and that the oocyte that can cleave well tends to be associated with a low testosterone concentration. Furthermore, the LH surge seems to provide a better hormonal condition for cleavage than does hCG injection.

[The influence of various molecules on the occurrence of the human sperm acrosome reaction].

The acrosome reaction is essential for fertilization, but the mechanism of the acrosome reaction of human spermatozoa is not clear at the present time. We studied the mechanism to analyze the cause of unexplained infertility, the appropriate timing of insemination, and the environment of spermatozoa prior to fertilization. For this study, we examined the effects of Ca++, Mg++, Kallikrein, Phospholipase A2, p-bromophenacyl bromide (Phospholipase A2 specific inhibitor), Lysophosphatidyl choline, Arachidonic acid, and Glyceryl monooleate using in vitro penetration assay employing zona- free hamster eggs. Results obtained were as follows. When human spermatozoa were incubated in mBWW with Ca++ or (and) Mg++ free medium, the acrosome reaction was inhibited. When human spermatozoa were incubated in mBWW with Kallikrein (1.0-4.0 KU ml), the acrosome reaction was promoted. When Phospholipase A2 was used at concentrations of 0.2 and 2.0 unit/ml, penetration rates showed the same tendency as in the control. But when p-bromophenacyl bromide was tested at concentrations of 1 X 10(-5) - 1 X 10(-3)M, penetration rates were inhibited when compared with the control. When human spermatozoa were incubated in medium containing Lysophosphatidyl choline (50 micrograms/ml), Arachidonic acid (5-50 micrograms/ml), and Glyceryl monoleate (300-400 micrograms/ml), the acrosome reaction was accelerated.

[Effects of the addition of serum albumin on penetration of human spermatozoa into zona-free hamster eggs].

Since Yanagimachi et al. (1976) suggested that human spermatozoa were capable of penetrating into zona-free hamster eggs, this in vitro assay has been used to analyse the fertilizing ability of human spermatozoa. Serum albumin is an important constituent of the medium used for the assay. However, a great variation in the rate of sperm penetration was observed in the use of different albumin preparations at different concentrations. Therefore we examined the effects of three different kinds of albumin preparations on the rate of human sperm penetration into zona-free hamster eggs. The results obtained were as follows. The percentages of eggs being penetrated by spermatozoa from three fertile donors A,B, and C, were assessed. When Fraction V, Globulin Free and Fatty Acid Free albumin preparations were tested at a concentration of 3.5% (W/V) by the assay using sperm from donor A, penetration rates were 13.3%, 97.4%, and 8.7% respectively. Dilution of the albumin concentration to 0.3% considerably changed the penetration rates to 64.4%, 78.8% and 12.1% in that order. In cases B and C, penetration rates showed the same tendency as in case A. alpha 1 and alpha 2 globulin fractions contaminated in the Fraction V preparation possibly inhibit the human sperm penetration into zona-free hamster eggs. An appropriate quantity of fatty acid is necessary for human spermatozoa to penetrate into zona-free hamster eggs, because penetration rates were low in the percentage of Fatty Acid Free albumin regardless of the concentration added. It is concluded that use of the same preparation of good quality is mandatory for human sperm penetration tests using zona-free hamster eggs to evaluate the results with reproducibility and accuracy.

Effects of phospholipase A2, lysophosphatidyl choline, and fatty acid on the acrosome reaction of human spermatozoa.

An in vitro penetration assay employing zona-free hamster eggs was used to study the effects of phospholipase A2, lysophosphatidyl choline, and fatty acid on the acrosome reaction of human spermatozoa. Human spermatozoa were preincubated for 4 hr in modified Biggers, Whitten, and Whittingham's medium (mBWW) containing a specific phospholipase A2 inhibitor, p-bromophenacyl bromide (p-BPB: 1 X 10(-5) - 1 X 10(-3) M), lysophosphatidyl choline (LC: 5-500 micrograms/ml), and arachidonic acid (AA: 5-500 micrograms/ml), prior to the addition of zona-free superovulated hamster eggs. Eggs were examined microscopically 2 or 4 hr later for evidence of swelling or decondensing sperm heads in the cytoplasm. Lysophosphatidyl choline increased penetration rates of spermatozoa from 43.2% (control) to 91.4% (LC: 50 micrograms/ml). Arachidonic acid also increased penetration rates from 51.6% (control) to 87.0% (AA: 5 micrograms/ml) and 80.5% (AA: 50 micrograms/ml). p-BPB decreased penetration rates from 90.6% (1% dimethyl sulfoxide) to 16.0% (1% dimethyl sulfoxide+p-BPB 1 X 10(-4) M). These results suggest that endogenous phospholipase A2 may break membrane phosphatidyl choline into lysophosphatidyl choline and fatty acid, when the acrosome reaction occurs.