The significance of donor cell differentiation status for successful cloning by somatic cell nuclear transfer (SCNT) is unclear. Here, we cloned a new species, red deer (Cervus elaphus), from multipotent antler stem cells and their differentiated progeny. Cultured donor cell lines from male antlerogenic periosteum (AP) were left undifferentiated or chemically induced to initiate osteogenesis or adipogenesis. Based on their morphology and marker gene expression profile, donor cells were classified as undifferentiated AP cells, presumptive osteoblasts, or adipocytes. Adipocytes upregulated adipogenic markers procollagen type I alpha 2 (COL1A2), peroxisome proliferator-activated receptor gamma 2 (PPARG), and gylceraldehyde-3-phosphate dehydrogenase (GAPDH), and downregulated antlerogenic transcripts POU-domain class 5 transcription factor (POU5F1) and parathyroid hormone (PTH)-like hormone (PTHLH). Despite differences prior to NT, transcript abundance of donor-specific markers COL1A2, PPARG, GAPDH, and POU5F1 did not differ significantly in cloned blastocysts (P = 0.10, 0.50, 0.61, and 0.16, respectively). However, donor cell and blastocyst expression levels were completely different for most genes analyzed, indicating their successful reprogramming. The type of donor cell used for NT (AP, bone, and fat cells), had no effect on in vitro development to blastocysts (93 [38%] of 248 vs. 32 [44%] of 73 vs. 59 [32%] of 183, respectively). Likewise, development to weaning was not significantly different between the three cell types (2 [4%] of 46 vs. 2 [29%] of 7 vs. 4 [13%] of 31, for AP vs. bone vs. fat, respectively). Microsatellite DNA analysis confirmed that the eight cloned red deer calves were genetically identical to the cells used for NT.

It has been postulated that mammalian nuclear transfer (NT) cloning efficiency is inversely correlated with donor cell differentiation status. To test this hypothesis, we compared genetically identical and increasingly differentiated donors within the myogenic lineage. Bovine male fetal muscle cells were cultured for 1–6 days in vitro. The proportion of cells displaying the following antigens was quantified by immunofluorescence microscopy: MYOD1, MYF5, PAX7, MYOG, DES, MYH, and 5-Bromo-2-deoxyuridine. Based on the antigen profile of both bulk populations and individually size-selected cells prepared for NT, donors serum-starved for 1, 4, and 5 days were classified as myogenic precursors (MPCs), myotubes (MTs), and muscle-derived fibroblasts (MFs) with purities of 92%, 85%, and 99%, respectively. Expression of the following transcripts was measured by RT-PCR in 1) cells selected for NT, 2) metaphase II oocytes, 3) NT couplets, 4) NT reconstructs, 5) NT two-cell embryos, and 6) NT blastocysts: MYOD1, MYF5, PAX7, MYOG, MYF6, ACTB, and 18S rRNA. Muscle-specific genes were silenced and remained undetectable up to the blastocyst stage, whereas housekeeping genes 18S and ACTB continued to be expressed. Differentiation status affected development to transferable embryos (118 [23%] of 520 vs. 93 [11%] of 873 vs. 66 [38%] of 174 for MPC vs. MT vs. MF, respectively, P < 0.001). However, there were no significant differences in pregnancy rate and development to weaning between the cell types (pregnancy rate: 14 [64%] of 22 vs. 8 [35%] of 23 vs. 10 [45%] of 22, and development: 4 [18%] of 22 vs. 2 [9%] of 23 vs. 3 [14%] of 22 for MPC vs. MT vs. MF, respectively).

We recently demonstrated that mouse spermatozoa contain a mechanism to degrade their DNA into loop-sized fragments of about 50 kb, mediated by topoisomerase IIB, termed sperm chromatin fragmentation (SCF). SCF is often followed by a more complete digestion of the DNA with a sperm nuclease. When SCF-induced spermatozoa are injected into oocytes, the paternal pronuclei degrade their DNA after the initiation of DNA synthesis, but the maternal pronuclei are unaffected and replicate normally. Here, we tested whether the nuclease activity changes in spermatozoa of different maturation stages, and whether there is a functional relationship between the initiation of DNA synthesis and paternal DNA degradation induced by SCF in the zygote. We found that spermatozoa from the vas deferens have a much higher level of SCF activity than those from the cauda epididymis, suggesting that spermatozoa may acquire this activity in the vas deferens. Furthermore, paternal pronuclei formed in zygotes from injecting oocytes with SCF-induced vas deferens spermatozoa degraded their DNA, but this degradation could be inhibited by the DNA synthesis inhibitor, aphidicolin. Upon release from a 4 h aphidicolin-induced arrest, DNA synthesis was initiated in maternal pronuclei, while the paternal pronuclei degraded their DNA. Longer aphidicolin arrest resulted in the paternal pronuclei replicating their DNA, suggesting that delaying the initiation of DNA synthesis allowed the paternal pronuclei to overcome the SCF-induced DNA degradation pathway. These results suggest that the paternal DNA degradation, in oocytes fertilized with SCF-induced spermatozoa, is coupled to the initiation of DNA synthesis in newly fertilized zygotes.

Experiments were conducted to characterize the effects of oxytocin (OT) and vasopressin (VP) on epithelial cells isolated from human (1°HVD) and porcine (1°PVD) vas deferens and an immortalized epithelial cell line derived from porcine vas deferens (PVD9902 cells). Cultured monolayers were assessed in modified Ussing flux chambers and the OT- or VP-induced change in short circuit current (I_SC) was recorded. All cell types responded to basolateral OT or VP with a transient increase in I_SC that reached a peak of 3–5 μA cm⁻². Concentration-response curves constructed with 1°PVD and PVD9902 cells revealed that the apparent K_D (k_app) for OT was ∼100-fold less than the k_app for VP. Amplicons for the OT receptor (OXTR) and vasopressin type 2 and type 1a receptors (AVPR2 and AVPR1A) were generated with RT-PCR and the identification of each amplicon confirmed by sequence analysis. A selective antagonist for OXTR and AVPR1A fully blocked the effects of OT and partially blocked the effects of VP when assessed in both 1°PVD and PVD9902 monolayers. APVR2 antagonists blocked the effects of low (≤30 nM) but not high concentrations of VP, indicating that VP was affecting both AVPR2 and a second receptor subtype, likely OXTR or AVPR1A. Experiments employing chelerythrine demonstrated that OT stimulation of vas deferens monolayers requires PKC activity. Alternatively, VP (but not OT) increased the accumulation of cytosolic cAMP in vas deferens epithelial cells. Results from this study demonstrate that OT and VP can modulate ion transport across vas deferens epithelia by independent mechanisms. OT and VP have the potential to acutely change the environment to which sperm are exposed and thus, have the potential to affect male fertility.

Reduced litter sizes in mice missing pentraxin 3 (Ptx3) have been attributed to fertilization failure. However, our global gene expression studies showed high uterine Ptx3 expression at the implantation site in mice, suggesting its role in blastocyst implantation. We initiated molecular and genetic studies in mice to explore the importance of uterine Ptx3 in this process. We found that Ptx3 is expressed in a unique and transient fashion at implantation sites. With the initiation of implantation on midnight of Day 4 of pregnancy, Ptx3 is expressed exclusively in stromal cells at the site of blastocysts. On Day 5, its expression is more intense in decidualizing stromal cells, but it disappears on Day 6. The expression again becomes evident in the deciduum on Day 7, followed by a more robust expression on Day 8, particularly at the antimesometrial pole. From Day 9, with the initiation of placentation, Ptx3 expression becomes undetectable. These results suggest a role for PTX3 in implantation and decidualization. Indeed, deletion of Ptx3 results in both compromised implantation and decidualization. Interleukin 1B (IL1B), a known inducer of Ptx3, is also transiently expressed in stromal cells at the implantation site, suggesting that IL1B is an inducer of uterine Ptx3 expression. In fact, uterine Ptx3 expression follows that of Il1b induced by lipopolysaccharide treatment on Day 7 of pregnancy. Collectively, these findings provide evidence for an important role for PTX3 in implantation and decidualization. This study has clinical implications, since PTX3 is expressed in the receptive endometrium, and trophoblast cells influence decidual Ptx3 expression in humans.

Embryo implantation is a complex process that involves interactions between cell-surface and extracellular components of the blastocyst and the uterus, including blastocyst adhesion to the uterine luminal epithelium, epithelial basement membrane penetration and stromal extracellular matrix remodeling, angiogenesis, and decidualization. These processes all involve interactions with heparan sulfate (HS) proteoglycans, which harbor various growth factors and cytokines and support cell adhesion. Heparanase (HPSE) is an endo-beta-glucuronidase that cleaves HS at specific sites. HPSE also can act as an adhesion molecule independent of its catalytic activity. Thus, HPSE is a multifunctional molecule contributing to and modulating HS-dependent processes. Exogenously added HPSE improves embryo implantation in mice; however, no information is available regarding the normal pattern of HPSE expression and activity during the implantation process in any system. Using several approaches, including real-time RT-PCR, in situ hybridization, and immunohistochemistry, we determined that uterine HPSE expression increases dramatically during early pregnancy in mice. Heparanase mRNA and protein were primarily expressed in decidua and were rapidly induced at the implantation site. Uterine HPSE activity was characterized and demonstrated to increase >40-fold during early pregnancy. Finally, we demonstrate that the HPSE inhibitor PI-88 severely inhibits embryo implantation in vivo. Collectively, these results indicate that HPSE plays a role in blastocyst implantation and complements previous studies showing a role for HS-dependent interactions in this process.

The polycyclic aromatic hydrocarbon (PAH) 9,10-dimethyl-1,2-benzanthracene (DMBA) destroys primordial, primary, and secondary ovarian follicles in rodents, but its effects on antral follicles have received limited attention. PAHs are metabolized to reactive species, some of which can undergo redox cycling to generate reactive oxygen species (ROS). We previously showed that ROS initiate apoptosis in preovulatory follicles cultured without gonadotropin support and that glutathione (GSH) depletion induces apoptosis in the presence of gonadotropins. In the present study, we tested the hypothesis that DMBA induces apoptosis in preovulatory follicles, which is mediated by ROS and prevented by GSH. Preovulatory follicles were isolated from ovaries of 25-day-old rats 48 h after the injection of 10 IU of eCG and were cultured with DMBA in the presence of FSH for 2 to 48 h. DMBA induced granulosa cell (GC) and theca cell (TC) apoptosis at 48 h, as judged by TUNEL and activated caspase-3 immunostaining. DMBA treatment also increased the numbers of GCs and TCs that immunostained for the proapoptotic protein BAX. Follicular ROS levels were significantly increased in DMBA-treated follicles at 12, 24, and 48 h. GSH supplementation protected against and GSH depletion enhanced the induction of apoptosis in GCs and TCs by DMBA. These findings suggest that GSH is a critical protective mechanism against DMBA-induced apoptosis in antral follicles and that ROS generation may mediate DMBA-induced GC apoptosis.

The 5′AMP-activated protein kinase (AMPK) activation is involved in the meiotic maturation of oocytes in the ovaries of mice and pigs. However, its effects on the oocyte appear to be species-specific. We investigated the patterns of AMPK and mitogen-activated protein kinases (MAPK3/1) phosphorylation during bovine in vitro maturation (IVM) and the effects of metformin, an AMPK activator, on oocyte maturation in cumulus-oocyte complexes (COCs) and denuded bovine oocytes (DOs). In bovine COCs, PRKAA Thr172 phosphorylation decreased, whereas MAPK3/1 phosphorylation increased in both oocytes and cumulus cells during IVM. Metformin (5 and 10 mM) arrested oocytes at the GV stage in COCs but not in DOs. In COCs, this arrest was associated with the inhibition of cumulus cell expansion, an increase in PRKAA Thr172 phosphorylation, and a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. However, the addition of compound C (10 μM), an inhibitor of AMPK, accelerated the initiation of the GV breakdown (GVBD) process without any alteration of MAPK3/1 phosphorylation in oocytes from bovine COCs. Metformin decreased AURKA and CCNB1 protein levels in oocytes. Moreover, after 1 h of IVM, metformin decreased RPS6 phosphorylation and increased EEF2 phosphorylation, suggesting that protein synthesis rates were lower in oocytes from metformin-treated COCs. Most oocytes were arrested after the GVBD stage following the treatment of COCs with the MEK inhibitor, U0126 (100 micromoles). Thus, in bovine COCs, metformin blocks meiotic progression at the GV stage, activates PRKAA, and inhibits MAPK3/1 phosphorylation in both the oocytes and cumulus cells during IVM. Moreover, cumulus cells were essential for the effects of metformin on bovine oocyte maturation, whereas MAPK3/1 phosphorylation was not.

Immunohistochemistry was used to examine GCNA1, a germ cell-specific protein, together with DMRT1 (Doublesex and Mab-3-related transcription factor-1), a transcription factor implicated in Sertoli cell and germ cell function, in order to resolve DMRT1′s cellular profile during pre- and postnatal gonad development in the mouse. In the indifferent gonad (10.5–11.5 days postcoitus [dpc]), DMRT1 localized to somatic cells and GCNA1 germ cells and was indistinguishable in males and females. By 12.5 dpc, a clear sexual preference for DMRT1 in male somatic cells was observed, with male DMRT1 localized to testicular cords and more abundant in Sertoli cells than in germ cells and female DMRT1 diffusely labeled and markedly lower in somatic cells than in germ cells. A male somatic preference continued throughout development, with DMRT1 evident in Sertoli cells at all ages examined and absent in ovarian somatic cells from 13.5 dpc onward. In contrast, expression in primordial germ cells was not sexually distinct, and both sexes showed DMRT1 increasing through 13.5 dpc and absent by 15.5 dpc. Notably, sexual differences in germ cell DMRT1 were detected after birth, when it was detected only in spermatogonia of the testis. Colocalization of DMRT1 with proliferation markers KI67 and proliferating cell nuclear antigen (PCNA) and stem cell markers OCT4 (also known as POU5F1) and NGN3 indicated that, in postnatal testes, DMRT1 was present in both stem and proliferating spermatogonia. Together, the findings implicate opposite functions for DMRT1 in somatic and germ cells of the testis. In Sertoli cells, DMRT1 expression correlated with differentiation, whereas in germ cells, it suggested a role in expansion and maintenance of undifferentiated spermatogonia.

Eppin (SPINLW1; serine peptidase inhibitor-like with Kunitz and WAP domains 1 (eppin); epididymal protease inhibitor) coats the surface of human ejaculate spermatozoa and originates from Sertoli and epididymal epithelial cells. In this study, we have isolated native eppin from ejaculate supernatants (seminal plasma) and washed ejaculate spermatozoa using column chromatography and two-dimensional SDS-PAGE, and identified by mass spectrometry and Western blots an eppin protein complex (EPC) containing lactotransferrin (LTF; also known as lactoferrin), clusterin (CLU), and semenogelin (SEMG1). To confirm the association of eppin with LTF, CLU, and SEMG1, antibodies to CLU and LTF were used to immunoprecipitate CLU and LTF from human sperm lysates. In both cases identical results were obtained, namely, the immunoprecipitate of the EPC. Additionally, we localized eppin, LTF, and CLU in human Sertoli cells and on human testicular and ejaculate spermatozoa, implying that the EPC is present on spermatozoa from the time they leave the seminiferous tubule. On ejaculate spermatozoa eppin, LTF, and CLU colocalize on the tail. The identification of the EPC components suggests that LTF, CLU, and/or eppin receptors may function as sperm plasma membrane receptors for the EPC, implicating the complex as a central player in a network of protein-protein interactions on the human sperm surface. The EPC may provide a surface network with microbicidal properties that protects spermatozoa as well as regulates the sperm's transition to a motile, capacitated sperm.

Insulin-like growth factor (IGF)-binding protein (IGFBP) 7 is a secreted protein that regulates cellular proliferation, adhesion, and angiogenesis, and has low affinity for IGF compared with that of IGFBP1-IGFBP6. We sought to determine whether IGFBP7 is present in follicular fluid and to elucidate whether IGFBP7 participates in the steroidogenesis of rat mature follicles. Follicular fluid and granulosa cells (GCs) were collected from immature rats 2 days after their treatment with equine chorionic gonadotropin (eCG). IGFBP7 protein was detected in the follicular fluid and the conditioned medium of cultured ovarian GCs by immunoblot analysis. When subconfluent GCs were cultured and treated with FSH and activin, coincubation with FSH and activin markedly increased GC expression of Cyp19a1 (aromatase) mRNA and 17beta-estradiol (E₂) secretion. The addition of recombinant murine IGFBP7 to these cultures decreased in the activin-enhanced, FSH-stimulated Cyp19a1 mRNA levels in the cells and suppressed the 17beta-E₂ levels in the culture medium. Treatment of GCs with Igfbp7-specific small interfering RNA (siRNA), which knocked down Igfbp7 expression, increased the FSH-stimulated levels of Cyp19a1 but not Cyp11a1 expression. Basal and FSH-stimulated 17beta-E₂ secretion into the culture medium was also enhanced by Igfbp7 siRNA. These results suggest that IGFBP7 suppresses estrogen production in GCs. These observations support the notion that this protein, which is secreted into the follicular fluid, may serve as an intraovarian factor that negatively regulates GC differentiation.

Estradiol and progesterone induction of the LH surge in ovariectomized female rats requires concurrent activation of brain insulin-like growth factor 1 (IGF1) receptors. The present study determined whether brain IGF1 receptor signaling is required for estrous cyclicity in gonadally intact female rats. A selective IGF1 receptor antagonist (JB-1) or vehicle was continuously administered into the third ventricle by osmotic minipumps. Following surgical placement of the minipumps, all rats temporarily reduced food intake, lost weight, and suspended estrous cycles. Control rats resumed cycles within a few days and exhibited compensatory hyperphagia until they returned to presurgical body weight. Animals receiving JB-1 had severely delayed or absent estrous cycles, failed to show rebound feeding, and regained body weight more slowly. Vehicle-infused animals pair fed to JB-1-treated rats had even lower body weights but resumed estrous cycles sooner than those given drug alone. Chronic infusion of IGF1 alone had no effect on any of these parameters, but coinfusion of IGF1 with the antagonist completely reversed JB-1 effects on food intake and estrous cyclicity and partially reversed the effects on body weight. There were no significant differences in the expression of galanin-like peptide (Galp) or Kiss1 mRNA in the arcuate or periventricular hypothalamic area of control and JB-1-treated animals at a time point when food intake and estrous cycles were different between controls and JB-1-treated rats. These data suggest that brain IGF1 signaling is necessary for normal estrous cycles as well as compensatory hyperphagia and that IGF1 modulation of the reproductive axis is not secondary to reduced food intake.

Transcripts encoding a fatty acid-binding protein (FABP), Fabp11, and two isoforms of very low-density lipoprotein receptor (Vldlr; vitellogenin receptor) were characterized from the ovary of Senegalese sole (Solea senegalensis). Phylogenetic analyses of vertebrate FABPs demonstrated that Senegalese sole Fabp11, as zebrafish (Danio rerio) homologous sequences, is part of a newly defined teleost fish FABP subfamily that is a sister clade of tetrapod FABP4/FABP5/FABP8/FABP9. RT-PCR revealed high levels of vldlr transcript splicing variants in the ovaries and, to a lesser extent, in somatic tissues, whereas fabp11 was highly expressed in the ovaries, liver, and adipose tissue. In situ hybridization analysis showed vldlr and fabp11 mRNAs in previtellogenic oocytes, whereas no hybridization signals were detected in the larger vitellogenic oocytes. Transcript expression of fabp11 was strongly upregulated in somatic cells surrounding atretic follicles. Real-time quantitative RT-PCR demonstrated that ovarian transcript levels of vldlr and fabp11 had a significant positive correlation with the percentage of follicles in previtellogenesis and atresia, respectively. These results suggest that the expression level of vldlr transcripts may be used as a precocious functional marker to quantify the number of oocytes recruited for vitellogenesis and that fabp11 mRNA may be a very useful molecular marker for determining cellular events and environmental factors that regulate follicular atresia in fish.

17-beta hydroxysteroid dehydrogenase type 2 (HSD17B2) oxidizes estradiol to estrone, testosterone to androstenedione, and 20 alpha-dihydroprogesterone to progesterone. HSD17B2 is highly expressed in human placental tissue where it is localized to placental endothelial cells lining the fetal compartment. The aim of this study was to investigate the effects of potential regulatory factors including progesterone, estradiol, and retinoic acid (RA) onHSD17B2 expression in primary human placental endothelial cells in culture.HSD17B2 mRNA expression was not regulated by progesterone, the progesterone agonist R5020, or estradiol treatment. RA significantly induced HSD17B2 mRNA levels and enzyme activity in a dose- and time-dependent manner. Maximal stimulation occurred at Hour 48 at an RA concentration of 10⁻⁶ M. Both retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA) were readily detected by immunoblotting in isolated placental endothelial cells. RNA interference directed against RARA or RXRA led to reduced basal levels of HSD17B2 mRNA levels and significantly abolished RA-stimulated HSD17B2 expression. Together, these data indicate that regulation of HSD17B2 mRNA levels and enzymatic activity by RA in the placenta is mediated by RARA and RXRA.

Endothelin 1 (EDN1) plays a primary role in the pathophysiology of hypoxia-induced fetal growth restriction in the rat. In this study we evaluated the effects of chronic maternal hypoxia on the expression of endothelin and its receptors and on receptor binding activity in the uterus and placenta of the rat, in order to elucidate their roles in hypoxia-induced fetal growth restriction. Timed-pregnant Sprague-Dawley rats were maintained in either a normoxic or a normobaric hypoxic (12% O₂) atmosphere from Gestational Days 18–21. Uterine and placental tissues collected on Gestational Day 21 were assayed for Edn1, Ednra, and Ednrb (endothelin receptors) mRNA expression by real-time quantitative RT-PCR, for localization of EDN1 and its receptors by immunohistochemistry, for EDNRA and EDNRB protein expression by Western blot, and for receptor binding activity by homologous competitive binding assays. EDN1 mRNA expression was significantly increased in the hypoxic placenta, but not in the uterus, compared with normoxic controls. Immunohistochemistry revealed increased EDN1 specifically in the labyrinth of the placenta. Receptor mRNA levels were not significantly affected by hypoxia, but EDNRA protein expression was significantly decreased specifically in the uterine placental beds. Receptor binding decreased significantly in response to hypoxia in all tissues investigated, compared with controls. These results suggest that chronic maternal hypoxia results in increased expression of EDN1 in the placenta but not in the uterus, and that reduced binding activity, rather than regulation of receptor expression, is a mechanism by which these tissues regulate the local hemodynamic response to increased endogenous placental EDN1 in the setting of hypoxia.

A proteomics screen of human placental microvillous syncytiotrophoblasts (STBs) revealed the expression of dysferlin (DYSF), a plasma membrane repair protein associated with certain muscular dystrophies. This was unexpected given that previous studies of DYSF have been restricted to skeletal muscle. Within the placenta, DYSF localized to the STB and, with the exception of variable labeling in the fetal placental endothelium, none of the other cell types expressed detectable levels of DYSF. Such restricted expression was recapitulated using primary trophoblast cell cultures, because the syncytia expressed DYSF, but not the prefusion mononuclear cells. The apical plasma membrane of the STB contained ∼4-fold more DYSF than the basal membrane, suggesting polarized trafficking. Unlike skeletal muscle, DYSF in the STB is localized to the plasma membrane in the absence of caveolin. DYSF expression in the STB was developmentally regulated, because first-trimester placentas expressed ∼3-fold more DYSF than term placentas. As the current literature indicates that few cell types express DYSF, it is of interest that the two major syncytial structures in the human body, skeletal muscle and the STB, express this protein.

The negative effect of estradiol-17β (E2) on LH, based on exogenous E2 treatments, and the reciprocal effect of LH on endogenous E2, based on hCG treatments, were studied throughout the ovulatory follicular wave during a total of 103 equine estrous cycles in seven experiments. An initial study developed E2 treatment protocols that approximated physiologic E2 concentrations during the estrous cycle. On Day 13 (ovulation = Day 0), when basal concentrations of E2 and LH precede the ovulatory surges, exogenous E2 significantly depressed LH concentrations to below basal levels. Ablation of all follicles ≥10 mm when the largest was ≥20 mm resulted in an increase in percentage change in LH concentration within 8 h that was greater (P < 0.03) than for controls or E2-treated/follicle-ablated mares. Significant decreases in LH occurred when E2 was given when the largest follicle was either ≥25 mm, ≥28 mm, ≥35 mm, or near ovulation. Treatment with 200 or 2000 IU of hCG did not affect E2 concentrations during the initial portion of the LH surge (largest follicle, ≥25 mm), but 2000 IU significantly depressed E2 concentrations before ovulation (largest follicle, ≥35 mm). Results indicated a continuous negative effect of E2 on LH throughout the ovulatory follicular wave and may be related to the long LH surge and the long follicular phase in mares. Results also indicated that a reciprocal negative effect of LH on E2 does not develop until the E2 surge reaches a peak.

Many Ca² channel proteins have been detected in mammalian sperm, but only the four CATSPER channels have been clearly shown to be required for male fertility. Ca² entry through the principal piece-localized CATSPER channels has been implicated in the activation of hyperactivated motility. In the present study, we show that the Ca² entry also triggers a tail-to-head Ca² propagation in the mouse sperm. When activated with 8-Br-cAMP, 8-Br-cGMP, or alkaline depolarization, a CATSPER-dependent increase in intracellular Ca² concentration starts in the principal piece, propagates through the midpiece, and reaches the head in a few seconds. The Ca² propagation through the midpiece leads to a Ca² -dependent increase in NADH fluorescence. In addition, CatSper1-mutant sperm have lower intracellular ATP levels than wild-type sperm. Thus, a Ca² influx in the principal piece through CATSPER channels can not only initiate hyperactivated motility, but can also trigger a tail-to-head Ca² propagation that leads to an increase in [NADH] and may regulate ATP homeostasis.

The activation of AKT (also called protein kinase B) is thought to be a critical step in the phosphoinositide 3-kinase pathway that regulates cell growth and differentiation. In this report, we investigated the role of AKT in the regulation of mouse early embryo development. Injection of mRNA coding for a constitutively active myristoylated AKT (myr-Akt1) into one-cell stage fertilized eggs induced cell division more effectively than injection of wild-type AKT (Akt1-WT) mRNA, whereas microinjection of mRNA of kinase-deficient AKT (Akt1-KD) delayed the first mitotic division. Meanwhile, microinjection of different kinds of mRNA of AKT affected the phosphorylation status of CDC2A-Tyr15 and the activation of M-phase promoting factor (MPF). To investigate the intermediate factor between AKT and MPF, we then injected one-cell stage eggs first with Akt1-WT mRNA or myr-Akt1 mRNA and then with mRNA encoding either wild-type CDC25B (Cdc25b-WT) or a AKT-nonphosphorylatable Ser351 to Ala CDC25B mutant (Cdc25b-S351A). Cdc25b-S351A strongly inhibited the effect of AKT. Therefore, AKT causes the activation of MPF and strongly promotes the development of one-cell stage mouse fertilized eggs by inducing AKT-dependent phosphorylation of CDC25B, a member of the CDC25 phosphatase family. Our finding that CDC25B acts as a potential target of AKT provides new insight into the effect of AKT in the regulation of early development of mouse embryos.

Cytoplasmic transfer is an assisted reproductive technique that involves the infusion of ooplasm from a donor oocyte into a recipient oocyte of inferior developmental competence. Although this technique has shown some success for couples with recurrent in vitro fertilization failure, it results in mitochondrial heteroplasmy in the offspring, defined as the presence of two different mitochondrial genomes in the same individual. Because the long-term health consequences of mitochondrial heteroplasmy are unknown, there is a need for appropriate animal models to evaluate any physiological changes of dual mtDNA genotypes. This longitudinal study was designed as a preliminary screen of basic physiological functions for heteroplasmic mice (NZB mtDNA on a BALB/cByJ background). The mice were tested for cardiovascular and metabolic function, hematological parameters, body mass analysis, ovarian reserve, and tissue histologic abnormalities over a period of 15 mo. Heteroplasmic mice developed systemic hypertension that corrected over time and was accompanied by cardiac changes consistent with pulmonary hypertension. In addition, heteroplasmic animals had increased body mass and fat mass compared with controls at all ages. Finally, these animals had abnormalities in electrolytes and hematological parameters. Our findings suggest that there are significant physiological differences between heteroplasmic and control mice. Because ooplasm transfer appears to be consistently associated with mitochondrial heteroplasmy, children conceived through ooplasm transfer should be closely followed to determine if they are at risk for any health problems.

The amnion is the inner of two membranes surrounding the fetus. That it arises from embryonic epiblast cells prior to gastrulation suggests that it may retain a reservoir of stem cells throughout pregnancy. We found that human amniotic epithelial cells (hAECs) harvested from term-delivered fetal membranes express mRNA and proteins present in human embryonic stem cells (hESCs), including POU domain, class 5, transcription factor 1; Nanog homeobox; SRY-box 2; and stage-specific embryonic antigen-4. In keeping with possible stem cell-like activity, hAECs were also clonogenic, and primary hAEC cultures could be induced to differentiate into cardiomyocytic, myocytic, osteocytic, adipocytic (mesodermal), pancreatic, hepatic (endodermal), neural, and astrocytic (neuroectodermal) cells in vitro, as defined by phenotypic, mRNA expression, immunocytochemical, and/or ultrastructural characteristics. However, unlike hESCs, hAECs did not form teratomas upon transplantation into severe combined immunodeficiency mice testes. Last, using flow cytometry we have shown that only a very small proportion of primary hAECs contain class IA and class II human leukocyte antigens (HLAs), consistent with a low risk of tissue rejection. However, following differentiation into hepatic and pancreatic lineages, significant proportions of cells contained class IA, but not class II, HLAs. These observations suggest that the term amnion, an abundant and easily accessible tissue, may be a useful source of multipotent stem cells that possess a degree of immune privilege.