Effect of Prolactin on Gonadotropin Regulation of Mouse Ovarian Function

We have demonstrated that prolactin inhibits gonadotropin-induced ovulation in PMSG-primed mice.The number of ova in oviducts considerably decreased in the group of hCG plus prolactin (PRL)(19.7 + 4.9) as compared with that of hCG alone (31.7 + 6.7). PRL inhibition of hCG-inducedovulation in mice may be through decreasing the ovarian plasminogen activator (PA) activity on onehand, and inhibiting the preovulatory increase in estrogen (E) secretion on the other.

Two Different Forms of Gonadotropin-releasing Hormone in Amphioxus

Since a GnRH was isolated from mammalian hypothalamus and purified in 1971 and 1972, severalvariants have been identified in various forms of lower vertebrates. However, the presence of GnRHin amphioxus is still an open question. Chang et al. (1984) observed the presence of immunopositivegranules for GnRH in Hatschek's pit of amphioxus. In this paper, HPLC was used for the isolationand purification of GnRH-like peptide from amphioxus tissues, while radioimmunoassay was appliedto determine the immunoreactivity of the peptide. Based on the immunological and chromatographiccharacteristics, two kinds of GnRH (mGnRH and sGchH) were identified in amphioxus and theseGnRH-like peptide were found to be present in the 'head', 'middle' and 'tail' regions ofamphioxus.

Effect of Gossypol on Bovine Blastocyst Attachment and Trophoblastic outgrowth in Vitro

The presont study was carried out to investigate the effect of different amounts of gossypol on bovineblastocyst attachment and trophoblastic outgrowth in vitro. Bovine oocyte were collected from theovaries of slaughtered cows and were matured and fertilized in vitro. Cleaved oocyte were culturedin CRlaa + BOEC and TC-199 + 10% FCS combined in an 1:1 ratio. After 8 days of co-culture,the hatched blastocysts were randomly allotted to different treatment groups. All were cultured ona fetal fibroblast monolaycr (prepared from bovine fetuses) in TC-199 culture medium supplementedwith 10% fetal calf serum (TCFCS). But the groups differed from one another in the dose ofgossypol given: 0.01 μg, 0.1 μg, 1 μg, 10 μs/ml, and no gossypol as control. All cultures wereperformed in 24-well culture plates at 39℃ with 5% CO_2 in air. The results indicate that the ratesof attached and outgrowing blastocysts in the medium containing 1 μg/ml gossypol were significantlylowcr than the control group (p<0.01) and outgrowth were inhibited by gossypol in a dose-dependent manner.

Structural Analysis of Chloroplast rbcL Gene from Grape (Vitis vinifera L.)

The 3.1 kb BamHI fragment containing the chloroplast rbcL gene from grape (Vitis vinifera L) hasbeen cloned and its restriction map and nucleotide sequence determined. The complete nucleotidesequence is 20O4 bp long with a coding sequence of 1428 bp, which cncode a polypeptide of 475amino acids with a predicted molecular weight of 53 kd. The 5' upstream sequence including theputative promoter is 358 bp, with a -10 sequence (TAAAAT), a -35 sequence (TTGCGC) and theSD sequence (GGAGG). The 218 bp long 3' downstream sequence contains three transcription stem-loop tendnation structures. The homologies of this gene with those of tobacco, petunia, spinach,alfalfa, rice and maise are 91.5%, 91.4% 90.2%, 89.8%, 86.3% and 84.5% respectively while thehomologies between their putative polypeptide sequences are 92.2% 91.6%, 92.2%, 93.7%, 93.5%and 90.1% respectively.

Plasminogen Activator and Plasminogen Activator Inhibitor in Mouse Ovaries during Periovulatory Periods

Changes of ovarian tPA,uPA and PA inhibitor activities were examined in PMSG-and hCG-treatedimmature mice during periovulatory periods.The results show that 15% of the gonadotropin-treatedanimals ovulated 8 hrs after hCG administration,about 6-8 hrs earlier than in rat.It is also shownthat not only tPA activity,but also uPA activity,was regulated by gonadotropins in ovarianhomogenates and granulosa cells,and they reached maximum prior to ovulation.No measurableamount of PAI-1 activity could be detected in mouse granulosa cell conditioned medium andfollicular fluid,but considerable amount of α_2-antiplasmin,a specific inhibitor for plasmin,wasfound in follicular fluid.Cumulus-oocyte complexes contain mainly tPA.Since the ovulated eggsstill have high tPA activity,it is thought that the enzyme in the oocyte may play an important rolein implantation.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF LUTEINIZING HORMONE (LH) IN HATSCHEK'S PIT OF AMPHIOXUS (BRANCHIOSTOMA BELCHERI GRAY)

Hatschek’s pit is a groove-like structure located in the frontal area of the wheel organ of Amphioxus. It has long been postulated, with no convincing physiological evidence, to be homologous with the anterior pituitary gland of vertebrates. Recently, we have demonstrated, by means of an immunocytochemical technique, that the

Secretion of Plasminogen Activator Inhibitor Type 1 by Cultured Ovarian Cells Obtained From Gonadotropin-treated Immature Rats

It is demonstrated that i) theca-interstitial compartment synthesizes the majority ofplasminogen activator inhibitor type 1 (PAI-1) in the ovary before ovulation,and the follicular wall maytherefore serve as a specific barrier with the presence of PAI-1 activity to prevent the secretion of tPA intothe extrafollicular compartments;ii) granulosa cells secrete only a small amount of ovarian PAI-1,butsynthesize the most of tissue-type plasminogen activator tPA involved in the processes leading to ovula-tion;iii) since only matured cumulus-oocyte complexes secrete a large amount of tPA and PAI-1,bothtPA and PAI-1 activity in the conditioned medium may be used as reliable markers for evaluating oocytequality for in vitro fertilization.

A COMPARATIVE STUDY ON INVOLVEMENT OF tPA ACTIVITY IN OVULATION INDUCED BY hCG AND GnRH AGONIST IN HYPOPHYSECTOMIZED RATS

A GnRH agonist (5-50 μg) is capable of inducing ovulation in PMSG-primed hypophy-sectomized immature rats, as is the case in hCG-induced ovulation, but 2-4 h earlier than hCG. GnRH-induced ovulation is effectively blocked by the concomitant administration of the GnRH-antagonist which failed to interfere with hCG-induced ovulation, indicating that GnRH and its agonists do not share a receptor with LH/hCG. Like hCG, GnRH is also capable of inducing tissue type (tPA), but not urokinase type (uPA) PA. The plasminogen activator activity in ovarian homogenates and the granulosa and theca-interstitial cells increase in a time-dependent manner, reaching maximum levels just prior to ovulation. Similar to hCG, GnRH also increases tPA activity in cumulus-oocyte complexes in a time-dependent fashion.

REGULATION OF STEROIDOGENESIS OF INFANT AND ADULT RHESUS MONKEY GRANULOSA CELLS in vitro

The present studies have demonstrated that infant monkey granulosa cells, like the adult ones, have the potential of responding markedly in vitro to human FSH, cyclic-AMP and forskolin, resulting in the increase of progesterone and estrogen production. Exogenous hCG was also capable of increasing FSH-stimulated progesterone biosynthesis in both infant and adult granulosa cells, but did not stimultate the infant granulosa cells to secrete estrogen. Addition of a synthetic estrogen, diethylstilestrol, to the culture of monkey granulosa cells enhanced the FSH-stimulated progesterone and estrogen production. The esteroidogenesis of monkey granulosa cells was also dramatically stimulated by a gonadotropin-releasing hormone agonist. Monkey granulosa cells, unlike the other animal cells, secrete measurable amount of estrogen in the absence of androgen substrate. The findings reported here are significant in regard to understanding of the mechanism of hormonal regulation of primate ovarian function.

Coordinated Regulation of Tissue Type Plasminogen Activator and Plasminogen Activator Inhibitor Type-1 Gene Expression in Hypophysectomized Rat Ovaries During GnRHa-Induced Ovulation

In this study we have demonstrated that both granulosa and theca-interstitial cells of hy-pophysectomized rat ovaries are capable of synthesizing tPA and PAI-1.Injection of a GnRH agonist canmarkedly induce these gene expressions in the ovary in a cell-specific and time-coordinated manner,so that asurge of tPA mRNA and its activity in both granulosa and theca-interstitial cells was obtained just prior toovulation.Theca-interstitial cells make PAI-1 become the most active in the ovary.Both the amount PAI-1mRNA and its activity in the cells reach the maximum level 6 h before the tPA peak.By contrast,granulosacells produce only a little amount of PAI-1 (most increase tPA activity),and both PAI-1 mRNA and activityin the cells reach the maximum after ovulation.The coordinated regulation of tPA and PAI-1 in the ovarymay fine-tune the peak of tPA activity which may be important for the regulation of the ovulatory process.The changes of tPA and PAI-1 in the ovarian cells of hypophysectomized rats during GnRHa-induced ovula-tion are similar to that in intact rats during hCG-induced ovulation,suggesting that the ovulatory process canbe modulated by different regulatory signals mediated by influencing the coordinated expression of both tPAand PAI-1.

Hormonal Regulation of Expression of Tissue Type Plasminogen Activator and Plasminogen Activator Inhibitor Type 1 in Cultured Rat Granulosa Cells

In the present study, gonadotropin and gonadotropin-releasing hormone (GnRH) regulation of tPA and PAI-1 expression in PMSG-primed granulosa cells has been investigated, (i) Addition of go-nadotropins (FSH and LH) and GnRH agonist (GnRHa) or PMA to the culture increases tPA activity) FSH (or LH) plus GnRHa (or PMA) in the culture further enhances the enzyme production to such an extent that a more obvious effect than the additive effect caused by these hormones used alone has been observed; (ii) in contrast, FSH and LH decrease PAI-1 activity, whereas GnRHa and PMA alone markedly increase PAI-1 mRNA level and PAI-1 activity. Because FSH and LH stimulate tPA production and have no significant effect on PAI-1 mRNA induction, the observed inhibition of PAI-1 activity by gonadotropins may be due to the occurrence of neutralization of PA and PAI-1 proteins in the conditioned media by the formation of complexes between PA and PAI-1 ; (iii) increases in PAI-1 mRNA level and activity by GnRH and PMA are completely inhibited by the co-addition of FSH or LH to the culture. It is, therefore, suggested that the mechanism of gonadotropin and GnRHa regulation of tPA and PAI-1 expression in granulosa cells is different.

PARADOXICAL EFFECT OF A GnRH AGONIST ON STEROIDOGENESIS IN CULTURED MONKEY GRANULOSA CELLS

In this study we demonstrate: (i) The GnRH agonist exerts a direct dose-dependet stimulative effect on the aromatase activity and progesterone production in cultured monkey granulosa cells; (ii)the stimulative effect on steroidogenesis can be completely blocked by concomitant treatment with a GnRH antagonist, suggesting that the actions of GnRH are mediated through stringent stereospecific recongnition sites; (iii) in addition to the stimulative effect, the GnRH agonist in the presence of gonadotropins also exerts an inhibitory effect, even though the peptide by itself is more effective in the stimulation of steroidogenesis, and the stimulation of gonadotropin on steroidogenesis could be gradually restored by decreasing the concentration of the GnRH agonist in the culture; and (iv) paradoxical effect can also be observed in the presence of cAMP-inducing agents, suggesting that the inhibitory action of the peptide on gonadotropin-induced steroidogenesis is localized at a step distal to the stringent recognition sites.

VARIATIONS OF LECTIN-BINDING GLYCOPROTEINS IN EARLY PREGNANT RABBIT EMBRYO

In the present study, the quantitative and qualitative changes of three kinds of lectinbinding glycoproteins of early pregnant rabbit embryos (D4—D12) were analyzed. The technique of Western blot, as well as video densitometer scanning and its analytic software was used for analysis. Results found that there were five specific lectin-binding glycoproteins in Day 4 to Day 6 blastocyst fluids: one ConA-binding glycoprotein about 70 kD, two WGA-binding glycoproteins respectively about 42 kD and 25kD and two PNA-binding glycoprotein respectively about 180kD and 75kD. They disappeared immediately after implan tation, It is demonstrated that there are stage-specific glycoproteins in rabbit blastocyst fluid which might be relevant to the recognition of pregnancy and implantation.

Hormonal Regulation of Tissue-type Plasminogen Activator and Plasminogen Activator Inhibitor Type-1 Gene Expression in Cultured Mouse Sertoli Cells

We have demonstrated for the first time that (ⅰ) mouse Sertoli cells predominantly secrete tPA under the action of FSH and cAMP-generating agents, whereas Leydig cells mainly produce uPA; (ⅱ) Sertoli cells are also capable of secreting PAI-1, as well as FSH, growth factors and GnRH increase PAI-1 gene expression; (ⅲ) the increases in tPA and PAI-1 activities by different hormones in the conditioned media of Sertoli cells correspond to the increases in the levels of tPA and PAI-1 mRNA in the cultured cells, suggesting that the synthesis of the activator-inhibitor in mouse Sertoli cells is regulated at a transcriptional level and the tPA secreted by Sertoli cells may be involved in the spermatogenesis.

VISUALIZATION OF THE CHROMOSOME SCAFFOLD IN A PRIMITIVE EUKARYOTE DINOFLAGELLATE Crypthecoainium cohnii

The chromosome scaffolds in higher eukaryotic nuclei have been described elsewhere. Butit is unknown when they evolved. The dinoflagellates are the primitive organisms that maybe the intermediate between prokaryotes and eukaryotes. Combining chromosome scaffold prep-aration methods with embedment-free section microscopy,we demonstrate that the dino-flagellate Crypthecodinium cohnii chromosome retains a protein scaffold after the depletionof DNA and soluble proteins. This scaffold preserves the morphology characteristic of thechromosome. Two-dimensional electrophoreses show that the chromosome scaffolds are mainlycomposed of acidic proteins. Our results suggest that a framework similar to the chromosomescaffold in the mammalian cell appeared in the primitive eukaryote. We propose that thechromosome scaffold possibly originated from the early stages of eukaryote evolution.

DETECTION OF HUMAN TROPHOBLAST INTERFERON WITH ENZYME-LINKED IMMUNOSORBENT ASSAY

Although it is well known that luteal lifespan in early pregnancy is prolonged by human chorionic gonadotropin (hCG), recent evidence has suggested that proteins other than hCG, especially interferon-like substances resembling trophoblast protein-1 of domestic ruminants, may also be involved in the maintenance of corpus luteum.