Certain fish, such as rainbow trout (Oncorhynchus mykiss), are seasonal breeders. Spermatogenesis in rainbow trout is synchronous; therefore, at any time point during this process, germ cells are predominantly at the same stage of development. As such, rainbow trout represent an excellent model in which to study spermatogenesis. Gap junctions are composed of connexons, which are themselves formed by six transmembrane proteins termed connexins (Cxs). The objectives of this study were to assess which Cxs are expressed in the rainbow trout testis, and if their expression was stage specific during gonadal maturation. Rainbow trout were killed at various stages of maturation, and total cellular RNA was isolated from the testes. RT-PCR using degenerate primers recognizing all vertebrate Cxs indicates that there are several different Cxs in trout testes. Amplicons were cloned and sequenced. Homology comparisons indicate that these were cx43, cx43.4, cx31, and cx30. Immunolocalization of these Cxs indicate that Cx43 was localized primarily to Sertoli cells, while Cx43.4 was localized along the lateral plasma membranes between adjacent spermatocytes. Cx30 was localized to the interstitial Leydig cells, and Cx31 was localized primarily to the endothelium of interstitial blood vessels. The expression of each Cx varied as a function of the stage of spermatogenesis, suggesting that the expression of these proteins is highly regulated. Together, these results indicate that intercellular communication in the testis is complex, involves several different Cxs, and is a highly regulated process.

The egg envelope, referred to as zona pellucida (ZP) in mammalian eggs, is a fibrous and noncollagenous extracellular matrix surrounding vertebrate eggs, and composed of three to four homologous glycoproteins with a common ZP domain. In birds, a liver-derived ZP glycoprotein (ZP1/ZPB1) is transported through the bloodstream to ovarian follicles and joins the egg-envelope matrix construction together with the other ZP glycoproteins, such as ZPC and ZPD/ZPX2, both secreted from follicular granulosa cells. We report here that, through its ZP domain, ZPB1 specifically associates with ZPC, which might lead to the construction of egg-envelope matrix. The ZPB1 in laying hen's serum specifically bound to ZPC, but not to ZPX2, separated by SDS-PAGE and blotted on a membrane. Hemagglutinin (HA)-tagged ZPC expressed in a mammalian cell line (COS-7) cells was processed and secreted as a mature-form into the culture medium. From the culture supernatant of ZPC-expressing transfectants cultured in the presence of ZPB1, both ZPB1 and ZPC were recovered as heterocomplexes by immunoprecipitation using either anti-HA or anti-ZPB1 antibody. Interestingly, a monoclonal antibody, 8E1, which immunoprecipitated free ZPB1, did not immunoprecipitate the ZPB1-ZPC heterocomplexes. An 8E1 epitope was mapped on a C-terminal region of the ZP domain in a ZPB1 molecule by identifying an 8E1-positive peptide using mass spectroscopy. Furthermore, by laser scanning confocal microscopy, ZPB1 and ZPC were observed to colocalize on the surface of ZPC-expressing transfectants cultured in the presence of ZPB1, whereas almost no ZPC was detected on the surface of the transfectants cultured in the absence of ZPB1. Taken together, these results suggest that ZPB1 transported into ovarian follicles encounters and associates with ZPC secreted from granulosa cells, resulting in the formation of heterocomplexes around an oocyte. In addition, it appears that such ZPB1-ZPC complexes accumulated on the oocyte surface act as a scaffold for subsequent matrix construction events including ZPX2 association.

Thirty to eighty-seven percent of patients treated by radical prostatectomy experience erectile dysfunction (ED). The reduced efficacy of treatments in this population makes novel therapeutic approaches to treat ED essential. We propose that abundant apoptosis observed in penile smooth muscle when the cavernous nerve (CN) is cut (mimicking the neural injury which can result from prostatectomy) is a major contributing factor to ED development. We hypothesize that decreased Sonic hedgehog (SHH) signaling is a cause of ED in neurological models of impotence by increasing apoptosis in penile smooth muscle. We examined this hypothesis in a bilateral CN injury model of ED. We found that the active form of SHH protein was significantly decreased 1.2-fold following CN injury, that SHH inhibition causes a 12-fold increase in smooth muscle apoptosis in the penis, and that SHH treatment at the time of CN injury was able to decrease CN injury-induced apoptosis (1–3-fold) in a dose-dependent manner. These results show that SHH stabilizes the alterations of the corpora cavernosal smooth muscle following nerve injury.

To study mechanisms governing fetoplacental vascular function, we have established a primary ovine fetoplacental artery endothelial (OFPAE) cell line. These OFPAE cells produce nitric oxide (NO), proliferate, and migrate in response to fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF). To overcome the senescence crisis that this primary OFPAE cell line will eventually enter, we attempted to establish a functional OFPAE cell line with a prolonged life span by transfecting cells with plasmids containing a neomycin resistance gene and a simian virus 40 gene (SV40) expressing large T (T) and small t (t) antigens. The OFPAE cells at passage 8 were transfected. After neomycin selection, the surviving OFPAE (designated SV40 OFPAE) cells were expanded up to passage 80. Up to passage 30, these SV40 OFPAE cells maintained a morphology similar to untransfected OFPAE cells. Expression of T and t antigens in SV40 OFPAE cells was confirmed by immunocytochemistry. These SV40 OFPAE cells exhibited positive uptake of acetylated low-density lipoprotein (Ac-LDL) and positive staining for NO synthase 3 (NOS3) and formed capillary-like tube structures on Matrigel. Up to passages 20–23, these SV40 OFPAE cells proliferated (P < 0.05) and produced (P < 0.05) NO in response to both FGF2 and VEGF. Moreover, this cell proliferation stimulated by FGF2 and VEGF was dose-dependently inhibited (P < 0.05) by PD98059 (a selective mitogen-activated protein kinase 1 and 2 [MAP2K1/2, also termed MEK1/2] inhibitor) or by LY294002 (a selective phosphoinositide 3-kinase [PI3K] inhibitor). These data indicate that SV40 OFPAE cells, at least at passage 23, retain endothelial phenotypes and functions similar to their parental, untransfected OFPAE cells. Thus, a functional OFPAE cell line with an extended life span has been successfully established, potentially providing a valuable cell model for studying fetoplacental endothelial function.

Until now, no primate animals have been successfully cloned to birth with somatic cell nuclear transfer (SCNT) procedures, and little is known about the molecular events that occurred in the reconstructed embryos during preimplantation development. In many SCNT cases, epigenetic reprogramming of the donor nuclei after transfer into enucleated oocytes was hypothesized to be crucial to the reestablishment of embryonic totipotency. In the present study, we focused on two major epigenetic marks, DNA methylation and histone H3 lysine 9 (H3K9) acetylation, which we examined by indirect immunofluorescence and confocal laser scanning microscopy. During preimplantation development, 67% of two-cell- and 50% of eight-cell-cloned embryos showed higher DNA methylation levels than their in vitro fertilization (IVF) counterparts, which undergo gradual demethylation until the early morula stage. Moreover, whereas an asymmetric distribution of DNA methylation was established in an IVF blastocysts with a lower methylation level in the inner cell mass (ICM) than in the trophectoderm, in most cloned blastocysts, ICM cells maintained a high degree of methylation. Finally, two donor cell lines (S11 and S1–04) that showed a higher level of H3K9 acetylation supported more blastocyst formation after nuclear transfer than the other cell line (S1–03), with a relatively low level of acetylation staining. In conclusion, we propose that abnormal DNA methylation patterns contribute to the poor quality of cloned preimplantation embryos and may be one of the obstacles to successful cloning in primates.

During mammalian development, morphogenesis of the testis requires the coordinated interplay of somatic cells to form seminiferous cords in which the primitive germ cells reside. These cords are the precursor of the functional male gonad and as such form the basis of male fertility. Cell migration during mammalian organogenesis and formation of complex tissues, such as the testis, are difficult to study in situ. Herein, we report extensive rearrangement of cells to regenerate complete functional testis tissue after implantation of isolated neonatal porcine testis cells under the skin of immunodeficient mice. Somatic cells and germ cells reorganized into structures that have remarkable morphologic and physiologic similarity to normal testis tissue, forming the endocrine and spermatogenic compartment of the testis. This unique in vivo system provides an accessible model for the study of testicular morphogenesis that could be especially useful in nonrodent species.

MSY2 is a member of the Y-box family of proteins solely expressed in male and female germ cells. In the male, MSY2 serves as a coactivator of transcription by binding to a consensus promoter element present in many germ cell-specific genes. In the nucleus, MSY2 marks specific mRNAs for cytoplasmic storage, stabilization, and suppression of translation. The inactivation of MSY2 by gene targeting leads to spermatogenic arrest and infertility. In testes of mice lacking MSY2, incomplete nuclear condensation is prominent in later-stage spermatids at the time of massive spermatid loss. Because MSY2 interacts with DNA and mRNAs, there are several distinct sites of action, which could be disrupted in mice that lack MSY2, resulting in the arrest of spermatogenesis. To define the molecular cause(s) of the spermatogenic arrest in mice lacking MSY2, transcriptional and posttranscriptional processes were assayed. Transcription, mRNA processing, and mRNA intracellular transport appear normal in the absence of MSY2. However, a redistribution of mRNAs from ribonucleoprotein particles to polysomes and marked decreases were detected for many meiotic and postmeiotic germ cell mRNAs, including the mRNAs encoding the transition proteins and protamines. This suggests that increased mRNA instability is a likely cause of the male infertility in Msy2-null mice.

Spermatogonial stem cells continuously divide in the testis to support spermatogenesis throughout the life of adult male animals. Although very few spermatogonial stem cells are present in vivo, we recently succeeded in expanding these cells in vitro. Germ cells from postnatal testes were able to proliferate in the presence of several types of cytokines, and they formed uniquely shaped colonies of spermatogonia (germline stem or GS cells). These cells reinitiated normal spermatogenesis when transplanted into seminiferous tubules. However, much remains unknown about the contributions of cytokines to successful stem cell culture. In the present study, we examined the role of leukemia inhibitory factor (LIF) in GS cell culture. We found that the addition of LIF to newborn testis cell culture enhances the formation of germ cell colonies. Ciliary neurotrophic factor, but not oncostatin M, had the same effect, although they both bind to the IL-6ST (gp130) receptor. On the other hand, GS cells could be established from pup or adult testes in the absence of LIF. No phenotypic or functional difference was found between GS cells established from different stages, and normal offspring were born from pup-derived GS cells that had been maintained in the absence of LIF, indicating that LIF per se is not involved in the self-renewal of GS cells. These results demonstrate that LIF is useful in the initiation of GS cell culture and suggest that LIF or a related cytokine is involved in the maturation of gonocytes into spermatogonia.

Members of the RNase superfamily participate in a diverse array of biological processes, including RNA degradation, antipathogen activities, angiogenesis, and digestion. In the present study, we cloned the rat RNase9 gene by in silico methods and genome walking based on homology to the Macaca mulatta (rhesus monkey) epididymal RNase9. The gene is located on chromosome 15p14, spanning two exons, and is clustered with other members of the RNase A superfamily. It contains 1279 bp and encodes 182 amino acids, including a 24-amino acid signal peptide, and it has unique features known from other RNases. Unlike those other members, the rat RNase9 mRNA was specifically expressed in the epididymis, especially in the caput and corpus, and exhibited an androgen-dependent expression pattern but was downregulated in an epididymitis animal model. The RNASE9 was expressed in a principal cell-specific pattern. Interestingly, most of the principal cells in the caput expressed the RNASE9; however, in the distal caput, the principal cells showed a checkerboard-like pattern of immunoreactivity. We also observed that the RNASE9 was bound on the acrosomal domain of sperm. Its potential roles in sperm maturation are discussed.

The cDNA sequence encoding orange-spotted grouper lhb (LHbeta) and cga (GTHalpha) subunits were cocloned into baculovirus transfer vectors and expressed in insect Sf9 cells. The results showed that two bands of 15.6 kDa and 11.4 kDa could be detected by SDS-PAGE and a band of 30 kDa could be detected by native PAGE. The recombinant grouper Lh (rgLh) could stimulate the secretion of testosterone (T) and estradiol-17beta (E₂) from the gonad in a static incubation system in a time-dependent, but not a dose-dependent, manner. Using in vivo bioassay, the mRNA levels of two aromatases (cyp19a1a [P450aromA] and cyp19a1b [P450aromB]), gnrh (GnRH), lhb, and cga in the pituitary, gonad, and hypothalamus were determined in different groups of orange-spotted groupers treated respectively with rgLh, human chorionic gonadotropin (hCG), and a culture medium of insect cells transformed with an expression vector without lhb and cga subunits. The mRNA levels of cyp19a1a and cyp19a1b rose dramatically after injecting rgLh intraperitoneally, which was consistent with the secretion of sex steroid hormones. Interestingly, the mRNA levels of gnrh dropped in the pituitary, hypothalamus, and gonad, and the mRNA levels of lhb and cga in the pituitary of the experimental group expressed at a higher level than that of the hCG group. These results are in accord with the long positive feedback loop of Lh on gonad sex steroid hormones and the short negative feedback loop of Lh on gnrh mRNA levels. These results indicate that the rgLh is successfully expressed by the baculovirus-insect expression system and that the rgLh has biological activity.

The GATA4 transcription factor is an important developmental determinant for many organs, such as the heart, gut, and testis. Despite this pivotal role, our understanding of the transcriptional mechanisms that control the proper spatiotemporal expression of the GATA4 gene remains limited. We have generated transgenic mice expressing a green fluorescent protein (GFP) marker under the control of rat Gata4 5′ flanking sequences. Several GATA4-expressing organs displayed GFP fluorescence, including the heart, intestine, and pancreas. In the gonads, while GATA4 is expressed in pregranulosa, granulosa, and theca ovarian cells, and Sertoli, Leydig, and peritubular testicular cells, the first 5 kb of Gata4 regulatory sequences immediately upstream of exon 1 were sufficient to direct GFP reporter expression only in testis and, specifically, in Sertoli cells. Onset of GFP expression occurred after Sertoli cell commitment and was maintained in these cells throughout development to adulthood. In vitro studies revealed that the first 118 bp of the Gata4 promoter is sufficient for full basal activity in several GATA4-expressing cell lines. Promoter mutagenesis and DNA-binding experiments identified two GC-box motifs and, particularly, one E-box element within this −118-bp region that are crucial for its activity. Further analysis revealed that members of the USF family of transcription factors, especially USF2, bind to and activate the Gata4 promoter via this critical E-box motif.

Recent studies have demonstrated that fetal cells can be detected in the maternal circulation during virtually all human pregnancies. These fetal cells can engraft and may be isolated for many decades after pregnancy, leading to a state that may be maintained by the passage of pregnancy-associated progenitor cells. The clinical consequences of fetal cell microchimerism are unclear but may be potentially detrimental or valuable to the mother. One possibility is the generation of an alloreactive immune response by the mother to antigens expressed by the fetus; for example, the HY protein encoded by the Y chromosome. To test this we have screened a cohort of women with a range of parity histories within 8 yr of their last pregnancy for the presence of an HY-specific CD8 T-cell response. Fluorescent HLA-peptide (HY) tetramers were used to stain short-term T-cell cultures from these women for analysis by flow cytometry. Responses were detected in 37% of women with a history of pregnancies that produced males, and this value rose to 50% in women with two or more pregnancies that produced males. HY-specific CD8 T cells also could be detected directly in the peripheral blood of women with a history of at least two pregnancies that produced males. These HY-specific CD8 T cells produced interferon gamma (IFNG) following peptide stimulation, demonstrating their functional capacity. In conclusion, our data indicate that alloreactive CD8 T cells are generated frequently following normal pregnancy and retain functional capability for years following pregnancy.

During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast differentiation and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important for successful embryonic implantation, including establishing the placental vasculature, anchoring the placenta to the uterine wall, and promoting the immunoacceptance of the fetal allograph. To our knowledge, global crosstalk between the trophoblast and the decidua has not been elucidated to date, and the present study used a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and further treated with conditioned media from human trophoblasts (TCM) or, as a control, with control conditioned media (CCM) from nondecidualized stromal cells for 0, 3, and 12 h. Total RNA was isolated and processed for analysis on whole-genome, high-density oligonucleotide arrays containing 54 600 genes. We found that 1374 genes were significantly upregulated and that 3443 genes were significantly downregulated after 12 h of coincubation of stromal cells with TCM, compared to CCM. Among the most upregulated genes were the chemokines CXCL1 (GRO1) and IL8,CXCR4, and other genes involved in the immune response (CCL8 [SCYA8], pentraxin 3 (PTX3), IL6, and interferon-regulated and -related genes) as well as TNFAIP6 (tumor necrosis factor alpha-induced protein 6) and metalloproteinases (MMP1, MMP10, and MMP14). Among the downregulated genes were growth factors, e.g., IGF1, FGF1, TGFB1, and angiopoietin-1, and genes involved in Wnt signaling (WNT4 and FZD). Real-time RT-PCR and ELISAs, as well as immunohistochemical analysis of human placental bed specimens, confirmed these data for representative genes of both up- and downregulated groups. The data demonstrate a significant induction of proinflammatory cytokines and chemokines, as well as angiogenic/static factors in decidualized endometrial stromal cells in response to trophoblast-secreted products. The data suggest that the trophoblast acts to alter the local immune environment of the decidua to facilitate the process of implantation and ensure an enriched cytokine/chemokine environment while limiting the mitotic activity of the stromal cells during the invasive phase of implantation.

Cardiovascular adaptation is a hallmark of pregnancy. Here we report on vascular hyperresponsiveness to an endogenous vasodilator, adrenomedullin (ADM), during pregnancy. Intravenous administration of ADM dose dependently decreased the mean arterial pressure, and the decrease was significantly greater in pregnant compared with nonpregnant rats without affecting the heart rate. In endothelium-intact mesenteric artery precontracted by ED₇₀ concentration of norepinephrine, the potency and efficacy of ADM in causing the vasodilation of mesenteric arterial rings from pregnant rats are significantly higher compared with nonpregnant females at diestrus. The magnitude of inhibition of concentration-dependent response to ADM by the inhibition of either soluble guanylate cyclase or adenylate cyclase was greater in pregnant rats. Moreover, ADM-induced cyclic nucleotide generation, both cGMP and cAMP, in the mesenteric artery was elevated during pregnancy and was sensitive to the receptor antagonist, ADM_22–52. These findings suggest that during pregnancy the vasodilatory effects of ADM are greater and are associated with increased generation of cyclic nucleotides in resistance vessels, and these changes may be part of the cardiovascular adaptations that occur during pregnancy.

The use of transcervical artificial insemination in sheep is limited because of the anatomy of the cervix, which restricts the passage of an inseminating pipette into the uterine lumen. There is a degree of natural cervical relaxation at estrus that enables greater penetration with an inseminating pipette. We hypothesize that this relaxation may be regulated by cervical prostaglandin synthesis and remodeling of the cervical extracellular matrix. The present study investigated the changes in prostaglandin endoperoxide synthase 2 (PTGS2) mRNA expression and the proportion of smooth muscle and collagen in the sheep cervix during the estrous cycle. Sheep cervices were collected at four stages of the estrous cycle: prior to the LH surge, during the LH surge, after the LH surge, and during the luteal phase. The expression of cervical PTGS2 mRNA was determined by in situ hybridization, and the proportion of smooth muscle and collagen in the cervix was investigated by Masson trichrome staining. The expression of PTGS2 mRNA in the sheep cervix was greatest prior to the LH surge, when estradiol concentrations were also greatest. The increase in PTGS2 mRNA expression was associated with an increase in the proportion of collagen in the sheep cervix. We propose that prior to the LH surge, estradiol may stimulate PTGS2 mRNA expression and hence prostaglandin E₂ synthesis in the sheep cervix to regulate cervical relaxation, most likely through the rearrangement of collagen bundles within the cervical extracellular matrix.

In the mammalian testis, spermatogenesis is initiated from a subset of stem cells belonging to undifferentiated type A spermatogonia. In spite of the biologic significance of undifferentiated type A spermatogonia, little is known about their behavior and properties because of a lack of specific cell surface markers. Here we show that CDH1 (previously known as E-cadherin) is expressed specifically in undifferentiated type A spermatogonia in the mouse testis. Histologic analysis showed that CDH1-positive cells had all the characteristics of undifferentiated type A spermatogonia. Whole-mount immunohistochemistry showed that CDH1-positive cells made clusters mainly comprising one, two, four, or eight cells. They survived after administration of the cytotoxic agent busulfan to mice, and then regenerated seminiferous epithelia. Transplantation experiments showed that only CDH1-positive cells had colonizing activity in the recipient testis. Our data clearly demonstrated that spermatogenic stem cells reside among undifferentiated type A spermatogonia, which express CDH1.

The testis is a tightly controlled dynamic tissue. In mammals, there is growing evidence that estrogen plays a role in the regulation of testicular functions. In teleosts, high levels of 17beta-estradiol (E₂) in serum correlate with the end of spermatogenesis, spawning, and the initiation of postspawning stages when spermatogonia are the main cell types in the testis. Moreover, E₂ modulates leukocyte functions in several teleost species. We hypothesized, therefore, that E₂ would induce the infiltration of acidophilic granulocytes and cause a resumption of testicular cell proliferation in spermatogenically active gilthead seabream males. Several studies of this species have reported that supraphysiological doses of E₂ are needed to induce histological and developmental changes in males. In fact, as gilthead seabream is a protandrous hermaphrodite teleost, long exposures (6–14 wk) to high doses of E₂ result in feminization of the males. Taking all this into account, we sharply increased E₂ levels during short times by i.p. injecting E₂ diluted in coconut oil as the vehicle and sampled the fish after 7, 13, and 18 days to assess the effects that E₂ had on spermatogenesis. It was observed that E₂ levels in plasma increased, while 11-ketotestosterone (11-KT) and testosterone (T) levels remained unaltered. However, 11-KT and T levels strongly increased in control fish 18 days postinjection. The most relevant result of our study was that E₂ accelerates the final events of spermatogenesis, inhibits the proliferation of spermatogonia in early stages, and induces some of the processes that usually occur during postspawning, such as the infiltration of acidophilic granulocytes and the apoptosis of primary spermatogonia. Strikingly, neither the shedding of spermatozoa nor an increase in the proliferative rate of spermatogonia stem cells was observed, probably because of the lack of other necessary stimuli, such as the increase in T levels that takes place during normal postspawning.

Theophylline (THP) and 1,3-dinitrobenzene (DNB) are thought to induce infertility by incapacitating the nurturing Sertoli cells and causing germ cell apoptosis in the testicular seminiferous epithelium, respectively. We hypothesized that THP and DNB exposure would alter the expression of the genes within the ubiquitin-proteasome pathway (UPP), implicated in spermatogenesis and epididymal sperm quality control. Rats were fed 0 or 8000 ppm of THP and necropsied on Days 18, 30, and 42 or administered 0, 2, or 6 mg/kg DNB via oral gavage and necropsied on Day 7. Tissues were collected from the testis and the caput, corpus, and cauda regions of the epididymis for transcriptional profiling by semiquantitative RT-PCR, real-time RT-PCR, and histopathology. Target UPP genes included those encoding for constitutive the 20S proteasomal core subunits Psmb1 (beta1), Psmb2 (beta2), and Psmb5 (beta5); the inducible 20S core subunits Psmb9 (LMP2), Psmb8 (LMP7), and Psmb10 (LMP10); and Ube1 (ubiquitin-activating enzyme E1), Ube2d3 (ubiquitin-conjugating enzyme E2), and Uchl1 (ubiquitin C-terminal hydrolase PGP9.5). Spermatozoa were collected from the cauda epididymis for analysis by light microscopy and flow cytometric evaluation of sperm surface ubiquitin. These data show that reprotoxic exposure alters the tissue-specific expression of UPP genes in the testis and epididymis, which may contribute to the aberrant spermatogenesis and epididymal processing of both normal and defective spermatozoa. Transcriptional profiling and flow cytometric analysis of the UPP thus captures the prodromal effects of reproductive toxicity not captured by conventional histology and functional cytology. Complementing seminal analysis with these measures may be useful in screening drug-induced toxicity or environmental infertility.

Chorionic villi in the human placenta serve as essential structures in fetomaternal exchanges. According to the embryology and placentology literature, during the first trimester, the cytotrophoblast (CTB) layer that is subjacent to the syncytiotrophoblast (STB) and supported by a basal lamina is nearly complete, but later, it becomes discontinuous. In the present study, we investigated the structural integrity of the CTB layer in the normal villous tree by advanced microscopy techniques using an antibody to hepatocyte growth factor (HGF) activator inhibitor type 1 (SPINT1), a potent inhibitor of HGF activators expressed exclusively on villous CTB. In full-term placenta, the cell surface of the CTB layer was spread over the basal lamina but was not interrupted. Morphometric analysis showed that throughout the villous tree, 80% of the continuity of the CTB layer of full-term placenta and 90% of that of first-trimester placenta were preserved. Gestation was accompanied by unique structural change in the basal domain of the trophoblast layer. The initially cuboidal-shaped CTB cells were transformed to flat cells with many cellular processes that, together with those of the adjacent STB, eventually covered the trophoblast basal lamina in a complex network of interdigitations. In addition, the expression levels of SPINT1, ST14, HGF, and MET mRNAs in the villous tree increased over the course of gestation. These results suggest that the structural integrity of the SPINT1-positive CTB layer may play an important role in villous differentiation and in maintenance of the villous tree via the HGF signaling system during gestation.

We recently succeeded in inducing germline transmission by transferring chicken testicular cells into heterologous testes. This study was designed subsequently to identify pluripotent cells in the testicular cells, which would induce the germline transmission. Testicular cells retrieved from juvenile (4-wk-old) or adult (24-wk-old) White Leghorn (WL) chickens were stained with germ cell-specific markers anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-EMA1, anti-ITGA6, and anti-ITGB1 antibodies; 2C9; and lectin-Solanum tuberosum agglutinin (STA). The percentages of the cells that were positive for each marker were within the ranges of 0.33%–0.44% and 0.029%–0.072% of the total testicular cell population in the juvenile and adult, respectively, and significant (P < 0.0002) differences were detected between the ages. When 1 × 10⁶ testicular cells were cultured in Dulbecco minimum essential medium-based medium supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (FGF2), and/or insulinlike growth factor 1 (IGF1), colony formation was detected only in LIF FGF2-containing or LIF FGF2 IGF1-containing medium during primary culture, and the supplementation of LIF FGF2 IGF1 was the most efficient for maintaining the colony-forming cells through subculture. The established cells retrieved at the end of the primary culture or the 20th subpassage were positive for chicken germ cell-specific periodic acid-Schiff (PAS), EMA1, 2C9, SSEA1, SSEA3, SSEA4, ITGA6, and ITGB1; and lectin-STA markers (evaluated after 11th subpassage). Double staining of lectin-STA with anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-ITGA6, and anti-ITGB1 also was possible. They differentiated spontaneously into embryoid bodies after being cultured in LIF-free medium. We conclude that germline stem cell-like cells are present in chicken testicular cells retrieved from both juvenile and adult testes, which can be identified with the specific markers for primordial germ cells or embryonic germ cells.