Expression and localization of matrix metalloproteinases (MT1-MMP, MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) during synepitheliochorial placentation of goats (Capra hircus).

Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) play key roles during the placentation of highly invasive haemochorial type. Our knowledge is yet scanty, however, regarding the roles played by MMPs and TIMPs in the placentation of non-invasive synepitheliochorial type. In the present study, expression patterns of MT1-MMP, MMP-2 and TIMP-2 mRNAs as well as the encoded proteins in the endometrium and the placenta were examined on Days 35, 75, and 100 of pregnancy, representing roughly the 1st, 2nd and 3rd trimesters of caprine gestation, by means of quantitative RT-PCR analysis, in situ hybridization, immunoblotting, gelatin zymography and immunohistochemistry. In the endometrium and the intercotyledonal trophoblast, the expression levels of the 3 genes remained relatively uniform throughout the period of gestation examined. Curiously, however, in the placentomes, the relative expression levels of MT1-MMP mRNA increased linearly from Day 35 to Day 100, while those of MMP-2 and TIMP-2 were clearly down-regulated in Day 100 placentae. The expression levels of MT1-MMP and TIMP-2 proteins in placentomes were well correlated with those of the respective mRNAs. In the case of MMP-2, the total amount of MMP-2 protein (the combined values of the latent, the intermediate and the active forms) decreased slightly, while the levels of the active form increased markedly from Day 35 to Day 100. Immunohistochemical analysis of the placentome revealed that MT1-MMP and TIMP-2 proteins were co-localized in the binucleate trophoblast cells; expression of these 2 proteins was not detected in the uninuclear principal trophoblast cells. MMP-2 expression was detected both in the binucleate and in the uninuclear principal cells of the trophoblast and in the endometrial stromal cells of the uterine septum, regardless of the stages of gestation examined. The co-localization of MT1-MMP, MMP-2 and TIMP-2 in binucleate trophoblast cells, the cotyledonal trophoblast cells and the subsyncytial stromal cells is likely to reflect the functional coordination of the 3 proteins in these cells during trophoblastic invasion and the placental tissue remodeling in the placentome.

Analysis of transcriptional control elements in the 5'-upstream region of ovine interferon-tau gene using feeder-independent caprine trophoblast cell line, HTS-1.

Interferon-tau (IFNtau) is a protein secreted from the embryonic trophectoderm of ruminant ungulates during peri-implantation period. This protein acts on the uterine endometrium, which indirectly maintains corpus luteum function, and is therefore considered essential for the process of maternal recognition of pregnancy. Transcriptional regulation of IFNtau genes had been examined using human choriocarcinoma cell lines, JEG-3 or JAR, however, molecular mechanisms by which cell and term specific IFNtau expression are regulated have not been elucidated. Recently, a feeder cell free-trophoblast cell line derived from Shiba-goat placenta, termed HTS-1, was established. In the present investigation, the 5'-upstream region of ovine IFNtau (oIFNtau) gene was analysed using this cell line, which would provide a more suitable system for studies of the ovine trophoblast specific gene than human choriocarcinoma cells. Variously modified 5'-upstream sequences of the oIFNtau gene fused to a luciferase reporter gene were transiently transfected into HTS-1 cells, and human JEG-3 cells were used as a control. These results and co-transfection with expression vectors revealed that Ets-2 binding site in the promoter region was important in HTS-1, whereas AP-1 that binds to the enhancer region was a major activator in JEG-3. By electrophoretic mobility shift assay, a nuclear protein from HTS-1 cells was confirmed to bind specifically to the Ets-2 site of oIFNtau promoter region. Differences in amounts of AP-1 and Ets-2 protein were demonstrated in nuclear extracts from HTS-1, JEG-3 and ovine conceptuses. Substantial differences on oIFNtau gene transcriptions found between caprine HTS-1 and human JEG-3 cells suggest that this cell line could be valuable in the elucidation of a molecular mechanism(s) by which oIFNtau gene expression is regulated in a cell specific manner.

Establishment of feeder-independent cloned caprine trophoblast cell line which expresses placental lactogen and interferon tau.

A feeder-independent cloned trophoblast cell line, HTS-1, was established from a mature placenta of Shiba goat (Capra hircus). During the growth phase, single HTS-1 cells exhibited ruffled membranes or lamellipodia often accompanied by elongated cell shape, indicating highly motile nature of the cells. At or near confluence, HTS-1 cells formed monolayers with few sign of cellular overlapping. Binucleate cells were found at a high frequency especially in the peripheral regions of monolayers. In small colonies and the monolayers, majority of HTS-1 cells assumed polygonally shaped cobble-stone like morphology characteristic to epithelial cells, although considerable variations in cellular morphology were observed despite of repeated cloning. Time-lapse video recordings of HTS-1 cells during culture revealed that not only the small colonies but also the monolayers near or at confluence were remarkably motile, often causing extreme elongation of the cells within them. The extremely plastic nature of HTS-1 cells in vitro is likely to be the reflection of the extraordinary capacity of caprine trophoblast cells to be stretched to extreme thinness in vivo as shown by electron microscopy. HTS-1 cells cultured on matrigel are highly invasive, and express MT1-MMP which, in the mouse, has been known to be expressed at the invasive edge of trophoblast both in vitro and in vivo. HTS-1 cells express placental lactogen (PL) and interferon-tau (IFNtau), as confirmed by immunocytochemistry, Western blotting and RT-PCR analysis. Both PL and IFNtau expression in the cells appeared to be down-regulated by cell-cell contact. In the medium conditioned by HTS-1 cells, the presence of secretory form of PL and IFNtau was confirmed by Western blotting. The HTS-1 cell line will serve as a useful in vitro model for the analysis of the molecular and/or cellular mechanisms underlying synepitheliochorial placentation in bovidae animals.

The trophinin gene encodes a novel group of MAGE proteins, magphinins, and regulates cell proliferation during gametogenesis in the mouse.

Trophinin is a membrane protein that mediates apical cell adhesion between trophoblastic cells and luminal epithelial cells of the endometrium and is implicated in the initial attachment during the process of human embryo implantation. The present study identified novel trophinin gene transcripts, which encode proteins structurally distinct from trophinin protein in the mouse. We designated these proteins "magphinins," because they share consensus amino acid sequences with MAGE (melanoma-associated antigen) superfamily proteins. Among many MAGE proteins, magphinins are closely related to NRAGE, which mediates p75 neurotrophin receptor-dependent apoptosis, and necdin, which is a strong suppressor of cell proliferation in post-mitotic neurons. There are three major forms of magphinins, i.e. magphinin-alpha, -beta, and -gamma, in the mouse, which are formed due to alternative usage of different exons. Northern blot analysis revealed that magphinins are expressed in brain, ovary, testis, and epididymis. In addition, Western blot analysis and in vitro translation experiments showed that magphinins expressed in the mouse ovary and testis are translation products utilizing the second initiation AUG codon and contain an active nuclear localization signal. Ectopic expression of magphinins in mammalian cells resulted in nuclear localization of magphinin and suppressed cell proliferation. Immunohistochemistry of the mouse ovary and testis showed that magphinin proteins are distributed in the cytoplasm of the male and female germ cells, whereas these proteins are translocated to the nucleus at a specific stage of gametogenesis. These results strongly suggest that magphinins regulate cell proliferation during gametogenesis in the mouse.

A transgenic mouse model for investigating the response of the upstream region of whey acidic protein (WAP) gene to various steroid hormones.

The limitations of studies of clarification of response elements of whey acidic protein (WAP) gene to hormones using mammary cell lines has been shown. We studied the response of the upstream region (2.6 kb) of WAP to various steroid hormones using gonadectomized mWAP/hGH transgenic mice. Ovariectomy or castration for transgenic mice was performed at 10 days or 30 days post partum. Various steroid hormones were administered daily for 10 days to the gonadectomized transgenic mice after they reached 2 months of age. Prior to the hormonal administration and 24 hr after the final administration, blood was collected and the hGH levels in the plasma was measured by RIA. Daily doses of estradiol-17 beta were significantly more effective at increasing hGH levels in transgenic females ovariectomized at 10 days post partum than progesterone of an equal dose. A combined dose of progesterone and of estradiol-17 beta significantly amplified the increase of hGH levels accompanied by the great development of mammary glands, compared to a dose of progesterone alone. Corticosterone induced only a slight increase of hGH, while testosterone had no effect. The doses of gonadal steroid hormones did not induce an increase in hGH levels and development of mammary glands in the castrated transgenic males. The results showed that the response of 5' region of WAP requires at least some extended development of the mammary gland and that the 2.6 kb upstream region of the exogenous WAP gene contained the element responsive to ovarian hormones.

Possible role for the c-ski gene in the proliferation of myogenic cells in regenerating skeletal muscles of rats.

Skeletal muscle regeneration after injury involves various processes, such as infiltration by inflammatory cells, the proliferation of satellite cells and fusion to myotubes. The c-ski nuclear protein has been implicated in the control of cell proliferation and/or terminal differentiation in the growth of skeletal muscle. However, there have been no reports concerning the involution of c-ski in the regeneration of injured skeletal muscle in mammals. A possible role for c-ski in the proliferation of myogenic cells in rat skeletal muscle during regeneration has been investigated with the assistance of in vitro experiments with L6 skeletal muscle cells. The expression levels of c-ski mRNA in regenerating tissues increased to approximately threefold that of intact tissues at 2 days after injury and decreased to normal levels at 2 weeks after injury. Many mononuclear cells among the Ski-positive cells expressed desmin and proliferating cell nuclear antigen, indicating that Ski-producing cells include the proliferating myogenic cells. The proliferation of L6 cells was significantly retarded by expression of the antisense ski gene. The results of the present study reveal that the c-ski gene plays an important role in the proliferation of myogenic cells in the regeneration of injured skeletal muscle.

Differential gene expression of beta-1,4-galactosyltransferases I, II and V during mouse brain development.

Since most brain glycoproteins from beta-1,4-galactosyltransferase (beta-1,4-GalT) I knockout mice were galactosylated without apparent reduction the gene expression of novel beta-1,4-GalTs II and V which are involved in N-linked oligosaccharide biosynthesis in addition to beta-1,4-GalT I was studied during mouse brain development. Isolation and characterization of beta-1,4-GalT II and V cDNAs from mouse brains indicates that they are also functioning in the brain. Northern blot analysis revealed that the beta-1,4-GalT I gene is expressed mainly in mid-embryonic stages, while the expression level of beta-1,4-GalT II transcript remains constant and of beta-1,4-GalT V transcript increases during mouse brain development after birth. In situ hybridization revealed that beta-1,4-GalT II and V signals are present in most neural cells, with a marked difference between them in the hippocampus of adult mouse brain tissue. The differential gene expression of beta-1,4-GalTs I, II and V during mouse brain development could affect the differential galactosylation of brain glycoproteins, as revealed by lectin blot analysis.

Content and localization of myostatin in mouse skeletal muscles during aging, mechanical unloading and reloading.

Changes in myostatin content and localization in mouse skeletal muscles were investigated during aging, hindlimb suspension (HS) and reloading after HS. During aging, the content of myostatin among solubilized proteins in gastrocnemius and plantaris muscles (Gast/Plant) was initially low and increased until their wet weight/body weight ratio reached a peak. It remained unchanged with further aging, although gradual atrophy of the muscles was seen to occur. Also, the myostatin content did not change significantly during HS (up to 14 days) in both Gast/Plant and soleus muscles, though the muscles showed morphological signs of atrophy. However, reloading for 2 days after a 14-day HS caused significant decreases in the myostatin content in both of these muscles. Immunohistochemical observations showed the sarcoplasmic existence of myostatin, the amount of which appeared to decrease after reloading. The results suggest that myostatin plays a part in the processes of muscular growth and loading-induced hypertrophy, but is not involved in either aging-related or unloading-induced muscular atrophy.

Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) mRNA in trophoblast and endometrial epithelial cell populations of the synepitheliochorial placenta of goats (Capra hircus).

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a membrane-bound matrix metalloproteinase, plays crucial roles in cellular migration through the matrix during embryogenesis, wound healing, and the invasion of host tissues by cancer cells. Mammalian trophoblast cells exhibit different degrees of invasiveness towards the endometrium in different species during gestation. The highly invasive trophoblast cells of primates and rodents which form hemochorial placentae have often been compared to metastatic cancer cells, and are known to express MT1-MMP at their invasive edge. So far, however, little is known about MT1-MMP expression in the placenta of non-invasive type including the synepitheliochorial placenta of bovidae. As an approach to assess the role played by MT1-MMP in the non-invasive synepitheliochorial placentation, we determined the open reading frame (ORF) base sequence of caprine MT1-MMP (DDBJ/EMBL/GenBank database: AB010921); this sequence is the first registered MT1-MMP ORF sequence of artyodactyls which develop placentae of the non-invasive type. The deduced amino acid sequence of caprine MT1-MMP exhibited 92, 87 and 89% identity with its human, mouse and rat counterparts, respectively. Availability of the cloned caprine MT1-MMP cDNA allowed us to carry out Northern blot analysis which revealed that in the placentome, the expression levels of MT1-MMP mRNA were very low on Day 35 of gestation (peri-implantation stage), while the levels gradually increased from Day 75 to Day 100. In the interplacentome regions of the placenta and the uterus, the signal levels were higher than those in the placentome, and increased from Day 35 onward, peaking on Day 75. In situ hybridization experiments revealed that the binucleate trophoblast cells reacted with the MT1-MMP cRNA probe throughout the period examined while the uninuclear principal trophoblast cells did so only on Day 100. Of particular interest is the expression of MT1-MMP transcripts in the luminal and glandular epithelial cells of the gestational endometrium, since epithelial cells in general have been noted to lack MMP expression, including MT-MMPs. The high levels of MT1-MMP expression in the endometrial epithelial cell populations might reflect extensive remodeling during gestation.

In vitro studies on PGC or PGC-like cells in cultured yolk sac cells and embryonic stem cells of the mouse.

The present study aims: 1) to determine those conditions which promote the proliferation of primordial germ cells (PGCs) of the migratory phase in the yolk sac; and 2) to examine the effects of yolk sac cells as a feeder layer under the conditions mentioned above upon the embryonic stem (ES) cells (R1) with high potential for entering the germ line in vivo in chimeras. In murine yolk sac cells obtained on Day 10.5-11.5 of pregnancy and cultured in a modified Dulbecco's modified Eagle's medium (DMEM-plus/20: the postfix represents the concentration of FBS added in percentage), many cells exhibited strong immunoreactivities to the monoclonal antibodies 4C9 and 2C9 which are known to react with PGC specifically. Both the 4C9- and the 2C9-positive cells were sensitive to the treatment with busulfan added in vitro, supporting the supposition that they were PGCs. The respective numbers of the 4C9- and the 2C9-positive cells increased approximately 4 and 12 times when they were cultured in DMEM-plus/20 fortified with SCF, LIF, bFGF and TNF-alpha (DMEM-NT/20). When the R1 cells were cultured in the yolk sac-conditioned DMEM-NT/20 medium on the laminin substratum, the entire colonies were faintly stained with 4C9 but not with 2C9. At times solitary ES cells migrated out from the colonies, and reacted strongly with 4C9. When yolk sac cells and R1 cells were cultured on the two sides of a collagen-coated membrane, the yolk sac cells being feeder cells, some R1 cell colonies were intensely stained as a whole with either the 4C9 or the 2C9 antibody, suggesting that these colonies might be composed of cells clonally derived from stem cells which either had been destined to become the germ line cells or had already acquired cellular characteristics close to PGCs. It was tentatively concluded that the R1 cell population contained, as judged from the immunoreactivities, germ-cell-like cells, and that the yolk sac cells and/or their secretory products might facilitate the proliferation of, or the conversion of R1 cells to, the germ-cell-like cells.

Co-injection of restriction enzyme with foreign DNA into the pronucleus for elevating production efficiencies of transgenic animals.

The microinjection method for production of transgenic farm animals requires specialized techniques and results in intolerably low production efficiencies. We investigated whether or not co-injection of foreign DNA constructs with restriction endonuclease into the pronucleus of mouse zygotes would improve the integration frequencies of foreign DNA into the host genome. Two kinds of DNA constructs that have no EcoRI site in their sequences were used for co-microinjection. With reference to the results of experiments in which EcoRI alone was injected at various amounts varying from 10(-9) to 10(-5) U/nucleus, the amount of 5x10(-8) U/nucleus that showed survival rate of 60.6% was used for the co-injection with DNA. Successful transgenesis of co-injected embryos was identified by DpnI-Bal31 digestion method for single embryos and by PCR method for pups born, respectively. The overall efficiency for the integration of foreign DNA in single embryos and live-born pups obtained by the co-injection procedures were 17.9% compared with 9.1% obtained by the injection of DNA alone. The results suggest that co-injection of foreign genes with restriction enzyme may elevate the integration rate of foreign genes into host genomes.

Comparative analysis of HOXC-9 gene expression in murine hemochorial and caprine synepitheliochorial placentae by in situ hybridization.

Mammalian placentae exhibit wide structural diversity among different species and are formed under intricate interplay between the embryonic trophoblast and the maternal endometrial cells. Increasing evidence in the literature indicates a possible role played by homeobox genes in the complex placental organogenesis. Although the expression of all HOX 9 paralogs has been demonstrated both in highly invasive murine hemochorial placentae and in non-invasive caprine synepitheliochorial placentae, no reports so far published in the literature described the patterns of gene expression of Hoxc-9 in the murine nor those of HOXC-9 in the caprine placenta at cellular levels. We carried out comparative analyses of the location and identity of the cells expressing Hoxc-9/HOXC-9 during various stages of placentation in the murine hemochorial and caprine synepitheliochorial placentae by means of in situ hybridization using murine Hoxc-9 or caprine HOXC-9 cRNA probe, respectively. The results demonstrated that Hoxc-9 mRNA was expressed at high levels in giant trophoblast cells of murine placentae on Days 12-19, but not on Day 8. Similar analysis of caprine Day 75 and Day 100 placentae revealed that the binucleate trophoblast cells that penetrate the uterine luminal epithelial cell layer, strongly expressed HOXC-9 mRNA. Although the functional significance of Hoxc-9/HOXC-9 gene expression in trophoblast cells remains to be elucidated, it was suggested that it might play a role in the regulation of invasiveness or endocrine activities in the murine giant trophoblast cells and/or the caprine binucleate trophoblast cells.

Expression of milk protein genes is induced directly by exogenous STAT5 without prolactin-mediated signal transduction in transgenic mice.

STAT5 is a member of the family of STAT (signal transducer for activating transcription), which was isolated from nuclear extracts of the lactating gland of sheep. It was reported that, in vitro, STAT5 could be activated directly by prolactin (PRL) without PRL-mediated signal transduction. The present study was conducted to investigate above the possibility in vivo, using the transgenic mice overexpressing ovine STAT5 (oSTAT5) under the control of MMTV promoter. Three transgenic mouse lines that expressed the exogenously introduced oSTAT5 in their mammary glands were established. The expression levels of exogenous oSTAT5 were higher than those of endogenous STAT in the mammary glands of all of the three transgenic lines. Although the expression levels of endogenous milk protein genes, WAP, and beta-casein genes, were not correlated with oSTAT5 expression, the expression levels of WAP and beta-casein were induced by exogenous oSTAT5 in the transgenic mice. The present study demonstrated, at very least, that STAT5 expression can directly activate the expression of milk protein genes, and particularly the WAP gene without PRL-mediated signal transduction.

Molecular cloning of cDNA for equine follistatin and its gene expression in the reproductive tissues of the mare.

A cDNA clone encoding equine follistatin was isolated from an equine ovarian cDNA library. Out of 1.2 x 10(5) independent clones screened, one positive clone was isolated and its cDNA sequence determined. The isolated clone, named EQ-FS-1, contained a complete open reading frame encoding 344 amino acid residues. The similarity of its deduced amino acid sequence to these of other mammalian species was greater than 95%. Although its expression level varied among the tissues examined, follistatin mRNA was detected in the equine uteroplacental tissues, follicles and corpora lutea by Northern blot analysis. In situ hybridization revealed that the expression of follistatin mRNA in the equine follicle was restricted exclusively to granulosa cells. When the expression pattern of follistatin mRNA in the equine uteroplacental tissues from mid- to late-pregnancy was examined, it was shown that its expression level tended to decrease after mid-pregnancy. These results suggest that follistatin acts in the reproductive tissues of the mare in maintaining pregnancy.

Expression and localization of membrane type matrix metalloproteinase-1 (MT1-MMP) in trophoblast cells of cultured mouse blastocysts and ectoplacental cones.

Membrane type matrix metalloproteinases (MT-MMPs) possess a C-terminal transmembrane domain and are expressed on the cell membrane. It was suspected, therefore, that MT1-MMP might play an important role in the trophoblastic invasion during implantation. The patterns of expression and localization of membrane type matrix metalloproteinase-1 (MT1-MMP) were examined immunocytochemically in cultured mouse blastocysts and excised extoplacental cones (EPCs). MT1-MMP immuno-reactivity was present in the giant trophoblast cells located at the periphery of the spreading trophoblast of cultured blastocysts and the outgrowths of cultured EPCs, but not in the densely packed trophoblast cells in both the blastocysts and the EPCs. It appears likely that MT1-MMP expressed on the edge of the invading trophoblast facilitates the trophoblastic invasion by cleaving proMMP-2, a known substrate of MT1-MMP, in the decidua. Immunohistochemical examination of early conceptuses confirmed that the trophoblast cells actively invading the endometrium in vivo express MT1-MMP strongly. It is suggested, furthermore, that the expression of MT1-MMP might be downregulated by cell-cell contact in mouse trophoblast cells, as in the mouse mammary epithelial cell line HC11.

Wilms' tumor suppressor gene (WT1) as a target gene of SRY function in a mouse ES cell line transfected with SRY.

With the aim of identifying the gene(s) located downstream from SRY, we transfected an ES cell line with XX karyotype, TMA-18, with a Sry DNA construct and established cell lines, TS18-1 and TS18-2, where the transfected Sry was expressed in the functional linear mRNA form. Among the five potential SRY-target genes examined, i.e., MIS, SF1, P450arom, Sox9 and WT1, only the expression of WT1 was induced de novo by the unscheduled expression of Sry in the transfected cell lines. No clear indication of Sry-induced enhancement of Sox9 expression was obtained in the present series of experiments. Function of a yet unidentified gene(s) located on the Y chromosome might be needed for the up-regulation of Sox 9 expression which takes place during the development of male gonads. Quantitative RT-PCR analysis of the patterns of WT1 expression in developing fetal gonads revealed that although both male and female fetal gonads express WT1, male gonads invariably expressed WT1 mRNA at higher levels than female ones after the Sry expression. Immunohistochemical analysis of the male fetal gonads between 10.5 and 13.5 dpc demonstrated the presence of strong WT1 immunoreactivity in Sertoli cells of the primordial testes. Suggestions were made in the past indicating that both SF1 and WT1 proteins might be active in a common pathway upstream from Sry. Our results showed that WT1 is located downstream, rather than upstream from Sry and behaves independently from SF1. Analysis using an appropriate in vitro system will be essential to understand the molecular mechanisms of SRY action within cells.

Developmental fate of single embryonic stem cells microinjected into 8-cell-stage mouse embryos.

Embryonic stem (ES) cells are pluripotent and capable of differentiating into somatic as well as germ cell lineages when conjoined with blastomeres of early mouse embryos. However, the developmental potential of single ES cells has not been fully investigated. We injected single murine ES cells (A3-1 cell line) of 129 origin into 8-cell mouse embryos (B6xBDF1) and examined the patterns of distribution of ES-cell-derived cells in the blastocysts as well as in the fully grown chimeric mice. The ES cells underwent 1-2 cycles of mitosis between the 8-cell and the blastocyst stage when they were introduced as single cells, whereas those introduced as groups of 2-5 cells did not proliferate during the same period of development. The ES cells and their daughter cells were predominantly incorporated into the ICM. From the 63 8-cell embryos which received single ES cells microinjected into the perivitelline space, 24 newborns were obtained, and 4 (2 fertile males, 1 sterile female and 1 hermaphrodite) of them (16.6%) were chimeric. The test breeding studies revealed that all the progeny of the two chimeric males were derived from spermatozoa of 129 genotype. The relative contribution of the host-derived and the ES-cell-derived cells in different tissues of the chimeric mice was assessed by PCR analyses of the microsatellite polymorphism of genomic DNA extracted from the tissues. In two male germ line chimeras, the testes, the kidneys and the dorsal skeletal muscles exhibited exceptionally high 129 contents. Our results demonstrated that single ES cells which maintain totipotency or pluripotency of high degree are present in a colony of ES cells, and that single ES cells conjoined with the blastomeres of 8-cell-stage embryos may colonize, if the circumstances allow, almost exclusively the germ cells and concomitantly the urogenital cell lineages. Possible correlation between the allocation of the germ line and the urogenital lineages is discussed.

Growth hormone-releasing hormone (GHRH)-GH-somatic growth and luteinizing hormone (LH)RH-LH-ovarian axes in adult female transgenic mice expressing human GH gene.

We have examined alterations in the hypothalamo-pituitary GH-somatic growth axis and the hypothalamo-pituitary LH-ovarian axis in a line of transgenic ICR mice expressing human GH (hGH) under the influence of the whey acid protein promoter. Transgenic female mice weighed twice as much as control females and were infertile. The size of the anterior pituitary (AP) was 1/3 that of the controls. In transgenic mice, acinar cells in the mammary and mandibular glands displayed hGH-immunoreactivity, and plasma hGH was detected by radioimmunoassay. In the medial basal hypothalamus (MBH) of transgenic females, the immunoreactive-GHRH level was decreased (P<0.01). There was a corresponding reduction in the number of GHRH-immunoreactive neurons in the arcuate nucleus (ARC) and in the immunostaining of GHRH nerve terminals in the median eminence. The level of somatostatin (SRIH) in the MBH was increased (P<0.05), and SRIH-immunoreactive neurons in the periventricular nucleus (PeV) were increased in size and number in transgenic mice. The MBH level of LHRH in transgenic animals was greater (P<0.01) than in controls, although there was no apparent difference in the number of LHRH-immunoreactive neurons or in LHRH level in the preoptic area. There are fewer SRIH- and LHRH-immunoreactive neurons in the ARC in transgenic mice. Cells in the AP for GH, PRL, and LH were fewer in transgenic mice. The ovary suffered disturbance of follicular development and of corpora lutea formation. These results demonstrate that chronic overproduction of hGH may profoundly affect the organization of the GHRH/SRIH-GH-somatic growth axis and the LHRH-LH-ovarian axis due to reduction of GHRH-, SRIH- and LHRH-neurons in the ARC and increase of SRIH-neurons in the PeV.

Equine inhibin/activin beta A-subunit mRNA is expressed in the endometrial gland, but not in the trophoblast, during pregnancy.

The expression of both inhibin alpha- and inhibin/activin beta A-subunit mRNA was examined in equine uteroplacental tissues collected during pregnancy (days 90 to 300). Northern blot analysis revealed that 5 transcripts (7.0, 4.1, 3.4, 2.6, 1.5 kb) of beta A-subunit were present, and the most abundantly expressed transcript was the 1.5 kb one. Relatively high levels of the 1.5 kb transcript were seen in the second trimester of pregnancy compared to what was found in the third trimester. To identify the tissue localization of beta A-subunit mRNA, in situ hybridization was performed, and the positive signal was observed exclusively in the endometrial glands, but not in the fetal placental tissue (trophoblast) at days 150, 210, and 300 of pregnancy. On the other hand, inhibin alpha-subunit transcript could not be detected at any stage of pregnancy examined either by Northern blot analysis or in situ hybridization. Although the factor(s) regulating the gene expression of beta A-subunit in this equine tissue is currently unknown, these results suggest that activin, but not inhibin, is predominantly produced in the endometrial glands of the pregnant mare, and thus produced activin may play a paracrine or endocrine role during pregnancy in this species.

Expression and cellular localization of inhibin alpha-subunit mRNA in equine fetal gonads.

The expression of inhibin alpha-subunit mRNA in equine fetal gonads during pregnancy (Days 90 to 300) was examined by means of Northern blot analysis. In all samples examined, a single species of transcript was detected at the size of 1.5 kb. A digoxigenin-labeled antisense cRNA probe specific to equine inhibin alpha-subunit was synthesized and in situ hybridization analysis to locate the inhibin alpha-subunit mRNA positive cells was performed using frozen tissue sections of equine fetal ovary (day 150 of pregnancy) and equine fetal testis (day 180 of pregnancy). In the fetal ovary, positive cells were seen throughout the interstitial area but did not show any particular localization. In the fetal testis, on the other hand, the antisense cRNA hybridized almost exclusively to the interstitial cells surrounding developing seminiferous cords and Sertoli cells within the cords. Positive signals were also detected in a limited number of the interstitial cells located away from the cords. These results suggest that in equine fetal gonads, inhibin and/or inhibin alpha-subunit related molecules such as the monomeric form are produced and these molecules may have a paracrine/autocrine role within the gonads.