Expression of regulator of G-protein signalling protein 2 (RGS2) in the mouse uterus at implantation sites.

Establishment of an active dialogue between the maternal endometrium and the implanting blastocyst is essential for successful implantation. The aim of this study was to identify genes that are explicitly expressed at implantation sites of the mouse uterus by subtractive hybridization. One expressed sequence tag of the genes identified showed 92% identity to the regulator of G-protein signalling protein 2 (RGS2). The full cDNA sequence of this gene was amplified by PCR and subsequently registered in GenBank. The sequence of its open reading frame encoding 211 amino acids was the same as that of mouse RGS2, with the exception of four amino acids. Northern blot analysis showed that the expression of this gene was much higher at implantation sites than at inter-implantation sites on days 5-8 of pregnancy. In situ hybridization localized this mRNA predominantly to the stromal cells at the implantation sites. These results indicate that RGS2 has a role during implantation, possibly by regulating the intracellular Ca(2+) mobilization and T-cell proliferation at the maternal-fetal interface.

[Immunocytochemical localization of fertilin beta on human sperm].

Fertilin is a kind of sperm plasma membrane protein that mimics snake venom protein. It belongs to the ADAMs family of surface proteins that contain a disintegrin and a metalloprotease domain. Fertilin functions in the sperm-egg binding process by connecting the sperm to the egg plasma membrane via a binding site in the disintegrin domain of fertilin beta (HF93). Its localization on the sperm is in the change. In this study, the monoclonal antibody against human fertilin beta was prepared and used to analyze the localization of fertilin beta on capacitated and acrosome-reacted sperm by immunofluorescence and immunoelectron microscopy techniques. The results were as follows: (1) fertilin beta became restricted to the anterior head during the course of capacitation. (2) During the course of acrosome reaction, the expression and localization of fertilin beta changed immensely on the anterior head and restricted to the lateral of posterior head at last. The restrictions of fertilin beta to the anterior head of capacitated sperm of human beings indicated that fertilin beta may be involved in the binding the sperm to the epithelial cells of the oviduct; the restrictions of fertilin beta to the posterior head domain of acrosome-reacted sperm implied its function in sperm-egg binding and fusion.

Temperature dependent expression of cdc2 and cyclin B1 in spermatogenic cells during spermatogenesis.

p34cdc2 and Cyclin B1 are key components of cell cycle controlling machine and are believed to play a fundamental role in gametogenesis. It is also well known that, in scrotal mammals, spermatogenesis depends greatly on the maintenance of comparatively low temperature in the scrotum. To investigate whether the expression of cdc2 and cyclin B1 in spermatogenic cells during spermatogenesis is actually a temperature dependent event, in situ hybridization, Western blotting and immunohistochemistry analysis were used to study the expression of cdc2 and cyclin B1 in normal and cryptorchid testis. Results showed that the abdominal temperature had no significant influence on the transcription of cdc2 and cyclin B1 in the spermatogonia and pachytene/diplotene primary spermatocytes, but it blocked the translation of them. Due to the deficiency of p34cdc2 and Cyclin B1, the spermatogonia and pachytene/diplotene primary spermatocytes were unable to form MPF, hence, they couldn't undergo karyokinesis. The development of primary spermatocytes was arrested at the G2 to M phase transition. We also found that testosterone could regulate the Cyclin B1 expression in spermatogenic cells. Muscular injection of testosterone could recover spermatogenesis in the unilateral scrotal testis which was influenced by the contralateral cryptorchid testis, but it could not salvage the spermatogenesis block in the cryptorchid testis.

[Cloning and identification of recombinant cDNA to a rabbit oviductin "DPF-1"].

A recombinant cDNA library to polyA + RNA isolated from rabbit oviduct epithelial cells was constructed, and screened with a polyclonal antibody against DPF-1 (64 kDa). 4 immunopositive plaques (DPF-1.1, DPF-1.2, DPF-1.3 and DPF-1.4) were purified. The polyclonal antibodies were epitope-selected respectively against the fused proteins produced by these positive recombinant plaques. Identification of recombinant clones by epitope selection revealed that the epitope-selected antibodies from DPF-1.1, DPF-1.2 and DPF-1.3 could recognise not only DPF-1, but 44 kDa protein also (Fig. 2). By using EcoRI-Not1 digestion method, the insert cDNA fragment size of these three recombinants was revealed to be 0.8 kb, 1.2 kb and 1.2 kb respectively (Fig. 3). These cDNA fragments were then isolated and subcloned into pBluescriptKS, and recombinant plasmids (pDPF-1.1, pDPF-1.2 and pDPF-1.3) were constructed (Fig. 4). Dot blot hybridization with a 32p-labeled 1.2 Kb-insert of cDNA from pDPF-1.3 indicated that these recombinant plasmids could cross-hybridized (Fig. 5), further indicating that they all possessed a common nucleic acid sequence. Dot and Northern blotting analysis of total RNA prepared from eight different tissues (skeleton muscle, heart, kidney, oviduct, liver, spleen, lung and small intestine) showed that the gene encoding DPF-1 was expressed specifically in the oviduct tissue (Fig. 6, Fig. 7).

[Preliminary studies on the rabbit oviductin "DPF-1"].

Anti-rabbit 64 kDa oviductin (named Development Promoting Factor-1, DPF-1) antibody could inhibit totally the early development of mouse fertilised eggs cultured in the conditioned medium derived from the rabbit oviduct mucosa epithelial cells, revealed that DPF-1 synthesized and secreted from rabbit oviduct mucosa has a function to overcome the developmental block of early mouse embryos. It seems that DPF-1 consists of a group of polypeptide isoforms, since its isoelectric points are ranging from 7.2 to 8.1 (Fig. 3). The synthesis and secretion of DPF-1 was not dependent on either 17 beta-estradiol or progesterone (Fig. 7), it can pass through zona pellucida easily and associate tightly with the early embryonic cell membrane (Fig. 6). By using Western blotting method, we found that DPF-1 was not appeared in the tissues of liver, heart, lung, spleen, uterus, ovary, small intestine, skeleton muscle and brain, but in that of oviduct (Fig. 4): some DPF-1 homologous molecules were also revealed in the oviduct tissues of mouse and golden hamster, their apparent molecular weights were 32 kDa, 72 kDa in mouse, and 49 kDa, 68 kDa in golden hamster (Fig. 5). Results obtained from the in vivo anti-fertility experiment, namely to analyse the anti-fertility effect in adult female mice after active immunization with DPF-1, showed that the fertility decreased significantly as compared to those of controls (p < 0.01) (Table 1). DPF-1 and its in vivo "loss of function" evidence we obtained will encourage us to study the mechanism of DPF-1 in overcoming the developmental block of early embryos, and its role in transition from maternal to embryonic control of early development.

[Emancipation of the developmental block of early rat embryos by protein factors excreted from rabbit oviduct epithelial cells in culture].

Experiments were designed to evaluate the ability of rabbit oviduct epithelial cells (ROEC) or ROEC conditioned medium to promote the development of rat eggs fertilized in Vivo or in Vitro. 61.73% of the eggs fertilized in Vitro and 73.33% of the eggs fertilized in Vivo cocultured with ROEC overcame the 2-cell block (Plate I, tables 1 and 2). Similarly, 67.99% of the eggs fertilized in Vitro cultured in ROEC conditioned medium developed over the 2-cell stage, and nearly half of them developed to morula and blastocysts stage (Table 3). By using 35S-methionine incorporation and autoradiography methods, several polypeptides, with molecular weight of 135 Kd, 68 Kd, 55 Kd, 51 Kd and 44 Kd respectively (Fig. 1), were excreted from rabbit oviduct epithelial cells and found in the ROEC conditioned medium. The possibility of entrance of the ROEC proteins into the developing embryo was tested by determining whether any of the secreted proteins bound to the zona pellucida. The results of iodination by using 125I-containing acylating agent labelling method showed that the 68 Kd and 55 Kd proteins were bound onto the zona pellucida of rat eggs co-cultured with ROEC in vitro for 24 h (Fig. 2). Studies concerning the problem that whether these two secreted proteins are the key factors to promote the development of early embryos and the transition of maternal to zygotic control of embryo development are undertaking.

[Experimental analysis of mechanism concerning the environmental temperature effect on acquisition of capability in toad oocyte to resume meiotic division].

Full-grown oocytes derived from Bufo bufo gargarizans rearing in high temperature environment (28-30 degrees C), called high temperature oocytes, never underwent germinal vesicle breakdown (GVBD) after progesterone stimulation, no MPF was detected in their ooplasm, but some events which manifested normally at the beginning of progesterone induced maturation process were revealed in these oocytes. It is worth notice an another kind of maturation promoting substance(s) appeared in the ooplasm of high temperature oocyte after the hormone treatment, which was capable of triggering the resumption of meiotic division of the full-grown oocytes derived from hibernating toad (called low temperature oocytes). It is a hibernation factor-dependent maturation promoting substance (HF-MPS), which appeared after decrease of the oocyte endogenous cAMP level. Its appearance depended upon the oocyte protein synthesis, and its activity to inducing GVBD of low temperature oocytes did not inhibited by puromycin. HF-MPS differs from MPF in maturation promoting activity, as low temperature (10 degrees C) delayed obviously HF-MPS' activity but did not influence the rate of GVBD induced by MPF. Further more, probably due to the lack of "hibernation factor(s)", no expression of p34cdc2 gene was detected in high temperature oocytes (unpublished data), neither HF-MPS nor MPF could amplify autocatalytically in the oocytes. So the low temperature (below 15 degrees C) was found to be indispensable for the toad oocyte maturation. If one day we can prove HF-MPS appeared also in the course of oocyte maturation induced by progesterone, the relationship between HF-MPS and MPF may be: [formula: see text] All these discoveries indicated above make a reasonable explanation of the geographical distribution of the toad which was restricted in the region north to the 23 degrees north latitude and east to the 100 degrees east longitude in China.

[The expression of kainate and GABA receptors in oocytes of Bufo bufo gargarizans].

Oocytes of Bufo bufo gargarizans were used as an expression system for analyzing structure of exogenous membrane protein and their functions. Poly (A)+ mRNA isolated from rat brain was injected into Toad (Bufo bufo gargarizans) oocytes (50 ng/oocyte). Rat brain kainic acid and gamma-aminobutyric acid(GABA) receptors expressed by the injected mRNA were integrated in the oocyte membrane 48 h after the injection at 19 degrees C. Membrane currents induced by kainic acid (5 x 10(-5) mol/L) and GABA (10(-4) mol/L) were 294.0 +/- 6.4 nA (n = 5) and 309.5 +/- 4.9 nA (n = 4) respectively. The kainic acid induced current reached its maximum value at about 10(-3) mol/L. Moreover, it was observed that the 36Cl- influx of the oocytes microinjected with mRNA was one-fold more rapid than the control oocytes. These results indicate that the oocytes of Bufo bufo gargarizans as those of Xenopus laevis can be used to express membrane proteins (receptors & transports) to acquire their proper functions from exogenous mRNA.

[A voltage-dependent potassium channel of outward rectifier type in plasma membrane of oocyte from toad, Bufo bufo gargarizans].

Membrane properties of the fully-grown oocytes from toad, Bufo bufo gargarizans, were studied by using voltage-clamp technique. It was found that a sustained outward current was elicited by membrane depolarization to -30 mV or more positive value. The increase of the current was nearly proportional to the degree of depolarization. The peak value of the current ranged 2-5 microA at a membrane potential of 20 mV in oocytes from different toads. The current was inhibited by antagonists of potassium channel, TEA and 4-AP. The concentration of TEA capable of inhibiting half of the current was 2.6 mmol/L. Chloride channel antagonist 9-AC (2.5 mmol/L) had no effect on the current. Triple the extracellular calcium concentration did not show any effect either. The reversal potential of the current varied with an increase of 47.3 mV per decade change of the extracellular potassium concentration. Changing extracellular concentration of sodium or chloride did not shift the reversal potential. It was concluded that the outward current was a voltage-activated potassium current. The voltage-dependent potassium current decreased after treatment of the oocytes with progesterone to a state of maturation. A large decrease of the current (to about 1/20 of the control) occurred to the oocytes obtained from hibernating toads while a less striking decrease of the current (to about 1/3 of the control) was observed in the oocytes from toads all year round reared at 25-30 degrees C.

[Increased ornithine decarboxylase activity is not essential for progesterone-induced Bufo oocyte maturation].

Ornithine decarboxylase (ODC) activity increases by 2 times in the process of progesterone-induced Bufo oocyte maturation (Table 1). Tumor promotor phorbol ester (PMA) is unable to affect both basal and stimulated ODC activity (Fig. 5) although it is capable of elevating the rate of steroid-induced maturation (Fig. 4). Spermine can inhibit significantly ODC activity of oocytes (Fig. 3). Hormone-stimulated ODC activity falls by 17% when Bufo oocytes are cultured in the alkaline Ringer's solution containing 5 mM spermine (pH 11.6) (Fig. 2). The period, however, is shortened by more than 50% during which the oocytes undergo GVBD (Fig. 1). Otherwise, spermine is found to repress ODC activity in dose dependent manner when microinjected in Bufo oocytes (Fig. 3). But oocytes undergo GVBD with a frequency of more than 80% when progesterone-induced increment of the enzyme activity is totally inhibited in the oocytes injected with approximately 50 nl 4.0 mM spermine. The conclusion may emerge from the above-stated results that increased ornithine decarboxylase activity is not essential for progesterone-induced Bufo oocyte maturation. In addition, ODC activity begins to increase rapidly when endogenous spermine level has been lowered to the largest extent in the maturation process. Therefore the endogenous spermine probably acts as a physiologically negative regulator of ODC activity since exogenous spermine inhibits seriously ODC activity of Bufo oocytes.

[Experimental analysis of membrane potential changes during maturation of Bufo bufo gargarizans oocytes].

The full-grown oocytes obtained from toad (bufo bufo gargarizans) submitted in hibernation state or reared at 25-30 degrees C for several months, named hibernation oocyte or high temperature oocyte, had a membrane potential of -41.51 +/- 0.77 mV and -43.83 +/- 1.39 mV in Ringer's solution respectively. The hibernation oocytes underwent GVBD (germinal vesicle breakdown) and membrane depolarization at 19 +/- 1 degree C after progesterone stimulation. The membrane potential was about -20 mV at the period of GVBD, and -10 mV or so at 20 hours after the hormone treatment. However, the high temperature oocytes did not undergo GVBD, their membrane potential decreased before the fourth hour after treatment with progesterone and then recovered. If the hibernation oocytes were preincubated at 37-38 degrees C for 13 hours prior to the culture in the medium containing progesterone (10(-6)M, 37-38 degrees C), no GVBD was observed and the membrane depolarized before the fourth hour after treatment with progesterone then recovered, but MPF was detectable in the cytoplasm (unpublished). Both GVBD and membrane depolarization appeared in the hibernation oocytes and high temperature oocytes after injection of MPF. The time required for the hibernation oocytes injected MPF to attain the membrane potential about -20 mV was 4 hours earlier than that of progesterone treatment. It was just the time required for the appearance of MPF in the cytoplasm of oocytes treated with the hormone. It was noticed in our precedent article that a factor which appeared in the cytoplasm of high temperature oocytes differed from MPF. The factor was called Hibernation Oocyte Mature Promoting Factor (HOMPF).(ABSTRACT TRUNCATED AT 250 WORDS)