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The functions of organs decrease as the age increases. The fecundity of women also decreases due to mainly the decreasing quality of oocytes. The recent change of life style makes the age of infertility patients elder and infertility treatments more difficult. The therapeutic strategy for the elder infertility patients is still chaotic. The precise evaluation for the ageing in the female genital organs should be developed and the treatment for the failure of fecundity due to aging must be overcome.

This review article summarizes the current knowledge on the development of gonadal dysgenesis in Turner syndrome. The degree of gonadal dysfunction is well correlated with the size of unpaired region of the sex chromosomes and is independent of the dosage of gene(s) on the sex chromosomes. Thus, it is deduced that sex chromosome aberrations result in meiotic pairing failure of homologous chromosomes, leading to accelerated oocyte loss and resultant gonadal dysgenesis. Although the underlying factor responsible for the oocyte loss remains to be determined, it is likely that activation of apoptotic mechanism to prevent the generation of abnormal gametes plays an essential role in the rapid oocyte degeneration.

In this study I attempted to elucidate the depressant effects of volatile anesthetics on inositol trisphosphate (IP3)-mediated signal transduction pathway and to identify the site of action. For this purpose, we used Xenopus laevis oocytes which translated and expressed 5-HT receptors after injection of mRNA isolated from the rat brain. In this system, binding of the agonist to G-protein coupled receptors activates phospholipase C that produces IP3. Mobilization of Ca2+ by IP3 from the storage finally opens Ca2+ dependent Cl- channels. Halothane, isoflurane and methoxyflurane depressed Cl- current elicited by 5-HT. For the further quantitative study, methoxyflurane was used because of its better solubility and less vapor pressure that avoided evaporation of the agent. The 5-HT elicited Cl- current was depressed in a non-competitive fashion. Response were 75, 60, 20% of control in the presence of 0.5, 1 and 3 mM methoxyflurane, respectively. Responses elicited by a pressure-injection of Ca2+ or IP3 remained unchanged in the presence of high concentrations of either halothane, isoflurane or methoxyflurane. These results suggest that the depressant mechanism by volatile anesthetics on the signal transduction pathway involves neither Ca2+ dependent Cl- channel dynamics nor intracellular Ca2+ mobilization by IP3. Changes of microdomain characteristics of the membrane in the presence of anesthetic molecules including membrane-bound proteins and enzyme system may be a main mechanism of action of volatile anesthetics.

Steroid hormones in preovulatory follicular fluid, maturity of the oocyte-corona-cumulus complex (OCCC), and oocyte fertilizability were studied in 55 hyperstimulated cycles in 40 patients who were indicated for in vitro fertilization and embryo transfer due to tubal infertility. Aspirated follicles were categorized into four groups according to the degree of oocyte maturation and fertilizability, i.e. follicle (Fol)-1 (non-fertilized, intermediate maturation), Fol-2 (fertilized, intermediate maturation), Fol-3 (fertilized, full maturation), and Fol-4 (non-fertilized, full maturation). Estradiol (E2), progesterone (P), and delta 4- androstenedione (delta 4A) concentrations were highest in Fol-3, being lower in Fol-2 and Fol-1 sequentially. Fol-3 contained significantly more E2 and P than did Fol-1, showing that the degree of oocyte maturation and fertilizability correlate with concentrations of E2 and P in the follicles. A comparison of Fol-3 and Fol-4 revealed significantly higher concentrations of delta 4A in Fol-4, whereas differences in E2 and P concentrations were not significant although Fol-4 values were lower. This comparison indicated that Fol-4 were already undergoing atresia at the time of oocyte retrieval, containing morphologically mature OCCC which had lost fertilizability. This study shows that steroid hormones in the follicular fluid reflect oocyte viability and fertilizability, and suggests that preovulatory follicles with mature oocytes can also undergo atresia.

In strains LT/Sv and LTXBJ mice, susceptible to spontaneous ovarian teratocarcinogenesis, ovarian teratomas are derived from parthenogenetically activated ovarian eggs. The follicles containing the parthenotes are the abnormal ones in which granulosa cells show histochemically high activity of beta-glucuronidase (beta-G). To investigate whether ovarian teratocarcinogenesis depends on the genetic nature of eggs themselves or the microenvironment in the abnormal follicles, we used chimeras derived from aggregates of eight-cell embryos of strains LTXBJ or (LTxLTXBJ) F1 and of normal nonsusceptible strain C3H/HeJ which shows low activity of beta-G genetically. The original strain of teratomas and follicles in chimeras were histochemically determined by activity of beta-G as a marker. The results showed that both teratomas and the follicles containing parthenotes originate from the susceptible strains. Then, we examined for presence of activated oocytes, teratomas and corpora lutea in LT ovaries untreated or treated with PMSG-HCG implantation. These results showed that parthenogenetic blastocysts closely adhere to the follicle tissue as if a kind of and develop to post implantation stages in the ovaries in which corpora lutea were formed near the parthenotes. In conclusion, it was suggested that ovarian parthenogenesis in mice occurs most frequently when eggs with genetically susceptible nature, were exposed to the microenvironment produced by the abnormal follicles, and these parthenotes adhere to the ovarian tissues and differentiate three germ layers by some roles of corpora lutea and undergo teratocarcinogenesis.

The possible existence of granulosa-cell growth factor (GGF) in the oocyte has been investigated using the labelling index (LI) with 3H-Thymidine and the mitotic index (MI) of granulosa cells (G-cells) in mice. The following results were obtained. In preantral follicles the LI increased with the development of the oocyte until its diameter reached the maximum (approximately 90 mu). In preantral follicles with the oocyte diameter below 69 mu, the LI was low and the follicular diameter was small. As the oocyte developed, the LI increased and reached the maximum value when the follicular diameter was approximately 200 mu. Then it decreased a little but maintained a high value. The antrum formation was seen when the follicular diameter was about 300 mu showing no change in LI. In preantral follicles with the oocyte diameter over 80 mu the G-cell near the oocyte had a much higher LI than the distant one. After the antrum formation, the LI and MI of cumulus layers adjacent to the oocyte were three times as great as those of the mural layers. Among the mural cell layers in the antral follicle the layer adjacent to the antrum showed remarkably high LI in comparison with the distant layers. These results suggest that GGF which seems to play a role in the formation of cumulus is secreted with the development of the oocyte. The GGF is transported through two systems the preantral follicle and cumulus layer through the G-cell gap junction and mural layers through the follicular fluid.

Granulosa cells (G-C) collected from mature follicles of human and bovine ovaries were cultured with an oocyte from mature follicles of human, bovine and porcine ovaries to investigate the influence of the oocyte upon the proliferation of G-C as well as the differentiation of G-C. For this purpose, G-C were compared in mitotic index (MI) between those cultured with oocyte and without oocyte control at the fourth day of culture. 1) G-C cultured with an autogenous oocyte--A bovine oocyte cultured with bovine G-C increased in MI 2.8 times compared with the control. Human G-C cultured with a human oocyte also appeared to raise the MI 2.7 times compared with the control. 2) G-C culture with heterogeneous oocyte--Moreover, a porcine oocyte cultured with bovine G-C acted to increase the MI to 2.7 times that of the control. An addition of a porcine oocyte to human cultured G-C also almost doubled the MI as compared with that of the control. 3) Within 2 days after culture of G-C with an auto- or hetero-genetic oocyte from which the cumulus oophorous had been removed, the oocyte was surrounded by a dense mass of small type of G-C, which resembled follicular G-C morphologically and indicated a high value of MI. The cells on the periphery from the oocyte, however, were clearly enlarged differing from those close to the oocyte and began to resemble lutein cells as the culture days went on. 4) Instead of the oocyte, sterilized muscle, cartilage and bone tissues were cultured with the G-C as controls. However, no binding of G-C with these tissues were found as was verified between G-C and the oocyte. These results suggest that the oocyte may characteristically bind with G-C to accelerate the multiplication and inhibit the luteinization of G-C.

The mechanism of cumulus oophorus formation with the follicular growth has been little observed. In order to make clear about it, the possible existence of a granulosa cell growth factor (GGF) in the oocyte was investigated by using the Mitotic Index (MI) (%0) of granulosa cells (GC) in human antral follicles. In each follicle, GC were divided into two groups, cumulus and mural GC, on the basis of the distance from the oocyte. The MI of cumulus GC was 8.2 +/- 0.5 (M +/- SE), constantly high in value with no relation to the follicular diameter, and two fold higher than that of mural GC, 4.7 +/- 0.4 (p less than 0.001). In general, Estradiol (E2) is well known as a granulosa cell growth factor. Hence, we investigated whether oocyte had E2 to know how E2 participates in the cumulus oophorus formation, by using the enzyme histochemical study. E2 content of the oocytes measured by Multi-Micro-Spectrophotometer in preantral and small antral follicles in which GC actively proliferate were 13.6 +/- 0.8% and 10.2 +/- 0.7% respectively, and higher than that of in large antral follicle, 7.6 +/- 1.0%. To examine the origin of E2 in oocytes, oocytes, GC and follicular fluid were measured using porcine ovaries. E2/protein of oocytes was 34.1 +/- 6.4 (pg/mg. prot) and that of GC was 10.2 +/- 2.8. It means that the origin of E2 may be from the oocyte itself. These results suggest, E2 in oocytes is estimated to be one of the oocyte factor to stimulate the granulosa cell proliferation and E2 plays a crucial role in the cumulus oophorus formation.

The correlations with maturity of gametes and fertilizability was studied. Specimens of semen were treated by several methods for induction of capacitation and acrosome reaction (AR). Follicular oocytes were recovered with endoscopic aspiration procedures, and divided to two groups: 1, maturing in vivo (preovulatory) and 2, immature. Immature oocytes were divided further into two subgroups: 1, germinal vesicle breakdown (GVBD) and 2, first polar body formation (FPB), after maturation culture. The sperms treated with the centrifuge-trypsinization-layering method showed a higher incidence of in-vitro fertilization (69.2%) and cleavage (51.3%) of human preovulatory eggs compared with other capacitation inducing methods. It was found that human spermatozoa that have undergone the acrosome reaction prior to zona attachment are capable of fertilizing eggs under the in vitro conditions. On the estimation of fertilizability with reference to egg maturity, 52.5% (21/40) of IMIV-GVBD and 64.7% (22/34) of IMIV-FPB eggs were fertilized. However, only 12.5% (5/40) of IMIV-GVBD and 11.8% (4/34) of IMIV-FPB eggs cleaved. These results showed that IMIV eggs had fair fertilizability, but no developmental capacity after the first cleavage. This may due to prematurity of the cytoplasm in IMIV eggs.

The possible existence of a granulosa cell growth factor (GGF) in the oocyte was investigated by using the mitotic index (%) of granulosa cells (GC) in human antral follicles. 50 follicles (0.4-13 mm in diameter) in 35 ovaries obtained from 35 women were used for the experiments. In each follicle, GC were divided into two groups, cumulus and mural GC, according to the distance from the oocyte. The following results were obtained: The Mitotic Index (MI) of cumulus GC was 8.2 +/- 0.5 (M +/- SE) and was two fold higher than that of mural GC, 4.7 +/- 0.4 (p less than 0.001). As follicles developed, MI of mural GC increased until their diameter reached 3mm but beyond 3mm, MI decreased. On the other hand, there was little relationship between MI of cumulus GC and the follicular size, and MI was constantly high in value. MI of both cumulus and mural GC was higher in the late follicular phase than in other phases of the cycle. These results suggest that the proliferation of GC in growing follicle might be regulated by some unknown substance (GGF) secreted from the oocyte as well as the endocrinological environment. It is also suggested that the presence of the oocyte plays a crucial role in the cumulus oophorus formation in the antral follicles.