Expression of pluripotency-related genes during bovine inner cell mass explant culture.

Recent findings identifying the transcription factors involved in the regulation of pluripotency and self-renewal in embryonic stem cells (ESC) may provide keys that enable the derivation of ESC in domestic species. In this study we monitored the expression of pluripotency-related genes in bovine inner cell mass (ICM) explants during the critical first steps in establishment of primary cultures. The expression of NANOG and POU5F1 transcripts and proteins in intact, in vitro produced (IVP) blastocysts was confirmed by quantitative RT-PCR and fluorescent immunocytochemistry. NANOG was localized to the nucleoplasm as well as the nucleoli in the ICM, whereas it appeared to be restricted to the nucleoli in trophectoderm cells. POU5F1 was localized in the nuclei of ICM and trophectoderm cells. ICM explants were analyzed by quantitative PCR and semiquantitative RT-PCR. The three major pluripotency-related transcription factors, NANOG, POU5F1, and SOX2, were expressed initially in the ICM explants, but were downregulated with subsequent culture. Markers of differentiation (BMP4, HNF4, NCAM, CDX2) and genes involved in LIF, BMP, and WNT signaling pathways were also expressed. ICM explants were cultured in the presence of various concentrations of cytokines belonging to the TGF-beta superfamily. Noggin, a cytokine inhibiting the BMP4 pathway, successfully upregulated the relative expression of NANOG mRNA in the ICM explants with respect to controls.

Small RNA molecules in the regulation of spermatogenesis.

Small RNA molecules (small RNAs), including small interfering RNAs (siRNAs), microRNAs (miRNAs), and piwi-interacting RNAs (piRNAs), have recently emerged as important regulators of gene expression at the post-transcriptional or translation level. Significant progress has recently been made utilizing small RNAs in elucidating the molecular mechanisms regulating spermatogenesis. Spermatogenesis is a complex process that involves the division and eventual differentiation of spermatogonial stem cells into mature spermatozoa. The process of spermatogenesis is composed of several phases: mitotic proliferation of spermatogonia to produce spermatocytes; two meiotic divisions of spermatocytes to generate haploid round spermatids; and spermiogenesis, the final phase that involves the maturation of early-round spermatids into elongated mature spermatids. A number of miRNAs are expressed abundantly in male germ cells throughout spermatogenesis, while piRNAs are only present in pachytene spermatocytes and round spermatids. In this review, we first address the synthesis, mechanisms of action, and functions of siRNA, miRNA, and piRNA, and then we focus on the recent advancements in defining the small RNAs in the regulation of spermatogenesis. Concerns pertaining to the use of siRNAs in exploring spermatogenesis mechanisms and open questions in miRNAs and piRNAs in this field are highlighted. The potential applications of small RNAs to male contraception and treatment for male infertility and testicular cancer are also discussed.

Pluripotent stem cells derived from adult human testes.

Recent reports have demonstrated that adult tissue cells can be induced to pluripotency, the iPS cells, mostly with the addition of genes delivered using viruses. Also, several publications both in mouse and in human have demonstrated that spermatogonial stem cells (SSCs) from testes can convert back to embryonic stem (ES)-like cells without the addition of genes. Furthermore, these pluripotent ES-like cells can differentiate into all three germ layers and organ lineages. Thus, SSCs have great potential for cell-based, autologous organ regeneration therapy for various diseases. We obtained testes from organ donors and using 1 g pieces of tissue (biopsy size) we demonstrate that testis germ cells (putative SSCs and/or their progenitors) reprogram to pluripotency when removed from their stem cell niche and when appropriate growth factors and reagents in embryonic stem cell medium are added. In addition, our method of obtaining pluripotent ES-like cells from germ cells is simpler than the described methods and may be more suitable if this procedure is developed for the clinic to obtain pluripotent cells to cure disease.

Placental vascularity and growth factor expression in singleton, twin, and triplet pregnancies in the sheep.

For singleton, twin, and triplet pregnancies, uteri were collected on day 140 of pregnancy. For each ewe (n = 18), placentomes were fixed by arterial perfusion supplying the fetal (cotyledon) and maternal placenta (caruncle). Tissue sections were stained for determination of vascularity by image analysis. Further, protein expression for factor VIII, vascular endothelial growth factor (VEGF) and its receptor, VEGFR1, as well as basic fibroblast growth factor (FGF2) and its receptor, FGFR, in tissue sections was determined by immunohistochemistry and image analyses. Cotyledonary and caruncular samples were analyzed for expression of mRNA for Vegf and its two receptors, Vegfr1 and Vegfr2, as well as Fgf2 and Fgfr. Fetal number did not affect placental capillary density or factor VIII expression, whereas increased fetal number reduced total cotyledon and caruncle capillary volume. While expression of Vegf, Vegfr1, Vegfr2, and Fgfr mRNA in cotyledonary but not caruncular tissue was greater in twin pregnancies compared to singleton and triplet pregnancies, protein expression of VEGF in the placentome decreased with increasing numbers of fetuses, VEGFR1 did not change, and FGFR was greater in twin versus singleton and triplet pregnancies. Fetal number did not affect the expression of Fgf2 mRNA in placental tissues, whereas FGF2 protein expression was less in triplet compared to singleton and twin pregnancies. Reduced fetal and placental weights in twins and/or triplet pregnancies are associated with an overall decrease in total placental vascularity, VEGF and FGF2 and/or FGFR protein expression, but not in angiogenic factor mRNA expression or VEGFR1 protein expression in sheep.

Lymphoid enhancer factor 1-mediated Wnt signaling promotes the initiation of trophoblast lineage differentiation in mouse embryonic stem cells.

Embryonic stem (ES) cells can differentiate into all three embryonic germ layers but rarely into trophectoderm (TE) lineages that contribute to the placenta, although TE differentiation can be initiated by genetic manipulation of key genes involved in TE development. We demonstrate that Wnt signaling can initiate TE lineage differentiation by triggering an appropriate cue, caudal-related homeobox 2 (Cdx2). Overexpression and RNA interference knockdown studies indicate that Cdx2 induction in response to Wnt3a is mediated by lymphoid enhancer factor 1, whose expression is regulated by leukemia inhibitory factor (LIF) and bone morphogenetic protein. Removal of LIF, along with addition of Wnt3a, stimulated Cdx2 expression and induced formation of trophoblast stem (TS) cells. These TS cells were able to differentiate into cells with characteristics of spongiotrophoblast and trophoblast giant cells. This is, to our knowledge, the first evidence that TE lineage differentiation can be induced by Wnt signaling in mouse ES cells.

Developmental expression of pluripotency determining factors in caprine embryos: novel pattern of NANOG protein localization in the nucleolus.

Transcription factors, POU5F1/OCT4 and NANOG, whose expression is restricted to the inner cell mass (ICM) in mouse and human blastocysts, are used to characterize undifferentiated embryonic stem cells (ESC) in vitro. However, POU5F1 may not be a useful marker in domestic animals due to its expression in both ICM and trophectoderm (TE), while NANOG mRNA and protein expression have only been described fully in mice. In an effort to identify ESC markers for domestic animals, expression patterns of NANOG, POU5F1, and the cell surface markers (SSEA1, SSEA4, TRA-1-60, TRA-1-81) were examined in preimplantation goat embryos, a species that has proven to be a superior choice for the production of transgenic proteins in milk (biopharming). Our results indicate that while goat embryos express POU5F1, SSEA1, and SSEA4 proteins, their expression is not strictly restricted to the ICM. In a unique staining pattern, NANOG protein was localized to the nucleoplasm and nucleoli in ICM cells, but was localized strictly to nucleoli in TE. This pattern may reflect down-regulation of protein by sequestration/degradation utilizing a nucleolar mechanism known to operate in stem cells. Furthermore, NANOG mRNA in TE was also significantly down-regulated as compared with that in ICM. Taken together, this novel expression pattern of NANOG in goat preimplantation embryos suggests that NANOG could serve as marker of pluripotency in goats and may be useful in derivation and characterization of caprine ESC. This study is the first to characterize both NANOG mRNA and protein expression in any species other than the mouse.

Pregnancy rates and gravid uterine parameters in single, twin and triplet pregnancies in naturally bred ewes and ewes after transfer of in vitro produced embryos.

The objectives of this study were to: (1) evaluate the pregnancy rates after transfer of embryos produced in the presence or absence of epidermal growth factor (EGF) during in vitro maturation, and (2) compare several variables of the gravid uterus on day 140 after fertilization in single, twin and triplet pregnancies in ewes (n = 12) bred naturally and in ewes (n = 18) after transfer of embryos produced in vitro. Oocytes collected from FSH-treated ewes (n = 18) were collected from all visible follicles and cultured in maturation medium with or without EGF. Oocytes were then fertilized in vitro by frozen-thawed semen. On day 5 after fertilization, embryos with > or = 16 cells were transferred to recipient ewes (n = 39). In addition 12 ewes were bred naturally. Pregnancy was verified by real-time ultrasonography on day 45 or later after embryo transfer (ET) or breeding. On day 140 of pregnancy, the reproductive tract was collected from all ewes and the following parameters were determined: the number, sex, weight and crown to rump length (CRL) of fetuses, weights of gravid uterus and fetal membranes, and weight and number of placentomes. Presence of EGF in maturation medium increased (P < 0.04) cleavage rates (78% versus 59%) and percentage of > or = 16 cell embryos on day 5 after fertilization (62% versus 40%). Pregnancy rates tended to be greater (P < 0.1) after transfer of embryos matured in the presence of EGF (52%) than in the absence of EGF (39%). EGF presence in maturation medium did not affect any variables of gravid uterus or fetal weight. For single pregnancies in naturally bred ewes and ewes after ET all uterine variables were similar. For twin pregnancies, weight of gravid uterus, weight of uterus plus fetal membranes, total weight of placentomes/ewe, mean weight of individual placentome, mean weight of fetus, total fetal weight/ewe and CRL were greater (P < 0.0001-0.04) for ewes after ET than for ewes bred naturally. The weights of gravid uterus, fluid, uterus plus fetal membranes, fetal membranes, total placentomes/ewe, mean weight of individual placentome and total fetal weight/ewe were greater (P < 0.0001-0.08) for triplet pregnancies in ewes after ET than single and twin pregnancies in ewes naturally bred or after ET. The number of placentomes/fetus was greatest (P < 0.0001-0.06) in single pregnancies in ewes bred naturally and after ET fewer in twin pregnancies in ewes bred naturally and after ET and fewest in triplet pregnancies in ewes after ET. The total number of placentomes/ewe was greatest (P < 0.0001-0.06) for twin pregnancies in ewes naturally bred, fewer in single pregnancies in ewes naturally bred and twin and triplet pregnancies after ET, and fewest in single pregnancies in ewes after ET. The mean weight of fetus was greater (P < 0.0001-0.07) in single pregnancies in ewes naturally bred or after ET than in twin or triplet pregnancies in ewes naturally bred or after ET. The CRL was the lowest (P < 0.01) in twin pregnancies in ewes bred naturally. For pregnancies after natural breeding and after ET, the number of fetuses/ewe was negatively correlated (P < 0.03-0.0001) with the weight of placentomes/fetus, the number of placentomes/fetus, the mean weight of the fetus and CRL, and was positively correlated (P < 0.0001-0.05) with weight of gravid uterus, the total number of placentomes/ewe and total fetal weight/ewe. These data demonstrate that the presence of EGF in maturation medium increases the rates of cleavage and early embryonic development, and has a tendency to enhance rates of pregnancy but does not affect variables of the gravid uteri in ewes after transfer of in vitro produced embryos. Transfer of embryos produced in vitro affected some uterine variables in twin but not single pregnancies to compare with pregnancies after natural breeding. In addition, culture conditions in the present experiment did not create large offspring syndrome. The low number of placentomes/fetus seen in triple pregnancies appears to be compensated for by the increase in the weight of each individual placentome.

Ovarian follicular development and oocyte quality in anestrous ewes treated with melatonin, a controlled internal drug release (CIDR) device and follicle stimulating hormone.

The objective of the current study was to determine the effects of hormonal treatments on ovarian follicular development and oocyte quality in anestrous ewes. Multiparous crossbred (RambouilletxTarghee) ewes were given melatonin implants (MEL) and/or controlled internal drug release (CIDR) devices in conjunction with follicle stimulating hormone (FSH) during anestrus (March-May). In Experiment 1, ewes (n=25) were assigned randomly to four groups (n=4-7/group) in a 2x2 factorial arrangement [+/-MEL and +/-CIDR], resulting in Control (no treatment), CIDR, MEL, and MEL/CIDR groups, respectively. Ewes received an implant containing 18 mg of melatonin (Melovine) on Day 42 and/or a CIDR from Days 7 to 2 (Day 0: oocyte collection). In Experiment 2, ewes (n=12) were assigned randomly to two groups (n=6/group; 1CIDR or 2CIDR) and received the same type of melatonin implant on Day 60. All ewes received a CIDR device from Days -22 to -17 and 2CIDR ewes received an additional CIDR device from Days -10 to -2. In both experiments, ewes were given FSH im twice daily (morning and evening) on Days -2 and -1 (Day -2: 5 units/injection; Day -1: 4 units/injection). On the morning of Day 0, ovaries were removed, follicles>or=1 mm were counted, and oocytes were collected. Thereafter oocytes were matured and fertilized in vitro. In Experiment 1, the number of visible follicles and the rates of oocyte recovery and in vitro maturation were similar (P>0.10) for Control, CIDR, MEL and MEL/CIDR (overall 29.7+/-2.9%, 89.9+/-7.1% and 95.0+/-2.0%, respectively). The rates of in vitro fertilization (IVF) were lower (P<0.01) for CIDR and MEL/CIDR than for Control and MEL groups (10.3% and 10.1% versus 20.0% and 18.5%, respectively). In Experiment 2, the number of visible follicles, and the rates of oocyte recovery and in vitro maturation were similar (P>0.10) for 1CIDR and 2CIDR groups (overall 27.3+/-3.2%, 92.1+/-2.7% and 90.2+/-1.9%, respectively). However, the rates of IVF were lower (P<0.01) for 2CIDR than 1CIDR group (30.2% versus 58.0%, respectively). In summary, when treatment with P4 commenced only 2 d before oocyte collection, rates of IVF were reduced in both experiments. Therefore, progestin treatment protocols used in ovine IVF programs should be carefully designed to minimize adverse effects on fertilization rates. In addition, melatonin treatment did not affect follicular development and oocyte quality for anestrous ewes.

Expression of connexin 43 and gap junctional intercellular communication in the cumulus-oocyte complex in sheep.

To evaluate the effects of FSH, LH and/or cAMP on expression of connexin 43 (Cx43) in the ovine cumulus-oocyte complex (COC) and gap junctional intercellular communication (GJIC) of cumulus cells, two experiments were carried out. In experiment 1, Cx43 was immunodetected in the COC, before or after maturation, obtained from non-treated or FSH-treated ewes. The expression of Cx43 in the COC was greater (P < 0.01) on day 16 than on day 15 of the estrous cycle. In vivo FSH treatment decreased (P < 0.02) Cx43 expression on day 16 but not on day 15 of the estrous cycle. In experiment 2, intact COCs or isolated cumulus cells obtained from small and large follicles from FSH-treated ewes were cultured with or without FSH, LH or cAMP agonist and evaluated for GJIC by laser cytometry. For large follicles, the basal rate of GJIC was greater (P < 0.01) for cumulus cells in intact COCs than for isolated cumulus cells. FSH increased (P < 0.04) GJIC in cumulus cells in intact COCs and tended to increase (P < 0.1) GJIC in isolated cumulus cells from small follicles but decreased (P < 0.01) GJIC in cumulus cells in intact COCs from large follicles. LH also increased (P < 0.01) GJIC in isolated cumulus cells from small follicles but decreased GJIC in intact COCs (P < 0.01) and isolated cumulus cells (P < 0.02) from large follicles. cAMP increased (P < 0.01) the GJIC in both intact COCs and cumulus cells from small and large follicles. These results indicate that day of estrous cycle, stage of maturation and duration of FSH treatment affect expression of Cx43 in ovine COCs. In intact COCs, GJIC in cumulus cells was enhanced, probably due to the presence of the oocyte. In addition, the effects of FSH and LH, but not cAMP, on GJIC of cumulus cells depended on the stage of follicular development and on the presence of the oocyte.