MicroRNAs are a class of noncoding small RNAs that regulate the expression of nearly 30% of all the human genes and participate in all fundamental cell processes. Genome-wide analysis has revealed that human placenta expresses more than 600 miRNA species, including placenta-specific ones with high levels of expression. Comparative analysis also has revealed many differentially expressed miRNAs with either high or low levels of expression in human placentas from normal versus preeclamptic pregnancies, indicating an important role of miRNAs in normal and pathological placental physiology. Although limited information is currently available as to how miRNA regulates human placental development and function, there are studies suggesting that preeclampsia-associated differentially expressed miRNAs possess critical roles in regulating placental development and function via targeting specific genes with diverse known functions. Herein we summarize the current findings regarding the expression of placental miRNAs and their function, especially in the trophoblast cells. We have recently found that the angiogenesis-associated miR-17-family miRNAs are upregulated in preeclamptic compared with normotensive placentas and they target the ephrin-B2/Eph receptor B4 (EPHB4) system. Because ephrin-B2 and EPHB4 has been previously shown to play a crucial role in trophoblast invasion into maternal spiral artery and vascular patterning during early human placental development, the miR-17-ephrin-B2/EPHB4 pathway seems to be a novel miRNA pathway for regulating normal and aberrant placental development during preeclampsia.

Recent evidence has linked human phthalate exposure to abnormal reproductive and hormonal effects. Phthalates are plasticizers that confer flexibility and transparency to plastics, but they readily contaminate the body and the environment. In this study, timed pregnant CD1 outbred mice were treated with di-(2-ethylhexyl) phthalate (DEHP) from Embryonic Day 7 (E7) to E14. The subsequent generation (F1) offspring were then bred to produce the F2, F3, and F4 offspring, without any further DEHP treatment. This exposure scheme disrupted testicular germ cell association and decreased sperm count and motility in F1 to F4 offspring. By spermatogonial transplantation techniques, the exposure scheme also disrupted spermatogonial stem cell (SSC) function of F3 offspring. The W/W^V recipient testes transplanted with F3 offspring germ cells from the DEHP-treated group had a dramatically lower percentage of donor germ cell-derived spermatogenic recovery in seminiferous tubules when compared to the recipient testes transplanted with CD1 control germ cells. Further characterization showed that the major block of donor germ cell-derived spermatogenesis was before the appearance of undifferentiated spermatogonia. Interestingly, the testes transplanted with the F3 offspring germ cells from the DEHP-treated group, when regenerated, replicated testis morphology similar to that observed in the testes from the F1 to F3 offspring of the DEHP-treated group, suggesting that the germ cell disorganization phenotype originates from the stem cells of F3 offspring. In conclusion, embryonic exposure to DEHP was found to disrupt testicular germ cell organization and SSC function in a transgenerational manner.

Considerable effort has been invested in searching for less invasive methods of diagnosing endometriosis. Previous studies have indicated altered levels of the CALD1 gene (encoding the protein caldesmon) in endometriosis. The aims of our study were to investigate whether average CALD1 expression and caldesmon protein levels are differentially altered in the endometrium and endometriotic lesions and to evaluate the performance of the CALD1 gene and caldesmon protein as potential biomarkers for endometriosis. Paired biopsies of endometrial tissue (eutopic endometrium) and endometriotic lesions (ectopic endometrium) were obtained from patients with endometriosis to evaluate CALD1 gene expression and caldesmon protein levels by real-time PCR and Western blot analysis, respectively. In addition, immunostaining for caldesmon to determine cellular localization was also performed. Endometrium from women without endometriosis was used as a control. Increased CALD1 expression and caldesmon levels were detected in the endometriotic lesions. The electrophoretic profile of caldesmon by Western blot analysis was clearly different between the control group (endometrium of women without endometriosis) and the group of women with endometriosis (eutopic endometrium and endometriotic lesions). Caldesmon expression as determined by immunostaining showed no variation among the cell types in endometriotic lesions and eutopic endometrium. Stromal cells marked positively in eutopic endometrium from control patients and in the endometriotic lesions. The presence of caldesmon in the endometrium of patients with and without endometriosis permitted diagnoses with 95% sensitivity (specificity 100%) and 100% sensitivity (specificity 100%) for the disease and for minimal to mild endometriosis in the proliferative phase of the menstrual cycle, respectively. In the secretory phase, minimal to mild endometriosis was detected with 90% sensitivity and 93.3% specificity. Caldesmon is a possible predictor of endometrial dysregulation in patients with endometriosis. A potential limitation of our study is the fact that other endometrial diseases were not excluded, and therefore prospective studies are needed to confirm the potential of caldesmon as a biomarker exclusively for endometriosis.

Bovine uterine infections are the most important cause of economic losses in the cattle industry. Although the etiology of uterine diseases is mainly ascribed to bacterial infection, they can also be associated with viral infection, such as bovine herpesvirus 4 (BoHV-4), which is often a secondary agent following bacteria. Besides microbial infection, many inflammatory molecules belonging to the innate immune response orchestrate the outcome of the infection. In the present study, the interaction between BoHV-4-infected bovine endometrial stromal cells and tumor necrosis factor alpha (TNF-alpha) was investigated. Bovine herpesvirus 4 possesses a special tropism toward endometrial stromal cells. For this reason, a simian virus 40 (SV40) immortalized endometrial stromal cell line (SV40BESC) was established; it was proven that it was stable, it expressed toll-like receptors (TLRs; from 1 to 10) and TNF-alpha receptors I and II, and it was responsive to exogenous TNF-alpha. Further, an increase of BoHV-4 replication and cytopathic effect was observed in BoHV-4-infected and TNF-alpha-treated SV40BESCs. This increase of viral replication was associated with BoHV-4 immediate early 2 (IE2) gene promoter trans-activation through the interaction of the nuclear factor KB (NFKB) with the putative NFKB-responsive elements found within BoHV-4 IE2 gene promoter, and this interaction was abolished when NFKB-responsive elements were deleted. These data shed light on two important and rather controversial issues: the role of TNF-alpha receptor, which is weakly expressed in the stromal layer of the bovine uterus, as well as the possible interactions between proinflammatory molecules, viral replication, and chronic uterine disease.

It is well accepted that oocyte meiotic resumption is mainly regulated by the maturation-promoting factor (MPF), which is composed of cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDC2). Maturation-promoting factor activity is regulated by the expression level of CCNB1, phosphorylation of CDC2, and their germinal vesicle (GV) localization. In addition to CCNB1, cyclin O (CCNO) is highly expressed in oocytes, but its biological functions are still not clear. By employing short interfering RNA microinjection of GV-stage oocytes, we found that Ccno knockdown inhibited CDC2 (Tyr15) dephosphorylation and arrested oocytes at the GV stage. To rescue meiotic resumption, cell division cycle 25 B kinase (Cdc25b) and Ccnb1 were overexpressed in the Ccno knockdown oocytes. Unexpectedly, we found that Ccno knockdown did not affect CDC25B entry into the GV, and overexpression of CDC25B was not able to rescue resumption of oocyte meiosis. However, GV breakdown (GVBD) was significantly increased after overexpression of Ccnb1 in Ccno knockdown oocytes, indicating that GVBD block caused by cyclin O knockdown can be rescued by cyclin B1 overexpression. We thus conclude that cyclin O, as an upstream regulator of MPF, plays an important role in oocyte meiotic resumption in mouse oocytes.

The objective of these experiments was to evaluate the importance of fatty acid beta-oxidation (FAO) in the cumulus oocyte complex (COC) during in vitro maturation (IVM) to oocyte nuclear maturation and gene expression in both the oocyte and cumulus cells in three species with differing amounts of oocyte intracellular lipids (mouse, low; bovine, moderate; porcine, high). We inhibited FAO using etomoxir at 0, 10, 25, 100, or 250 μM. Completion of oocyte nuclear maturation was inhibited after COC exposure to 250 μM etomoxir in mouse oocytes, 100 μM etomoxir in bovine oocytes, and as little as 10 μM etomoxir in porcine oocytes (P < 0.05). When FAO was inhibited in mouse and porcine COCs resulting in inhibition of meiosis, the abundance of FAO, glycolytic, and oxidative stress gene transcripts were decreased in oocytes and cumulus cells (P < 0.05), although to a much greater extent in the pig. In bovine oocytes and cumulus cells, FAO gene transcripts were increased and glycolytic gene expression altered following meiotic inhibition due to etomoxir. Etomoxir, at doses that did not inhibit nuclear maturation in bovine and murine COCs, increased glucose consumption (P < 0.05), suggesting glucose metabolism is increased to meet the metabolic demands of the COCs when fatty acid metabolism is compromised. Our data demonstrates that FAO is essential to oocyte nuclear maturation in all three species. Sensitivity of nuclear maturation to FAO inhibition reflects the amount of lipid present in the ooplasm and may suggest a relative reliance on this metabolic pathway.

Maternal diabetes has adverse effects not only on oocyte quality but also on embryo development. However, it is still unknown whether the DNA imprinting in oocytes is altered by diabetes. By using streptozotocin (STZ)-induced and nonobese diabetic (NOD) mouse models we investigated the effect of maternal diabetes on DNA methylation of imprinted genes in oocytes. Mice which were judged as being diabetic 4 days after STZ injection were used for experiments. In superovulated oocytes of diabetic mice, the methylation pattern of Peg3 differential methylation regions (DMR) was affected in a time-dependent manner, and evident demethylation was observed on Day 35 after STZ injection. The expression level of DNA methyltransferases (DNMTs) was also decreased in a time-dependent manner in diabetic oocytes. However, the methylation patterns of H19 and Snrpn DMRs were not significantly altered by maternal diabetes, although there were some changes in Snrpn. In NOD mice, the methylation pattern of Peg3 was similar to that of STZ-induced mice. Embryo development was adversely affected by maternal diabetes; however, no evident imprinting abnormality was observed in oocytes from female offspring derived from a diabetic mother. These results indicate that maternal diabetes has adverse effects on DNA methylation of maternally imprinted gene Peg3 in oocytes of a diabetic female in a time-dependent manner, but methylation in offspring's oocytes is normal.

In order to understand the mechanisms of mammalian fertilization, studies using genetically manipulated animals have provided us with plenty of interesting and valuable information on the genetic factors affecting male fertility. In the present work, we demonstrate for the first time that Prss37, a previously uncharacterized putative trypsin-like serine protease, is required for male fertility. Prss37 is highly and exclusively expressed in the testis of adult mice, especially in the elongating spermatids during spermiogenesis, and almost vanishes in the mature sperm of mice. Mice deficient for Prss37 show male infertility, but their mating activity, spermatogenesis, sperm morphology, and motility remain unaffected. In vivo fertilization assays revealed that Prss37^−/− mice exhibited a markedly decreased fertilization rate (2.3% vs. 70% of that in control mice) accompanied by the defect in sperm migration from uterus into oviduct. In vitro study further showed sperm were incapable of sperm-egg recognition/binding when zona-intact eggs were exposed to Prss37^−/− sperm, in which mature Adam3 was completely undetectable. Interestingly, however, Prss37^−/− sperm were able to fertilize cumulus-intact oocytes in vitro. These data clearly indicate that Prss37 deficiency causes the absence of mature Adam3 in sperm and a defect in sperm migration from uterus into oviduct, which mainly accounts for male infertility of Prss37-null mice, while the defect in sperm-zona binding seems irrelevant to the fertilizing ability of Prss37^−/− sperm.

DNA methylation is a central epigenetic event that regulates cellular differentiation, reprogramming, and pathogenesis. Genomewide DNA demethylation occurs in preimplantation embryos and in embryonic germ cell precursors called primordial germ cells (PGCs). We previously showed that Dppa3, also known as Stella and PGC7, protects the maternal genome from tet methylcytosine dioxygenase 3 (Tet3)-mediated conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in zygotes. Here, we demonstrated that retrotransposon genes, such as long interspersed nuclear element-1 (Line-1) and intracisternal A particle (IAP), showed higher 5mC levels in Dppa3-null PGCs. In contrast, oxidative bisulfite sequence analysis revealed that the amounts of 5hmC in Line-1 and IAP were slightly reduced in the Dppa3-deficient PGCs. From our findings, we propose that Dppa3 is involved in the Tet-mediated active demethylation process during reprogramming of PGCs.

The gonadotropin surge is the essential trigger to stimulate ovulation and luteinization of ovarian follicles. While the hormone signals from the brain that initiate ovulation are known, the specific targets which regulate this process are not well known. In this study, we assessed the suitability of the Rhox homeobox gene cluster to serve as the master regulators of folliculogenesis. In superovulated (equine chorionic gonadotropin [eCG]/human chorionic gonadotropin [hCG]) mice, the Rhox genes exhibited four distinct windows of peak expression, suggesting that these genes may regulate specific events during the ovulatory cycle. Like many members of the cluster, Rhox8 mRNA and protein were induced by follicle stimulating hormone [FSH]/eCG in granulosa cells. However, Rhox8 displayed unique peak expression at 8 h post-hCG administration, implying it might be the lone member of the cluster regulated by progesterone. Subsequent promoter analysis in granulosa cells revealed relevant homeobox binding and progesterone response elements within Rhox8's 5′-flanking region. In superovulated mice, progesterone receptor (PGR) is recruited to the Rhox8 promoter, as assessed by chromatin immunoprecipitation. In Rhox5-null mice, Rhox8 mRNA was reduced at 2 h and 4 h post-hCG administration but recovered once the follicles passed the antral stage of development. Conversely, in progesterone receptor knockout mice, Rhox8 exhibited normal stimulation by eCG but failed to reach its peak mRNA level at 8 h post-hCG found in wild-type mice. This suggests a model in which Rhox8 transcription is dependent upon RHOX5 during early folliculogenesis and upon progesterone during the periovulatory window when RHOX5 normally wanes. In support of this model, transfection of RHOX5 and PGR expression plasmids stimulated, whereas dominant negative and mutant constructs inhibited, Rhox8 promoter activity.

Oogenesis is a complex process requiring the coordinated sequential expression of specific genes and ultimately leading to the release of the female gamete from the ovary. In the present study we aimed to investigate the contribution of miRNAs to the regulation of this key biological process in teleosts using a model in which growing oocytes develop simultaneously. Taking advantage of the strong sequence conservation of miRNAs among phylogenetically distant species, we designed a generic microarray displaying most known chordate miRNAs. It allowed us to provide an overview of the ovarian miRNome during oogenesis for the first time in any vertebrate species. We identified 13 differentially expressed miRNAs, and a differential expression of at least one miRNA was observed at each step of oogenesis. A surprisingly high differential expression of several miRNAs was observed at several stages of oogenesis and subsequently confirmed using quantitative PCR. By refining in silico prediction of target genes with gene expression data obtained within the same sample set, we provide strong evidence that miRNAs target major players of oogenesis, including genes involved in rate-limiting steps of steroidogenesis and those involved in gonadotropic control of oocyte development, as well as genes involved in ovulation, oocyte hydration, and acquisition of the ability of the oocyte to support further development once fertilized (i.e., oocyte developmental competence). Together, these observations stress the importance of miRNAs in the regulation and success of female gamete formation during oogenesis.

During the peri-implantation and early placentation periods in pigs, conceptuses (embryo and its extra-embryonic membranes) undergo dramatic morphological changes and differentiation that require the exchange of nutrients (histotroph) and gasses across the trophectoderm and a true epitheliochorial placenta. Of these nutrients, arginine (Arg), leucine (Leu), and glutamine (Gln) are essential components of histotroph; however, little is known about changes in their total amounts in the uterine lumen of cyclic and pregnant gilts and their effects on cell signaling cascades. Therefore, we determined quantities of Arg, Leu, and Gln in uterine luminal fluids and found that total recoverable amounts of these amino acids increased in pregnant but not cyclic gilts between Days 12 and 15 after onset of estrus. We hypothesized that Arg, Leu, and Gln have differential effects on hypertrophy, hyperplasia, and differentiated functions of trophectoderm cells that are critical to conceptus development. Primary porcine trophectoderm (pTr) cells treated with either Arg, Leu, or Gln had increased abundance of phosphorylated RPS6K, RPS6, and EIF4EBP1 compared to basal levels, and this effect was maintained for up to 120 min. When pTr cells were treated with Arg, Leu, and Gln, low levels of pRPS6K and pEIF4EBP1 were detected in the cytosol, but the abundance of nuclear pRPS6K increased. Immunofluorescence analyses revealed abundant amounts of pRPS6 protein in the cytoplasm of pTr cells treated with Arg, Leu, and Gln. These amino acids also increased proliferation of pTr cells. Furthermore, when Arg, Leu, and Gln were combined with siRNAs for either MTOR, RPTOR, or RICTOR, effects of those amino acids on proliferation of pTr cells were significantly inhibited. Collectively, these results indicate that Arg, Leu, and Gln act coordinately to stimulate proliferation of pTr cells through activation of the MTOR-RPS6K-RPS6-EIF4EBP1 signal transduction pathway.

Endothelial cells chronically reside in low-O₂ environments in vivo (2%–13% O₂), which are believed to be critical for cell homeostasis. To elucidate the roles of this physiological chronic normoxia in human endothelial cells, we examined transcriptomes of human umbilical vein endothelial cells (HUVECs), proliferation and migration of HUVECs in response to fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA), and underlying signaling mechanisms under physiological chronic normoxia. Immediately after isolation, HUVECs were cultured steadily under standard cell culture normoxia (SCN; 21% O₂) or physiological chronic normoxia (PCN; 3% O₂) up to 25 days. We found that PCN up-regulated 41 genes and down-regulated 21 genes, 90% of which differed from those previously reported from HUVECs cultured under SCN and exposed to acute low O₂. Gene ontology analysis indicated that PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from benchtop assays that showed that PCN significantly enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. Interestingly, preexposing the PCN cells to 21% O₂ up to 5 days did not completely diminish PCN-enhanced cell proliferation and migration. These PCN-enhanced cell proliferations and migrations were mediated via augmented activation of MEK1/MEK2/ERK1/ERK2 and/or PI3K/AKT1. Importantly, these PCN-enhanced cellular responses were associated with an increase in activation of VEGFR2 but not FGFR1, without altering their expression. Thus, PCN programs endothelial cells to undergo dramatic changes in transcriptomes and sensitizes cellular proliferative and migratory responses to FGF2 and VEGFA. These PCN cells may offer a unique endothelial model, more closely mimicking the in vivo states.

Increased litter size and within-litter uniformity in birth weight would improve pig reproductive efficiency. This study compared the location and gene and protein expression of secreted phosphoprotein 1 in placental and uterine tissues supplying a normally sized and the smallest fetus carried by hyperprolific Large White and Meishan gilts on Days 41–42 of pregnancy. Immunohistochemistry and in situ hybridization showed that the protein and gene encoding secreted phosphoprotein 1 were located in the glandular and luminal epithelium of the endometrium and in the placenta. Secreted phosphoprotein 1 protein levels were higher in glandular epithelium, luminal epithelium, and placenta from Meishan gilts compared to corresponding tissues from hyperprolific Large White gilts. Reverse transcription quantitative PCR demonstrated secreted phosphoprotein 1 mRNA levels were higher in endometrium, but not placenta, from Meishan compared to hyperprolific Large White gilts. In hyperprolific Large White gilts, secreted phosphoprotein 1 protein levels were higher in glandular epithelium and placenta surrounding small fetuses than corresponding tissues supplying normal-sized fetuses. Similarly, in Meishan gilts, secreted phosphoprotein 1 protein levels were higher in luminal epithelium surrounding small compared to normal-sized fetuses. Within hyperprolific Large White, but not Meishan, gilts secreted phosphoprotein 1 mRNA was higher in endometrium surrounding the normal-sized fetus than the control fetus. The contradictory relationship between fetal size and secreted phosphoprotein 1 protein and mRNA in the hyperprolific Large White is intriguing and may reflect breed differences in posttranslational modification. The striking breed differences in secreted phospoprotein 1 expression suggest that SPP1 may be associated with placental efficiency.

Pregnancy is a complex process that can be jeopardized when associated with cancer, because of the coexistence of two complex metabolic conditions: a fetus and cancer. The aim of this study was to evaluate fetal growth in association with cancer development as well as the indirect effects produced by tumors in pregnant mice subjected to a leucine-rich diet, knowing that leucine supplementation can minimize the tumor effects by acting as a cell signaling agent to improve the protein synthesis process. We evaluated fetuses (n = 6) from NMRI pregnant mice fed either a control or a leucine-rich diet in either the presence or absence of an MAC16 colon adenocarcinoma or ascitic fluid inoculation. The fetal serum amino acids were separated using high-performance liquid chromatography, and fetal cytokine levels were analyzed using a microsphere-based multiplex immunoassay (Luminex xMAP). Fetal body composition was measured as the water, fat, and protein total content and total serum protein, albumin, and glucose content. Tumor growth resulted in a severe reduction in fetal body weight and protein content and increased fetal resorption, associated with placental weight decrease; these effects were minimized by a leucine-rich diet. Serum total protein and glucose content were reduced in fetuses from tumor-bearing dams but were reverted by nutritional supplementation. The serum amino acid profiles differed significantly between the tumor-bearing mice fed with a leucine-rich diet and controls. Certain tumor effects were reproduced in fetuses from ascitic fluid-injected dams, suggesting indirect effects of tumor growth. We conclude that certain effects of tumor growth can be mimicked by ascitic fluid injection and can be modulated by a leucine-rich diet.

Among primates, the common marmoset is suitable for primate embryology research. Its small body size, however, has delayed the technical development of efficient embryo transfer. Furthermore, three factors have been determined to adversely affect the performance of marmoset embryo transfer: nonsurgical approaches, the use of cryopreserved embryos, and the use of late-stage embryos. Here we performed embryo transfer under conditions that included the above three factors and using either a small (1 μl or less) or a large volume (2–3 μl) of medium. The pregnancy and birth rates were 50% (5/10) and 27% (3/11), respectively, when using the large volume, and 80% (8/10) and 75% (9/12), respectively, when using the small volume. The latter scores exceed those of previous reports using comparable conditions. Thus, it appears that these three previously considered factors could be overcome, and we propose that reducing the transfer volume to 1 μl or less is essential for successful marmoset embryo transfer.

The aim of this study was to compare the proteome profiles of the chorioamnion and corresponding caruncle for buffalo embryos that had either normal or retarded development on Day 25 after artificial insemination (AI). In experiment 1, embryos that were to subsequently undergo late embryonic mortality had a smaller width on Day 25 after AI than embryos associated with pregnancy on Day 45 after AI. In experiment 2, 25 Italian Mediterranean buffaloes underwent transrectal ultrasonography on Day 25 after AI, and pregnant animals were categorized as one of two groups based on embryonic width: normal embryos (embryonic width > 2.7 mm) and retarded embryos (embryonic width < 2.7 mm). Three buffaloes of each group were slaughtered on Day 27 after AI to collect chorioamnion and caruncle tissues for subsequent proteomic analyses. Two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrometer analysis were used to ascertain the proteomic profiles. To confirm 2D-DIGE-results, three selected proteins were analyzed by Western blot. The proteomic profiles of the chorioamnion of retarded embryos and the corresponding caruncles showed differences in the expression of several proteins compared to normal embryos. In particular, a down-regulation was observed for proteins involved in protein folding (HSP 90-alpha, calreticulin), calcium binding (annexin A1, annexin A2), and coagulation (fibrinogen alpha-chain) (P < 0.05), whereas proteins involved in protease inhibition (alpha-1-antiproteinase, serpin H1, serpin A3-8), DNA and RNA binding (heterogeneous nuclear ribonucleoproteins A2/B1 and K), chromosome segregation (serine/threonine-protein phosphatase 2A), cytoskeletal organization (ezrin), cell redox homeostasis (amine oxidase-A), and hemoglobin binding (haptoglobin) were up-regulated (P < 0.05).

As advanced reproductive technologies have become routine for domesticated species, they have begun to be applied in the field of endangered species conservation. For avian conservation, the most promising technology is the transfer of germ stem cells of exotic species to domestic hosts for the production of gametes. In this study, adult quail (model for exotic species) spermatogonial stem cells were xenogeneically transferred to stages 14–17 chicken host embryos. Fluorescent cellular dyes, quail-specific antibodies, and quail-specific quantitative PCR confirmed donor cell migration to and colonization of the host gonadal ridge. Donor-derived cells were observed by fluorescent microscopy in the caudal area as early as 2 h after injection, in the gonadal ridge at 4 h after injection, as well as in the gonads of stages 35–38 host embryos. Four of eight donor-derived cell flow cytometry-positive host gonads were confirmed by quantitative PCR using quail-specific primers. There was no statistically significant effect of host stage of injection, host gonad isolation stage, or host sex on the number of hosts positive for donor cells or the percent of donor-derived cells per positive gonad. Donor-derived cells isolated from stages 35–38 host gonads costained with the germ stem cell marker SSEA-1, indicating that the donor-derived cells have maintained stem cell-ness. This is the first study to suggest that it is feasible to rescue adult germ stem cells of deceased birds to prolong the reproductive lifespan of critically endangered species or genetically valuable individuals by transferring them to an embryonic chicken host.

Spermatogenesis is a complex process that generates spermatozoa; its molecular mechanisms are not completely understood. Here we focused on the functions of three testis-specific serine proteases: Prss42/Tessp-2, Prss43/Tessp-3, and Prss44/Tessp-4. These protease genes, which constitute a gene cluster on chromosome 9F2-F3, were presumed to be paralogs and were expressed only in the testis. By investigating their mRNA distribution, we found that all three genes were expressed in primary and secondary spermatocytes. However, interestingly, the translated proteins were produced at different locations. Prss42/Tessp-2 was found in the membranes and cytoplasm of secondary spermatocytes and spermatids, whereas Prss43/Tessp-3 was present only in the membranes of spermatocytes and spermatids. Prss44/Tessp-4 was detected in the cytoplasm of spermatocytes and spermatids. To assess the roles of these proteases in spermatogenesis, we used organ culture of mouse testis fragments. Adding antibodies against Prss42/Tessp-2 and Prss43/Tessp-3 resulted in meiotic arrest at the stage when each protease was beginning to be translated. Furthermore, the number of apoptotic cells dramatically increased after the addition of these antibodies. These results strongly suggest that the three paralogous Prss/Tessp proteases play different roles in spermatogenesis and that Prss42/Tessp-2 and Prss43/Tessp-3 are required for germ cell survival during meiosis.

Cryopreservation of testicular tissue can be used for ex situ conservation of male germplasm of avian species. The possibility of using vitrification and transplantation of testicular tissue for fertility preservation and recovery was tested in Japanese quail. Testes were removed from 1-wk-old Japanese quail; transfixed on acupuncture needles; equilibrated with dimethyl sulphoxide, ethylene glycol, and sucrose; plunged into liquid nitrogen; and stored in 2-ml straws. Cryopreserved tissue was warmed in sucrose solution at room temperature or at 40°C. Fresh and cryopreserved tissue were transplanted subcutaneously into castrated, 1-wk-old recipients. Twenty of 21 recipients survived the surgery, and 18 had viable transplants at maturity, with no difference in transplantation success between fresh and cryopreserved tissue. Fluid extrusion from 11 of the transplants was collected and inseminated surgically into the magnum of 22 quail hens, and 10 inseminations included foam from the proctodeal gland of the same recipients. Egg production in the 2 wk after insemination was reduced, and none of the hens inseminated with foam produced fertile eggs. Five hens inseminated without foam produced a total of eight live offspring; four of these hens had been inseminated with fluid extrusion from cryopreserved tissue. Histological examination showed spermatogenesis in the transplants, and the tubules, lumens, and epithelium of the seminiferous tubules were of comparable size to those of testicular tissue from intact males. These results demonstrate that testicular tissue of Japanese quail can be preserved using vitrification procedures and recovered through transplantation.

A computer program has been developed that simulates the behavior of spermatogonial stem cells (SSCs) and their offspring inside and outside of the stem cell niche. Various parameters derived from previous morphological and cell kinetic studies have been used to set up an Excel-based computer program that simulates the proliferative activity of SSCs during the seminiferous epithelial cycle. SSCs and their offspring are depicted in a virtual piece of seminiferous tubule in which the daughter cells of self-renewing divisions of SSCs migrate away from each other, while after SSC differentiation a pair of cells is formed. Those SSC daughter cells that migrate out of the niche will very likely differentiate at their next division. Putting in physiologically acceptable parameters, the program renders numbers of spermatogonial cell types similar to those previously counted in whole mounts of seminiferous tubules. In this model, SSC numbers and numbers of differentiating cells remain constant for more than 50 virtual epithelial cycles, i.e., more than 1 yr of a mouse life and 2 yr of that of a Chinese hamster. The program can simulate various recent cell kinetic experiments and confirms, or offers alternative explanations for, the results obtained, showing its usefulness in spermatogenesis research.

Sertoli cells provide nutritional and physical support to germ cells during spermatogenesis. Sox8 encodes a member of the high mobility group of transcription factors closely related to Sox9 and Sox10. Sertoli cells express SOX8 protein, and its elimination results in an age-dependent dysregulation of spermatogenesis, causing adult male infertility. Among the claudin genes with altered expression in the Sox8^−/− testes, was claudin-3, which is required for the regulation and maintenance of the blood-testes barrier (BTB). Because the BTB is critical in restricting small molecules in the luminal compartment of the seminiferous tubules, the aim of this study was to analyze the level of tight junction proteins (claudin-3, claudin-11, and occludin) and BTB permeability in Sox8^−/− adult testes. The acetylation level of alpha-tubulin and microtubule organization was also evaluated because microtubules are critical in maintaining the microenvironment of the seminiferous epithelium. Western blot analysis shows that claudin-3 protein is decreased in Sox8^−/− testes. Chromatin immunoprecipitation confirmed that SOX8 binds at the promoter region of claudin-3. Claudin-3 was localized to the Sertoli cell tight junctions of wild-type testes and significantly decreased in the Sox8^−/− testes. The use of biotin tracers showed increased BTB permeability in the Sox8^−/− adult testes. Electron microscopy analysis showed that microtubule structures were destabilized in the Sox8^−/− testes. These results suggest that Sox8 is essential in Sertoli cells for germ cell differentiation, partly by controlling the microenvironment of the seminiferous epithelium.

Perinatal estrogen exposure elicits a wide range of abnormalities in the female genital tract. Since angiogenesis is essential for morphogenesis, we investigated the vascular density, integrity of vasculatures, and expression of angiogenic factors and their receptors in the uteri of mice treated with diethylstilbestrol (DES) neonatally (DES-mice); the uteri were collected from Day 4 to Day 20. DES treatment reduced the number and density of vasculatures immunostained with PECAM1 (platelet and endothelial cell adhesion molecule 1) in the stroma. Horseradish peroxidase injected into the left ventricle leaked into the endometrium and myometrium on Day 10 in the DES-mice but not in the controls. Electron microscopy confirmed the immaturity of the capillaries, which had an incomplete basal lamina and fewer pericytes. Immunohistochemical studies demonstrated that VEGFA (vascular endothelial growth factor A) expression and ANGPT1 (angiopoietin 1) expression were down-regulated in the stromal cells until Days 20 and 10, respectively. The number of vasculatures with ANGPT2 immunoreaction was reduced in the DES-mice. DES treatment suppressed the expression of VEGFR2 (VEGF receptor 2) and the co-receptor NRP1 (neuropilin 1) as well as TEI2 in the vasculatures. The results of RT-PCR and Western blotting supported the down-regulation of the expression of angiogenic factors and their receptors in DES-mice, whereas the VEGFR1 protein expression was up-regulated. These results suggested that the low concentration of angiogenic factors in the stroma was primarily responsible for the low vascular density in the stroma of the DES-mice, and that the low vascular density and immature vasculatures resulted in uterine malformations.